Ethics Of Animal Experimentation Research Articles

Application of Fish Embryo Assay Using Zebrafish and Oryzias latipes for Toxicity Testing and Deriving Water Quality Criteria

To protect aquatic organisms in ecosystems, each country and continental union has established guidelines for deriving the water quality standards (WQS) of specific substances. These guidelines mandate the use of acute and chronic toxicity data for fish, which are high-trophic-level organisms. However, due to increasing concerns about animal welfare and experimental ethics, there is a growing need for alternative methods to determine substance toxicity in fish. Fish toxicity tests using early life stages, such as embryos or larvae, have been utilized as alternative methods for adult fish toxicity assays. This review of the WQS guidelines and relevant test protocols confirmed the classification of acute and chronic toxicity in fish assays using different developmental stages. Fish toxicity data derived from exposure periods longer than one week using embryonic- or larval-stage organisms can be considered as indicative of chronic toxicity. There is a high correlation between fish embryo toxicity and adult effects, suggesting that fish embryo toxicity tests with appropriate exposure durations could replace adult fish toxicity tests, addressing experimental animal ethics concerns.

Hesperidin activates the GLP-1R/cAMP-CREB/IRS2/PDX1 pathway to promote transdifferentiation of islet α cells into β cells Across the spectrum

Background and aimsIn all age, FoShou as a Chinese medicinal herb has been active in various kinds of Traditional Chinese medicine formula to treating diabetes. Hesperidin (HES), the main monomeric component of FoShou, has been extensively investigated for interventions with pathogenic mechanism of diabetes as well as subsequent treatment of associated complications. Islet β-cells have an essential effect on dynamically regulating blood sugar. Functional abnormalities in these cells and their death are strongly associated with the onset of diabetes. Therefore, induction of islet endocrine cell lineage re-editing for damaged βcell replenishment would be a promising therapeutic tool. Previously, it has been found that HES can protect islet β-cells in vivo, But, the regenerative function of HES in islet β cells and its role in promoting differential non-β cells transdifferentiation into β cells and cell fate rewriting associated mechanisms remain unclear.This work focused on investigating whether HES can induce islet α cells transdifferentiation into β cells for achieving damaged β cell regeneration and the causes and possible mechanisms involved in the process. Materials and methodsIn brief, 60 mg/kg/d streptozotocin (STZ) was administered intraperitoneally in each male C57bL/6J mouse raised by the high-sugar and high-fat diet (HFD) to create a diabetic mouse model with severe β-cell damage. After 28 consecutive days of HES treatment (160 mg/kg; 320 mg/kg; once daily, as appropriate). Tracing the dynamics of α as well as β cell transformation, together with β cells growth and apoptosis levels during treatment by cell lineage tracing. The self-enforcing transcriptional network on which the cell lineage is based is used as a clue to explore the underlying mechanisms. Guangdong Pharmaceutical University's Animal Experiment Ethics Committee (GDPulac2019180) approved all animal experiments. ResultsLocalization by cell lineage we find that transdifferentiated newborn β-cells derived from α cells appeared in the islet endocrine cell mass of DM mice under HES'action. Compared to the model group, expressed by Tunel staining and CXCL10 levels the overall apoptosis rate of β-cells of the pancreas were reduced,the inflammatory infiltration feedback from HE staining were lower.Ki-67 positive cells showed enhanced β-cell proliferation. Decreased HbA1c and blood glucose contents, elevated C-Peptide and insulin contents which respond to ability of nascent beta cells. Also upregulated the mRNA levels of MafA, Ngn3, PDX-1, Pax4 and Arx. Moreover, increased the expression of TGR5/cAMP-CREB/GLP-1 in mouse intestinal tissues and GLP-1/GLP-1R and cAMP-CREB/IRS2/PDX-1 in pancreatic tissues. ConclusionsHES directly affects β-cells, apart from being anti-apoptotic and reducing inflammatory infiltration. HES promotes GLP-1 release by intestinal L cells by activating the TGR5 receptor in DM mouse and regulating its response element CREB signaling. GLP-1 then uses the GLP-1/GLP-1R system to act on IRS2, IRS2 as a port to influence α precursor cells to express PDX-1, with the mobilization of Pax4 strong expression than Arx so that α cell lineage is finally reversed for achieving β cell endogenous proliferation.

Need for Ethical Governance on the Implementation and Use of Patient-derived Xenograft (PDX) Models for Anti-cancer Drug Discovery and Development: Ethical Considerations and Policy Proposal.

Patient-derived xenograft (PDX) models, in which tumor tissues resected from cancer patients are transplanted into immunocompromised mice, have been recently considered the most reliable preclinical models that quite accurately stimulate the real-world characteristics and microenvironments of tumors in patients. The ethical uniqueness of the PDX model, which lies in the fact that it is a hybrid of living human tumor tissue and a carrier mouse, raises several ethical concerns. This study presents four ethical points for consideration and a model ethical governance policy for the implementation and use of PDX models for research. First, PDX models carrying living tumor tissues originating from individual patients with dignity must be treated ethically as materials and data in compliance with the principle of respect for persons. Second, although PDX models themselves are patentable and can be commercialized, it is a standard view, as represented by the Oviedo Convention by the Council of Europe, that those living tumor tissues carried by PDX models shall not give rise to financial gain since those tissues are human body parts; therefore, they should be treated according to a recent ethical approach with the custodianship model as the trust property of patients of origin and shall not be subjected directly to monetary transactions. Third, PDX models must be treated with due care in an ethical manner in line with experimental animal ethics. Finally, the implementation and use of PDX models for research purposes must comply with national and international regulations on both animal experimentation and human subject research. These four points should be carefully examined and properly institutionalized as an ethical governance policy in each institution that plans on utilizing or implementing PDX models for research.

Experimenting with Humans and Animals: from Aristotle to Crispr, second edition

Experimenting with Humans and Animals: from Aristotle to Crispr, second edition

Inovo chorioallantoic membrane assay as a xenograft model for pediatric rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common highly malignant pediatric soft tissue sarcoma. While recent multidisciplinary treatments have improved the 5‑year survival rate of low/intermediate‑risk patients to 70‑90%, there are various complications that arise due to treatment‑related toxicities. Immunodeficient mice‑derived xenograft models have been widely used in cancer drug research; however, these models have some limitations, including i)they are time‑consuming and expensive, ii)their use needs to be approved by animal experimental ethics committees, and iii)the inability to visualize where tumor cells or tissues were engrafted. The present study performed a chorioallantoic membrane (CAM) assay in fertilized chicken eggs, which is time‑saving, simple, and easy to standardize and handle because of the high vascularization and the immature immune system of the fertilized eggs. The present study aimed to examine the usability of the CAM assay as a novel therapeutic model for the development of precision medicine for pediatric cancer. A protocol was developed for constructing cell line‑derived xenograft (CDX) models using a CAM assay by transplanting RMS cells on the CAM. It was then examined as to whether these CDX models could be used as therapeutic drug evaluation models using vincristine (VCR) and human RMS cell lines. After grafting and culturing the RMS cell suspension on the CAM, three‑dimensional proliferation over time was observed visually and by comparing volumes. VCR reduced the size of the RMS tumor on the CAM in a dose‑dependent manner. Currently, treatment strategies based on patient‑specific oncogenic backgrounds have not been adequately developed in the field of pediatric cancer. The establishment of a CDX model with the CAM assay may lead to the advancement of precision medicine and help formulate novel therapeutic strategies for intractable pediatric cancer.

Single-cell transcriptome analysis of the mouse and primate ovaries reveals oocyte-specific expression patterns of risk genes in ovarian aging.

Single-cell transcriptome analysis of the mouse and primate ovaries reveals oocyte-specific expression patterns of risk genes in ovarian aging.

Considerations for experimental animal ethics in the research planning and evaluation process

Research using experimental animals has substantially contributed to advances in science and medicine. Animal experiments are nearly essential for biomedical research and development efforts. Because many animals are sacrificed, researchers should consider the welfare of experimental animals and related ethical issues, along with the successful results of their experiments. This review introduces the criteria that should be considered in terms of experimental animal ethics, based on the principles of the 3 R’s: replacement, representing careful consideration of the need for animal experiments; reduction, representing the use of the minimal number of animals to obtain meaningful experimental results; and refinement, representing continuous effects to find alternative methods to reduce pain and distress in experimental animals. Based on these principles, the following points should be considered when planning experiments: the necessity of animal experiments; alternatives to animal experiments; the relevance of the species and numbers of experimental animals; appropriate assessment and management of pain; the proper usage of sedatives, painkillers, and anesthesia; and valid timing for humane endpoints and euthanasia. These criteria are beneficial for both experimental animals and researchers because careful handling to ensure experimental animal welfare guarantees that scientific research will yield convincing, repeatable, and accurate results.

Development of a Point-of-Care Test Based on Selenium Nanoparticles for Heart-Type Fatty Acid-Binding Proteins in Human Plasma and Blood.

PurposeA rapid, convenient, cost-effective in-home test method for identifying heart-type fatty acid-binding protein (H-FABP) in plasma and blood by a lateral-flow immunoassay (LFIA) based on selenium nanoparticles (SeNPs) was developed.MethodsSeNPs were synthesized by using L-ascorbic acid to reduce seleninic acid at room temperature and conjugated with an anti-H-FABP monoclonal antibody. The limit of detection, specificity, and stability were measured, and clinical samples were analyzed.ResultsThe SeNPs were spherical with a diameter of 39.48 ± 3.72 nm and were conjugated successfully with an anti-H-FABP antibody, resulting in a total diameter of 46.52 ± 2.95 nm. The kit was designed for the determination of H-FABP in plasma specimens and whole blood specimens. The limit of detection was 1 ng/mL in plasma and blood, and the results could be determined within 10 min. No cross-reaction occurred with cardiac troponin I, creatine kinase-MB or myoglobin. The kits were stored at 40 °C for up to 30 days without significant loss of activity. The sensitivity was determined to be 100%, the specificity 96.67%, and the overall coincidence rate 97.83%.ConclusionThis SeNP assay kit can conveniently, rapidly, and sensitively detect H-FABP in plasma or blood with a readout of a simple color change visible to the naked eye with no special device, and can be used as an auxiliary means for the early screening of AMI.Clinical Trial RegistrationPlasma and blood samples were used under approval from the Experimental Animal Ethics committee of the Joint National Laboratory for Antibody Drug Engineering, Henan University. The clinical trial registration number was HUSOM-2019-047.

Optogenetics stimulates nerve reorganization in the contralesional anterolateral primary motor cortex in a mouse model of ischemic stroke.

The anterolateral motor cortex of rodents is an important motor auxiliary area, and its function is similar to that of the premotor area in humans. Activation and inhibition of the contralesional anterolateral motor cortex (cALM) have been shown to have direct effects on motor behavior. However, the significance of cALM activation and inhibition in the treatment of stroke remains unclear. This study investigated the role of optogenetic cALM stimulation in a mouse model of cerebral stroke. The results showed that 21-day optogenetic cALM inhibition, but not activation, improved neurological function. In addition, optogenetic cALM stimulation substantially altered dendritic structural reorganization and dendritic spine plasticity, as optogenetic cALM inhibition resulted in increased dendritic length, number of dendritic spines, and number of perforated synapses, whereas optogenetic activation led to an increase in the number of multiple synapse boutons and the number of dendritic intersections. Furthermore, RNA-seq analysis showed that multiple biological processes regulated by the cALM were upregulated immediately after optogenetic cALM inhibition, and that several immediate-early genes (including cFOS, Erg1, and Sema3f) were expressed at higher levels after optogenetic inhibition than after optogenetic activation. These results were confirmed by quantitative reverse transcription-polymerase chain reaction. Finally, immunofluorescence analysis showed that the c-FOS signal in layer V of the primary motor cortex in the ischemic hemisphere was higher after optogenetic cALM activation than it was after optogenetic cALM inhibition. Taken together, these findings suggest that optogenetic cALM stimulation promotes neural reorganization in the primary motor cortex of the ischemic hemisphere, and that optogenetic cALM inhibition and activation have different effects on neural plasticity. The study was approved by the Experimental Animal Ethics Committee of Fudan University (approval No. 201802173S) on March 3, 2018.

Neuroprotective effects of long noncoding RNAs involved in ischemic postconditioning after ischemic stroke.

During acute reperfusion, the expression profiles of long noncoding RNAs in adult rats with focal cerebral ischemia undergo broad changes. However, whether long noncoding RNAs are involved in neuroprotective effects following focal ischemic stroke in rats remains unclear. In this study, RNA isolation and library preparation was performed for long noncoding RNA sequencing, followed by determining the coding potential of identified long noncoding RNAs and target gene prediction. Differential expression analysis, long noncoding RNA functional enrichment analysis, and co-expression network analysis were performed comparing ischemic rats with and without ischemic postconditioning rats. Rats were subjected to ischemic postconditioning via the brief and repeated occlusion of the middle cerebral artery or femoral artery. Quantitative real-time reverse transcription-polymerase chain reaction was used to detect the expression levels of differentially expressed long noncoding RNAs after ischemic postconditioning in a rat model of ischemic stroke. The results showed that ischemic postconditioning greatly affected the expression profile of long noncoding RNAs and mRNAs in the brains of rats that underwent ischemic stroke. The predicted target genes of some of the identified long noncoding RNAs (cis targets) were related to the cellular response to ischemia and stress, cytokine signal transduction, inflammation, and apoptosis signal transduction pathways. In addition, 15 significantly differentially expressed long noncoding RNAs were identified in the brains of rats subjected to ischemic postconditioning. Nine candidate long noncoding RNAs that may be related to ischemic postconditioning were identified by a long noncoding RNA expression profile and long noncoding RNA-mRNA co-expression network analysis. Expression levels were verified by quantitative real-time reverse transcription-polymerase chain reaction. These results suggested that the identified long noncoding RNAs may be involved in the neuroprotective effects associated with ischemic postconditioning following ischemic stroke. The experimental animal procedures were approved by the Animal Experiment Ethics Committee of Kunming Medical University (approval No. KMMU2018018) in January 2018.

Inactivation of NF-κB Signaling Facilitates the Occurrence of Premature Ovarian Failure via Accelerating Granulosa Cell Death

Background: Premature ovarian failure (POF) is defined as deployment of amenorrhea because of the cessation of ovarian function prior to woman’s age of 40 years. The POF pathological mechanism has not yet well understood although genetic aberrations, autoimmune damage, and environmental factors have been identified. Methods: In this study, we assume that NF-κB inactivation is closely associated with the development of POF based on the data from previous literatures and CTX-induced POF mouse model. By establishing a NF-κB inactivated mouse model, using histomorphology and molecular biology methods to analyze. Findings: In the successfully established NF-κB-inactivated mouse model, we found the reduced expression of nuclear P65 and the increased expression of IκBα in ovarian granulosa cells; the reduced numbers of antral follicles; the reduction of Ki67/PCNA-labeled cell proliferation and enhanced Fas/FasL-dependent apoptosis in granulosa cells; the increase of phosphorylated H2AX-labelled DNA damage; the elevated level of FSH and declined E2 and AMH; the decreased expressions of FSHR and Cyp19a1 in granulosa cells, which was reversed in the context of blocking NF-κB signaling with Bay 11-7082. Dual-luciferase reporter assay demonstrated that p50 could stimulate the transcription of GRP78. Interpretation: Lack of NF-κB signaling normal response (i.e., inactivation of NF-κB signaling) would increase the incidence of granulosa cell death, which further causes an accelerated antral follicular atresia. Another pathway to initiate the death of granulosa cells might result from the imbalance of ER stress caused by GRP78. Funding Information: This study was supported by NSFC grants (31771331, 31971108) Declaration of Interests: The authors declare that there are no conflict of interests. Ethics Approval Statement: This study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Jinan University.

Exploration of the mechanism by which icariin modulates hippocampal neurogenesis in a rat model of depression

Icariin (ICA) has a significant capacity to protect against depression and hippocampal injury, but it cannot effectively cross the blood-brain barrier and accumulate in the brain. Therefore, the mechanism by which ICA protects against hippocampal injury in depression remains unclear. In this study, we performed proteomics analysis of cerebrospinal fluid to investigate the mechanism by which ICA prevents dysfunctional hippocampal neurogenesis in depression. A rat model of depression was established through exposure to chronic unpredictable mild stress for 6 weeks, after which 120 mg/kg ICA was administered subcutaneously every day. The results showed that ICA alleviated depressive symptoms, learning and memory dysfunction, dysfunctional neurogenesis, and neuronal loss in the dentate gyrus of rats with depression. Neural stem cells from rat embryonic hippocampi were cultured in media containing 20% cerebrospinal fluid from each group of rats and then treated with 100 μM corticosterone. The addition of cerebrospinal fluid from rats treated with ICA largely prevented the corticosterone-mediated inhibition of neuronal proliferation and differentiation. Fifty-two differentially expressed proteins regulated by chronic unpredictable mild stress and ICA were identified through proteomics analysis of cerebrospinal fluid. These proteins were mainly involved in the ribosome, PI3K-Akt signaling, and interleukin-17 signaling pathways. Parallel reaction monitoring mass spectrometry showed that Rps4x, Rps12, Rps14, Rps19, Hsp90b1, and Hsp90aa1 were up-regulated by chronic unpredictable mild stress and down-regulated by ICA. In contrast, HtrA1 was down-regulated by chronic unpredictable mild stress and up-regulated by ICA. These findings suggest that ICA can prevent depression and dysfunctional hippocampal neurogenesis through regulating the expression of certain proteins found in the cerebrospinal fluid. The study was approved by the Experimental Animal Ethics Committee of Guangzhou University of Chinese Medicine of China in March 2017.

Testicle histomorphometry in senescence/senility under use of homeopathy

Aging is an extremely complex and multifactorial process. Over time, the elderly begin exhibiting signs of function reduction. The aim of the present study was to assess the effects of homeopathic medicines on testicle histomorphometry in senescence/senility. The study was approved by the animal experimentation ethics committee of Santo Amaro University (07/2015). The rats (Wistar, all male, with 22-23 months of age were used) were caged with access to food and water ad libitum and 12-hour light cycle (7:00 am to 7:00 pm). The homeopathic medicines were prepared following the Brazilian Homeopathic Pharmacopoeia. The animals were randomly distributed in 4 groups (n= 6): control; hydroalcoholic solution; Calcarea carbonica 30cH (Calc) and Baryta muriatica 30cH (Bar-m). 5 globules of the medicines were added to each water dispenser; the containers were changed twice per week along 40 days. All investigators were blinded to the allocation groups. After 40 days all the animals were euthanized and the testicles collected. Based on their width and length, the testicles’ volume was calculated by means of the ellipsoid formula. Slides were prepared and stained with PAS. Photographs were taken of the slides with 40x magnification under microscope; software Image J was used to measure the diameter of 50 seminiferous tubules per slide (animal), diameter of the parenchyma and lumen of each tubule. Statistical analysis was performed by means of ANOVA followed by Tukey’s test; the significance level was set to p≤ 0.05. Increase of the testicles’ volume, increased parenchymal diameter and decrease of the tubular lumen was found in groups Calc and Bar-m compared to controls and the group treated with hydroalcoholic solution (p ≤ 0.05). We conclude that the tested homeopathic medicines changed the testicle histomorphometry of animals in senescence/senility. 
 
 Keywords: Testicular histomorphometry, homeopathy, Calcarea carbonica, Baryta muriatica, Senescence/Senility

America’s Public Philosopher: Essays on Social Justice, Economics, Education, and the Future of Democracy by John Dewey

Reviewed by: America’s Public Philosopher: Essays on Social Justice, Economics, Education, and the Future of Democracy by John Dewey John R. Shook DEWEY, John. America’s Public Philosopher: Essays on Social Justice, Economics, Education, and the Future of Democracy. Edited by Eric Thomas Weber. New York: Columbia University Press, 2021. xii + 335 pp. Cloth, $95.00; paper, $28.00 John Dewey conducted his philosophy in public as a public philosopher, concerned with public questions that crucially mattered in America. How may integration ever happen? When shall women ever vote? Will religion control schooling, or not? Shall teachers be professionals, or not? Will labor gain protections, or not? Shall [End Page 622] science lead policy, or not? Are civil liberties truly for everyone, or not? Can technocratic management be trusted, or not? Is capitalism more democratic, or is socialism? Shall America be an empire, or a peacemaker? If answers seem obvious now, or at least debate-worthy, that is because you live in Dewey’s America. That progressivism was pragmatism, not just “publicity.” Dewey was constantly in the public eye and on the public’s mind during several of its most transformative decades because his philosophy was of, by, and for the American people. Dewey was always on the march, often literally as well as in literary form. There was hardly any agenda of the liberal, civil rights, and progressive movements from the 1890s through the 1940s that proceeded without his name as a cofounder, signatory, delegate, advisor, or advocate. If a single affordable volume could capture the ethical, educational, social, economic, legal, and political philosophy of such a wide-ranging epochal thinker, this must be it. Eric Thomas Weber’s fine general introduction and concise prefaces to each selection (forty-six in total) keeps up with that pace. This volume’s writings are sometimes more academic than popularly written, but there was not a big difference when Dewey offered his explanations of social problems, dissected rival theories, and proposed practical solutions. We are to be reminded of his prolificity; it is hardly an exaggeration to say that no matter the topic, a hundred or more items stand alongside any single piece represented here, filling the bulk of the thirty-seven volumes of his Collected Works. His open letters, talks, testimonies, speeches, and addresses to innumerable meetings, councils, leagues, committees, and associations were broadcast and printed everywhere, from local radio and city newspapers to national and international magazines and journals, along with pamphlets and tracts printed in uncountable numbers. Dewey did not have to compete for public attention; his philosophy attended to America’s struggles. Part 1, on democracy and the United States, brings together “Democracy Is Radical,” “Address to National Negro Conference,” “A Symposium on Woman’s Suffrage,” “Pragmatic America,” and “The Basic Values and Loyalties of Democracy,” along with three more pieces on democracy’s duty and destiny. Part 2, on politics and power, has eight essays, including “Politics and Culture,” “Force, Violence, and the Law,” “Why I Am Not a Communist,” and “A Liberal Speaks Out for Liberalism.” The eight essays in part 3, on education, explain the essential role of public education and professionalized teachers for democracy’s growth. Part 4, on social ethics and economic justice, has eight essays, including “Capitalistic or Public Socialism?”, “Does Human Nature Change?”, “The Ethics of Animal Experimentation,” “Ethics and International Relations,” and “The Jobless—A Job for All of Us.” Part 5, on science and society, has “The Influence of Darwinism on Philosophy,” “Science, Belief and the Public,” “Social Science and Social Control,” “Education and Birth Control,” “The Supreme Intellectual Obligation,” and “The Revolt against Science.” Part 6, on philosophy and culture, has “The Case of the Professor and the Public Interest,” “Social Absolutism,” “Some Factors in [End Page 623] Mutual National Understanding,” “The Basis for Hope,” “Art as Our Heritage,” “The Value of Historical Christianity,” and “What Humanism Means to Me.” Although his stand-alone books such as Democracy and Education and Reconstruction in Philosophy should be required reading, the essentials of Dewey’s entire philosophy are all here in their full vitality. Fame for this Columbia University professor was inescapable. A cover of Time magazine came in...

Three‐Month Graft Survival of Kidneys Retrieved 4,5 Hours After Circulatory Death

Background: The availability of an increased proportion of organs from donors after circulatory death (DCD) could solve the organ shortage globally. We present a model of extended uncontrolled DCD (uDCD), allowing sufficient time for the consent procedure in most countries. A novel reconditioning technique allows successful transplantation after prolonged warm ischemia of kidneys from uDCD donors. Methods: Two hours after circulatory death, ice slush was inserted into the abdomen of pigs. Donor organs were then retrieved 4.5 hour after death, and treated on the back-table by injecting Lys-plasminogen, antithrombin-III (AT-III), and alteplase (tPA) through the renal arteries. The kidneys were then perfused with an albumin-rich solution at 15 °C and 20 mmHg for 3 hours. Washed erythrocytes were added to the solution after increasing temperature to 32 °C and pressure to 30 mmHg, continuing the perfusion for another three hours. Argatroban and abciximab were added to inhibit re-thromboses. Reconditioning (study group) kidneys were transplanted into pigs using a novel auto-transplant technique with a single functioning autologous kidney and observed for three months. Sham-treated pigs underwent contra-lateral transplantation of the left kidney after removing the right kidney under the same anesthesia conditions and trauma as study group pigs. Findings: During perfusion, vascular resistance decreased to 100ml/100g/min, significantly higher (p < 0.00018) than kidneys not receiving thrombotlytic treatment. At 3 months post-transplant, 4/6 study and 4/6 control pigs survived. Serum creatinine and renal histology did not differ between the groups. Interpretation: We present a novel method to salvage kidneys from extended uDCD, enabling subsequent transplantation after ex-vivo reconditioning, in a clinically acceptable manner. Funding Information: Supported by grants from IngaBritt and Arne Lundbergs Research Foundation; LUA ALF Grant; The Gelin Foundation and Hans-Gabriel and Alice Trolle Wachtmeister Foundation for Medical Research. Declaration of Interests: MO is the inventor of several patents. None of the other authors reported any potential conflicts of interest Ethics Approval Statement: The Regional (Malmo/Lund) Animal Experiment Ethics Committee approved the study (Dnr 5.8.18‐13977/2018; Dnr 5.8.18‐09474/2019; Dnr 5.8.18‐09182/2020). All animals were housed and cared for in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Resources and published by the National Institute of Health (NIH, Publication No 86‐23, revised 1996).

Rapid Detection of Ractopamine and Salbutamol in Swine Urine by Immunochromatography Based on Selenium Nanoparticles.

PurposeThe purpose of this study was to establish a lateral flow immunoassay using selenium nanoparticles (Se-NPs) as a probe to detect ractopamine (RAC) and salbutamol (SAL) in swine urine.MethodsSDS and PEG were used as templates to prepare Se-NPs; anti-RAC monoclonal antibodies or anti-SAL monoclonal antibodies were labelled with Se-NPs; and rapid detection kits were prepared. The sensitivity, specificity, and stability were measured, and actual samples were analysed.ResultsThe Se-NPs were spherical with a diameter of 40.63 ± 5.91 nm, and were conjugated successfully with an anti-RAC antibody to give a total diameter of 82.33 ± 17.91 nm. The detection limit of a RAC kit in swine urine was 1 ng/mL, and that of a SAL kit was 3 ng/mL. Both procedures could be completed within 5 minutes. No cross-reaction occurred with clenbuterol, bambuterol and phenylethanolamine A. Samples were tested consistently across different batches of kits for swine urine. The results of the kits were identical to those of actual clinical samples analysed by ELISA, and the coincidence rate was 100%.ConclusionThe assay kit does not require any special device for reading the results, and the readout is a simple colour change that can be evaluated with the naked eye. It is easy to operate, sensitive, specific, and stable This kit is suitable for the rapid and real-time detection of RAC and SAL residues in swine urine samples.Clinical Trial RegistrationSwine urines samples were used under approval from the Experimental Animal Ethics committee of the Joint National Laboratory for Antibody Drug Engineering, Henan University.

Neutralizing Antibody-Independent SARS-CoV-2 Control Correlated with Intranasal Vaccine-Induced CD8 &lt;sup&gt;+&lt;/sup&gt; T-Cell Responses

Effective vaccines are essential for the control of the COVID-19 pandemic. Currently-developed vaccines inducing SARS-CoV-2 spike (S) antigen-specific neutralizing antibodies (NAbs) are effective, but the appearance of NAb-resistant S variant viruses is of great concern. A vaccine inducing S-independent or NAb-independent SARS-CoV-2 control may contribute to containment of these variants. Here, we investigated the efficacy of an intranasal vaccine expressing viral non-S antigens against intranasal SARS-CoV-2 challenge in cynomolgus macaques. Nine vaccinated macaques exhibited significantly reduced viral load in nasopharyngeal swabs on day 2 post-challenge compared to nine unvaccinated controls. The viral control in the absence of SARS-CoV-2-specific NAbs was significantly correlated with vaccine-induced viral antigen-specific CD8+ T-cell responses. Our results indicate that CD8+ T-cell induction by intranasal vaccination can result in NAb-independent control of SARS-CoV-2 infection, highlighting a potential of vaccine-induced CD8+ T-cell responses to contribute to COVID-19 containment.Funding: This work was supported by Japan Agency for Medical Research and Development (AMED [JP19fk0108104 to A.K.-T. and JP20nk0101601, JP20jm0110012, JP21fk0410035, JP21fk0108125, and JP21jk0210002 to T.M.]) and the Ministry of Education, Culture, Sports, Science and Technology (MEXT) in Japan (JSPS Grants-in-Aid for Scientific Research [21H02745 to T.M.]).Declaration of Interests: H.I., K.K., R.S., and T.M are the inventors on Patent Cooperation Treaty (PCT) application for SeV-NME vaccine. Authors have no other conflicts of interest to declare.Ethics Approval Statement: Approval by the Committee on the Ethics of Animal Experiments in NIID (permission number: 520001) under the guidelines for animal experiments in accordance with the Guidelines for Proper Conduct of Animal Experiments established by the Science Council of Japan.

Theophylline Extracted from &lt;i&gt;Fu Brick&lt;/i&gt; Tea Affects the Metabolism&amp;nbsp;Of&amp;nbsp;Preadipocytes&amp;nbsp;And Body Fat in Mice as a Pancreatic&amp;nbsp;Lipase Inhibitor

The dramatic increase in obesity has put people under increasing pressure. Lipase inhibitors, have received increasing attention from researchers because of their advantages of acting in the intestinal tract and having no side effects on the central nervous system. In this paper, the molecular dynamics, cellular level and zoological model of lipase inhibitors for anti-obesity drug screening and inhibition mechanisms were analysed and discussed. FBT was identified as a mixed reversible inhibitor with an IC 50 of 1.02 ± 0.03 μg/mL. In vitro experiments showed that FBT reduce ROS production and mRNA expression of related inflammatory factors during cell differentiation. In animal experiments, we confirmed that FBT can effectively lessen the level of fatty liver, prevent liver fibrosis and the accumulation of lipid droplets, improve mitochondrial membrane structure damage. The inhibitory mechanism of FBT was discussed to provide a theoretical basis for the screening and clinical treatment of lipase inhibitors. Funding: Supported by Xiamen Municipal Bureau of Science and Technology (3502Z20193006)； Postdoctoral Science Foundation of China (2021M691283). Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: The animal experiment was conducted in accordance with the “Laboratory Animal Guideline for Ethical Review of Animal Welfare” (GB/T 35892–1272018) and the Experimental Animal Management and Ethics Committee of Xiamen University (Certificate no. SYXK2013–0006).

Biocompatibility and biosafety of butterfly wings for the clinical use of tissue-engineered nerve grafts.

In a previous study, we used natural butterfly wings as a cell growth matrix for tissue engineering materials and found that the surface of different butterfly wings had different ultramicrostructures, which can affect the qualitative growth of cells and regulate cell growth, metabolism, and gene expression. However, the biocompatibility and biosafety of butterfly wings must be studied. In this study, we found that Sprague-Dawley rat dorsal root ganglion neurons could grow along the structural stripes of butterfly wings, and Schwann cells could normally attach to and proliferate on different species of butterfly wings. The biocompatibility and biosafety of butterfly wings were further examined through subcutaneous implantation in Sprague-Dawley rats, intraperitoneal injection in Institute of Cancer Research mice, intradermal injection in rabbits, and external application to guinea pigs. Our results showed that butterfly wings did not induce toxicity, and all examined animals exhibited normal behaviors and no symptoms, such as erythema or edema. These findings suggested that butterfly wings possess excellent biocompatibility and biosafety and can be used as a type of tissue engineering material. This study was approved by the Experimental Animal Ethics Committee of Jiangsu Province of China (approval No. 20190303-18) on March 3, 2019.

Transcriptomic analysis reveals essential microRNAs after peripheral nerve injury.

Studies have shown that microRNAs (miRNAs) mediate posttranscriptional regulation of target genes and participate in various physiological and pathological processes, including peripheral nerve injury. However, it is hard to select key miRNAs with essential biological functions among a large number of differentially expressed miRNAs. Previously, we collected injured sciatic nerve stumps at multiple time points after nerve crush injury, examined gene changes at different stages (acute, sub-acute, and post-acute), and obtained mRNA expression profiles. Here, we jointly analyzed mRNAs and miRNAs, and investigated upstream miRNAs of differentially expressed mRNAs using Ingenuity Pathway Analysis bioinformatic software. A total of 31, 42, 30, and 23 upstream miRNAs were identified at 1, 4, 7, and 14 days after rat sciatic nerve injury, respectively. Temporal expression patterns and biological involvement of commonly involved upstream miRNAs (miR-21, let-7, miR-223, miR-10b, miR-132, miR-15b, miR-127, miR-29a, miR-29b, and miR-9) were then determined at multiple time points. Expression levels of miR-21, miR-132, miR-29a, and miR-29b were robustly increased after sciatic nerve injury. Biological processes involving these miRNAs include multicellular organismal response to stress, positive regulation of the epidermal growth factor receptor signaling pathway, negative regulation of epithelial cell differentiation, and regulation of myocardial tissue growth. Moreover, we constructed mechanistic networks of let-7, miR-21, and miR-223, the most significantly involved upstream miRNAs. Our findings reveal that multiple upstream miRNAs (i.e., let-7, miR-21, and miR-223) were associated with gene expression changes in rat sciatic nerve stumps after nerve injury, and these miRNAs play an important role in peripheral nerve regeneration. This study was approved by the Experimental Animal Ethics Committee of Jiangsu Province of China (approval No. 20190303-18) on March 3, 2019.