Resistant Animals Research Articles

Lactobacillus murinus alleviates insulin resistance via promoting L-citrulline synthesis.

The role of Lactobacillus murinus as a potential probiotic is being explored. Our objectives were to explore the effects of Lactobacillus murinus on insulin resistance and the underlying mechanism. Insulin resistance animal models were applied to study the effect of L. murinus and the underlying mechanism by six weeks of treatment. Metformin was administered in vitro to analyze the growth and metabolites of L. murinus. Serum metabolites were further analyzed after L. murinus administration. The effect of L-citrulline and the underlying mechanism in alleviating insulin resistance were evaluated. L. murinus not only reduced body weight gain and postprandial blood glucose (PBG) but improved impaired glucose tolerance (IGT) and insulin resistance. Moreover, L. murinus inhibited the secretion of pro-inflammatory factors (IL-1β, IL-6 and TNF-α) while promoted the secretion of anti-inflammatory factor (IL-10). Further, L. murinus promoted the expression of carnitine palmitoyl transferase 1 (CPT1) while inhibited phosphoenolpyruvate carboxykinase (PCK) and glucose-6-phosphatase (G6Pase). A total of 147 metabolites of L. murinus were identified, in which the content of L-citrulline increased to 7.94 times after metformin regulation. Further, the serum concentration of L-citrulline significantly increased after L. murinus administration. Similarly, L-citrulline reduced body weight gain and PBG, improved IGT and insulin resistance. Additionally, L-citrulline improved inflammation, promoted CPT1 while inhibited PCK and G6Pase. L. murinus mediated by L-citrulline alleviated insulin resistance via promoting fatty acid oxidation and inhibiting gluconeogenesis, suggesting that supplementation of L. murinus could be a potential therapeutic approach for type 2 diabetes related to insulin resistance.

Myocardial contractile function in the post-traumatic period of cardiac contusion in rats with different stress resistance: A preclinical experimental randomized trial

Background. Pathogenesis of cardiac contusion involves primary traumatic and secondary hypoxic mechanisms of myocardial contractile function depression as well as body reactions aimed at adapting to altered environment. A significant part of these reactions is realized in the context of stress. The intensity of the stress component in myocardial dysfunction is largely determined by the individual stress reactivity of the body. Objectives. To assess the contractile function and functional reserves of the myocardium of rats with a high and low level of stress resistance in the post-traumatic period of cardiac contusion. Methods. A preclinical experimental randomized trial involved 134 white mature nonlinear male rats weighing about 270 g. The animals were divided by sequentially performed Open Field and Porsolt Forced Swim tests into 2 groups: group 1 — animals with low stress resistance, group 2 — animals with high stress resistance. Within each group, the animals were randomized into control and experimental subgroups. In the experimental subgroups, 6, 12 and 24 hours after simulating cardiac contusion, the force and rate indicators of myocardial contractility were evaluated using the Fallen isolated heart model. The summary measures of the study included assessing the contractile force and rate of isolated hearts of rats with low and high stress resistance, particularly under conditions of high-frequency rhythm load (ranging from 4.0 to 8.3 Hz) during the post-traumatic period of cardiac contusion. Data analysis was performed using software packages MS Office 2013 (Microsoft Corporation, USA) and Statistica, v. 10 (StatSoft, USA). The differences were considered to be statistically significant at p < 0.05. Results. 6, 12 and 24 hours after simulating a cardiac contusion, contractile force and rate of isolated hearts decreased in group 1 and group 2. In low stress-resistant animals, immediately following the stabilization period and during high-frequency rhythm test, the contractility force and rate in isolated hearts were statistically significantly lower (p = 0.0008) compared to those recorded in highly stress-resistant individuals. During the stimulation of a high-frequency rhythm, isolated hearts in the experimental group revealed diastolic dysfunction at all time points. In highly stress-resistant animals, diastolic dysfunction occurred at a heart rate of 300 min-1 and above, whereas in low stress-resistant animals, it manifested at a heart rate of 240 min-1 and above. At the same heart rate, diastolic dysfunction in low stress-resistant animals was statistically significantly greater (p = 0.0008) compared to that of highly resistant animals. Conclusion. The post-traumatic period following experimental myocardial contusion is characterized by a reduction in the force and rate of myocardial contractility, as well as a decrease in functional reserves of the myocardium, regardless of stress resistance. High stress resistance is associated with better preservation of cardiac contractile function and contractility reserves, whereas low stress resistance correlates with a more pronounced degree of myocardial dysfunction and a significant reduction in functional reserves of the contused heart. Differences in the severity of contractile dysfunction under conditions of high and low body resistance to stress can be attributed to varying degrees of secondary myocardial damage in the contused area, resulting from the misbalance between stress-activating and stress-limiting mechanisms involved in the development of secondary damage.

Selection of antibiotic-resistant bacterial populations in the dairy cow gut following intramuscular ceftiofur treatment for metritis

Third-generation cephalosporins such as ceftiofur are critically important antibiotics because human pathogens with resistance to these drugs contribute to high mortality rates. These antibiotics are also frequently given to dairy cattle for treating infections, emphasizing the critical role they play in both human and veterinary medicine. To investigate the impact of intramuscular ceftiofur treatment on the concentration of resistant bacteria in the gut, we focused on cows with metritis, a common bacterial infection that frequently requires antibiotic intervention. Twelve cows with metritis (cases) were enrolled and treated with intramuscular ceftiofur for 5 d along with 12 matched healthy cows that were not given ceftiofur (controls). Fecal samples were collected weekly from cows in both the case and control groups for 4 weeks, starting before the treatment of the case group. Five fecal samples per cow were used for analysis (n = 120 samples). The abundance of Gram-negative bacteria was quantified per sample after plating on MacConkey agar, which was also used to quantify the abundance of Gram-negative bacteria with resistance to ceftiofur, ampicillin, and tetracycline. Interestingly, the case cows with metritis had a greater abundance of Gram-negative bacteria than the control cows just before treatment, but no difference in abundance was observed between groups at wk 1-4. The abundance of ceftiofur-resistant Gram-negative bacteria was also similar between the case and control cows immediately before treatment of the cases. However, a significant increase in abundance of ceftiofur-resistant Gram-negative bacteria was observed in the case cows 1-week after treatment that persisted through wk 3. Although the recovery of ampicillin- and tetracycline-resistant bacteria was similar between the 2 groups post-treatment, cases had significantly higher levels of ampicillin-resistant bacteria before treatment. Collectively, these findings demonstrate that intramuscular ceftiofur treatment can affect the abundance of cultivable Gram-negative bacteria and select for ceftiofur-resistant populations that can persist for up to 3 weeks. Judicious use practices are needed to ensure that ceftiofur and other critically important antibiotics are administered only when necessary to minimize the spread of resistance and safeguard public and animal health.

A model proposal for sustainable Nevşehir local cuisine

Local cuisine is a valuable cultural-gastronomical asset for any tourism destination as well as local people. In order not to lose this asset, it is vital to manage the changes triggered by climate change and transmitted through agriculture. Climate change is likely to impact different regions at different levels; thus, some regions have to be ready to challenge a harsher situation. Nevşehir is a Central Anatolian province which is likely to suffer from quite harsh impacts of climate change and its local cuisine is prone to many risks unless an effective plan which considers the relevant issues is executed. With this end in view, representatives of 5 key institutions were interviewed. Beside climate issues, the interviewees note other relevant matters such as economy, ethics, and consumer and producer awareness issues. After a conceptual model draft, this study proposes a conceptual and a practical model for a more sustainable Nevşehir local cuisine. Focusing on sustainability, the final practical model developed is based on climate change issues, agriculture, local cuisine, and their close relationships as well as the factors reminded by interviewed representatives. Main risks associated with climate change include drought, decrease in agricultural efficiency, related quality issues in products and agricultural assets such as soil, and – at the extreme end – a global food crisis. Despite this negative outlook, the province has also some advantages such as resistant native plant and animal species, potential for renewable energy, and slow city possibilities. Proposed recommendations for a more sustainable local cuisine focus on managing change, preserving authenticity, taking the advantage of strengths, increasing efficiency, and climate change mitigation efforts. Final part of the study includes a discussion and some possible future research themes.

The genetic resistance of sows to Escherichia coli F4 adhesion reduces their response to a vaccine containing F4 fimbriae but does not affect the preweaning performance of their susceptible piglets.

Pigs without intestinal receptors for F4 fimbriae are congenitally resistant to F4 fimbriae-bearing enterotoxigenic Escherichia coli (ETEC F4). In general, 50 % and 100 % of piglets born to resistant (RR) sows crossed with hetero- or homozygous susceptible (SR, SS) boars, respectively, are susceptible but do not receive colostral antibodies against F4 fimbriae unless the sows have been vaccinated. The question arises as to whether resistant sows produce protective amounts of F4 antifimbrial antibodies after vaccination. The serum and colostrum antibody titres of 12 resistant and 12 susceptible vaccinated gilts were compared. The effect of the receptor status of the dam and sire on the preweaning performance of 5027 piglets was evaluated using Agroscope's recordings. The sows of the experimental herd, where ETEC F4 was circulating, were vaccinated against ETEC twice during the first pregnancy and once during each following pregnancy. The log2 transformed F4 antibody titres in the serum obtained after the second vaccine injection as well as in the colostrum of the 12 resistant animals were lower than the titres of the susceptible animals (serum: F4ab 11,19 ± 1,44 vs. 12,18 ± 1,33, P = 0,096; F4ac 10,03 ± 1,58 vs. 11,59 ± 1,43, P = 0,019; colostrum: F4ab 12,20 ± 2,41 vs. 14,02 ± 1,31, P = 0,033; F4ac 10,93 ± 2,46 vs. 13,03 ± 5,21, P = 0,006). The heat labile enterotoxin (LT) antibody titres after vaccination did not differ between susceptible and resistant animals (p > 0,10). Preweaning mortality in the offspring of RR sows × SS boars was slightly lower than in the offspring of SS sows × RR boars (P = 0,04), suggesting that the disease risk of susceptible piglets born to vaccinated resistant sows was not increased, even though they received colostrum with a slightly reduced content of antibody against F4 fimbriae.

Phenotypic and Molecular Characterization of Escherichia coli Isolated from Food Animals for Antimicrobial Resistance

Background: The use of antimicrobials in food animals significantly contributes to the development of antimicrobial resistance in nature. This study aimed to determine the prevalence of ESBL-producing E. coli isolated from cattle, buffalo, sheep and goat fecal samples. Methods: In this study out of 2025 healthy animals from 10 different farms, fecal samples were randomly collected by rectal swabs from 200 animals (50 each from cattle, buffalo, sheep and goat). E. coli was isolated and further studied for AMR and ESBL production by phenotypic assays and PCR targeting ESBL genes. Result: The overall prevalence of E. coli was 81.50% with the highest isolation rate from cattle and goat samples. Out of 163 isolates, 45.39% were ESBL-producers. Genes encoding beta-lactam resistance viz. blaSHV, blaTEM, blaCTX-M and blaOXA were detected in 44.59% ESBL-positive isolates. The blaSHV was present in only cattle isolates, while blaCTX-M was present in E. coli isolated from all animals. The prevalence of beta-lactam genes was found as blaSHV (2.70%), blaTEM (12.16%), blaCTX-M (22.97%) and blaOXA (6.75%). A high degree of resistance to multiple antimicrobials was observed. Maximum susceptibility of E. coli was observed for trimethoprim, imipenem and chloramphenicol. This study demonstrated that food animals could be the source of ESBL-producing multidrug-resistant E. coli. The present findings are significant in the context of AMR monitoring in India.

The Transcript Levels and the Serum Profile of Biomarkers Associated with Clinical Endometritis Susceptibility in Buffalo Cows.

Determining the gene expression and serum profile of the indicators linked to clinical endometritis susceptibility in Egyptian buffalo cows was the aim of this investigation. The buffalo cows that were enrolled were divided into two groups: forty infected buffalo cows with clinical endometritis and forty seemingly healthy buffalo cows that served as the control group. For the purposes of gene expression and biochemical analysis, ten milliliters of blood was obtained via jugular venipuncture from each buffalo cow. TLR4, IL-8, IL-17, NFKB, SLCA11A1, NCF4, Keap1, HMOX1, OXSR1, ST1P1, and SERP1 were manifestly expressed at much higher levels in the buffaloes with endometritis. On the other hand, the genes that encode SOD, CAT, NDUFS6, Nrf2, and PRDX2 were down-regulated. There was a significant (p < 0.05) elevation of the serum levels of non-esterified fatty acids (NEFAs), beta hydroxy butyric acid (BHBA), triglycerides (TGs), globulin, creatinine, and cortisol, along with a reduction in the serum levels of glucose, cholesterol, total protein albumin, urea, estrogen (E2), progesterone (P4), follicle-stimulating hormone (FSH), luteinizing hormone (LH), thyroxine (T4), prostaglandin F2 α (PGF2α), calcium, iron, and selenium, in the endometritis group in comparison with the control. However, no significant change was observed in the values of phosphorus, magnesium, copper, or zinc in either group. Within the selective breeding of naturally resistant animals, the variation in the genes under study and the changes in the serum profiles of the indicators under investigation may serve as a reference guide for reducing endometritis in Egyptian buffalo cows.

The multiple faces of footshock punishment in animal research on addiction

Continued drug use despite negative consequences is a hallmark of addiction commonly modelled in rodents using punished drug intake. Over the years, addiction research highlighted two subpopulations of punishment sensitive and resistant animals. While helpful to interrogate the neurobiology of drug-related behaviors, these procedures carry some weaknesses that need to be recognized and eventually defused. Mainly focusing on footshock-related work, we will first discuss the criteria used to define punishment-resistant animals and how their relative arbitrariness may impact our findings. With the overarching goal of improving our interpretation of the punishment-resistant phenotype, we will evaluate how tailored punishment protocols may better apprehend resistance to punishment, and how testing the robustness of punishment resistance could yield new results and strengthen interpretations. Second, we will question whether and to what extent punishment sensitivity, as currently defined, is reflective of abstinence and suggest that punishment resistance is, in fact, a prerequisite to model abstinence from addiction. Again, we will examine how challenging the robustness of the punishment-sensitive phenotype may help to better characterize it. Finally, we will evaluate whether diminished relapse-like behavior after repeated punishment-induced abstinence could not only contribute to better understand the mechanisms of abstinence, but also uniquely model progressive recovery (i.e., after repeated failed attempts at recovery) which is the norm in people with addiction. Altogether, by questioning the strengths and weaknesses of our models, we would like to open discussions on the different ways we interpret punishment sensitivity and resistance and the aspects that remain to be explored.

Resistance of juvenile Nile tilapia Oreochromis niloticus from Brazilian populations to Streptococcus agalactiae (serotype Ib and ST-NT)

Nile tilapia is the main farmed fish in Brazilian aquaculture. Streptococcus agalactiae (GBS) is responsible for high mortality rates in fish farms. Genetic improvement is considered an effective alternative for producing resistant animals in aquaculture. The objective of this study was to estimate genetic parameters for resistance to GBS infection in a breeding population of Nile tilapia from Brazil, based on disease challenge using a circulating serotype (Ib and ST-NT). Furthermore, genetic correlations between resistance to GBS and average daily gain (ADG) were estimated to determine whether these traits can be included in selective breeding programs. Survival (SS) and time until death (TD) were the evaluated traits. The survival of the most resistant families was approximately 30%, indicating high phenotypic variation in resistance to the infection. Low to moderate heritability values for resistance traits in the Nile tilapia population were estimated, ranging from 0.14 to 0.27. Heritability for ADG was moderate to high (0.40) based on a growth trial involving 43 families (735 animals). Phenotypic correlations between the resistance traits (SS and TD) and ADG were positive and low, ranging from 0.09 to 0.18, whereas genetic correlations were close to zero. Phenotypic and genetic correlations between SS and TD were considered high and positive, ranging from 0.38 to 0.72. The results suggest that selection for resistance against GBS does not negatively affect juvenile weight gain in Nile tilapia.

Abstract 2726: A novel trispecific antibody HC010 targeting PD-1/CTLA-4/VEGF for the potential treatment in anti-PD-1 antibody resistant NSCLC patients

Abstract Background and Aim: Immune checkpoint inhibitors (anti-PD-1/CTLA-4 antibody) have been approved and widely used for the first line treatment of non-small cell lung cancer (NSCLC). However, most patients are experiencing resistant to PD-1 blockade. In recent clinical trials, anti-VEGF antibody has shown to improve the clinical outcomes of anti-PD-1 antibodies and overcome resistance to checkpoint blockade. We developed a PD-1/CTLA-4/VEGF trispecific antibody (HC010) by site-specifically fusing anti-PD-1 scFv and anti-CTLA-4 nanobody to the defined site of anti-VEGF antibody. The aim of this study was to evaluate the potential use of HC010 for treatment of anti-PD-1 antibody resistant NSCLC patients. Method: Humanized mice were established in NCG mice by reconstituting human PBMC before inoculating the PDX tissue, which was derived from a NSCLC patient treated with two cycles of an anti-PD-1 antibody sintilimab. When tumors were approx. 170mm3, mice were dosed with HC010 twice weekly for 3 weeks. Pembrolizumab, sintilimab and a Standard of Care (SoC) agent docetaxel were included as reference. For IHC analysis, tumor samples were harvested 24h post final dose and fixed with 10% NFB overnight and subsequently stained with CD31 and CD8 to explore the change in tumor microenvironment after HC010 treatment. For preclinical toxicity assessment, HC010 was intravenously administered once weekly for four weeks in naïve cynomolgus monkeys and immunogenicity/local tolerance/clinical pathology were evaluated during the dosing and recovery phase. Results: HC010 showed a significant tumor inhibition (TGI=81%) when compared with the vehicle control. Pembrolizumab and sintilimab alone exhibited limited anti-tumor effect. Docetaxel only showed a partial response during the dosing phase. By analyzing tumor samples, there was a significant increase in CD8+ positive T cells in HC010 treated tumors in contrast to the vehicle group and pembrolizumab/sintilimab treated groups, suggesting that HC010 can significantly improve CD8+ T cell infiltration. Expression of CD31 as an angiogenic and vasculogenic marker, was significantly reduced in HC010 treated tumors. In the toxicity study, HC010 at selected doses showed no obvious toxicity in cynomolgus monkeys. Conclusion: Our trispecific antibody HC010 targeting PD-1/CTLA-4/VEGF showed a superior anti-tumor effect in a sintilimab resistant PDX animal model when compared with pembrolizumab and sintilimab as well as SoC. By analyzing tumor microenvironment, HC010 overcame acquired resistance by modulating tumor microenvironment such as increase in intra-tumoral cytotoxic T cell infiltration and normalization of blood vessels. Together with preclinical efficacy and safety data, HC010 showed potential benefits in treatment of anti-PD-1 antibody resistant NSCLC patients. Citation Format: Diandong Jiang, Yue Xi, Chen Xu, Xingding Wang, Qiaoshan Yin, Zhaohui Li, Teddy Yang. A novel trispecific antibody HC010 targeting PD-1/CTLA-4/VEGF for the potential treatment in anti-PD-1 antibody resistant NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2726.

Abstract 2826: Examples of translational preclinical models to mimic cancer drug resistance

Abstract Drug resistance is becoming a major obstacle when treating patients with cancer. Cancer is characterized by an uncontrolled growth of abnormal cells with permanent genome alterations, which lead to severe mutations including resistance mechanisms. Mutations can occur in different resistance pathways, such as inhibition of drug transport, DNA damage repair, drug efflux, EMT (epithelial-mesenchymal transition), drug target alteration and cell death inhibition. Resistance mechanisms are now well studied in vitro, based on mutated cell lines for specific resistance pathways. There is however a gap in our knowledge of resistance mechanisms observed in patient tumor cells. We are challenged to discover cancer resistance treatments in patients and need predictive models of clinical situations. Animal tumor models are the best translational tools to study cancer drug resistance, especially when they closely mimic human pathologies. Such models can bridge the gap between our understanding of cancer resistance in vitro and in tumor patients. In this poster we show different pre-clinical experiments, simulating clinical translational approaches, that led to novel and unique tumor resistant models. Patient Derived Xenograft (PDX) models: Using our breast cancer PDX collection we showed that after repeated treatment with a spindle poison, tumor-bearing mice started to develop resistance and continued tumor growth under drug pressure. Following this successful approach, we initiated the development of a novel ER2-expressing PDX breast tumor model, resistant to hormone therapy by repeated Fulvestrant treatment. Using our colon cancer PDX collection we were able to recapitulate clinical situations by correlating PDX molecular profiles and drug sensitivity. We confirmed the key role of a KRAS mutation in cetuximab resistance. Syngeneic model: In this model, animals were dosed with anti-PDL1 as first line of treatment. All animals were then defined as initial responders or non-responders at the end of the dosing schedule. Following this, a new treatment protocol was initiated with a Tyrosine Kinase inhibitor to study the effect of second line drug treatment in responder and non-responder arms. Human model: In this experiment, we performed a first line Avastin treatment on human colon carcinoma tumor-bearing mice. A tumor resistance to Avastin was observed. During the second line of treatment with a tyrosine kinase inhibitor, we observed an antitumor activity on Avastin resistant animals. In each of these case studies, we developed a novel tumor-resistant mechanism model. In some tumor models, we showed the added value of treating animals with new drugs as second line treatment, after establishing resistance to the first line standard of care treatment. Citation Format: Marc Hillairet de Boisferon, Nicolas Hoffmann, Elodie Rajon, Ismahène Benzaid. Examples of translational preclinical models to mimic cancer drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2826.

Abstract 4103: Novel mRNA-encoded HPV vaccine with APC maturation signal and endolysosomal trafficking domain demonstrates superior T cell-mediated immunogenicity and efficacious tumor clearance in vivo

Abstract Human Papillomavirus (HPV), particularly HPV16, is a known causative agent of cancers in various anatomical sites, notably the anogenital and oropharyngeal regions. Despite the availability of prophylactic HPV vaccines for almost two decades, vaccination rates among young females, who are at a heightened risk for cervical intraepithelial neoplasia (CIN) and subsequent invasive cancer, remain suboptimal. The current standard of care for cervical cancer, involving invasive surgery and chemotherapy-based treatments, underscores the pressing need for safer and more effective therapeutics to enhance patient survival and quality of life. In this context, while several HPV16-E6/E7 targeted therapeutic vaccines are in development and clinical trials, their clinical efficacy has been limited due to inadequate antigen presentation and suboptimal immunogenicity. Addressing this, our team has developed a novel, highly immunogenic mRNA therapeutic cancer vaccine, engineered to activate a robust T cell-mediated adaptive immune response and exhibit strong anti-tumor potential. Our innovative approach includes the design of an HPV16-E6/E7 mRNA vaccine (ABO2101) that incorporates a unique antigen-expressing cassette. This cassette features endolysosomal trafficking domains and antigen presenting cell (APC) activation stimulators, enhancing both Class I and Class II antigen presentation and APC maturation, thereby amplifying antigen-specific T cell responses. In both murine and human-derived experimental models, ABO2101 has demonstrated a significantly more potent adaptive T cell response against HPV16 antigens compared to other HPV16 mRNA therapeutic vaccines. In antitumor efficacy evaluations using TC-1 tumor-bearing mice, ABO2101 single-dose administration successfully eradicated both early-stage (50 mm³) and advanced (500 mm³) tumors. This was accompanied by a marked increase in HPV16-specific CD8+ cytotoxic T lymphocytes within the tumor microenvironment. Moreover, in prophylactic and adjuvant therapy models, ABO2101 prevented tumor growth across various dosages, indicating its robust preventative capabilities. Further, the combination of ABO2101 with immune checkpoint inhibitors, including PD-1 and CTLA-4 antibodies, displayed a synergistic effect in both immune-oncology sensitive and resistant animal models, suggesting a potential for combinational clinical therapy. In conclusion, our development of ABO2101, a mRNA-based HPV16-E6/E7 therapeutic cancer vaccine inclusive of APC maturation signals, represents a groundbreaking advancement in HPV-associated cancer treatment, as supported by our compelling preclinical data. Citation Format: Liang Du, Hongya Han, Jingshu Ma, Zhenxing Yang, Jinjuan Mao, Jijun Yuan, Bo Ying. Novel mRNA-encoded HPV vaccine with APC maturation signal and endolysosomal trafficking domain demonstrates superior T cell-mediated immunogenicity and efficacious tumor clearance in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4103.

Abstract 4890: In silico study of enzymes involved in heparan sulfate catabolic process in cancer resistant mammals

Abstract Some mammals such as elephants, bats, whales, and blind mole rats (Spalax) are resistant to cancer. One of the cancer protective mechanisms present in such species includes a reduced degradation of heparan sulfate (HS), an important component of the extracellular matrix (ECM), present in Spalax. Such effect occurs due to a splicing variant of heparanase, called variant 36, that lacks enzymatic activity. Consequently, both HS and ECM are very stable in Spalax’s tissues. If HS is stabilized, in cancer resistant mammals, due to the decrease of heparanase or other enzymes involved in HS catabolic process is unknown. We studied total of 9 enzymes (HPSE2, GusB, IDUA, IDS, NAGLU, SGSH, GNS, Sulf-2, HGSNAT) that regulate HS catabolism and homologs in 9 mammals. Multiple sequence alignments were done by MEGA to identify substitutions present in cancer resistant species in comparison with cancer susceptible animals. Such amino acid variants were further analyzed and both their energy stability and their functional effects were predicted by using online in silico tools including MAESTROweb, Condel, PANTHER, REVEL, and CADD. Finally, homologs with deleterious predicted variants were modelled with SWISSMODEL and their 3D structures were analyzed with Pymol and ChimeraX to identify structural features likely related to its loss of function. We found two loss of function missense variants, K264Q and W626L, in Alpha-L-iduronidase (IDUA) of Spalax. In addition, most of Brandt's bat enzymes and the N-sulphoglucosamine sulphohydrolase (SGSH) of the bowhead whale were very energy destabilizing enzymes with positive and higher ΔΔG values compared to human homologs. In the other hand, although some catalytic residues in the study enzymes are conserved in cancer resistant animals, their spatial arrangement and conformation in the 3D structure are different compared to human homologs, including E182 of IDUA which is one of the catalytic residues. In general, the secondary structure is conserved across the 9 enzymes studied but some differences were observed in cancer resistant animals including one of the beta strands near the catalytic domain in human and mice IDUA that appears as a loop in Spalax. In summary, HS stabilization could raise in cancer resistant mammals through the existence of lack of enzymatic activity not only in heparanase but also in other enzymes that participate in HS catabolic process, via a set of loss of function variants and changes in secondary and tertiary structure as well as a reduction of energy stabilization. More interestingly, such mechanism could be present in other cancer resistant mammals such as bats and bowhead whales. This study demonstrates that computational approaches are useful to study biological mechanisms of cancer resistance present in some mammals. This knowledge could be translated to humans and HS catabolic process could offer a new source of therapeutic anticancer targets. Citation Format: Fabrizio Oliveri Payet, Claudia Machicado Rivero. In silico study of enzymes involved in heparan sulfate catabolic process in cancer resistant mammals [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4890.

Sink-source connectivity for restocking of Pinna nobilis in the western Mediterranean Sea

The critically endangered endemic bivalve Pinna nobilis from the Mediterranean Sea suffered a sudden population decline after a mass mortality event in early autumn 2016. Conservation efforts aimed at preventing extinction included safeguarding resistant individuals and implementing a breeding plan to contribute to the repopulation of the species. This study utilized a model combining Lagrangian dispersion and connectivity analyses to pinpoint optimal restocking sites in the Western Mediterranean. Our approach allowed to identify locations capable of sustaining and generating larvae for broader repopulation in key areas of the Western Mediterranean Sea prior to the mass mortality event. Six important repopulation locations from Murcia, Valencia and Balearic Islands were selected for reintroduction efforts. The results obtained in this study show how the network could be self-sufficient and able to self-replenish itself of recruits. Overall, our work can be used to direct the reintroduction of resistant animals in the Western Mediterranean Sea.

Doppler ultrasonographic scan, gene expression and serum profile of immune, APPs and antioxidant markers in Egyptian buffalo–cows with clinical endometritis

The objective of this study was to elaborate Doppler ultrasonographic scan, genetic resistance and serum profile of markers associated with endometritis susceptibility in Egyptian buffalo–cows. The enrolled animals were designed as; twenty five apparently healthy buffalo–cows considered as a control group and twenty five infected buffalo with endometritis. There were significant (p < 0.05) increased of cervical diameter, endometrium thickness, uterine horn diameter, TAMEAN, TAMAX and blood flow through middle uterine artery with significant decrease of PI and RI values in endometritis buffalo–cows. Gene expression levels were considerably higher in endometritis-affected buffaloes than in resistant ones for the genes A2M, ADAMTS20, KCNT2, MAP3K4, MAPK14, FKBP5, FCAMR, TLR2, IRAK3, CCl2, EPHA4, and iNOS. The RXFP1, NDUFS5, TGF-β, SOD3, CAT, and GPX genes were expressed at substantially lower levels in endometritis-affected buffaloes. The PCR-DNA sequence verdicts of healthy and affected buffaloes revealed differences in the SNPs in the amplified DNA bases related to endometritis for the investigated genes. However, MAP3K4 elicited a monomorphic pattern. There was a significant decrease of red blood cells (RBCs) count, Hb and packed cell volume (PCV) with neutrophilia, lymphocytosis and monocytosis in endometritis group compared with healthy ones. The serum levels of Hp, SAA, Cp, IL-6, IL-10, TNF-α, NO and MDA were significantly (P˂0.05) increased, along with reduction of CAT, GPx, SOD and TAC in buffalo–cows with endometritis compared to healthy ones. The variability of Doppler ultrasonographic scan and studied genes alongside alterations in the serum profile of investigated markers could be a reference guide for limiting buffalo endometritis through selective breeding of natural resistant animals.

Epigenetic Investigation Related to Gastrointestinal Helminth Resistance and Performance in Cattle

Abstract The objective was to characterize a herd of 73 Nellore heifers, identifying resistant, resilient, and susceptible animals to gastrointestinal helminths, relating the global methylation of the DNA of these animals with the degree of helminthiasis and factors that interfere with performance. Individual count of eggs per gram of feces (EPG), fecal culture for gender identification, weighing and blood sampling were carried out to determine PCV, STP, EOS, IgG, followed by DNA extraction and methylation analysis. The results were: 47% resistant animals, 34% resilient, and 19% susceptible to gastrointestinal helminth infections, with EPG counts of 53, 216, and 841, respectively, showing a statistical difference between all groups. The quantification of DNA methylation was 0.311, 0.245 and 0.178, respectively, for resistant, resilient, and susceptible animals, with a correlation being found between resistance to gastrointestinal helminths and overall DNA methylation. For weight gain, resistant and resilient animals showed higher values than susceptible ones, with a correlation between weight gain and EPG. The same was observed for VG; however, there was no statistical difference to the EOS, PPT, and IgG values. A significant correlation was found between PCV and EPG; quantifications of PCV and methylated DNA, STP and EPG; VG and STP. Therefore, the methodologies used made it possible to identify the animals regarding the degree of infection by gastrointestinal nematodes, making it possible to correlate the resistance of cattle to helminths with the amount of global DNA methylation and its performance.

Exercise training improves diabetic renal injury by reducing fetuin-A, oxidative stress and inflammation in type 2 diabetic rats

BackgroundDiabetic kidney disease (DKD) stands as a primary contributor to end-stage renal disease, associated with heightened mortality in cardiovascular diseases. This study aimed to explore the impact of an eight-week high-intensity interval training (HIIT) on renal injury in diabetic rats. MethodsTwenty-eight male Wistar rats were randomly allocated into four groups: healthy control (CTL), diabetic control (DC), exercise (EX), and diabetes-exercise (D + EX). Induction of diabetes in the DC and D + EX groups occurred through a two-month high-fat diet followed by a single dose of 35 mg/kg streptozotocin (STZ). Rats in the EX and D + EX groups underwent 4–10 intervals of HIIT (80–100% Vmax) over 8 weeks. Subsequently, pathological and biochemical parameters were assessed in the serum and kidney tissue of the experimental groups. ResultsIn the DC group, diabetes led to elevated kidney damage, glomerulosclerosis, fasting blood glucose (FBG), Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) index, animal weight, kidney dysfunction, albuminuria, and glomerular filtration rate. Additionally, serum and kidney levels of fetuin-A increased, along with kidney levels of KIM-1. Mechanistically, diabetes induction resulted in kidney inflammation by elevating levels of tumor necrosis factor-alpha (TNF-α), transforming growth factor beta (TGF-β), and interleukin 6 (IL-6), while reducing IL-10 levels and increasing the IL-6/IL-10 ratio. Furthermore, diabetes triggered renal oxidative stress, evidenced by increased Malondialdehyde (MDA) levels and decreased levels of glutathione peroxidase (GPx), catalase, and superoxide dismutase (SOD). HIIT mitigated the adverse effects of diabetes in the D + EX group compared to the DC group. ConclusionOur findings suggest that HIIT ameliorates type 2 diabetes (T2D)-induced kidney damage by mitigating inflammation, lowering serum levels of fetuin-A, and bolstering antioxidant defenses. This study highlights the potential of HIIT as a time-efficient intervention for diabetic nephropathy.

Multi-omics data elucidate parasite-host-microbiota interactions and resistance to Haemonchus contortus in sheep

BackgroundThe integration of molecular data from hosts, parasites, and microbiota can enhance our understanding of the complex biological interactions underlying the resistance of hosts to parasites. Haemonchus contortus, the predominant sheep gastrointestinal parasite species in the tropics, causes significant production and economic losses, which are further compounded by the diminishing efficiency of chemical control owing to anthelmintic resistance. Knowledge of how the host responds to infection and how the parasite, in combination with microbiota, modulates host immunity can guide selection decisions to breed animals with improved parasite resistance. This understanding will help refine management practices and advance the development of new therapeutics for long-term helminth control.MethodsEggs per gram (EPG) of feces were obtained from Morada Nova sheep subjected to two artificial infections with H. contortus and used as a proxy to select animals with high resistance or susceptibility for transcriptome sequencing (RNA-seq) of the abomasum and 50 K single-nucleotide genotyping. Additionally, RNA-seq data for H. contortus were generated, and amplicon sequence variants (ASV) were obtained using polymerase chain reaction amplification and sequencing of bacterial and archaeal 16S ribosomal RNA genes from sheep feces and rumen content.ResultsThe heritability estimate for EPG was 0.12. GAST, GNLY, IL13, MGRN1, FGF14, and RORC genes and transcripts were differentially expressed between resistant and susceptible animals. A genome-wide association study identified regions on chromosomes 2 and 11 that harbor candidate genes for resistance, immune response, body weight, and adaptation. Trans-expression quantitative trait loci were found between significant variants and differentially expressed transcripts. Functional co-expression modules based on sheep genes and ASVs correlated with resistance to H. contortus, showing enrichment in pathways of response to bacteria, immune and inflammatory responses, and hub features of the Christensenellaceae, Bacteroides, and Methanobrevibacter genera; Prevotellaceae family; and Verrucomicrobiota phylum. In H. contortus, some mitochondrial, collagen-, and cuticle-related genes were expressed only in parasites isolated from susceptible sheep.ConclusionsThe present study identified chromosome regions, genes, transcripts, and pathways involved in the elaborate interactions between the sheep host, its gastrointestinal microbiota, and the H. contortus parasite. These findings will assist in the development of animal selection strategies for parasite resistance and interdisciplinary approaches to control H. contortus infection in sheep.Graphical

Disease resistance and infectivity of virus susceptible and resistant common carp strains

Infectious diseases challenge health and welfare of humans and animals. Unlike for humans, breeding of genetically resistant animals is a sustainable solution, also providing unique research opportunities. Chances to survive a disease are improved by disease resistance, but depend also on chances to get infected and infect others. Considerable knowledge exists on chances of susceptible and resistant animals to survive a disease, yet, almost none on their infectivity and if and how resistance and infectivity correlate. Common carp (Cyprinus carpio) is widely produced in aquaculture, suffering significantly from a disease caused by cyprinid herpes virus type 3 (CyHV-3). Here, the infectivity of disease-resistant and susceptible fish types was tested by playing roles of shedders (infecting) and cohabitants (infected) in all four type-role combinations. Resistant shedders restricted spleen viral load and survived more than susceptible ones. However, mortality of susceptible cohabitants infected by resistant shedders was lower than that of resistant cohabitants infected by susceptible shedders. Virus levels in water were lower in tanks with resistant shedders leading to lower spleen viral loads in cohabitants. Thus, we empirically demonstrated that disease resistant fish survive better and infect less, with implications to epidemiology in general and to the benefit of aquaculture production.

Antimicrobial resistance and public and animal health risks associated with pathogenic Escherichia coli isolated from calves

We aimed to determine the antimicrobial susceptibility profile of pathogenic Escherichia coli strains isolated from fecal samples of calves and buffalo calves (2008–2013), in Minas Gerais, Brazil, as well as the frequency of O157 gene and strains carrying extended-spectrum beta-lactamases (ESBL) and mobile colistin resistance (mcr) genes. E. coli strains (n=518) were tested for susceptibility against ten antimicrobials. Tetracycline was the antimicrobial with the highest resistance rate (382/518), followed by ampicillin (321/518), sulfamethoxazole/trimethoprim (312/518), chloramphenicol (192/518), gentamicin (126/518), ciprofloxacin (148/518), cefazolin (89/518), colistin (54/518) and cefoxitin (34/518). Multidrug resistance (MDR) was observed in 381/518 isolates. No strain harbored mcr or O157 genes, whereas 19/99 were ESBL positive. The most prevalent pathotype and phylogroup were STEC and B1, respectively. Age, EHEC pathotype and resistance to aminoglycoside and cephem were significantly associated with MDR in the multivariate model. Overall, E. coli strains showed high rates of resistance to penicillin, tetracyclines and folate inhibitors, in addition to an alarming rate of MDR and ESBL-producing strains.