Significant Decrease In Gene Expression Research Articles

Acceleration of Wound Healing in Diabetic Rats through a Novel 3D Organo-Hydrogel Nanocomposite of Polydopamine/TiO2 Nanoparticles and Cu (PDA-TiO2@Cu).

Diabetic wound represents a serious issue with a substantial impact and an exceptionally complex pathology affecting patients' mental health and quality of life. So, we have developed a novel 3D organo-hydrogel nanocomposite of polydopamine/TiO2 nanoparticles and cu (PDA-TiO2@Cu) and examined its efficacy in diabetic wound healing. Forty-five adult male albino rats were divided into normal control rats (non-diabetic rats with non-treated skin wounds), diabetic control rats (diabetic rats with non-treated skin wounds), and organo-hydrogel-treated rats (diabetic wounds treated with topically applied organo- hydrogel once daily). Macroscopic changes of the wound were observed on days 0, 3, 5, 7, and 10 to measure wound diameters. Skin specimens from the wound tissue were taken on days 3, 7, and 10, respectively, and examined histologically and immunohistochemically. Also, the gene expressions of collagen I, Matrix Metalloproteinase-9 (MMP-9), and Epidermal Growth Factor (EGF), and levels of Interleukin 6 (IL-6) and Superoxide Dismutase (SOD) were assessed. Our observed results indicated that the developed patch significantly accelerated the healing time compared to the normal control and diabetic control groups. Moreover, the patchloaded group revealed complete re-epithelization and a highly significant increase in the mean area % of CD31 immunostaining on day 7. The organo-hydrogel-loaded group displayed a significant decrease in gene expression of MMP-9 and a significant increase in gene expression of EGF and collagen I. Additionally, the organo-hydrogel-loaded group exhibited a significant decrease in levels of IL-6 and a significant increase in levels of SOD, compared to the normal diabetic control groups. The organo-hydrogel can be used for treating and decreasing the healing period of diabetic wounds.

Antibacterial and antibiofilm activities of diclofenac against levofloxacin-resistant Stenotrophomonas maltophilia isolates; emphasizing repurposing of diclofenac

Background and Objectives: Stenotrophomonas maltophilia is an opportunistic pathogen causing nosocomial infections. Diclofenac is an anti-inflammatory drug that is considered a non-antibiotic drug. This study assessed the antibacterial and antibiofilm effects of diclofenac and levofloxacin/diclofenac combination against levofloxacin resistant isolates. Materials and Methods: Minimum inhibitory concentration was determined using broth microdilution method for levo- floxacin, diclofenac, and levofloxacin/diclofenac combination. Biofilm forming capacity and biofilm inhibition assay were determined. Relative gene expression was measured for efflux pump genes; smeB, and smeF genes and biofilm related genes rmlA, spgM, and rpfF without and with diclofenac and the combination. Results: Diclofenac demonstrated MIC of 1 mg/ml. The combination-with ½ MIC diclofenac- showed synergism where levofloxacin MIC undergone 16-32 fold decrease. All the isolates that overexpressed smeB and smeF showed a significant decrease in gene expression in presence of diclofenac or the combination. The mean percentage inhibition of biofilm for- mation with diclofenac and the combination was 40.59% and 46.49%, respectively. This agreed with biofilm related genes expression investigations. Conclusion: Diclofenac showed an antibacterial effect against Stenotrophomonas maltophilia. The combination showed in-vitro synergism, significant reduction in biofilm formation and in the relative level of gene expression. Furthermore, it can potentiate the levofloxacin activity or revert its resistance.

Effect of PCV-2 Vaccination on Cytokines Gene Expression Profile in Wild Boar Peripheral Blood Mononuclear Cells after Stimulation with Mycobacteria Antigens

Wild boar (Sus scrofa) is a common wild ungulate known as the most important reservoir of tuberculosis (TB) in Spain. The severity of TB lesions in this species and the high prevalence of porcine circovirus type 2 (PCV-2) have been related. PCV-2 is ubiquitous in swine populations, being usual for the free-living ones the contact with this agent. Recent studies found a correlation between a decrease of generalised TB prevalence in wild boar populations and the PCV-2-vaccination. The aim of this study was to find out if PCV-2 vaccination modulates the gene expression of cytokines from immune cells after its exposition with mycobacterial antigens using an in vitro methodology. A total of 46 wild boars from a PCV-2 infection endemic area were blood-sampled before and after the PCV-2 vaccination of 22 of them. Peripheral blood mononuclear cells (PBMC) were obtained and isolated from these samples. Aliquots of the cells were in vitro cultured and respectively stimulated with PPDa, PPDb, and a mitogen. A complete analysis of the gene expression of cytokines from the cultured PBMC was carried out. Also, Mycobacterium bovis and PCV-2 contacts were revealed by ELISA and/or qPCR. The results demonstrated that the animals which have had contact with PCV-2 and had been vaccinated, manifested a significant decrease in gene expression of proinflammatory cytokines, like interleukin 1 beta, interleukin 6, and tumour necrosis factor-alpha, possibly related with the severity of TB lesions, and also a significant decrease of interleukin 10, a key cytokine. In conclusion, in case of possible infection or contact events with the virus, PCV-2 vaccination could be an effective measure to reduce the TB severity in wild boar populations, which could decrease the intra and interspecies transmission of TB.

Dietary restriction plus exercise change gene expression of Cxcl12 abundant reticular cells in female mice.

Low energy availability due to excessive exercise lowers bone mass and impairs various physiological functions, including immunity and hematopoiesis. We focused on Cxcl12 abundant reticular (CAR) cells, which are bone marrow mesenchymal stem cells and are essential for the maintenance of hematopoietic and immune cells in bone marrow. We examine the functional changes in CAR cells resulting from dietary restriction combined with exercise. Five-week-old wild-type female mice were divided into an ad libitum group (CON), a 60% dietary restriction group (DR), an ad libitum with exercise group (CON + ex), and a 60% dietary restriction with exercise group (DR + ex). Blood parameters, bone structure parameters, and bone marrow fat volume were evaluated after 5weeks. In addition, bone marrow CAR cells were isolated by cell sorting and analyzed for gene expression by RT-qPCR. Bone mineral density (BMD) was significantly decreased in DR and DR + ex compared to CON and CON + ex. Especially, cortical bone mass and thickness were significantly decreased in DR and DR + ex groups, whereas trabecular bone mass was significantly increased. Bone marrow fat volume was significantly increased in DR and DR + ex groups compared to CON and CON + ex. The number of leukocytes in the blood was significantly decreased in the DR + ex group compared to the other three groups. RT-qPCR showed a significant decrease in gene expression of both Foxc1 and Runx2 in CAR cells of the DR + ex group compared to CON. Dietary restriction combined with exercise promotes CAR cell differentiation into bone marrow adipocyte and suppresses osteoblast differentiation.

RET enhancer haplotype-dependent remodeling of the human fetal gut development program.

Hirschsprung disease (HSCR) is associated with deficiency of the receptor tyrosine kinase RET, resulting in loss of cells of the enteric nervous system (ENS) during fetal gut development. The major contribution to HSCR risk is from common sequence variants in RET enhancers with additional risk from rare coding variants in many genes. Here, we demonstrate that these RET enhancer variants specifically alter the human fetal gut development program through significant decreases in gene expression of RET, members of the RET-EDNRB gene regulatory network (GRN), other HSCR genes, with an altered transcriptome of 2,382 differentially expressed genes across diverse neuronal and mesenchymal functions. A parsimonious hypothesis for these results is that beyond RET's direct effect on its GRN, it also has a major role in enteric neural crest-derived cell (ENCDC) precursor proliferation, its deficiency reducing ENCDCs with relative expansion of non-ENCDC cells. Thus, genes reducing RET proliferative activity can potentially cause HSCR. One such class is the 23 RET-dependent transcription factors enriched in early gut development. We show that their knockdown in human neuroblastoma SK-N-SH cells reduces RET and/or EDNRB gene expression, expanding the RET-EDNRB GRN. The human embryos we studied had major remodeling of the gut transcriptome but were unlikely to have had HSCR: thus, genetic or epigenetic changes in addition to those in RET are required for aganglionosis.

Genetic Variations and Altered Blood mRNA Level of Circadian Genes and BDNF as Risk Factors of Post-Stroke Cognitive Impairment Among Eastern Indians.

Post-stroke cognitive impairment (PSCI) is a clinical outcome in around 30% of post-stroke survivors. BDNF is a major gene in this regard. It is regulated by circadian rhythm. The circadian genes are correlated with stroke timings at molecular level. However, studies suggesting the role of these on susceptibility to PSCI are limited. We aim here to determine: (a) genetic risk variants in circadian clock genes, BDNF and (b) dysregulation in expression level of CLOCK, BMAL1, and BDNF that may be associated with PSCI. BDNF (rs6265G/A, rs56164415C/T), CLOCK (rs1801260T/C, rs4580704G/C), and CRY2 (rs2292912C/G) genes variants were genotyped among 119 post-stroke survivors and 292 controls from Eastern part of India. In addition, we analyzed their gene expression in Peripheral blood Mononuclear cells (PBMC) from 15 PSCI cases and 12 controls. The mRNA data for BDNF was further validated by its plasma level through ELISA (n = 38). Among the studied variants, only rs4580704/CLOCK showed an overall association with PSCI (P = 0.001) and lower Bengali Mini-Mental State Examination (BMSE) score. Its 'C' allele showed a correlation with attention deficiency. The language and memory impairments showed association with rs6265/BDNF, while the 'CC' genotype of rs2292912/CRY2 negatively influenced language and executive function. A significant decrease in gene expression for CLOCK and BDNF in PBMC (influenced by specific genotypes) of PSCI patients was observed than controls. Unlike Pro-BDNF, plasma-level mBDNF was also lower in them. Our results suggest the genetic variants in CLOCK, CRY2, and BDNF as risk factors for PSCI among eastern Indians. At the same time, a lowering expression of CLOCK and BDNF genes in PSCI patients than controls describes their transcriptional dysregulation as underlying mechanism for post-stroke cognitive decline.

Possible role of corticosterone on behavioral, physiological, and immune responses in chicks

Glucocorticoids are one of steroid hormone and have a variety of functions including stress response, carbohydrate metabolism, and modulation of immune system in vertebrates. Corticosterone is the main glucocorticoid in birds, although the precise role of the glucocorticoid during immune challenge is not fully understood. Therefore, the purpose of the present study was to determine if a single subcutaneous injection of corticosterone could affect inflammation-related gene expressions in the spleen and liver of chicks (Gallus gallus). In addition, the effects of corticosterone injection on the food intake, cloacal temperature, formation of conditioned visual aversion, and plasma constituents were also measured. Corticosterone did not affect the food intake or cloacal temperature and did not cause conditioned visual aversion in chicks. The corticosterone injection was associated with a significant decrease in gene expression of several pro-inflammatory cytokines including inducible nitric oxide synthase and cyclooxygenase-2 in the spleen and liver at 1 and 3 h post-injection. Corticosterone increased the plasma glucose and uric acid concentrations and the antioxidant capacity. In summary, the present study suggests that corticosterone is likely not associated with food intake, cloacal temperature or the development of aversive sensation, but suppresses the synthesis of inflammation-associated bioactive molecules and increases the antioxidant capacity in chicks.

Effects of Black Cumin Seed (Nigella sativa) and Coconut Meals (Cocos nucifera) on Broiler Performance and Cecal Microbiota.

The objective of this study was to investigate the effect of dietary supplementation with black cumin seed meal (BCSM) and coconut meal (CM) on the performance and cecal microbiota of Cobb 500 hybrid broilers. The study was conducted on 600 chicks on the first day of hatching; the chicks were randomly distributed equally into 12 equal-sized floor pens. Four dietary treatments (C, T1, T2 and T3) were replicated three times (50 chicks/replicate): C was the control group; T1 was supplemented with 10% BCSM; T2 was supplemented with 10% CM; T3 was supplemented with 5% BCSM and 5% CM. At slaughter age on day 35, our findings showed that treatment T2 increased significantly body weight and feed conversion ratio (FCR) compared to C, T1 and T3. In addition, the hot carcass dressing percentages in treatments T1, T2, and T3 were significantly higher than that of the C group. The results of relative normalized comparative gene expression of Clostridioides difficile, Roseburia and Streptococcus were not significantly changed in all treatments (p > 0.05). Treatment T1 resulted in a significant decrease in gene expression of the entire microbiota, while treatment T2 resulted in a significant increase in gene expression of all microbes, leading to an enriched and diverse microbial community. It can be concluded that supplementation with 10% BCSM is beneficial in inhibiting pathogenic microbes during early post-hatch days. In contrast, CM may promote and enhance the diversity of microbial communities during broiler growth. The inclusion of non-conventional feed ingredients in poultry diets may improve growth performance and may reduce the cost of broiler feed.

Analysis of Promoter Methylation of the Bovine FOXO1 Gene and Its Effect on Proliferation and Differentiation of Myoblasts.

This study aimed to explore the regulatory role of FOXO1 promoter methylation on its transcriptional level and unravel the effect of FOXO1 on the proliferation and differentiation of bovine myoblasts. Bisulfite sequencing polymerase chain reaction (BSP) and real-time quantitative PCR were performed to determine the methylation status and transcript levels of the FOXO1 promoter region at different growth stages. BSP results showed that the methylation level in the calf bovine (CB) group was significantly higher than that in the adult bovine (AB) group (p < 0.05). On the other hand, qRT-PCR results indicated that the mRNA expression level in the AB group was significantly higher than that in the CB group (p < 0.05), suggesting a significant decrease in gene expression at high levels of DNA methylation. CCK-8 and flow cytometry were applied to determine the effect of silencing the FOXO1 gene on the proliferation of bovine myoblasts. Furthermore, qRT-PCR and Western blot were conducted to analyze the expression of genes associated with the proliferation and differentiation of bovine myoblasts. Results from CCK-8 revealed that the short hairpin FOXO1 (shFOXO1) group significantly promoted the proliferation of myoblasts compared to the short-hairpin negative control (shNC) group (p < 0.05). Flow cytometry results showed a significant decrease in the number of the G1 phase cells (p < 0.05) and a significant increase in the number of the S phase cells (p < 0.05) in the shFOXO1 group compared to the shNC group. In addition, the expression of key genes for myoblast proliferation (CDK2, PCNA, and CCND1) and differentiation (MYOG, MYOD, and MYHC) was significantly increased at both mRNA and protein levels (p < 0.05). In summary, this study has demonstrated that FOXO1 transcription is regulated by methylation in the promoter region and that silencing FOXO1 promotes the proliferation and differentiation of bovine myoblasts. Overall, our findings lay the foundation for further studies on the regulatory role of epigenetics in the development of bovine myoblasts.

RNA Sequencing of Intestinal Enterocytes Pre- and Post-Roux-en-Y Gastric Bypass Reveals Alteration in Gene Expression Related to Enterocyte Differentiation, Restitution, and Obesity with Regulation by Schlafen 12.

Background: The intestinal lining renews itself in a programmed fashion that can be affected by adaptation to surgical procedures such as gastric bypass. Methods: To assess adaptive mechanisms in the human intestine after Roux-en-Y gastric bypass (RYGB), we biopsied proximal jejunum at the anastomotic site during surgery to establish a baseline and endoscopically re-biopsied the same area 6–9 months after bypass for comparison. Laser microdissection was performed on pre- and post-RYGB biopsies to isolate enterocytes for RNA sequencing. Results: RNA sequencing suggested significant decreases in gene expression associated with G2/M DNA damage checkpoint regulation of the cell cycle pathway, and significant increases in gene expression associated with the CDP-diacylglycerol biosynthesis pathway TCA cycle II pathway, and pyrimidine ribonucleotide salvage pathway after RYGB. Since Schlafen 12 (SLFN12) is reported to influence enterocytic differentiation, we stained mucosa for SLFN12 and observed increased SLFN12 immunoreactivity. We investigated SLFN12 overexpression in HIEC-6 and FHs 74 Int intestinal epithelial cells and observed similar increased expression of the following genes that were also increased after RYGB: HES2, CARD9, SLC19A2, FBXW7, STXBP4, SPARCL1, and UTS. Conclusions: Our data suggest that RYGB promotes SLFN12 protein expression, cellular mechanism and replication pathways, and genes associated with differentiation and restitution (HES2, CARD9, SLC19A2), as well as obesity-related genes (FBXW7, STXBP4, SPARCL1, UTS).

Decreasing TNFα in serum and increasing mitotic index rate in albino mice treated with melatonin

Melatonin, as an endogenous hormone, is associated with the moderation of the circadian rhythm. Besides controlling the sleep-wake cycle, many physiological functions of melatonin have been identified, such as antioxidant, immune modulating, and anti-inflammatory. The current study was designed to estimate the effect of different doses of melatonin on gene expression of (TNF) in serum and mitotic index (MI) in bone marrow stem cells as cytogenetic test. This study was conducted at Wasit University, College of Science, Department of Biology, Cytogenetic Laboratory, from the period 2/11/2021 to 14/4/2022 using albino mice. The mice were divided into four groups, a control group and three groups divided according to melatonin concentration (5, 10, &15 mg/kg), and were treated for seven consecutive days for every dose. Results showed a significant decrease in gene expression (protein) of TNF α- at P≤0.05 for all three doses of melatonin that were used and mitotic index numbers increased at P≤0.05 for the same doses above of melatonin, when all results above were compared with the control group.

FliA, flrB, and fliR regulate adhesion by controlling the expression of critical virulence genes in Vibrio harveyi

Bacteria adhesion to fish mucus is a crucial virulence mechanism. As the initial step of bacterial infection, adhesion is impacted by bacterial motility and environmental conditions. However, its molecular mechanism is yet unclear. In this study, a significant decrease in gene expression of adhesion-deficient Vibrio harveyi was observed when the bacteria were subjected by Cu2+(50 mg/L), Pb2+(100 mg/L), Hg2+(25 mg/L), and Zn2+(50 mg/L). The genes fliA, fliR, and flrB were responsible for flagellation; being crucial for adhesion, these genes were identified and silenced via RNAi. After silencing of these genes by RNAi technology, the ability of adhesion, biofilm formation, motility, and flagella synthesis of V. harveyi were considerably reduced. Compared with the control group, it was observed that the expression levels of fliS, fliD, flgH, and flrC were significant down-regulated in fliR-RNAi, flrB-RNAi, and fliA-RNAi. This data indicates that the expression levels of most virulence genes are affected by fliA, fliR, and flrB. Also, the expression of fliA, fliR, and flrB can be influenced by the salinity, temperature, and pH. The results show that: (1) fliA, fliR, and flrB have important roles in the adhesion of V. harveyi; (2) fliA, fliR, and flrB can regulate bacterial adhesion by affecting its motility, and biofilm formation; (3) fliA, fliR, and flrB can regulate adhesion ability of V. harveyi in different environments.

27-OR: Fatty Acid Uptake and Metabolism in Human Adipose Tissue Is Decreased upon Consuming an Isocaloric High–Saturated Fat Diet and Partially Genetically Determined

CD36 transports fatty acids (FA) into adipocytes and is increased in white adipose tissue (WAT) in obesity. In rodents, however, it was shown that CD36 knock out is protective for diet induced adiposity. In humans a hypocaloric high fat diet decreased CD36 gene expression in WAT. This study investigated the response of gene expression of FA uptake and metabolism in sub cutaneous WAT after a sudden switch from a healthy isocaloric low fat diet to an isocaloric high fat diet (HF) in healthy human twins. 35 monzyogotic and dizygotic healthy pairs of twins (women and men, age 18 - 70) were investigated for 12 weeks. An isocaloric diet rich in carbohydrates (55% carbohydrates, 30% fat, 15% protein, LF) was applied for 6 weeks, followed by an isocaloric diet rich in saturated fat (40% carbohydrates, 45% fat, 15% protein, HF) for another 6 weeks. Body weight (BW) , BMI, serum FA and gene expression of CD36, PDK4, PGC1α and CPT1α by qPCR were analyzed after the LF period, after one week (HF1) and at the end (HF6) of the HF period. BW did not change during the study. Serum FA were unchanged at HF1 but decreased at HF6 by 18% (P<0.compared to LF) . Gene expression of CD 36 was strongly decreased by 33% at HF1 and by 26% at HF6, respectively, compared to LF (both P<0.001) . A significant decrease of gene expression of PDK4 by 18% was observed at HF1 and by 27% at HF6 (both P<0.0compared to LF) . Gene expression of PGC1α and CPT1α were not changed at HF1 but decreased both at HF6 by 14% (both P<0.0compared to LF) and were highly heritable: 64% for PGC1α and 48% for CPT1α. Gene expression was positively correlated with BMI for CD36 at HF1 and for PDK4 at LF, HF1 and HF6 (P<0.05) . These data indicate that gene expression of transport (CD36) and re-esterification (PDK4) of FA are not only dependant on the amount of nutritional fat, but also on energy content. Moreover, expression of genes controlling mitochondrial (PGC1α) and FA (CPT1α) metabolism appear to be genetically determined. Disclosure M.Kruse: None. A.Ost: None. S.Hornemann: None. D.Hoffmann: None. T.Frahnow: None. A.Busjahn: None. A.F.Pfeiffer: None. Funding German Federal Ministry of Education and Research (0315424)

Inhibition of DNA-Pkcs As Novel Therapy for the Prevention of Acute Gvhd Following Hematopoietic Stem Cell Transplantation

Hematopoietic stem cell transplantation (HCT) is an effective therapeutic option for many cancers and disorders of immune dysfunction. However, acute graft versus host disease (GVHD) remains a morbid complication of HCT, caused by donor T cells attacking the recipient's organs. GVHD can be severe, resulting in multiorgan failure and death. Current immunosuppressive therapies remain marginally effective. The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is emerging as a master regulator of the immune system. It is required for lymphocyte maturation and immune system diversity due to its role in V(D)J recombination. Our published data and others have determined that DNA-PKcs is required for cytokine expression, namely IL2, IL4, IL6, IL10, IFN, and TNF. Therefore, we hypothesized that DNA-PKcs inhibitors could serve as effective immunosuppressants for transplant patients to prevent rejection, which we tested in a murine allogeneic skin graft model. Ear skin from Balb/c mice was transplanted onto the backs of C57bl/6 or DNA-PKcs null mice (KO). C57bl/6 mice were treated with saline, FK506, or NU7441, a potent and selective DNA-PKcs inhibitor. Treatment with NU7441 significantly reduced graft rejection compared to both saline and FK506. Similar levels were observed in the KO mice, with less than 10% rejection after 10 days when a majority of the controls were completely rejected. These results led us to hypothesize that DNA-PKcs is required for cytotoxic activity of T cells, highlighting the potential use of DNA-PKcs inhibitors to prevent and/or treat GVHD in HCT patients .In order to evaluate the role of DNA-PKcs on T cell activation, we isolated CD8+ T cells from mouse spleens using negative selection via magnetic column. The T cells were then activated with anti-CD3/CD28 for 24-48 hours in the presence of NU7441 or vehicle (DMSO), and the cells were analyzed by flow cytometry. A fully activated T cell expresses both CD69 (early) and CD25 (later) on the surface. T cells that had been activated with NU7441 had a higher expression of CD69 and lower expression of CD25 (figures 1&2). These results are even more pronounced after 48 hours, with a much lower percent of cells expressing both CD69 & CD25 compared to T cells that had been activated in the presence of DMSO alone (figure 3). This suggests that DNA-PKcs plays a role in the activation pathway of cytotoxic T cells - the T cells are blocked from becoming completely activated as evidenced by a significant decrease in cells expressing both CD69 and CD25. If DNA-PKcs is required for the complete activation of CD8+ T cells, this may have therapeutic implications in the prevention of GVHD as cytotoxic T cells are required to cause the pathophysiologic manifestations of GVHD.We then addressed whether DNA-PKcs affects the expression of cytotoxic genes in CD8+ T cells. The T cells were isolated and activated in the presence of NU7441 or vehicle as described above. Total RNA was then isolated from the activated T cells, and qPCR was performed to assess the difference in cytotoxic gene expression. Several genes required for cytotoxic function of CD8+ T cells, including perforin-1, granzyme, and eomes. IL2Ra is the gene coding the CD25 surface marker/IL2 receptor. CTLA4 and TGFb are associated with immune tolerance. A statistically significant decrease in gene expression was seen when treating with NU7441 in the expression of granzyme (p-value 0.01), eomes (p-value <0.01), and IL2Ra (p-value 0.04). While other data was not statistically significant, an interesting trend was seen in increased expression of TGFb. Overall, the significant data suggests that DNA-PKcs plays a role in the expression of certain cytotoxic genes, and may lead to overall decreased cytotoxic activity, perhaps by both decreasing expression of certain genes required for cytotoxic activity/cytokine production as well as increasing the expression of other genes involved in immune tolerance. Both of these effects could potentially lead to a favorable outcome in prevention of acute GVHD.This data serves as an excellent proof of concept for testing the hypothesis that DNA-PKcs inhibitors can prevent acute GVHD in an animal model. This preclinical model would potentially lay the foundation to move into preclinical studies and eventually phase I clinical trials to prevent and/or treat GVHD, creating a new therapeutic option for a morbid complication for HCT patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 16: Altered Testicular Macrophage Polarization Is Associated With Reproductive Dysfunction In Hypertensive Mice

Elevated circulating proinflammatory (M1) and decreased anti-inflammatory (M2) macrophages contribute to hypertension (HTN) and end-organ damage. HTN is associated with reproductive dysfunction in men. However, the impact of HTN on testicular macrophages and inflammation is unknown. We hypothesized that HTN increases M1 and decreases M2 testicular macrophages, which is associated with inflammation and reproductive dysfunction. Male mice were made hypertensive by either providing L-arginine methyl ester hydrochloride (L-NAME) (0.5 mg/mL) in the drinking water for 3 weeks [L-NAME-induced HTN (LHTN)] or L-NAME water for 2 weeks, followed by a 2-week washout period and a subsequent 3-week 4% high salt diet [salt-sensitive hypertension (SSHTN)]. Control (C) mice received tap water and normal diet. Flow cytometry analysis revealed a significant increase in both M1 (C: 15%±1, LHTN: 22%±2; p&lt;0.05) and M2 (C: 10%±1, LHTN: 21%±2; p&lt;0.05) macrophages in testes from LHTN mice. Similarly, testes from SSHTN mice had a significant increase in M1 (C: 17%±1, SSHTN: 28%±2; p&lt;0.05) but had a significant decrease in M2 (C: 14%±1, SSHTN: 7%±1; p&lt;0.05) macrophages. Testes from both hypertension models had a significant increase in gene expression of the proinflammatory cytokines TNFa, IFNg, IL-1b, IL-6, and IL-17. Sperm concentration (C: 8.5±0.7, LHTN: 6.5±0.2, SSHTN: 4.7±0.5; both p&lt;0.05) and the percentage of sperm mitochondrial activity (C: 88%±3, LHTN: 71±5, SSHTN: 64%±3; both p&lt;0.05) were decreased significantly in both hypertension groups. Hypertensive mice presented a significantly increased percentage of sperm with abnormal morphology (C: 5%±1, LHTN: 8%±1, SSHTN: 13%±2; both p&lt;0.05) and damaged acrosome (C: 1.4%±0.2, LHTN: 2.8%±0.2, SSHTN: 4%±0.5; both p&lt;0.05). There was a significant decrease in gene expression of the hormone receptors AR, ERa, and LHR, and the steroidogenic enzymes StAR, 3bHSD, 17bHSD, and CYP17a1 in the testes of LHTN and SSHTN mice. These data demonstrate that HTN alters testicular macrophage polarization which is associated with inflammation and impaired reproductive health. Therapeutic strategies may be developed to improve reproductive health in male hypertensive patients by targeting testicular macrophage imbalance.

H2A.B is a cancer/testis factor involved in the activation of ribosome biogenesis in Hodgkin lymphoma.

Testis-specific regulators of chromatin function are commonly ectopically expressed in human cancers, but their roles are poorly understood. Examination of 81 primary Hodgkin lymphoma (HL) samples showed that the ectopic expression of the eutherian testis-specific histone variant H2A.B is an inherent feature of HL. In experiments using two HL cell lines derived from different subtypes of HL, H2A.B knockdown inhibited cell proliferation. H2A.B was enriched in both nucleoli of these HL cell lines and primary HL samples. We found that H2A.B enhanced ribosomal DNA (rDNA) transcription, was enriched at the rDNA promoter and transcribed regions, and interacted with RNA Pol I. Depletion of H2A.B caused the loss of RNA Pol I from rDNA chromatin. Remarkably, H2A.B was also required for high levels of ribosomal protein gene expression being located at the transcriptional start site and within the gene body. H2A.B knockdown reduced gene body chromatin accessibility of active RNA Pol II genes concurrent with a decrease in transcription. Taken together, our data show that in HL H2A.B has acquired a new function, the ability to increase ribosome biogenesis.

Food Enrichment with Glycyrrhiza glabra Extract Suppresses ACE2 mRNA and Protein Expression in Rats-Possible Implications for COVID-19.

Angiotensin converting enzyme 2 (ACE2) is a key entry point of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus known to induce Coronavirus disease 2019 (COVID-19). We have recently outlined a concept to reduce ACE2 expression by the administration of glycyrrhizin, a component of Glycyrrhiza glabra extract, via its inhibitory activity on 11beta hydroxysteroid dehydrogenase type 2 (11betaHSD2) and resulting activation of mineralocorticoid receptor (MR). We hypothesized that in organs such as the ileum, which co-express 11betaHSD2, MR and ACE2, the expression of ACE2 would be suppressed. We studied organ tissues from an experiment originally designed to address the effects of Glycyrrhiza glabra extract on stress response. Male Sprague Dawley rats were left undisturbed or exposed to chronic mild stress for five weeks. For the last two weeks, animals continued with a placebo diet or received a diet containing extract of Glycyrrhiza glabra root at a dose of 150 mg/kg of body weight/day. Quantitative PCR measurements showed a significant decrease in gene expression of ACE2 in the small intestine of rats fed with diet containing Glycyrrhiza glabra extract. This effect was independent of the stress condition and failed to be observed in non-target tissues, namely the heart and the brain cortex. In the small intestine we also confirmed the reduction of ACE2 at the protein level. Present findings provide evidence to support the hypothesis that Glycyrrhiza glabra extract may reduce an entry point of SARS-CoV-2. Whether this phenomenon, when confirmed in additional studies, is linked to the susceptibility of cells to the virus requires further studies.

Effect of 8 weeks high-intensity interval training (HIIT) with and without caloric restriction on gene expression of myocardial Bax and Bcl2 in mice

Background: Apoptosis is the physiological cell death that in natural conditions leads to the elimination of old, damaged, waste, and harmful cells. The aim of this study was the effect of eight weeks of high-intensity interval training (HIIT) with and without caloric restriction on gene expression of myocardial Bax and Bcl2 in mice. Methods: Present study was an experimental multi-group design with a control group conducted on 30 two-month old male mice. Subjects were divided into five homogenous groups including base control, control, caloric restriction, interval exercise training, and caloric restriction + interval exercise training. Training groups participated in interval exercise training five sessions per week for 8 weeks. The level of gene expression of myocardial Bax and Bcl2 was evaluated by real-time PCR. Data were analyzed using the one-way ANOVA at the level of (P&lt;0.05). Results: The results showed that the training group had a significant increase in gene expression of myocardial Bcl2 in comparison with caloric restriction + exercise training (P&lt;0.05) and a significant decrease in gene expression of myocardial Bax compared to the caloric restriction group (P&lt;0.05). Also, exercise training and exercise training + caloric restriction significantly increased the gene expression of myocardial Bcl2 and significantly decreased Bax/Bcl2 ratio compared to caloric restriction, base control, and control (P&lt; 0.05). Conclusion: It seems that high-intensity interval training without caloric restriction would provide a suitable environment for increasing the integrity of the mitochondrial membrane of myocardial cells and possibly apoptosis.

Anticancer Activity and In Vitro to In Vivo Mechanistic Recapitulation of Novel Ruthenium-Based Metallodrugs in the Zebrafish Model.

Ruthenium is popular as a metal core for chemotherapeutics, due to versatile molecular coordination. Because new metallodrugs are synthesized at high rates, our studies included assays in zebrafish to expedite the initial evaluation as anticancer agents. Here we evaluated novel metallodrugs (PMC79 and LCR134), and cisplatin, a widely used platinum-based chemotherapeutic. We hypothesized that this model could characterize anticancer properties and recapitulate previous in vitro results in vivo. Our findings suggest anticancer properties of PMC79 and LCR134 were similar with less toxicity than cisplatin. Exposures from 24 to 72 h at or below the LOAELs of PMC79 and LCR134 (3.9 µM and 13.5 µm, respectively), impaired blood vessel development and tailfin regeneration. Blood vessel examination through live imaging of larvae revealed distinct regional antiangiogenic impacts. The significant decrease in gene expression of the VEGF-HIF pathway and beta-actin could explain the morphological effects observed in the whole organism following exposure. Tailfin amputation in larvae exposed to PMC79 or LCR134 inhibited tissue regrowth and cell division, but did not impact normal cell proliferation unlike cisplatin. This suggests Ru drugs may be more selective in targeting cancerous cells than cisplatin. Additionally, in vitro mechanisms were confirmed. PMC79 disrupted cytoskeleton formation in larvae and P-glycoprotein transporters in vivo was inhibited at low doses which could limit off-target effects of chemotherapeutics. Our results demonstrate the value for using the zebrafish in metallodrug research to evaluate mechanisms and off-target effects. In light of the findings reported in this article, future investigation of PMC79 and LCR134 are warranted in higher vertebrate models.

ANGIOTENSIN AT2 RECEPTORS IN THE NUCLEUS OF THE SOLITARY TRACT REDUCE BLOOD PRESSURE VIA INHIBITION OF GABA SIGNALING

Objective: Sustained CNS administration of angiotensin AT2 receptor (AT2R) agonists lowers blood pressure (BP) in normal and hypertensive rodents, but the location(s) and mechanism(s) of action are unknown. One locus may be the intermediate solitary tract nucleus (intNTS), which contains a high density of AT2R located on GABA neurons, and it is well-known that increased GABA signaling in the intNTS contributes to increased BP and hypertension. Our studies have revealed that reductions in BP (∼8 mmHg) induced by 2-week intracerebroventricular (icv) infusion of the AT2R agonist C21 (7.5 ng/kg/h) in normotensive mice is associated with significant decreases in gene expression of GABA synthetic enzymes (GAD-1 &amp; GAD-2) in the intNTS. From this, we developed the hypothesis that the depressor action of C21 is mediated through AT2R located on intNTS GABA neurons and a reduction of GABA signaling. Design and method: To test this, we engineered a novel mouse line that has Cre-recombinase expression directed to the AT2R gene (AT2R-Cre), and demonstrated that Cre recombinase activity mirrors AT2R mRNA expression within the NTS. Next, we used the Cre-LoxP system and virally (AAV)-mediated gene transfer to direct expression of the light-sensitive excitatory opsin channelrhodopsin-2 (ChR2) and/or eYFP specifically to neurons in the NTS that synthesize AT2R. Using these mice, we performed cardiovascular recordings during optogenetic manipulations (blue light) that selectively excite AT2R-expressing neurons in the intNTS. Results: These studies revealed that: (i) Optogenetic stimulation of the AT2R intNTS neurons expressing ChR2 in isoflurane anesthetized mice elicited significant increases in BP compared with control mice that express only eYFP; (ii) The AT2R-containing neurons that were activated by blue light stimulation are GABAergic, as indicated by increased expression and co-localization of c-Fos mRNA with GAD-1 mRNA and eYFP; (iii) Chronic icv infusion of C21 as above significantly blunted the light-induced increases in BP, an effect that was present across all patterns of optical stimulation. Conclusions: Collectively, these data indicate that AT2R-mediated decreases in BP involve a blunting of GABA signaling in the intNTS, and suggest that neurons expressing AT2R at this site may be an access point for cardiovascular control.