bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of the GPR30 receptor inhibits aldosterone‐induced cardiac hypertrophy
Authors: Orlowski Alejandro , Di Mattía Romina Alejandra , Aiello Ernesto Alejandro
Published: November 2023

The hormone aldosterone (Ald) plays classic role acting through mineralocorticoids receptors (MR) located in the cytosol, which act as ligand‐induced transcription factors. Many cardiac deleterious effects of Ald are mediated by the activation of the MR. However, activated MR can also elicit additional non‐genomic effects. In addition, it has been recently proposed that certain non‐genomic effects of Ald were due to the activation of the G protein‐coupled receptor 30 (GPR30). It has been reported that the activation of GPR30 is cardioprotective. The administration of the GPR30 synthetic agonist G1 reduced the infarct size, diminished left ventricular wall thickness, attenuated heart failure and induced a decrease in perivascular fibrosis. The aim of this study was to investigate the potential cardioprotective effects of GPR30 on Ald‐induced hypertrophy in neonatal cardiac myocytes. Consistent with previous reports, dose‐dependent response to 24 hs Ald treatment increased cell size (A.U., control: 186±11, n=20; Ald (0.1nM): 185±12, n=22; Ald (1 nM): 202±12, n=17; Ald (10 nM): 260±21*, n=23; Ald (100 nM): 268±17*, n=29; Ald (1000 nM): 275±15*, n=14; * indicates p<0.05), while no significant effects were found with the synthetic agonist of GPR30 (G1) (A.U., control: 186±11, n=20; G1 (0.1 nM): 189±10, n=26; G1 (1 nM): 192±13, n=30; G1(10 nM): 188±7, n=24; G1 (100 nM): 197±11, n=30; G1 (1000 nM): 196±14, n=22). The Ald‐induced hypertrophy was cancelled when the expression of MR was silenced with a small hairpin interference RNA directed against it (A.U., Ald (10nM) + siControl: 419.28±14.19, n=54; Ald(10nM) + siMR: 423.48±14.64, n=62). Interestingly, the Aldo‐induced cell hypertrophy was significantly inhibited with the treatment with G1 (1000 nM), (A.U., control: 524±9, n=96; Ald (10 nM): 737±9, n=93; Ald (10 nM)+G1 (1000 nM): 351±10, n=96; *p<0.05). We examined the intracellular signaling pathways induced by G1 and Ald. The results show that G1 (1000 nM) or G1+Ald (10 nM) incubation increases AKT phosphorylation. However, no significant changes were observed with the incubation with Ald alone. In summary, these results showed that G1 was able to prevent the hypertrophy induced by Ald, indicating that activation of GPR30 prevents the hypertrophy produced by the stimulation of MR induced by the hormone.
Proteomic Analysis of Titin Phosphorylation and Sarcomere Stiffness in the Human Heart
Authors: Heroux Maxime
Published: November 2023

Heart failure (HF) is a major clinical problem with extremely poor prognosis. Increased sarcomere stiffness is one of the major contributors to HF progression. Differential phosphorylation of the sarcomeric protein Titin and its domains N2‐Bus and PEVK differentially affect sarcomere stiffness. Phosphorylation of specific sites within Titin's N2‐Bus domain by kinases PKA and PKG reduces passive tension whereas phosphorylation of specific sites within Titin's PEVK domain by kinases PKCα and CaMKIIδ increases passive tension and sarcomere stiffness. The quantitative extent of site‐specific Titin phosphorylation within these two domains and their contribution to sarcomere stiffness in the human heart and HF are not known. Thus, the goal of this proposal is to define the quantitative relation between Titin domain phosphorylation and sarcomere stiffness in health and disease. Using an in‐house developed Titin phosphorylation multiple‐reaction monitoring mass spectrometry (MRM‐MS) approach, we propose to test the quantitative hypothesis that the N2‐Bus and PEVK specific phosphorylation sites independently and uniquely contribute to sarcomere stiffness in the human heart in health and HF. We will use the MRM‐MS Titin phosphorylation assay to quantify the site‐specific phosphorylation of the N2‐Bus and PEVK regions in cardiac tissue isolated from donors, dilated cardiomyopathy (DCM), and ischemic cardiomyopathy (ICM) samples. In addition, skinned myocytes from these samples will be isolated and mechanical measurements of passive tension as a function of sarcomere length will be performed to determine the relationship between the extent of Titin site‐specific phosphorylation and sarcomere stiffness. Understanding the link between the quantitative extent of site‐specific Titin N2‐Bus and PEVK phosphorylation and sarcomere stiffness would further our understanding of the physiology of the myocardium and the pathogenesis of HF. Results from this study may provide novel quantitative diagnostic, prognostic, or therapeutic targets for improved management and treatment of HF.
Protective Effects of mTOR Against Ferroptosis, a Form of Iron‐dependent Regulated Cell Death, in Cardiomyocytes
Authors: Baba Yuichi , Shimada Briana K , Horiuchi Kate M , Higa Jason K , Matsui Takashi
Published: November 2023

While iron is important for maintaining physiological metabolism, excess iron causes tissue damage in multiple organs, including the heart. Ferroptosis was recently reported as a new form of iron‐dependent necrosis. Although ferroptosis is a key etiological factor in cancer and other human diseases, the role of ferroptosis in the heart is less understood. Previous studies from our group and others demonstrated that the mechanistic target of rapamycin (mTOR) is sufficient and necessary to prevent cardiomyocyte (CM) cell death induced by pathological stresses, including ischemia and pressure overload. To define the role of mTOR on ferroptosis‐mediated cell death in CMs, we treated mouse adult CMs with erastin, a ferroptosis inducer, and examined whether mTOR overexpression prevents ferroptosis‐mediated cell death in CMs. Mouse adult CMs were isolated from hearts of either male transgenic mice with cardiac‐specific overexpression of mTOR (mTOR‐Tg) or littermate negative (WT) mice at 12–20 weeks of age, and cultured in conditioned medium (Medium 199, 100 μg/ml bovine serum albumin, insulin‐transferrin‐sodium selenite, 25 μM blebbistatin, 100 U/ml penicillin, 100 μg/ml streptomycin). One day after plating, CMs were treated with erastin or DMSO vehicle. Cell viability was assessed by Live/Dead Cell Viability Assay (2 μM calcein AM for survival, and 4 μM ethidium homodimer‐1 for cell death). Treatment with excess iron [1 mM iron (III) citrate] induced 4.4‐fold higher cell death in WT cells compared to vehicle controls (p<0.01, n=6). Sixteen‐hour treatment with 40 μM erastin induced significant cell death (control 18.1±0.7% vs. erastin 32.0±0.5%, p<0.01, n=4), and co‐treatment with 5 μM of ferrostatin‐1, a ferroptosis inhibitor, attenuated erastin‐induced cell death (p<0.05, n=4). Intriguingly, the number of dead cells induced by erastin was significantly lower in CMs isolated from mTOR‐Tg mice compared to that from WT mice (p<0.05, n≥4). Ferroptosis is also mediated by an increase in reactive oxygen species (ROS) generation. We measured ROS production with 25 μM 2′,7′dichlorofluorescein acetate (DCFDA). Erastin‐induced ROS production was ~50% lower in mTOR‐Tg than WT. These findings suggest that another form of regulated cell death, ferroptosis, occurs in CMs, and that overexpression of mTOR inhibits ferroptosis at least in part by attenuating ROS production. Understanding mechanisms of ferroptosis in CMs will provide a novel therapeutic strategy for many cardiac diseases such as heart failure, in which preventing CM cell death plays an important role in preserving cardiac function.

Support or Funding Information

This work was supported in part by grants from the NIH (P30GM103341) to TM and Kochi Organization for Medical Reformation and Renewal to YB.
Intravenous Delivery of Cardiosphere‐Derived Cells Improves Striated Muscle Function and Structure in a Murine Model of Duchenne Muscular Dystrophy
Authors: Rogers Russell G , Aminzadeh Mark A , Fournier Mario , Gouin Kenneth , Sanchez Lizbeth , Li Liang , Sakoda Chris , Marban Eduardo
Published: November 2023

Duchenne muscular dystrophy (DMD), the most common inherited muscular disease, affects approximately 1 in 3,500 live‐born males worldwide. Loss of dystrophin results in a myriad of dysfunctional cellular processes that ultimately manifest as severe and progressive muscle‐wasting in both cardiac and skeletal muscle. DMD patients are clinically characterized by an early loss of ambulation followed by dilated cardiomyopathy in the second decade of life. There is no cure for DMD and current treatments marginally slow the progression of the disease course. Cardiosphere‐derived cells (CDCs) are cardiac progenitor/stromal cells with regenerative, angiogenic, anti‐inflammatory, and anti‐fibrotic properties. Previous research has demonstrated that CDCs are efficacious in treating both ischemic and non‐ischemic heart disease, including Duchenne cardiomyopathy in mdx mice – a mouse model of DMD. The latter work motivated the ongoing HOPE‐Duchenne clinical trial to treat DMD‐associated cardiomyopathy. Here, we sought to explore the potential of CDCs to treat both cardiac and skeletal myopathies when delivered systemically. A single bolus of syngeneic CDCs or vehicle were administered via the femoral vein of 10‐month‐old mdx mice. Baseline and three week follow up measurements of ambulatory capacity, in vivo cardiac function, and isolated skeletal muscle function were recorded. Systemic delivery of CDCs partially reversed the dystrophic phenotype in the heart and skeletal muscle of mdx mice. Specifically, CDC treated mice ran 34.6% (p = 0.01) farther than vehicle‐treated mice. Moreover, CDC treatment improved cardiac ejection fraction by 18.7% (p = 0.008) and isometric force developed by the soleus muscle by 47.8% (p < 0.001), to levels comparable to those in age‐matched wild type controls. Histological examination of the hearts and solei revealed fewer necrotic lesions in CDC treated mice. Interestingly, solei from CDC treated mice contained more centrally nucleated myofibers (208.7 ± 20.7 vs 106.8 ± 9.0, p = 0.004), consistent with enhanced active regeneration. Further, immunohistological staining of the soleus demonstrated that CDC treatment promoted nascent myotube formation, indicating replenishment of a pool of fusion‐competent myoblasts. These data demonstrate that a single intravenous infusion of syngeneic CDCs is sufficient to impact favorably on the phenotype of the mdx mouse. Given that CDCs are already in advanced clinical testing, the present work motivates clinical testing of CDCs delivered systemically to DMD patients, looking for improvements in cardiac and skeletal muscle function and biomarkers.

Support or Funding Information

NIH T32 HL116273
Prenatal Androgen Exposure of Single‐Injection of Testosterone Induces Cardiovascular and Metabolic Dysfunction in Adult Female Wistar Rats.
Authors: Sherman Shermel B , Sarsour Nadeen , Hill Jennifer
Published: November 2023

Cardiovascular disease is the leading cause of death in men and women within the United States. Individuals at risk for developing cardiovascular disease possess morbidity characteristic of metabolic dysfunction including diabetes, hypertension, and obesity. Previous studies have found that the origins of cardiovascular disease may arise from the prenatal environment and exposure to excess sex steroids such as testosterone produces adverse consequences that place male and female adult offspring at risk for the development of insulin resistance, infertility, obesity, and heart disease. However, the effects of excess testosterone on female cardiovascular function remains poorly understood. In this study, we hypothesized that a single‐injection of testosterone in the late gestation of Wistar rats would establish a metabolic phenotype characteristic of hypertension in adult female rats. Sixty‐six female Wistar rats were separated into two groups, vehicle and prenatal androgen (PNA), after weaning. We investigated the effect of excess testosterone on cardiovascular function and morphology with telemetry, echocardiogram, and immunohistochemistry. We also assessed insulin and glucose tolerance and utilized quantitative gene expression analysis to determine differential gene and protein expression. Our data suggest that the prenatal exposure to excess androgen alters systemic expression of genetic markers associated with diabetes and hypertension and negatively impacts cardiovascular function by increasing systolic and diastolic blood pressure. Overall, our results confirm that prenatal androgen exposure may negatively impact cardiovascular health of females in adulthood.
The anti‐obesity effect of FGF19 occurs independent of the thermogenic protein UCP1
Authors: Samms Ricardo J , Antonellis Patric , Bauer Steven , Smith Dennis , O'Farrell Libbey , Culver Alexander , Hamang Matthew , Perfield James , Coskun Tamer , Yaden Benjamin , Gimeno Ruth , Brozinick Joseph , Adams Andrew
Published: November 2023

Fibroblast growth factor 19 (FGF19) is a postprandial hormone reported to play diverse roles in the regulation of bile acid, glucose and lipid metabolism. The administration of FGF19 to obese/diabetic mice has been reported to lower body weight, improve insulin sensitivity and glucose homeostasis. Classically, the primary target organ of FGF19 was considered to be the liver, where in the postprandial period it has been shown to regulate bile acid homeostasis in response to nutrient absorption. More recently, it has been proposed that the therapeutic benefits associated with FGF19 administration are due, in part, to the binding of its receptor complex (FGFR1 and β‐klotho) in white and brown adipose tissue and recruitment of the thermogenic protein uncoupling protein 1 (UCP1). However, the precise contributions of UCP1‐dependent thermogenesis to the therapeutic actions of FGF19 have not been critically evaluated. Here we report that FGF19 does indeed potently induce the expression and activity UCP1 in white adipose tissue and this effect is required for FGF19 to increase caloric expenditure. Surprisingly, using UCP1KO mice, we determined that neither UCP1 nor increased energy expenditure are required for the anti‐obesity/diabetic effects of FGF19. In both wild‐type and UCP1 null mice, FGF19‐mediated weight loss was associated with reduced expression of key regulators bile acid production as well as increased fecal lipid content which are known physiological actions of FGF19 and indicative of reduced postprandial lipid absorption. In preclinical models of metabolic disease, FGF19 is a potent anti‐obesity/diabetic agent, whose effects were presumed to be driven in part by the recruitment of UCP1 in adipose tissue. In contrast to these beliefs, we now show for the first time that the anti‐obesity effect of FGF19 occurs independent of UCP1 and is rather due to its classical role in the regulation of hepatic bile acid production.
GPCR signaling in brown/beige adipose tissue (Symposium: Adipose Tissue and the Cardiovascular System: Interactions with Sleep and Cardiometabolic Risk Factors)
Authors: Pfeifer Alexander
Published: November 2023

Overweight and obesity are associated with a high risk for cardiovascular disease, diabetes and certain tumors. Sleep disorders have been linked to obesity in multiple way: on one hand obesity increases the risk for obstructive sleep apnea on the other hand sleep disorders are associated with obesity. Brown adipose tissue (BAT) is a special type of fat that dissipates energy to produce heat. In human adults, BAT activity inversely correlates with body‐mass index and is therefore considered as a possible target for treatment of overweight and obesity. Moreover, brown‐fat like cells have been described in white fat depots; these cells are also knonw as brite (brown‐in‐white) or beige cells and their number and activity can be induced by cold exposure and pharmacological stimulation in a process described as ‘browning’.

G protein‐coupled receptors (GPCRs) regulate a variety of cellular functions. Importantly, Gs‐coupled GPCRs activate BAT energy expenditure. Our aim is to identify the GPCRs involved in BAT activation and/or recruitment. To this end, we profiled the GPCR expression profile in preadipocytes and mature brown adipocytes. Unexpectedly, we found that a major part of GPCRs in brown adipocytes.

Using a variety of pharmacological and genetic tools, we found that Gq‐coupled receptors inhibit brown and beige adipocyte differentiation. Further studies revealed that that Gq signaling is linked to RhoA/ROCK signaling in these cells.

Taken together, Gq signaling is an important regulator of brown adipocyte differentiation.

Support or Funding Information

Funding: DFG
Nox1 Inhibition Attenuates Non‐Alcoholic Fatty Liver Disease and De Novo Lipogenesis
Authors: Larion Sebastian , Thompson Jennifer , Butcher Josh , Haigh Stephen , Mintz James , Khurana Sandeep , Fulton David , Stepp David
Published: November 2023

Objective

Investigate the role of NADPH oxidase‐1 (Nox1) in the development of non‐alcoholic fatty liver disease (NAFLD).

Methods

Nox1 mRNA expression was investigated in human liver repository data [GDS4881] and confirmed in hepatic tissue of db/db mice. Deletion studies investigating the role of Nox1 in the development of NAFLD were conducted by generating constitutive Nox1 knockout mice bred on a db/db background. Metabolic syndrome was assessed using biochemical and histological indices. Hepatic mRNA expression was assessed using microarray (Affymetrix) with genes of interest confirmed using RT‐PCR. Lipid species accumulation was assessed by GC‐MS with ROS production evaluated by 8‐OHdG and nitrotyrosine immunofluorescence.

Results

Nox1 expression was significantly increased in human liver biopsies from patients with simple steatosis over healthy or obese patients without NAFLD. Increased expression of Nox1 and associated subunits was also evident in liver from db/db mice vs. lean (all p<0.05). Mouse studies revealed that obese mice had significantly greater food intake, anthropomorphic (body weight, visceral adiposity, lean muscle) and biochemical evidence of metabolic syndrome including dyslipidemia (serum triglycerides, cholesterol, free fatty acids), and insulin resistance (insulin, fasting blood glucose, HgbA1c, glucose tolerance test) than lean controls, with no difference vs obese mice with Nox1 deletion. Liver mass was increased in obese vs lean controls, but significantly attenuated (−36%; p<0.05) in obese mice with Nox1 deletion. Obese mice with Nox1 deletion also had a striking reduction in macroscopic nodularity and pale yellow coloring, with reduced ORO staining and triglyceride content (−69%; p<0.05) than controls. NMR revealed significantly increased fat accumulation in obese mice vs lean controls, which was reduced (−23%; p<0.05) in obese mice with Nox1 deletion. Similarly, SFA species and ROS production were increased in obese mice, but significantly attenuated in obese mice with Nox1 deletion (all p<0.05). Measurement of mRNA expression via RT‐PCR revealed increased expression of hepatic SREBF1 and its downstream effector SCD1, the rate limiting enzyme of hepatic MUFA production, in obese mice vs controls, which were both significantly decreased in obese mice with Nox1 deletion. Pathway analysis disclosed SREBF1 as a major regulator in the highest scoring network: Lipid metabolism (score 35).

Conclusion

Nox1 inhibition attenuates hepatic steatosis in a genetically obese mouse model, likely through alterations in de novo lipogenesis. Nox1 signaling may prove to be a viable therapeutic target in NAFLD.

Support or Funding Information

NIHHL124691
Pharmacologic activation of the sarco‐endoplasmic reticulum ATPase (SERCA) reduces palmitate‐mediated endoplasmic reticulum stress in liver cells
Authors: Estrada Andrea L , Stewart Claire M , Kim Paul Y , Wang Dong , Wei Yuren , Pagliassotti Michael
Published: November 2023

Purpose

Elevated fatty acids, in particular saturated fatty acids, have been shown to induce endoplasmic reticulum (ER) stress in numerous cell types including hepatocytes. However, the specific mechanisms that link saturated fatty acids to ER stress are poorly understood. Calcium is a critical cofactor for chaperone‐mediated protein folding in the ER. Previous studies have demonstrated that palmitate, a saturated fatty acid, reduces ER lumenal calcium stores, which are maintained by the activities of three ER‐bound proteins: the inositol triphosphate receptor, the ryanodine receptor, and SERCA. In the present study, we examined whether palmitate‐induced ER stress could be mitigated via activation of SERCA.

Methods

H4IIE liver cells (n=3) were treated with control media or control media supplemented with palmitate (250μM), a saturated fatty acid that induces ER stress, oleate (250μM), an unsaturated fatty acid that does not induce ER stress, or a combination of both palmitate and oleate that does not induce ER stress. Treatments were administered in the presence or absence of CDN1163 (10μM), a SERCA pump activator, for 6 hours.

Results

Only palmitate treated cells were characterized by increased ER stress. Co‐incubation with CDN1163 reduced palmitate‐mediated ER stress. These data suggest that palmitate‐mediated ER stress involves a reduction in SERCA activity.

Support or Funding Information

NIH: DIK072017
Repeated exposure to food limitation earlier in life enables rats to spare lipid stores during prolonged starvation
Authors: McCue Marshall D
Published: November 2023

The risks of food limitation and ultimately starvation date back to the dawn of heterotrophy; yet, starvation remains a major factor in regulating modern animal populations. Researchers studying starvation over a century ago suggested that animals subjected to sublethal periods of food limitation were somehow more tolerant of subsequent starvation events. This possibility has received little attention over the past decades, yet is highly relevant to modern science for two reasons. First, animals in natural populations are likely to be exposed to bouts of food limitation once or more before they face prolonged starvation during which the risk of mortality becomes imminent. Second, our current approach to studying starvation physiology in the laboratory focuses on nourished animals with no previous exposure to nutritional stress. In this study we examined the relationship between previous exposure to food limitation and potentially adaptive physiological responses to starvation in adult rats and found several significant differences. Rats that had been fasted until they lost 20% of their body mass on two previous occasions maintained lower body temperatures (as measured by passive integrated transponders implanted into their bodies) and lower plasma glucose levels when subjected to prolonged starvation (where they lost 30% of their body mass) than their naïve cohort that never experienced food limitation. These trends were particularly evident during the lattermost stages of starvation. These rats had also been raised to adulthood on diets spiked with either 13C‐labeled palmitic acid or 13C‐leucine to isotopically enrich either their body lipids or proteins, respectively. This approach allowed us to use 13CO2 ‐breath testing to demonstrate that the rats with prior experience with food limitation significantly reduced their reliance on the oxidation of endogenous lipids; we did not observe differences in oxidation rates of endogenous proteins. In short, food limitation affected the starvation strategies used by the rats. Ultimately, these findings underscore 1) the need for biologists to revisit the classic hypothesis that animals can adapt to starvation using a modern set of research tools and 2) the need to design controlled experiments of starvation physiology that more closely resemble the dynamic nature of food availability.
Fecal Supernatants from Active Ulcerative Colitis Patients Impair Colonic Epithelial Barrier Integrity and Activate Pelvic Mucosal Extrinsic Afferent Nerves
Authors: Wardill Hannah R , Dmochowska Nicole , Campaniello Melissa A , Mavrangelos Chris , Bowen Joanne M , Andrews Jane M , Costello Sam P , Hughes Patrick A
Published: November 2023

Introduction

Ulcerative Colitis is characterised by colonic mucosal inflammation. Microbial dysbiosis is well documented in active UC, but little is understood regarding the physiological implications this has on epithelial barrier integrity or colonic extrinsic afferent signalling. We aimed to determine the effects of fecal supernatants from active UC on epithelial barrier and colo‐rectal mucosal extrinsic pelvic afferent nerves.

Methods

Excreted fecal samples from 10 patients with active UC (endoscopic MAYO subscore ≥2) were mixed with Ringers (Ussing) or Krebs solution (electrophysiology) at 1.5gm / 3 ml, centrifuged, and the supernatant filtered and pooled. Pooled fecal supernatants (FSN) were run over a 30kDa size exclusion column. Neat, high and low molecular weight FSN were applied to CACO‐2 monolayers or healthy mouse colon tissue in Ussing chamber experiments over a 2 hour period. Extracellular electrophysiological responses from pelvic colonic extrinsic afferent nerves were recorded from healthy mice. Mucosal afferents were characterised as responding to fine mucosal stroking with 10 mg Von Frey Hair. Neat FSN was applied to mucosal afferents for 5 min and mechanical sensitivity to graded mucosal stroking with Von Frey Hair re‐assessed.

Results

Neat UC FSN potently decreased the resistance of CACO‐2 monolayers and mouse colon over a 2 hour period compared to vehicle. Low molecular weight FSN also decreased the resistance of mouse colon, yet increased the resistance of CACO‐2 monolayers. High molecular weight FSN had no effect on either mouse tissue or CACO‐2 monolayers resistance. Neat UC FSN directly activated mucosal colonic afferent endings immediately following addition, but had no effect on mechanical sensitivity to mucosal stroking.

Discussion

Mediators present in faecal samples impair the resistance of the epithelial barrier and directly activate mucosal afferent nerve endings. The <30kDa fraction had the most potent activity on epithelial barrier. Further characterisation of the active mediator(s) and mechanisms has the potential to lead to new targets for the treatment of barrier dysfunction and nerve activation in active UC.

Support or Funding Information

Supported by NHMRC Australia
Control of lupus nephritis by the gut microbiota
Authors: Mu Qinghui , Zhang Husen , Luo Xin
Published: November 2023

Dysbiosis of gut microbiota promotes autoimmune disorders that damage extraintestinal organs. Here we report the role of gut microbiota in the pathogenesis of renal dysfunction in systemic lupus erythematosus. In a classical model of lupus nephritis, MRL/lpr, we found marked depletion of Lactobacilli in the gut microbiota. Increasing Lactobacilli in the gut improved renal function of these mice. Further studies revealed that MRL/lpr mice possessed a “leaky” gut, which was reversed by Lactobacilli. Lactobacillus treatment decreased IL‐6 and increased IL‐10 in the gut, thereby providing an anti‐inflammatory environment. It also increased IL‐10 and decreased IgG2a in the circulation, the latter a major immune deposit in the kidney of MRL/lpr mice. Inside the kidney, Treg‐Th17 balance was skewed towards Treg with Lactobacillus treatment. These effects of Lactobacilli were present in female and castrated male mice, but not unaltered males, suggesting that gut microbiota controls lupus nephritis in a sex hormone‐dependent manner.

Support or Funding Information

This work was supported by Xin Luo's start‐up funds provided by Virginia Tech College of Veterinary Medicine.
Lactobacillus casei Cell‐Surface Components and Their Impact on Immunomodulation and Cecal Microbiata Composition
Authors: Aktas Busra , De Wolfe Travis J. , Safdar Nasia , Darien Benjamin J. , Steele James L.
Published: November 2023

Lactobacillus casei are commonly utilized as a probiotic in a wide‐range of fermented and unfermented dairy products. Previously it has been shown that L. casei have extensive strain‐to‐strain genetic variation and that significant strain‐to‐strain differences are present in their ability to alter the gut microbiota and modulate the host innate immune system. The objective of this study is to evaluate L. casei for potential mechanisms resulting in the observed probiotic function. We utilized comparative in silico analysis to investigate genes putatively encoding surface components, including: proteins exposed to the surface; pili; wall teichoic acids (WTA); and lipoteichoic acids (LTA). We also evaluated in vitro the ability of six L. casei strains to modulate cytokine production (IL‐10 and IL12) by intestinal epithelial cells (IEC‐6). Additionally, we investigated the contribution of cell surface components and small molecules produced by L. casei to their probiotic function by examining the effect of live, UV‐killed, and supernatants from L. casei strains on cytokine production by IEC‐6. To evaluate the ability of UV‐killed L. casei to alter the gut microbiota and modify the immune system in vivo, we fed mice L. casei 32G at dose of 108 CFU/day/mouse for seven days and sacrificed 3.5h after the last administration. The cecal content and the ileum tissue were collected for microbiota analysis and immune profiling, respectively. The in silico results demonstrated that there is an extensive genetic diversity within L. casei cell surface components. The in vitro cytokine assay revealed that the strains modulate cytokine expression in a strain specific manner. The cell surface component of all the strains examined reduced the IL‐10 expression while small molecules were determined to modulate IL‐12 expression in some strains, indicating that both cell surface components and small molecules participate in immunomodulation. The in vivo results demonstrated that only live 32G significantly altered the overall microbiota. The effect of live and UV‐killed 32G on the expression of genes involved in innate immune system was gene specific. In conclusion, significant variation occurs between L. casei strains in the mechanism by which they signal with the host innate immune system and hence their mechanisms of probiotic action.

Support or Funding Information

This work was funded by the DuPont Inc (Grant no. PRJ19MQ) and by the WARF (Grant no. PRJ66JZ)
Feeding obese diabetic mice a genistein diet induces thermogenic and metabolic change
Authors: Rockwood Schuyler , Sandoval‐Skeet Noemy , Broderick Thomas L , Al‐Nakkash Layla
Published: November 2023

Obesity is associated with elevated plasma levels of glucocorticoids, which is known to affect food intake and energy expenditure. Further, tissue specific glucocorticoid metabolism is altered in obesity, and may increase insulin resistance and cardiometabolic risk. We have previously demonstrated that obese (ob/ob) female mice fed a genistein‐based diet resulted in a significant decrease in body weight, fat pad weight, and improved cardiovascular risk markers. The goal of this study was to examine if those changes were sex‐dependent. In order to test our hypothesis, lean and ob/ob male mice were fed either a genistein‐rich diet (600 mg genistein/kg) or a genistein‐free diet, over a 4‐week period. During the 4‐week study, measures of daily diet intake, body weights, and body temperatures were taken. At the conclusion of the diet study, mice were euthanized and organs were removed, weighed and immediately frozen for further analysis. Ob/ob mice weighed significantly more (51.50±0.72 g, n=6, P<0.05) than leans (30.38±2.08 g, n=5) and this was associated with significant increases in liver and total fat pad weight. While there was no genistein effect on weight loss, genistein‐fed ob/ob mice showed a significant increase in overall food consumption in weeks 3 and 4 compared to control ob/ob mice. Interestingly, body temperatures of ob/ob mice after 4‐weeks on the diets were significantly less (30.80±0.33 °C, n=6, P<0.05) than those of lean controls (33.30±0.28 °C, n=5), and genistein diet reversed this (32.66±0.36 °C, n=5, P<0.05), indicating a thermogenic effect. Western blot analysis of liver samples revealed that 11βHSD‐I, which converts inactive corticosterone into active corticosterone, was significantly decreased in ob/ob mice (0.34±0.05, n=6, P<0.05) compared to leans (0.79±0.18, n=4), and there was no genistein‐effect. Protein analysis of kidney 11βHSD‐II, which converts corticosterone into in its inactivated form, was increased with genistein (0.93±0.03, n=5) compared to ob/ob controls (0.68±0.06, n=5, P<0.05). Based on these results, we predict a corresponding improvement in serum corticosterone levels as well as a potential rescue effect from excessive glucocorticoid production by the kidney in genistein‐fed ob/ob mice.

Support or Funding Information

Financial support: MWU intramural funds (LA and TLB), DAREF (LA) and Department of Biomedical Sciences (SR, NS‐S)
G protein‐coupled receptor 30 (GPR30), a novel estrogen receptor, plays an independent role in the pathogenesis of estrogen‐induced cholesterol gallstones in female mice
Authors: Wang David Q , de Bari Ornella , Wang Helen H
Published: November 2023

Background

Estrogen is an important risk factor for gallstone formation because gallstone prevalence is significantly higher in women than in men. The classical estrogen receptor α (ERα), but not ERβ, in the liver plays a critical role in estrogen‐induced gallstones in female mice. Understanding the molecular mechanisms underlying the lithogenic effect of estrogen on gallstone formation became more complicated with the identification of GPR30 as a new gallstone gene Lith18. Our aim is to explore whether GPR30 produces an independent lithogenic action, or works in conjunction with ERα, to promote estrogen‐induced gallstone formation in female mice.

Methods

The biliary and gallstone phenotypes were studied by physical‐chemical methods in ovariectomized (OVX) female GPR30 (−/−), ERα (−/−), and WT mice injected daily with the potent GPR30‐selective agonist G‐1 at 0 or 200 ng/day and fed a lithogenic diet for 8 wk. Gene expression was studied by qPCR and Western blotting.

Results

Activation of GPR30 by G‐1 enhanced cholelithogenesis by suppressing expression of cholesterol 7α‐hydroxylase (CYP7A1), the rate‐limiting enzyme for the classical pathway of bile salt synthesis. This abnormality led to biliary cholesterol hypersecretion associated with reduced bile salt secretion, causing cholesterol‐supersaturated gallbladder bile and accelerating cholesterol crystallization. The prevalence of gallstones was 80% in OVX ERα (−/−) and WT mice treated with G‐1 compared to 10% in OVX WT mice receiving no G‐1. Moreover, G‐1 treatment did not influence gallstone formation in OVX GPR30 (−/−) mice. G‐1 treatment led to a significant increase in mRNA levels and protein concentrations of the epidermal growth factor receptor (EGFR) and a significant decrease in expression of liver receptor homolog‐1 (LRH‐1), coupled with reduced expression of CYP7A1 in OVX ERα (−/−) and WT, but not GPR30 (−/−) mice. In vitro studies using cultured primary hepatocytes isolated from ERα (−/−) and WT mice showed that when EGFR was inhibited by AG1478, a highly potent EGFR kinase inhibitor, expression of CYP7A1 was unchanged even though GPR30 was activated by G‐1.

Conclusions

GPR30 plays an independent role in the pathogenesis of estrogen‐induced gallstones in female mice. EGFR is required for GPR30 to exert a non‐transcriptional regulatory role in suppressing hepatic expression of CYP7A1 and the classical pathway of bile salt synthesis, leading to the availability of excess amounts of cholesterol for hepatic hypersecretion and the formation of supersaturated bile. Both GPR30 and ERα are potential targets for preventing cholesterol gallstones, particularly in women and patients who expose to high levels of estrogen.

Support or Funding Information

This work was supported in part by research grants DK101793 and DK106249, both from the National Institutes of Health (US Public Health Service).
Inhibition of p53 with its specific inhibitor pifithrin‐α prevents hepatic oxidative stress and inflammation, probably via up‐regulating Sirt1/Nrf2 pathway
Authors: Qian Lingbo , Gu Junlian , Ji Honglei , Cai Lu
Published: November 2023

Background and objective

Up‐regulation of the tumor suppressor p53 has been reported to mediate the diabetic hepatic injury, including oxidative stress and inflammation. However, its underlying mechanisms remain unclear. Nuclear factor erythroid‐derived 2‐like 2 (Nrf2) is a potent factor against diabetic oxidative stress by inducing the expression of its downstream antioxidants. It is revealed that Nrf2 activates its target genes through an antioxidant‐response element (ARE) when Sirtuin 1 (Sirt1) triggers the release of Nrf2 from Nrf2/Keap1 complex. Furthermore, inverse relationship between Sirt1 signaling and p53 protein abundance has been found in high glucose‐exposed hepatic cells. The aim of present study was to explore whether the hepatic protection from diabetes by p53 inhibition with pifithrin‐α is associated with up‐regulation of Sirt1/Nrf2 pathway in type 1 diabetic mice.

Methods and Results

FVB mice were treated with five daily injections of 50 mg/kg streptozotocin to induce type 1 diabetes. Both hyperglycemic and age‐matched control mice were treated with or without pifithrin‐α five times a week for 2 months and then kept without treatment until 3 months. At three months, diabetes significantly increased serum ALT activity, hepatic 3‐NT, 4‐HNE, IL‐6 and TNF‐α expressions, which was significantly prevented by inhibition of p53 expression with pifithrin‐α in diabetic mice. Mechanistically, the inhibition of p53 reversed diabetes‐increased hepatic expression of Keap1 and p47 phox and decreased Sirt1 expression and Nrf2 expression and function.

Conclusions

These results demonstrate that the inhibition of p53‐mediated down‐regulation of Sirt1/Nrf2 pathway by administration of diabetic mice with p53 specific inhibitor can effectively prevent the development of hepatic oxidative stress and inflammation. Therefore, intervention targeting to p53 may be a potential therapy for diabetic hepatic injury.

Support or Funding Information

This work was supported in part by the Scholarship awarded by China Scholarship Council (No. 201508330753), and the grant awarded by National Natural Science Foundation of China (No. 81470495).
Presence and Variations of the Palmaris Longus in a Small Population
Authors: Monin Lydia , Hurley Mary , Olivieri Marion P.
Published: November 2023

Dr. Frank Jobe first performed the wezl‐known “Tommy John Surgery” on a Major League baseball player in 1974. This surgical procedure involves the reconstruction of the ulnar collateral ligament (UCL) of the elbow. The Palmaris longus, a tendon located in the forearm, has been successfully used as a replacement tendon for the reconstructive surgery. Part of the preoperative plan of the reconstruction includes examination of the Palmaris longus presence, whether bi‐lateral, unilateral or absent; variations may occur that lead to a change in the surgical steps taken during the procedure. More knowledge on the different possible variations of the tendon involved with this surgery will allow medical professionals to have a better understanding of the orientation of the muscle/tendon involved in Tommy John reconstruction. The Palmaris longus normally arises from the medial epicondyle of the humerus, and inserts to the palmar aponeurosis. It exhibits several variations including its overall presence. Thompson did a clinical study on 300 subjects observing the presence of the muscle/tendon in 2001 that concluded forty‐nine subjects had unilateral absence, and bilateral absence in 26 subjects. In those subjects with unilateral absence, the side more commonly affected was the right side. In this study, 12 cadavers were examined for the general presence of the Palmaris longus. Out of the 12 cadavers examined only 3 presented with an absence of the tendon, of those three cadavers all expressed a unilateral absence of the tendon in the left arm. This study indicates that unilateral absence was considerably more prevalent in the left arm than what was originally reported reported by Thompson in 2001, continuing studies will examine additional cadavers and assess significance.

Support or Funding Information

Supported and Funded by the Department of Biology and Mathematics of D'Youville College, Buffalo NY
Anatomical Variation of Arterial Supply to the Prostate in a Cadaver Population
Authors: Danovskis Daniel , Gervasi Megan E , Hurley Mary , Olivieri Marion P
Published: November 2023

For men in the United States, aside from the skin, the prostate is the most common cancer site. Roughly 1 of 7 males will be diagnosed with prostate cancer. This does not include the numerous undocumented signs of prostate cancer identified postmortem. In 2015, it was estimated that in 2016 there would be180,890 new cases of prostate cancer and the disease would cause 26,120 deaths. The vasculature of the prostate differs among individuals, and is not completely understood. Typically, the prostate is supplied by arteries arising from the internal pudendal artery, however there are many undefined variations. The position of the prostate makes it difficult to study during standard gross anatomy course dissection. This study involved development of a protocol for isolation and examination of cadaveric prostates.

It was found that observation of the prostate gland was enhanced by isolating the pelvic region. This involved careful removal of the sigmoid colon followed by cuts through both thighs including the femur just inferior to the inguinal region ligament. The vertebral column was detached from the sacrum by cutting through the intervertebral disc between L5 and the sacrum. Once the pelvis was isolated it could be positioned to allow access to the internal iliac artery. The branches of internal iliac were then identified. This was then followed by a midsaggital cut through the entire pelvis enabling greater access to the prostate gland. This also allowed for confirmation of the targets of the branches of the internal iliac and for further dissection revealing how those branches continued into the prostate. Future studies will involve examination of the arterial supply of the prostates of 12 male cadavers. Further understanding of the prostate arterial supply may lead to innovation regarding treatment of prostate cancer.

Support or Funding Information

Work supported by the Department of Biology and Mathematics, D'Youville College.
Quantitative Characterization of Cadaveric Larynges Using 3‐Dimensional Modeling
Authors: Lewis Christina C
Published: November 2023

Airway management procedures are a fundamental aspect of anesthetic practice of emergency and critical care medicine. Performing these procedures requires an excellent working knowledge of the typified gross anatomy of the airways, as well as many of the normal variations in proportionality that often occur. While some variations in laryngeal anatomy have been described, such as the gender dimorphisms for the thyroid laminae and the volume of preepiglottic and paraglottic spaces, the scope of anatomical variation is not well appreciated. The present study seeks to expand the working knowledge of variations in laryngeal anatomy and geometry, using 3‐dimensional modeling and high powered quantitative analysis tools. To this end, 7 adult larynges were excised from embalmed cadavers (4F: 3M, ages 64–93, mean 82) and carefully dissected. The causes of death varied, but none directly involved the airways or larynx. The larynges were subsequently scanned using an HDI Advance R1X 3D Scanner (GoMeasure 3D, Amherst, VA) and the resultant 3‐dimensional models were analyzed via Geomagic Free Form software (3D Systems; Rock Hill, South Carolina). Quantitative analyses of the laryngeal cartilages is currently underway, and a combination of gross parameters, including length, height, width, internal dimension, and angulation will be determined for the thyroid, cricoid, arytenoid, and epiglottic cartilages. Additionally, the laryngeal musculature will be analyzed for volume, fiber bundle length, and pennation angle. Results from the current study will expand the knowledge base of the clinical anatomy of the airways, and potentially inform individual airway management procedures and approaches.
ALFABISABOLOL REVERSES THE MORPHOLOGICAL CHANGES INDUCED BY 5‐FU IN A MODEL OF INTESTINAL MUCOSITIS
Authors: Cerqueira Gilberto Santos , de Godoy José Roberto Pimenta , Lopes José , Sobrinho Osvaldo Pereira Costa , Medeiros Jand Venes Rolim , Martins Conceição Silva , da Silva Costa Deiziane Viana
Published: November 2023

Introduction

5‐Fluorouracil (5‐FU) is broadly used to treatment of breast and colorectal cancer. However, it induces side effects as intestinal mucositis. Studies several has been performed to reduce the 5‐FU‐induced intestinal injury. In this regard, the α‐bisabolol (1‐methyl‐4‐(1,5‐dimethyl‐1‐hydroxhex‐4 (5)‐enyl)‐cyclohexen‐1) (BISA) has not yet been investigated in treatment of the 5‐FU‐induced intestinal mucositis. BISA is a monocyclic sesquiterpene found in essential oils of a variety of plants such as chamomile (Matricaria chamomilla, Compositae, syn. M. recutita), arnica (Arnica amplexicaulis and A. chamissonis), sage (Salvia stenophylla) incanus Eremanthus, Vanillosmopsis arborea among others. The aim of this study was to investigate the effect of BISA in the 5‐FU‐induced intestinal mucositis.

Methods and Results

Swiss Mice (weighing 25 to 30 grams) received DMSO 2% (control) or 5‐FU (450 mg/kg, i.p., single dose). After 24h, BISA (50, 100 or 200 mg/kg, dissolved at DMSO 2%) was injected by orogastric gavage during three days and the animals were euthanized four hours after last dose of BISA. The mice's body weight were evaluated daily. The ileum segments were collected to evaluate following parameters: histopatological analysis, count number of mast cells, reduced glutathione (GSH) and myeloperoxidase (MPO) levels. The ileum segments were extracted, measured and processed by histology routine and dyed with Hematoxiline eosine, Masson' Trichrome with toluidine blue 1% for mast cell counting and with picrosirius red for the total collagen fibers. 5‐FU promoted accentuated weight loss, shortening villus, loss of crypt architecture, intense inflammatory cells infiltrate along ileum layer. Moreover, 5‐FU increased the number of mast cells (53,80±3.426); DMSO (22,20±2.21); BISA‐50 (53,80±3.426);BISA‐100 (31,60±1.621); BISA‐200(79,20±4.923) and MPO levels (6,532±1,354);DMSO (2,402±0,2190);BISA 50 (4,678±0,3424);BISA 100(3,382±0,3239); BISA 200 (4,330±0,6093) in ileum, as well as decreased GSH levels compared with control group. The experimental group presented increased vascular density compared to the control group.

Conclusion

These results show that BISA had a protective effect against intestinal injury induced by 5‐FU. Moreover, we also showed that BISA reduces 5‐FU‐induced the mastocytosis. However, more tests are needed to evaluate the BISA mechanism in the 5‐FU‐induced intestinal mucositis.

Support or Funding Information

Footnotes: This study received funding of CAPES.