Animal Care Guidelines Research Articles

Effect of oncolytic herpes simplex virus on fusion-positive rhabdomyosarcoma to radiotherapy.

10063 Background: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Despite multimodal interventions including surgery, chemotherapy, and radiotherapy, nearly a third of patients with localized disease will experience tumor recurrence. Patients with tumors harboring an oncogenic FOXO1 fusion are at greater risk of relapse and are most in need of novel therapies. Oncolytic viruses are being explored as treatments for pediatric tumors, and data suggest that oncolytic viruses may enhance responses to chemoradiotherapy. M002 is a genetically engineered oncolytic herpes simplex virus (oHSV) that exhibits aneurovirulence in normal cells. M002 selectively replicates in malignant cells, eliminating these cells through cellular burst during the lytic cycle. In the present study, we explored the efficacy of the M002 virus alone and in combination with low-dose ionizing radiotherapy in a human patient-derived xenograft (PDX) model of fusion-positive RMS (FP-RMS). Methods: A human FP-RMS PDX was established and maintained by injection into the quadriceps femoris muscle of athymic nude mice under Institutional Animal Care and Use Committee guidelines (IACUC-09363). Tumors were dissociated and cells were treated with increasing multiplicity of infection (MOI) of either the oHSV, R3659 or its genetically engineered variant, M002. A colorimetric assay evaluated the cytotoxicity of oHSV treatments on the RMS PDX cells after 24, 48, and 72 hours of infection. In separate experiments, cells treated with vehicle (control) or infected with M002 were exposed to irradiation (2 Gy), and viability was determined at 24, 48, 72, and 96 hours. Results: Treatment with M002 or its parent construct, R3659, significantly reduced RMS PDX cell viability in a dose-dependent manner at all time points. M002 showed slightly higher cytotoxicity than R3659 as measured by lethal dose 50% (LD50, 37±1.25 vs 44±0.95 PFU/cell, M002 vs R3659, at 24 hours, p=0.02). When sub-lethal doses of M002 were combined with low-dose irradiation, RMS PDX cell viability was significantly decreased compared to cells treated with either M002 or irradiation alone (26% decrease at 10 PFU/cell). Conclusions: Our preliminary data highlight that M002 exhibits cytotoxicity in a PDX model of FP-RMS. Treatment with M002 further increased the cytotoxicity of ionizing radiation compared to individual treatments, suggesting that oHSV sensitized the cells to radiotherapy. Future studies in vivo may provide more pre-clinical evidence for the radio-sensitizing and therapeutic potential of M002 in FP-RMS.

Shenqi Pill Mitigates Renal Interstitial Fibrosis ThroughWeakening Notch1/Jag1 Pathway.

Shenqi pill (SQP) can be used to treat various kidney related diseases, but its exact mechanism of action remains unclear. We intended to analyze the role and mechanism of SQP on renal interstitial fibrosis (RIF). After performing unilateral ureteral obstruction (UUO) surgery following the Institutional Animal Care and Use Committee guidelines, all rats were assigned into the sham group, UUO group, UUO + SQP 1.5 g/kg, UUO + SQP 3 g/kg, and UUO + SQP 6 g/kg groups. After treatment with SQP for 4 weeks, the appearance of kidney, serum creatinine (SCr), and blood urea nitrogen (BUN) levels were monitored in each group. The pathological injury, extracellular matrix (ECM), and Notch1 pathway-related protein levels were measured using H&E staining, Masson staining, immunohistochemistry, and Western blot, respectively. SQP could obviously ameliorate the appearance of the kidney as well as the levels of SCr and BUN in UUO rats (SCr: 67.6 ± 4.64 μM, 59.66 ± 4.96 μM, 48.76 ± 4.44 μM, 40.43 ± 3.02 μM for UUO, low, medium, and high SQP treatment groups; BUN: 9.09 ± 0.97 mM, 7.72 ± 0.61 mM, 5.42 ± 0.42 mM, 4.24 ± 0.34 mM for UUO, low, medium, and high SQP treatment groups; P < .05). SQP also effectively mitigated renal tissue injury in UUO rats (P < .05). Moreover, we uncovered that SQP significantly inhibited Collagen I, α-SMA, Collagen IV, TGF-B1, Notch1, and Jag1 protein expressions in UUO rats kidney (P < .05). Our data elucidated that SQP can alleviate RIF, and the mechanism may be related to the Notch1/Jag1 pathway. DOI: 10.52547/ijkd.7703.

Stakeholder Perceptions of Animal Welfare as a Component of Sustainable Beef Programs in the United States-A Pilot Study.

This study aimed to investigate how stakeholders in the United States beef industry incorporate animal welfare into their sustainability programs. A survey was administered online to the U.S. Roundtable for Sustainable Beef membership. Twenty-seven surveys were analyzed. Most respondents (n = 26, 96%) had sustainability programs that incorporated animal welfare. Most respondents believed that welfare positively impacted environmental (n = 25/26, 96%), economic (n = 25/26, 96%), and social (n = 26/26, 100%) sustainability. The thematic analysis of five free response questions identified ten themes: Animal Care, Regulations and Guidelines, Responsibility, Consumers and Stakeholders, Performance and Efficiency, Financial Impact, Connectedness, Critical Component, Animal-based Outcomes, and Employees. When asked to define welfare, the most common themes were Animal Care and Regulations and Guidelines. When asked why welfare was a component of their sustainability program, the top factors from a provided list were: cattle health (n = 20, 74%), cattle performance (n = 12, 44%), and consumer perceptions (n = 12, 44%). Findings suggest a widespread recognition of animal welfare's importance within sustainable beef production.

Veterinary management protocol for non-human primates: quarantine, anesthesia, and postoperative care for mastoidectomy at animal research institutions

Non-human primate (NHP) research faces challenges due to zoonosis risk and complex veterinary management yet lacks standardized guidelines for animal care. Therefore, we developed an advanced veterinary management protocol for NHP quarantine, anesthesia, and postoperative care. Three female 4 to 5-year-old cynomolgus monkeys were anesthetized and underwent various tests, including body weight, temperature, blood tests, urinalysis, microbiological monitoring, and physical and dental examinations. Ivermectin and medicated baths were administered to eradicate ectoparasites and endoparasites, and testing was repeated 30 days later. Following quarantine, we performed computed tomography and anesthesia maintenance for mastoidectomy. To relieve pain and maintain body weight, we administered tramadol intramuscularly 4 times/day for 3 days and meloxicam subcutaneously twice daily for 14 days. Feed replacements were provided. During the 33-day quarantine period, physical examinations revealed no abnormalities indicative of infectious diseases, and no specific clinical symptoms were observed. Through a preliminary test of anesthesia time, we selected ketamine 4 mg/kg + medetomidine 50 µg/kg for short experiments such as computed tomography, and ketamine 8 mg/kg + medetomidine 50 µg/kg for intubation. Ten days after mastoidectomy, NHPs consumed 100 kcal/kg and recovered their body weight. This study offers advanced veterinary management guideline for NHP research. Such protocols can lead to more standardized and ethical practices in NHP research, thereby enhancing the quality of studies on NHPs and the translation of findings to human health and disease.

Phytochemistry, Proximate Analysis and Acute Toxicity Study of Ethanolic Moringa Oleifera Root Bark Extract.

Moringa Oleifera is a fast growing deciduous tree with its different plant parts commonly used in herbal medicine, also with good socio-economic and industrial values. The ethanolic extract of M.Oleifera root bark is shown to be rich in nutrients, owing to the presence of essential phytochemicals in the root bark. The phytochemicals or secondary metabolites of the ethanolic extract of the root bark were screened and identified. Also, acute toxicity study of the ethanolic extract of the root bark was carriedout in Albino Rats according to Institutional Animal Care and Use (IACU) guidelines. The fresh M.Oleifera root was harvested at the inception of the rainy season at Pil Gani District of Langtang North LGA and excess soil washed off as the root bark was properly peeled, air dried, pulverized to powdered form and stored for subsequent use. Serial extractions using water, acetone, ethanol, and ethyl acetate were carriedout by Simple Maceration. The ethanolic extract was seen to have the best yield of the phytochemicals qualitatively identified as tannins, saponins, carbohydrates, flavonoids, alkaloids, steroids, terpenes, cardiac glycosides and anthraquinones. Acute toxicity test was determined by Lorke’s method and the LD50 was shown to be at the highest limit dose of 5000mg/kg, which signifies that the extract is non toxic at such single dosage. The ethanolic M.Oleifera root bark extract contains nutritive phytochemicals with vast medicinal uses. No sign of acute toxicity was observed on oral administration as the median lethal dose was shown to be safe at the highest limit single dose of 5000mg/kg.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: N6-methyladenosine (m6A) is the most abundant form of chemical modification in eukaryotes. m6A methylation is dynamically modulated by diverse types of regulators, including methyltransferases (‘writers’), RNA binding proteins (‘readers’), and demethylases (‘erasers’). Growing evidence has shown that m6A methylation plays an essential role in the development and progression of multiple cancers. However, the functions of m6A methyltransferase KIAA1429 in diffuse large B-cell lymphoma (DLBCL) remain undefined. Here, we aimed to unravel an epitranscriptomic mechanism mediated by the m6A methyltransferase KIAA1429 in DLBCL. Methods: Collecting lymph node biopsy samples from 60 de novo DLBCL patients and 20 reactive hyperplasia cases after informed consent. The biological function of KIAA1429 was evaluated using CRISPR/Cas9 mediated genomic deletion and CRISPR/dCas9-VP64 activation. RNA-seq, MeRIP-seq, RIP assays, luciferase activity assay, RNA stability experiments, and co-immunoprecipitation were performed to explore the regulatory mechanism of KIAA1429 in DLBCL. DLBCL cells were subcutaneously injected into SCID-Beige mice to establish xenograft models. The Institutional Animal Care guidelines were followed during the animal experiments. Results: m6A regulators were dysregulated in DLBCL, and a predictive model based on m6A score was developed. KIAA1429 expression was found to be elevated and associated with poor prognosis. KIAA1429 knockout inhibited DLBCL cell proliferation, induced cell cycle arrest in the G2/M phase, promoted apoptosis in vitro, and repressed tumor growth in vivo. Furthermore, KIAA1429 mediated m6A modification of CHST11 mRNA and recruited YTHDF2 to reduce the stability and expression of CHST11. By decreasing MOB1B expression, CHST11 inhibited Hippo-YAP signaling, reprogramming the expression of Hippo target genes (Figure 1). Conclusions: In summary, we identified for the first time that m6A methylation regulators were dysregulated, and its risk score could exert as an independent prognostic indicator in DLBCL. More importantly, our study identified a novel mechanism in which KIAA1429/YTHDF2-coupled epitranscriptional repression of CHST11 inactivates the Hippo-YAP pathway in DLBCL, highlighting the potential of KIAA1429 as a novel predictive biomarker and therapeutic target for DLBCL progression. Keywords: aggressive B-cell non-Hodgkin lymphoma, bioinformatics, computational and systems biology, genomics, epigenomics, and other -omics No conflicts of interests pertinent to the abstract.

Prophylactic Glatiramer Acetate Treatment Positively Attenuates Spontaneous Opticospinal Encephalomyelitis.

Background: Glatiramer acetate (GA) is a well-established treatment option for patients with clinically isolated syndrome and relapsing-remitting multiple sclerosis (MS) with few side effects. The double transgenic mouse model spontaneous opticospinal encephalomyelitis (OSE), based on recombinant myelin oligodendrocyte glycoprotein35-55 reactive T and B cells, mimicks features of chronic inflammation and degeneration in MS and related disorders. Here, we investigated the effects of prophylactic GA treatment on the clinical course, histological alterations and peripheral immune cells in OSE. Objective: To investigate the effects of prophylactic glatiramer acetate (GA) treatment in a mouse model of spontaneous opticospinal encephalomyelitis (OSE). Methods: OSE mice with a postnatal age of 21 to 28 days without signs of encephalomyelitis were treated once daily either with 150 µg GA or vehicle intraperitoneally (i. p.). The animals were scored daily regarding clinical signs and weight. The animals were sacrificed after 30 days of treatment or after having reached a score of 7.0 due to animal care guidelines. We performed immunohistochemistry of spinal cord sections and flow cytometry analysis of immune cells. Results: Preventive treatment with 150 µg GA i. p. once daily significantly reduced clinical disease progression with a mean score of 3.9 ± 1.0 compared to 6.2 ± 0.7 in control animals (p < 0.01) after 30 d in accordance with positive effects on weight (p < 0.001). The immunohistochemistry showed that general inflammation, demyelination or CD11c+ dendritic cell infiltration did not differ. There was, however, a modest reduction of the Iba1+ area (p < 0.05) and F4/80+ area upon GA treatment (p < 0.05). The immune cell composition of secondary lymphoid organs showed a trend towards an upregulation of regulatory T cells, which lacked significance. Conclusions: Preventive treatment with GA reduces disease progression in OSE in line with modest effects on microglia/macrophages. Due to the lack of established prophylactic treatment options for chronic autoimmune diseases with a high risk of disability, our study could provide valuable indications for translational medicine.

Detection of brown adipose tissue in rats with acute cold stimulation using quantitative susceptibility mapping.

Detection of brown adipose tissue in rats with acute cold stimulation using quantitative susceptibility mapping.

Feasibility of simultaneous multichannel magnetocardiographic mapping, multiple monophasic action potential recording and laser photocoagulation of isolated and perfused rabbit ventricular myocardium

Abstract Background Multichannel magnetocardiographic mapping (MCG) has been validated as an accurate non-invasive method for 3D localization of arrhythmogenic substrates suitable for ablation treatment and of intracardiac electrophysiology catheters (1,2). High resolution (HR) MCG-guided multiple simultaneous monophasic action potential (multi-MAP) recording and pacing with a single patented amagnetic catheter (AC) detects local arrhythmogenic mechanisms (3). Laser photocoagulation (LP) providing more controlled and homogeneous myocardial lesions (4), may be an alternative to radiofrequency (RF) ablation, without unwanted electromagnetic noise effect during real-time MCG. Purpose This study aimed to experimentally test the feasibility of simultaneous multi-MAP recording and LP guided by real-time MCG. Methods An unshielded 36 channels SQUID system (sensitivity of 30 fT/Hz1/2, at 1 Hz) (CMI, USA) was used to study Langendorff-perfused isolated rabbit heart with MCG (Figure 1A). Four epicardial MAPs were simultaneously recorded (inter MAP distance: 1 mm) with a single multi-MAP AC (bandpass: DC-500 Hz; sampling frequency: 2 kHz) (GE-Prucka Cardiolab). LP was performed with a NdYAG laser (Dornier Medilas 5100) and a catheter system for safe myocardium LP (5). The study was carried out according to NIH guidelines for animal care and use. Results MCG imaging of rabbit ventricular magnetic field distribution and inverse source localization with the magnetic dipole model are shown in Figure 1B. The difference in morphology of local HR ventricular multi-MAP signals was compatible with myocardial anisotropy (Figure 1C). With appropriate cooling of the optic fiber-tissue interface, no epicardial damage was induced by LP, whereas deeper well defined intramural myocardial lesions were appreciable, of different size as a function of the energy delivered (Figure 1D). Multi-MAP recording was not significantly disturbed by LP. Keeping the Medilas unit at least 5 meters away from the MCG sensors, no electromagnetic artifacts were observed during MCG. Conclusion MCG, single catheter multi-MAP recording and well-defined LP lesions of ventricular myocardium were simultaneously feasible on Langendorff-perfused isolated rabbit heart, without major electromagnetic interferences, in an unshielded catheterization laboratory. With appropriate technological developments, safe MCG-guided single-catheter laser ablation of focal arrhythmogenic substrates is foreseen as a possible minimally invasive alternative to current interventional approaches. Funding Acknowledgement Type of funding sources: None.

The Antiarrhythmic Effect of Inhibition of the Mitochondrial Calcium Uniporter under Catecholamine Overload is Partly Dependent on Mitochondrial Function Preservation

IntroductionVentricular arrhythmias are a major cause of mortality in cardiovascular patients. Catecholamines have been associated with the development of ventricular arrhythmias and no new intervention has demonstrated efficacy in reducing mortality since the use of β‐blockers. Mitochondrial calcium transport has been deemed necessary for an adequate adrenergic response and constant adrenergic stimulation leads to mitochondrial calcium overload with subsequent mitochondrial dysfunction. Inhibition of the mitochondrial calcium uniporter (MCU) have been described to reduce asynchronized contraction in cellular and animal models. In this study, we assessed the effects that MCU inhibition by administration of Ru360, a MCU inhibitor, has on ventricular arrhythmia incidence in a model of catecholamine toxicity and characterize cellular and mitochondrial function to describe the subcellular mechanisms involved.MethodsThe study followed the national guidelines for laboratory animal use and care and was approved by the Animal Care and Use Committee. 12‐15‐week‐old C57bl/6 male mice received Ru360 or normal saline via IV, and a baseline ECG was recorded. Afterwards, Isoproterenol (400mg/kg) was administrated subcutaneously, and ECG recording was kept for another 20 minutes. Finally, hearts were excised, and cardiomyocytes and mitochondria isolated for further characterization. Data is presented as mean ± SEM. Fisher exact test was performed to evaluate arrhythmia incidence and one‐way ANOVA followed by Holm‐Sidak method for mitochondrial experiments and heart rate measurements.ResultsRu360 completely prevented the development of ventricular arrhythmias at a dose of 50nmol/kg. Mitochondrial Ca2+ transport inhibition preserved mitochondrial function and membrane integrity demonstrated by a preserved respiratory control and calcium retention capacity and a partially preserved mitochondrial membrane potential. MCU inhibition also prevented mitochondrial membrane potential loss upon the addition of a Ca2+ insult. The effects shown appear to be caused by a reduced oxidative stress, shown by a higher cytochrome c oxidase activity, that helps preserve cellular Ca2+ homeostasis as seen in confocal microscopy.ConclusionRu360 prevents arrhythmias possibly by reducing mitochondrial Ca2+ overload, preserving mitochondrial function, preventing mitochondrial ROS formation and oxidative damage, and conserving cellular Ca2+ homeostasis.

Promoting the Resolution of Thromboinflammation

BackgroundThrombosis is viewed as a multi‐step, multi‐cellular process driven by inflammatory stimuli and leukocyte (e.g. neutrophil) activation, and is a major pathophysiological contributor of stroke. Accumulating data supports the hypothesis that anti‐inflammatory therapies may limit thrombosis and anti‐thrombotic therapies may reduce vascular inflammation. One such target is the anti‐inflammatory and pro‐resolving endogenous mediator Annexin A1 (AnxA1). The development of therapies that temper inflammation and enhance resolution offer potential therapeutic strategies for the treatment and management of stroke and other thromboinflammatory diseases. Here we sought to comprehensively elucidate the functional significance of targeting the AnxA1/Fpr2/ALX pathway in thrombosis.Material and MethodsWe used a well‐characterized light/dye thrombosis model, coupled with pharmacological and genetic approaches. Male and female adult wild type (WT, C57/BL6) or AnxA1 knock‐out (AnxA1‐/‐) mice were used. Fluorescence intravital microscopy (IVM) was used to monitor cerebral thrombosis, along with flow cytometry. Mice were treated with AnxA1 (1 µg/mouse) or saline vehicle 20 minutes before the onset of cerebral thrombus formation. Thrombogenesis and blood flow cessation times were quantified. (All animal experiments were approved by relevant institutional animal welfare panels and were conducted in accordance with the American Association for Accreditation of Laboratory Animal Care guidelines and complied with ARRIVE [Animal Research: Reporting In Vivo Experiments]).ResultsIVM revealed that AnxA1 treatment was able to prolong blood flow cessation times in both cerebral arterioles and venules, via regulation of the key inflammation resolution receptor: FPR2/ALX, as evidenced by the co‐administration of the FPR2/ALX antagonist WRW4. Using AnxA1−/− mice, we found that a deletion of AnxA1 impacts thrombosis as shown by the accelerated cerebral microvascular thrombus formation (decrease in blood flow cessation time) observed in both arterioles and venules of these mice compared to their WT counterparts (arterioles: 17.9 ± 2.3 vs 33.2 ± 1.9 min and venules: 13.2 ± 2.4 vs 20.9 ± 2.2 min. p&lt;0.05). Furthermore, we found that in an inflammatory backdrop (induced by the administration of lipopolysaccharide [LPS]), AnxA1 treatment was able to suppress both platelet αIIbβ3 and P‐selectin expression.ConclusionHerein, we found a previously unknown phenomenon that the anti‐inflammatory protein AnxA1 can modify a thromboinflammatory environment to promote resolution. These findings have far‐reaching applicability for drug discovery programs focused on resolution biologics targeting thromboinflammatory diseases such as cardiovascular disease and stroke.

Characterization of CARM1 Inhibition in Skeletal Muscle

Coactivator‐associated arginine methyltransferase 1 (CARM1) catalyzes the methylation of arginine residues of target proteins critical for health and disease. CARM1 has recently emerged as an important regulator of skeletal muscle biology. The purpose of this study was to further investigate CARM1 biology in skeletal muscle. For our first experiment, we utilized 3‐month‐old male and female mice (n = 3/sex) that were housed and cared for in accordance with Canadian Council for Animal Care guidelines. Mice were treated with either a CARM1 inhibitor (150 mg/kg EZM2302; Epizyme, Inc.) or vehicle via oral gavage BID for 2, 4, or 8 days. Tissues were collected 6 hours following the final dose and muscle mass was recorded. Western blotting was performed to evaluate protein expression in the tibialis anterior (TA) muscle and liver. To assess CARM1 activity, we investigated the arginine methylation status (i.e., the methylated form of the protein relative to its total amount) of known CARM1 substrates BAF155 and PABP1, as well as the myocellular level of arginine methylated proteins preferentially marked by CARM1. We observed similar outcomes between males and females, so the data were collapsed by sex (n = 6). CARM1 inhibition significantly reduced BAF155 methylation status in the TA muscle by 70‐80% between 2‐8 days of treatment. Muscle PABP1 methylation status was also attenuated (p &lt; 0.05) by 60‐65% after 4 and 8 days of exposure. Similarly, arginine methylation of CARM1‐specific substrates across all muscle proteins was reduced by ~50% (p &lt; 0.05) between 2‐8 days of CARM1 inhibitor administration. The attenuation of BAF155 and PABP1 methylation status, as well as arginine methylated CARM1‐specific substrates, was greater in liver as compared to muscle. Next, we completed additional experiments to assess the effect of pharmacological CARM1 inhibition on skeletal muscle function. After 11 days of CARM1 inhibitor treatment, we observed a sex‐specific reduction (p &lt; 0.05) in exercise capacity and muscular endurance such that muscle function in female mice was unaffected by CARM1 suppression. Collectively, these results suggest that global, pharmacological CARM1 inhibition preferentially impacts liver enzyme activity over muscle. Furthermore, CARM1 inhibitor treatment may affect muscle performance with males being more sensitive to the functional outcomes of CARM1 suppression than females. Future work in our laboratory will examine the mechanisms and functional consequences of sex‐based differences in CARM1 biology within skeletal muscle.

An increase in spontaneous activity mediates visual habituation.

SUMMARYThe cerebral cortex is spontaneously active, but the function of this ongoing activity remains unclear. To test whether spontaneous activity encodes learned experiences, we measured the response of neuronal populations in mouse primary visual cortex with chronic two-photon calcium imaging during visual habituation to a specific oriented stimulus. We find that, during habituation, spontaneous activity increases in neurons across the full range of orientation selectivity, eventually matching that of evoked levels. This increase in spontaneous activity robustly correlates with the degree of habituation. Moreover, boosting spontaneous activity with two-photon optogenetic stimulation to the levels of visually evoked activity accelerates habituation. Our study shows that cortical spontaneous activity is linked to habituation, and we propose that habituation unfolds by minimizing the difference between spontaneous and stimulus-evoked activity levels. We conclude that baseline spontaneous activity could gate incoming sensory information to the cortex based on the learned experience of the animal.

Abstract P5-01-12: Physical and visual (social) isolation increases tumor aggressiveness in thermoneutral housing temperatures in murine mammary cancer models

Abstract Correct interpretation of tumor progression data , including the influence of host biology, in mouse models of breast cancer requires models and conditions that faithfully recapitulate human disease and human host status. Our previous attempts to investigate the effects of social isolation have proven inconclusive due to premature mortality in tumor-bearing animals. Those studies were completed in standard temperature (ST), which commonly is 70-72°F (21-22°C) for in vivo murine research based on laboratory animal care and use guidelines. Previous work from the Repasky lab (Kokulus, 2013) , which we have validated (Gaymon, 2020), demonstrates that ST housing results in chronic cold stress and immune suppression mediated by an increase in norepinrephrine (NE) levels, leading to increased tumor aggressiveness. Based on these findings, we investigated the effects of social isolation on BALB/cJ-4T1-luc and C57BL/6J/E0771 tumor progression and metastasis in thermoneutral housing conditions (84-85°F). Mice were first acclimatized to thermoneutral temperature and/or isolation for two weeks in cages that were unilaterally draped to provide physical and visual isolation. In BALB/cJ mice, 4T1-luc tumors were significantly larger in isolated mice compared to group-housed (GH) mice at day 18 (p&amp;lt;.0001). Statistically larger tumors were observed in isolated mice compared to GH mice through day 24 and final tumor masses were also significantly different (p=.004). Spleen masses were not statistically different. In C57BL/6J mice, E0771 tumors were significantly larger in isolates at Day 25 (p=.002). Final tumor masses were statistically (p=.002) different while no difference in spleen sizes were observed. Data on metastasis will be presented at the meeting. We hypothesized that social isolation may perturb immune function and next investigated the growth of 4T1-luc xenograft tumors in NSG mice. 4T1-luc/NSG tumor progression and metastasis data will also be presented at the meeting. We conclude that syngeneic breast tumor growth in immunocompetent BALB/cJ and C57BL/6J mice demonstrates that social isolation is a bona fide stress with sufficient influence to exacerbate breast cancer growth. These data are potentially clinically important due the known relationship of social support to survivorship outcomes in patients and the high-risk of depression and isolation in patients following breast cancer diagnosis. The data may provide additional insight into possible effects of COVID-19 isolation on breast cancer progression. Citation Format: Darius O Gaymon. Physical and visual (social) isolation increases tumor aggressiveness in thermoneutral housing temperatures in murine mammary cancer models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-01-12.

Fall in the ATP levels in the red blood cells in ApoE-LDLR double-deficient mice model prior to atherosclerosis development

Abstract Background It was previously reported that red blood cells (RBCs) regulate blood flow via RBC-derived ATP [1]. Nevertheless, to the best of our knowledge, no study was performed to characterize possible alterations in RBC ATP levels in ApoE/LDLR−/− mice [2] which constitute a reliable model of human atherosclerosis, displaying distinct erythropathy [3]. Interestingly, young ApoE/LDLR−/− mice display higher exercise capacity and higher O2 carrying capacity of RBCs, as compared to their age-matched control [3,4]. However, it is not known whether increased exerise capacity in ApoE/LDLR−/− mice is linked to altered ATP release from RBCs. It was previously reported that prostacyclin analogs, known atheroprotective agents, which preserve vascular endothelium functions in various diseases [5,6], induce ATP release from human RBCs [1,7]. Purpose To characterize intra- and extra-cellular ATP levels in RBCs isolated from ApoE/LDLR−/− mice in comparison to control mice. Methods All experiments were conducted according to the Guidelines for Animal Care and Treatment of the EU and to the Local Ethical Committee on Animal Testing at our University. For experiments, 8- and 24-week-old C57BL/6 control mice (N=4–6 and N=5–8, respectively) and ApoE/LDLR−/− mice (N=4–7 and N=4–5, respectively) were used. The complete blood count, RBC morphology, biochemistry of blood plasma, RBC deformability, and RBC phosphatidylserine exposure were assessed. The intra- and extra-cellular ATP levels and ATP release from RBCs due to administration of iloprost (100 nM, 1 μM, 10 μm) were studied. Results Intracellular ATP level in RBCs isolated from 8-week-old ApoE/LDLR−/− mice was considerably lower as compared to their age-matched control (7.72±0.77 and 21.23±3.40 pmoles/1x106 RBCs, respectively). In 24-week-old mice, intracellular ATP in RBCs was low not only in ApoE/LDLR−/− mice but also in control mice (8.70±1.30 and 6.27±0.96 pmoles/1x106 RBCs, respectively). Basal extracellular ATP released from RBCs over 30 min incubation was 400 times lower than corresponding intracellular level, and mirrored intracellular ATP levels in all studied groups. Iloprost (100 nM–10 μM) did not produce robust ATP release in any of the studied groups, with only some effects when the highest concentration was used (10 μM). Conclusion(s) Hypercholesterolemia-dependent changes in young ApoE/LDLR−/− mice prior to atherosclerotic plaque development may induce a severe fall in intracellular ATP levels in the RBCs that might be linked to a possible diversion of glycolysis to 2,3-DPG to increase oxygen delivery, and might contribute to the alterations in RBC-dependent regulation of blood flow in ApoE/LDLR−/−. Further studies are required to mechanistically explain these findings. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): 1) National Science Centre, Poland (UMO 2016/23/B/ST4/00795) 2) the Innovation Incubator 4.0 project funded by the Ministry of Science and Higher Education, Poland (POIR 2014-2020)

Water-in-Water Emulsions, Ultralow Interfacial Tension, and Biolubrication

Water-in-Water Emulsions, Ultralow Interfacial Tension, and Biolubrication

Open Transparent Communication about Animals in Laboratories: Dialog for Multiple Voices and Multiple Audiences.

Simple SummaryOver the past 10 years, animal research support groups in Europe have committed to greater openness and transparency of research institutions and scientists, a commitment that US labs could also take up. For openness initiatives to satisfy animal welfare advocates’ concerns, openness must be more than just showing more; it must invite feedback on what to show. In this article, I propose going further in the US, inviting animal welfare advocates into laboratories, onto ethics committees, and into any initiatives to update guidelines for animal care practices.In this article, I offer insights and proposals to the current movement for increased openness and transparency about animal use in laboratories. Increased transparency cannot be total transparency—as no story or picture can ever be complete. When research advocates share their stories, they must decide which words and pictures to edit out. I ask here: Who of the listening “public” gets a chance to revisit this editing, and find the information that is important to them? To the extent that (what I call) the “new openness” attempts to speak to a “lay public” and exclude animal activists, I suggest that refinement-focused animal protectionists deserve enhanced avenues of openness and inclusion—which some research advocates might fear giving to more extreme activists and which a less invested “lay public” may not want or need. I conclude with some specific examples and suggestions to not just invite inquiry from animal advocates, but to bring them in as witnesses and participants, to learn from and incorporate their concerns, priorities, expertise, and suggestions. This can bring a diversity of ideas and values that could improve the quality of science, the credibility of animal researchers, and the welfare of the animals in laboratories.

Male Sex and Age Biases Viral Burden, Viral Shedding, and Type 1 and 2 Interferon Responses During SARS-CoV-2 Infection in Ferrets

Background: The SARS-CoV-2 (Severe Acute Respiratory Syndrome coronavirus 2) has led to more than 73 million COVID-19 cases and ~1.6 million deaths worldwide. Epidemiological analysis of COVID-19 severity has revealed a male sex bias which increases with age. Understanding the mechanisms underlying this disparity will not only be beneficial to host-specific treatment but will give insight into the pathogenesis of the virus in general. Here we investigated the male sex and age bias as well as the corresponding immunological regulation contributing to COVID-19 using ferrets as a model of SARS-CoV-2 infection.Methods: We investigated infected adult male, aged male, and adult female ferrets with SARS-CoV-2 at 106 TCID50 intranasally. Ferrets were monitored for clinical signs post-inoculation and in-life samples and end-point tissues were collected. The samples were analyzed for viral dynamics by qRT-PCR and live viral load by TCID 50 assays. Respiratory tissue was investigated for tissue destruction and viral antigen presence by histopathology and immunohistochemistry. RNA sequencing of isolated RNA from the upper and lower respiratory tract was performed using the NovaSeq 6000 50 base-pair, paired-end run for a minimum of 30 million reads per sample. Sequence analysis was used to determine differential gene expression. Findings: Clinically, all groups of ferrets developed mild disease although all males had consistent temperature decreases while females developed a fever. Viral load analysis revealed that aged male ferrets shed virus from their nares for longer and had higher viral loads in the upper respiratory tract associated with increased pathology. Host response analysis indicated that female ferrets had significant increases in antiviral and interferon response genes as soon as day 2 post-infection characterized by the upregulation of OASL, MX1, and ISG15 as well as others, whereas upregulation of antiviral genes was delayed until day 7 in aged males. In the later time points, males had significant increases in genes associated with neurological signalling and sensory detection (taste and smell) such as RTP1, CHGA, and CHGA1.Interpretation: Older male ferrets had increased viral shedding and burden compared to females which may have been due to the dysregulation of the male antiviral responses. Increases in olfactory-associated genes during the infection time course may suggest mechanisms regulating anosmia, which has been associated with COVID-19. These results provide insight into COVID-19 epidemiological findings and suggest that the male sex bias in hospitalizations has a biological component and is not solely due to cultural and behavioural factors. Considering the threat of SARS-CoV-2 mutations in hosts that have prolonged viral replication and shedding, understanding why older males have increased viral durability is of public health importance.Funding Statement: Canadian Institutes of Health Research (CIHR), NIH/NIAID, Genome Atlantic, and Canadian Foundation for Innovation through the Major Science Initiates Fund and by Innovation Saskatchewan.Declaration of Interests: Authors declare no competing interests.Ethics Approval Statement: All work was conducted in accordance with the Canadian Council of Animal Care (CCAC) guidelines, AUP number 20200016 by the University Animal Care Committee (UACC) Animal Research Ethics Board (AREB) from the University of Saskatchewan.

Detection and whole genome sequencing of murine norovirus in animal facility in Italy

Viruses belonging to the genus Norovirus (NoV) of the family Caliciviridae are the major cause of acute viral gastroenteritis worldwide. NoVs are classified into 10 genogroups (GI-GX), and those belonging to the genogroup GV are able to infect several species of rodents. To evaluate the circulation of MNV among mice housed in an Italian facility, sampling was performed over two separate periods, in 2011, and 3 years later in 2014. During the two samplings, 75 fecal samples were collected from healthy mice housed in the animal facility and subjected to RT-PCR for viral detection. After the analysis, 41/75 animals (54.6%) resulted positive for the presence of MNV in feces. Nucleotide sequencing revealed the presence of two MNV variants co-circulating in both 2011 and 2014. One MNV strain was isolated on RAW264.7 cell line, and subjected to full genome sequencing. Our study showed that the murine noroviruses are widespread in the investigated animal facility, despite guidelines for animal care and maintenance. Full genome sequence analysis of the MNV strain described in this study showed a correlation with other strains circulating in Europe. Understanding the molecular epidemiology of this virus should give insight into its natural history and evolution in mice.

Research Ethics: The Bane of the unethical researcher.

Every human research creates generalizable information which is an integral part of health improvement &amp; understanding of disease. It also causes some risk or inconvenience to research participants. Codes of conduct for physicians have existed since ancient period. Declaration of Helsinki ICMR, Human subjects’ protection; Animal care guidelines etc provide a policy for conduct of research involving research participants in India. The Why &amp; the How in research are vital questions. The ‘Why’ is very clear to define as improvement of public health. What matters more is the ‘How’: while performing it is necessary to adhere to research ethics for promotion of knowledge, truth and avoidance of errors. Research being team work, involves principles of team work such as trust, mutual respect, even handedness, and accountability. Upholding confidentiality, data sharing policies, guidelines of authorship, peer review policies, copyright and patenting policy promotes security of intellectual property and gives confidence for collaborations. Ethical research outlines standard guidelines for institutions and professionals for their behavior towards the specified goals and helps to develop the trust of community further promoting funding.