IACUC Research Articles

From the Slaughterhouse to the Laboratory Bench: On the Ethics of Using Slaughtered Animals for Biomedical Research.

Is it ethically permissible to acquire biological materials from a slaughterhouse for biomedical research? This essay examines this question, using a recent, high-profile research program as a case study. Using roughly 300 decapitated pig heads acquired from a slaughterhouse, researchers reperfused the animals' brains and observed a variety of cellular and molecular activities. The study was exempted from review and oversight by the Institutional Animal Care and Use Committee (IACUC), on the grounds that the animals were already dead. This essay argues that the IACUC was mistaken in exempting the study from their oversight, and furthermore, that acquiring animals or their parts from a slaughterhouse for research purposes is unethical and should not occur again. Examination of the study within a broader societal context helps to illuminate why each of us has an ethical obligation to do what we can to abolish the vicious and cruel treatment endured by billions of animals every year on factory farms and in slaughterhouses.

SARS-CoV-2 Variants of Concern Have Acquired Mutations Associated With an Increased Spike Cleavage

Several SARS-CoV-2 lineages have emerged leading to the divergence of more transmissible variants termed Variants of Concern (VOCs). The natural selection of mutations in the spike protein can impact viral cell entry, transmission, and pathogenesis. Here, we characterized emerging SARS-CoV-2 spike polymorphisms in vitro and in vivo to demonstrate that the substitution S:655Y, included in the highly prevalent Gamma variant, enhances viral replication and spike protein cleavage. Moreover, viral competition experiments demonstrate that the S:655Y transmits more efficiently than the ancestor 655H in the hamster model. Finally, we analyze a set of emerging SARS-CoV-2 variants to investigate how different sets of mutations may impact spike processing. All VOCs tested exhibit an increased spike cleavage and fusogenic capacity. This study demonstrates that the S:655Y is an important adaptative mutation that increases viral cell entry, transmission, and host susceptibility. Moreover, SARS-CoV-2 VOCs show a convergent evolution that promotes the spike protein processing.Funding Information: This research was partly funded by CRIPT (Center for Research on Influenza Pathogenesis and Transmission), a NIAID funded Center of Excellence for Influenza Research and Response (CEIRR, contract #75N93021C00014) (AGS), NCI SeroNet grant U54CA260560 (AGS), NIAID grants U19AI135972 and U19AI142733 (AGS), DARPA grant HR0011-19-2-0020 (AGS), JPB Foundation (AGS), Open Philanthropy Project (research grant 2020-215611 (5384) (AGS), anonymous donors to AGS, NBAF Transition Funds from the State of Kansas (JAR), NIAID Centers of Excellence for Influenza Research and Surveillance under contract number HHSN 272201400006C (JAR), AMP Core of the Center for Emerging and Zoonotic Infectious Diseases of the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health under award number P20GM130448 (JAR) and Department of Homeland Security Center of Excellence for Emerging and Zoonotic Animal Diseases under grant number HSHQDC 16-A-B0006 (JAR). AGR is funded by Marion Alban MSCIC Scholars Award and the 2020 Robin Chemers Neustein Postdoctoral fellowship. ML is funded by a fellowship of the Belgian American Education FoundationDeclaration of Interests: The A.G.-S. laboratory has received research support from Pfizer, Senhwa Biosciences, Kenall Manufacturing, Avimex, Johnson & Johnson, Dynavax, 7Hills Pharma, N-fold LLC, Pharmamar, ImmunityBio, Accurius, Nanocomposix, Hexamer and Merck, outside of the reported work. A.G.-S. has consulting agreements for the following companies involving cash and/or stock: Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Vaxalto, Pagoda, Accurius, Esperovax, Farmak, Applied Biological Laboratories and Pfizer, outside of the reported work. A.G.-S. is inventor on patents and patent applications on the use of antivirals and vaccines for the treatment and prevention of virus infections, owned by the Icahn School of Medicine at Mount Sinai, New York. The Icahn School of Medicine at Mount Sinai has filed a patent application relating to SARS-CoV-2 serological assays, which lists Viviana Simon as co-inventor. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2. All other authors have nothing to declare. Ethics Approval Statement: Human SARS-CoV-2: Nasopharyngeal swab specimens were collected as part of the routine SARS-CoV-2 surveillance conducted by the Mount Sinai Pathogen Surveillance program (IRB approved, HS#13-00981).All hamster animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Icahn School of Medicine at Mount Sinai (ISMMS).The Institutional Animal Care and Use Committee (IACUC) of the Icahn School of Medicine at Mount Sinai (ISMMS) reviewed and approved the mink model of COVID-19.

Mitochondria-Targeted Antioxidant SS-31 Attenuates Mitochondrial Dysfunction, Reduces Pyroptosis and Improves Locomotor Functional Recovery After Spinal Cord Injury

Background: Oxidative stress, neuroinflammation, neuronal loss, and demyelination contribute to mechanisms of s pinal cord injury (SCI). Reactive oxygen species (ROS) have been reported to be associated with SCI , which can trigger the pathways toward cellular pyroptosis. Accumulating evidence has suggested that oxidative stress caused by mitochondrial dysfunction play a vital role in the pathophysiology of SCI. In the present study, we explore the effects and mechanisms of SS-31, a mitochondria-targeted antioxidant , on SCI in vivo and vitro. Methods: Functional recovery was assessed using Basso Mouse Scale (BMS) and BMS subscore. Oxidative stress was evaluated using detection of malonaldehyde (MDA) and 3-nitrotyrosine(3-NT). Neuronal loss was evaluated by immunochemistry staining of NeuN. Pyroptosis was assessed by flow cytometry and western blot. Findings: Administration of SS-31 to mice significantly improved locomotor functional recovery up to 28 days after SCI and suppressed neuronal loss and demyelination in mice. Moreover, SS-31 attenuated oxidative stress, inhibited nucleotide-binding oligomerization domain-like receptor 3 (NLRP3) inflammasome activation and pyroptosis occurrence, and decreased proinflammatory cytokines levels in mice. Furthermore, SS-31 in oxygen-glucose deprivation neurons promoted the survival , alleviated mitochondrial dysfunction, decreased oxidative stress, and suppressed NLRP3 inflammasome activation and pyroptosis . Interpretations: Together, our findings reveal that mitochondria-targeted antioxidant SS-31 can improves locomotor functional recovery in mice after SCI and that SS-31-mediated neuroprotection presumably depends on the attenuation of pyroptosis, mitochondrial dysfunction, neuroinflammation and oxidative stress. Funding Statement: This study was supported by National Natural Science Foundation of China (grant.81702163), Zhejiang Provincial Public Welfare Research Project (grant.LGF20H060014), Zhejiang Provincial Medical and Health Technology Project (grant.2020KY699), Science and Technology Development Project of Hangzhou (grant.20170533838), Zhejiang Provincial Public Welfare Research Project (grant.LGF21H060004). Declaration of Interests: The authors declare no competing financial interest. Ethics Approval Statement: All animal studies (including the mice euthanasia procedure) were done in compliance with the regulations and guidelines of Hangzhou First People's Hospital's institutional animal care and conducted according to the Association for Assessment and Accreditation of Laboratory Animal Care international (AAALAC) and the Institutional Animal Care and Use Committee (IACUC) guidelines.

Injectable Self-Healing Cellulose Hydrogel Based on Host-Guest Interaction and Acylhydrazone Bond for Sustained Cancer Therapy

Tumor local chemotherapy employing injectable hydrogels reservoirs is a promising platform to achieve precise drug administration. However, balanced injectability, pH-responsiveness and long-term hydrolysis resistance of self-healing hydrogels remains an appealing challenge. Herein, a modular preassembly strategy combining host-guest interactions with dynamic acylhydrazone bonds, was exploited to fabricate injectable cellulose-based hydrogels dressed with outstanding self-healing property, pH-responsiveness and hydrolytic degradation resistance. Attributed to the host-guest interaction between β-cyclodextrin and 1-adamantane, the hydrogels exhibited excellent injectability, self-healing property (healing efficiency of 97.5 %) and rapid recovery ( 30 days) and pH-responsive behavior, endowing the hydrogels with precisely spatio-temporal drug release administration. When used for osteosarcoma therapy, the average tumor weight (25 mg) treated with doxorubicin (DOX) loaded hydrogel was much smaller than DOX group (161 mg) and control group (553mg), demonstrating the DOX-loaded hydrogels achieved high inhibition efficacy against tumor recurrence without obvious long-term toxicity. Funding Information: This work was supported by the Major International (Regional) Joint Research Project of National Natural Science Foundation of China (21620102004), the National Natural Science Foundation of China (51973166), the Key Research and Development Program of HubeiProvince (2020BCA079), and Hubei Province Scientific and Technical Innovation Key Project (No. 2019ACA136). Declaration of Interests: The authors declare no known conflict of interest. Ethics Approval Statement: All Animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Wuhan University (No. WP2020-08082).

<i>Phodopus roborovskii </i>SH101 as a Systemic Infection Model of SARS-CoV-2

SARS-CoV-2 frequently causes systemic infection by breaking the bounds of the respiratory systems. Although the development of animal models recapitulating the clinical manifestations of systemic COVID-19 is of utmost importance to understand its pathogenesis, there has not been developed an animal model for systemic infection of SARS-CoV-2. Here we report that Phodopus roborovskii SH101, a laboratory inbred hamster strain of P. roborovskii, infected with SARS-CoV-2 breached the bounds of the respiratory system infection causing systemic infection as in the case of the human counterpart. P. roborovskii SH101 post-infection of SARS-CoV-2 represented most clinical symptoms of COVID-19 such as snuffling, dyspnea, cough, labored breathing, hunched posture, progressive weight loss, and ruffled fur, in addition to high fever accompanied by shaking chills. Histological examinations also revealed a serious right-predominated pneumonia as well as slight organ damages in the brain and liver as in the case of human systemic COVID-19 cases. Funding Statement: This research was supported by JINIS BDRD Research Institute of JINIS Biopharmaceuticals Inc. (Korea). Declaration of Interests: Authors declare no competing interests. Ethics Approval Statement: All procedures involving the mice and hamsters were accredited with the approval of the Institutional Animal Care and Use Committee (IACUC), in compliance with the guidelines of the Ethics Committee of Jeonbuk National University Laboratory Animal Center Guidelines on the Care and Use of Animals for Scientific Purposes. All animal experiments in this study were in accordance with the ARRIVE guidelines and checklist. All the animals used in this research were handled in a manner consistent with CDC/ABSA/WHO guidelines for the prevention of human infection with the SARS-CoV-2 virus.

Biomaterial Encapsulation of Human Mesenchymal Stromal Cells Modulates Paracrine Signaling Response and Enhances Efficacy for Treatment of Established Osteoarthritis

Mesenchymal stromal cells (MSCs) have shown promise as osteoarthritis (OA) treatments; however, effective translation has been limited by high variability and heterogeneity of MSCs, suboptimal delivery strategies, and poor understanding of critical quality and potency attributes. Furthermore, most pre-clinical studies of MSC therapeutics for OA have focused on delaying OA development and not on treating established OA, which brings added clinical relevance. Thus, the objective of the current study was to assess the effects of biomaterial encapsulation on human MSC (hMSC) secretion of immunomodulatory cytokines in an OA microenvironment and therapeutic efficacy in treating established OA. A Medial Meniscal Transection (MMT) pre-clinical model of OA was implemented. Three weeks post-surgery, after OA was established, intra-articular injections of encapsulated hMSCs or nonencapsulated hMSCs were administered. Six weeks post-surgery, microstructural changes in the knee joint were quantified using microCT. Encapsulated hMSCs reduced articular cartilage degeneration and subchondral bone remodeling. A multiplexed immunoassay panel was used to profile the in vitro secretome of hMSCs in response to IL-1β. Nonencapsulated hMSCs showed an indiscriminate increase in all cytokines in response to IL-1β while encapsulated hMSCs showed a targeted secretory response with increased expression of pro-inflammatory (IL-1β, IL-6, IL-7, IL-8), anti-inflammatory (IL-1RA), and chemotactic (G-CSF, MDC, IP10) cytokines. These data show that biomaterial encapsulation can modulate hMSC paracrine signaling and enhance the therapeutic efficacy of the hMSCs in treating established OA. This cytokine profile provides a foundation for the identification of key factors affecting the overall potency of hMSC therapeutics for OA. Funding Information: This work was supported in part by VA (SPiRE) Grant [I21RX002372-01A1] from the United States (U.S.) Department of Veterans Affairs Rehabilitation Research and Development Service. The research was also supported in part by the DOD PRMRP Grant [PR171379] and PHS Grant [UL1TR000454] from the Clinical and Translational Science Award Program, National Institutes of Health, National Center for Advancing Translational Sciences. Declaration of Interests: There are no conflicts to declare. Ethics Approval Statement: All animal care and experiments were conducted in accordance with the institutional guidelines and approved by the Atlanta Veteran Affairs Medical Center (VAMC) with experimental procedures approved by the Atlanta VAMC Institutional Animal Care and Use Committee (IACUC).

Micro-computed tomography utility for estimation of intraparenchymal spinal cord cystic lesions in small animals.

Precise assessment of spinal cord cystic lesions is crucial to formulate effective therapeutic strategies, yet histological assessment of the lesion remains the primary method despite numerous studies showing inconsistent results regarding estimation of lesion size via histology. On the other hand, despite numerous advances in micro-computed tomography (micro-CT) imaging and analysis that have allowed precise measurements of lesion size, there is not enough published data on its application to estimate intraspinal lesion size in laboratory animal models. This work attempts to show that micro-CT can be valuable for spinal cord injury research by demonstrating accurate estimation of syrinx size and compares between micro-CT and traditional histological analysis. We used a post-traumatic syringomyelia rat model to compare micro-CT analysis to conventional histological analysis. The study showed that micro-CT can detect lesions within the spinal cord very similar to histology. Importantly, micro-CT appears to provide more accurate estimates of the lesions with more measures (e.g., surface area), can detect compounds within the cord, and can be done with the tissue of interest (spinal cord) intact. In summary, the experimental work presented here provides one of the first investigations of the use of micro-CT for estimating the size of intraparenchymal cysts and detecting materials within the spinal cord. All animal procedures were approved by the University of Akron Institutional Animal Care and Use Committee (IACUC) (protocol # LRE 16-05-09 approved on May 14, 2016).

Culture of Care Enhancement in Egypt: The Impact of Laboratory Animal Science Training on Participants' Attitudes.

Cairo University was the first academic institution in Egypt to establish an Institutional Animal Care and Use Committee (IACUC), as mandated by the World Organisation for Animal Health (OIE). Animal-based research should be performed in accordance with international regulations to monitor the humane care and use of the laboratory animals. Until 2018, the formal training of researchers in the appropriate and correct methods of animal handling during sampling and administration, as well as their husbandry demands, was an uncommon practice in Egypt. In 2018, the Egyptian Association for Animal Research Advancement (EAARA) organised the first international course in laboratory animal science (LAS), in collaboration with Utrecht University (The Netherlands) and the Faculty of Science, Cairo University, to raise researchers' awareness and increase their knowledge of the principles that govern the humane use and care of laboratory animals. A total of 26 researchers from a number of fields (veterinary medicine, dentistry, science, medicine, pharmacy and agriculture) enrolled in the course. In the responses to the post-course questionnaire, 24 (92.3%) participants stated that the principles of animal welfare (Three Rs) were well explained. In addition, 18 (69%) participants found that the course improved their skills in animal sampling and handling. Of the 26 participants, 22 (84.6%) became aware of their responsibility towards their experimental animals and agreed that the different methods of euthanasia were well explained. In conclusion, the general assessment of the course revealed a positive outcome regarding the culture of animal care; the course was repeated a year later, and several participants were enlisted as trainers in this second course.

β2 and α2 adrenergic receptors mediate the proneurogenic in vitro effects of norquetiapine.

Positive modulation of adult hippocampal neurogenesis may contribute to the therapeutic effects of clinically relevant antidepressant drugs, including atypical antipsychotics. Quetiapine, an antipsychotic which represents a therapeutic option in patients who are resistant to classical antidepressants, promotes adult hippocampal neurogenesis in preclinical studies. Norquetiapine, the key active metabolite of quetiapine in humans, has a distinctive receptor profile than the parent compound. The drug is indeed a high affinity norepinephrine transporter inhibitor and such activity has been proposed to contribute to its antidepressant effect. At present, no information is available on the effects of norquetiapine on adult neurogenesis. We extensively investigated the activity of quetiapine and norquetiapine on adult murine neural stem/progenitor cells and their progeny. Additionally, selective antagonists for β2/α2 adrenergic receptors allowed us to evaluate if these receptors could mediate quetiapine and norquetiapine effects. We demonstrated that both drugs elicit in vitro proneurogenic effects but also that norquetiapine had distinctive properties which may depend on its ability to inhibit norepinephrine transporter and involve β2/α2 adrenergic receptors. Animal care and experimental procedures were approved by the Institutional Animal Care and Use Committees (IACUC) at University of Piemonte Orientale, Italy (approval No. 1033/2015PR) on September 29, 2015.

Retracted article: Tranilast attenuates neuropathic pain during type-2 diabetes by inhibiting hypoxia-induced pro-inflammatory cytokines in Zucker diabetic fatty rat model

We, the Editors and Publisher of the journalArchives of Physiology and Biochemistry, have retracted the following article: Wei Zhang, Jun Ma, Shan Wang, Tao Huang & Min Xia (2020) Tranilast attenuates neuropathic pain during type-2 diabetes by inhibiting hypoxia-induced pro-inflammatory cytokines in Zucker diabetic fatty rat model, Archives of Physiology and Biochemistry, DOI: 10.1080/13813455.2020.1854309 Since publication, the authors informed the Publisher and Editor that the authors had not received ethics approval from the Institutional Animal Care and Use Committee (IACUC) at the time that their research was conducted. As the Editor and Publisher also have concerns about the integrity of the reported results which the authors have been unable to address, all parties have agreed to retract the article to ensure correction of the scholarly record. We have been informed in our decision-making by our policy on publishing ethics and integrity and the COPE guidelines on retractions. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.

Retraction: Yu, X., Hu, Y., Freire, M., Yu, P., Kawai, T., Han X. (2017) Role of toll-like receptor 2 in inflammation and alveolar bone loss in experimental peri-implantitis versus periodontitis. Journal of Periodontal Research 53(1), 98-106. https://doi.org/10.1111/jre.12492.

The above article, published online on 05 September 2017 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the authors, the journal's Editor-in-Chief, Dr. Shinya Murakami, and John Wiley & Sons A/S. The retraction has been agreed as the Institutional Animal Care and Use Committee (IACUC) approval for the experimental peri-implantitis procedure used in this article was still pending at the time that the procedure was performed. The IACUC approval for the peri-implantitis procedure was received in 2017 after the article had been submitted. Although all the research design, methods, and results are valid, IACUC approval was not received at the time the research was performed, which is a requirement for all research submitted to the journal. The authors have agreed to retract the article.

341 How the IACUC uses the Ag Guide to ensure the best care

Abstract All Institutional Animal Care and Use Committees (IACUC) are all subject to the same regulatory requirements. Membership requirements (Scientist, Nonscientist, Attending Veterinarian, Public Members, and Chairperson) are established by the regulatory agencies and appointed by the Institutional Official (IO). Today we will review the basic role of the IACUC: reviewing animal use protocols, inspecting animal facilities and procedure areas, evaluating the program of animal care, investigating allegations of non-compliance, making recommendations to the institutional official about needed changes, and utilizing the Ag Guide as a guidance document. The role of the IACUC at Land Grant Institutions is confounded often by the variety of research that is conducted. At the University of Nebraska, we have traditional food and fiber, biomedical and wildlife research, which requires a diverse set of committee members to adequately review the animal care and use. All IACUC protocols, regardless of the species or funding mechanism, are required to address several common questions. As a principal investigator, you can help yourself tremendously by providing all the required information in a transparent, logical and organized manner. If you can anticipate your audience and provide key information in multiple formats, it will help streamline the review process. Based on membership requirements, most committee members will not come from agriculture backgrounds, so it is imperative you help the committee to understand standard agriculture practices. You can best accomplish this with standard operating procedures, herd management protocols, disaster plans, and veterinary care processes and provide photographs or short video clips of your proposed activities. Additionally, if you are asked to serve on this committee, be willing to proactively educate your peers on what is considered appropriate management, housing, experimental and humane endpoints. By acting as your own advocate, you can ensure good animal care as well as achieve timely approvals.

342 Progress Report - AgGuide 2020

Abstract The Guide for the Care and Use of Agricultural Animals in Research and Teaching, commonly known as the Ag Guide, is published jointly by the American Dairy Science Association®, the American Society of Animal Science, and the Poultry Science Association under an open access creative commons license. At present, its 4th edition is under development and out for public comment. When finalized, this edition is intended to replace previous editions from 1988, 1999, and 2010. The first five chapters of the Ag Guide cover subjects that are relevant across species. These are followed by species-specific chapters, which are under the purview of the respective associations dedicated to the species. Authors of the individual chapters, recruited by the associations, were scientists, veterinarians, or engineers with expertise in the species or subjects covered in the respective chapters. All chapters are grounded in the scientific literature. The board of directors of each association approved final drafts of the general chapters and their respective species chapters. The content is intentionally broad so as to cover the diverse research and teaching institutions that use agricultural animals, yet be clear enough to give unambiguous guidance to institutional animal care and use committees (IACUCs) responsible for the care and use of animals in their respective institutions. It is viewed as essential that IACUCs and others who oversee animal care practices use sound professional judgment to achieve appropriate animal welfare outcomes, recognizing that too prescriptive an approach to animal care and use can stifle research and teaching opportunities. The 4th edition of the Ag Guide constitutes a timely and robust updating of guidance pertinent to animal care and use that will be released in 2020.

Newcastle disease virus (NDV) expressing the spike protein of SARS-CoV-2 as a live virus vaccine candidate.

BackgroundDue to the lack of protective immunity of humans towards the newly emerged SARS-CoV-2, this virus has caused a massive pandemic across the world resulting in hundreds of thousands of deaths. Thus, a vaccine is urgently needed to contain the spread of the virus.MethodsHere, we describe Newcastle disease virus (NDV) vector vaccines expressing the spike protein of SARS-CoV-2 in its wild type format or a membrane-anchored format lacking the polybasic cleavage site. All described NDV vector vaccines grow to high titers in embryonated chicken eggs. In a proof of principle mouse study, the immunogenicity and protective efficacy of these NDV-based vaccines were investigated.FindingsWe report that the NDV vector vaccines elicit high levels of antibodies that are neutralizing when the vaccine is given intramuscularly in mice. Importantly, these COVID-19 vaccine candidates protect mice from a mouse-adapted SARS-CoV-2 challenge with no detectable viral titer and viral antigen in the lungs.InterpretationThe results suggested that the NDV vector expressing either the wild type S or membrane-anchored S without the polybasic cleavage site could be used as live vector vaccine against SARS-CoV-2.FundingThis work is supported by an NIAID funded Center of Excellence for Influenza Research and Surveillance (CEIRS) contract, the Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract, philanthropic donations and NIH grants.

Molecular Modeling of the FOXO4-TP53 Interaction to Design Senolytic Peptides for the Elimination of Senescent Cancer Cells

Background: Senescent cells accumulate in tissues over time as part of the natural aging process and the removal of senescent cells has shown promise for alleviating many different age-related diseases in mice. Cancer is an age-associated disease and there are numerous mechanisms driving cellular senescence in cancer that can be detrimental to recovery. Thus, it would be beneficial to eliminate senescent cancer cells to prevent cancer recurrence or progression. Methods: We used molecular modeling to develop a series of rationally designed peptides to mimic and target FOXO4 disrupting the FOXO4-TP53 interaction and releasing TP53 to induce apoptosis. We then tested these peptides as senolytic agents for the elimination of senescent cancer cells both in cell culture and in vivo. Findings: Here we show that these peptides can act as senolytics for eliminating senescent human cancer cells both in cell culture and in orthotopic mouse models. We then further characterized one peptide, ES2, showing that it co-localizes with FOXO4 foci and binds FOXO4 to a greater extent compared to TP53. Finally, we show that concomitant intra-tumoral injection of ES2 plus a BRAF inhibitor results in a significant increase in apoptosis in a transgenic mouse model of melanoma. Interpretation: Taken together, our results reveal that peptides can be generated to specifically eliminate FOXO4+ senescent cancer cells, which has implications for eradicating residual disease and as a combination therapy for frontline treatment of cancer. Funding Statement: This work was supported by the Cancer Early Detection Advanced Research Center at Oregon Health & Science University. Declaration of Interests: S.S. Cinaroglu, Y. Ahiska, U. Sezerman, G.B. Akcapinar, E. Timucin have a patent for the ES2 structure and are members of a company, Eternans Ltd. that is working on ES2 for therapeutic use. No potential conflicts of interest were disclosed by the other authors. Ethics Approval Statement: All experiments were approved by Oregon Health and Science University (OHSU) Institutional Animal Care and Use Committee (IACUC) (TR01_I0674) and conformed to the guidelines set by United States Animal Welfare Act and the National Institutes of Health.

The Rigor and Transparency Index Quality Metric for Assessing Biological and Medical Science Methods.

SummaryThe reproducibility crisis is a multifaceted problem involving ingrained practices within the scientific community. Fortunately, some causes are addressed by the author's adherence to rigor and reproducibility criteria, implemented via checklists at various journals. We developed an automated tool (SciScore) that evaluates research articles based on their adherence to key rigor criteria, including NIH criteria and RRIDs, at an unprecedented scale. We show that despite steady improvements, less than half of the scoring criteria, such as blinding or power analysis, are routinely addressed by authors; digging deeper, we examined the influence of specific checklists on average scores. The average score for a journal in a given year was named the Rigor and Transparency Index (RTI), a new journal quality metric. We compared the RTI with the Journal Impact Factor and found there was no correlation. The RTI can potentially serve as a proxy for methodological quality.

A Circular Network of Coregulated Sphingolipids Dictates Lung Cancer Growth and Progression

Sphingolipid metabolism is one of the top dysregulated pathways in NSCLC. However, the molecular control of sphingolipid metabolic reprogramming in cancer progression remains unknown. Here, we first provide clinical evidence that expression of sphingolipid metabolic enzymes, B3GNT5 and GAL3ST1, but not the others, is most significantly associated with patient prognosis, whilst sphingolipidomics analysis of NSCLC patient sera identifies their metabolites, neolacto-series glycosphingolipid and sulfatide, as biomarkers that outperformed current clinical biomarkers for staging patients. Further network analysis of the sphingolipidomes reveals a circular network of coregulated sphingolipids, indicating that the neolacto-series glycosphingolipid/sulfatide balance functions as a checkpoint to determine sphingolipid metabolic reprograming during patient progression. Importantly, sphingolipidomics analysis of B3GNT5/GAL3ST1 genetically perturbed NSCLC cell lines confirms their key regulatory role in sphingolipid metabolism, while B3GNT5 and GAL3ST1 expression has an opposite role on tumorigenesis in vitro and in vivo. Overall, our results provide new insights whereby B3GNT5 and GAL3ST1 differentially regulate sphingolipid metabolism in lung cancer growth and progression. Funding Statement: This work was supported by the Natural Science Foundation of China (81920108028, 81872142); Guangzhou Science and Technology Program (201904020008); Young Teacher Foundation of Sun Yat-sen University (18ykzd07); Guangdong Science and Technology Department (2020B1212060018, 2019A1515011802, 2020A1515011280, 2020A0505100029); China Postdoctoral Science Foundation (2019M650226, 2019M650227). Declaration of Interests: No potential conflicts of interest were disclosed. Ethics Approval Statement: Serum samples were collected from healthy individuals and NSCLC patients (age-matched and body-mass index –matched) with informed consent, and the research program and all the related procedures were approved by the Ethics Committee of Sun Yat-sen Memorial Hospital (Guangzhou, China) and stored according to standard operating procedures for Good Clinical Laboratory Practice standards. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Sun Yat-Sen University.

Timing of Proteasome Inhibition for Prevention of Muscle Contractures in Neonatal Brachial Plexus Injury

Background: Neonatal brachial plexus injury (NBPI) causes disabling and incurable contractures, or limb stiffness, which result from proteasome-mediated protein degradation impairing the longitudinal growth of neonatally denervated muscles. We recently showed in a mouse model that the 20S proteasome inhibitor, bortezomib, prevents contractures after NBPI. Given that contractures uniquely follow neonatal denervation, the current study tests the hypothesis that proteasome inhibition during a finite window of neonatal development can prevent long-term contracture development. Methods: We used a mouse model of NBPI in which neonatal forelimb denervation at P5 causes shoulder and elbow contractures 4 weeks post-denervation. We first outlined the minimum period for proteasome inhibition to prevent contractures 4 weeks post-NBPI by treating mice with saline or bortezomib for varying durations between P8 and P32. We then compared the ability of varying durations of longer-term proteasome inhibition to prevent contractures at 8 and 12 weeks post-NBPI. Elbow and shoulder contractures, elbow flexor muscle length, and proteasome catalytic subunit activity were assessed. Findings: Proteasome inhibition can be delayed 3-4 days after denervation but is required throughout skeletal growth to prevent contractures long-term. Proteasome inhibition becomes less effective in preventing contractures beyond the neonatal period. The therapeutic effect of bortezomib is associated with attenuation of 20S proteasome β1 subunit activity. Interpretation: Temporary neonatal proteasome inhibition does not prevent contractures long-term. Instead, neonatal denervation causes a permanent longitudinal growth deficiency that must be continuously ameliorated during skeletal growth. Additional mechanisms must be explored to reduce the risk of toxicity from long-term proteasome inhibition. Funding Statement: This work was supported by grants to RC from the National Institutes of Health (NIH) (R01HD098280-01) and Pediatric Orthopaedic Society of North America (POSNA), as well as funding from the Cincinnati Children’s Hospital Division of Orthopaedic Surgery and Junior Cooperative Society. Declaration of Interests: The authors have declared that no conflict of interest exists. Ethics Approval Statement: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All mice were handled according to approved institutional animal care and use committee (IACUC) protocols (#2017-0084) of the Cincinnati Children’s Hospital Medical Center. All surgeries were performed under isoflurane anesthesia, and every effort was made to minimize suffering.

LACK OF HUMAN THROMBOMODULIN GENE EXPRESSION IN LIFE SUPPORTING PIG-TO-BABOON CARDIAC XENOTRANSPLANTATION MODELS ACCELERATE ANTIBODY MEDIATED REJECTION

Introduction: Transplantation of organs from animals to humans (xenotransplantation) has been proposed to address the critical shortage of organs for transplantation. The use of genetically engineered organ xenografts, including knock-out (KO) of alpha 1-3 galactosyltransferase (GTKO) along with insertion of a human complement regulator genes (hCD46-human CD46, hDAF-human decay accelerating factor) and other cell surface carbohydrate KOs, has been helpful in circumventing acute and subacute rejection. However, thrombotic complications still remain. Xenografts with additional human transgenes, including human thrombomodulin (hTBM), have been generated to address this barrier. Here, we studied the importance of hTBM in preventing microvascular and intramural thrombosis and rejection by comparing two transplanted groups utilizing GE donors with or without hTBM. Methods: Specific pathogen-free baboons of either sex weighing 15-30 kg (2-3 years of age) were used as recipients. 6 to 8-week-old genetically modified pigs with hTBM (GTKO.hCD46.hTBM) or without hTBM (triple knock out-GTKO.B4KO.CMAHKO, with or without hCD46 and hDAF) were used as donors. Expression of transgenes were consistent and high level across all pigs. All animals were used in compliance with guidelines provided by the Institutional Animal Care and Use Committee (IACUC). Results: GTKO.hCD46.hTBM donors (n=4) were found to have no intracardiac thrombus with survival up to 30 days post-transplant, but one with ongoing survival at the time of this abstract submission. However, non-hTBM donors (n=2) had an average survival of 7 days and were found to have a large intracardiac thrombus burden with propagation into major vascular structures such as the aorta, pulmonary artery and coronary sinus. Gross examination of thrombi indicated acute and subacute components, suggesting early formation of the thrombus postoperatively. Additionally, early clinical signs of thrombus formation included profound hypocalcemia requiring continuous calcium infusions in the first 48-72 hours postoperatively. These phenomena were not prevented by continuous heparin infusion. Histologic myocardial examination revealed microscopic and macroscopic thrombotic phenomena, likely related to accelerated antibody mediated rejection in non-hTBM containing grafts. Conclusion: Non-hTBM containing xenografts exhibited accelerated antibody mediated rejection. While immunologic rejection has been circumvented with novel immunosuppression regimens and GTKO, it is likely that hTBM or other transgenes involved in regulation of coagulation, may be needed for prevention of thrombotic complications and accelerated antibody mediated rejection.

The astute resident and the IACUC.

The astute resident and the IACUC.