Biomedical Research Laboratories Research Articles

Effects of CD8 T-cell transfer on biomechanics of post-myocardial infarction scar formation

CD8+ T-cells are adverse regulators of post-MI remodeling, resulting in attenuated cardiac function and overall survival. Based on previous studies, we hypothesize that CD8+ T-cells impair cardiac function by altering scar composition. MI was induced by ligating the left anterior descending coronary artery on C57BL6/J wildtype (WT; 3-7 months of age, n≥2/sex) and CD8atm1mak (CD8−/−; 3-7 months of age, n≥2/sex/ group) mice. CD8−/− mice were injected with either vehicle or naïve splenic CD8+ T-cells via tail vein, 4-hours post-coronary artery ligation to assess potential effects on the scar. On day 7 post-MI, infarct tissue was collected, undergoing either passive stretch biomechanical analysis or histological and biochemical assays for collagen composition. Effects of granzyme (Gzm) B and K on collagen cleavage were tested using a fluorogenic collagen cleavage assay to examine possible mechanisms of scar alteration. Mice lacking CD8+ T-cells had improved ejection fraction and decreased dilation compared to WT mice at post-MI Day 7. This protective effect was lost in CD8−/− mice that received splenic CD8+ T-cells. Biomechanical analysis demonstrated CD8−/− mice had a 2-fold increase in regional stiffness compared to WT mice. Re-supplementation with splenic CD8+ T-cells decreased scar tissue stiffness mimicking biomechanics of WT mice. Picrosirius red staining surprisingly showed no significant differences in total collagen levels (p=0.51). Immunoblotting for Collagen 1 trended an increase in the 250 kDa band in CD8−/− mice that decreased after supplementation indicating a loss of pro-collagen formation (p=0.051). Ex-vivo cleavage assay demonstrated GzmK cleaved collagen in a concentration (0-50 AU) and temporal-dependent manner (0-24 hrs). Moreover, GzmK demonstrated a 10-fold greater capacity in collagen cleavage compared to GzmB at 25 AU, indicating the role GzmK plays during post-MI remodeling may be distinctly separate from that of GzmB. In conclusion, our data demonstrates that CD8+ T-cells regulate cardiac fibrosis potentially in part through the release of granzymes, leading to alterations in biomechanical capability of the left ventricle. This work was supported by the National Institutes of Health T32GM123055, R25GM113278; the Biomedical Laboratory Research and Development Service of the Veterans Affairs Offce of Research and Development Award IK2BX003922 and BX003922; and South Carolina Translational Research Center UL1TR001450. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

PTSD-like mice display negative cardiac characteristics post-inescapable foot shock

Post-traumatic stress disorder (PTSD) is a risk factor for cardiovascular disease, however the mechanism behind this correlation is unknown. We hypothesized that PTSD alters cardiac homeostasis by increasing macrophage numbers and initiating cardiac fibrosis. To induce experimental PTSD, male and female C57BL/6 mice (4-weeks: n=16-17/sex, males=4.7±0.4 months old; females=4.8±0.3 months old; 8-weeks: n=13-16/sex, males=6.0±0.3 months old; females=6.3±0.5 months old) were exposed to 5 separate foot-shock incidences (IFS; 1.0 mA, 1 sec duration) in 6 min. Control mice (n=8-9/sex) were placed in the same chambers but experienced no foot shocks. Behavioral tests (16 parameters based on the DSM-5 clinical standards) were performed to characterize mice that do not demonstrate PTSD-like behavioral characteristics (NR) and PTSD-like mice. Doppler echocardiography measurements were collected serially up to 8-weeks post-IFS. Histological and gene assessments were collected 4- and 8-weeks post-IFS to measure markers of adverse cardiac remodeling that resulted in later functional changes. Over time PTSD-like mice demonstrated a decline in cardiac function becoming significantly impaired by 8-weeks as shown by an increase in E/e’ ratio along with left atrial diameter compared to controls, indicative of impaired LV filling pressure and diastolic dysfunction (p<0.05 for all). Beginning at 4-weeks post-IFS, PTSD-like mice had increased picrosirius red staining (PSR) compared to control mice (p<0.05) that remained elevated at 8-weeks. PTSD-like mice had increased macrophage numbers (Mac3) at 4-weeks post-IFS compared to control mice (p<0.05) and showed an increased trend when compared to controls 8-weeks post-IFS (p=0.09). To determine potential signaling mechanisms, Ccl2, Ccl5, and discoidin domain receptor tyrosine kinase 2 (DDR2) mRNA levels were assessed. Gene expression of Ccl2 demonstrated a decreased trend in PTSD-like mice (p=0.13), while Ccl5 (p=0.08) and DDR2 (p=0.06) mRNA demonstrated an increased trend in PTSD-like mice when compared to controls. Our data indicates that in a mouse model of PTSD, animals that demonstrate PTSD-like behaviors show signs of increased cardiac inflammation stimulating cardiac remodeling and cardiac dysfunction. This work was supported by the National Institutes of Health T32GM123055; the American Heart Association Innovator Project IPA35260039, GM113278; the Biomedical Laboratory Research and Development Service of the Veterans Affairs Offce of Research and Development Award IK2BX003922, I01BX00584; and South Carolina Translational Research Center UL1TR001450. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Chronic kidney disease alters tryptophan metabolite concentrations in male and female Sprague Dawley rats

Kynurenine is the first stable metabolite of tryptophan degradation. An increased ratio of kynurenine (KYN) to tryptophan (TRP) has been implicated in many disease states including chronic kidney disease (CKD) in humans. We hypothesized that we would see a similar increase in KYN/TRP in a CKD model in male and female rats. We induced CKD with a 0.25% adenine diet for 15 weeks and then measured a variety of renal parameters. Serum and kidneys were collected at sacrifice. We found that GFR was better preserved in CKD female rats compared to males (0.232 ± 0.056 vs. 0.081 ± 0.019 ml/min/100g BW) but was decreased significantly compared to controls (0.739 ± 0.048 female, 0.635 ± 0.099 male). Urine volume was elevated significantly in CKD rats compared to controls (80.0 ± 6.9 ml/day male CKD, 72.5 ± 4.7 female CKD vs. 13.3 ± 1.1 male CTL and 22.0 ± 1.8 female CTL). However, male CKD rats consumed significantly more water than female CKD (104.1 ± 7.3 ml/day male vs. 81.0 ± 5.1 female), although water consumption was similar in controls in both sexes (30.9 ± 2.6 male and 30.1 ± 2.5 female). Weekly food consumption and body weight were similar between same sex groups. Both male and female rats showed increased KYN/TRP in CKD compared to controls (0.102 ± 0.010 in male and 0.105 ± 0.011 in female CKD vs. 0.049 ± 0.005 and 0.033 ± 0.002 CTL). Similarly, kynurenic acid was elevated significantly in both sexes in CKD (305.7 ± 76.7 nmol/L male and 669.2 ± 140.7 female) compared to controls (92.3 ± 17.7 and 118.1 ± 14.7) respectively. These data show that the adenine feeding model of CKD reproduces many of the characteristics of human disease including alterations in TRP metabolites. This model will allow study of TRP metabolites in CKD progression. This work was supported by Career Development Award IK2 BX004997 from the United States Department of Veteran Affairs, Biomedical Laboratory Research and Development Service. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Endothelin-1 Correlates with Blood Pressure and Pulse Pressure in Females with Lupus Associated Hypertension

Systemic Lupus Erythematosus (SLE) is a common autoimmune disease that is classified by IgG complex deposition in end organs including the kidney, heart, and vasculature. SLE disproportionally affects women to men (9:1 ratio), with a high prevalence in African American women. Importantly, patients with an SLE diagnosis have common cardiovascular complications including hypertension. Importantly, numerous studies have reported that plasma and urinary endothelin-1 (ET-1) levels are elevated in patients with SLE or hypertension. However, few studies have investigated the role of the synergistic effects of SLE and hypertension on ET-1. Therefore, we hypothesized that women with SLE and hypertension would have elevated plasma ET-1 levels compared to SLE or hypertension alone. To investigate this, we recruited non-SLE non-HTN controls (n=8), non-SLE HTN (n=7), SLE non-HTN (n=5), and SLE-HTN (n=6) female human subjects and analyzed clinical and demographic characteristics, plasma ET-1, c-reactive protein (CRP), soluble vascular adhesion molecule-1 (sVCAM-1), and anti-Smith autoantibodies. We found that plasma ET-1 levels are significantly higher in African American compared to European American patients independent of SLE or blood pressure status (p=0.0182). When plasma ET-1 levels were correlated with age and body mass index (BMI), ET-1 had a positive association with age (r=0.4861;p=0.0118), but no correlation with BMI (r=0.2137;p=0.2945). When subjects we characterized by SLE diagnosis, we found that female subjects with SLE had elevated plasma ET-1 levels compared to non-SLE controls (p=0.0141). Next, we stratified subjects by blood pressure status and found that only SLE-HTN subjects had significantly elevated plasma ET-1 levels compared to non-SLE non-HTN controls (p=0.0005), and a trend for elevated ET-1 levels over SLE non-HTN (p=0.0799). Lastly, we correlated plasma ET-1 levels to offce systolic blood pressure, diastolic blood pressure and calculated pulse pressure. We found that plasma ET-1 positively associated with systolic blood pressure (r=0.5621;p=0.0035) and pulse pressure (r=0.5810;p=0.0023) but not diastolic blood pressure (r=0.3177;p=0.1217). These data demonstrate that plasma ET-1 levels associate with SLE diagnosis and blood pressure status in our cohort of female SLE human subjects. Further investigation of ET signaling should be done in SLE and SLE-HTN human subjects. Veterans Affairs Biomedical Laboratory Research & Development Career Development Award (CDA-2) 1IK2BX005605-01 to J.P.V.B, Medical University of South Carolina College of Medicine Program Project Grant to J.C.O and J.P.V.B., and Medical University of South Carolina Core Centers for Clinical Research Project Award P30AR072582 to M.A.C and J.P.V.B. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sex Differences of the Endothelin System in the Cardio-Renal Axis of a Lupus Prone Mouse Model

Systemic Lupus Erythematosus (SLE) is the most common form of lupus autoimmunity. It is characterized by overactivation of toll-like receptor 7 (TLR7) and immune complex deposition in target organs including the kidney and vasculature. A lupus diagnosis disproportionately affects women at a rate of 9:1 compared to men. Clinically, patients present with heterogenous symptoms including chronic hypertension, heart failure, renal failure, vascular disease, and neuropathies. Despite a variety of clinical presentations of lupus, elevated endothelin-1 (ET-1) has been detected in both male and female lupus patients' plasma compared to healthy controls. However, the exact sex differences in ET-1 in the development of lupus and its associated cardiovascular complications remain unknown. Therefore, we hypothesized that acceleration of SLE through TLR7 activation promotes sex-dependent regulation of the ET system within the cardiorenal axis. To investigate this, female and male lupus-prone B6.Nba2 mice were treated with a TLR7/8 agonist (R848; 100ug/30ul) or acetone as a control topically twice a week for 4 weeks. After the R848 treatment, mice were followed for 12 more weeks until the 16-week terminal timepoint where the kidney and heart tissues were collected and tissue ET-1, endothelin receptor A (ETA) and endothelin receptor B (ETB) were analyzed by ELISA, RTqPCR, and immunoblotting. At the RNA level, cardiac Ednra expression trended towards upregulation in females, whereas males remained unchanged compared to their respective controls. At the protein level, both sexes tended to decrease ETA receptor levels. Renal Ednra expression trended towards downregulation in females but was unchanged in treated males, which was opposite of renal protein ETA where females were unchanged, and males trended towards reduction. Interestingly, Ednrb and Edn1 trended towards an increase in both the cardiac and renal system in both sexes. However, at the protein level only female cardiac ET-1 was significantly elevated (p<0.0001) while renal ET-1 levels were unchanged. Males demonstrated a significant increase in both cardiac (p=0.0004) and renal ET-1 (p=0.0127). Analysis of female cardiac ETB receptor levels yielded a significant reduction (p=0.0025) and an increasing trend in renal levels. Both cardiac and renal ETB was unchanged in males. The sex differences in the cardiorenal ET system regulation warrants further exploration and possible therapeutic treatments into cardiorenal lupus associated pathologies. Veterans Affairs Biomedical Laboratory Research & Development Career Development Award (CDA-2) 1IK2BX005605-01 to J.P.V.B, Medical University of South Carolina College of Medicine Program Project Grant to and J.P.V.B., and Medical University of South Carolina Core Centers for Clinical Research Project Award P30AR072582 to M.A.C and J.P.V.B. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sex Differences in Temporal Regulation of Piezo1 in Resiquimod Accelerated Systemic Lupus

Lupus disproportionally affects women (9:1), particularly women of color, but men with this disease often have more pronounced symptoms than their female counterparts. Individuals with lupus are anywhere from two to fifty times as likely to experience a cardiovascular (CV) event than the rest of the population. Though such complications are widely known, the mechanisms these disease processes are unknown. One well established pathogenesis of many CV diseases stems from an alteration of mechanical forces. The cation channel, Piezo1, is a mechanosensitive channel and is known to play a critical role in CV diseases like heart failure. Therefore, we hypothesized that inducing lupus-associated cardiac pathology in a lupus-prone mouse model would increase the expression of Piezo1. To investigate this, lupus-prone female and male B6.Nba2 mice aged 10-14 weeks were subjected to Resiquimod (R848; a TLR7/8 agonist;100μg/30μl) treatment or acetone vehicle twice a week for four weeks. Echocardiograms were performed every four weeks. Mice were sacrificed and their tissues harvested at 4- and 16-weeks after the start of R848 treatment. Piezo1 levels were subsequently analyzed in the heart by real time quantitative polymerase chain reaction (RTqPCR), immunoblot, and immunofluorescent staining. By echocardiography, female mice treated with R848 showed an increase in left ventricle (LV) wall thickness was seen at four weeks, followed by an increase in internal diameters over the following 12 weeks, culminating in cardiac dysfunction by 16 weeks via significantly decreased ejection fraction (EF; p<0.05) Interestingly, males showed no such cardiac dysfunction at any timepoint. By RTqPCR, female Piezo1 levels were higher overall than their male counterparts (p<0.05). Cardiac Piezo1 gene expression at 4 weeks was shown to be decreased in the R848-treated groups of both sexes. At 16 weeks, this trend was reversed in both male and female mice, showing R848-treatment induced an an upregulation of cardiac Piezo1 compared to each sex’s respective vehicle controls. Interestingly, while Piezo1 protein expression followed the same 4-week trend of downregulation in R848-treated mice regardless of sex, protein expression in the female R848-treated heart continued in a trending increase at the 16-week timepoint, while the male R848 heart showed unchanged. We found that this increase in cardiac Piezo1 in female, R848-treated is significantly correlated with higher left ventricular internal diameters (both LV-EDV (p<0.05) and -ESV (p<0.01)). This data demonstrates a sex difference phenotype in lupus-associated murine cardiac dysfunction, potentially through a Piezo1-mediated pathway. Further investigation is warranted to elucidate the Piezo1-dependent mechanisms involved in lupus-associated cardiovascular disease. Veterans Affairs Biomedical Laboratory Research & Development Career Development Award (CDA-2) 1IK2BX005605-01 to J.P.V.B, Medical University of South Carolina College of Medicine Program Project Grant to and J.P.V.B., and Medical University of South Carolina Core Centers for Clinical Research Project Award P30AR072582 to M.A.C and J.P.V.B. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Effect of Curcuma and Garcinia Peel on Rat PPARα Gene Expression Induced by High-Fat Diet

High levels of fat in the blood are a risk factor for nonalcoholic steatohepatitis liver disease. Indonesian medicinal plants that can decrease blood fat levels are turmeric and mangosteen peel. One of the mechanisms of blood fat-lowering drugs is to increase the expression of the PPARα gene. The purpose of this study was to assess the effect of turmeric and mangosteen peel on the expression of the PPARα gene in the rat liver induced by a high-fat diet. This research was conducted at Maranatha Biomedical Research Laboratory in February–October 2021, using male Wistar rats that were divided into 5 groups (n=5): negative control groups (no treatment), positive control groups (high-fat diet), turmeric, mangosteen, and fenofibrate groups, that was given high-fat diet continued by ethanol extract of turmeric, ethanol extract of mangosteen peel, and fenofibrate. At the end of the study, the animals were terminated, and the liver was extracted for RNA extraction and semi-quantitative PCR. The results showed that there was an increase in PPARα gene expression in the turmeric group and fenofibrate group, which were significantly different from the positive control group that received a high-fat diet (p<0.05) and between the fenofibrate group compared to negative controls that received standard chow diet (p<0.05). In conclusion, turmeric and fenofibrate are suggested to increase the expression of the PPARα gene in the liver induced by a high-fat diet.

A challenge-based interdisciplinary undergraduate concept fostering translational medicine.

Translational medicine (TM) is an interdisciplinary branch of biomedicine that bridges the gap from bench-to-bedside to improve global health. Fundamental TM skills include interdisciplinary collaboration, communication, critical thinking, and creative problem-solving (4Cs). TM is currently limited in undergraduate biomedical education programs, with little patient contact and opportunities for collaboration between different disciplines. In this study, we developed and evaluated a novel interdisciplinary challenge-based educational concept, grounded in the theoretical framework of experimental research-based education, to implement TM in undergraduate biomedicine and medicine programs. Students were introduced to an authentic clinical problem through an interdisciplinary session with patients, medical doctors, and scientists. Next, students collaborated in groups to design unique laboratory-based research proposals addressing this problem. Stakeholders subsequently rewarded the best proposal with funding to be executed in a consecutive interdisciplinary laboratory course, in which mixed teams of biomedicine and medicine students performed the research in a fully equipped wet laboratory. Written questionnaires and focus groups revealed that students developed 4C skills and acquired a 4C mindset. Working on an authentic patient case and the interdisciplinary setting positively contributed to communication, collaboration, critical thinking, and creative problem-solving skills. Furthermore, students were intrinsically motivated by (i) the relevance of their work that made them feel taken seriously and competent, (ii) the patient involvement that highlighted the societal relevance of their work, and (iii) the acquisition of a realistic view of what doing science in a biomedical research laboratory is. In conclusion, we showcase a widely applicable interdisciplinary challenge-based undergraduate concept fostering TM.

1747-P: Islet Ca2+ Dynamics, Heterogeneity, and Consistency in Three Dimensions with Activators of Pyruvate Kinase

Pancreatic β-cell heterogeneity is important for Ca2+ triggering and insulin secretion and is affected in diabetes. Organization and consistency of β-cell subpopulations are poorly understood despite being key in understanding how individual cells contribute to islet function and diabetes. Existing studies analyze single 2D planes of the islet. However, subpopulation organization cannot be fully understood without examining β cell dynamics throughout the entire islet in 3D. Here, we imaged islets from β cell specific GCaMP6s Ca2+ reporter mice using high-speed light-sheet microscopy to analyze the 3D organization and consistency in high glucose and with pharmacologic activators of pyruvate kinase (PK), a glycolytic enzyme that controls βcell excitability. Phase analysis revealed that cells which initiated islet depolarization (leaders) are not highly consistent across oscillations, but the 3D region where the calcium wave originates is consistent, according to a metric based upon the KL-divergence (P<0.0001 over 11 oscillations). Using functional network analysis, β-cell hubs which drive islet synchrony are more consistent over an islet region than across individual cells (P<0.0001). The wave origin was more regionally dependent than the β-cell hubs (P<0.0001). We also found that network analysis is similar (P=0.04) and phase analysis is different between 2D and 3D (P<0.0001). Following quantification of heterogeneity in the 3D islet, certain sub-populations of β cells are similar throughout the islet, while others are not. Acute PK activation decreased the consistency of wave initiators (P<0.0001), but not the consistency of hub cells in the functional network (P=0.15), shedding new light onto the mechanism behind these subpopulations. Islet regions, rather than individual cells, are more consistent and thus likely responsible for initiating Ca2+ triggering and insulin secretion. These mechanisms help render the islet robust to single β cell failures. Disclosure J.Briggs: None. E.Jin: None. M.J.Merrins: None. R.K.Benninger: None. Funding William S. Middleton Memorial Veterans Hospital; U.S. Department of Veterans Affairs Biomedical Laboratory Research and Development Service (I01B005113); National Institutes of Health (R01DK113103, R01DK127637, R01DK102950, R01DK106412); National Science

IL-17 Receptor A protects against Helicobacter pylori-induced gastric cancer

Abstract Infection with Helicobacter pylori is the predominant risk factor for the development of gastric cancer. Risk is enhanced by specific H. pylori virulence factors, diet, and the inflammatory response. Chronic activation of T helper (Th) 1 and Th17 pathways contribute to inflammation. Previous work suggests that while IL-17 activates several antimicrobial components of the innate immune response, IL-17 is also required to prevent a hyper-inflammatory adaptive immune response. To investigate if IL-17 receptor signaling contributes to the development of carcinogenesis, the transgenic InsGAS mouse, which is susceptible to H. pylori-induced gastric cancer, was used. InsGAS mice and InsGAS Il17ra −/−mice were infected with a cag T4SS positive strain for up to 6 months. By 6 weeks post infection, IL-17RA deficiency led to increased bacterial burden and increased gastritis, concomitant with an exacerbated Th1/Th17 cytokine profile, increased B cell infiltration and enhanced production of IgA despite reduced Pigr expression. Paragastric lymph nodes of InsGAS Il17ra −/−mice, while enlarged relative to InsGAS mice, exhibited altered gene expression profiles and potential loss of fibroblastic reticular cells. Gastric pathologies, including intramucosal carcinoma and adenocarcinoma with submucosal invasion, developed more frequently by 3 and 6 months post infection in H. pylori-infected InsGASIl17ra −/−mice. These increases in severe gastric disease were accompanied by loss of parietal cells, loss of mucin-producing cells and enhanced expression of ceacam1. These data suggest that IL-17RA signaling activates a protective pathway to prevent excessive inflammation which can drive gastric carcinogenesis after H. pylori infection. This work was supported in part by Merit Review I01BX000915 (Algood) from the U.S. Department of Veterans Affairs, Biomedical Laboratory Research and Development Service. We acknowledge the Translational Pathology Shared Resource supported by NCI.NIH Cancer Center Support Grant 5P30 CA68485-19. Core Services performed through Vanderbilt University Medical Center's Digestive Disease Research Center supported by NIH grant P30DK058404.

Effect of Mechanical Stretch on Sex-Dependent Gene Expression by RNA Sequencing in Mouse Renal Endothelial Cells

Increased mechanical endothelial cell stretch contributes to the development of numerous cardiovascular and renal pathologies by orchestration of immune cell activation. While endothelial cells are not recognized as professional antigen presenting cells, they can express both major histocompatibility complexes (MHC) I and II. MHCs are essential to form part of the immunological synapse during antigen presentation to adaptive immune cells. The mechanisms by which increased endothelial cell stretch regulates renal endothelial antigen processing and presentation is unknown. We hypothesized that increased mechanical endothelial stretch promotes upregulation of key antigen processing and presentation genes in mouse renal endothelial cells (MRECs) in a sex-dependent manner. MRECs derived from both sexes underwent 5%, 10%, and 15% (1 Hz) uniaxial cyclical stretch for 48 hours. After stretch, RNA sequencing was performed and bioinformatic and pathway analysis was executed to determine potential inflammatory mechanisms of action. We found that 10% stretch induced downregulation in MHC and costimulatory genes ( H2-Da, H2-K1, H2-T23, H2-T24) and upregulation of antigen processing associated genes ( tap1, psmb2, psmb4, and psmb8) in male MRECs. Females undergoing 10% stretch exhibited inverse gene expression profiles with upregulation of MHC genes and downregulation of antigen processing and presentation genes. At 15% stretch, the sex-specific gene expression tendencies were inverse from 10% stretch. To understand the function of differential gene expression we performed gene ontology enrichment pathway analysis. Male MRECs undergoing 10% stretch exhibited an enrichment for renal and cardiovascular development processes and inflammatory processes including chemokine-mediated signaling and cytokine-mediated signaling. When we compared female MRECs at 10% stretch, we found enrichment for renal and cardiovascular development processes including endothelial cell migration, angiogenesis and glomerular vascular, kidney, and artery development. Interestingly, 10% mechanical stretch enriched pro-inflammatory processes including positive regulation of cell migration and leukocyte migration. These data provide novel insight into how mechanical stretch regulates key antigen processing and presentation pathways in renal endothelial cells in a stretch and sex-dependent manner. This work was funded by MUSC Core Centers for Clinical Research Project Award to JPVB and Biomedical Laboratory Research and Development Service of the VA office of Research and Development Awards IK2BX003922 to KYDP and IK2BX005605 to JPVB. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ion transport basis of norovirus diarrhea in a neonatal mouse model

Background: Human Norovirus (HuNoV) is the leading cause of infectious gastroenteritis and food-borne outbreaks, responsible for over 220K deaths annually. Given difficulties studying HuNoV, Murine Norovirus (MNV) has been used as a model for norovirus (NoV) pathophysiology. A major barrier to understand how NoV causes diarrhea is the absence of diarrhea in adult wild-type (WT) mice, which have asymptomatic MNV infections. While several KO mouse strains (e.g., STAT1) have been employed to recapitulate disease, these mice often have exaggerated, systemic, and lethal disease courses that imperfectly match human disease. Very recently, a new model of MNV that appears to recapitulate symptomatic HuNoV infection has been established. This model involves the oral gavage of three- or four-day-old (d.o.) WT BALB/c mice with high-concentrations of MNV resulting in a diarrheal phenotype. Current studies were undertaken to understand the ion transport basis of MNV diarrhea using this novel model. Methods: Three to four d.o. BALB/c pups were gavaged with either 1x10 8 plaque-forming units (PFU) of MNV-1 (CW3) or PBS alone followed by sacrifice at 48 hours-post-gavage (hpg). Small and large intestines were harvested for RNA and immunofluorescence (IF) analysis of multiple ion transporters that have been linked to diarrhea in other disease models. Results: MNV-gavaged pups had loose, orange stools, consistent with a diarrheal phenotype observed previously with this model. MNV RNA was detectable in small and large intestinal tissue for CW3-gavaged vs mock-gavaged pups (p-values <0.05) demonstrating successful infection with MNV. MNV infection had no significant effect on transcript levels of various electrolyte and nutrient transporters including DRA, NHE3, PAT-1, Na-K-ATPase, or SGLT-1. However, we found significant increases in CFTR and Vasoactive intestinal peptide receptor 1 (VPAC1) transcripts (p <0.05 and 0.01, respectively) and a significant increase in CFTR IF staining in frozen sections of the SI over mock (p<0.01). We did not find a significant increase in VIP transcript, VPAC1 protein expression or immunofluorescent staining, nor nuclear localization of pCREB in SI frozen sections. Conclusion: Using this novel neonatal WT BALB/c MNV mouse model to evaluate the effect of infection on gene transcription, and protein expression and localization of ion transporters commonly implicated in diarrhea, we observed significant increases in CFTR and VPAC1 SI gene expression in CW3-gavaged pups with negligible changes in transcription of all other transporters examined in this study. In addition, apical and total CFTR staining increased significantly without changes in VPAC1 or pCREB nuclear localization in frozen SI sections of CW3-gavaged pups compared to mock, suggesting that MNV causes a CFTR-mediated secretory diarrhea in the absence of changes in VPAC1 protein expression, localization, or activation. These studies were supported by the Department of Veterans Affairs, Veterans Heath Administration, Office of Research and Development, Biomedical Laboratory Research and Development: Merit Review Award: BX002011 (PKD), and VA Senior Research Career Scientist Award:1IK6BX005242 (PKD). The studies were also supported by NIH/NIDDK grants, R01 DK54016 and DK92441 (PKD). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Tartaric acid induces toxicity in Madin–Darby canine kidney cells, but not human kidney‐2 cells in vitro, and is prevented by organic anion transporter inhibition and human OAT‐4 transfection

To evaluate the effect of tartaric acid (TTA) on Madin-Darby canine kidney (MDCK) cells compared to human kidney (HK)-2 cells. Secondarily, to evaluate the effects of probenecid, an organic anion transporter (OAT)-1 inhibitor, as well as human (h)OAT-4 transfection into MDCK cells to prevent TTA-induced cytotoxicity through decreasing accumulation via OAT-1 uptake inhibition or increasing OAT-4-mediated TTA efflux. Seventy-two-hour TTA concentration response and inhibitor studies in immortalized cell lines. School of Pharmacy biomedical research laboratory and tissue culture facility. MDCK and HK-2 immortalized cell lines. Both cell lines were treated with increasing concentrations of TTA for 72hours. Additionally, MDCK cells were co-incubated with TTA and increasing concentrations of probenecid or had been transfected with hOAT-4 and subsequently treated with TTA for 72hours. Media and samples were collected and lactate dehydrogenase (LDH) release was measured. LDH release was measured to assess TTA-induced cytotoxicity after 72hours. LDH was not significantly increased in the HK-2 cells at any concentration but was significantly increased in the MDCK cells from 10 to 100mM. LDH concentrations were significantly decreased (61%) in MDCK cells incubated with 50mM TTA and probenecid when compared to TTA alone. hOAT-4 MDCK cell transfection also significantly reduced LDH release (57%) when comparing the transfected MDCK cells to the nontransfected MDCK cells treated with 50mM TTA. TTA is a species-specific nephrotoxicant in dogs due to an interspecies difference in OAT-4 expression. Inhibiting TTA uptake in MDCK cells in vitro using the OAT-specific inhibitor, probenecid, prevents TTA-induced cytotoxicity.

Herpes simplex virus type 2 (HSV-2) and its association with HIV, HCV, HBV, HTLV-1&2 and syphilis among men who have sex with men in Burkina Faso.

This study aimed to estimate herpes simplex virus type 2 (HSV-2) seroprevalence and its association with HIV, HBV, HCV, HTLV-1&2 and syphilis among men who have sex with men (MSM) in Ouagadougou, Burkina Faso, West Africa. We screened MSM sera for HSV-2 antibodies. A total of 329 sera were collected from an HIV and syphilis behavioral and biological cross-sectional survey conducted among MSM in Ouagadougou from January to April 2013. Serum samples were tested using Enzyme Linked Immuno-Sorbent Assay (ELISA) for the detection of IgG antibodies to HSV-2. Also, antibodies to HTLV-1&2, HBsAg and anti-HCV antibodies were screened by ELISA. Laboratory assays were performed according to manufacturers' instructions at the Biomedical Research Laboratory at the "Institut de Recherche en Sciences de la Sante" (IRSS) in Burkina Faso. The seroprevalence of HSV-2 infection among MSM was 14.3%(95% CI: 10.6-18.1), with disparities according to age and occupation. HSV-2 seroprevalence was high among MSM who were seropositive for HIV (40% versus 13.9%), for syphilis (42.9% versus 13.3%), for HCV (32.5% versus 11.7%) and for HTLV-1&2 (38.5% versus 12.9%) compared to people seronegative for these pathogens. Multivariate analysis showed that HIV-positive (ORa​=​5.34, p​=​0.027), anti-HCV-positive (ORa​=​4.44, p​=​0.001), and HTLV-1&2 positive (aOR​=​4.11, p​=​0.046) were associated with HSV-2 infection among MSM. However, no significant statistical association between HSV-2 and syphilis was found. HSV-2 seroprevalence among MSM in Burkina Faso is relatively high. Positive associations between sexual transmitted infections including HIV with HSV-2 suggest that HSV-2 infection's prevention should be strengthened through HIV transmission control programs.

Essential Oils of Rosmarinus officinalis and Eucalyptus globulus Cultivated in the Mountainous Region of the State of Rio de Janeiro (Brazil): Chemical Profile and Antileishmanial Activity

Pharmacognosy Research,2023,15,2,297-306.DOI:10.5530/pres.15.2.032Published:February 2023Type:Original Article Authors:Rogéria Cristina Zauli, Tarcísio Martins Santos, Alberto Wisniewski Jr, Patrícia Xander, and Aislan Cristina Rheder Fagundes Pascoal Author(s) affiliations:Rogéria Cristina Zauli1, 2, 3, Tarcísio Martins Santos4, Alberto Wisniewski Jr4, Patrícia Xander1, 3, Aislan Cristina Rheder Fagundes Pascoal5,* 1Department of Pharmaceutical Sciences, Institute of Environmental, Chemical and Pharmaceutical Sciences, Laboratory of Cellular Immunology and Biochemistry of Fungi and Protozoa, Federal University of São Paulo, Diadema, São Paulo, BRAZIL. 2Technical Support Center for Teaching, Research and Extension (NATEPE), Federal University of São Paulo, Campus Diadema, Diadema, São Paulo, BRAZIL. 3Biology-Chemistry Graduate Program, Federal University of São Paulo, Campus Diadema, Diadema, São Paulo, BRAZIL. 4Department of Chemistry, Federal University of Sergipe, Av. Marechal Rondon, São Cristóvão, SE, BRAZIL 5Department of Basic Sciences, Multi-user Biomedical Research Laboratory, Fluminense Federal University (ISNF-UFF), Nova Friburgo, Rio de Janeiro, BRAZIL. Abstract:The present study aimed to characterize the chemical profile of essential oils of Rosmarinus officinalis (EORo) and Eucalyptus globulus (EOEg) cultivated in the mountainous region of the state of Rio de Janeiro, Brazil, and to verify the leishmanicidal potential, as well as to determine the cytotoxicity in vitro in mammalian cells. EORo had α-Pinene (37.99%), Cineol (21.48%), D-Verbenone (5.78%) and Limonene (3.28%) as major constituents. The EOEg had as major components Cineol (75.52%), Limonene (8.82%) and o-Cymeno (7.81%). The EOEg had a lower IC50 value of 14.03 ± 2.08 μg/mL, while EORo had an IC50 value of 31.12 ± 4.6 μg/mL, both during the 24-hr exposure period. In the 48-hr period, the IC50 value was almost twice as high with rosemary oil, while in eucalyptus oil there was no significant difference when exposed in this period. The selectivity of EOs was demonstrated in murine cells, showing almost 30 times less toxic to the cell than to the parasite. It can be concluded that this is a pioneering study on the potential of these essential oils against the leishmania parasite. Furthermore, the low toxicity effect on the host cell encourages future studies and in vivo applications. Keywords:cytotoxicity, E. globulus, Essential oil, Leishmanicidal, R. officinalisView:PDF (359.76 KB)

Lost in transduction: Critical considerations when using viral vectors.

The application of retroviral vectors in the laboratory requires considerations that often go overlooked but are often easy to circumvent. Here, we discuss the relationship between the observed transduction efficiency of a cell population and per-cell viral insertions-and describe how differential cell-type susceptibilities can confound results. We consider the math underlying this problem and review an alternative approach to the commonly used "multiplicity of infection" (MOI) method of titering and using viral vectors in the biomedical research laboratory.

High-throughput FastCloning technology: A low-cost method for parallel cloning.

FastCloning, a reliable cloning technique for plasmid construction, is a widely used protocol in biomedical research laboratories. Only two-step molecular manipulations are required to add a gene (cDNA) of interest into the desired vector. However, parallel cloning of the gene into multiple vectors is still a labor-intensive operation, which requires a range of primers for different vectors in high-throughput cloning projects. The situation could even be worse if multiple fragments of DNA are required to be added into one plasmid. Here, we describe a high-throughput FastCloning (HTFC) method, a protocol for parallel cloning by adding an adaptor sequence into all vectors. The target gene and vectors were PCR amplified separately to obtain the insert product and linear vectors with 18-base overlapping at each end of the DNAs required for FastCloning. Furthermore, a method for generating polycistronic bacterial constructs based on the same strategy as that used for HTFC was developed. Thus, the HTFC technique is a simple, effective, reliable, and low-cost tool for parallel cloning.

Core competencies for a biomedical laboratory scientist – a Delphi study

BackgroundAfter completing university education, biomedical laboratory scientists work in clinical laboratories, in biomedical research laboratories, in biotech, and in pharmaceutical companies. Laboratory diagnostics have undergone rapid development over the recent years, with the pace showing no signs of abatement. This rapid development challenges the competence of the staff and will most certainly influence the education of future staff. This study aimed to examine what was considered the necessary competencies needed to pursue a career as a biomedical laboratory scientist.MethodsA modified Delphi technique was used, with the panel of experts expressing their views in a series of three questionnaire. Consensus was defined as the point which 75 % or more of the panel participants agreed that a particular competency was necessary.ResultsThe study highlights the perceived importance of mostly generic competencies that relate to quality, quality assurance, and accuracy, as well as different aspects of safety, respect, trustworthiness (towards patients/clients and colleagues), and communication skills. The results also stress the significance of self-awareness and professionality.ConclusionsWe identified important competencies for biomedical laboratory scientists. Together with complementary information from other sources, i.e., guidelines, laws, and scientific publications, the competencies identified can be used as learning outcomes in a competency-based education to provide students with all the competencies needed to work as professional biomedical laboratory scientists.

Mod3D: A low-cost, flexible modular system of live-cell microscopy chambers and holders

Live-cell microscopy imaging typically involves the use of high-quality glass-bottom chambers that allow cell culture, gaseous buffer exchange and optical properties suitable for microscopy applications. However, commercial sources of these chambers can add significant annual costs to cell biology laboratories. Consumer products in three-dimensional printing technology, for both Filament Deposition Modeling (FDM) and Masked Stereo Lithography (MSLA), have resulted in more biomedical research labs adopting the use of these devices for prototyping and manufacturing of lab plastic-based items, but rarely consumables. Here we describe a modular, live-cell chamber with multiple design options that can be mixed per experiment. Single reusable carriers and the use of biodegradable plastics, in a hybrid of FDM and MSLA manufacturing methods, reduce plastic waste. The system is easy to adapt to bespoke designs, with concept-to-prototype in a single day, offers significant cost savings to the users over commercial sources, and no loss in dimensional quality or reliability.

Biomedical Research and Informatics Living Laboratory for Innovative Advances of New Technologies in Community Mobility Rehabilitation: Protocol for Evaluation and Rehabilitation of Mobility Across Continuums of Care.

BackgroundRapid advances in technologies over the past 10 years have enabled large-scale biomedical and psychosocial rehabilitation research to improve the function and social integration of persons with physical impairments across the lifespan. The Biomedical Research and Informatics Living Laboratory for Innovative Advances of New Technologies (BRILLIANT) in community mobility rehabilitation aims to generate evidence-based research to improve rehabilitation for individuals with acquired brain injury (ABI).ObjectiveThis study aims to (1) identify the factors limiting or enhancing mobility in real-world community environments (public spaces, including the mall, home, and outdoors) and understand their complex interplay in individuals of all ages with ABI and (2) customize community environment mobility training by identifying, on a continuous basis, the specific rehabilitation strategies and interventions that patient subgroups benefit from most. Here, we present the research and technology plan for the BRILLIANT initiative.MethodsA cohort of individuals, adults and children, with ABI (N=1500) will be recruited. Patients will be recruited from the acute care and rehabilitation partner centers within 4 health regions (living labs) and followed throughout the continuum of rehabilitation. Participants will also be recruited from the community. Biomedical, clinician-reported, patient-reported, and brain imaging data will be collected. Theme 1 will implement and evaluate the feasibility of collecting data across BRILLIANT living labs and conduct predictive analyses and artificial intelligence (AI) to identify mobility subgroups. Theme 2 will implement, evaluate, and identify community mobility interventions that optimize outcomes for mobility subgroups of patients with ABI.ResultsThe biomedical infrastructure and equipment have been established across the living labs, and development of the clinician- and patient-reported outcome digital solutions is underway. Recruitment is expected to begin in May 2022.ConclusionsThe program will develop and deploy a comprehensive clinical and community-based mobility-monitoring system to evaluate the factors that result in poor mobility, and develop personalized mobility interventions that are optimized for specific patient subgroups. Technology solutions will be designed to support clinicians and patients to deliver cost-effective care and the right intervention to the right person at the right time to optimize long-term functional potential and meaningful participation in the community.International Registered Report Identifier (IRRID)PRR1-10.2196/12506