StemCell Therapy

Today marked some wheeling and dealing in the life sciences industry as several companies licensed products or invested in other companies. Heres a look.

Eli Lillyand Asahi Kasei Pharma Eli Lilly and Company inked a license agreement with Tokyos Asahi Kasei Pharma Corporation. In it, Lilly picks up exclusive rights to AK1780 from Asahi. The drug is an oral P2X7 receptor antagonist that recently finished a Phase I dosing study. P2X7 receptors are associated with neuroinflammation that drives chronic pain conditions.

Under the terms of the deal, Lilly will handle future global development and regulatory activities. Lilly is paying Asahi Kasei Pharma $20 million up front and the Japanese company is eligible for up to $210 million in development and regulatory milestones. Asahi Kasei will retain the rights to promote the drug in Japan and China, including Hong Kong and Macau. If it makes it to market, Asahi Kesei will also be eligible for up to $180 million in sales milestones and tiered royalties from the mid-single to low-double digits.

Lilly is committed to developing novel medicines that may provide relief for patients suffering with various pain conditions, said Mark Mintun, vice president of pain and neurodegeneration research at Lilly. We are pleased to license this molecule from Asahi Kasei Pharma, and look forward to developing it further as a potential treatment for neuroinflammatory pain conditions.

Artiva Biotherapeutics and Merck San Diego-based Artiva Biotherapeutics announced an exclusive global collaboration and license agreement with Merck to develop novel chimeric antigen receptor (CAR)-NK cell therapies against solid tumor-associated antigens. They will leverage Artivas off-the-shelf allogeneic NK cell manufacturing platform and its proprietary CAR-NK technology. At first, the collaboration will include two CAR-NK programs with an option for a third. None of them are currently part of Artivas current or planned pipeline. Artiva will develop the programs through the first GMP manufacturing campaign and to preparation for the Investigational New Drug (IND) application, where Merck will take over clinical and commercial development.

Merck is paying Artiva $30 million upfront for the first two programs and another $15 million if Merck chooses to go ahead with the third. Artiva will be up for development and commercial milestones up to $612 million per program and royalties on global sales. Merck also is ponying up research funding for each program.

Our NK platform has been developed to be truly off-the-shelf and we believe it will be further validated by this exclusive collaboration with Merck, as we work together to bring cell therapies to all patients who may benefit, said Peter Flynn, chief operating officer of Artiva.

NeuBase Therapeutics and Vera Therapeutics Pittsburgh-based NeuBase Therapeutics announced a binding agreement to acquire infrastructure, programs and intellectual property for several peptide-nucleic acid (PNA) scaffolds from Vera Therapeutics, formerly called TruCode Gene Repair. Vera is based in South San Francisco. On January 19, Vera announced its launch with a $80 million Series C financing led by Abingworth LLP and joined by Sofinnova Investments, Longitude Capital, Fidelity Management & Research Company, Surveyor Capital, Octagon Capital, Kliner Perkins, GV and Alexandria Venture Investments. Veras lead clinical candidate is atacicept, a novel B cell and plasma cell inhibitor being developed for patients with IgA nephropathy (IgAN).

The technology acquired by NeuBase has shown the ability to resolve disease in genetic models of several disease indications. NeuBase is focused on genetic medicine.

With this acquisition, we enhance our PATrOL platform, furthering our unique ability to directly engage and correct malfunctioning genes with exquisite precision to address the root causes of a wide variety of human diseases, said Dietrich A. Stephan, chief executive officer of NeuBase. These assets extend and refine our PATrOL platforms capabilities and accelerates, through our Company, to bring the rapidly growing genetic medicines industry toward a single high-impact focal point. We are committed to advancing our pipeline and candidates to the clinic and to exploiting the full potential of PNA technology to continue creating value for our shareholders and importantly, for patients.

Bio-Techne Corporation and Changzhou Eminence Biotechnology Co Minneapolis-based Bio-Techne Corporation announced an initial minority strategic equity investment in Chinas Changzhou Eminence Biotechnology Co. Eminence plans to use the financing to expand its manufacturing capacity and increase the service capabilities of its China-based GMP media production facility. Eminence, based in Changzhou City, Jiangsu, China, launched in 2016 and initially focused on manufacturing and selling best-in-class media to life science companies, including Chinese Hamster Ovary (CHO) cells and other serum-free media products and services. The company is currently finishing and scaling its GMP production facility, which it plans to complete by the end of this year.

With our protein analysis instruments and expanding GMP protein capabilities, Bio-Techne continues to expand its offering of products and tools critical for bioprocessing, said Chuck Kumeth, president and chief executive officer of Bio-Techne. Investing in Eminence not only gives Bio-Techne a foothold in providing additional products and services to support the critical needs of the rapidly growing Chinese biopharmaceutical industry, but also fits extremely well with our existing high-growth product portfolio in China. We look forward to working with the Eminence team.

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4 New Life Sciences Licensing Deals and Investments to Watch - BioSpace

Dr. Li has over 30 years of experience in basic and applied biomedical research. She joins Neurophth from the University of Louisville School of Medicine, where she was a professor in the department of Ophthalmology and Visual Sciences for over 14 years. Her research focuses on the role of Hippo/YAP1 signaling pathway on different stages of ocular development, NF-kB/IKK2 inhibition of neovascularization, and gene discovery screening for eye diseases using mouse models.

Throughout her career, Dr. Li has contributed to more than 45 publications in journals including Investigative Ophthalmology & Visual Science (IOVS), Proceedings of the National Academy of Sciences of the United States of America(PNAS), Nature Review Immunology, and Science. She is currently the editorial board member of Scientific Reportsand Source Journal of Ophthalmology. Dr. Li holds a Ph.D. in cell biology from the Washington University in St. Louis and obtained both her Bachelor's and Master of Science degrees in Genetics from Beijing University.

"We are thrilled to have Dr. Li on our team, bringing over 3 decades of her diverse experience in basic and applied biomedical research," said Bin Li, M.D., Ph.D., Founder and Chairman of the Board of Neurophth. "Given her prior experience at Baylor College of Medicine mentored by Dr. Savio Woo, an internationally recognized expert in molecular human genetics and gene therapy, and Dr. Mark Kay, a leading researcher in the fields of AAV gene therapy and the current Head of Division of Human Gene Therapy at the Stanford University School of Medicine, Dr. Li has extensive knowledge in gene therapy for hepatic deficiencies, ocular diseases, and viral vector reconstruction."

"We are excited to have Qiutang join and expand our exceptional research and development team. She brings a wealth of experience in gene therapies for ocular diseases to Neurophth," said Alvin Luk, Ph.D., M.B.A., C.C.R.A., Chief Executive Officer at Neurophth. "Her deep understanding of viral vector design and animal models in the inhibition of neovascularization for ocular diseases, such as age-related macular degeneration and diabetic retinopathy, further bolsters our ability to deliver on our growing pipeline of clinical programs and platform capabilities."

"It has been captivating to watch the scale, scope, and speed with which Neurophth has successfully transformed itself into an innovative and diversified gene therapy company," said Dr. Li. "I look forward to being a part of Neurophth team as the company executes the next stage of its growth strategy and expands its pipeline of gene therapy candidates focused on ocular and non-ocular diseases, building a brighter future for patients worldwide."

About Neurophth

Neurophth is China's first gene therapy company in ophthalmic diseases.Headquartered in Wuhan with subsidiaries in Shanghai, Suzhou, and the U.S., Neurophth, a fully integrated company, is striving to discover and develop gene therapies for patients suffering from blindness and other eye diseases globally. Our AAV validated platform which has been published in Nature - Scientific Reports, Ophthalmology, and EBioMedicine, successfully delivered proof-of-concept data with investigational gene therapies in the retina. Our most advanced investigational candidate, NR082 (rAAV2-ND4), in development for the treatment ofND4-mutated LHON, has received orphan drug designations in theU.S. The pipeline also includesND1-mutated LHON, autosomal dominant optic atrophy, glaucoma, wAMD/DME, and other preclinical candidates. Neurophth has initiated the scaling up in-house process in single-use manufacturing technologies to support future commercial demand at the Suzhou facility. To learn more about us and our growing pipeline, visitwww.neurophth.com.

SOURCE Neurophth Therapeutics, Inc.

http://www.neurophth.com

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Neurophth Therapeutics Further Expands Ocular Gene Therapy Expertise with Appointment of Qiutang Li, Ph.D., as Chief Scientific Officer - PRNewswire

Over the next 10 years, gene therapies are expected come into their own as a treatment option for a variety of diseases. So far, two such therapies have snagged regulatory approval, Novartis Zolgensma for spinal muscular atrophy, and Sparks Luxturna for a rare form of genetic blindness. More are waiting their turn.

Multiple companies are delving into gene therapy research with hopes of developing a one-time treatment for devastating genetic diseases. Gene therapies offer great reward in the form of treating various devastating diseases, but there are also significant risks. Over the past year, several clinical studies have been halted or scrapped due to safety concerns.

Bay Area-based Audentes Therapeutics had a temporary hold placed on the gene therapy under development for X-linked myotubular myopathy following reports of several patient deaths. That hold has since been lifted by the U.S. Food and Drug Administration. Uniqure also saw a hold placed on its hemophilia B trial after a patient in the study developed liver cancer. The hold was placed weeks after the company announced promising Phase III results at a conference in December.

Despite those risks, hundreds of millions of dollars in research dollars are being invested in gene therapies because of the potential near-curative capabilities the technology could offer. In December, life sciences giant Bayer launched a cell and gene therapy platform within its pharmaceutical division in order to become a leading company within a rapidly emerging and evolving field that offers the potential of life-saving therapies. Eli Lilly also dove into the field in December with the acquisition of Prevail Therapeutics. That deal was expected to extend Eli Lillys research efforts through the creation of a gene therapy program that will be anchored by Prevail's portfolio of clinical-stage and preclinical neuroscience assets.

This week, German scientists reported they were able to use gene therapy to help paralyzed mice run again. The researchers were able to genetically engineer a unique protein dubbed hyper-interleukin-6, which was then able to stimulate the regeneration of nerve cells in the visual system. A few weeks after the treatment, the injured animals were able to walk again.

Scientists in China announced the development of a gene therapy that could potentially reverse the effects of ageing. Initial research was conducted with mice, but if it is proven to be safe, human testing could begin. As Reuters reported, the method involved inactivating a gene called kat7 which the scientists found to be a key contributor to cellular ageing. Researchers used CRISPR/Cas9 to screen thousands of genes for those which were particularly strong drivers of cellular senescence, the term used to describe cellular ageing, Reuters said.

Earlier this month, a public-private partnership in Boston formed to open a new facility to boost advances in cell and gene therapies. This creation of this new facility is being helmed by Harvard University and the Massachusetts Institute of Technology. Those prestigious universities are partnering with industry members such as Fujifilm Diosynth Biotechnologies, Cytivia and Alexandria Real Estate Equities, as well as multiple research hospitals. Part of the goal of this new institute, which is still unnamed at this point, is to boost the supply of materials for research and early clinical studies, provide space for some research and also offer training in equipment used for gene therapies, The Harvard Gazette reported this week.

On Monday, Curadigm, a subsidiary of France-based Nanobiotix, forged a collaboration with Sanofi to assess if that companys Nanoprimer technology is a promising option to significantly improve gene therapy development. The goal of the project is to establish proof-of-concept for the Nanoprimer as a combination product that could improve treatment outcomes for gene therapy product candidates.

Many promising nucleic acid-based therapeutics administered intravenously are limited in their efficacy due to rapid clearance in the liver, which prevents these therapies from reaching the necessary accumulation in target tissues to generate their intended outcomes. Additionally, accumulation in the liver, rather than in the target tissues, can lead to dose-limiting hepatic toxicity, Nanobiotix said in its announcement. The Nanoprimer is designed to precisely and temporarily occupy therapeutic clearance pathways in the liver. Delivered intravenously, immediately prior to the recommended therapy, the technology acts to prevent rapid clearancethereby increasing bioavailability and subsequent accumulation of therapeutics in the targeted tissues.

The Nanoprimer is a combination product candidate that does not alter or modify the therapies it is paired with, which means if the research with Sanofi is successful, Curadigm could seek out other opportunities for its technology.

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Are Gene Therapies the Medicine of the Future? - BioSpace

Two different gene therapies have been used to mitigate a mechanism underlying development of sickle cell disease (SCD) and transfusion-dependent -thalassemia (TDT), and both have demonstrated clinical success in separate, concurrently published trials.

The hemoglobinopathies manifest after fetal hemoglobin synthesis is replaced by adult hemoglobin in individuals who have inherited a mutation in the hemoglobin subunit gene (HBB).Identifying factors in the conversion from fetal to adult hemoglobin synthesis, however, has provided potential targets for therapeutic intervention.

Gene therapy that can safely arrest or reduce the conversion offers the potential for a one-time treatment to obviate the need for lifetime transfusions and iron chelation for patients with TDT, and the pain management, transfusions and hydroxyurea administration for those with SCD.

Two groups of investigators have now reported in The New England Journal of Medicine that, using different gene therapy techniques that target the transcription factor, BCL11a, involved in the globin switching, they have improved clinical outcomes in patients with TDT and with SCD.

In an editorial in the issue featuring the 2 studies, Mark Walters, MD, Blood and Marrow Transplant Program, University of California, San Francisco-Benioff Children's Hospital, welcomed the breakthroughs.

"These trials herald a new generation of broadly applicable curative treatments for hemoglobinopathies," Walters wrote.

In one clinical trial with 2 patients, one with TDT and the other with SCD, Haydar Frangoul, MD, MS, Medical Director, Pediatric Hematology/Oncology, Sarah Cannon Center for Blood Cancer at the Children's Hospital at Tristar Centennial, and colleagues administered CRISPR-Cas9 gene edited hematopoietic stem and progenitor cells (HSPCs) with reduced BCL11A expression in the erythroid lineage.

The product, CTX001, had been shown in preclinical study to restore -globulin synthesis and reactivate production of fetal hemoglobin. Both patients underwent busulfan-induced myeloablation prior to receiving the treatment.

The investigators suggested that the CRISPR-Cas9-based gene-edited product could change the paradigm for patients with these conditions, if it was found to successfully and durably graft, produce no "off-target" editing products, and, importantly, improve clinical course.

"Recently approved therapies, including luspatercept and crizanlizumab, have reduced transfusion requirements in patients with TDT and the incidence of vaso-occlusive episodes in those with SCD, respectively, but neither treatment addressed the underlying cause of the disease nor fully ameliorates disease manifestations," Frangoul and colleagues wrote.

The investigators reported that both patients had "early, substantial, and sustained increases" in pancellularly distributed fetal hemoglobin levels during the 12-month study period. Further, the patients no longer required transfusions, and the patient with SCD no longer experienced vaso-occlusive episodes after the treatment.

In commentary accompanying the report, Harry Malech, MD, Genetic Immunotherapy Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Disease (NIAID), National Institutes of Health (NIH), Bethesda, MD, described the investigators' application of the gene-editing technology as a "remarkable level of functional correction of the disease phenotype."

"With tangible results for their patients, Frangoul et al have provided a proof of principle of the emerging clinical potential for gene-editing treatments to ameliorate the burden of human disease," Malech pronounced.

In the other published trial, with 6 patients with SCD, Erica Esrick MD, Dana-Farber/Boston Children's Cancer and Blood Disorders Center, Harvard Medical School, and colleagues described results with infusion of gene-modified cells derived from lentivirus insertion of a gene that knocks down BCL11a by encoding an erythroid-specific, inhibitory short-hairpin RNA (shRNA).

The severity of SCD that qualified patients for enrollment included history of stroke (n = 3), frequent vaso-occlusive events (n = 2) and frequent episodes of priapism (1).Patients were followed for 2 years, and offered enrollment in a 13-year long-term follow-up study.The infusion of the experimental drug BCH-BB694, from the short hairpin RNA embedded within an endogeonous micro RNA scaffold (termed a shmiR vector), was initiated after myeloablation with busulfan.

Esrick and colleagues reported that, at median follow-up of 18 months (range, 7-29), all patients had engraftment and a robust and stable HbF induction broadly distributed in red cells.Clinical manifestations of SCD were reduced or absent during the follow-up period; with no patient having a vaso-occlusive crisis, acute chest syndrome, or stoke subsequent to the gene therapy infusion.Adverse events were consistent with effects of the preparative chemotherapy.

"The field of autologous gene therapies for hemoglobinopathies is advancing rapidly," Esrick and colleagues reported, "including lentiviral trials of gene addition in which the nonsickling hemoglobin is formed from an exogenous -globin or modified -globin gene."

Walters agreed that gene therapy is rapidly progressing, but expressed concern about the large gap that looms between laboratory bench and clinical bedside, particularly for this affected population.

"Access to and delivery of these highly technical therapies in patients with sickle cell disease will be challenging and probably limited to resource-rich nations, at least in the short term," Walters commented.

The studies, CRISPR-Cas9 Gene Editing for Sickle Cell Disease and -Thalassemia, as well as, Post-Transcriptional Genetic Silencing of BCL11A to Treat Sickle Cell Disease, were published online in The New England Journal of Medicine.

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Two Gene Therapies Fix Fault in Sickle Cell Disease and -thalassemia - MD Magazine

With the rising demand for cell and gene therapies, the need for manufacturing innovation has never been higher. A surge of deals and expansions in the last year is fuelling the push to truly make these therapies widely available and affordable.

Cell and gene therapies offer huge potential to treat a wide range of diseases including cancer, neurological, and genetic diseases. They have even shown promise to treat the symptoms of Covid-19.

The amount of academic and early-stage biotech research in this area has skyrocketed over the last few years. According to the Alliance for Regenerative Medicine, there are currently 1,220 ongoing clinical trials in this space, 152 of which are at phase III. Despite the global pandemic, investment in this area is also at a record high around the world, with the equivalent of 15.7B invested in 2020, a figure double that of 2019.

But research alone cannot get these complex treatments to patients. The sharp discrepancy between the high number of products in early-stage development and the still very small number that have made it onto the market, as well as their cost, speaks to the impact and importance of cost-effective and scalable manufacturing, Ryan Cawood, CEO of Oxgene (previously Oxford Genetics), told me. Oxgene is a UK biotech aiming to improve manufacturing for cell and gene therapies.

To meet this challenge, cell and gene therapy producers are exploding into motion. With 2021 only just getting started, weve seen manufacturing deals between Vigeneron and Daiichi Sankyo, Sirion Biotech and Cellectis, and Cevec and Biogen. The giant Thermo Fisher Scientific absorbed the Belgian viral vector producer Henogen for 724M. And CDMO heavyweights like Cognate BioServices and Polyplus Transfection have announced expansions to their manufacturing capacity.

Thedifficulties with manufacturing the recently approved Covid-19 mRNA vaccines in high enough quantities has really highlighted the importance of having a solid manufacturing strategy in place. This lesson applies equally to companies trying to take cell and gene therapies to market.

Stuck in the first generation

Despite the huge increase in development of cell and gene therapies over the past couple of years, manufacturing technology for these therapies is largely still at the first-generation stage. This can make scaling up a challenge.

Often cell and gene therapy manufacturing processes are highly manual stemming from the early academic or process development stage and, without adequate technology solutions available currently, these processes often remain this way through clinical trials and then into commercial manufacturing, said Jason Foster, CEO of Ori Biotech, a London- and New Jersey-based company focusing on cell and gene therapy manufacturing.

These first-generation processes cause manufacturing to be expensive, highly variable and low-throughput, which reduces the ability of patients to access these potentially life-saving therapies.

Another problem common to all bio-based therapeutics is that any product sourced from a live cell or a component of one is subject to a lot more variation than a simpler pharmaceutical product.

Most gene therapies are built on viruses found in nature. They have not evolved for very high productivity in a large-scale, animal component-free bioreactor, said Cawood.

The more complicated the biologic becomes, the more parts of it require optimization, and the more analytics you require.

According to Kevin Alessandri, the cofounder and CEO/CTO of the French company TreeFrog Therapeutics, there is also a lot of waste in cell therapy manufacturing.

Yields are impaired by high cell death at every passage, and genetic alterations inevitably arise, said Alessandri. When it comes to producing commercial batches to treat thousands of patients, scaling out 2D cell culture processes is far too expensive and poses batch-to-batch reproducibility issues.

While many in the industry are now turning to bioreactors to produce cells on a bigger scale, this is also not without problems. Impeller-induced shear stress is damaging the cells, thus negatively impacting cell viability and triggering undesired genetic mutations, explained Alessandri.

Taking manufacturing up a gear

What are companies in this space doing to make scaling up cell and gene therapies easier, quicker, and cheaper?

Ori Biotech raised24.8M in Series A funding in October last year to develop an automated and robotic manufacturing system to minimize the number of manual steps needed to produce a given cell or gene therapy. This speeds up the process as well as making it more accurate. Another advantage of the technology is that it can tailor the production capacity according to demand.

This is impossible to do in most current processes, which involve manual tube welding and transfers from flask to bag to bigger bag to bioreactor, said Foster, adding that this increases cost and variability while constraining throughput. Oris technology, in contrast, could take years off the production timeline and cut costs by as much as 80%.

London-based Synthace is one of several companies trying to improve advanced therapeutic manufacturing by developing software and computer systems to optimize the process, rather than industrial machinery.

Peter Crane, Corporate Strategy Manager for the company, said that in-depth data analysis and planning before starting the manufacturing process can make a big difference to outcomes, and that connected software can help make this task easier.

The best way to remove some of the risk associated with biomanufacturing of these products is to solve as many problems as possible before manufacturing.

In addition to making the process quicker, cheaper, and more accurate, computing tools can also help with quality control and tracking. In cell therapy manufacturing, especially autologous products, line of sight around electronic batch records, as well as the vein-to-vein supply chain, is incredibly important, emphasized Crane.

Another company specifically focusing on logistics and quality control is the Cardiff- and San Francisco-based TrakCel, which nailed deals with Ori Biotech in February and the UKs National Health Service in November.

The company TreeFrog Therapeutics works with cell encapsulation technology to improve quality and reduce waste, albeit from a more mechanical viewpoint. The company launched an industrial demonstration plant in June last year, followed by two co-development deals with undisclosed big pharma partners.

Encapsulated stem cells spontaneously self-organize in an in vivo-like 3D conformation promoting fast and homogeneous growth, as well as genomic stability, said Alessandri. The resulting 3D stem cell colony can then be differentiated in the capsule into functional microtissues ready for transplantation.

With our technology, which is based on high-throughput microfluidics capable of generating over 1,000 capsules per second, it becomes possible to expand and differentiate stem cells at a large scale, in industrial bioreactors, with best-in-class cell quality and reduced operating costs.

Oxgene has a focus on scaling up production for manufacturers. In September, the company launched a technology to scale up manufacturing of viral vector production with less contamination and a 40-fold improvement in yield compared to current methods. Oxgenes expertise with viral vectors also prompted a collaboration deal in April with the CDMO Fujifilm Diosynth Biotechnologies.

Innovation in new manufacturing technologies just hasnt kept pace with the level of discovery around genetic disease and potential avenues open to treat them, or even development of the viral vectors themselves, said Cawood. This is definitely changing though.

Enter the second generation of manufacturing

Cell and gene therapy manufacturing is definitely hot right now, boosted by increased needs from biotech and pharma companies developing Covid-19 vaccines and therapies, and by notable increases in investment.

Huge advances in gene and cell therapies over the last few years, such as the approval of the eye gene therapy Luxturna and the first CAR T-cell therapies, mean the demand for new manufacturing technologies has also increased exponentially.

A lot of very promising programs are now in the pipeline, and patients are waiting for their approval, said Alessandri. Industry urgently needs robust manufacturing technology, capable of serving millions of patients.

European biotechs are busy developing second-generation technologies to allow easier and cheaper scale up, producing higher quality products with less waste. They could start to phase out first-generation methods very soon.

The realm of cell manufacturing in industrial and food biotech is also likely to see big breakthroughs in the coming years. Earlier this month, for instance, the nutrition and health giant Royal DSM set up a lab in the Netherlands dedicated to applying artificial intelligence (AI) to the challenge of growing microbial strains at a commercial scale.

Rapid improvements in advanced computing options such as AI and machine learning technology, as well as robotics, are already having an effect on the industry, but this will only get bigger as time goes on.

Cover image from Elena Resko. Body text image from Shutterstock

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Cell and Gene Therapy Firms Gear up to Revolutionize Manufacturing - Labiotech.eu

Introduction

The arrival and prevalence of drug-resistant tuberculosis has become a major problem in global tuberculosis (TB) control. In 2019, it was estimated that there were 500,000 cases of rifampicin-resistant TB (RR-TB) worldwide, of which 78% were multidrug-resistant TB (MDR-TB), resistant to both isoniazid (INH) and rifampicin (RIF). China has a high burden of TB and RR/MDR-TB and accounts for 14% of global RR/MDR-TB cases.1 INH and RIF are the core first-line drugs in the treatment of TB, but treatment of RR/MDR-TB with these first-line regimens will have poor effect. It is important to quickly identify the results of drug susceptibility tests (DST) in patients, especially tests relating to INH and RIF, to enable appropriate drugs to be chosen based on DST profiles.

Detection of drug-resistant target gene mutation can help to detect drug resistance earlier than phenotypic DST. Molecular DST in MTB has been widely used in clinical work to evaluate resistance to INH and RIF. KatG and inhA gene mutations are the main mechanism of INH resistance in MTB.2 Gene inhA (including promoter and coding areas) is one of important molecular markers of INH resistance, and inhA is also the molecular basis of cross resistance to ethionamide (ETH) prothionamide (PTH),24 a group C drug recommended by the WHO for the treatment of MDR-TB.5 ETH/PTH and INH are activated by monooxygenase EthA and catalase-peroxidase KatG. The activated forms of the two drugs act on a common targetthe NADH-dependent enoyl-ACP reductase inhA (Rv1484) bindingwith a bactericidal effect that affects cell wall synthesis.2 ETH/PTH has obvious adverse reactions such as nausea and drug-induced liver injury,6 and care is needed when choosing this drug. However, due to the convenience and ease of oral administration, ETH/PTH is still recommended in MDR-TB treatment in China.6 Based on the correlation between ETH/PTH resistance and inhA, clinicians may refer to inhA gene detection to guide the use of ETH/PTH.3,4,7

A variety of reports suggest that mutations in the inhA gene in TB strains can predict ETH/PTH resistance, although some studies have also shown that clinical strains with inhA mutations are sensitive to ETH.3,4,7 MeltPro TB assay utilizes the real-time polymerase chain reaction (PCR) probe-based melting curve analysis technique8 to detect the common drug-resistant mutation sites of katG, inhA and rpoB genes in MTB and rapidly diagnose INH and RIF resistance. This technique is widely used in clinical work.9,10 In this study, we analyze the correlation between inhA mutation test results and phenotypic ETH susceptibility through MeltPro TB assay and evaluate whether the inhA test can be used to guide the clinical application of ETH where phenotypic DST results are unavailable.

In this retrospective study, patients undergoing treatment at Beijing Chest Hospital, Capital Medical University with positive MeltPro TB assay results for inhA and katG genes (mutated or not mutated) were screened from February 2015 to February 2016. Samples were tested for katG and inhA genes with culture and phenotypic DST and the dissociation curve method. Patients who met the following conditions were subsequently included in the analysis: cultured clinical specimens were positive for MTB; DST results were available for INH, RIF, Levofloxacin (Lfx), Amikacin (Am), Capreomycin (Cm), and ETH; and test results were positive for katG and inhA mutations. If two or more samples from the same patient were positive, the first sample was recorded. The basic information collected for each patient included their age, gender, disease diagnosis, initial treatment, and subsequent treatment.

The study was conducted in accordance with the Declaration of Helsinki (revised 2013). The study was approved by Beijing Chest Hospital, Capital Medical University (No.201986) and informed consent was obtained from all the patients.

Sample processing, culture, and drug susceptibility detection were conducted in accordance with the Laboratory Inspection Procedure of Tuberculosis Diagnosis.11 The clinical samples were treated and cultured on a modified LowensteinJensen culture medium (Zhuhai intkr Co. Ltd., China). Positive colonies were cultured for DST and strain identification using the LowensteinJensen proportion method. The critical concentration references were as follows: low-concentration INH 0.2 g/mL, high-concentration INH 1.0 g/mL, RIF 40 g/mL, Levofloxacin2 g/mL, Amikacin 30g/mL, Capreomycin 40 g/mL, and ETH 40g/mL. Growth (cultivation) at this concentration was defined as indicating drug resistance.3

An automatic DNA extraction machine (Zeesan Biotech, Xiamen, China) and a paramagnetic particle method were used to extract crude DNA (1 mL) from the decontaminated samples according to the MeltPro TB assay instructions. The amplification program was used to analyze the melting. The fluorescence signal intensity was collected on the LightCycler 480 System (Indianapolis Roche Group) in the FAM and TET channels, and the melting temperature TM value was obtained by identifying the peak of the melting.7,12 The detection sites of INH resistance included inhA94, inhA promoter region 17 ~ 8 mutation, and katG315 codon mutation. The katG and inhA mutation results were recorded.

Data collection was carried out using Excel 2007 and the statistical analysis employed SPSS 17.0 software. The count data were represented by rate (%), 2 TEST and Fishers exact test to compare the differences between the groups. The parameters of the continuous measurements were expressed as mean standard deviation and compared using a t-test. P < 0.05 indicated that a result was statistically significant.

A total of 704 clinical specimens were tested using the dissociation curve method and found to be positive for inhA and katG genes. Specimens of MeltPro TB assay detect negative, specimens without phenotypic DST results and repeated samples were removed. Following this, 382 patients were enrolled in the study. These patients included 283 (74.1%) cases of sputum, 58 (15.2%) cases of bronchial lavage fluid, 1(0.3%) case of cerebrospinal fluid, and 40 (10.5%) cases of sanious; 292 were initial treatment patients and 90 were re-treatment patients. And 28 (7.3%) cases were resistant to ETH. The proportion of re-treatment patients with resistance to ETH was higher than the proportion of initial treatment patients (P < 0.001; Table 1). In addition, 11.0% (42/382) were MDR-TB, 10.2% (39/382) were pre-extensively drug resistant tuberculosis (pre-XDR-TB); 4.2% (16/382) were extensively drug resistant tuberculosis (XDR-TB).

Table 1 Demographic and Clinical Characteristics of Patients

Of the 382 bacterial strains, 118 strains (30.9%) were resistant to INH. Among these INH resistant strains, 22.9% (27/118) were also resistant to ETH, and all of these strains were MDR-TB. Of the 118 INH-resistant strains, katG mutation accounted for 52.5% (62/118), inhA mutation accounted for 20.3% (24/118), and inhA+katG mutation accounted for 4.2% (5/118); strains with no mutation accounted for 22.9% (27/118). Of the 28 phenotypic ETH-resistant strains, 27 (96.4%) resistant to INH resistance. Of the same 28 strains, inhA mutation accounted for 42.9% (12/28). Of the 34 inhA mutant strains, 85.3% (29/34) had an inhA mutation without a katG mutation; among this group, 34.5% (10/29) showed low resistance to INH, 48.3% (14/29) showed high resistance to INH, and 13.8% (5/29) were sensitive to INH. The rates of ETH-resistance in low- and high-level INH-resistant strains showed no statistical differences (2 = 2.264; P = 0.132; Fishers test). The rate of single inhA mutations (without katG mutation) in strains with low INH-resistance was higher than the rate in strains with high INH-resistance (2 = 13.076; P < 0.001; Fishers test). All of 21 INH-resistant but non-MDR-TB strains were sensitive to ETH; four of these were inhA mutant strains. In addition, eight strains (four INH-resistant and four INH-sensitive) with single inhA mutation (without katG and rpoB mutations) were sensitive to ETH, and the patients carrying those eight strains were not initially treated with anti-TB drugs (Figure 1 and Table 2).

Table 2 Frequency of Ethionamide Resistance in Low-and High-Level Isoniazid Resistant and Isoniazid Susceptible Mycobacterium tuberculosis Isolates

Figure 1 Phenotypic DST results of ETH in MTB with inhA gene mutation enrolled in this study.

China has a high burden of TB and one of the highest incidences of MDR-TB in the world.13 Particularly in recent years, the incidence of DR-TB has been on the rise in China. Rifampicin (RIF) and isoniazid (INH) are the leading first-line anti-TB drugs, playing an important role in the treatment of TB. MDR-TB is widely regarded as an important factor in the failure of chemotherapy in treating TB. The resistance of genes to INH is more complicated, and is mainly caused by mutations in genes such as katG and inhA. Each mutation site has a certain correlation with drug resistance. ETH/PTH is a second-line drug treatment for TB, used mostly in MDR-TB and XDR-TB. According to data from domestic and overseas research, the majority of ETH/PTH-resistant strains also show INH resistance.10,14,15 In the present study, almost all ETH-resistant strains were also found to be resistant to both INH and RIF (96.4%), and the ETH-resistance rate in MDR-TB was 27.8%, which is consistent with our previous studies and similar data (20%24.8%) from TB treatment institutions in China.1619

The main molecular mechanisms underlying INH resistance are inhA and katG mutations, reported to account for 8%43% and 50%95% of drug-resistant strains, respectively.20 Tests for these two genetic mutations are used to diagnose the majority of instances of MTB resistance to INH. Mutations in the inhA gene are the molecular basis of cross resistance to ETH/PTH and INH. Therefore, the inhA gene can also aid in diagnosing ETH resistance. This study analyzed inhA and katG mutations and phenotypic INH and ETH susceptibility in clinical strains. Of the 118 INH-resistant strains analyzed, 56.7% (67/118) were katG mutations, and 24.5% (29/118) were inhA mutations. Of the 28 phenotypically ETH-resistant strains, inhA mutations accounted for 42.9%, which is consistent with previous reports.10,20 However, in this study, only 35.3% of the 34 inhA mutant strains were resistant to ETH, and only 42.9% of ETH-resistant strains had inhA mutations. A recent study in South Korea found that only 23 (67%) of 34 PTH-resistant strains had an inhA mutation, while data from a study in Guangzhou, China indicates that, of 46 PTH-resistant strains, 43.2% had an inhA promoter (12 strains were c-15t and 4 strains t-8c), and 6.2% had a coding gene mutation (all were S94A).10,21 The results of the present study show an inhA mutation rate in ETH-resistant strains (42.9%) similar to that in the study in China referenced above.

The question of whether inhA mutations can indicate phenotypic ETH resistance remains. Most previous research has focused on inhA mutations in INH- or ETH/PTH-resistant strains (mainly MDR-TB), while inhA mutations in sensitive strains have rarely been studied. There is a moderate level of evidence for an association between c-15t inhA promoter mutations and low-to-moderate INH resistance.22 This study shows that only 35.3% of 34 inhA mutant strains were resistant to ETH. The possible reasons for the inconsistency between inhA mutation and the ETH-resistant phenotype include the dissociation curve detection of mutant codons on inhA94 from 17 to 8 in the inhA promoter region. In addition, some positive mutations may be synonymous mutations and will not cause protein changes or ETH resistance.15,17,23 The ETH-resistant phenotype may have other regulatory mechanisms that cause strains with inhA mutations not to generate ETH resistance. This study also found that all strains with single inhA mutations (without katG or rpoB mutations) were sensitive to ETH. Taking the results of the present study in combination with those of other domestic studies, it can be argued that inhA mutations are not a reliable indicator of ETH resistance in China;17,24 the detection of inhA mutations is not necessarily a sign of resistance to ETH, and genotypic and phenotypic drug susceptibility must be detected simultaneously to guide clinical use of ETH.

Although inhA mutations have been shown to be associated with low INH resistance, this study shows that, of 29 inhA mutant strains, 48.3% (14/29), mainly INH- and RIF-resistant strains, had high INH resistance.2 InhA mutations were not a good indicator of low INH resistance. Other studies have also shown that inhA mutant (non-katG mutation) strains are highly resistant to INH, as are some strains combined with furA, oxyr-ahpc, or inhA double (c-15t combined with S94A or I194T) mutations.25 The data in this study were derived from clinical data, and no particular type of inhA mutation was specified. In addition, apart from inhA and katG315, no other INH-resistant genes were examined. Hence, the specific mechanism underlying the high drug resistance of nearly half of inhA remains unclear. Nonetheless, we found that the single inhA mutant strain (without katG and rpoB mutation) showed low-level resistance and susceptibility to INH, and these strains were sensitive to ETH. Hence, ETH and high-dose INH treatment may be effective for the majority of single inhA mutated INH-resistant strains.

However, this study has certain limitations. First, the present study is a summary of clinical data. Laboratory tests only reported whether or not the isolates had katG and inhA mutations; they did not provide detailed descriptions including the mutation sites. In addition, we did not discuss the mechanism of drug resistance in isolates where the inhA genotype and ETH-resistant phenotype were inconsistent. This will be the focus of further study, and we will evaluate the feasibility of using the targets identified by the melting curve analysis in the Chinese population. Third, the sample size was small, and all included patients came from the hospital where the author worked. The research findings may therefore contain some bias; however, they are still encouraging.

Although inhA mutations are associated with mechanisms of joint INH and ETH resistance, they may not be a reliable indicator of ETH resistance. In particular, TB strains with single inhA mutations (without katG or rpoB mutations) may remain sensitive to ETH. This is a preliminary study,future work is required to explore the mechanism of ETH resistance, to look for the reasons of inconsistency of phenotype and gene mutation.

We are particularly grateful to all the people who have given us help on our article.

There is no funding to report.

The authors declare that they have no competing interests.

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2. Vilchze C, Jacobs JR. WR. Resistance to isoniazid and ethionamide in Mycobacterium tuberculosis: genes, mutations, and causalities. Microbiol Spectr. 2014;2(4):MGM22013. doi:10.1128/microbiolspec.MGM2-0014-2013

3. Vadwai V, Ajbani K, Jose M, et al. Can inhA mutation predict ethionamide resistance? Int J Tuberc Lung Dis. 2013;17(1):129130. doi:10.5588/ijtld.12.0511

4. Niehaus AJ, Mlisana K, Gandhi NR, Mathema B, Brust JC. High prevalence of inhA promoter mutations among patients with drug-resistant tuberculosis in KwaZulu-Natal, South Africa. PLoS One. 2015;10(9):e0135003. doi:10.1371/journal.pone.0135003

5. World Health Organization. WHO consolidated guidelines on tuberculosis. Module 4: treatment - drug-resistant tuberculosis treatment. 2020. Availble from: https://www.who.int/tb/publications/global_report/TB20_Exec_Sum_20201014.pdf. Accessed December 23, 2020.

6. Tuberculosis society of Chinese Medical Association. Chinese expert consensus on multidrug-resistant tuberculosis and Rifampicin-resistant tuberculosis treatment. Chin J Tuberc Respir. 2019;42(10):733749.

7. Lee JH, Jo KW, Shim TS. Correlation between genoType MTBDRplus assay and phenotypic susceptibility test for prothionamide in patients with genotypic isoniazid resistance. Tuberc Respir Dis (Seoul). 2019;82(2):143150. doi:10.4046/trd.2018.0027

8. Pang Y, Dong H, Tan Y, et al. Rapid diagnosis of MDR and XDR tuberculosis with the MeltPro TB assay in China. Sci Rep. 2016;6:25330. doi:10.1038/srep25330

9. Haeili M, Fooladi AI, Bostanabad SZ, Sarokhalil DD, Siavoshi F, Feizabadi MM. Rapid screening of rpoB and katG mutations in Mycobacterium tuberculosis isolates by high-resolution melting curve analysis. Indian J Med Microbiol. 2014;32(4):398403. doi:10.4103/0255-0857.142245

10. Darban-Sarokhalil D, Nasiri MJ, Fooladi AA, Heidarieh P, Feizabadi MM. Rapid detection of rifampicin- and isoniazid-resistant Mycobacterium tuberculosis using TaqMan allelic discrimination. Osong Public Health Res Perspect. 2016;7(2):127130. doi:10.1016/j.phrp.2016.01.003

11. Basic Professional Committee of China National Defense Tuberculosis Association. TB diagnostic laboratory test procedures. Beijing: China Education Press; 2006.

12. Wang G, Dong W, Lan T, et al. Diagnostic accuracy evaluation of the conventional and molecular tests for Spinal Tuberculosis in a cohort, head-to-head study. Emerg Microbes Infect. 2018;7(1):109. doi:10.1038/s41426-018-0114-1

13. Jou R, Lee WT, Kulagina EV, et al. Redefining MDR-TB: comparison of Mycobacterium tuberculosis clinical isolates from Russia and Taiwan. Infect Genet Evol. 2019;72:141146. doi:10.1016/j.meegid.2018.12.031

14. Morlock GP, Metchock B, Sikes D, Crawford JT, Cooksey RC. ethA, inhA, and katG loci of ethionamide-resistant clinical Mycobacterium tuberculosis isolates. Antimicrob Agents Chemother. 2003;47(12):37993805. doi:10.1128/AAC.47.12.3799-3805.2003

15. Rueda J, Realpe T, Mejia GI, et al. Genotypic analysis of genes associated with independent resistance and cross-resistance to isoniazid and ethionamide in Mycobacterium tuberculosis clinical isolates. Antimicrob Agents Chemother. 2015;59(12):78057810. doi:10.1128/AAC.01028-15

16. Song YH, Wang GR, Huo FM, et al. Correlation analysis of inhA gene mutation in MTB and propioniazid resistance. Chin J Def Consumpt. 2018;40(8):821824.

17. Liu YP, Wang J, Zhang JX, et al. Detection of clinical isolates of Mycobacterium tuberculosis resistant to isoniazid and propioniazid and study on related gene mutation. Chin J Def Consumpt. 2016;38(9):718721.

18. Chen HF, Huang QS, Gao AX, et al. Observation on the sensitivity of mDR-MYCObacterium tuberculosis to second-line anti-tuberculosis drugs. J Nanjing Med Univ. 2014;34(1):6971.

19. Li XD. Analysis of resistance of 174 mDR-Mycobacterium tuberculosis strains to second-line anti-tuberculosis drugs. Int J Lab Med. 2014;13:17321733,1748.

20. Zhang Y, Yew WW. Mechanisms of drug resistance in Mycobacterium tuberculosis: update 2015. Int J Tuberc Lung Dis. 2015;19(11):12761289. doi:10.5588/ijtld.15.0389

21. Tan Y, Su B, Zheng H, Song Y, Wang Y, Pang Y. Molecular characterization of prothionamide-resistant mycobacterium tuberculosis isolates in Southern China. Front Microbiol. 2017;8:2358. doi:10.3389/fmicb.2017.02358

22. Organization W.H. The use of next-generation sequencing technologies for the detection of mutations associated with drug resistance in Mycobacterium tuberculosis complex: technical guide. 2018.

23. Malinga L, Brand J, Jansen van Rensburg C, Cassell G, van der Walt M. Investigation of isoniazid and ethionamide cross-resistance by whole genome sequencing and association with poor treatment outcomes of multidrug-resistant tuberculosis patients in South Africa. Int J Mycobacteriol. 2016;5(Suppl 1):S36S37. doi:10.1016/j.ijmyco.2016.11.020

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25. Machado D, Perdigo J, Ramos J, et al. High-level resistance to isoniazid and ethionamide in multidrug-resistant Mycobacterium tuberculosis of the Lisboa family is associated with inhA double mutations. J Antimicrob Chemother. 2013;68(8):17281732. doi:10.1093/jac/dkt090

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[Full text] The Value of the inhA Mutation Detection in Predicting Ethionamide Res | IDR - Dove Medical Press

A research report on Regenerative Medicine Market features a succinct analysis on the latest market trends. The report also includes detailed abstracts about statistics, revenue forecasts and market valuation, which additionally highlights its status in the competitive landscape and growth trends accepted by major industry players.

Regenerative Medicine Market: Increased in bone and joint surgeries and increased prevalence of neurodegenerative, orthopedic, oncology, and genetic diseases are the key drivers for Global Regenerative Medicine Market.

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Regenerative medicines are those medicines which are used to repair, regenerate, and replace the tissues or organs damaged due to disease, injury or natural aging. Regenerative medicines are used in the treatment of various disorders such as orthopedic, neurodegenerative, oncology and others. These medicines help in the restoration of natural functioning of the organs and tissues. Regenerative medicines can also be used for the treatment of various chronic and genetic disorders, it also helps in the treatment of organ transplant which has reduced the rejection cases to a major extent.

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Regenerative Medicine MarketKey Players

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January 29, 2021 I COVID-19 may become seasonal, severe infection associated with myeloid immune cells, potential Achilles heel of coronaviruses identified, melatonin synthesized in lungs could have protective effect, and plitidepsin outperforms remdesivir in preclinical trials. Plus: NSAID use during COVID-19 is time-dependent on its harm or benefit and NAU to test Allarity drug against Coronavirus Variant B117.

Research News

COVID-19 may be seasonal, like the flu, suggests a new paper published in Evolutionary Bioinformatics. Authors of the paper show that COVID-19 cases and mortality rates, among other epidemiological metrics, are significantly correlated with temperature and latitude across 221 countries. They also explain that our own immune systems could be partially responsible for the pattern of seasonality. For example, our immune response to the flu can be influenced by temperature and nutritional status, including vitamin D, a critical nutrient to our immune defenses. The researchers add that it is, however, too soon to say how seasonality and our immune systems interact in the case of COVID-19. DOI:10.1177/1176934321989695

SARS-CoV-2 independently entered Russia at least 67 times, primarily at the end of February and beginning of March 2020, according to a new study published in Nature Communications. Researchers of the study used 211 virus genomes, which were sequenced at Smorodintsev Research Institute of Influenza, and all genomes had been obtained from patients from 25 Russian regions during mid-March to April 2020. They determined that the vast majority of introductions came from European countries, and no cases of introduction from China were registered, which they attribute to the timely closure of borders with the country. Currently, nine local virus lineages are circulating in Russia, which are not present elsewhere in the world. DOI:10.1038/s41467-020-20880-z

Research led at Vanderbilt University Medical Center has discovered a proofreading exoribonuclease, called nsp14-ExoN, which can correct errors in the RNA sequence that occur during replication, when copies of a virus are generated. They believe that this may be the Achilles heel of the coronavirus, a finding that could help close the door on COVID-19 and possibly head off future pandemics. Using cutting-edge technologies and novel bioinformatics approaches, the researchers discovered that this ExoN also regulates the rate of recombination, which is the ability of the coronavirus to shuffle parts of its genome and even pull genetic material from other viral strains while it replicates in order to gain evolutionary advantage. These patterns of recombination are conserved across multiple coronaviruses, including SARS-CoV-2. They believe that the coronavirus ExoN is therefore a conserved, important target for inhibition and attenuation in the ongoing pandemic. This research is published in PLOS Pathogens. DOI:10.1371/journal.ppat.1009226

Also at Vanderbilt University Medical Center (VUMC), researchers have identified genetic factors that increase the risk for developing pneumonia to help identify patients with COVID-19 at greatest risk for this life-threatening complication. The researchers conducted genome-wide association studies (GWAS) of more than 85,000 patients whose genetic information is stored in VUMCs BioVU biobank. They identified nearly 9,000 cases of pneumonia in patients of European ancestry and 1,710 cases in patients of African ancestry. After further analysis, the research team linked the gene that causes cystic fibrosis (CF) and European ancestry and the mutation that causes sickle cell disease (SCD) in patients of African ancestry as the strongest pneumonia associations. After removing patients with CF and SCD, they then pinpointed a pneumonia-associated variation in a gene called R3HCC1L in patients of European ancestry, and one near a gene called UQCRFS1 in patients of African ancestry. They believe these findings could be applied to identifying patients with high risk of severe pneumonia to enable early interventions. They have published this work in the American Journal of Human Genetics. DOI:10.1016/j.ajhg.2020.12.010

Melatonin produced in the lungs acts as a barrier against SARS-CoV-2, blocking the expression of genes that encode proteins in cells serving as viral entry points, finds researchers at the University of So Paulo (USP). The hormone, therefore, prevents infection of these cells by the virus and inhibits the immune response so that the virus remains in the respiratory tract for a few days and then leaves the host, say the researchers. They used RNA sequencing data to quantify the level of expression of 212 COVID-19 signature genes in 288 samples from healthy human lungs. The researchers correlated these gene expression levels with a gene index that estimated the capacity of the lungs to synthesize melatonin (MEL-index). They then were able to determine that when the MEL-index was high, the entry points for the virus in the lungs were closed, and vice-versa. The research team suggests the potential for nasal administration of melatonin to prevent disease from developing in pre-symptomatic COVID-19 patients. This study is published in Melatonin Research. DOI:10.32794/mr11250090

In a new study, published in Cell Reports Medicine, La Jolla Institute for Immunology (LJI) researchers suggest that T cells can mount attacks against many SARS-CoV-2 targets, beyond the key sites on the viruss spike protein. They believe that by attacking the virus from many angles, the body is equipped to potentially recognize different SARS-CoV-2 variants. The researchers examined T cells from 100 people who had recovered from COVID-19 to take a closer look at the genetic sequence of the virus to separate the potential epitopes from the epitopes that these T cells would recognize. Their analysis revealed that not all parts of the virus induce the same strong immune response in everyone, and T cells can recognize dozens of epitopes on SARS-CoV-2 that vary from person to person. They determined that each study participant had the ability to recognize about 17 CD8+ T cell epitopes and 19 CD4+ T cell epitopes. DOI:10.1016/j.xcrm/2021/100202

John Hopkins Medicine researchers, in collaboration with Immunoscape, have published a complete profile of the response of T cells in people who have recovered from SARS-CoV-2 infection. The paper, published in The Journal of Clinical Investigation, better defines which T cells interact with which specific portion of the SARS-CoV-2 virus and how those interactions can provide long-lasting immunity against COVID-19. The researchers collected blood samples from 30 convalescent patients who had recovered from mild cases of COVID-19 and the Immunoscape team, a U.S.-Singapore biotechnology company, used its highly sensitive human leukocyte antigen (HLA)-SARS-CoV-2 tetramers to tag and identify the types of virus-recognizing CD8+ T cells. The researchers found that as levels of neutralizing antibodies increased in the convalescent plasma, so did the number of memory CD8+ T cells that recognized SARS-CoV-2 epitopes. They believe this means lasting protection against reinfection, and this knowledge will guide COVID-19 vaccine design to produce a strong immune response that could provide years of protection. DOI:10.1172/JCI145476

Severe COVID-19 patients have significantly elevated levels of a certain type of immune cell in their blood, call monocytic myeloid-derived suppressor cells (M-MDSC), according to a new study published in the Journal of Clinical Investigation. Karolinska Institutet researchers studied 147 patients with mild to fatal COVID-19 who were sampled repeatedly from blood and respiratory tract. These samples were then compared with patients who had influenza and healthy individuals. They found that the patients with severe COVID-19 had significantly higher levels of M-MDSCs in their blood when compared to milder cases and healthy participants. COVID-19 patients also had fewer T cells in their blood than healthy individuals that showed signs of impaired function. Additionally, their analysis revealed that the levels of M-MDSCs early in the course of infection seemed to reflect subsequent disease severity. DOI:10.1172/JCI44734

Researchers have engineered an antibody that effectively neutralizes SARS-CoV-2 and that also acts against multiple SARS-like viruses. Their antibody, ADG-2, was studied in mice. To engineer this broadly neutralizing antibody (bnAb), the researchers started with antibodies from the memory B cells of a 2003 SARS survivor that cross-neutralized multiple SARS-related viruses with modest potency. They then selectively engineered the binding affinities of several of these bnAbs, creating improvements in their abilities to bind the virus. The researchers then studied the engineered antibodies for SARS-CoV-2 neutralizing activity in mouse cell lines. ADG-2 was particularly effective. It showed broad binding activity to more than a dozen SARS-related coronaviruses. This research is published in Science. DOI:10.1126/science.abf4830

Plitidepsin has shown a potent efficacy against SARS-CoV-2 in preclinical trials, outperforming the antiviral remdesivir. These results, published in Science, show that in studies in human cells, plitidepsin demonstrated potent anti-SARS-CoV-2 activity: 27.5-fold more so than remdesivir as tested in the same cell line. In a model of human lung cells, plitidepsin greatly reduced viral replication. In further experiments involving both plitidepsin and remdesivir in vitro, the researchers suggest that plitidepsin has an additive effect with the approved drug and would be a potential candidate for a combined therapy. Authors of the research article believe that this promising treatment, which has limited clinical approval for the treatment of multiple myeloma, should be strongly considered for expanded clinical trials for the treatment of COVID-19. DOI:10.1126/science.abf4058

Oregon Health & Science University (OHSU) researchers have demonstrated that antibodies generated by the SARS-CoV-2 virus react to other strains of coronavirus and vice-versa. They determined, however, that antibodies generated by the 2003 SARS outbreak had only limited effectiveness in neutralizing SARS-CoV-2. The researchers believe that these findings have implications on both vaccine effectiveness and diagnosis of COVID-19. They believe that more work needs to be done to determine the lasting effectiveness of COVID-19 vaccine, given the speed of mutations. The team believes their study also suggests that efforts to accurately discern a previous COVID-19 infection, by analyzing antibodies in the blood, may be complicated by the presence of antibodies reacting to other strains of coronavirus including the common cold. This study is published in Cell Reports. DOI:10.1016/j.celrep.2021.108737

A new method to mapping viral mutations that escape leading clinical antibodies against COVID-19 has revealed mutations in the SARS-CoV-2 virus that allow it to evade treatments, including a single amino-acid mutation that fully escapes Regenerons antibody cocktail. University of Washington researchers and colleagues developed this scanning method to map how mutations to the receptor-binding domain (RBD) affect its recognition by antibodies. Their maps identified mutations that escape antibody binding, including a single mutation that escapes both antibodies in the Regeneron antibody cocktail. To further investigate, the team examined deep sequencing data from a persistently infected patient who was treated with the antibody cocktail at day 145 after diagnosis with COVID-19, and their analysis identified resistance mutations that arose in the patient. Furthermore, after they examined all human-derived SARS-CoV-2 sequences available as of mid-January 2021, the researchers report a substantial number of RBD mutations that escaped one or more of the antibodies that are in circulation. This paper is published in Science. DOI:10.1126/science.abf9302

Monash University researchers have discovered two new molecules that provide profound protection in experimental models of asthma, as well as protection from acute respiratory distress syndrome (ARDS) that is seen in some patients with severe COVID-19. In their study, originally designed to investigate how the immune system impacts gut bacteria, the researchers found that p-cresol sulfate (PCS), a gut bacteria by-product, led to a striking protection against asthma. They then determined that PCS was produced by enhanced bacterial metabolism of L-tyrosine, a well-known amino acid found in dietary supplements. The researchers saw significant protection against lung inflammation in mice given either L-tyrosine or PCS, as well as protection from ARDS. The researchers now aim to test one of the molecules in a clinical trial in asthmatics this year. These new findings are published in Nature Immunology. DOI:10.1038/s41590-020-00856-3

Non-steroidal anti-inflammatory drugs (NSAIDs) reduced both antibody and inflammatory responses to SARS-CoV-2 infection in mice, a new study finds that is published in the Journal of Virology. The authors of the study highlight that the timing of NSAID use during COVID-19 is important. They explain that NSAIDs anti-inflammatory activity could be detrimental early in SARS-CoV-2 infection because inflammation is usually helpful during this stage. This changes at later stages of COVID-19, particularly if the patient experiences intense inflammation known as cytokine storm. The researchers also note that a reduction in neutralizing antibodies caused by NSAIDs could be benign, or it might hinder the immune systems ability to fight the infection in its early stages. It could also reduce the magnitude or duration of protection from either natural infection or vaccination. DOI:10.1128/JVI.00014-21

Rhesus macaque monkeys infected with SARS-CoV-2 developed protective immune responses that could be reproduced with a vaccine, according to University of California, Davis (UC Davis) researchers. The team infected eight rhesus macaques at the California National Primate Research Center (CNPRC) with SARS-CoV-2 virus isolated from the first human patient treated at UC Davis, and they followed the immune responses in the monkeys over two weeks. The animals showed signs of lasting immunity and, most importantly, structures called germinal centers developed in the lymph nodes near the lungs. These germinal centers contained cells call T follicular helper (Tfh) cells. Germinal centers and Tfh cells are associated with generating plasma cells that remain in the body for many years to produce antibodies against pathogens the immune system has seen before, the researchers explain. They believe these results suggest that vaccines that induce this response will support immunity against COVID-19. This study is published in Nature Communications. DOI:10.1038/s41467-020-20642-x

Patients who have recovered from severe COVID-19 infection could be left with more protective T cells needed to fight reinfection, finds a team of researchers led at La Jolla Institute for Immunology (LJI). For their study, published in Science Immunology, the team analyzed CD8+ T cells from 39 COVID-19 patients and 10 individuals who had never been exposed to the virus. Of the COVID-19 patients, 17 had a mild case that did not require hospitalization, 13 had been hospitalized, and nine needed intensive care support. Surprisingly, the researchers saw weaker CD8+ T cell responses in patients with milder COVID-19 cases and saw the strongest CD8+ T cell responses in the patients who required hospitalization or intensive care. The team now hopes to study how T cells in tissues hit hardest by SARS-CoV-2, such as the lungs, react to the virus. They explain the importance of this as the memory T cells that provide long-term immunity need to live in the tissues. DOI:10.11260/sciimmunol.abe4782

In a new study published in Science Signaling, scientists discovered that SARS-CoV-2 may enter and replicate in human cells by exploiting newly identified sequences within cell receptors. They also suggest that these sequences could potentially serve as targets for new therapies against COVID-19. After analyzing the Eukaryotic Linear Motif database, the team of scientists discovered that ACE2 and various receptors contained several short linear motifs (SLiMs), or small amino acid sequences, that they predict plays a role in endocytosis and autophagy, or the entering of human cells and cellular housekeeping. The team determined that two SLiMs in ACE2 bound to endocytosis-related proteins, and one SLiM in the integrin beta-3 (3) bound to two proteins involved in autophagy. They believe that their prediction models could help identify similar SLiMs that assist with the replication of not only SARS-CoV-2, but other viruses that cause disease. DOI:10.1126/scisignal.abd0334

Ohio University researchers have published the first structural biology analysis of a section of the COVID-19 viral RNA called the stem-loop II motif, which they believe could be a potential target for anti-viral drugs to combat the virus. The research team identified this non-coding section of the RNA that is likely key to SARS-CoV-2 replication. Interestingly, they determined that the structural flexibility of this noncoding RNA motif differs by only a single nucleotide when compared to that in the early 2000s SARS-CoV outbreak, and the team also identified FDA-approved drugs that bind to the RNA motif and alter its flexibility. Since the structure and flexibility of noncoding RNA affects its function, the researchers suggest that it may be possible to develop antiviral drugs that specifically target this RNA motif to battle the virus. This research is published in Biochemical and Biophysical Research Communications. DOI:10.1016/j.bbrc.2021.01.013

Innate immunity may play a larger role in controlling SARS-CoV-2 viral load than adaptive immunity, according to a new study published in ACS Pharmacology & Translational Science. Researchers of the study developed a mathematical model that predicts viral load over time in organs that express the ACE2 receptor, which allows SARS-CoV-2 entry into human cells. They then used this model to simulate different conditions to determine this key role for innate immunity in controlling viral load. The researchers suggest the importance of starting antiviral or interferon therapy as soon as possible after the onset of COVID-19 symptoms. DOI:10.1021/acsptsci.0c00183

Industry News

Allarity Therapeutics in Denmark plans to further test the antiviral activity of stenoparib, its Poly ADP-Ribose Polymerase (PARP) inhibitor, against the B.1.1.7 variant of SARS-CoV-2. Stenoparib is a small molecule, targeting inhibitor of PARP, a key DNA damage repair enzyme active in tumors, which was originally developed by the pharmaceutical company Eisai. Results of previous pre-clinical studies for SARS-CoV-2 demonstrated that stenoparib inhibits SARS-CoV-2 as a single agent, and stenoparib in combination with remdesivir was active in inhibiting coronavirus in vitro. Allarity will now work with scientists at Northern Arizona Universitys Pathogen and Microbiome Institute (PMI) to test the similar ability of stenoparib to block the infection and replication of Coronavirus Variant B117. Press Release

Clear Labs announced the availability of the Clear Dx Whole Genome Sequencing (WGS), the first automated WGS solution that determines the complete RNA sequence of the SARS-CoV-2 genome in less than 24-hours with only minutes of hands-on time. The Clear Dx platform is powered by next generation sequencing (NGS), robotics and cloud-based analytics, and as a result, their WGS can more easily determine the nature of virus transmission by differentiating virus strains and monitoring mutations that lead to variants. In addition to WGS, the platform also features the Clear Dx SARS-CoV-2 Diagnostic Assay, which has received EUA, that allows labs to perform diagnostic screening and genomic surveillance simultaneously. Press Release

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Profile of T Cells, Broadly Neutralizing Antibodies, Anti-Viral Targets: COVID-19 Updates - Bio-IT World

BOSTON--(BUSINESS WIRE)--Vertex Pharmaceuticals Incorporated (Nasdaq: VRTX) today announced that the U.S. Food and Drug Administration (FDA) has accepted its supplemental New Drug Application (sNDA) to expand the use of TRIKAFTA (elexacaftor/tezacaftor/ivacaftor and ivacaftor) to include children ages 6 through 11 years old who have at least one F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene or a mutation in the CFTR gene that is responsive based on in vitro data. The FDA has granted Priority Review of the sNDA and assigned a Prescription Drug User Fee Act (PDUFA) target action date of June 8, 2021. The submission was supported by data from a global Phase 3 study of TRIKAFTA in children ages 6 through 11 years old with cystic fibrosis (CF) who have either two copies of the F508del mutation or one copy of the F508del mutation and one minimal function mutation.

If approved for this expanded use, we will have the opportunity to treat the underlying cause of the disease earlier in life with TRIKAFTA and potentially benefit approximately 1,500 additional children with CF, said Carmen Bozic, M.D., Executive Vice President and Chief Medical Officer at Vertex. Since our initial approval of TRIKAFTA in 2019, we have continued to work tirelessly to bring this medicine to those waiting as quickly as possible. We look forward to working with the Agency as they review the application over the course of the coming months.

Vertex plans to submit a Marketing Authorization Application (MAA) variation for the triple combination in the European Union in the first half of 2021 for children ages 6 through 11. Global regulatory filings in additional markets, including Canada and Australia, are planned in the coming months for this age group.

About Cystic Fibrosis

Cystic Fibrosis (CF) is a rare, life-shortening genetic disease affecting more than 80,000 people globally. CF is a progressive, multi-system disease that affects the lungs, liver, GI tract, sinuses, sweat glands, pancreas and reproductive tract. CF is caused by a defective and/or missing CFTR protein resulting from certain mutations in the CFTR gene. Children must inherit two defective CFTR genes one from each parent to have CF. While there are many different types of CFTR mutations that can cause the disease, the vast majority of all people with CF have at least one F508del mutation. These mutations, which can be determined by a genetic test, or genotyping test, lead to CF by creating non-working and/or too few CFTR proteins at the cell surface. The defective function and/or absence of CFTR protein results in poor flow of salt and water into and out of the cells in a number of organs. In the lungs, this leads to the buildup of abnormally thick, sticky mucus that can cause chronic lung infections and progressive lung damage in many patients that eventually leads to death. The median age of death is in the early 30s.

INDICATION AND IMPORTANT SAFETY INFORMATION FOR TRIKAFTA (elexacaftor/tezacaftor/ivacaftor and ivacaftor)

What is TRIKAFTA?

TRIKAFTA is a prescription medicine used for the treatment of cystic fibrosis (CF) in patients aged 12 years and older who have at least one copy of the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene or another mutation that is responsive to treatment with TRIKAFTA. Patients should talk to their doctor to learn if they have an indicated CF gene mutation. It is not known if TRIKAFTA is safe and effective in children under 12 years of age.

Patients should not take TRIKAFTA if they take certain medicines or herbal supplements, such as: the antibiotics rifampin or rifabutin; seizure medications such as phenobarbital, carbamazepine, or phenytoin; or St. Johns wort.

Before taking TRIKAFTA, patients should tell their doctor about all of their medical conditions, including if they: have kidney problems; have or have had liver problems; are pregnant or plan to become pregnant because it is not known if TRIKAFTA will harm an unborn baby; or are breastfeeding or planning to breastfeed because it is not known if TRIKAFTA passes into breast milk.

TRIKAFTA may affect the way other medicines work, and other medicines may affect how TRIKAFTA works. Therefore, the dose of TRIKAFTA may need to be adjusted when taken with certain medications. Patients should especially tell their doctor if they take antifungal medications such as ketoconazole, itraconazole, posaconazole, voriconazole, or fluconazole; or antibiotics such as telithromycin, clarithromycin, or erythromycin.

TRIKAFTA can cause dizziness in some people who take it. Patients should not drive a car, operate machinery, or do anything that needs them to be alert until they know how TRIKAFTA affects them.

Patients should avoid food or drink that contains grapefruit while they are taking TRIKAFTA.

TRIKAFTA can cause serious side effects, including:

High liver enzymes in the blood, which is a common side effect in people treated with TRIKAFTA. The patient's doctor will do blood tests to check their liver before they start TRIKAFTA, every 3 months during the first year of taking TRIKAFTA, and every year while taking TRIKAFTA. Patients should call their doctor right away if they have any of the following symptoms of liver problems: pain or discomfort in the upper right stomach (abdominal) area; yellowing of the skin or the white part of the eyes; loss of appetite; nausea or vomiting; dark, amber-colored urine.

Abnormality of the eye lens (cataract) in some children and adolescents treated with TRIKAFTA. If the patient is a child or adolescent, their doctor should perform eye examinations before and during treatment with TRIKAFTA to look for cataracts.

The most common side effects of TRIKAFTA include headache, diarrhea, upper respiratory tract infection (common cold) including stuffy and runny nose, stomach (abdominal) pain, inflamed sinuses, increase in liver enzymes, increase in a certain blood enzyme called creatine phosphokinase, rash, flu (influenza), and increase in blood bilirubin.

These are not all the possible side effects of TRIKAFTA. Please click the product link to see the full Prescribing Information for TRIKAFTA.

About Vertex

Vertex is a global biotechnology company that invests in scientific innovation to create transformative medicines for people with serious diseases. The company has multiple approved medicines that treat the underlying cause of cystic fibrosis (CF) a rare, life-threatening genetic disease and has several ongoing clinical and research programs in CF. Beyond CF, Vertex has a robust pipeline of investigational small molecule medicines in other serious diseases where it has deep insight into causal human biology, including pain, alpha-1 antitrypsin deficiency and APOL1-mediated kidney diseases. In addition, Vertex has a rapidly expanding pipeline of genetic and cell therapies for diseases such as sickle cell disease, beta thalassemia, Duchenne muscular dystrophy and type 1 diabetes mellitus.

Founded in 1989 in Cambridge, Mass., Vertex's global headquarters is now located in Boston's Innovation District and its international headquarters is in London. Additionally, the company has research and development sites and commercial offices in North America, Europe, Australia and Latin America. Vertex is consistently recognized as one of the industry's top places to work, including 11 consecutive years on Science magazine's Top Employers list and a best place to work for LGBTQ equality by the Human Rights Campaign. For company updates and to learn more about Vertex's history of innovation, visit http://www.vrtx.com or follow us on Facebook, Twitter, LinkedIn, YouTube and Instagram.

Special Note Regarding Forward-Looking Statements

This press release contains forward-looking statements as defined in the Private Securities Litigation Reform Act of 1995, including, without limitation, statements made by Dr. Carmen Bozic in this press release, statements regarding our plans to submit an MAA in the EU and other global regulatory filings in additional markets, our expectations regarding the number of patients newly eligible for TRIKAFTA, and statements regarding the potential benefits of TRIKAFTA. While Vertex believes the forward-looking statements contained in this press release are accurate, these forward-looking statements represent the company's beliefs only as of the date of this press release and there are a number of risks and uncertainties that could cause actual events or results to differ materially from those expressed or implied by such forward-looking statements. Those risks and uncertainties include, among other things, that the sNDA to expand the use of TRIKAFTA to include children ages 6 through 11 could not be approved on a timely basis, or at all, that data from the company's development programs may not support registration or further development of its compounds due to safety, efficacy or other reasons, and other risks listed under the heading Risk Factors in Vertex's most recent annual report and subsequent quarterly reports filed with the Securities and Exchange Commission (SEC) and available through the company's website at http://www.vrtx.com and on the SECs website at http://www.sec.gov. You should not place undue reliance on these statements. Vertex disclaims any obligation to update the information contained in this press release as new information becomes available.

(VRTX-GEN)

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Vertex Announces U.S. FDA Acceptance of Supplemental New Drug Application for TRIKAFTA (elexacaftor/tezacaftor/ivacaftor and ivacaftor) in Children...

When the French Emmanuelle Charpentier and the American Jennifer Doudna, won the Nobel Prize in Chemistry 2020, the award fell short compared to the importance of their contribution to the gene editing. These girls, yeah they discovered the black thread with their scissors CRISPR to alter and repair DNA, contributing to the health of living beings. A revolutionary technique, thanks to which mice with damage to the spinal cord They have walked again, hoping that one day it could be applied to humans.

Although it seems inspired by the Lucy movie, gene editing also known as biohacking, it is nothing other than correct and repair the cells of the DNA Or, insert the missing cells, which cause genetic diseases as simple as myopia to the diabetes. In addition, they have been shown to have an important benefit in other types of patients such as HIV. Being a new miracle in medicine.

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In a simple explanation, one of the theories of CRISPR, is that genetic diseases are due to damage or absence in the genomic sequence. What the experts do is all engineering work, repairing or inserting the genetic sequence. For example, some genetic diseases such as sclerosis, diabetes or muscular dystrophy are believed to be due toin the absence or damage de a gene X, while biohackers take care of repair it. It is truly fascinating.

However, gene editing therapies in humans are illegal in many countries, in addition to being a potentially controversial issue because it questions the objective of prolonging life and practically playing god. In fact, a group of UNESCO scientists requested the prohibition of any edition of the human germ line since they could lead to actions of baby design .

Twitter: @N_Neandertalien

However, a study of the Ruhr University, in Germany, revives the debate on the future of genetic medicine, by getting a group of hamsters with spinal cord damage to walk again dfter two to three weeks of receiving a new gene therapy, while there is a huge chance that this new treatment will have the same success in humans.

The scientists worked with mice with spinal damage and consequently lost all mobility on both legs. They first began by stimulating the nervous system to identify the damaged gene, after a series of studies, they developed the gene hyperinterleukin-6genetically modified and injected it into the sensory motor cortex and did what they could at the time: wait.

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Being an unpublished study, the consequences could have been diverse, but once the hyper-interleukin-6, achieved adapt to the genetic sequence, for the production of the protein within the cells. Lto protein, or hyperinterleukin-6 (hIL-6), acts assuming a key characteristic Disabling spinal cord injuries, which is damage to nerve fibers known as axons. What is special about our study is that the protein is not only used to stimulate nerve cellss that produce it themselves, but also takes it further (through the brain) , Gave DailyMail, Dr. Dietmar Fischer, who led the study.

Nature

There is no miracle, everything is thanks to science and like all treatment, genetic editing would take time to show the first results. But, after a couple of weeks, the CRISPR worked its magic. All mice managed to recobrar movement body and not only that, Little time they all walked again.

Now the scientists will have to wait to analyze if the mice do not suffer from serious side effects, if so, they will take the next step to study if this gene-editing therapy can be applied in humans, which could save more than five million people in the world who suffer from partial or total paralysis.

It sounds spectacular, we know. But please dont try to play scientist. Gene editing may have grave consequences in humans, if it is practiced in a way experimental. While modified genes are not found in nature, they are designed specifically for genetic engineers with all the knowledge and within a laboratory. Better, if you are interested in the subject and you would like to enter the world of CRISPR, we recommend the Netflix documentary entitled Unnatural Selection

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The story of the hamster that walked again thanks to genetic editing - Explica

Introduction

Elizabethkingia anophelis (E. anophelis) is an aerobic, immotile, oxidase-positive, indole-positive, Gram-negative, non-fermenting bacillus, belonging to the genus Elizabethkingia and the family Flavobacteriaceae, which was first isolated from the midgut of the mosquito Anopheles gambiae in 2011.1 The first clinically significant E. anophelis infection was associated with a case of neonatal meningitis in Bangui, Central African Republic in 2011.2

Thereafter, within the genus Elizabethkingia, E. anophelis has emerged as a major nosocomial pathogen, which can cause severe pneumonia, meningitis, infections of the bloodstream, osteomyelitis, endocarditis, endophthalmitis, skin and soft tissue infection, urinary tract infection and abdominal infection.310,14,15 Whole genome sequencing (WGS) has recently uncovered a wide range of virulence factors contributing to the pathogenesis of E. anophelis, including products of the capsule, lipopolysaccharides, endopeptidases, lipid biosynthesis and metabolism, magnesium transport proteins, macrophage infectivity, heat shock proteins, catalase, peroxidases, superoxide dismutase, two-component regulatory system, and more.5,1820

Extensive research has shown that E. anophelis isolates are resistant to most -lactams, carbapenems and aminoglycosides.4,5,1417 The emergence of multidrug-resistant (MDR) bacterial pathogens is considered a potential public health hazard, they are widely detected in the environment and their transmission to humans is either by the food chain or via infected animals, poultry, and fish.2629 Furthermore, MDR bacterial pathogens often pose a therapeutic dilemma for clinicians and are therefore associated with a high mortality rate and poor prognosis.

Recently, the incidence of infections caused by the genus Elizabethkingia has increased continuously worldwide, especially those caused by E. anophelis species. A single-hospital study from South Korea reported that there was an increase in the prevalence of Elizabethkingia genus infections among hospitalized patients from 0.02 in 2009 to 0.88 in 2017.9 The first recorded outbreak of E. anophelis infection was from Singapore in 2012, in which three out of five patients died of septicemia.3 Furthermore, during 20142016, several outbreaks have occurred in the Midwestern United States, including Wisconsin, Illinois, and Michigan, in which the patient fatality rate related to E. anophelis infections ranged from 30.8% to 70%.58 Therefore, E. anophelis infection is regarded as profoundly serious and important and should be taken seriously by clinicians.

A recent study indicated that E. anophelis has been continuously misidentified as Elizabethkingia meningoseptica (E. meningoseptica) using conventional methods (API/ID32, Phoenix 100 ID/AST, Vitek 2 and Vitek MS).13 Therefore, most of the previously reported data regarding clinical characteristics, antimicrobial susceptibility patterns and carbapenem resistance mechanisms of E. anopheles, may be incorrect. To date, however, the susceptibility patterns of E. anophelis isolates have not been reported in Mainland China, especially with data collated using the more robust broth dilution method. Genome-wide analysis has revealed that this multidrug-resistant pathogen carries a class A serine--lactamase, CME, 2 metallo--lactamases, GOB and BlaB, in addition to numerous genes encoding for putative efflux pumps.5,1820 However, no studies have focused on the function of these putative efflux pumps in E. anophelis isolates. In addition, data reporting on risk factors associated with infection and mortality in E. anophelis infected patients may potentially help clinicians identify high-risk patients and help guide future therapeutic strategies.

The present study was therefore initiated to: (i) identify the risk factors associated with E. anophelis infection and in-hospital mortality, (ii) investigate the antimicrobial susceptibility patterns and carbapenem resistance mechanisms of E. anophelis isolates and (iii) characterize the function of -lactamases and putative efflux pumps expressed in E. anophelis isolates.

This study used the clinical microbiology database from a 3200-bed university-affiliated medical center (Chongqing, China) to retrospectively collect those strains that were identified as the genus Elizabethkingia between January 2015 and December 2019. Sampling and isolation of bacterial strains were a part of the routine hospital laboratory procedures and microbial identification was performed in the microbiology laboratory using the VITEK2 compact (bioMrieux, Inc., NC, USA) and the VITEK MS (bioMrieux, MO, USA) systems. All strains from the genus Elizabethkingia were stored at 80C in 15% glycerol until use. Complete 16S rRNA gene sequencing was used to reconfirm the identity of all isolates. The primers used for amplification and sequencing of the 16S rRNA gene are listed in Table S1 in Additional file 1. The sequences were assembled using SeqMan (DNAStar) and compared with publicly available sequences in the NCBI (http://www.ncbi.nlm.nih.gov) using the BLAST algorithm. Strains were considered to be accurately identified when a strain shared >99.0% 16S rRNA sequence with a type of strain in GenBank. The sequences of 16S rRNA were performed using ClustalW, and the phylogenetic trees were constructed in MEGA7 software using the Neighbor-Joining method.

Electronic medical records of the patients were collected retrospectively and we excluded subjects with the following characteristics: patients with polymicrobial infection and patients admitted for <48 hours. Only the first episode was considered for patients with more than one positive E. anophelis culture. To evaluate the risk factors associated with E. anophelis infection, controls were defined as randomly selected patients with non-E. anophelis infections during the same time (at a 3:1 ratio to the case group). Selected epidemiological, demographic, clinical, laboratory, treatment and outcome data were obtained from the electronic medical records. Empirically administering agents to isolates that were not susceptible was defined as inappropriate empirical antimicrobial therapy. Shock was defined as the coexistence of a systolic pressure of <90 mm Hg and organ dysfunction of the respiratory system, liver, or kidneys. Serum total protein content of <60 g/L or albumin content <25 g/L was the criteria used to define hypoproteinemia. Hypokalemia was diagnosed for a serum potassium level <3.5 mmol/L. Systemic steroid use was defined as oral or intravenous administration of at least 20 mg/day of a steroid (prednisone, hydrocortisone, methylprednisolone, or dexamethasone) within 1 month of infection. We defined anemia as a hemoglobin level of <130 g/L in men and <120 g/L in women according to the World Health Organization (WHO) guidelines. The primary clinical outcome was in-hospital mortality.

The reference broth microdilution method was used to evaluate the minimum inhibitory concentrations (MICs) of all antibiotics in E. anophelis and recombinant strains according to the Clinical and Laboratory Standards Institute (CLSI) M07-Ed11 (2019). The criterion suggested by the CLSI for other non-Enterobacteriaceae was used to determine the susceptibility of isolates to antibiotics except for ceftazidime/avibactam, aztreonam/avibactam, vancomycin, tigecycline, rifampicin, colistin and fosfomycin. The US Food and Drug Administration (FDA) Enterobacteriaceae criteria were used to interpret isolate susceptibility to tigecycline (resistant MIC 8 g/mL, susceptible MIC 2 g/mL and intermediate MIC = 4 g/mL). The MIC breakpoint applied to vancomycin and rifampicin was adapted from the CLSI criteria for Staphylococcus spp. A MIC of 16/4 g/mL was considered resistant for the combination of ceftazidime/avibactam and aztreonam/avibactam. MICs for colistin were interpreted at susceptible breakpoints of 2 g/mL and resistant breakpoints of >2g/mL according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) Enterobacteriaceae criteria. Likewise, for fosfomycin, we elected to use the susceptible breakpoint of 32 g/mL and resistant breakpoint of >32 g/mL based on EUCAST Enterobacteriaceae criteria. The reference strains Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) were used as negative controls and quality controls for antibiotic susceptibility testing.

The presence of the carbapenemase genes (blaKPC, blaIMP, blaVIM, blaNDM, blaBlaB, blaGOB and blaOXA-48-like) and extended-spectrum beta-lactamase (ESBL) genes (blaTEM, blaSHV, blaCME and blaCTX-M) were confirmed by touch-down PCR assays and sequencing. Touch-down PCR amplification was performed using a thermal cycler (Applied Biosystems VeritiPro PCR, CA, USA) under the following cycling conditions: Initial step of 95C for 3 min, followed by 8 cycles of 92C for 20 sec, with annealing temperatures starting at 68C for 20 sec (decreasing 2C/cycle), and with a final extension at 72C for 30 sec; this step was followed by 21 cycles of 92C for 20 sec, 55C for 20 sec, 72C for 30 sec, and finally, 72Cfor 5 min. DNA sequencing was performed using an Applied Biosystems 3730 DNA Analyzer.

We amplified the full-length coding sequences (CDSs) of the genes using specific primers flanked by restriction sites (EcoRI, XhoI or BamHI). These included genes encoding for blaCME, blaBlaB, blaGOB, CzcABC family efflux RND transporter, Efflux ABC transporter (ATP-binding protein), the MATE family of MDR efflux pumps, small multidrug resistance family (SMR) proteins and MFS-type transporter. Amplified PCR fragments were purified and cloned into the corresponding sites within the pET-28a plasmid and then electroporated into BL21 competent E. coli. Positive clones were verified by PCR and sequencing. The sets of primers used for amplification and sequencing of target genes are provided in Table S1 in Additional file 1.

The data were evaluated using SPSS statistical software (version 22.0, IBM). Data are presented as counts (proportions) for categorical variables. Direct comparisons between two groups were determined using the 2 test or Fishers exact test. Means ( standard deviation) were used to express normally distributed continuous variables and the median ( inter-quartile range) was calculated for non-normally distributed variables. Comparisons between two groups were conducted using Students t-test for normally distributed variables or a MannWhitney U-test for non-normally distributed variables. To evaluate independent risk factors for the infection and in-hospital mortality of E. anophelis isolates, we examined all plausible variables using a univariate analysis. Risk factors with a P value <0.1 as detected by the univariate analysis were included in a multivariate logistic-regression model with the enter method. Odds ratios (ORs) and 95% confidence intervals (CIs) were determined for each risk factor of infection and in-hospital mortality. A two-tailed P value <0.05 was considered statistically significant.

The Institutional Review Board and Ethics Committee of Chongqing Medical University approved this study (approval number: 2020703). The study was conducted in accordance with the Declaration of Helsinki. The collection of culture isolates and collation of anonymous clinical data was in accordance with the approved clinical practice guidelines. The need to give informed consent for this study was waived by the Institutional Review Board due to the nature of the retrospective analysis with no individual patient identifiers.

A total of 59 non duplicated Elizabethkingia isolates were collected from the clinical microbiology laboratory for microbial trait investigation. Full 16S rRNA gene sequencing was performed on 59 Elizabethkingia clinical strains. After comparison with available sequences in the NCBI using BLAST, 16S rRNA sequencing showed that 39 (69.6%) isolates were identified as Elizabethkingia anophelis R26. The phylogenetic tree based on 16S rRNA showed the genetic relationship among Elizabethkingia anophelis (Figure 1).

Figure 1 Phylogenetic tree showing the relationship of the 39 E. anophelis isolates using 16S rRNA gene sequence. Bootstrap support value above branches, the scale bar indicates the number of substitutions per site.

Of the E. anophelis isolates with a documented site of infection, 18 (46.2%) were from the respiratory tract, eight (20.5%) were from the urinary tract, six (15.4%) from blood, three (7.7%) from cerebrospinal fluid, and four were from peritoneal fluid, amniotic fluid, conjunctival sac, and the cornea (Table 5). From January 2015 to December 2019, 39 E. anophelis samples were isolated from 39 consecutive patients who were enrolled in the study. These patients consisted of 21 males (53.8%) and 18 females (46.2%) with a median age of 61.

Notably, chronic obstructive pulmonary disease was the most frequent comorbidity (76.9%), followed by anemia (66.7%). The empirical antibiotics used in the 39 patients included carbapenems (38.5%), -lactam/lactamase inhibitors (33.3%), -lactams (30.8%), teicoplanin (25.6%), levofloxacin (17.9%), aminoglycosides (17.9%), minocycline (12.8%) and antibiotics administered either alone or in combination (Table 5), and 84.6% of these empirical antibiotic therapies were deemed as inappropriate antibiotic use. Overall, the in-hospital mortality rate of patients with E. anophelis infection was 51.3%. Furthermore, -lactam/lactamase inhibitor antibiotics were used significantly more in patients who did not survive (P = 0.041).

Compared to the non-E. anophelis infection controls, the potential risk factors for the acquisition of E. anophelis infections are shown in Tables 1 and 2. Univariate analysis indicated that hypertension, cerebrovascular diseases, chronic obstructive pulmonary disease, renal diseases, surgery in the past 6 months, anemia, hypoproteinemia, and hypokalemia were significantly more frequent in patients with E. anophelis infections (P < 0.05). There was also a positive correlation between patient age and their likelihood of acquiring an E. anophelis infection (P < 0.05). In the multivariate analysis, coronary artery diseases (OR 5.81, 95% CI: 1.0930.93, P = 0.039), chronic obstructive pulmonary disease (OR 6.71, 95% CI: 1.5528.99, P = 0.011), surgery in the past 6 months (OR 18.04, 95% CI: 3.2998.87, P = 0.001), anemia (OR 6.72, 95% CI: 1.1240.42, P = 0.038) and systemic steroid use (OR 9.87, 95% CI: 1.3074.94, P = 0.027) were independent risk factors for the acquisition of an E. anophelis infection.

Table 1 Univariate Analysis of Clinical Features of Patients Infected with E. anophelis Isolates

Table 2 Multivariate Analysis of Clinical Features of Patients Infected with E. anophelis Isolates

Univariate and multivariate logistic-regression analysis results for the factors associated with in-hospital mortality are shown in Table 3. Univariate analysis showed that cerebrovascular disease (P = 0.035), chronic obstructive pulmonary disease (P = 0.020), nasogastric tube insertion (P = 0.008) and anemia (P = 0.002) were associated with a higher mortality rate. Using further multivariate analysis, anemia (OR 86.38, 95% CI: 1.425251.29; P = 0.033) was identified as the only independent risk factor for in-hospital mortality in patients with E. anophelis infections.

Table 3 Risk Factors Associated with In-Hospital Mortality

The susceptibility of the 39 E. anophelis isolates to the antimicrobial agents tested in this study is shown in Table 4. The isolates showed high in-vitro susceptibility towards minocycline (100%), and piperacillin/tazobactam (71.8%) but with lower in-vitro susceptibility towards levofloxacin (38.5%), ciprofloxacin (30.8%), rifampicin (20.5%), piperacillin (17.9%) and tigecycline (10.3%). The MIC50, MIC90 and MIC range for minocycline on the isolates were 0.5, 1, and 0.251 g/mL, respectively, whereas the MIC50, MIC90 and MIC range for piperacillin/tazobactam were 16, 32, and 464 g/mL, respectively. All isolates displayed resistance to vancomycin, ceftazidime, cefepime, aztreonam, ceftazidime/clavulanic acid, cefepime/clavulanic acid, colistin and fosfomycin according to the breakpoints used. It is worth noting that while all isolates produced the MBLs BlaB and GOB, aztreonam/avibactam could not further inhibit growth.

Table 4 Antimicrobial Susceptibilities of E. Anophelis Isolates Determined by the Broth Microdilution Method

PCR experiments were used to detect the presence of blaGOB and blaBlaB in 37 isolates, and blaCME -lactamase genes in 36 isolates from the original 39 E. anophelis isolates (Table 5 and Figure 2). Sequence alignments showed that E. anophelis strains harbored two types of the blaCME gene, blaCME-1 (n = 34) and blaCME-2 (n = 2), eight types of the blaBlaB gene, including blaBlaB-29 (17), blaBlaB-2 (7), blaBlaB-1 (5), blaBlaB-17 (3), blaBlaB-3 (1), blaBlaB-9 (1), blaBlaB-33 (1) and blaBlaB-34 (1), and eight types of the blaGOB gene, including blaGOB-38 (19), blaGOB-20 (8), blaGOB-32 (3), blaGOB-50 (3), blaGOB-39 (1), blaGOB-4 (1), blaGOB-40 (1) and blaGOB-45 (1). The most detected combination of -lactamases was CME-1 + BlaB-29 + GOB-38 (n = 17).

Table 5 Empirical Antimicrobial Therapy in Patients Infected with E. anophelis Isolates

Figure 2 (A) Electrophoretic pattern of BlaB gene (759 bp); M: 1002000 bp DNA ladder; Lanes 1, 2, 3, 5, 6, 7, 8, 9: positive E. anophelis strains; Lanes 4: negative E. anophelis strain. (B) Electrophoretic pattern of CME gene (912 bp); M: 1002000 bp DNA ladder; Lanes 1, 2, 3, 4, 6, 7, 8: positive E. anophelis strains; Lanes 5, 9: negative E. anophelis strains. (C) Electrophoretic pattern of GOB gene (885 bp); M: 1002000 bp DNA ladder; Lanes 1, 2, 4, 5, 7, 8, 9: positive E. anophelis strains; Lanes 3, 6: negative E. anophelis strains.

We also investigated the presence of other -lactamase genes, however, none of the 39 E. anophelis isolates harbored carbapenemase genes (blaKPC, blaIMP, blaVIM, blaNDM and blaOXA-48-like) or ESBL genes (blaTEM, blaSHV and blaCTX-M).

To further evaluate the function of -lactamases, the most prevalent forms of blaCME, blaBlaB and blaGOB genes from E. anophelis isolates were cloned into a pET28a(+) plasmid vector. We also amplified and cloned genes encoding putative efflux pump proteins including the CzcABC family efflux RND transporter, Efflux ABC transporter (ATP-binding protein), MATE family members of the MDR efflux pump, small multidrug resistance family (SMR) proteins and MFS-type transporter. These plasmids were transformed into BL21 (DE3) E.coli and the MICs of the common antibiotics were tested in the resultant strains. The strain transformed with pET-CME displayed an increased MIC for ampicillin, piperacillin, cefazolin, cefuroxime, ceftazidime, ceftriaxone and aztreonam when compared to the pET28a(+) vector construct. This suggests that the extended-spectrum serine--lactamase CME is functionally involved in cephalosporin and monobactam resistance (Table 6). The MIC for imipenem increased 32-fold (from 0.125 to 4 g/mL) in the presence of the pET-BlaB construct and 16-fold (from 0.125 to 2 g/mL) in the presence of the pET-GOB construct. This suggests that the MBLs BlaB and GOB, are responsible for increased imipenem resistance (Table 6). Along with the increase in imipenem resistance, the pET-BlaB and pET-GOB constructs also conferred an increased MIC for ampicillin, piperacillin, cefazolin, cefuroxime, and ceftazidime. This indicates that the MBLs BlaB and GOB can also degrade ampicillin, piperacillin, cefazolin, cefuroxime, and ceftazidime (Table 6). All the efflux pump transformants tested, including pET-ABC, pET-MFS, pET-MATE, pET-SMR and pET-RND did not result in increased MICs for any of the antibiotics tested (Table 6).

Table 6 The Antibiotic Susceptibilities of BL21 (DE3) E. coli Expressing CME, BlaB, GOB, Putative Efflux Proteins or the pET28a(+) Vector

Infection with E. anophelis in humans is increasing in many countries and there have been several reports of E. anophelis outbreaks in the community and nosocomial environment in Singapore and the Midwestern United States, including Wisconsin, Illinois, and Michigan.310 However, as suggested previously, E. anophelis is commonly misidentified as E. meningoseptica when using biochemical identification methodologies or automated identification systems in clinical settings. Therefore, data regarding the clinical features, clinical prognosis, and the antimicrobial susceptibility profiles of E. anophelis could be skewed. For these reasons, we have, for the first time identified the risk factors associated with the acquisition of E. anophelis and we have found that anemia is an independent risk factor for in-hospital mortality in patients with E. anophelis infections. Moreover, we demonstrate for the first time that various putative efflux pumps found in E. anophelis do not alter antimicrobial resistance and therefore, do not possess a drug efflux function. -Lactamases were commonly found in E. anophelis isolates and the MBLs BlaB and GOB, are responsible for carbapenem resistance, whereas the ESBL, CME is functionally involved in resistance to cephalosporins and monobactams.

In the present study, we initially explored risk factors associated with E. anophelis infection and using multivariate analysis, we found that coronary artery diseases, chronic obstructive pulmonary disease, surgery in the past 6 months, anemia and systemic steroid use were independently associated with E. anophelis infection. Previous studies have suggested that patients with E. anophelis infection could have greater underlying comorbidities and this study is the first to provide a statistical analysis to support this hypothesis.310 Moreover, surgery in the past 6 months, anemia and systemic steroid use are known to compromise the immune status of the patients. Therefore, these patients are more vulnerable to the acquisition of E. anophelis infection in the same hospital environment.

In previous reports, the case fatality rate of patients with E. anophelis infection ranged from 24% to 60% in different countries.414 In line with this, our study showed that the in-hospital mortality rate of patients with an E. anophelis infection was 51.3%. We, therefore, went on to explored the factors influencing this mortality. When compared with the only previous study investigating risk factors for mortality,10 our study demonstrated that anemia was the only independent predictor of mortality in patients infected with E. anophelis, a factor which has not been reported previously and these patients may present as anemic because of the hemolytic activity of E. anophelis. Several studies have reported that this bacterium can lyse erythrocytes to access essential nutrients (such as amino acids) using hemolysins and heme-degrading proteins.2022 This process may alter the host's physiological status and compromise the immune system, thereby worsening the patient prognosis with E. anophelis. Therefore, anemic patients with a confirmed E. anophelis infection should be considered as higher risk and should be given greater scrutiny and special care. Unexpectedly, inadequate antibiotic therapy was not associated with mortality, this is probably because of the small sample size of infected patients in our study.

Published information concerning the antimicrobial susceptibility patterns of E. anophelis, identified using reliable methods is limited. Studies from Singapore and Taiwan have demonstrated that E. anophelis was resistant to carbapenems, -lactams, -lactam/-lactam inhibitor and aminoglycosides.1416 However, previous research showed that the susceptibility of E. anophelis to fluoroquinolones, tigecycline, piperacillin, piperacillin-tazobactam and trimethoprim-sulfamethoxazole was variable.46,10,17 Using a disk diffusion or agar dilution test, studies from Hong Kong, South Korea and the USA reported the following susceptibilities of E. anophelis to ciprofloxacin (22%100%), levofloxacin (29%96%), piperacillin (41.1%100%), piperacillin-tazobactam (92%), vancomycin (0%100%) and trimethoprim-sulfamethoxazole (22%70.6%).4,5,17 However, when using the broth microdilution test, researchers from Taiwan and Singapore showed the following susceptibilities of E. anophelis to ciprofloxacin (1%21.5%), levofloxacin (16%78.5%), piperacillin (19.4%), piperacillin-tazobactam (30.6%92.4%), vancomycin (0%), tigecycline (5.1%26.4%), minocycline (97.5%100%) and trimethoprim-sulfamethoxazole (4%92.4%).1416 As shown above, there are huge discrepancies in the susceptibility levels of E. anophelis to ciprofloxacin, levofloxacin, piperacillin, and vancomycin when comparing the disk diffusion or agar dilution tests and the broth microdilution test. This suggests that the susceptibility of E. anophelis seen using the disk diffusion test or agar dilution test may be unreliable and inaccurate, as the broth microdilution test represents the gold-standard method for antimicrobial susceptibility testing.

There are also obvious differences in the susceptibility of E. anophelis to antibacterial agents when the standard broth microdilution test is used. A possible explanation for this inconsistency is that natural geographical differences cause variation in the susceptibility patterns observed in previous studies. It is therefore necessary to investigate the antimicrobial susceptibility of E. anophelis in local areas as a guide to antibiotic selection. Our study showed the following susceptibility of E. anophelis to various treatments: minocycline (100%), piperacillin-tazobactam (71.8%), levofloxacin (38.5%), ciprofloxacin (30.8%), piperacillin (17.9%), rifampicin (20.5%) and tigecycline (10.3%). All isolates displayed resistance to ceftazidime, cefepime, aztreonam, ceftazidime/clavulanic acid, cefepime/clavulanic acid, colistin and fosfomycin, according to the breakpoints used. Most of the antimicrobial susceptibility results in this study are consistent with those of previous studies performed using the broth microdilution test. These results suggest that antimicrobial therapy for E. anophelis should prioritize minocycline or piperacillin-tazobactam. However, in our study, patients in the non-survival group were treated with significantly more -lactam/lactamase inhibitor antibiotics as compared to the survival group (P = 0.041). This observation indicates that piperacillin-tazobactam is not an effective treatment for E. anophelis infections. This study was limited by the fact that antimicrobial susceptibility in-vitro does not equate to in-vivo clinical efficacy. Therefore, large prospective clinical trials are urgently needed to validate therapeutic recommendations.

In this study, we screened for the presence of carbapenemase genes (blaKPC, blaIMP, blaVIM, blaNDM, blaBlaB, blaGOB and blaOXA-48-like) and ESBL genes (blaTEM, blaSHV, blaCME and blaCTX-M) in all 39 E. anophelis isolates. No carbapenemase genes (blaKPC, blaIMP, blaVIM, blaNDM and blaOXA-48-like) or ESBL genes (blaTEM, blaSHV and blaCTX-M) were detected in any isolates. However, we identified 35 (89.7%) isolates co-harboring blaGOB, blaBlaB and blaCME -lactamase genes. The most detected combination of -lactamases was CME-1, BlaB-29, and GOB-38 (n = 17).

To further evaluate the function of -lactamases, recombinant strains harboring either blaCME, blaBlaB or blaGOB were constructed. The transformed strain expressing CMEs displayed an increased MIC for ampicillin, piperacillin, cefazolin, cefuroxime, ceftazidime, ceftriaxone and aztreonam as compared to the pET28a(+) vector construct. This suggests that the ESBL CME is functionally involved in resistance to cephalosporins and monobactams. The MIC for imipenem increased 32-fold (from 0.125 to 4 g/mL) in the presence of the BlaB-expressing construct and 16-fold (from 0.125 to 2 g/mL) in the presence of the GOB-expressing construct. This suggests that the metallo--lactamases BlaB and GOB are responsible for the observed carbapenem resistance.

Aztreonam/avibactam is a novel class of combinational -lactamase-inhibitor, designed to treat serious infections of metallo--lactamase (MBL)-producing Gram-negative bacteria, which is currently in Phase I clinical trials (NCT01689207). Aztreonam is relatively stable against MBL hydrolysis, however, it is easily inactivated by class A (eg, KPC), class C (eg, AmpC) and certain class D (eg, OXA-48) serine--lactamase enzymes.23 Avibactam potently inhibits class A, class C and certain class D serine--lactamase enzymes and displays a broader -lactamase inhibition profile than other -lactamase inhibitors.23 When in combination, aztreonam/avibactam is effective against isolates co-producing ESBLs and MBLs with porin loss/deficiency.24 However, it was quite unexpected that E. anophelis is resistant to aztreonam/avibactam according to our experimental results.

Genomic annotation of all Elizabethkingia spp. reveals that besides -lactamases, there are also numerous putative efflux pump proteins including CzcABC family efflux RND transporter, Efflux ABC transporter (ATP-binding protein), MATE family of MDR efflux pumps, small multidrug resistance family (SMR) proteins and MFS-type transporter. Interestingly, however, none of these transporters have been phenotypically characterized.5,1820 It was, therefore, critical to investigate the function of these putative efflux pumps. Our data showed that all recombinant efflux pump strains including pET-ABC, pET-MFS, pET-MATE, pET-SMR and pET-RND did not result in increased MICs for -lactam and non--lactam antibiotics. These results suggest that the putative efflux pump genes from E. anophelis are not responsible for antimicrobial drug resistance. Similarly, Schindler et al cloned and expressed 21 putative efflux pump genes in Staphylococcus aureus which had no effect on any of the antibiotics tested.25 In summary, we demonstrate for the first time that the various putative efflux pumps found in E. anophelis do not possess antimicrobial drug efflux function.

There were some limitations to our study; however, firstly, the small sample size from the single-center study prevented its translation to the wider population. However, the identification of both a carbapenem resistance mechanism and the susceptibility profile of the drug-resistant E. anophelis are of great clinical importance and warrant an urgent, wider, in-depth study. Secondly, no further investigation into the clonality of these isolates was performed, so that the possibility of infection outbreaks cannot be ruled out.

In conclusion, this study provided a detailed report of risk factors, antimicrobial susceptibility patterns and carbapenem resistance mechanisms in E. anophelis clinical isolates from one medical center in Southwest China. Our data showed that patients with anemia, coronary artery diseases, chronic obstructive pulmonary disease or patients who have received systemic steroids or surgery in the past 6 months are more likely to acquire an E. anophelis infection. Furthermore, patients with anemia have a worse prognosis and therefore require more attention and special care from clinicians. The collected clinical isolates exhibited remarkable multidrug resistance to colistin, fosfomycin, aztreonam/avibactam and tigecycline, which are all regarded as last-resort treatments for carbapenem-resistant Enterobacteriaceae, while minocycline is the most effective antibiotic against E. anophelis in-vitro. Mechanistic analysis revealed that carbapenem resistance is associated with the hydrolytic activity of the MBLs BlaB and GOB and is not associated with various putative efflux pumps expressed in E. anophelis. Future in-vivo and prospective clinical trials are urgently needed to determine optimal antimicrobial agent efficacies based on in-vitro drug susceptibility testing results and resistance mechanisms.

This study was supported by the Natural Science Foundation of Chongqing (No.cstc2019jcyj-msxmX0253). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

The authors report no conflicts of interest in this work.

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2. Frank T, Gody JC, Nguyen LB, et al. First case of Elizabethkingia anophelis meningitis in the Central African Republic. Lancet. 2013;381(9880):1876. doi:10.1016/S0140-6736(13)60318-9

3. Teo J, Tan SY, Tay M, et al. First case of E anophelis outbreak in an intensive-care unit. Lancet. 2013;382(9895):855856. doi:10.1016/S0140-6736(13)61858-9

4. Lau SK, Chow WN, Foo CH, et al. Elizabethkingia anophelis bacteremia is associated with clinically significant infections and high mortality. Sci Rep. 2016;6:26045. doi:10.1038/srep26045

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9. Choi MH, Kim M, Jeong SJ, et al. Risk factors for Elizabethkingia acquisition and clinical characteristics of patients, South Korea. Emerg Infect Dis. 2019;25(1):4251. doi:10.3201/eid2501.171985

10. Lin JN, Lai CH, Yang CH, Huang YH, Lin HH. Clinical manifestations, molecular characteristics, antimicrobial susceptibility patterns and contributions of target gene mutation to fluoroquinolone resistance in Elizabethkingia anophelis. J Antimicrob Chemother. 2018;73(9):24972502. doi:10.1093/jac/dky197

11. Janda JM, Lopez DL. Mini review: new pathogen profiles: Elizabethkingia anophelis. Diagn Microbiol Infect Dis. 2017;88(2):201205. doi:10.1016/j.diagmicrobio.2017.03.007

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13. Lin JN, Lai CH, Yang CH, Huang YH, Lin HF, Lin HH. Comparison of four automated microbiology systems with 16S rRNA gene sequencing for identification of Chryseobacterium and Elizabethkingia species. Sci Rep. 2017;7(1):13824. doi:10.1038/s41598-017-14244-9

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22. Kukutla P, Lindberg BG, Pei D, et al. Insights from the genome annotation of Elizabethkingia anophelis from the malaria vector Anopheles gambiae. PLoS One. 2014;9(5):e97715. doi:10.1371/journal.pone.0097715

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24. Zou H, Xiong SJ, Lin QX, Wu ML, Niu SQ, Huang SF. CP-CRE/non-CP-CRE stratification and CRE resistance mechanism determination help in better managing CRE bacteremia using ceftazidime-avibactam and aztreonam-avibactam. Infect Drug Resist. 2019;12:30173027. doi:10.2147/IDR.S219635

25. Schindler BD, Frempong-Manso E, DeMarco CE, et al. Analyses of multidrug efflux pump-like proteins encoded on the Staphylococcus aureus chromosome. Antimicrob Agents Chemother. 2015;59(1):747748. doi:10.1128/AAC.04678-14

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27. Algammal AM, Mabrok M, Sivaramasamy E, et al. Emerging MDR-Pseudomonas aeruginosa in fish commonly harbor oprL and toxA virulence genes and blaTEM, blaCTX-M, and tetA antibiotic-resistance genes. Sci Rep. 2020;10(1):15961. doi:10.1038/s41598-020-72264-4

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[Full text] MBLs, Rather Than Efflux Pumps, Led to Carbapenem Resistance in Fosfom | IDR - Dove Medical Press

Attempts to identify the genetic causes of neuropsychiatric diseases such as post-traumatic stress disorder (PTSD) through large-scale genome-wide analyses have yielded thousands of potential links. The challenge is further complicated by the wide range of symptoms exhibited by those who have PTSD. For instance, does extreme arousal, anger, or irritation experienced by some have the same genetic basis as the tendency to re-experience traumatic events, another symptom of the disorder?

A new study led by researchers at Yale and the University of California-San Diego (UCSD) provides answers to some of these questions and uncovers intriguing genetic similarities between PTSD and other mental health disorders such as anxiety, bipolar disorder, and schizophrenia.

The findings also suggest that existing drugs commonly used for other disorders might be modified to help treat individual symptoms of multiple disorders.

The complexity is still there, but this study helped us chip away at it, said co-senior author Joel Gelernter, theFoundations Fund Professor of Psychiatry and professor of genetics and neurobiology at Yale.

The study was publishedJan. 28 in the journal Nature Genetics.

For the study, the researchers analyzed the complete genomes of more than 250,000 participants in theMillion Veteran Program, a national research program of the U.S. Veterans Administration that studies how genes, lifestyle, and military experiences affect the health and illness of military veterans. Among those participants were approximately 36,000 diagnosed with PTSD.

But instead of looking just for gene variants shared by PTSD patients, they also searched for variants that have been linked to three kinds of clinical symptoms that are experienced, to varying degrees, by those diagnosed with the disorder. These symptom groups, or subdomains, include the re-experience of a traumatic event, hyperarousal or acute anger and irritability, and the avoidance of people or subjects that might be related to past trauma.

While the researchers found underlying genetic commonalities among all three symptom groups, they also discovered specific variants linked to only one or two of the symptoms.

We found a remarkably high degree of genetic relatedness between these three symptom subdomains. But we also wouldnt expect them to be genetically identical, and they are not, Gelernter said. We found biological support for different clinical presentations of PTSD.

The research also showed that some these variants found in subgroups of patient symptoms are also linked to other disorders such as major depression. The results suggest drugs used to treat other disorders might also help treat of PTSD.

Our research pointed to some medications that are currently marketed for other disease states and could be repurposed for PTSD, said co-senior author Murray Stein, Distinguished Professor of Psychiatry and Public Health at UC-San Diego.

Intriguingly, some of the variants linked to all PTSD symptoms have been associated with other neuropsychiatric disorders. For instance, PTSD-associated variants of the geneMAD1L1,which helps regulate cell cycling, have also been linked to schizophrenia and bipolar disorder.

These observations, and the recent finding of GWS [genomewide-significant] association with anxietysuggest thatMAD1L1may be a general risk factor for psychopathology, the authors write.

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Genetic analysis of symptoms yields new insights into PTSD - Yale News

BETHESDA, Md., Jan. 27, 2021 /PRNewswire/ --The ACMG Annual Clinical Genetics Meeting will be a fully virtual meeting in 2021 and continues to provide groundbreaking research and the latest advances in medical genetics, genomics and personalized medicine. To be held April 1316, experience four days of professional growth, education, networking and collaboration with colleagues from around the world and discover what's shaping the future of genetics and genomics, including several sessions on COVID-19. The 2021 ACMG Meeting Virtual Experience is designed to offer a variety of engaging and interactive educational formats and types of sessionsfrom Scientific Sessions and Workshops to TED-Style Talks, Case-based Sessions, Platform Presentations and Short Courses. The 2021 ACMG Meeting Virtual Experience has something for everyone on the genetics healthcare team and will be available to participate in from the convenience of your home or office.

Interview those at the forefront in medical genetics and genomics, connect with new sources, and get story ideas on the clinical practice of genetics and genomics in healthcare today and for the future. Learn how genetics and genomics research is being integrated and applied into medical practice. Topics include COVID-19, gene editing, cancer genetics, molecular genomics, exome sequencing, pre- and perinatal genetics, diversity/equity and inclusion, biochemical/metabolic genetics, genetic counseling, health services and implementation, legal and ethical issues, therapeutics and more.

Credentialed media representatives on assignment are invited to cover the ACMG Annual Meeting A Virtual Experience on a complimentary basis. Contact Kathy Moran, MBA at [emailprotected]for the Press Registration Invitation Code, which will be needed to register at http://www.acmgmeeting.net.

Abstracts of presentations will be available online in February.

A few 2021 ACMG Annual Meeting highlights include:

Program Highlights:

Two Short Courses Available Starting on Tuesday, April 13:

Cutting-Edge Scientific Concurrent Sessions:

Social Media for the 2021 ACMG Meeting Virtual Experience: As the ACMG Annual Meeting approaches, journalists can stay up to date on new sessions and information by following the ACMG social media pages on Facebook,Twitterand Instagramand by usingthe hashtag #ACMGMtg21 for meeting-related tweets and posts.

The ACMG Annual Meeting website has extensive information at http://www.acmgmeeting.net and will be updated as new information becomes available.

About the American College of Medical Genetics and Genomics (ACMG) and the ACMG Foundation for Genetic and Genomic Medicine (ACMGF)

Founded in 1991, the American College of Medical Genetics and Genomics (ACMG) is the only nationally recognized medical society dedicated to improving health through the clinical practice of medical genetics and genomics and the only medical specialty society in the US that represents the full spectrum of medical genetics disciplines in a single organization. The ACMG is the largest membership organization specifically for medical geneticists, providing education, resources and a voice for more than 2,400 clinical and laboratory geneticists, genetic counselors and other healthcare professionals, nearly 80% of whom are board certified in the medical genetics specialties. ACMG's mission is to improve health through the clinical and laboratory practice of medical genetics as well as through advocacy, education and clinical research, and to guide the safe and effective integration of genetics and genomics into all of medicine and healthcare, resulting in improved personal and public health. Four overarching strategies guide ACMG's work: 1) to reinforce and expand ACMG's position as the leader and prominent authority in the field of medical genetics and genomics, including clinical research, while educating the medical community on the significant role that genetics and genomics will continue to play in understanding, preventing, treating and curing disease; 2) to secure and expand the professional workforce for medical genetics and genomics; 3) to advocate for the specialty; and 4) to provide best-in-class education to members and nonmembers. Genetics in Medicine, published monthly, is the official ACMG journal. ACMG's website (www.acmg.net) offers resources including policy statements, practice guidelines, educational programs and a 'Find a Genetic Service' tool. The educational and public health programs of the ACMG are dependent upon charitable gifts from corporations, foundations and individuals through the ACMG Foundation for Genetic and Genomic Medicine.

Kathy Moran, MBA[emailprotected]

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http://www.acmg.net

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Press Registration Is Now Open for the 2021 ACMG Annual Clinical Genetics Meeting - A Virtual Experience - PRNewswire

Huntsville Hospital and Kailos Genetics are teaming up to offer COVID-19 testing for businesses. Basically, all they have to do is sign up and get tested.

Troy Moore with Kailos Genetics said they're making it easy for businesses to reduce outbreaks in an office.

"The Huntsville hospital staff comes on sight and they perform the collection, and then, they bring the samples back to Kailoss lab over at HudsonAlpha," said Moore.

Businesses can sign up for weekly sentinel testing. Kailos Genetics created a viral wash as a less invasive way to get tested for COVID-19. You will get the results back within four days.

The CEO of Huntsville Hospital, David Spillers, said testing for the virus is vital.

If anything, I think testing has become more important going forward than it has been in the past, particularly with these new strains," he said.

The testing Kailos Genetics uses is able to detect both the COVID-19 we've been seeing and the new variants. Moore said consistent testing is key to reducing COVID-19 in a workplace.

What were looking for is to identify those that are carrying the virus, or have been exposed but arent showing symptoms yet, take them back out before they spread it to others, or catch those people that have been exposed very early so they dont, you know, obviously dont spread it to more," Moore said.

Moore said they will discuss with the businesses how frequently they should do sentinel testing based on individual risk factors.

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Huntsville Hospital, Kailos Genetics work to prevent coronavirus outbreaks in the workplace - WAAY

New York, Jan. 26, 2021 (GLOBE NEWSWIRE) -- Reportlinker.com announces the release of the report "Animal Genetics Market Forecast to 2027 - COVID-19 Impact and Global Analysis By Product, Genetic Material, and Services and Geography." - https://www.reportlinker.com/p06010023/?utm_source=GNW However, the market is likely to get impacted by the limited number of skilled professionals in veterinary research and stringent government regulations for animal genetics during the forecast period.

The branch of genetics that deals with the study of gene variation and inheritance in companion, domestic and wild animals is called as animal genetics.Animal genetics are used for genetic trait testing, DNA testing, and genetic disease treatment.

Animal genetics is one of the best mainstays of livestock development (alongside animal nutrition, animal health, and husbandry concerns such as housing). According to the Food and Agriculture Organization of the United Nations, it is a wide field, ranging from characterization to maintenance to genetic improvement, and involves activities at local, national, regional, and global scales.Increasing population and rapid urbanization across the world has resulted in growing preference for animal derived food products such as dairy products and meat that contain high protein.The demand for animal derived proteins and food products, which, in turn drives the growth of animal genetics market.

Growing focus on developing superior animal breeds using genetic engineering to obtain high reproduction rates for large-scale production of modified breeds is expected to drive animal genetics market during the forecast period.Based on product, the market is segmented into poultry, porcine, bovine, canine, and others.The porcine segment held the largest share of the market in 2019, whereas the same segment is anticipated to register the highest CAGR in the market during the forecast period.

Growth of this segment is attributed to rise in production of porcine and increase in pork consumption across the globe.Based on genetic material, the market is segmented into semen and embryo. The embryo segment held the largest share of the market in 2019, and the semen segment is anticipated to register the highest CAGR in the market during the forecast period.COVID-19 pandemic has become the most significant challenge across the world.This challenge would be frightening, especially in developing countries across the globe, as it may lead to reducing imports due to disruptions in global trade, which further increases the shortages of meat and dairy product supplies, resulting in a considerable price increase.

Asian countries such as China, South Korea, and India are severely affected due to COVID-19 outbreak.The World Health Organization, Food and Drug Administration, American Pet Products Association, American Veterinary Medical Cattle Health, and Welfare Group for Disease Control and Prevention are among the major primary and secondary sources referred for preparing this report.Read the full report: https://www.reportlinker.com/p06010023/?utm_source=GNW

About ReportlinkerReportLinker is an award-winning market research solution. Reportlinker finds and organizes the latest industry data so you get all the market research you need - instantly, in one place.

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Animal Genetics Market Forecast to 2027 - COVID-19 Impact and Global Analysis By Product, Genetic Material, and Services and Geography. -...

Fiona the hippopotamus could play a major role in her species' rebound. The world-famous hippo, who turns 4 this week at the Cincinnati Zoo, has genetics that are pretty valuable, her zookeepers said. She could play a critical role in bringing back a threatened species. Hippos are listed as a vulnerable, meaning they face a high risk of extinction in the wild. Officially, threatened species are those listed as critically endangered, endangered or vulnerable. Hippos are listed as vulnerable due to widespread poaching for meat and ivory, as well as human encroachment. It is the eventual goal to have Fiona breed in an effort to increase her species' numbers, but the timeline on when she will be able to breed remains murky.We anticipate that she will not be sexually mature until about 5 or 6 years old maybe even later than that because Fiona was a preemie, said Wendy Rice, head keeper of Africa Department at Cincinnati Zoo.Fiona was thrust into the spotlight due to her remarkable survival story. Born six weeks premature at the Cincinnati Zoo on Jan. 24, 2017, Fiona weighed only 29 pounds at birth 25 pounds less than the lowest recorded birth weight for her species. But she has rebounded from near-death, now weighing a whopping 1,600 pounds, consistent with a normal hippo her age. Fiona has a long way to go until shes considered full grown. But shes on track and making gains every day, Rice said.Already, Cincinnati's once-baby hippo has reached a certain level of maturity. And, when she's ready, Fiona will likely attempt to breed.Her fate and her love interest will likely be determined by the Hippo Species Survival Plan, a cooperation of all zoos across the United States that house hippos and breed them. The group shares information about captive populations in order to maintain genetic diversity.With Fiona being Henrys only living offspring, her genetics are fairly valuable in that theyre not well represented in the population that we have," Rice said. "Its very likely that she will get a recommendation to breed someday.So what happens then? It's highly unlikely that Fiona would move away from Cincinnati, Rice said. Instead, expect a male suitor to arrive in the Queen City.If and when she gets a recommendation for a breeding partner, theres a really good chance that the boy would have to come to Cincinnati. We do not want to have our princess leave Cincinnati, and the whole city would probably riot if she moved away.But we're still talking at least a year -- probably more -- down the road. In the meantime, Fiona will focus on growing. Right now, Rice said Fiona is probably the human equivalent of a pre-teen girl. She's growing out of her sassy phase and becoming more and more independent of her mother. In the past, wherever Bibi was, thats where Fiona was. Just this past year, shes gotten a little bit braver and bolder. Shes also starting to read boundaries a little bit better with mom. She was pushing the envelope, trying to see what she could get away with. But shes kind of settled down a bit and matured, and she can now read mama really well, Rice said. Even as the hippo matures, Rice said her personality is here to stay.Shes still full of personality and shell still come out here and put a show on for her guests," Rice said." Shell come right up to the glass and make eye contact with people. She understands that theyre here for her and that shes kind of a big deal. I think she appreciates her fandom and tries to give them the best experience possible.

Fiona the hippopotamus could play a major role in her species' rebound.

The world-famous hippo, who turns 4 this week at the Cincinnati Zoo, has genetics that are pretty valuable, her zookeepers said. She could play a critical role in bringing back a threatened species.

Hippos are listed as a vulnerable, meaning they face a high risk of extinction in the wild. Officially, threatened species are those listed as critically endangered, endangered or vulnerable. Hippos are listed as vulnerable due to widespread poaching for meat and ivory, as well as human encroachment.

It is the eventual goal to have Fiona breed in an effort to increase her species' numbers, but the timeline on when she will be able to breed remains murky.

We anticipate that she will not be sexually mature until about 5 or 6 years old maybe even later than that because Fiona was a preemie, said Wendy Rice, head keeper of Africa Department at Cincinnati Zoo.

Fiona was thrust into the spotlight due to her remarkable survival story. Born six weeks premature at the Cincinnati Zoo on Jan. 24, 2017, Fiona weighed only 29 pounds at birth 25 pounds less than the lowest recorded birth weight for her species.

But she has rebounded from near-death, now weighing a whopping 1,600 pounds, consistent with a normal hippo her age.

Fiona has a long way to go until shes considered full grown. But shes on track and making gains every day, Rice said.

Already, Cincinnati's once-baby hippo has reached a certain level of maturity. And, when she's ready, Fiona will likely attempt to breed.

Her fate and her love interest will likely be determined by the Hippo Species Survival Plan, a cooperation of all zoos across the United States that house hippos and breed them. The group shares information about captive populations in order to maintain genetic diversity.

With Fiona being Henrys only living offspring, her genetics are fairly valuable in that theyre not well represented in the population that we have," Rice said. "Its very likely that she will get a recommendation to breed someday.

So what happens then? It's highly unlikely that Fiona would move away from Cincinnati, Rice said. Instead, expect a male suitor to arrive in the Queen City.

If and when she gets a recommendation for a breeding partner, theres a really good chance that the boy would have to come to Cincinnati. We do not want to have our princess leave Cincinnati, and the whole city would probably riot if she moved away.

But we're still talking at least a year -- probably more -- down the road. In the meantime, Fiona will focus on growing.

Right now, Rice said Fiona is probably the human equivalent of a pre-teen girl. She's growing out of her sassy phase and becoming more and more independent of her mother.

In the past, wherever Bibi was, thats where Fiona was. Just this past year, shes gotten a little bit braver and bolder. Shes also starting to read boundaries a little bit better with mom. She was pushing the envelope, trying to see what she could get away with. But shes kind of settled down a bit and matured, and she can now read mama really well, Rice said.

Even as the hippo matures, Rice said her personality is here to stay.

Shes still full of personality and shell still come out here and put a show on for her guests," Rice said." Shell come right up to the glass and make eye contact with people. She understands that theyre here for her and that shes kind of a big deal. I think she appreciates her fandom and tries to give them the best experience possible.

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Fionas genetics are hugely valuable in species rebound - WLWT Cincinnati

MOUNTAIN VIEW, Calif., Jan. 26, 2021 /PRNewswire/ --Digbi Health, the first company with a clinically proven genetics and gut-microbiome based program to safely and sustainably treat and manage obesity and associated inflammatory gut, skin and cardiometabolic health issues, is now available to Blue Shield of California members, as a fully covered program, on the health plan'sWellvolution platform.

It's the first time over 60,000 physicians and clinicians practicing in California in the Blue Shield of California's network can prescribe a weight-loss program personalized on a person's genetic, gut microbiome and lifestyle. Through the Digbi Health solution, patients are supported by a team of caregivers, led by a physician and care experts trained in nutrition, cognitive behavior therapy, genetics and gut microbiome. Blue Shield of California offers access to Digbi Health through the Wellvolution platform as a fully covered program to members who qualify.

The Digbi Health Precision Care Network (PCN) is a network of physicians practicing precision medicine. As part of that network, physicians get marketing resources to educate their patients about Digbi Health on the Wellvoution platform, access to their patient's dashboard, with patient approval, and additional support from the Digbi Health care concierge team to support their patients between visits to help improve patient outcomes. Digbi Health program members without a physician can also get referred to a specialist in the PCN.

"The development of cardiovascular disease is a matter of genetic predisposition and gut microbiome composition interacting with acquired conditions, and factors such as diet, exercise, and exposure to damaging elements," said Cynthia Thaik, MD. Harvard-trained cardiologist at the Holistic Healing Heart Center and Digbi Health PCN member.

"I have already prescribed Digbi Health to a patient covered by Blue Shield of California for pre-diabetes and hypertension," she added.

Blue Shield of California is taking the lead on personalized and preventive care for their members.

Among participants participating in Wellvolution:

"We are an innovative telehealth company that supports overburdened physicians by redefining care for 100 million Americans who struggle under one-size-fits-all digital health programs, weight loss diets and stigma of "poor self-control" while fighting obesity and associated inflammatory comorbidities," said Ranjan Sinha, CEO and founder of Digbi Health.

About Digbi Health Precision Care NetworkOur network includes healthcare providers from all specialties and practice settings throughout the U.S., including bariatric surgeons, internal medicine, family medicine, chiropractitioners, nutritional experts, and others in the lifestyle and integrative medicine space using genetics, nutrigenomics, gut microbiome and lifestyle risk to treat the complexity of the multifactorial disease of obesity and its' related medical conditions. Providers can sign-up to the network at no charge here.

About Digbi HealthDigbi Health is a first-of-its-kind precision digital therapeutics company that offers a prescription-grade digitally enabled personalized obesity and obesity related gut, skin disorders, hypertension and other cardiometabolic health management programs based on an individual's gut biome, genetic risks, blood markers, and lifestyle factors. Digbi Health and members of its physician network are committed to empowering people to take control of their own health and wellness. Digbi Health is prescribed by doctors, health care providers, and insurance companies.

SOURCE Digbi Health

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Digbi Health's gut-microbiome and genetic-based obesity management program now allows 60,000 Doctors and Providers in Blue Shield of California's...

A team of researchers from the University of Maine Darling Marine Center in Walpole, Bigelow Laboratory for Ocean Sciences in East Boothbay and Maine Department of Marine Resources in West Boothbay Harbor recently published their research on the effects of ocean warming and acidification on gene expression in the earliest life stages of the American lobster.

The work was published in the scientific journal Ecology and Evolution with collaborators from the University of Prince Edward Island and Dalhousie University in Canada.

Leading the study was recent UMaine graduate student Maura Niemisto, who received her masters degree in marine science. Co-authors on the journal article were her advisers Richard Wahle, research professor in UMaines School of Marine Sciences and director of the Lobster Institute, and David Fields, senior research scientist at Bigelow Laboratory for Ocean Sciences.

Co-authors Spencer Greenwood of the University of Prince Edward Island and Fraser Clark of Dalhousie University brought the genetic expertise to the study. Jesica Waller of the Maine Department of Marine Resources conducted some of the initial studies that led to Niemistos experiments, also in the laboratories of Wahle and Fields.

The teams experiments examined the gene regulatory response of postlarval lobsters to the separate and combined effects of warming and acidification anticipated by the end of the 21st century. They found that genes regulating a range of physiological functions, from those controlling shell formation to the immune response, are either up- or down-regulated. Importantly, they observed that the two stressors combined induced a greater gene regulatory response than either stressor alone.

The results from the study indicate that changes in gene expression of postlarval lobster may act as a mechanism to accommodate rapid changes in the ocean environment. Niemisto noted that there is still need for further study to determine how rapidly populations of the species may be able to adapt to changing conditions. To better understand how gene regulation in response to environmental changes functions within the species, we should look at subpopulations and multigenerational studies to determine the extent of species capacity to respond to altered environmental conditions.

Mauras study reveals some of the hidden mechanisms species employ minute to minute and hour to hour at the cellular level to function normally in a variable environment, said Wahle. We need to gain these insights as we take on the larger challenge of understanding how species adapt on the much larger time scale of decades.

According to the National Marine Fisheries Service, the American lobster fishery is the most valuable in North America. The species holds particular socioeconomic importance in the Gulf of Maine, where sea surface temperatures are increasing at a rate faster than most of the worlds oceans and waters are more susceptible to higher rates of acidification.

The center of the American lobster range has been shifting northward in response to warming ocean temperatures. However, little is known about how the species will respond to the combined effects of increasing ocean temperatures and acidification. This study is a first step in answering that question. The species earliest life stages are thought to be especially vulnerable to these climate related challenges.

The research was supported by a grant from the NOAAs Ocean Acidification Program and the National Sea Grant Program. Additional funding for student internships came from Bigelow Laboratorys Research Experience for Undergraduates program, supported by the National Science Foundation.

Contact: Matt Norwood, matthew.norwood@maine.edu; 207.563.8220

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Research reveals genetic response of ocean warming and acidification in American lobster - UMaine News - University of Maine - University of Maine

LARGO There are roughly up to 500 boys in the world and only boys who suffer from NEMO deficiency syndrome, a rare genetic illness that affects the nervous system and leave one susceptible to infections.

Peyton Kudrnovsky and his brother Trevor are among them. Two weeks ago, 15-year-old Peyton lost his Golden Retriever Axl Rose, who had been a part of the family for 12 years. Axl always sat by Peytons side during his infusions.

Then on Saturday, his family brought home a new four-legged member of the family: Toby.

Toby is a Huskypoo a mix between a Siberian Husky and a poodle donated to the 15-year-old by the Petland Largo store.

Petland Largo manager Miranda Schimenek said they decided to donate Toby after they learned about Peytons story and the loss of Axl. The store at 10289 Ulmerton Road will also provide veterinary care and training for Toby, according to a press release.

The loss of Axl was particularly hard for Peyton, said his mother Tatiana Lee. Axl had been there every step of the way through the 15-year-olds medical treatments, she said. Axl was given to Peyton by his stepfather, Kyle Resler. But the stepfather died two years ago from cancer.

Over two years ago, we lost a loved one to terminal cancer, the mother said in a statement. Peyton had formed a very close bond with him. He was Peytons support system through the illness and we all miss Kyle very much.

They also miss Axl, who helped the family after they lost their stepfather.

He was there for us through the loss of my sons stepdad, Kyle, and would sit by Peyton as he had his infusions each week he was our rock, and we know he is looking down on us, she said. Toby will be an incredible addition to our family and I cannot thank Petland enough for their incredibly gracious gesture.

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Huskypoo puppy donated to teen with rare genetic disorder - Tampa Bay Times

Abstract

Genetic diversity and phylogenetic diversity reflect the evolutionary potential and history of species, respectively. However, the levels and spatial patterns of genetic and phylogenetic diversity of wildlife at the regional scale have largely remained unclear. Here, we performed meta-analyses of genetic diversity in Chinese terrestrial vertebrates based on three genetic markers and investigated their phylogenetic diversity based on a dated phylogenetic tree of 2461 species. We detected strong positive spatial correlations among mitochondrial DNA-based genetic diversity, phylogenetic diversity, and species richness. Moreover, the terrestrial vertebrates harbored higher genetic and phylogenetic diversity in South China and Southwest China than in other regions. Last, climatic factors (precipitation and temperature) had significant positive effects while altitude and human population density had significant negative impacts on levels of mitochondrial DNA-based genetic diversity in most cases. Our findings will help guide national-level genetic diversity conservation plans and a post-2020 biodiversity conservation framework.

Biodiversity loss and conservation are among the most concerning global issues. The Convention on Biological Diversity (CBD) was established to develop national strategies for the conservation and sustainable use of biological diversity. An endangerment status assessment of worldwide vertebrates showed that approximately 20% of vertebrates have become threatened (1). In China, the situation is even worse: 21.4% of vertebrates are threatened, including 43.1% of amphibians, 29.7% of reptiles, 26.4% of mammals, 20.4% of fishes, and 10.6% of birds (2). Thus, it is urgent to protect biodiversity regionally and globally. As the most fundamental dimension of biodiversity, genetic diversity is a key basis for species survival and ecosystem functions (3). Higher genetic diversity means higher evolutionary potential and a greater ability to respond to environmental changes (4). An increasing number of studies have shown that genetic factors play a critical role in species endangerment and extinction (57). Thus, assessment and protection of genetic diversity are becoming essential and high-priority strategies for biodiversity conservation (4). However, under the current CBD framework, the goal proposed for genetic diversity focuses mainly on the conservation of farmed and domestic animals and cultivated plants and neglects that of wild animals and plants, which would overlook genetic erosion and harm the evolutionary potential of wildlife (8). Therefore, to better conserve the genetic diversity of wildlife, it is necessary to assess genetic diversity at regional and global scales for use in the scientific designs of natural protected areas and biodiversity conservation strategies. Miraldo et al. (9) presented the first global distribution of genetic diversity for mammals and amphibians using mitochondrial cytochrome b (Cytb) and cytochrome oxidase subunit I (Co1) gene sequences. However, the grid cell size (~150,000 km2) that they used was so large that it was difficult to determine the national- or regional-level pattern of genetic diversity in detail, including in China.

Phylogenetic diversity is the sum of phylogenetic branch lengths for all of the species in an area (10). Phylogenetic diversity measures the time scale of species evolution and reflects the evolutionary history of species (11), which contributes to the selection of biodiversity conservation priority areas (1214). Higher phylogenetic diversity excluding the effect of taxonomic richness indicates a higher proportion of distantly related and anciently diverged taxa (11, 15). Previous studies have shown that regions with higher phylogenetic diversity may not necessarily have higher species diversity, which would result in neglecting the conservation of the regions (11, 16). In this case, the conservation of older evolutionary lineages might be neglected. Thus, monitoring the level and spatial distribution of phylogenetic diversity is also important for effective conservation of biodiversity.

China is one of the countries with the richest biodiversity in the world, harboring more than 3000 terrestrial vertebrates (2). In recent years, with the development of molecular genetics, genetic diversity of many species has been assessed and numerous DNA sequences have been accumulated. In this study, we focus on the patterns of genetic and phylogenetic diversity in Chinese terrestrial vertebrates, using meta-analyses of a large published dataset and a robust dated phylogenetic tree as well as species distribution. We aim to (i) reveal whether positive spatial correlation existed among species richness, genetic diversity, and phylogenetic diversity; (ii) identify hotspot regions of high genetic diversity and high phylogenetic diversity; and (iii) explore the influences of abiotic (precipitation, temperature, and altitude) and biotic (human population) factors on the levels of genetic and phylogenetic diversity. We found that, on the whole, species richness predicted phylogenetic diversity and mitochondrial DNA-based genetic diversity in a positive direction, and higher phylogenetic diversity predicted higher genetic diversity. We identified that the terrestrial vertebrates in South China and Southwest China harbored higher genetic and phylogenetic diversity than in other regions, and central South China was identified as an evolutionary museum, while the Hengduan Mountains was identified as an evolutionary cradle. We also revealed that both mean annual precipitation and temperature had significant positive effects, while altitude and human population density had significant negative impacts on levels of mitochondrial DNA-based genetic diversity in most cases. Our findings provide insights into the spatial patterns and influencing factors of genetic and phylogenetic diversity at a regional scale.

We surveyed the population-level genetic diversity data of Chinese terrestrial vertebrates (mammals, birds, reptiles, and amphibians) based on three molecular markers (mitochondrial Cytb gene sequence, mitochondrial D-loop sequence, and nuclear microsatellites). A total of 287 terrestrial vertebrate species (103 mammals, 59 birds, 31 reptiles, and 94 amphibians) were assessed for population-level genetic diversity with at least one molecular marker, accounting for 9.3% of the 3075 terrestrial vertebrates distributed in China (figs. S1 to S4 and tables S1 to S9). Two unbiased genetic diversity indices, nucleotide diversity () for the Cytb and D-loop sequences and expected heterozygosity (HE) for microsatellite, were used as measures of population-level genetic diversity. In this study, the Cytb-, D-loop, and microsatellite-based genetic diversity measures were analyzed separately (tables S1 to S9). Furthermore, the species-level genetic diversity for three genetic markers was obtained by averaging the population-level genetic diversity values (tables S10 to S12).

The species-level phylogenetic diversity of Chinese terrestrial vertebrates was surveyed on the basis of the coding sequences of five mitochondrial genes (Cytb, Co1, Nd1, 12S rRNA, and 16S rRNA). A total of 2461 terrestrial vertebrates were assessed for phylogenetic diversity with at least one available mitochondrial gene sequence, accounting for 80% of the Chinese terrestrial vertebrates (figs. S5 to S7 and table S13). On the basis of a constructed maximum likelihood phylogenetic tree and 391 available divergence times from the TimeTree database (table S14), we estimated the divergence times of these vertebrates. The results showed that the amphibians first diverged from the fishes and then the reptiles evolved from the amphibians. Both the mammals and birds evolved from the reptiles, with the mammals diverging first. These results are consistent with the general conclusion about the divergence order of the terrestrial groups (17). In this study, we used divergence time as the measure of phylogenetic diversity for further analysis.

We first divided the map of China into 0.5 0.5 (~50 km by 55 km) grid cells and then calculated the species richness, genetic diversity, and phylogenetic diversity within each grid cell. The spatial correlation tests showed that the genetic diversity measures based on mitochondrial Cytb and D-loop sequences were significantly correlated [correlation coefficient (r) = 0.385, P = 0.012]. However, no significant correlation was observed for Cytb versus microsatellites (r = 0.128, P = 0.475) and for D-loop versus microsatellites (r = 0.084, P = 0.463) (fig. S8 and table S15). The inconsistencies in spatial correlations among the three genetic markers were most likely due to different measure rationales (nucleotide diversity versus expected heterozygosity) and evolutionary rates (slowly versus rapidly evolving). The differences in correlation among the different markers were similar to that of Miraldo et al. (9).

The tests for spatial correlations between genetic diversity and species richness revealed a significant positive correlation for Cytb genetic diversity (r = 0.728, P = 0.008), and a marginally significant correlation for D-loop genetic diversity (r = 0.320, P = 0.072) (Fig. 1, A and B). These results were consistent with those of global terrestrial mammals (18) and global marine and freshwater fishes (19). However, a nonsignificant correlation for microsatellite genetic diversity (r = 0.138, P = 0.499) was detected (Fig. 1C and table S15), which was similar to AFLP marker-based genetic diversity assessment of alpine plant communities (20). The differences in correlation showed that the widely discussed correlation relationship between genetic and species diversity was genetic marker dependent.

(A to C) Correlation tests between species richness (SR) and Cytb-, D-loop, and microsatellite-based genetic diversity (GD). (D) Correlation test between SR and phylogenetic diversity (PD). (E to G) Correlation tests between PD and Cytb-, D-loop, and microsatellite-based GD.

The tests for spatial correlations between genetic diversity and phylogenetic diversity showed a significant positive correlation for Cytb (r = 0.722, P = 0.013) and a marginally significant positive correlation for D-loop (r = 0.306, P = 0.089) (Fig. 1, E and F). The results were similar to those of global terrestrial mammals (18). However, the correlation was not significant for microsatellites (r = 0.123, P = 0.566) (Fig. 1G and table S15). In addition, we selected a set of abundant terrestrial vertebrate species with a threatened status rank of LC (Least-Concern) (table S16) and tested the spatial correlations between genetic and phylogenetic diversity. The results were similar to those for all the terrestrial vertebrates (table S17).

A significant positive correlation was detected between phylogenetic diversity and species richness (r = 0.99, P < 0.001) (Fig. 1D and table S15), implying that the regions with high species richness often had high phylogenetic diversity. The significant positive correlation pattern between phylogenetic diversity and species richness may be common, as shown in different large-scale analyses focusing on birds, mammals, and angiosperms (16, 18, 21).

It is generally accepted that Chinas zoogeographical regionalization is divided into the Palaearctic and Oriental realms, including seven zoogeographical regions (22, 23). The Palaearctic realm includes the Northeast China, North China, Inner Mongolia-Xinjiang, and Qinghai-Tibet Plateau regions, while the Oriental realm consists of the Southwest China, Central China, and South China regions. We mapped the genetic diversity data onto the zoogeographical region map of China using a grid size of 0.5 0.5. Overall, the terrestrial vertebrates distributed in the Oriental realm had higher genetic diversity than those in the Palaearctic realm for all three markers (Fig. 2, A to C; fig. S9; and table S18). In the case of zoogeographical regions, the vertebrates in South China harbored the highest genetic diversity for Cytb and microsatellites, suggesting a hotspot region of genetic diversity, whereas those in North China had the lowest genetic diversity for D-loop and microsatellites (table S18). In addition, the Southwest China and west Central China harbored relatively high genetic diversity. The spatial pattern of species richness across the Palaearctic and Oriental realms was similar to that of genetic diversity (Fig. 2D). However, within the zoogeographical regions, the spatial patterns of species richness were somewhat different from those of genetic diversity. The South China region had the highest species richness, whereas the Qinghai-Tibet Plateau and Inner Mongolia-Xinjiang regions harbored the lowest species richness (Fig. 2D). These results suggest that regions with low species richness do not necessarily have low genetic diversity, such as the Qinghai-Tibet Plateau, which should be given more conservation attention. To determine the possible effects of different sample sizes of the grid cells, we examined the frequency distribution of the proportion of species with surveyed genetic diversity data in the grid cells based on the classification of seven zoogeographical regions and found similar frequency distributions on the whole across the seven regions (figs. S10 to S12).

Northeast China (NE), North China (NC), Inner Mongolia-Xinjiang (IX), Qinghai-Tibet Plateau (QT), Southwest China (SW), Central China (CC), and South China (SC). The red line indicates the boundary between the Palaearctic and Oriental realms. (A and B) Spatial patterns of Cytb- and D-loopbased GDs. measured by nucleotide diversity. (C) Spatial pattern of microsatellite-based GD measured by expected heterozygosity. (D) Spatial pattern of SR measured by number of species.

The province-level distributions of genetic diversity based on the three markers demonstrated similar patterns on the whole (figs. S13 and S14). The terrestrial vertebrates distributed in Yunnan, Guangxi, Sichuan, and Guizhou provinces harbored the highest genetic diversity. In contrast, the terrestrial vertebrates distributed in Shanxi, Shandong, Hebei, Liaoning, Jilin, Heilongjiang, and part of Xinjiang had lower genetic diversity. The terrestrial vertebrates in Qinghai and Tibet had intermediate genetic diversity. These results could help guide province-level conservation plans for genetic diversity.

The terrestrial vertebrates in the Oriental realm had significantly higher phylogenetic diversity (PD = 10,390.25 2029.43) than those in the Palaearctic realm (PD = 4942.60 1402.09) (Fig. 3, A and B). The terrestrial vertebrates in South China harbored the highest phylogenetic diversity (PD = 12,327.46 2111.27), and those in Central China and Southwest China had the second highest phylogenetic diversity. The terrestrial vertebrates on the Qinghai-Tibet Plateau had the lowest phylogenetic diversity (PD = 3936.66 1162.35) (Fig. 3B and table S18). The province-level distribution of phylogenetic diversity showed a clear pattern, in which the terrestrial vertebrates in south China had notably higher phylogenetic diversity than those in north China (fig. S15). Specifically, the vertebrates in Yunnan and Guangxi provinces had the highest phylogenetic diversity, and those in Tibet, Xinjiang, and Qinghai had the lowest phylogenetic diversity (fig. S15). These results could help guide province-level conservation plans for phylogenetic diversity.

(A) A dated phylogenetic tree of Chinese terrestrial vertebrates based on five mitochondrial genes (Cytb, Co1, Nd1, 12S rRNA, and 16S rRNA). Ma, million years. (B) Spatial pattern of PD measured by species divergence time. The red line indicates the boundary between the Palaearctic and Oriental realms. (C) Areas with significantly higher or lower PD after controlling for the confounding effect of SR. The red line indicates the boundary between the Palaearctic and Oriental realms.

As shown by the correlation analysis above, the phylogenetic diversity pattern was highly correlated with the species richness pattern (Fig. 1D). To control for the confounding effect of species richness, we detected areas with significantly higher or lower phylogenetic diversity than expected using a randomization method. The result showed that significantly higher phylogenetic diversity occurred in the central South China region, mainly including Hainan and Guangxi provinces, suggesting that these areas harbored many older terrestrial vertebrate lineages, serving as an evolutionary museum (Fig. 3C and fig. S16) (9). This result is similar to that for the phylogenetic diversity of genus-level angiosperms in China, in which the top 5% highest phylogenetic diversity and standard effective size of phylogenetic diversity were mainly located in Guangdong, Guangxi, Guizhou, and Hainan provinces (15). These results suggested that the above areas are phylogenetic diversity hotspots not only for terrestrial vertebrates but also for angiosperms in China, which deserve more conservation efforts. In contrast, significantly lower phylogenetic diversity occurred in the Southwest China region, i.e., the Hengduan Mountains, suggesting that these areas were the centers of recent speciation events and thus contained many younger lineages, serving as an evolutionary cradle (Fig. 3C and fig. S16) (15, 24). This divergence pattern is similar to that of a study on global terrestrial birds (16).

The above correlation results showed that the mitochondrial DNA-based genetic diversity was strongly correlated with species richness. Therefore, to reveal the effects of abiotic and biotic factors on genetic diversity, we performed the semi-part spatially explicit generalized linear mixed modeling (spaGLMM) analysis by regressing genetic diversity against species richness and then using the residuals of models to evaluate the effects of abiotic (mean annual precipitation, mean annual temperature, and altitude) and biotic (human population density) factors. The results showed that most of the genetic diversity measures were well predicted by these factors (Table 1). In detail, mean annual precipitation had a significant positive effect on Cytb-based genetic diversity; mean annual temperature had a significant positive effect on D-loopbased genetic diversity; and altitude and human population density had significant negative impacts on Cytb- and D-loopbased genetic diversity (Table 1). In addition, the spaGLMM analysis with the species richness included as an explanatory variable gave similar results to the semi-part spaGLMM analysis (table S19). Because the relationships between most of the factors and microsatellite-based genetic diversity were different from theoretically expected, here we did not discuss microsatellite-related results.

MAP, mean annual precipitation; MAT, mean annual temperature; ALT, mean altitude; HPD, human population density.

Because the phylogenetic diversity was very strongly correlated with species richness, we also performed the semi-part spaGLMM analysis for phylogenetic diversity. The results showed that the above abiotic and biotic factors had no significant impacts on phylogenetic diversity (Table 1), suggesting that the species richness had a much higher effect on phylogenetic diversity compared to other factors. To test this, we performed the spaGLMM analysis with species richness as an independent variable. The results showed that the importance of species richness was far more than those of other factors, indicating that phylogenetic diversity was mainly affected by species richness (table S19).

This is the first study to assess the correlation between genetic diversity and phylogenetic diversity for all the terrestrial vertebrate groups at a large spatial scale. The findings revealed a significant correlation between genetic and phylogenetic diversity for Cytb-based genetic diversity measure and a marginally significant correlation for D-loopbased measure at a grid cell scale, demonstrating the important role of phylogenetic diversity in predicting level of genetic diversity. In addition, we also found a significant positive correlation between genetic diversity and species richness for Cytb-based genetic diversity measure and a marginally significant correlation for D-loopbased measure. However, no significant correlations were detected between genetic diversity and phylogenetic diversity (or species richness) for microsatellite-based measure, suggesting that these correlations are genetic marker dependent.

Our study is also the first region-level survey and assessment of the genetic and phylogenetic diversity of Chinese terrestrial vertebrates that demonstrated the spatial distribution pattern of diversity and identified the regions of high and low genetic/phylogenetic diversity. The spatial patterns showed that the terrestrial vertebrates in South China and Southwest China harbored not only higher genetic diversity but also higher phylogenetic diversity, highlighting the high conservation priority for these hotspot regions. We also identified key areas with significantly higher or lower phylogenetic diversity after controlling for the effects of species richness and discerned the evolutionary museum and cradle for Chinese terrestrial vertebrates. In particular, we found inconsistencies among the regions in terms of genetic and species diversity. Although the terrestrial vertebrates on the Qinghai-Tibet Plateau had the lowest species richness, they had intermediate genetic diversity, possibly because of less human activity and heterogeneous abiotic effects in this region. The terrestrial vertebrates in North China and Northeast China, which are exposed to more human activity and located in north further in latitude, harbored intermediate species richness but lower genetic diversity. These results were supported by the semi-part spaGLMM analyses, which revealed that abiotic (precipitation, temperature, and altitude) and biotic factors (human population) played important roles in the spatial patterns of genetic diversity.

We investigated the effects of abiotic and biotic factors driving the spatial patterns of genetic and phylogenetic diversity at a grid cell scale. On the whole, the effects of these factors on Cytb- and D-loopbased genetic diversity were consistent with ecological and evolutionary expectations. Mean annual precipitation and temperature had significant positive effects on genetic diversity, because higher precipitation and temperature most likely provide more suitable conditions for species survival, population expansion, and speciation. In contrast, altitude had significant negative impacts on genetic diversity, because higher elevation means harsher living conditions especially for terrestrial vertebrates. For biotic factor, human population density had significant negative impacts on genetic diversity, because higher density means more human activities and more possible interference with wildlife and their habitats.

Our study summarizes the findings of genetic/phylogenetic diversity studies, revealing the basic background of genetic resources in Chinese terrestrial vertebrates, which could facilitate genetic resource protection under the CBD framework and guide future genetic/phylogenetic diversity research and conservation. In addition, compared with the total number of Chinese terrestrial vertebrates, the number of species with surveyed genetic diversity data is relatively small. To better conserve genetic diversity, scientists and managers should cooperate to perform genetic diversity surveys for more species, especially those with an unclear genetic status. Furthermore, the genetic and phylogenetic diversity of freshwater and marine vertebrates should be surveyed and assessed to protect gradually decreasing aquatic genetic resources. Last, our study is the first to use nuclear microsatellite markers to assess large-scale genetic diversity pattern and explore the relationship between genetic and phylogenetic diversity. However, it is worth noting that microsatellite-based correlation and model analyses produced different results from those based on mitochondrial DNA, which cautions us to carefully interpret results from different genetic markers.

We retrieved published literatures of population-level genetic diversity studies from public academic databases. For the English literature, we searched the Web of Science database (http://apps.webofknowledge.com/) using the search rule TS = (species Latin name OR species English name) AND TS = genetic diversity AND TS = population. For the Chinese literature, we searched the CNKI database (www.cnki.net), CQVIP database (www.cqvip.com), and Chinese Science Citation Database (http://sciencechina.cn) using the search rule species Latin name AND genetic diversity. Then, to search the literature as comprehensively as possible, we searched only the species Latin name again for species without related references or with few related references.

We screened the retrieved literature following several steps. First, we used only the literature about wild animal studies and discarded the literature studying captive populations. Second, we focused on population-level studies based on microsatellite, mitochondrial Cytb, or D-loop markers. These three markers have been widely used in population genetics and phylogeographic studies of vertebrates. For microsatellite-based studies, we extracted the expected heterozygosity (HE) values for each population of species as the measure of microsatellite genetic diversity. HE is an unbiased measure and thus insensitive to small sample sizes (25). For mitochondrial Cytb gene and D-loop sequence-based studies, we extracted Neis nucleotide diversity () values for each population of species as the measure of Cytb or D-loop genetic diversity (26). is also unbiased and thus insensitive to small sample sizes (26). If the same population had more than one HE or from different references, we used the mean value as the genetic diversity measure of this population. Last, on the basis of population-level genetic diversity data, we estimated species-level genetic diversity by averaging the population-level genetic diversity values (9). Mean genetic diversity metric has been widely applied in large-scale studies (9, 18, 19).

In total, we compiled a dataset of 287 terrestrial vertebrates, which included 103 mammals, 59 birds, 31 reptiles, and 94 amphibians, accounting for 15.6, 4.1, 6.7, and 18.6% of the respective total numbers of species (figs. S1 and S2). Overall, the assessment proportions for genetic diversity of mammals and amphibians were higher than those of birds and reptiles, with the proportion of birds being the lowest. The number of terrestrial vertebrate species with population-level genetic diversity data based on microsatellite marker (n = 151) was higher than those based on Cytb gene (n = 142) and D-loop (n = 105), accounting for 4.9, 4.6, and 3.4% of the 3075 Chinese terrestrial vertebrates, respectively (figs. S3 and S4).

Sequences of five mitochondrial genes (Cytb, Co1, 12S rRNA, 16S rRNA, and Nd1) were used to reconstruct the phylogeny of Chinese terrestrial vertebrates. The sequences of the five mitochondrial genes were searched in GenBank with the following steps. First, the available mitochondrial reference genomes were downloaded, and the corresponding coding sequences of these genes were extracted. Then, the available coding sequences for the remaining species were directly downloaded from GenBank using the species Latin name and gene name. If more than one sequence was available for the same locus of a species, the sequence with a length similar to that of the corresponding gene was selected. Last, the short genes whose coding sequence length was <300 base pairs were discarded from the dataset. After these steps, we compiled a total of 2461 species including 573 mammals, 1170 birds, 359 reptiles, and 359 amphibians, representing 87.0, 81.0, 77.2, and 71.0% of the respective total numbers of species. Our dataset covered 46 orders, 204 families, and 847 genera. For each gene, the coding sequences of 973 species were extracted from their mitochondrial genomes, while others were directly downloaded from the GenBank database. The numbers of species with Cytb and Co1 sequences were higher than those with Nd1, 12S rRNA, and 16S rRNA sequences (fig. S7).

The coding sequences of each gene were concatenated and aligned by MAFFT (27) with default parameters, and the poorly aligned sites at the beginning and the end were trimmed. Then, the aligned sequences of these five genes were imported into SequenceMatrix software (28) to construct a supermatrix with the gaps treated as missing data. A phylogenetic analysis was performed on this supermatrix using the maximum likelihood method implemented in RAxML 8.2.12 (29) with the ASC_GTRGAMMA model and 1000 bootstrap replicates. Each gene was treated as a partition, and the zebrafish was used as outgroup. On the basis of this phylogenetic tree, we used the penalized likelihood method implemented in treePL (30) to date the divergence times of these vertebrates. A total of 391 available divergence times from TimeTree (31) were selected as calibration points for the dating analysis (table S14). The prime option and through analysis were implemented with optimal parameters.

On the basis of our dated phylogenetic tree and species distribution data, we calculated Faiths phylogenetic diversity of Chinese terrestrial vertebrates using the picante package (32) in R, as widely used in phylogenetic diversity studies (33). In this study, we used divergence time as the measure of phylogenetic diversity of each species.

The distributional ranges of terrestrial vertebrate species (including mammals, amphibians, reptiles, and birds) were derived from the IUCN spatial database (www.iucnredlist.org/resources/spatial-data-download). The range of each species was originally in a vectorized shapefile format and was rasterized into a grid system with a 0.5 0.5 resolution (~50 km by 55 km). We double-checked the rasterized maps to confirm that they matched the original vectorized distributional range maps. The resultant rasterized map of each species was always conservative relative to the original vectorized map, as many margins of species fragmented distributions might not have been recorded as the presence of the species in our 0.5 0.5 grid cells. This is because the areas of these margins were too small in the corresponding grid cells. The map of China used in this study was from Resource and Environment Science and Data Center (www.resdc.cn/data.aspx?DATAID=200). The Latin name of each species was checked to avoid potential synonyms. In total, our gridded distribution database included the occurrence records for 1941 species. After matching with the genetic and phylogenetic data, the final distribution dataset used for the diversity assessment included a total of 180 species for the genetic diversity analysis and 1685 species for the phylogenetic diversity analysis.

Climate data with a 2.5 spatial resolution were collected from the WorldClim database (https://worldclim.org/). We used the two most important climatic variables, mean annual temperature and mean annual precipitation that were calculated for the climate data from 1970 to 2000, as predictors of spatial patterns of genetic and phylogenetic diversity of terrestrial vertebrates in China. Human population density in 2010 in China (in persons per square kilometer) was derived from the Gridded Population of the World collection (https://sedac.ciesin.columbia.edu/data/collection/gpw-v4). Digital elevation data with a 2.5 spatial resolution in China were originally derived from the NASA Shuttle Radar Topographic Mission and downloadable from the WorldClim database. Because we mapped the genetic and phylogenetic diversity using a grid cell size of 0.5 0.5 for each variable (including altitude), we took the average of all values within each grid cell as the variables value for the grid cell.

In many cases in which biodiversity data are collected associated with spatial information (e.g., sampling location coordinates), conventional correlation tests are not valid because the assumption of total independence of samples is violated. For spatial biodiversity data, neighboring locations can present similar biodiversity features (e.g., genetic diversity or phylogenetic diversity as investigated here), which is a phenomenon known as spatial autocorrelation, resulting in nonindependent association of biodiversity information between neighboring locations. To this end, conventional correlation tests can be misleading. To cope with this issue, we used a modified t test to account for spatial autocorrelation (34, 35) when testing the spatial associations between genetic diversity, phylogenetic diversity, and species richness. The test is based on the adjustment of the sample correlation coefficient between the two spatially correlated quantities and requires the estimation of an effective sample size (degrees of freedom).

We performed spatial correlation tests between genetic diversity based on different markers, between genetic diversity and species richness, between genetic diversity and phylogenetic diversity, and between phylogenetic diversity and species richness. In addition, we selected a set of abundant terrestrial vertebrate species with a threatened status rank of LC (2) to further explore the relationship between genetic diversity and phylogenetic diversity. The set of abundant terrestrial vertebrates included 39 species for Cytb, 25 species for D-loop, and 45 species for microsatellite (table S16). We performed the correlation analyses for Cytb-, D-loop, and microsatellite-based genetic diversity separately.

We divided the map of China into 0.5 0.5 grid cells using R software. Then, we mapped the spatial distributional patterns of species richness, genetic diversity, and phylogenetic diversity based on the diversity values calculated for each grid cell. For species richness, we summed the total number of species occurring in the grid cell. For genetic diversity, we summed the genetic diversity values of each species present within the grid cell and divided the total value by the number of species surveyed in the grid cell, as used in (9). For phylogenetic diversity, we summed the divergence times of all species surveyed within the grid cell following the definition of Faiths phylogenetic diversity (10, 15).

To detect grid cells with significantly higher or lower phylogenetic diversity than expected controlling for the confounding effect of species richness, we used a randomization protocol (36). In detail, we first computed the phylogenetic diversity for each grid cell and divided this value by the species richness found in the cell. Then, we used a random swapping algorithm to randomize the species-site binary matrix while fixing the species richness of each grid cell and the range size of each species. The randomization procedure was repeated 1000 times, and the following effective size of phylogenetic diversity-species richness was computedZPD=ObsPDMean(RandPD)SD(RandPD)where ObsPD is the observed phylogenetic diversity-species richness ratio for each grid cell. RandPD represents the random phylogenetic diversity-species richness ratio calculated for each grid cell derived from the randomized species-site matrix. Mean(RandPD) and SD(RandPD) denote the mean and standard deviation of the 1000 random phylogenetic diversity-species richness ratio values, respectively. ZPD approximately followed a standard normal distribution; as such, at the significance level of 0.05, a grid cell was identified as having statistically significantly high phylogenetic diversity given the associated species richness if ZPD > 1.96. Conversely, a grid cell was identified as having statistically significantly low phylogenetic diversity given the associated species richness if ZPD < 1.96.

Species richness might have strong associations with genetic and phylogenetic diversity (37, 38). To explore the effects of factors affecting the spatial patterns of genetic and phylogenetic diversity of Chinese terrestrial vertebrates, we performed a semi-part spaGLMM implemented in the spaMM package (39) in the R environment (40), in which the influence of species richness on genetic or phylogenetic diversity was explicitly partialled out. To do so, we firstly constructed a spaGLMM model in which species richness is the only explanatory variable of genetic or phylogenetic diversity and then we used the residuals of this model for evaluating the impacts of other abiotic and biotic factors on genetic or phylogenetic diversity. In addition, to assess the effect of species richness on genetic and phylogenetic diversity, we also performed the spaGLMM analyses with the species richness as an explanatory variable as well as other factors.

For all the above spaGLMM analyses, a correlation matrix according to the Matrn correlation function was assumed and fitted on the basis of the longitude and latitude information of the center point of each grid cell when fitting the mixed model. The Matrn correlation function, containing a scale parameter and a smoothness parameter, is widely applied to model spatial correlation by including exponential and squared exponential models as special cases (41, 42). For the modeling results of semi-part spaMM analyses, when the confidence interval of the estimated coefficient for an explanatory variable was significantly deviated from zero, the variable was considered to have a significant effect on levels of genetic or phylogenetic diversity.

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Acknowledgments: We thank Jiekun He for providing the map of zoogeographical regionalization. Funding: This study was supported by the National Natural Science Foundation of China (31821001); the Strategic Priority Research Program of Chinese Academy of Sciences (XDB31000000); the Biodiversity Survey, Monitoring and Assessment Project of Ministry of Ecology and Environment of China (2019HB2096001006); the National Natural Science Foundation of China (31672319); the Youth Innovation Promotion Association, CAS (2016082); and the Special Research Assistant Program of CAS. Author contributions: F.W. conceived and supervised the project. Y.H., H.F., J.C., X.Z., H.W., B.Z., L.Y., X.H., X.S., T.P., W.W., and J.L. performed the data collection. Y.H., H.F., Y.C., J.C., M.W., W.Z., L.Y., and H.H. performed the data analysis. Y.H., H.F., and Y.C. wrote the manuscript with input from F.W. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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