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Introduction Traumatic brain injury is a major public health concern. Among patients with severe traumatic brain injury, epidural hemorrhage is known to swiftly lead to brain herniation and death unless there is emergent neurosurgical intervention. However, immediate neurosurgeon availability is frequently a problem outside of level I trauma centers. In this context, the authors desired to test a novel device for the emergent management of life-threatening epidural hemorrhage. A review of existing animal models determined that all were inadequate for this purpose, as they were found to be either inappropriate or obsolete. Here, we describe the development of a new epidural hemorrhage model in swine (Sus scrofa, 18-26 kg) ideal for translational device testing. Materials and Methods Vascular access was achieved using an ultrasound-guided percutaneous Seldinger catheter-over-wire technique with 5 Fr catheters placed in the bilateral carotid arteries, for continuous blood pressure and to allow for withdrawal of blood for creation of epidural hemorrhage. To simulate an actively bleeding and life-threatening epidural hemorrhage, unadulterated autologous blood was infused from the vascular access point into the epidural space. To be useful for this application and clinical scenario, brain death needed to occur after the planned intervention time but before the end of the protocol period (if no intervention took place). An iterative approach to model development determined that this could be achieved with an initial infusion rate of 1.0 mL/min, slowed to 0.5 mL/min after the first 10 minutes, paired with an intervention time at 15 minutes. All experiments were performed at Oregon Health & Science University, an Association for Assessment and Accreditation of Laboratory Animal Care accredited facility. Oregon Health & Science University’s Institutional Animal Care and Use Committee, as well as the United States Army Animal Care and Use Review Office, reviewed and approved this protocol before the initiation of experiments (respectively, protocol numbers IP00002901 and 18116010.e001). Results The final developed model allows for the infusion of a known volume of autologous, unadulterated blood directly into the epidural space, without the use of a balloon or other restricting membranes, and is rapidly fatal in the absence of intervention. Conclusions This animal model is the first to mirror the expected clinical course of epidural hemorrhage in a physiologically relevant manner, while allowing translational testing of emergency devices. This model successfully allowed the initial testing of a novel interventional device for the emergent management of epidural hemorrhage that was designed for use in the absence of traditional neurosurgical capabilities.

Magnetic resonance imaging (MRI) is a clinically relevant, real-time imaging modality that is frequently utilized to assess stroke type and severity. However, specific MRI biomarkers that can be used to predict long-term functional recovery are still a critical need. Consequently, the present study sought to examine the prognostic value of commonly utilized MRI parameters to predict functional outcomes in a porcine model of ischemic stroke. Stroke was induced via permanent middle cerebral artery occlusion. At 24 hours post-stroke, MRI analysis revealed focal ischemic lesions, decreased diffusivity, hemispheric swelling, and white matter degradation. Functional deficits including behavioral abnormalities in open field and novel object exploration as well as spatiotemporal gait impairments were observed at 4 weeks post-stroke. Gaussian graphical models identified specific MRI outputs and functional recovery variables, including white matter integrity and gait performance, that exhibited strong conditional dependencies. Canonical correlation analysis revealed a prognostic relationship between lesion volume and white matter integrity and novel object exploration and gait performance. Consequently, these analyses may also have the potential of predicting patient recovery at chronic time points as pigs and humans share many anatomical similarities (e.g., white matter composition) that have proven to be critical in ischemic stroke pathophysiology. The study was approved by the University of Georgia (UGA) Institutional Animal Care and Use Committee (IACUC; Protocol Number: A2014-07-021-Y3-A11 and 2018-01-029-Y1-A5) on November 22, 2017.

Background: Hypothalamic astrocytes are the important contributors that activate gonadotropin-releasing hormone (GnRH) neurons and promote GnRH/LH (luteinizing hormone) surge. However, the underlying roles and mechanisms of astrocytes during the early age reproductive decline remain unclear. Methods: Immunofluorescence and immunohistochemical staining were applied to analyze the expression of p16, γ-H2AX, SA-β-Gal and peroxidase. Animal ovariectomized models and cell models receiving repeated estradiol intervention were adopted to investigate estradiol-induced astrocyte senescence. High-performance liquid chromatography (HPLC) was employed to detect the estradiol metabolites and progesterone. qPCR and western blot were performed to analyze the PKA-CYPs pathway, growth factors and proinflammatory cytokines. Findings: Intact middle-aged female mice, astrocytes within the hypothalamic RP3V accumulate senescence-related markers with increasing age. Molecular analysis revealed that ovarian estradiol activated PKA and up-regulated CYPs (cytochrome p450) expression, metabolizing estradiol into 2-OHE2 and 4-OHE2 and mediating the senescence of astrocytes in RP3V. Of note, the progesterone synthesis and the ability to promote GnRH release were significantly reduced in middle-aged mice. Besides, the expression of growth factors decreased and the mRNA levels of proinflammatory cytokines significantly increased in the aging astrocytes. Interpretation: The findings confirm that ovarian estradiol induces the senescence of hypothalamic astrocytes. The senescent astrocytes compromises the function for regulating progesterone synthesis and GnRH secretion, may be contributes to aging-related declines in female reproductive function. Funding Statement: The study was supported by National Natural Science Foundation of China to Jing Zhang (No.81671400) and Fujian Province Health and Family Planning Youth Research Project to Xiaoman Dai(No.2018-1-45). Declaration of Interests: All authors have no potential conflicts of interests to declare, financial or otherwise Ethics Approval Statement: All protocols and procedures used in these studies involving animals were approved by the Institutional Animal Care and Use Committee of Fujian Medical University and in compliance with NIH’s Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 85-23 Rev. 1985). This article does not contain any studies with human participants performed by any of the authors.

Background: Hyperbilirubinemia may increase the risk of Alzheimer's disease (AD) but its mechanistic role in AD pathogenesis remains obscure. Methods: Three animal models and primary hippocampal cells were used to explore in this study: (1) acute bilirubin treatment; (2) lateral ventricle injection; (3) long term observation. Results: Three days exposure to bilirubin in newborn rats could induce AD-like pathological changes in late life, including tau protein hyperphosphorylation at multiple sites, increased Aβ production in brain tissues, and spatial learning and memory injury. Bilirubin activated the activities of several protein kinases (GSK-3β, CDK5 and JNK), which were positively correlated with hyperphosphorylated tau; simultaneously increased the expression of APP γ-secretase PS2 and decreased the expression of α-secretase ADAM17, which were positively correlated with Aβ production. The above results were well replicated in primary hippocampal cells. Conclusions: Bilirubin may be an upstream activator to induce AD, and bilirubin encephalopathy is an AD-like disease. Funding Statement: The study was supported in part by the National High Technology Research and Development Program of China (2006AA02Z4B5); the National Natural Science Foundation of China(81170608, 81570278, 81602427, 81600089, 81670156); the Science and Technology Program of Guangzhou (201707010046, 201604020001); The National Funds for Developing Local Colleges and Universities (B16056001); Natural Science Foundation research team of Guangdong Province (2018B030312001); Guangzhou Education System (1201610014, 1201620494); Natural Science Foundation of Guangdong Province (2017A030313662). Declaration of Interests: All authors declare that there are no conflicts of interest. Ethics Approval Statement: The protocol for the care and use of all animals in this study was in accordance with the Guangdong Animal Center for the ethical treatment of animals and approved by the Institutional Animal Care and Use Committee of Guangzhou Medical University (SYXK2016-0168, Guangzhou, China).

Background: Nephrotic syndrome (NS) is associated with kidney podocyte injury and may occur as part of thyroid autoimmunity such as Graves’ disease. Therefore, the present study was designed to ascertain if and how podocytes respond to and regulate the input of biologically active thyroid hormone (TH), 3,5,3'-triiodothyronine (T3); and also to decipher the pathophysiological role of type 3 deiodinase (D3), a membrane-bound selenoenzyme that inactivates TH, in kidney disease. Methods: To study D3 function in healthy and injured (PAN, puromycin aminonucleoside and LPS, Lipopolysaccharide-mediated) podocytes, immunofluorescence, qPCR and podocyte-specific D3 knockout mouse were used. Surface plasmon resonance (SPR), co-immunoprecipitation and Proximity Ligation Assay (PLA) were used for the interaction studies. Findings: Healthy podocytes expressed D3 as the predominant deiodinase isoform. Upon podocyte injury, levels of DIO3 transcript and D3 protein were dramatically reduced both in vitro and in the LPS mouse model of podocyte damage. D3 was no longer directed to the cell membrane, it accumulated in the Golgi and nucleus instead. Further, depleting D3 from the mouse podocytes resulted in foot process effacement and proteinuria. Treatment of mouse podocytes with T3 phenocopied the absence of D3 and elicited activation of αvβ3 integrin signaling, which led to podocyte injury. We also confirmed presence of an active thyroid stimulating hormone receptor (TSH-R) on mouse podocytes, engagement and activation of which resulted in podocyte injury. Interpretation: The study provided a mechanistic insight into how D3-αvβ3 integrin interaction can minimize T3-dependent integrin activation, illustrating how D3 could act as a renoprotective thyrostat in podocytes. Further, injury caused by binding of TSH-R with TSH-R antibody, as found in patients with Graves’ disease, explained a plausible link between thyroid disorder and NS. Funding: Funding support from American Thyroid Association (ATA) to NJT (ATA-2018-050.R1). Declaration of Interest: Jochen Reiser has patents on novel strategies for kidney therapeutics and stands to gain royalties from their commercialization. He is the co-founder of Walden Biosciences (Cambridge, MA, USA), a biotechnology company in which he has financial interest, including stock. Other authors have nothing to disclose and there are no competing or conflicting interests. Ethical Approval: All animal experiments were carried out according to the NIH’s Guide for the Care and Use of Experimental Animals (National Academies Press, 2011), and approved by the Institutional Animal Care and Use Committee (IACUC) at Rush University (Chicago, Illinois, USA). Human biopsy kidney sections from healthy donors and patients with FSGS, DN and MCD were procured in accordance with guidelines on human research and with approval of the Institutional Review Board of Rush University Medical Center (Chicago, Illinois, USA).

Aim: Hyperleptinemia potentiates the effects of many atherogenic factors, such as inflammation, platelet aggregation, migration, hypertrophy, proliferation of vascular smooth muscle cells, and endothelial cell dysfunction. The present study analysed the effects of long-term hyperleptinemia in an in vivo myocardial ischemia-reperfusion model to demonstrate whether the in vivo deleterious effect also affects cardiac structure and function. Main methods: Rats by were subcutaneously administered leptin for 8 days to estimate the involvement of the JAK/STAT pathway. Data from 58 male Wistar rats were included in the final analysis. Myocardial infarction (MI) was modelled by the 30-minute ligation of the main left coronary artery followed by 120-minute reperfusion. Hemodynamic measurements, electrocardiography monitoring, echocardiography, myocardial infarct size and area at risk, blood biochemical parameters, leptin, IL-6, TNF-alpha, FGF-21, and cardiomyocyte morphology were measured. Statistical analyses were performed using IBM SPSS Statistics v.26. Key findings: Eight-day hyperleptinemia in rats led to increased an blood pressure and heart rate, myocardial hypertrophy, impaired LV function, an increased frequency of ischemic arrhythmias, dyslipidaemia, systemic inflammation, and an increased size of induced myocardial infarction. Significance: The blockade of the JAK/STAT signalling pathway effectively reversed the negative effects of leptin, including increased blood pressure and total cholesterol. Funding Information: The study has been supported by the grant from the Ministry of Science and Higher Education of the Russian Federation (agreement 075-15-2020-800). Declaration of Interests: None to declare. Ethics Approval Statement: This study was conducted in conformance with the policies and procedures detailed by the “Institutional Animal Care and Use Committee” (IACUC). All animal experimental protocols and procedures were reviewed and approved by the Committee for the Control of the Maintenance and Use of Laboratory Animals of the V. A. Almazov National Medical Research Center (date: 06-FEB-2020, application number: 20-01).

Melanoma is known to be a heterogeneous tumor, but the impact of this heterogeneity upon therapeutic response is not well understood. In the current study, we used single cell mRNA analysis and our Single Cell HETerogeneity (SinCHET) platform to define the transcriptional diversity of melanoma and its dynamic response to both BRAF inhibitor therapy and treatment holidays. Our analyses showed melanoma cell lines and patient specimens to be composed of >3 co-existent, transcriptionally distinct states. Among these, State #1 had increased expression of cyclin D1, ERBB3, STAT3/5, MITF and I²-catenin and lower expression of c-JUN and RTKs such as Axl and EGFR. State #2 was characterized by higher expression of ERBB3, Axl and the transcription factor c-JUN. State #3 was characterized by high Axl, c-JUN, E2F1, WEE1, c-MET and EGFR expression and lower expression of MITF, ERBB3 and SMAD9. The cell state composition was dynamically regulated in response to BRAF inhibitor therapy, with State #1 declining and States #2 and #3 increasing in response to drug. The percentage of cells in State #1 recovered following a drug holiday, allowing for a successful therapy rechallenge. Other melanomas that lacked State #1 had varying degrees of intrinsic drug resistance and were not amenable to rechallenge. We next leveraged the differences in fitness between the different transcriptional states in the absence and presence of drug to develop a mathematical model that optimized therapy schedules to retain the drug sensitive population. In vivo validation of the mathematical model demonstrated that the personalized adaptive dosing schedules were better at suppressing tumor growth than either continuous or fixed intermittent BRAF inhibitor schedules. Together our studies provide the first preclinical evidence that transcriptional heterogeneity at the single cell level predicts for the initial sensitivity to BRAF inhibitor therapy. We further demonstrate that manipulating transcriptional heterogeneity through personalized adaptive therapy schedules that can delay the time to resistance. Funding Statement: Supported by SPORE grant P50 CA168536, R21 CA198550, R21 CA216756 (to KSMS) K99 CA226679 (to IS), Moffitt Cancer Center PSOC, U54 CA193489 (EK & AA) and the Cancer Center Support Grant P30 CA076292 from the NIH (NCI/NIH). We also acknowledge the support Miami Center for AIDS Research (CFAR) at the University of Miami Miller School of Medicine funded by P30AI073961. Declaration of Interests: We have no competing interest to declare. Ethics Approval Statement: All animal experiments were carried out in compliance with ethical regulations and protocols approved by the University of South Florida Institutional Animal Care and Use Committee.

Calorie restriction (CR) has been well proved to be a powerful tool to improve metabolic health associated with aging; and many types of CR have been proposed. Intermittent CR has become a trend in recent years due to its better compliance than continuous CR every day. However, there are few studies that directly compare the interventional efficacy of intermittent CR to continuous CR in metabolic disorders such as diabetes. In this study, we analyzed two regimes of intermittent CR with the calorie-matched continuous CR in two diabetic mouse models including db/db and streptozotocin-treated mice. Intermittent CR was carried out by a fasting-mimicking diet (FMD, with 30% calorie intake of the control per day) for 2 days or 5 days (i.e., 2-5 or 5-9 regimes followed by free eating for 5 or 9 days respectively). In the two diabetic mouse models, both intermittent CR and continuous CR significantly reduced fasting blood glucose level and improved insulin sensitivity. However, intermittent CR performed significantly better than continuous CR in improving glycemic control and insulin sensitivity in db/db mice. In addition, intermittent CR improved the glucose homeostasis of the db/db mice without causing loss of body weight. Analyses with the pancreatic islets reveal that intermittent CR profoundly elevated the number of insulin-positive cells in both diabetic mouse models. In conclusion, our study indicated that both intermittent CR and continuous CR can lower fasting blood glucose level in the diabetic mice, while intermittent CR is better than the latter in improving insulin sensitivity. Funding Statement: This study was supported by research grants from the National Natural Science Foundation of China (31630036 to Y.C.), the Ministry of Science and Technology of China (2016YFA0500103 to Y.C.), and the Chinese Academy of Sciences (XDA12010102, QYZDJ-SSW-SMC008, ZDRW-ZS-2016-8, and Y817X11141 to Y.C Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS) with an approval number 2010-AN-8.

Although there are numerous Institutional Animal Care and Use Committee (IACUC) protocols aimed at standardizing the oral administration (gavage) of liquid chemicals to laboratory rodents, there is ample variability across protocols, and there are no protocols intended for the gavage of microbial communities to mice or rats. The objective of this study was to conduct a scoping review of publically available IACUC protocols from institutions in the United States, identify protocol criteria and deficiencies, and generate an ‘analytical consensus’ to unify such criteria into two revised protocols: one for chemicals, and another for microbes in mice. Eighteen (n = 18) written institutional protocols from prominent universities, and 26 demonstrations videos from various sources (accounting for >155,000 views) were identified in the World Wide Web. Although written protocols listed up to five criteria for consideration (dosing/volume, pregnancy, animal weight), collectively there was major variability and poor statistical agreement across methods (0% Kappa, p = 0.98). Because protocols also lacked details relevant for the bypassing and survival of live microorganisms in the gastric (antimicrobial) environment, we compiled two ‘analytical (Kappa-based) consensus’ protocols from available and new criteria, for the ‘gavage of chemicals’ and for the ‘gavage of delicate microscopic microbial communities’ to mice. A major difference lies in the volume of administration, (i.e., 20 mL/kg, without restrictions, after 4-6 h of fasting, for microbes), which was graphically illustrated simulating the predicted impact of administering large and small volumes on microbial distribution of simple microscopic communities. In conclusion, publicly available IACUC gavage protocols are highly variable, and do not reflect the need to adjust the dose volumes to ensure the rapid bypassing of the gastric environment which would impact the survival of microbes, especially if composing delicate microscopic communities. An ‘analytical consensus’ of IACUC-approved protocols is herein presented as a unifying baseline protocol for consideration. Impact statement Institutional protocols designed for the oral administration of live microbial communities, either complex or microscopic (microcosmic), to mice do not exist. However, this approach is increasingly employed by investigators focusing on the gut microbiome in experimental research. Herein, we propose two analytically Kappa-based consensus protocols to promote reproducibility and standardization in research practices and describe biologically relevant factors in achieving optimal microbial engraftment of communities in germ-free mice.

Anterior cruciate ligament (ACL) injuries are frequent, as >200,000 injuries occur in the United States alone each year. Owing to the risks for associated meniscus and cartilage damage, ACL injuries are a significant source of both orthopedic care and research. Given the extended recovery course after ACL injury, which often lasts 1-2 years, and is associated with limited participation in sports and activities of daily living for patients, there is a critical need for the evolution of new and improved methods for ACL repair. Subsequently, animal models of ACL reconstruction (ACLR) play a key role in the development and initial trialing of novel ACL interventions. This article provides a clear operative description and associated illustrations for a validated, institutional animal care and use committee, and veterinarian approved and facile model of ACLR to serve researchers investigating ACLR.

While animal models of controlled cortical impact often display short-term motor dysfunction after injury, histological examinations do not show severe cortical damage. Thus, this model requires further improvement. Mice were subjected to injury at three severities using a Pin-Point™-controlled cortical impact device to establish secondary brain injury mouse models. Twenty-four hours after injury, hematoxylin-eosin staining, Fluoro-Jade B histofluorescence, and immunohistochemistry were performed for brain slices. Compared to the uninjured side, we observed differences of histopathological findings, neuronal degeneration, and glial cell number in the CA2 and CA3 regions of the hippocampus on the injured side. The Morris water maze task and beam-walking test verified long-term (14–28 days) spatial learning/memory and motor balance. To conclude, the histopathological responses were positively correlated with the degree of damage, as were the long-term behavioral manifestations after controlled cortical impact. All animal procedures were approved by the Institutional Animal Care and Use Committee at Shanghai Jiao Tong University School of Medicine.

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).

COVID-19 has proven to be a metabolic disease resulting in adverse outcomes in individuals with diabetes or obesity. Patients infected with SARS-CoV-2 and hyperglycemia suffer from longer hospital stays, higher risk of developing acute respiratory distress syndrome (ARDS), and increased mortality compared to those who do not develop hyperglycemia. We find the prevalence of hyperglycemia in patients with COVID-19 ARDS to be equal to those with non-COVID-19 ARDS. Nevertheless, the pathophysiological mechanism(s) of hyperglycemia in COVID-19 remains poorly characterized. Here we show that insulin resistance rather than pancreatic beta cell failure is the prevalent cause of hyperglycemia in COVID-19 patients with ARDS, independent of glucocorticoid treatment. A screen of protein hormones that regulate glucose homeostasis reveals that the insulin sensitizing adipokine adiponectin is reduced in hyperglycemic COVID-19 patients. Hamsters infected with SARS-CoV-2 also have diminished expression of adiponectin. Together these data suggest that adipose tissue dysfunction may be a driver of insulin resistance and adverse outcomes in acute COVID-19. Funding Information: This work was supported by the following grants: NIH R01 DK121140 (J.C.L.) and R01 DK121844 (J.C.L.), NCI R01 CA234614 (R.E.S), NIAID 2R01 AI107301 (R.E.S), and NIDDK R01 DK121072 (R.E.S). R.E.S. and J.C.L are supported as Irma Hirschl Trust Research Award Scholars. In addition, this study received support from NewYork-Presbyterian Hospital (NYPH) and Weill Cornell Medical College (WCMC), including the Clinical and Translational Science Center (CTSC) (UL1 TR 0002384). Declaration of Interests: R.E.S. is on the scientific advisory board of Miromatrix Inc. R.E.S. is a speaker and consultant for Alnylam Inc. The other authors have no conflict of interest. Ethics Approval Statement: The registry was approved by the institutional review board of WCMC (1405015116, 20-05022072, 20-03021681). All animal experiment procedures, breeding, and ethical use were performed in accordance with the guidelines set by the Institutional Animal Care and Use Committee at Mount Sinai School of Medicine.

Background: Lumbar intervertebral disc (IVD) herniation causes severe low back pain (LBP), which results in substantial financial and emotional strains. Despite the effectiveness of discectomy, there is no existing treatment for post-operative LBP induced by progressive IVD degeneration. Two key factors of LBP are intradiscal inflammation, indicated by tumour necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), and sensory nerve ingrowth into the inner layer of the annulus fibrosus, triggered by nerve growth factor/high-affinity tyrosine kinase A (TrkA) signalling. In an animal models of discectomy, the bioresorbable ultra-purified alginate (UPAL) gel with an extremely low-toxicity has been effective in acellular tissue repair. Methods: We aimed to investigate whether UPAL gel can alleviate LBP using a rat nucleus pulposus (NP) punch model and a rabbit NP aspirate model. In both models, we assessed TNF-α and IL-6 production and TrkA expression within the IVD by immunohistochemistry. Further, histological analysis and behavioural nociception assay were conducted in the rat model. Findings: UPAL gel implantation suppressed TNF-α and IL-6 production, downregulated TrkA expression, inhibited IVD degeneration, and reduced nociceptive behaviour. Interpretation: Our results suggest the potential of UPAL gel implantation as an innovative treatment for IVD herniation by reducing LBP and preventing IVD degeneration after discectomy. Funding Statement: Ministry of Education, Culture, Sports, Science, and Technology of Japan, Akiyama Life Science Foundation, Mochida Pharmaceutical Co., Ltd., and Takeda Science Foundation. Declaration of Interests: The authors declare that no competing interests exist. Ethics Approval Statement: All animal procedures were performed in accordance with the ARRIVE guideline and National Institutes of Health guide for the care and use of Laboratory animals, and the study was approved by the Institutional Animal Care and Use Committee of Hokkaido University (17-0122).

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.

Background: The principal voltage-gated Nam+ channel, NaV1.5 governs heart excitability and conduction. NaV1.5 dysregulation is responsible for ventricular arrhythmias and subsequent sudden cardiac death in post-infarct hearts. The transcription factor Meis1 plays an important role in determining differentiation fate and regenerative capability of cardiomyocytes. However, the functions of Meis1 in ischemic arrhythmias following myocardial infarction (MI) are still largely undefined. Here we set out to study whether Meis1 could act as a key regulator to mediate cardiac Na+ channels and arrhythmogenesis and its underlying mechanisms. Methods: Heart-specific Meis1 overexpression was established by AAV9 virus injection in C57BL/6 mice. The QTc interval, the incidence of ventricular arrhythmias and cardiac conduction velocity were evaluated by ECG, programmed electrical stimulation and optical mapping techniques respectively. The conventional patch clamp technique was used to study the I Na characteristics of isolated mouse ventricular myocytes. Findings: We found that forced expression of Meis1 rescued the prolongation of heart rate corrected QT (QTc) interval, produced anti-arrhythmic activity and improved epicardial conduction velocity in infarcted mouse hearts. Mechanistically, Meis1 may ameliorate cardiac electrophysiological remodeling as characterized by the decrease of INa current density and NaV1.5 expression in marginal zone of infarcted mouse hearts. Furthermore, in vitro studies showed that E3 ubiquitin ligase CDC20 led to the ubiquitination and degradation of Meis1, which blocked the transcriptional regulation of SCN5A. Interpretation: This study demonstrates that Meis1 overexpression can effectively inhibit the ventricular arrhythmia by regulating the transcription of SCN5A and cardiac electrical conductance in mouse ischemic heart, which provides new therapeutic strategies for malignant arrhythmias and sudden cardiac death following MI. Funding: This work was supported by the National Nature Science Foundation of China [Grant No. 81573425 and 81773733 to NW]. Declaration of Interest: None to declare. Ethical Approval: All animal experiments were performed along with the NIH guidelines (Guide for the care and use of laboratory animals, NIH Publication No. 85-23, revised 1996) and were conducted in accordance with the protocols approved by the Institutional Animal Care and Use Committee of Harbin Medical University.

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).

Background Therapeutic options are limited for patients with liver metastases and hepatocellular carcinoma (HCC). Intratumoral and subcutaneous injections of CMP-001, a CpG-A TLR9 agonist packaged within a virus-like particle, have shown evidence of antitumor activity in patients with melanoma refractory to PD-1 blockade. In mice, CMP-001 intravenous distributes primarily to the liver, while CMP-001 subcutaneous is found mostly in local tissues and draining lymph nodes. The antitumor activity of CMP-001 intravenous and subcutaneous were compared with PD-1 blockade or sorafenib in two Hepa1-6 orthotopic mouse models of HCC. Methods Groups of 10–15 C57BL/6J mice were orthotopically implanted with syngeneic murine hepatoma cells using two different models. Model 1 used 1.5 x 106 Hepa1-6 cells injected into the spleen following a partial hepatectomy; Model 2 used 1 x 106 Hepa1-6-Luc cells injected into the upper left lobe of intact liver. Treatment was initiated 3–7 days later with either CMP-001 intravenous or subcutaneous Q4-5Dx3-4 doses, PD-1 blocking antibody intraperitoneal Q3-4Dx2 (Bio X Cell clone RPM1-14), or sorafenib QD oral. Antitumor activity was assessed by tumor imaging, liver weight, and/or survival. Results CMP-001 was compared with PD-1 blocking antibody therapy in Model 1, the more aggressive model. All animals were sacrificed at day 15 due to institutional welfare requirements. Tumor growth inhibition (TGI) was assessed by comparison of liver weight to body weight ratios, which relative to untreated control mice showed that CMP-001 intravenous achieved 85% mean TGI compared with 63% mean TGI for CMP-001 subcutaneous and 15% mean TGI for PD-1 blocking antibody intraperitoneal (table 1). CMP-001 intravenous was compared to sorafenib oral in Model 2, which utilized an engineered Hepa1-6 cell line that expresses luciferase to enable noninvasive monitoring of liver tumor growth. CMP-001 intravenous was active, with a 67% mean TGI, and survival that was comparable to sorafenib (table 2; figure 1). Conclusions In orthotopic mouse models of HCC, the antitumor activity of CMP-001 intravenous was greater than PD-1 blockade and comparable to sorafenib. CMP-001 intravenous was more active than CMP-001 subcutaneous in this model, which we hypothesize is due to increased liver exposure with intravenous infusion. Antitumor activity of CMP-001 monotherapy may be increased by combining it with standard of care or other therapies, as observed relative to historical benchmarks in ongoing CMP-001 clinical trials in patients with melanoma. CMP-001 intravenous may be a promising treatment option for patients with primary or metastatic liver cancers. Acknowledgements This work was supported by Checkmate Pharmaceuticals. Studies were performed at Oncodesign Biotechnology (Dijon, France) and Crown Bioscience UK Ltd (Osgathorpe, UK) and National Research Council Canada (Ottawa, Ontario, Canada) and funded by Checkmate Pharmaceuticals. Ethics Approval At Oncodesign Biotechnology, animal housing and experimental procedures were conducted according to French and European Regulations and the National Research Council Guide for the Care and Use of Laboratory Animals. The animal facility is authorized by the French authorities (Dijon: Agreement B21231011EA). The study and all animal procedures were approved by the Institutional Animal Care and Use Committee of Oncodesign (Oncomet) approved by French authorities (CNREEA agreement number 91). At Crown Bioscience, animal care and experimental procedures were compliant with the UK Animals Scientific Procedures Act 1986 (ASPA) in line with Directive 2010/63/EU of the European Parliament and the Council of 22 September 2010 on the protection of animals used for scientific purposes. At National Research Council Canada, animals were maintained in accordance with the guidelines of the Canadian Council on Animal Care, and all experimental procedures were performed in accordance with regulations and guidelines reviewed and approved by the NRC Human Health Therapeutics Ottawa Animal Care Committee.

Background: Treatment of acute pancreatitis (AP) and chronic pancreatitis (CP) remains problematic due to a lack of knowledge about disease-specific regulatory targets and mechanisms. The purpose of this study was to screen proteins related to endoplasmic reticulum (ER) stress and apoptosis pathways that may play a role in pancreatitis. Methods: Human pancreatic tissues including AP, CP and healthy volunteers were collected during surgery. Humanized PRSS1 transgenic mice were constructed and treated with caerulein to mimic the development of human AP and CP. Potential regulatory proteins in pancreatitis were identified by proteomic screen using pancreatic tissues of PRSS1 transgenic AP mice. Adenoviral shRNA-mediated knockdown of identified proteins, followed by functional assays was performed to validate their roles. Functional analyses included transmission electron microscopy for ultrastructural analysis; qPCR, western blotting, immunohistochemistry, immunofluorescence and immunoelectron microscopy for assessment of gene or protein expression, and TUNEL assays for assessment of acinar cell apoptosis. Findings: Humanized PRSS1 transgenic mice could mimic the development of human pancreatic inflammatory diseases. EMC6 and APAF1 were identified as potential regulatory molecules in AP and CP models by proteomic analysis. 1) increased expression of EMC6 and APAF1 was observed in patient and mouse AP/CP tissues; 2) inhibition of the two proteins resulted in attenuation of apoptosis and inflammation; 3) inhibition of EMC6 was associated with decreased expression of APAF1, suggesting a potential mechanistic connectivity. Interpretation: EMC6 and APAF1 are strongly associated with pancreatitis development, and may represent therapeutic targets for the treatment of pancreatic inflammatory diseases. Funding Statement: The article is supported by Guangdong Science and Technology Planning Project (2019A030317018) & Scientific Research Startup Program of Southern Medical University by High-level University Construction Funding of Guangdong Provincial Department of Education (CX2018N012) & Clinical Research Program of Nanfang Hospital, Southern Medical University (2018CR046) & College Students’ Innovative Entrepreneurial Training Plan Program (201812121128, 201812121263) & Clinical Research Startup Program of Southern Medical University by High-level University Construction Funding of Guangdong Provincial Department of Education (LC2016PY011) & National Natural Science Foundation of China (81801487). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: Written consent was obtained from each patient before the study, which were approved by the Ethics Committee of the Southern Medical University. The experimental operations were carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee of Southern Medical University.

Background: Sepsis-induced systemic inflammation response syndrome is the leading cause of morbidity and mortality among patients in intensive care units. While sepsis is associated with multiple organ damage, acute renal injury represents a hallmark of sepsis. Since systemic and renal inflammation is known to play a vital role in morbidity and mortality associated with sepsis, identifying a potent anti-inflammatory agent may help minimize morbidity and mortality associated with acute septic kidney injury. Since recent work have suggested that empagliflozin, a renal sodium-glucose cotransporter 2 (SGLT2) inhibitor, may assist in treatment of inflammatory diseases, our objective was to examine the effect of empagliflozin on acute sepsis-induced renal injury. Method: Mice were treated with empagliflozin or vehicle for 3 days prior to a single lipopolysaccharide (LPS) injection to induce sepsis. In another cohort, mice were injected with LPS for 3 hours to induce sepsis, then treated with empagliflozin. In both instances, mice were examined 24 hours after the injection of LPS. Findings: Our results show that empagliflozin improves survival in a mouse model of LPS-induced septic shock. We further demonstrate that the beneficial effects of empagliflozin are likely mediated via reducing LPS-induced acute renal injury. Moreover, our data indicate that empagliflozin significantly reduces systemic and renal inflammation to contribute to the improvements observed in our LPS-model. Interpretation: The findings of this study suggest that empagliflozin could be repurposed to reduce morbidity and mortality in patients with acute septic renal injury. Fund: This work was supported by a grant from CIHR to JRBD. Funding Statement: This work was supported by a grant from CIHR to JRBD. Declaration of Interests: The authors have declared that no conflict of interest exists. Ethics Approval Statement: All animal procedures were approved by the University of Alberta Institutional Animal Care and Use Committee, which conforms to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health and the principles for biomedical research involving animals developed by the Council for International Organizations of Medical Sciences.

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.

Background: Because the regenerative ability of intervertebral discs (IVDs) is restricted, defects caused by discectomy may induce insufficient tissue repair leading to further IVD degeneration. An acellular bioresorbable biomaterial based on ultra-purified alginate (UPAL) gel was developed to fill the IVD cavity and prevent IVD degeneration. However, an acellular matrix-based strategy may have limitations particularly in the elderly population, who exhibit low ability of self-repair. Therefore, further translational studies involving product combinations, such as UPAL gel plus bone marrow-derived mesenchymal stem cells (BMSCs), are required to evaluate the regenerative effects of BMSCs embedded in UPAL gel on degenerated IVDs. Methods: Rabbit BMSCs and nucleus pulposus cells (NPCs) were co-cultured in a three-dimensional (3D) system in UPAL gel. In addition, rabbit or human BMSCs combined with UPAL gel were implanted into IVDs following discectomy in rabbits with degenerated IVDs. Findings: Gene expression of NPC markers, growth factors, and extra cellular matrix was significantly increased in the NPC and BMSCs 3D co-culture compared to that in each 3D mono-culture. In vivo, whereas UPAL gel alone suppressed IVD degeneration as compared to discectomy, the combination of BMSCs and UPAL gel exerted a more potent effect to induce IVD regeneration. Similar IVD regeneration was observed using human BMSCs. Interpretation: These findings demonstrate the therapeutic potential of BMSCs combined with UPAL gel as a regenerative strategy following discectomy for degenerated IVDs. Funding Statement: This work was supported by a Grant-in-Aid for the Ministry of Education, Culture, Sports, Science, and Technology of Japan (16H03176), Japan, a “Project of Translational and Clinical Research Core Centers” from the Japan Agency for Medical Research and Development, AMED (18lm0203045h0001), Japan, and Mochida Pharmaceutical Co., Ltd Declaration of Interests: Patents pertaining to this work have been filed (inventors H. S., T. T., and N. I.). The other authors declare that they have no competing interests. Ethics Approval Statement: All animal procedures were approved by the Institutional Animal Care and Use Committee at Hokkaido University (approval number: 13-0051) and performed in accordance with the approved guidelines.

Background: The autoimmune mechanism in coronary heart disease (CHD) has been concerned recently. It is reported that the second extracellular loop of angiotensin II type 1 receptor (AT1R-ECII) autoantibody (AT1-AA) presents in the serum of CHD patients, but its clinical significance is unclear. The purpose of this study was to identify the diverse functional subclasses of AT1-AA in the patients of CHD. Methods: The AT1-AA levels in sera of 306 CHD patients were detected by ELISA. Then AT1-AA was purified from each patient’s serum (n=127) and their effects on intracellular calcium ([Ca2+]i) of mouse arterial smooth muscle cells (MASMC) was detected to classify the subclasses of AT1-AA from patients. Correlation analysis and logistic regression were used to analyze the clinical significances of different subclasses of AT1-AA in CHD. To further confirm AT1-AA classification, rats were subcutaneously injected with AT1R-ECII peptide. Findings: There were 4 [Ca2+]i change curves indicating different functional subclasses of AT1-AA (H1-, H2-, H3-, and H4-AT1-AA). Correlation analysis showed H1- and H2-AT1-AA levels were positively associated with endogenous and exogenous coagulation, respectively; H3-AT1-AA had no correlation; H4-AT1-AA was negatively correlated with leukocyte number and bile acid. Logistic regression showed H2-AT1-AA had predictive value for severe CHD. Moreover, H1- and H2-AT1-AA exerted cytotoxic effects in vitro, while H4-AT1-AA increased cell viability. 4 similar AT1-AA subclasses were found in AT1-AA-positive rats. Interpretation: This study demonstrates that there are 4 kinds of functional AT1-AA with different clinical significance in CHD patients, suggesting the importance of AT1-AA subclasses distinguishment in cardiovascular disease. Funding Statement: This work is supported by the Beijing Natural Science Foundation Program and Scientific Research Key Program of Beijing Municipal Commission of Education (No. KZ201810025039); the National Natural Science Foundation of China (No. 31771267); the Open Fund from Key Laboratory of Cellular Physiology (Shanxi Medical University), Ministry of Education, China (No. KLMEC/SXMU-201902). Declaration of Interests: The authors have declared that no conflict of interest exists. Ethics Approval Statement: All human studies had been conducted according to Declaration of Helsinki principles and approved by the local Research Ethics Committee (Beijing Anzhen Hospital, Capital Medical University, Beijing, China) (Ethics No.: 2019023X). All patients were identified by number and signed informed consent. All the animal experiments were approved by the Institutional Animal Care and Use Committee and Ethics Committee of Capital Medical University.

Currently, there has been growing attention to animal welfare in animal-assisted interventions (AAI). However, there is a lack of scrutiny regarding what steps to take to ensure animal wellbeing during AAI research. The Institutional Animal Care and Use Committee (IACUC) reviews, monitors, and approves all research involving animal subjects in order to ensure an animal's ethical and humane treatment. However, AAI publications rarely report IACUC approval. The purpose of this critical review was to determine the frequency of IACUC approval and to report the descriptions of animal use in AAI research publications. In the 139 articles reviewed, 14 (10%) studies reported attaining IACUC approval; 4/139 (3%) reported an adverse outcome on at least one animal used in the study. The publications inconsistently reported the training or certification and veterinary care of the animals. Lacking IACUC approval in AAI studies indicates that the field underutilizes this method of monitoring animal health and welfare. The IACUC should assess, approve, and monitor research protocols involving animals prior to conducting every study to safeguard AAI animal welfare.

Ketamine exposure can lead to selective neuroapoptosis in the developing brain. p66ShcA, the cellular adapter protein expressed selectively in immature neurons, is a known pro-apoptotic molecule that triggers neuroapoptosis when activated. Sprague-Dawley rats at postnatal day 7 were subcutaneously injected in the neck with ketamine 20 mg/kg, six times at 2-hour intervals. At 0, 1, 3, and 6 hours after final injection, western blot assay was used to detect the expression of cleaved caspase-3, p66ShcA, and phosphorylated p66ShcA. We found that the expression of activated p66ShcA and caspase-3 increased after ketamine exposure and peaked at 3 hours. The same procedure was performed on a different group of rats. At the age of 4 weeks, spatial learning and memory abilities were tested with the Morris water maze. Latency to find the hidden platform for these rats was longer than it was for control rats, although the residence time in the target quadrant was similar. These findings indicate that ketamine exposure resulted in p66ShcA being activated in the course of an apoptotic cascade during the neonatal period. This may have contributed to the deficit in spatial learning and memory that persisted into adulthood. The experimental protocol was approved by the Institutional Animal Care and Use Committee at the University of Texas at Arlington, USA (approval No. A13.008) on January 22, 2013.

Although animal research requires adherence to various regulations and standards, the manner in which compliance is maintained and the degree of additional constraints varies between institutions. Regulatory burden, particularly if institutionally imposed, has become a concern for institutions as increased regulatory expectations result in decreased resources available for research efforts. Faculty, research staff, and support staff engaged in animal research were surveyed to determine what institutional animal care and use committee (IACUC) processes were considered burdensome, the perceived value of some suggested modifications, and satisfaction with the IACUC administrative office and the animal resource unit. Although the results revealed overwhelming satisfaction with the IACUC administrative office and the animal resource unit, several IACUC processes were deemed burdensome, and therefore there would be value in modifying IACUC processes. When comparing the value of modifying IACUC processes, different groups within the animal care and use program (ACUP) tended to have different responses on many of the topics. This survey identified several perceived burdensome IACUC processes that would likely benefit individuals if modified. In today's environment of shrinking budgets for biomedical research, minimizing regulatory burden-particularly unnecessary, self-imposed burden-in the ACUP is particularly important to ensure that costs, time, and effort are appropriate to achieve animal welfare and quality of research endeavors.-Norton, J. N., Reynolds, R. P., Chan, C., Valdivia, R. H., Staats, H. F. Assessing the satisfaction and burden within an academic animal care and use program.

Laparoscopic surgery has become the standard option for gastrointestinal surgeries. However, laparoscopic procedures require extended training times and are difficult for inexperienced surgeons. Robot-assisted laparoscopic surgery facilitates easy adaptation of laparoscopic procedures, but robotic surgical systems are expensive. In addition, their cost has remained high because there is currently only one manufacturer of commercially available systems. Recently, a new Korean robotic surgical system, Revo-i, has been developed. The aim of this study was to evaluate the feasibility and safety of Revo-i by performing robotic cholecystectomy in a porcine model.After approval by the Institutional Animal Care and Use Committee of Yonsei University Health System, cholecystectomy was performed in four pigs using the Revo-i robotic surgical system. Operative time and perioperative complications were recorded, and all animals were observed for postoperative complications for 2 weeks after surgery RESULTS: Robotic cholecystectomy was completed successfully and without gallbladder perforation in all cases. The mean operative time was 78 ± 12 min, the mean docking time was 4.5 ± 2.52 min, and the mean console time was 49.8 ± 14.17 min. There were no perioperative complications, and none of the animal used for the in vivo models exhibited abnormal behavior during the postoperative observation period.These preliminary results verify the safety and efficacy of robotic cholecystectomy using the Revo-i robotic surgical system. Human trials are slated to begin accordingly.

Background: Barrett's esophagus (BE), a complication of gastroesophageal reflux disease (GERD), predisposes patients to esophageal adenocarcinoma (EAC). Reliable biomarkers for early detection and discovery of potential drug targets are urgently needed for improved BE and EAC patient outcomes. Methods: Patient biopsy samples were evaluated for COX1, COX2, and thromboxane A2 synthase (TBXAS) expression. Circulating prostaglandins biosynthesis was determined using enzyme immunoassay kits. Anchorage-independent cell growth assay, crystal violet staining assay, and xenograft experiments were conducted to assess BE and EAC cell growth. A surgical mouse model of reflux (i.e., esophagoduodenostomy) was established and samples were analyzed using an enzyme immunoassay kit, immunohistochemistry, immunoblotting, or RT-PCR. Esophageal biopsy samples (pre- and post-intervention) were obtained from a randomized clinical trial in which participants were administered esomeprazole (40 mg) twice daily in combination with an aspirin placebo or 81 or 325 mg aspirin for 28 days. Esophageal biopsy specimens before and after the intervention period were analyzed. Findings: COX2 and TBXAS are highly expressed in BE and EAC patients accompanied by a pronounced elevation of circulating TXA2 levels. Aspirin suppressed BE and EAC growth by targeting the TXA2 pathway. Additionally, biopsies from 49 patients (with similar baseline characteristics) showed that aspirin substantially decreased serum TXA2 levels, resulting in reduced inflammation. Interpretation: This study establishes the importance of the COX1/2-driven TXA2 pathway in BE and EAC pathophysiology and lays the groundwork for introducing a TXA2-targeting strategy for EAC prevention and early detection. Funding Statement: This work was supported by the Hormel Foundation and National Institutes of Health grants CA166011, CA187027, and CA196639 (Z. Dong). Declaration of Interests: The authors declare no potential conflicts of interest. Ethics Approval Statement: All animal studies were approved by the University of Minnesota Institutional Animal Care and Use Committee (IACUC).

Background: Intracavernous injection (ICI) of stem cells (SCs) has been demonstrated promsing for neurogenic erectile dysfunction (ED), but unexpected animal death after ICI was frequent and the therapeutic outcomes were inconsistent for unknown reasons. Here, our investigation was focused on whether cell escaping is responsible for attenuated therapeutic efficacy and unexpected animal death and its underlying mechanisms. Methods: Single-cell suspension of SCs and stem-cell spheroids with suitable size were intracavernously injected at doses of 0.5, 1, 2, or 4 million cells. The cell loss and safty after ICI, erectile function and histopathologic change, etc. were analyzed with multimodality of methods. Findings: Most free SCs escaped from sponge-like corpus cavernosum (CC) after ICI due to their small size, weakening stem-cell therapeutic efficacy. Worse still, the escaped cells were shown to cause widespread pulmonary embolism (PE), and even death in some animals. Further, it was founded that the limited effect of free SCs may be ascribed to the larger cell clusters which were spontaneously aggregated before ICI and were trapped within CC after ICI. In comparison, cell loss and PE were significantly avoided by transplanting stem-cell spheroids. Importantly, better therapeutic outcomes were detected with less stem cells by ICI of stem-cell spheroids. Interpretation: Transplantation of size-controlled stem-cell spheroids instead of single-cell suspension for neurogenic ED may be a wiser choice to achieve steady therapeutic outcome and minimize risks for the future clinical application. Funding Statement: This work was supported by the National Natural Science Foundation of China (81701432) (to Y. Xu). Declaration of Interests: None of the authors have any relationships/conditions/circumstances that present a potential conflict of interest. Ethics Approval Statement: All animal procedures were conducted following the guidelines of the Institutional Animal Care and Use Committee of the General Hospital of the People’s Liberation Army (Beijing, China).

Zika virus (ZIKV) has evolved into a global health threat because of its causal link to congenital Zika syndrome. Different case reports indicate that ZIKV infection of pregnant women may cause a spectrum of abnormalities in children. Here we evaluated a large cohort of children with perinatal exposure to ZIKV and identified a spectrum of neurodevelopmental abnormalities in the group. In addition, we show that infection of the mouse neonatal brain by a contemporary ZIKV strain instead of an Asian ancestral strain can cause microcephaly and various abnormal neurological functions. These include defects in social interaction and depression, impaired learning and memory, in addition to severe motor defects, which are present in adult mice as well as in our cohort of children. Importantly, although mouse brains infected later after birth do not have apparent abnormal brain structure, those mice still show significant impairments of visual cortical functions, circuit organization and experience-dependent plasticity. Thus, our study suggests that special attention should be paid to all children born to ZIKV infected mothers for screening of abnormal behaviors and sensory function during childhood. Funding Statement: This work was supported by the Strategic Priority Research Program and Innovation Program of the Chinese Academy of Sciences (No. XDB32020100, QYZDJ-SSW-SMC007), the National Natural Science Foundation of China (NSFC) (31730108, 31430037, U170220023, 81772176, 31670883, 91542201, 81590765, and 31500145), Beijing Municipal Science & Technology Commission (Z181100001518001), the Interdisciplinary Research Funds of Beijing Normal University, the CAMS Initiative Innovative Medicine (No. 2016-I2M-1-005), Major Project for "Significant New Drugs Innovation and Development" (2015ZX09102023), NIH R01 AI140718, AI069120, AI056154 and AI078389 502 grants, MOST (China, 2016YFD0500304). C.L. was supported by the PUMC Youth Fund (No. 3332016125). Declaration of Interests: The authors confirm that there are no known conflicts of interest associated with this publication. Ethics Approval Statement: The experimental procedures on mice were performed under biosafety level 2 (BSL-2) at Beijing Institute of Microbiology and Epidemiology with Institutional Biosafety Committee approval. The mouse care and experimentation were approved by the Animal Care and Use Committees of the Institutional Animal Care and Use Committee at Institute of Genetics and Developmental Biology (IGDB), Chinese Academy of Sciences, Beijing Institute of Microbiology and Epidemiology and the State Key Laboratory of Cognitive Neuroscience & Learning at Beijing Normal University. For the infant cohort, the study protocol was approved by the institutional review boards at Fundacao Oswaldo Cruz (Fiocruz) and the University of California, Los Angeles. All mothers provided written informed consent for themselves and for their children.

Numerous studies have suggested that dysregulated long noncoding RNAs (lncRNAs) contributed to development and progression of many cancers. LncRNA OIP5 antisense RNA 1 (OIP5-AS1) has been reported to be increased in several cancers. However, the roles of OIP5-AS1 in liver hepatocellular carcinoma (LIHC) remain to be investigated. In this study, we demonstrated that OIP5-AS1 was up-regulated in LIHC tissue specimens and its overexpression was associated with the poor survival of patients with LIHC. Furthermore, loss-of function experiments indicated that OIP5-AS1 promoted cell proliferation and inhibited cell apoptosis both in vitro and in vivo. Moreover, binding sites between OIP5-AS1 and hsa-miR-26a-3p as well as between hsa-miR-26a-3p and EPHA2 were confirmed by luciferase assays. Finally, rescue assay was performed to prove the effect of OIP5-AS1-hsa-miR-26a-3p-EPHA2 axis on LIHC cell biological behaviors. Based on all above findings, our results suggested OIP5-AS1 promoted LIHC cell proliferation and invasion via regulating of hsa-miR-26a-3p/EPHA2 axial. Funding Statement: This study was supported partly by grants from the National Natural Science Foundation of China (81972214, 81772932, 81472202, 81201535, 81302065, 81671716, 81301993, 81372175 and 81472209), The Fundamental Research Funds for the Central Universities (22120170212 and 22120170117), Shanghai Natural Science Foundation (12ZR1436000 and 16ZR1428900), Shanghai Municipal Commission of Health and Family Planning (201540228 and 201440398), Construction of Clinical Medical Center for Tumor Biological Samples in Nantong (HS2016004) and Jiangsu 333 Program (BRA2017205). Declaration of Interests: No potential conflicts of interest were disclosed. Ethics Approval Statement: This study protocol was in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Shanghai Tenth People's Hospital, Tongji University School of Medicine (Approval No: SHSY-IEC-2019K10). All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) guidelines at Tongji University School of Medicine (SYDW-19-215).

Background: Adult mammalian heart repair after myocardial damage is highly inefficient due to the post-mitotic nature of cardiomyocytes. Interestingly, in Traditional Chinese Medicine (TCM), there are reported effective treatments of myocardial infarction and heart failure in adult humans by oral intake of a TCM concoction named Gu Ben Pei Yuan San (GBPYS) . Methods: We fed the mice with GBPYS after myocardial damages through everyday self-feeding. We then examined the effect of everyday oral intake of GBPYS on improving cardiac function and myocardial repair in adult mice after apical resection or myocardial infarction. Findings: We found that long-term oral intake of GBPYS significantly improved cardiac function after myocardial damages in adult mice. Brdu, Phospho-histone 3, and AuroraB staining indicated increased cell proliferation at the border zone of MI after TCM feeding. GBPYS feeding reduced organ inflammation and is nontoxic to mice after long-term oral intake. Further, serum derived from TCM fed MI rats promoted division of both neonatal rat cardiomyocytes and human iPSC-derived cardiomyocytes in vitro. Interpretation: Oral intake of GBPYS improved heart repair after myocardial damages in adult mice. Our results suggest that there are substances present in GBPYS that help improve adult mammalian heart repair after MI. Funding Statement: This work was supported by the National Natural Science Foundation of China (NSFC No.31571527, N.S.; No.31501098, Q.L.; No.81500241, C.X.), the National Key R&D Program of China (2016YFC1000500, 2016YFC1305100), the Science and Technology Commission of Shanghai Municipality (No. 17XD1400300, No.17JC1400200), and the Haiju program of National Children's Medical Center EK1125180102. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: All the protocols in this study were approved by the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Publication No. 85-23, Revised) and was carried out under the supervision of the Fudan University Institutional Animal Care and Use Committee.

Background: Perioperative neurocognitive disorders (PND) are serious complications that afflict up to 50% of surgical patients and for which there are no disease-modifying therapeutic options. We developed the "broad spectrum" mixed lineage kinase 3 inhibitor URMC-099 to inhibit pathological innate immune responses that underlie neuroinflammation-associated cognitive dysfunction. Here, we test the hypothesis that prophylactic treatment with URMC-099 can prevent surgery-induced neuroinflammation and cognitive impairment in mice following orthopedic surgery-induced PND. Methods: We performed orthopedic surgery by fracturing the tibia of the left-hind limb with intramedullary fixation under general anesthesia and analgesia. We administered URMC-099 (10 mg/kg, i.p.) prophylactically via three intraperitoneal injections. We used two-photon microscopy and CLARITY with light-sheet microscopy to define surgery-induced changes in microglial dynamics and morphology. We assessed cognitive dysfunction following surgery using the "What-Where-When" task and the Memory Load Object Discrimination task. In surgically-treated samples, we analyzed fracture callouses using microcomputed tomography and histomorphometry analyses. Findings: Orthopedic surgery induced microglial activation in the hippocampus and cortex without overtly affecting microglial process dynamics. Surgically-treated mice exhibited impaired object place and identity discrimination, indicating cognitive dysfunction. URMC-099 prophylaxis prevented surgery-induced microgliosis and memory impairment. Analysis of fracture callouses showed no effect of URMC-099 on fracture healing. Interpretation: These findings show that prophylactic URMC-099 treatment is sufficient to prevent surgery-induced microgliosis and cognitive impairment without interfering with the fracture healing process. Funding Statement: Design, synthesis and validation of URMC-099 was supported by NIH grants P01MH64570 and RO1 MH104147 (HAG). The study was supported by the NIH RO1 AG057525 (NT). Declaration of Interests: URMC-099 is owned by URMC (HA Gelbard. lead inventor: patent nos. US 8,846,909 B2; 8,877,772; and 9,181,247 and associated international patents). HAG has no commercial disclosures regarding URMC-099 at present. Ethics Approval Statement: All animal procedures were carried out under protocols approved by Duke University Medical Center and University of Rochester Medical Center, Institutional Animal Care and Use Committee under the National Research Council Guide for the Care and Use of Laboratory Animals, 8th edition. Both Duke University and University of Rochester Medical Center are AAALAC accredited institutions.

Background: There are few effective treatment options for patients with recurrent or advanced cutaneous squamous cell carcinoma (CSCC). Acitretin demonstrated no therapeutic effect in patients with high-risk CSCC, though a preventive effect has been reported on new primary non-melanoma skin cancers in post-transplantation recipients. This study aimed to investigate the synergic efficacy of combined acitretin and clarithromycin for the treatment of high-risk CSCC. Methods: All patients of high-risk CSCC were treated with acitretin combined with clarithromycin in our department, and then anti-tumor effects and adverse drug reaction were followed up from 2008 to 2019. A. The potential synergistic mechanisms of acitretin and clarithromycin were further evaluated in vitro and in vivo using two human carcinoma cell lines A431 and KB. Findings: Five cases of high-risk CSCC were identified during the study period and complete remission were achieved in all lesions after 2 to 24 months of combination therapy. Four of five patients remain alive without recurrence of CSCC. During treatment, mucocutaneous dryness was noted in three patients and pseudotumor cerebri was found in one patient. The combination of acitretin and clarithromycin showed synergistic anti-tumor activity through inhibition of cell growth and induction of apoptosis in both A431 and KB cell lines. Tumor growth was significantly suppressed in a KB cell xenograft model by the combination therapy. Interpretation: Combined acitretin and clarithromycin therapy could represent a novel treatment approach with robust anti-tumor activity and a manageable safety profile in patients with unresectable high-risk CSCC. Funding Statement: This study was supported by grants from Fujian Provincial High-Tech research program (2016Y0036), the Key Research Program of Fujian Provincial Heath and Education Joint Committee (WKJ2016-2-24) and National Collaboration Center in Immuno-Oncology (No. 2016sysbz02). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The study was approved by the medical technology clinical application and ethics committee of the First Affiliated Hospital of Fujian Medical University (No. [2015]084). All procedures performed in studies involving animals were approved by the Fujian Medical University Institutional Animal Care and Use Committee (IACUC, NO. 2017-031) in accordance with the ethical standards.

Background: The natural carotenoid crocetin has been reported to suppress experimental myopia in mice. The purpose of this study is to investigate the minimum effective dosage and the preventive efficacy of crocetin with a murine model and human subjects, respectively. Methods: Three-week-old male mice (C57B6/J) were equipped with a -30 diopter (D) lens. Changes of refractive errors and axial lengths (AL) were evaluated to determine the minimum effective dosage. A multicenter randomized double-blind placebo-controlled clinical trial was performed with 69 participants aged 6 to 12 years, whose cycloplegic spherical equivalent refractions (SER) were between -1.5 and -4.5 D. We evaluated AL elongation and change of SER for 24 weeks. A mixed effect model was used for analysis using both eyes. Findings: As the minimum dose, 0.001% of crocetin containing extract chow showed a significant (P

Background: Central hemiplegia led by hypertensive intracerebral hemorrhage(HICH) is prevalent, devastating, and currently unable to intervene satisfactorily. Here,to move toward the clinic,we explored whether contralateral L4 nerve transfer,centralizing at flexion and extension, reanimates fine motor function for hemiplegic hindlimbs after HICH. Methods: Eligible rat models were manufactured by injecting autologous vein blood into the posterior limb of internal capsule (PLIC).Thirty rats were randomized into three groups.Rats in group A underwent sham operation(i.e.,exposure of bi-L4 roots without severance) after injection of saline. Rats in group B and group C were infused equal blood followed by bilateral L4 nerve roots transection and contralateral L4 nerve transfer, respectively. Behavioural, ultrastructural, immunofluorescent, and electromyographic assessments were applied at a scheduled timepoint in our study. fMRI was used as localization of PLIC impact. Findings: Through neurotization, skilled motion produced by dexterous flexion and extension in major joints (hip, knee, and ankle) of the paralyzed hindlimb recovered more sharply in group C than in group B (P

Background: Pneumoconiosis is a chronic occupational lung disease caused by long-term inhalation of dust in the workplace. Identifying accurate and reliable biomarkers would enable earlier detection before irreversible deteced radiology changes occurred in the lung. Methods: We created a rat model that closely approximates the exposure and development of pneumoconiosis in humans. iTRAQ coupled with LC-MS technology was used to screen differentially-expressed proteins and validate them with patient bronchopulmonary lavage fluid and serum samples to assess whether these differentially expressed proteins were appropriate biomarkers for the diagnosis of pneumoconiosis. Findings: Totally, 471 proteins were differentially expressed in the pneumoconiosis group compared to controls. Twenty proteins with increased expression levels and eight with decreased levels exhibited a ≥ 1.5 foldchange relative to controls. We next determined whether similar changes in protein expression were detectable in human bronchoalveolar lavage fluid and serum. The levels of PTPN2, VRK1 and factor B were significantly higher in pneumoconiosis patients. The highest area under the curve(AUC) and diagnostic accuracy was the combined diagnosis of all three proteins. Interpretation: We found PTPN2, factor B, and VRK1 could be predictive biomarkers for pneumoconiosis. The three-factor combined diagnosis was found to be the most efficient for early diagnosis. Funding Statement: This work was supported by the "precision medicine" projects under the national key research and development programme of China (No. 2016YFC0900605); the Hebei Medical Science Research Foundation (No. 20190108); The Science and Technology Foundation of Tangshan (No. 18130206a); Postgraduate Innovation Foundation of North China University of Technology (No. 2017B20). Declaration of Interests: The authors declare that they have no conflicts interest. Ethics Approval Statement: Animal studies were performed with a protocol approved by the Institutional Animal Care and Use Committee of the North China University of Science and Technology, Tangshan, China (No. LX201868), all animals were carefully handled in compliance with the guidelines. All participants provided written informed consent, including consent to provide bronchopulmonary lavage fluid and serum to test for biomarkers predictive of pneumoconiosis, and studies carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. All studies were approved by the Ethics Committee of North China University of Science and Technology, Tangshan, China (No.2018021).

Chronic inflammation of adipose tissues contributes to obesity-triggered insulin resistance. Unfortunately, the potential molecular mechanisms regarding obesity-associated systemic inflammation and metabolic disorder remain complicated. Here, we report that inactive rhomboid-like protein 2 (iRhom2) was increased in overweight mice with adipose inflammation. Mice with deletion of iRhom2 on a C57BL/6J background, mice without deletion of this gene (controls), and mice with deficiency of iRhom2 only in myeloid cells were fed a standard chow diet or a high-fat diet (HFD; 60% fat calories). Then the adipose tissues or bone-marrow cells were isolated for the further detection. After 16 weeks on a high-fat diet (HFD), obesity, chronic inflammation in adipose tissues, and insulin resistance were markedly mitigated in iRhom2-knockout (iRhom2 KO) mice, whereas these parameters were exaggerated in iRhom2-overactivated mice. The adverse influences of iRhom2 on adipose inflammation and associated pathologies were determined in db/db mice. Also, we further demonstrated that, in response to an HFD, iRhom2 KO mice and mice with deletion only in myeloid cells showed less severe adipose inflammation and insulin resistance than that of control groups. Conversely, transplantation of bone-marrow cells from normal mice to iRhom2 KO mice unleashed a severity of systemic inflammation and metabolic dysfunction after HFD ingestion. We identify iRhom2 as a key regulator that promotes obesity-associated metabolic disorders. Loss of iRhom2 from macrophages in adipose tissues inhibited inflammation and insulin resistance. Hence, iRhom2 may be a therapeutic target for obesity-induced metabolic dysfunction. Funding Statement: This work was jointly supported by (1) National Natural Science Foundation of China (NSFC Grant No.: 81703527); (2) Chongqing Research Program of Basic Research and Frontier Technology (Grant No.: cstc2017jcyjAX0356, cstc2018jcyjA3686, cstc2018jcyjAX0784, cstc2018jcyjA1472, cstc2018jcyjAX0811, cstc2018jcyjA3533 and KJZD-M201801601); (3) School-level Research Program of Chongqing University of Education (Grant No.: KY201710B and 17GZKP01); (4) Advanced Programs of Post-doctor of Chongqing (Grant No.: 2017LY39); (5) Science and Technology Research Program of Chongqing Education Commission of China (Grant No.: KJQN201901608, KJQN201901615, KJ1601402) and (6) Children's Research Institute of National Center for Schooling Development Programme and Chongqing University of Education (Grant No.: CSDP19FSO1108). Declaration of Interests: Minxuan Xu, Chenxu Ge, Yuting Qin, Deshuai Lou, Qiang Li, Jing Feng, Yekuan Wu , Linfeng Hu, Bochu Wang & Jun Tan declare that they have no conflict of interest. Ethics Approval Statement: All research procedures associating with mice were approved by the Institutional Animal Care and Use Committee in Chongqing Key Laboratory of Medicinal Resources in the Three Gorges Reservoir Region, School of Biological and Chemical Engineering, Chongqing University of Education, and were performed in accordance with the Guide for the Care and Use of Laboratory Animals, issued by the National Institutes of Health in 1996. The protocols used in this study were in accordance with the Regulations of Experimental Animal Administration issued by the Ministry of Science and Technology of the People's Republic of China.

Background: Corneal neovascularization (angiogenesis and lymphangiogenesis) compromises corneal transparency and transplant survival. The contribution and molecular mechanisms of three kinds of corneal host cells, including corneal epithelial, stromal, and endothelial cells, involved in corneal neovascularization are currently unclear. Methods: Liquid chromatography-mass spectrometry, immunoblotting, and ELISA were used to screen and identify potential neovascularization related factors in human full thickness vascularized corneal tissues. In vitro inflammation model, plasmid transfection, recombinant stimulation, tube formation assay, spheroid sprouting assay, and in vivo corneal neovascularization model were employed to clarify the contribution of three corneal host cells to neovascularization and identify the key proteins involved. Findings: All three kinds of corneal host cells could influence corneal neovascularization, but varied in degrees. Matrix metalloproteinase-9 (MMP-9) in human corneal epithelial cells, MMP-2, and alpha-crystallin A chain (CRYAA) in human corneal stromal cells, and MMP-2 and galectin-8 in human corneal endothelial cells might be key proteins participating into corneal neovascularization, respectively. Interpretation: These data highlight that both the effects of key proteins and cornea host cells involved should be considered for therapy of corneal neovascularization. Funding Statement: This study was supported by the Nature Science Foundation of China (81470618), the Scientific Research Foundation of First Affiliated Hospital of Harbin Medical University (2017B013), the Major Program of Applied Technology Research and Development Plan of Heilongjiang Province (GY2016ZB0159), the Scientific Research Fund of Heilongjiang Provincial Education Department (12521262), the Scientific Research Fund of Heilongjiang Provincial Health Bureau (2011-031), the Special Fund for the Doctoral Program of Higher Education (20132307120035), the Natural Science Foundation of China (81300728), the Natural Science Foundation of Heilongjiang Province of China (QC2010113), and the Postdoctoral Grant of Heilongjiang Province (LBH-Q12038). Declaration of Interests: The authors have no conflicts of interest to declare. Ethics Approval Statement: All experiments were conducted in accordance with Declaration of Helsinki Principles, and all experiments were performed with the approval of the Internal Review Board of Harbin Medical University. We obtained the informed consents from all patients prior to mortality or their family members following mortality for inclusion of autopsy specimens. The rats were housed in the Harbin Medical University Experimental Animal Resources Center in accordance with National Institutes of Health guidelines and operated upon using a research protocol approved by the Institutional Animal Care and Use Committee.

Background: Recurrent and metastatic head and neck squamous cell carcinoma (HNSCC) has a dismal prognosis with limited progressionfree survival and overall survival, even when treated with different combinations of chemotherapy, targeted therapies and immunotherapy. We explored in vitro and in vivo the effect of the epidermal growth factor (EGFR) inhibitor, osimertinib, alone and in combination with dihydroartemisinin (DHA) in HNSCC. Methods: The combination of osimertinib with DHA was tested in the FaDu and CAL27 HNSCC cell lines. Tumor cell proliferation assays were conducted in cultured cells and mouse xenografts. Western blotting analysis of related signal pathways was performed to investigate the molecular mechanisms of the inhibitory effect of DHA and the combination. Other compounds, which inhibit signal transducer and activator of transcription 3 (STAT3), Src-family kinases (SFKs), sphingosine kinase 1 (SPHK1), or the receptor tyrosine kinase (RTK) AXL were also combined with osimertinib in vitro. Results: Osimertinib exerted synergistic cytotoxicity toward FaDu and CAL27 HNSCC cells when combined with DHA. DHA reversed the osimertinibinduced STAT3 and Src phosphorylation. The double combination inhibited AXL expression. The anticancer potential of osimertinib plus DHA combination was validated in vivo on FaDu and CAL27 xenografts in mice with significant tumor regression. Interpretation: The results illustrate that the combinatory therapy of osimertinib and DHA, as a repurposing anticancer drug, could be a novel therapeutic strategy for recurrent and/or metastatic HNSCC patients. The findings strongly indicate that a clinical trial is warranted to confirm the benefit of the combination. Funding Statement: Work in Dr. Rosell's laboratory is partially supported by a grant from La Caixa Foundation, an Instituto de Salud Carlos III grant (RESPONSE, PIE16/00011), an Instituto de Salud Carlos III grant (PI14/01678), a Marie Sklodowska-Curie Innovative Training Networks European Grant (ELBA No 765492) and a Spanish Association Against Cancer (AECC) grant (PROYE18012ROSE). Work in Dr. Cao's laboratory is partially supported by the Major National Science and Technology Program of China for Innovative Drug (2017ZX09101002-002-006), the Priority Academic Program Development of Jiangsu Higher Education Institutions (Integration of Chinese and Western Medicine) grant and Key R&D Program of Jiangsu Province (BE2018755). Declaration of Interests: The authors declare no potential conflicts of interest. Ethics Approval Statement: All procedures were based on Guide for Care and Use of Laboratory Animals of National Institutes of Health and approved by Institutional Animal Care and Use Committee of Jiangsu Province Academy of Traditional Chinese Medicine (SYXK 2016-0018).

Fibroblast growth factor (FGF21) holds great promise as an effective therapeutic means for treating nonalcoholic steatohepatitis (NASH) which is a potential precursor for hepatocellular carcinoma (HCC). We aimed to explore the potential mechanism(s) of FGF21 during NASH-HCC transition using a FGF21 knockout (KO) mouse model. Lack of FGF21 caused significant up-regulation of the hepatocyte derived IL-17A via Toll-like receptor 4 (TLR4) and NF-κB signaling. Restoration of FGF21 alleviated the high NAFLD activity score (NAS) and attenuated the TLR4 triggered hepatocyte-IL-17A expression. The HCC nodule number and tumor size were significantly alleviated by treatments of anti-IL-17A antibody. Down-regulated FGF21 but up-regulated IL-17A expression was also found in the HCC samples from our clinical patients and consistent with data analysis of Gene Expression Profiling Interactive Analysis (GEPIA). In conclusion, lack of FGF21 contributed to FFA mediated induction of hepatocyte-TLR4 signal to up-regulate IL-17A expression in hepatocytes. The potential anti-inflammatory effect of FGF21 could be through a negative feedback on the hepatocyte-TLR4 signaling to inhibit IL-17A production in the liver. The negative feedback loop on the hepatocyte-TLR4 -IL-17A axis could be a potential anti-carcinogenetic mechanism for FGF21 to prevent NASH-HCC transition. Funding: Research reported in this publication was supported partly by an Institutional Development Award (IDeA) from the NIGMS of the National Institutes of Health under grant number P20GM113226. Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: The animal procedures were approved by the Institutional Animal Care and Use Committee of University of Louisville, which is certified by the American Association for Accreditation of Laboratory Animal Care. All the human sample collection procedures for this study were approved by the Institutional Review Board for Human Study at the University of Louisville.

Summary: Early-passage human mesenchymal stem cells (hMSCs) are typically used in clinical trials because of safety and efficacy issues. However, obtaining sufficient cells for treatment is difficult and expensive. Many more late-passage hMSCs can be obtained at lower cost, although efficacy is a large hurdle in clinical trials. The use of late-passage MSCs with better efficacy would be a revolutionary solution for reducing cost and facilitating clinical trials. In the present study, glia-like cells (ghMSCs) were induced from hMSCs and used for both in vitro and in vivo models of ischemia, effectively protecting neurons from ischemia and restoring brain tissue damaged by cerebral infarction. These beneficial effects were significantly blocked by insulin-like growth factor-binding protein-4 (IGFBP-4) antibody. The current study demonstrated that late-passage hMSCs can be efficiently induced into ghMSCs with a better neuroprotective effect against ischemic stroke and that IGFBP-4 may be a key neuronal survival factor secreted by ghMSCs. Funding Statement: This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI17C2160). Declaration of Interests: One of the corresponding authors, Mi-Sook Chang, has a patent related to this work, which is noted in the "Declaration of Interests" section of the manuscript. She obtained a Korean patent for glia-like human mesenchymal stem cells in 2012. Ethics Approval Statements: All procedures involving animals were approved by the Institutional Animal Care and Use Committee, Seoul National University (Seoul, Republic of Korea). In addition, all animal procedures were performed in accordance with the Hanyang University guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of Hanyang University. In accordance with Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines, all experiments were carried out in a strictly blinded fashion, inclusion and exclusion criteria were predetermined, and attrition due to mortality and other causes was reported

Background: Metabolic surgery has been increasingly recommended for obese diabetic patients, but questions remain as to its effectiveness for nonobese diabetic patients and its mechanism that leads to glucose homeostasis independently of weight loss. Methods: We evaluated the role of the Roux limb (RL) in Roux-en-Y gastric bypass (RYGB) surgery, which facilitates food outflow from the upper stomach pouch in nonobese diabetic rat models. Findings: Improvements in glucose metabolism were greater in the long-RL RYGB group than in the other operated RYGB groups or the Goto-Kakizaki (GK) controls. Moreover, diabetes remission by RYGB was found to be remarkably different from both the healthy and diabetic states in the analysis of transcriptome profiles of three rat models individually representing different physiological states. Furthermore, according to a functional analysis, RYGB surgery significantly affected one group of genes, i.e., the new-changed group, which was originally "normal" (not significantly different between GK and Wistar rats) but became "abnormal" in the RYGB group (significantly different between RYGB and Wistar rats), despite their diabetes remission. We further observed that novel dysfunctions of this new-changed group induced by RYGB played key roles in improved glycemic control to help rebalance intestinal glucose metabolism. In particular, an increase in newly synthesized cholesterol (CHOL) enriched by the new-changed group increased glucose consumption, which contributed to glucose homeostasis, as demonstrated by a biodistribution analysis using [14C]-labeled glucose. Interpretation: Our findings suggest glycometabolic rebalance by RYGB-induced "dysfunctions" in the new-changed group as a compensatory role contributes to diabetes remission. Additionally, we propose long-RL RYGB for diabetic patients without morbid obesity. Funding: National Key R&D Program of China (No. 2017YFA0505500), the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDB13040700), National Natural Science Foundation of China (Nos. 81471047, 31771476 and 81802354), and Shanghai Sailing Program (No. 18YF1420700). Author contributions Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: All experiments were in compliance with NIH guidelines and the Guide for the Care and Use of Laboratory Animals and were approved by the Chinese Academy of Science Institutional Animal Care and Use Committee.

Previous studies demonstrate an accumulation of transferrin and transferrin receptor 1 (TfR1) in regenerating peripheral nerves. However, the expression and function of transferrin and TfR1 in the denervated skeletal muscle remain poorly understood. In this study, a mouse model of denervation was produced by complete tear of the left brachial plexus nerve. RNA-sequencing revealed that transferrin expression in the denervated skeletal muscle was upregulated, while TfR1 expression was downregulated. We also investigated the function of TfR1 during development and in adult skeletal muscles in mice with inducible deletion or loss of TfR1. The ablation of TfR1 in skeletal muscle in early development caused severe muscular atrophy and early death. In comparison, deletion of TfR1 in adult skeletal muscles did not affect survival or glucose metabolism, but caused skeletal muscle atrophy and motor functional impairment, similar to the muscular atrophy phenotype observed after denervation. These findings suggest that TfR1 plays an important role in muscle development and denervation-induced muscular atrophy. This study was approved by the Institutional Animal Care and Use Committee of Beijing Institute of Basic Medical Sciences, China (approval No. SYXK 2017-C023) on June 1, 2018.

The mechanism and the type of hearing loss induced by cochlear implants are mostly unknown. Therefore, this study evaluated the impact and type of hearing loss induced by each stage of cochlear implantation surgery in an animal model.Original basic research animal study.The study was conducted in a tertiary, university-affiliated medical center in accordance with the guidelines of the Institutional Animal Care and Use Committee.Cochlear implant electrode array was inserted via the round window membrane in 17 ears of 9 adult-size fat sand rats. In 7 ears of 5 additional animals round window incision only was performed, followed by patching with a small piece of periosteum (control). Hearing thresholds to air (AC) and bone conduction (BC), clicks, 1 kHz and 6 kHz tone bursts were measured by auditory brainstem evoked potential, before, during each stage of surgery and one week post-operatively. In addition, inner ear histology was performed.The degree of hearing loss increased significantly from baseline throughout the stages of cochlear implantation surgery and up to one week after (p0.0001). In both operated groups, the greatest deterioration was noted after round window incision. Overall, threshold shift to air-conduction clicks, reached 61 dB SPL and the bone conduction threshold deteriorated by 19 dB SPL only. Similar losses were found for 1-kHz and 6-kHz frequencies. The hearing loss was not associated with significant changes in inner ear histology.Hearing loss following cochlear implantation in normal hearing animals is progressive and of mixed type, but mainly conductive. Changes in the inner-ear mechanism are most likely responsible for the conductive hearing loss.

Purpose 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, hDAF) 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 hTBM, have been generated to address this barrier. Here, we studied the importance of hTBM in preventing coagulation 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, provided by Revivicor. 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=3) were found to have no intracardiac thrombus with survival up to 30 days post-transplant. However, all non-hTBM donors (n=3) 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 examination revealed no signs of rejection. Conclusion hTBM prevents intracardiac thrombus in this model. 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.

Objective: Implantation of a ventricular assist device (VAD) is a seminal therapeutic option for patients with terminal cardiac failure. A growing number of VAD patients are successfully bridged to transplantation, or can even live permanently with the device. However, the success is restricted by frequent severe complications. Haemolysis is a relevant adverse effect of several VAD types, which is the result of destruction of red blood cells, reduced by wall shear stress, flow acceleration and interaction with artificial surfaces. The CH-VAD, a small implantable continuous-flow blood pump, featuring a magnetically levitated impeller and enough hydrodynamic performance, was under development and completed for a 60-days animal implantation experiment in 6 sheep. The goal of this study is to validate the hemolysis of the pump through in vitro and in vivo studies. Methods: A series of in vitro tests was quantified experimentally by using in vitro circulation loop system according to ASTM F1841, the standard practice for the assessment of hemolysis in continuous-flow blood pumps. The hemolysis test in vivo was performed during a 60-days ovine model implantation, which was being conducted under the Institutional Animal Care and Use Committee (IACUC) protocol 05-0600 1. Results in vitro tests showed that the average normalized index of hemolysis (NIH) value of the VAD was 0.007 mg/l. The hemolysis in vivo was evaluated based on the amount of free hemoglobin in the plasma, and which showed that the free hemoglobin level in plasma peaked at 0.95 mg/l on the fifth postoperative day and then returned to an acceptable range of 6.0 mg/dL. Conclusion The magnetic levitation left ventricular assist device has good hemolytic performance. These acceptable performance results supported proceeding initial clinical trail conditions.

Both anesthetic-induced and ischemic preconditioning are protective against hepatic ischemia–reperfusion injury. However, the effects of these preventive methods on the metabolic function remain to be elucidated. We investigated the anesthetic conditioning and ischemic preconditioning on the metabolic function of the rabbit model of hepatic ischemia–reperfusion. After approval by the institutional animal care and use committee, 36 Japanese White rabbits underwent partial hepatic ischemia for 90 min either under sevoflurane or propofol anesthesia. All the rabbits underwent 90 min of hepatic ischemia, and half of the rabbits in each group underwent additional 10-min ischemia and 10-min reperfusion before index ischemia. Hepatic microvascular blood flow was intermittently measured during reperfusion period, and galactose clearance, serum aminotransferase activities, and lactate concentrations were determined 180 min after reperfusion. Neither anesthetic conditioning with sevoflurane nor ischemic preconditioning altered hepatic microvascular blood flow during reperfusion and serum transaminase activities after reperfusion. However, galactose clearance of reperfused liver was significantly higher under sevoflurane anesthesia than propofol (0.016 ± 0.005/min vs. 0.011 ± 0.004/min). Statistically significant interaction between anesthetic choice and application of ischemic preconditioning suggests that the ischemic preconditioning is selectively protective under propofol anesthesia. Increase of blood lactate concentration was significantly suppressed under sevoflurane anesthesia compared to propofol (1.5 ± 0.8 vs. 3.9 ± 1.4 mmol/l) without any statistically significant interaction with the application of ischemic preconditioning. Sevoflurane attenuated the decrease of galactose clearance and increase of the blood lactate after reperfusion compared to propofol. Application of ischemic preconditioning was significantly protective under propofol anesthesia.

Background Impaired social interaction is one of the essential features of autism spectrum disorder (ASD). Our previous copy number variation (CNV) study discovered a novel deleted region associated with ASD. One of the genes included in the deleted region is ARHGEF10. A missense mutation of ARHGEF10 has been reported to be one of the contributing factors in several diseases of the central nervous system. However, the relationship between the loss of ARHGEF10 and the clinical symptoms of ASD is unclear. Methods We generated Arhgef10 knockout mice as a model of ASD and characterized the social behavior and the biochemical changes in the brains of the knockout mice. Results Compared with their wild-type littermates, the Arhgef10-depleted mice showed social interaction impairment, hyperactivity, and decreased depression-like and anxiety-like behavior. Behavioral measures of learning in the Morris water maze were not affected by Arhgef10 deficiency. Moreover, neurotransmitters including serotonin, norepinephrine, and dopamine were significantly increased in different brain regions of the Arhgef10 knockout mice. In addition, monoamine oxidase A (MAO-A) decreased in several brain regions. Conclusions These results suggest that ARHGEF10 is a candidate risk gene for ASD and that the Arhgef10 knockout model could be a tool for studying the mechanisms of neurotransmission in ASD. Trial registration Animal studies were approved by the Institutional Animal Care and Use Committee of National Taiwan University (IACUC 20150023). Registered 1 August 2015. Electronic supplementary material The online version of this article (10.1186/s13229-018-0197-5) contains supplementary material, which is available to authorized users.

The ApcMin/＋ mouse exhibits an intestinal cachexia accompanied by hypertriglyceridemia (HTG). Because the liver governs systemic energy demands through lipid metabolism regulation, the liver is a likely pathological target of HTG in the ApcMin/＋ mouse. Methods: On the basal diet, at 8, 14 and 20 weeks of age, the mice were fasted for 4 h prior to undergoing a hepatic triglyceride production test. Several rate-limiting enzymes and transcription factors participate in hepatic lipid metabolism were determined by RT-qPCR and western blot analysis. Findings: Hepatic de novo lipogenesis (DNL) and β-oxidation were found to be regulated by the AMPK pathway in the ApcMin/＋ mouse at 20 weeks of age. Although hepatic triglyceride production was significantly decreased at 20 weeks of age (p