bluuebunny/crossref_metadata_2025 huggingface dataset:
Garlic‐derived organosulfur compound diallyl trisulfide induces apoptosis in pancreatic cancer cells
Authors: Stan Silvia
Published: June 2021

Epidemiological observations have shown an inverse association between consumption of Allium vegetables (e.g., onions and garlic) and pancreatic cancer risk. The cancer preventive effect of Allium vegetables has been attributed to organosulfur compounds. The objective of this study was to determine the efficacy of diallyl trisulfide (DATS), a garlic‐derived organosulfur compound, against pancreatic cancer cells. Treatment with DATS inhibited the growth of MIAPaca2 and PL45 pancreatic cancer cells in a dose‐dependent manner. DATS induced apoptosis in pancreatic cancer cells as determined by cytoplasmic‐associated DNA fragmentation assay after exposure to DMSO (control) or DATS (20, 40, and 60 μmol/L) for 24 h. Exposure of pancreatic cancer cells to DATS increased levels of cleaved caspase 3 and induced proteolytic cleavage of poly‐(ADP‐ribose) polymerase (PARP). In addition, DATS treatment upregulated the expression of pro‐apoptotic Bim‐s and Bim‐L isoforms in MIAPaca 2 and PL45 cells. In conclusion, this study shows that DATS induces apoptotic cell death in pancreatic cancer cells and supports further investigation of DATS as a chemopreventive agent for pancreatic cancer.
Wolfberry water soluble extracts selectively induce leukemia cell apoptosis
Authors: Ji Hua , Lin Dingbo
Published: June 2021

Objective

Our aim was to determine whether wolfberry water soluble extracts (WWSE) selectively induce apoptosis of leukemia cells, but not normal T‐cells in culture.

Methods

Contents of taurine, betaine, and rutin of wolfberry water soluble extracts were measured by TLC and/or HPLC. Leukemia Jurkat cells and normal human T cells were treated with WWSE for 48 hours. Cell proliferation, apoptosis, and alteration of gene expression and protein phosphorylation were determined by BrdU assay, flow cytometry, Western blot, and/or immuneprecipitation.

Results

WWSE were enriched in carbohydrates (73.4 ± 4.5 % (w/w)), polyphenolics (1555 ± 112 mg quercetin equivalent/100 g freeze dry powder) including 213 mg rutin/100 g freeze dry powder, betaine (374 ± 25 mg/100 g freeze dry powder), and had enhanced antioxidant activity (7771 ±207 μmol Trolox equivalent/100 g freeze dry powder). WWSE had no effect on growth and proliferation of normal human T cells, but inhibited Jurkat cell proliferation, induced cycle arrest at the G2/M phase in a dose dependent manner starting at 1 mg/mL for 48 h. Furthermore, WWSE down‐regulated SIRT1 protein expression, inactivated cyclin‐dependent kinase 1 (CDK1), and disrupted the interaction of SIRT1 with CDK1 in Jurkat cells only. Knockdown of SIRT1 by specific siRNA caused inactivation of CDK1 leading to Jurkat cell apoptosis.

Conclusions

Data suggested that consumption of wolfberry could be an efficacious dietary strategy for preventing leukemia. Grant Funding Source: National Institutes of Health COBRE Grant P20‐RR‐017686 and the Kansas State University Cancer Center Research Initiative Award
Vitamin D inhibition of Has2 and hyaluronan synthesis in invasive breast cancer cells
Authors: LaPorta Erika , Welsh JoEllen
Published: June 2021

1,25‐Dihydroxyvitamin D (1,25D) exerts anti‐cancer actions through the vitamin D receptor (VDR) but the specific targets that mediate these effects remain to be defined. In this study, growth and genomic responses to 1,25D were evaluated in cells derived from mammary tumors generated in VDR knock‐out (KO) and wild‐type (WT) mice. WT145 cells (derived from WT tumors) expressed VDR and were growth inhibited by 1,25D, whereas KO240 cells (derived from VDRKO tumors) lacked VDR and were not growth inhibited by 1,25D. KO240 cell clones stably expressingVDR (KOhVDR cells) were sensitized to 1,25D mediated growth arrest. Genomic profiling of KO240, KOhVDR and WT145 cells identified 35 transcripts that were altered by 1,25D in relation to growth inhibition, with an enrichment of genes involved in heparin sulfate and proteoglycan binding (including Prelp, Hbegf, Fbn1, Postn and Has2). Currently we are focusing on VDR regulation of Has2 in mammary gland and breast cancer. Has2 is the rate‐limiting enzyme in the synthesis of hyaluronic acid (HA) which is strongly associated with tumor invasion and metastasis. To date, we have confirmed down‐regulation of Has2 protein by 1,25D and demonstrated enhanced HA accumulation in mammary gland and tumors of VDRKO mice. These data define a novel role for vitamin D in control of HA synthesis in epithelial tissues that likely contributes to its anti‐cancer actions.

NIH R01CA69700
Anticancer Effects of Bamboo Salt on Human Cancer Cells and on Buccal Mucosa Cancer in Mice
Authors: Park Kun‐Young , Zhao Xin , Mun Suk‐Hee
Published: June 2021

In vitro anti‐cancer effects of bamboo salt on HepG2 human hepatoma cells, AGS human gastric adenocarcinoma cells and TCA8113 human tongue carcinoma cells were investigated and compared with other salt samples. At the salt concentration of 1% in HepG2, AGS and TCA8113 cells, the growth inhibitory rate of 9× baked bamboo salt determined by MTT assay was 65%, 51% and 61%, respectively; however, sea salt showed lower inhibitory effects of 29%, 20% and 27%, respectively. Bamboo salt (9×) significantly (p<0.05) induced apoptosis, up‐regulated expression of Bax and down‐regulated Bcl‐2 expression. The bamboo salt also significantly down‐regulated inflammation‐related genes of iNOS and COX‐2. The ICR mice buccal mucosa cancer model was established by injecting the mice with U14 cells. Following the injection, the wound at the injection site was smeared with salt samples. It was observed that the tumor volumes for the group treated with 9× bamboo salt were smaller than those from the other samples. The buccal mucosa tissues of 9× bamboo salt group mice showed an increase in Bax and a decrease in Bcl‐2, iNOS and COX‐2 expressions by RT‐PCR and Western blot compared with the other treated samples.
ALOX5 gene variations modulate plasma triglyceride levels during an n‐3 polyunsaturated fatty acid supplementation
Authors: Cormier Hubert , Rudkowska Iwona , Julien Pierre , Couture Patrick , Lemieux Simone , Vohl Marie‐Claude
Published: June 2021

Single nucleotide polymorphisms (SNPs) in the arachidonate 5‐lipoxygenase (ALOX5) gene may play a role in lowering plasma triglyceride (TG) levels following supplementation with omega‐3 (n‐3) polyunsaturated fatty acids (PUFA).

Objective

To examine the effects of n‐3 PUFA supplementation on plasma TG levels in relation to the presence of SNPs in the ALOX5 gene.

Methods

208 subjects completed a 2‐wk run‐in period followed by a 6‐wk supplementation with 5g/d of fish oil (1.9g of EPA + 1.1g of DHA). Genotyping of 12 SNPs of the ALOX5 gene was performed covering 86% of common genetic variations (minor allele frequency ≥0.05).

Results

Gene*diet interaction effects were observed for plasma TG (rs1565096 and rs7913948). Significant differences in TG levels were observed for SNP rs1565096 where AA homozygotes (HMZ) showed a smaller reduction of plasma TG levels as compared to GG HMZ (p=0.04).

Conclusion

Results suggest that SNPs in the ALOX5 gene may modulate plasma TG levels in response to n‐3 PUFA supplementation. Funding : FAST‐NSERC
SELP Variant Modulates Plasma HDL‐C Responses in Subjects with Moderate Cardiovascular Risk after Skimmed Milk Consumption
Authors: Casas‐Agustench Patricia , Molina Susana , Espinosa‐Salinas Isabel , Olivares Monica , Reglero Guillermo , Ordovás José M , de Molina Ana Ramírez
Published: June 2021

P‐selectin is an adhesion molecule that contributes to atherosclerosis. The aim was to examine relationships between SELP (selectin P) variant rs2205895 and intake of two forms of milk for plasma lipid outcomes. In a parallel‐design, 12‐month feeding trial, 253 subjects (25–65 y) were randomly assigned to drink 500ml/d of semi‐skimmed (SS) (1.9% fat) or skimmed (S) milk (0.3% fat), in addition to their usual diet. Plasma lipids were determined at baseline and at completion of the study. We detected significant interaction terms between SELP genotype and type of milk for changes in HDL‐C (P=0.026). In carriers of the minor allele (T), intake of SS milk was associated with a greater increased HDL‐C (3.0%) compared to S milk (0.1%). In contrast, in CC carriers, SS milk intake was associated with a decreased HDL‐C (−2.0%), whereas S milk increased HDL‐C (4.6%). No other significant interaction terms between SELP genotype and type of milk for changes in total cholesterol, LDL‐C, triglycerides or LDL‐HDL ratio were detected. Daily intake of S milk may improve HDL‐C compared to SS milk in individuals with specific genotypes. The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007–2013) under grant agreement no. PIOF‐GA‐2010–272581 (to PC‐A).
Dietary protein diet prevents high fat induced intestinal adaptation and thereby increases intestinal lipid uptake rates in mice
Authors: Schwarz Jessica , Tomé Daniel , Müller Michael R , Hooiveld Guido JEJ
Published: June 2021

High saturated fat (HF) diets lead to obesity and an associated adaptation in the small intestine (SI), reflected by increased lipid uptake in the middle and distal part. We previously showed that a high protein (HP) diet prevents and reverses obesity and fatty liver even in the context of a HF diet. Our aim was to study if a HP diet can prevent obesogenic adaptation of the SI and consequently affect lipid handling by regional differences in gene expression.

Male C57BL/6 mice were fed either a high fat, normal protein (HF‐NP) or a HF‐HP diet for 1 week. After an overnight fast mice received a lipid load containing radio labeled fatty acid (FA) and triglyceride (TG). Appearance of labels in blood was followed over 5 hours. Changes in gene expression in SI were studied using microarray and subsequent gene set enrichment analysis.

The HF‐HP diet increased appearance rate of TG (35%, p=0.0407) and FA (29%, p=0.1085) in blood. Transcriptional changes were detected in the metabolism of amino acids along the SI while lipid processing and transport only increased in the proximal part of the SI.

Despite a more rapid appearance of lipids in the blood, mice fed the HF‐HP diet do not develop obesity and fatty liver. This suggests that during the HP diet lipids are taken up in the proximal part of the SI and HF diet induced uptake in the lower parts is prevented.

This research was supported by a FP7 Marie Curie ERG and by the Dutch Dairy Organisation (NZO).

Grant Funding Source: FP7 Marie Curie ERG and by the Dutch Dairy Organisation
High intestinal calcium (Ca) absorption efficiency is positively associated with bone mass in a genetically diverse population of mice
Authors: Replogle Rebecca A , Li Qiang , Fleet James C.
Published: June 2021

Habitual low Ca intake increases intestinal Ca absorption efficiency (Ca Abs) by a 1,25 dihydroxyvitamin D (1,25D)‐regulated mechanism. We investigated the role of Ca Abs on bone using a diverse population of 11 inbred mouse lines. Mice were fed a normal (0.5%) or low (0.25%) Ca diet from weaning to 3 mo of age and Ca Abs was determined by an oral gavage test. After correcting for the confounding effects of body size; Ca Abs, femur bone mineral density (BMD), trabecular bone volume fraction (BV/TV), and serum 1,25D varied significantly across the 11 lines at baseline and in low Ca adaptive response. Although Ca Abs was correlated to serum 1,25D levels (r=0.35, p<0.05), adaptation of Ca Abs to low Ca intake varied among the lines but was not strongly linked to diet‐induced changes in serum 1,25 D. Despite this heterogeneity, Ca Abs was significantly associated with femur BMD (r=0.17, p<0.05) and BV/TV (r=0.34, p<0.01). Duodenal transient receptor potential vanilloid member 6 and calbindin D9k mRNA levels were significantly correlated to each other (r=0.74), to serum 1,25D (r=0.44 and 0.39, respectively) and to Ca absorption (r=0.42 and 0.43, respectively), supporting the facilitated diffusion model. These results demonstrate the importance of Ca Abs and its adaptation to habitual low Ca intake for protecting bone mass during growth.

Grant Funding Source: NIH Award ES19103 to JCF
Epithelial transport of immunogenic and toxic gliadin peptides in vitro
Authors: Zimmermann Christian Alexander , Arampatzi Sevgi , Maison Wolfgang , Lochnit Günter , Zimmer Klaus‐Peter , Rudloff Silvia
Published: June 2021

In contrast to immunogenic gliadin peptides (GP) like P56–68, the toxic P31–43 may mediate direct toxic effects by activation of the intestinal immune system in celiac disease (CD). Epithelial translocation of immunogenic and toxic GP is considered as an important step in the pathogenesis of CD. Thus, we studied transepithelial passage of fluorescence labeled GP in a transwell system using differentiated enterocytes (Caco‐2 cells). Quantification of GP translocation was done by fluorometric measurement. Structural changes of GP were analyzed by mass spectrometry (MALDI‐MS); flow cytometry and confocal microscopy were used to investigate intracellular uptake. P31–43 and P56–68 were able to cross the epithelial barrier into the basolateral compartment. Fluorometric analysis revealed a higher basolateral fluorescence intensity when cells were incubated with P31–43 compared to P56–68. Flow cytometric analysis and confocal microscopy showed a cellular uptake of both GP which was markedly reduced at 4°C and by the endocytosis inhibitor methyl‐β‐cyclodextrin. MALDI‐MS revealed cleavage of GP on the apical side of the cells and the translocation of intact P31–43 and P56–68 as well as fragments thereof depending on incubation time and concentration. In conclusion, toxic and immunogenic GP were shown to pass the epithelial barrier and may thereby be able to interact with the intestinal immune system.
Inhibitory effects of resveratrol on IgE‐mediated degranulation and anaphylactic reaction in basophilic mast cells and mice
Authors: Han Seon‐Young , Kang Young‐Hee
Published: June 2021

Resveratrol is a phytoalexin abundantly found in red grape skin, and is effective in anti‐tumor and anti‐inflammation associated with immune responses. This study was conducted to test whether resveratrol suppressed IgE‐mediated allergic responses and passive cutaneous anaphylaxis (PCA) in RBL‐2H3 cells and in mice. RBL‐2H3 cells sensitized with anti‐DNP‐IgE and subsequently stimulated by DNP‐HSA increased the release of β– hexosaminidase and histamine. When RBL‐2H3 cells were pretreated with nontoxic resveratrol at 1–25 μM, such induction was dose‐dependently diminished. Syk, PLCγ, PKCμ and PKCθ of sensitized RBL‐2H3 cells were activated by stimulation with DNP‐HSA, which was dampened by ≥5 μM resveratrol. Furthermore, oral administration of 10 mg/kg resveratrol reduced serum histamine level enhanced in sensitized mice challenged to DNP‐HSA. Additionally, resveratrol decreased IgE‐mediated PCA and alleviated allergic edema in mice. Allergic skin inflammation, induction of MCP‐1 and MIP‐2, were inhibited by supplementing resveratrol to sensitized antigen‐challenged mice. Resveratrol inhibited mast cell‐derived immediate‐type allergic reactions, and these responses of resveratrol suggest possible therapeutic strategies in preventing allergic inflammatory diseases.
Chronic administration of low dose EGCG inhibits TNF‐α‐induced IL‐6 and IL‐8 production in rheumatoid arthritis synovial fibroblasts
Authors: Riegsecker Sharayah , Ahmed Salahuddin
Published: June 2021

Rheumatoid arthritis (RA) is a chronic inflammatory disease in which cytokines stimulate synovial fibroblast proliferation leading to the destruction of the joints. In this study we evaluated the efficacy of chronic low doses of epigallocatechin‐3‐gallate (EGCG), a polyphenol found in green tea, in regulating TNF‐α‐induced IL‐6 and and IL‐8 production. RA snyovial fibroblasts were treated with chronic low doses of EGCG (100–1000 nM) for 7 days followed by TNF‐α stimulation. Conditioned medium was used for the quantification of IL‐6 and IL‐8 by ELISA and cell lysates were prepared for the analysis of signaling proteins. Chronic administration of EGCG inhibited the ability of TNF‐α to induce IL‐6 and IL‐8 production in RA synovial fibroblasts in a dose‐dependent manner(p<0.05). Evaluation of the signaling pathways by Western blotting revealed that chronic exposure of EGCG selectively inhibited TNF‐α‐induced phosphorylation of JNK p54 and AKT, with no or little effect on p‐ERK or p‐P38 MAPKs. These results suggest that the chronic consumption of EGCG, which can be achieved physiologically by drinking green tea, has an ability to decrease the inflammatory millieu and consequently limit joint damage in RA.

Grant Funding Source: NIH
The Role of Insulin in Glucose Transporter Expression, Proliferation and Effector Function in Human CD4+ and CD8+ T Cells
Authors: Meng Huicui , Black Adam J , Rogers Connie J
Published: June 2021

Glucose can be transported into T cells via several glucose transporters. Our preliminary data shows the presence of GLUT‐1, 3 and 4 on human T cells, and significant enhancement in GLUT‐1 and 4 expression upon activation, suggesting increased glucose uptake and metabolism needed for proliferation. T cells are responsive to several hormones, including insulin, which may regulate cell proliferation via its effects on glucose uptake and metabolism in T cells. Insulin can induce GLUT‐4 expression in myocytes and adipocytes, and thus enhance glucose uptake in these cells. However, it is unknown if any of the glucose transporters on T cells are insulin sensitive. The goal of the current study is to determine if insulin can augment T cell activation and effector function via its effect on any of the glucose transporters. Peripheral blood was collected from healthy subjects (20–40 years old), PBMCs were isolated, and T cells were stimulated with anti‐CD3 antibody (1μg/mL) alone, or with insulin (1mU/mL). Following 3‐ day cultures, T cells were harvested, counted and GLUT‐1 and 4 expression was quantified on CD4+ T cells concurrently with CD4+ T cell proliferation. Our current data demonstrates that neither GLUT‐1 and 4 expression nor cell proliferation was altered by insulin in CD4+T cells. Further studies will measure the role of insulin in glucose transporter expression, proliferation and effector function in CD8+ T cells.

Grant Funding Source: Penn State Internal Funding
Immuno‐stimulating Activities of Polysaccharides from Korean Red Ginseng in Cyclophsphamide‐treated Immunosuppressed Mice
Authors: Cho Chang‐Won , Han Chun‐ji , Hong Hee‐Do , Rhee Young Kyoung , Nam So‐Hyun , Sung Su‐Kyung , Rho Jeonghae , Choi Sang Yoon , Lee Young‐Chul
Published: June 2021

In this work, the crude polysaccharide (RGW‐0) was isolated from Korean red ginseng by hot water extraction, ethanol precipitation and dialysis. The major constituents in RGW‐0 were neutral sugar (73.5%), acidic polysaccharide (26.3%). The neutral sugar was composed mainly of glucose (75.6 mole%), arabinose (9.9 mole%), and galactose (7.4 mole%). We also investigated the immuno‐stimulating activities of RGW‐0 in 30 male Chinese Kun Ming mice immunosuppressed by treatment with cyclophophamide. The mice were divided into three groups, and treated by oral administration with RGW‐0 into two dosages (100 mg/kg and 200 mg/kg body weight) for 4 weeks. The results showed that RGW‐0 stimulated proliferation of splenic lymphocytes (T and B cells) in cyclophophamide‐induced immunosuppressed mice. RGW‐0 also markedly increased the natural killer‐cell activity and peripheral white blood cell number. This result showed that RGW‐0 could restore declined immune functions in immunosupressed mice and can be used as an effective immuno‐stimulator.
Kinesin‐1 mediates targeted recycling of the lateral border recycling compartment
Authors: Cyrus Bita , Muller William
Published: June 2021

A critical step in the inflammatory response is transendothelial migration (TEM). Our lab has recently discovered a membrane compartment in endothelial cells (EC) that regulates TEM: The lateral border recycling compartment (LBRC). The LBRC contains a subset of membrane proteins, such as PECAM, CD99, and JAMA, which are required for TEM. During TEM, membrane from the LBRC is directed to the region of the EC where the leukocyte is transmigrating. This “targeted recycling” (TR) facilitates TEM by delivering LBRC membrane and proteins to the migrating leukocyte. Inhibiting targeted recycling of the LBRC blocks leukocyte TEM. Targeted recycling of the LBRC requires functional microtubules and kinesin motors. Microinjection of EC with an antibody against the conserved motor domain of kinesin inhibited TR and TEM to a similar magnitude as blocking PECAM or depolymerizing microtubules. We sought to identify the specific kinesin(s) that mediate targeted recycling. Preliminary experiments suggested the KIF5b gene product, kinesin‐1 as a likely candidate. Microinjection of EC with a kinesin‐1 mAb blocked TEM. Knocking down kinesin‐1 in EC decreased targeted recycling of LBRC and leukocyte TEM in preliminary experiments. This suggests that kinesin‐1 is a major molecular motor necessary for LBRC transport along microtubules during TR. Supported by an AHA Midwest Affiliate Predoctoral Fellowship and NIH R01 HL046849.
Activation of polymorphonuclear cells releases the chemotactic factor Ecrg4 into conditioned media
Authors: Costantini Todd W , Knowling Stu , Dang Xitong , Eliceiri Brian P , Baird Andrew , Coimbra Raul
Published: June 2021

Sentinel factors expressed on circulating cells monitor homeostasis and may initiate inflammatory signaling after injury. We identified Esophageal Cancer Related Gene‐4 (Ecrg4) as a candidate sentinel factor that resides on the surface of quiescent polymorphonuclear cells (PMNs) and responds to injury by releasing Ecrg4 protein from the cell surface. The objective of this study is to evaluate the kinetics of Ecrg4 release and the biological activity of Ecrg4 in chemotaxis migration assays. Peripheral blood was obtained from healthy volunteers and isolated cells were incubated with lipopolysaccharide (LPS) to characterize the release of Ecrg4 from the leukocyte surface. To establish the chemotactic potential of soluble recombinant Ecrg4, cells were subjected to in vitro migration assays using modified Boyden chambers. Ecrg4 was expressed at high levels on PMNs compared to other tissues surveyed, and is detected as a membrane‐anchored protein on the surface of resting PMNs. Exposure of cells to a dose curve of LPS led to shedding of Ecrg4 as determined by a reduction of Ecrg4+ PMNs and a concomitant increase in soluble Ecrg4 protein in conditioned media. Migration assays demonstrated that recombinant Ecrg4 increased the chemotaxis of primary fibroblasts. These results support a role for Ecrg4 as a novel chemokine expressed in a latent form on the surface of PMNs, and biologically active in a soluble form.
Polymorphisms in Toll‐Like Receptors 2 and 5 Are Associated with Legionella pneumophila infection
Authors: Zhang Fan , Wang Su‐ping , Gao Xiang‐dong , Wu Wei‐wei
Published: June 2021

Objective

we investigated whether single nucleotide polymorphisms (SNPs) in Toll‐like receptors2 (TLR2) and TLR5 affect the susceptibility of LD in a Han Chinese population by in vitro assay.

Method

Fifty four healthy subjects were genotyped for SNPs (TLR2 C597T [rs3804099] and TLR5 C1174T [rs5744168]). Peripheral blood mononuclear cells (PBMCs) were obtained from these subjects and were stimulated with live Legionella pneumophila (Lp) for 24 hours. The mRNA levels were determined using real‐time quantitative Polymerase Chain Reaction (RT‐qPCR) and the levels of TNF‐α, IL‐1β and IL‐6 were measured by enzyme‐linked immunosorbent assay (ELISA).

Results

After twenty four hours of Lp stimulation, the mRNA expression level of MyD88 was significantly lower in PBMCs with TLR2(C597T) CT/TT (P=0.0482) or with TLR5(C1174T)CC homozygotes (P=0.0223) comparing to PBMCs with other genotypes. Supernatant from culture of PBMCs with genotype TLR2(C597T) CT/TT or TLR5(C1174T)CC has higher levels of TNF‐α, IL‐1β and IL‐6 comparing to PBMCs with other genotypes after Lp‐stimulation.

Conclusion

Our findings suggest that both the TLR2(597CT/TT, rs3804099) and the TLR5 (1174CC, rs5744168) polymorphic variants are associated with resistance to LD in the evaluated Han Chinese population.

This study was supported by the National Natural Science Funds of China (grant no. 30872154)
Regulation of the Osmotic Tolerance Response in the Halophile Vibrio parahaemolyticus
Authors: Powell Laura Amanda
Published: June 2021

Vibrio parahaemolyticus is a Gram‐negative, rod‐shaped halophile bacterium present in marine environments worldwide. The bacterium has an absolute requirement for NaCl and can grow in up to 10% NaCl in complex media. Vibrio parahaemolyticus is also a pathogen of humans that causes gastroenteritis usually through the consumption of raw oysters. The V. parahaemolyticus genome contains a large number of compatible solute synthesis and transporter systems that allows it to grow in high NaCl concentrations. How these multiple systems and osmotic tolerance in general are regulated is unknown. In this study, we investigated the regulation of osmotic tolerance by examining the homologue of the EnvZ/OmpR two‐component signal transduction system, vp0155 and vp0154. In Escherichia coli EnvZ/OmpR regulates more than 100 genes, and significantly influences cell growth, metabolism, and motility. For example, in response to osmotic stress the EnvZ/OmpR system has been shown to regulate expression of outer membrane proteins (OMPs). We examine its role in V. parahaemolyticus by deleting a portion of vp0155, an envZ homologue and vp0154, the ompR homologue. Through splicing by overlap extension (SOE) PCR and homologous recombination, a deletion was created in both envZ and ompR. Through homologous recombination and selection, mutants harboring in‐frame deletions in the envZ and ompR genes were created, and confirmed by PCR and sequencing. We describe the effects of our knockout mutations in envZ/ompR in response to NaCl, acid, and alkaline stresses as well as swimming and swarming motility and OMPs and type VI secretion system expression.

Research supported by a grant from the Howard Hughes Medical Institute.
Giardia duodenalis genotype A and B are responsible for post infectious visceral hypersensitivity in an immunocompetent suckling rat model: Is commensal bacterial translocation one of the mechanisms leading to PI‐IBS?
Authors: HALLIEZ Marie Christiane , LE GOFF Laetitia , FRANCOIS Arnaud , GARGALA Gilles , FAVENNEC Loic , BURET Andre Gerald
Published: June 2021

Enteric pathogens such as Giardia duodenalis, common waterborne protozoan parasite, have been linked to Post‐infectious Irritable Bowel Syndrome (PI‐IBS) a functional intestinal disorder, in which visceral hypersensitivity is a key factor. We aimed to develop a model characterizing PI‐IBS triggered by Giardia infection and, reveal mechanisms responsible for the PI‐IBS‐like symptoms, such as the role of the commensal microbiota. Five day‐old Sprague‐Dawley rats gavaged with 104 trophozoites (genotypes A or B) were assessed for visceral hypersensitivity 50 days post‐infection (PI) via jejunal/rectal balloon distension. Changes in blood pressure were recorded as measurements of visceral hypersensitivity. Caco‐2 monolayers were used to assess if Giardia facilitates the translocation of a non‐invasive E. coli at various time points. On day 50 PI, jejunal and rectal hypersensitivity was observed with both genotypes. G. duodenalis also facilitated translocation of non‐invasive E. coli through Caco‐2 monolayers 3h PI. Hypersensitivity was detected in the jejunum, as well as in the rectum, a site beyond the infection area, indicating that the effect is not limited to the presence of the pathogen. Giardia facilitates the passage of non‐invasive bacteria through the intestinal epithelium, mechanism which could cause PI‐IBS. This study describe a new model for the study of protozoa‐induced PI‐IBS.

fundings: NSERC Create, FFCR (France‐Canada Research Fund), CCFC
Characterization of Debaryomyces hansenii strains as a potential treatment against superficial mycoses
Authors: Gordian Valerie , Serrano Yorki , Arroyo Nancy
Published: June 2021

Debaromyces hansenii is a marine yeast that inhibits the growth of certain types of fungi through the production of killer toxins. The potential of these killer toxins against human mycoses is the object of our study. We studied 100 patient samples with diverse superficial skin lesions. The samples were inoculated on Sabouraud Dextrose Agar to allow fungi growing and subsequently were inoculated on Dermatophytes Test Medium (DTM) to observe the growth of dermatophytes. There are three types of dermatophythes: Tricophyton sp., Microsporum sp. and Epidermophyton flucossum which commonly cause skin disorders such as secondary pyoderma or dermatophytosis that can spread from the affected area to feet, scalp, groin, or nails. If not treated, these organisms can cause more serious diseases. After isolating, identifying and characterizing these fungi species, the purified killer toxins activity from different D. hansenii strains were tested against these medically important fungi.
Stimulating innate immunity in feedlot cattle: strategies to induce tracheal antimicrobial peptide gene expression
Authors: Berghuis Lesley Michelle , Caswell Jeff , Sharif Shayan , Karrow Neil
Published: June 2021

Bovine respiratory disease (BRD) is an economically devastating complex of bacterial and viral infections that greatly affects the feedlot industry across North America. Studies have shown that viral infections such as BVDV and glucocorticoid production in stressed calves may inhibit the induction of tracheal antimicrobial peptide (TAP), a cationic β‐defensin that has direct microbicidal effects on invading microbes in the respiratory tract. Certain surface receptors of the innate immune system called the Toll‐Like‐Receptors (TLRs) recognize pathogen associated molecular patterns (PAMPs) to initiate intracellular signal transduction pathways that potentially induce TAP gene expression. PCR analyses of unstimulated tracheal epithelial cells show mRNA expression for TLRs 1,2,3,4, and 6. Therefore, stimulation of tracheal epithelial cells with agonists for these TLRs may induce TAP expression. Previous studies have shown that a TLR 4 agonist called Lipopolysaccharide (LPS) significantly induces TAP expression in epithelial cells after 16 hours of stimulation. This suggests that LPS induces TAP expression via the TLR 4 pathway. The present study examines TLR 2 agonists to determine their effect on TAP induction. Quantitative Real‐Time PCR analysis of epithelial cells stimulated by the TLR 2 agonist Lipoteichoic Acid has shown significant induction of TAP. However, the amount of induction is much less than LPS and occurs after the same amount of stimulation time. Future experiments will determine if other TLR 2 agonists can induce gene expression of TAP in a robust and rapid manner so that TAP production can be increased to ultimately reduce the incidence of BRD in feedlot calves.
Sublethal hypoxic injury increases intestinal permeability via disruption of sealing tight junction proteins, but not pore forming tight junction proteins in human intestinal epithelium
Authors: Jin Younggeon , Blikslager Anthony T.
Published: June 2021

Intestinal ischemia induces loss of barrier function which is followed by epithelial repair. The tight junction (TJ) has recently been characterized as having both leak and pore properties. We hypothesized that hypoxia would disrupt the TJ seal prior to the pore function followed by barrier recovery. Caco‐2 BBe1 confluent monolayers were subjected to hypoxia for 2 hours followed by recovery in 21% O2, after which barrier function was determined by measuring transepithelial electrical resistance (TER) and flux of 4 kDa FITC‐dextran. TJ proteins were studied by immunoblotting and immunolocalization. Following hypoxic injury, there was an increase in the paracellular permeability, as determined by dextran fluxes, but no change in TER compared to control cells. Permeability recovered with significant reduction in fluxes at 2 and 4 h. The sealing TJ proteins occludin and claudin‐4 increased expression after hypoxic injury. However, the occludin and claudin‐4 fluorescence was diffuse on confocal microscopy, but re‐localized their normal distribution after 4 h recovery. The expression and distribution of the pore forming TJ protein claudin‐7 was not changed. This correlated with a lack of change in TER. These data suggest that hypoxic injury increases paracellular permeability by disruption of sealing TJ proteins, creating a leak but not altering pore selectivity.
Adducin modulates intercellular keratinocyte adhesion
Authors: Roetzer Vera , Waschke Jens , Spindler Volker
Published: June 2021

Adducin is an actin‐binding protein necessary for proper assembly of the cortical actin belt. Although regulation of the membrane cytoskeleton by adducin is well established, a role for cell adhesion has not been investigated so far. We found that siRNA‐mediated silencing of adducin reduced desmoglein (Dsg) 3 protein levels and decreased intercellular adhesion of human keratinocytes in dispase‐based dissociation assays. Previous data indicated that the loss of keratinocyte cohesion by Dsg autoantibodies in the blistering skin disease pemphigus is paralleled by pronounced actin reorganization and blocked by RhoA activation. Since cortical actin polymerization is required for Rho GTPase‐mediated strengthening of cell adhesion, we studied the role of adducin in this context. AK23, a pathogenic Dsg3 antibody from a pemphigus mouse model, resulted in rapid adducin phosphorylation at Serin726, a residue important for regulation of adducin activity. AK23‐mediated cell dissociation was blocked by RhoA activation via Y. pseudotuberculosis cytotoxic necrotizing factor y (CNFy). However, under conditions of adducin silencing or after expression of a phosphorylation‐deficient adducin mutant, the protective effect of CNFy was inhibited. These data demonstrate that adducin is necessary for proper intercellular adhesion and suggest an involvement in the protective effect of RhoA in pemphigus vulgaris.
Differential Regulation of Class A Scavenger Receptor (SR‐A) Function by Compartmentalization
Authors: Vadali Shanthi , Post Steven
Published: June 2021

SR‐A is a macrophage receptor with roles in the development of atherosclerosis and tissue repair following myocardial infarction. SR‐A mediates ligand internalization and macrophage adhesion; however, the mechanisms that differentially regulate these functions are not well defined. In this study, we examined whether SR‐A function is regulated by localization in cholesterol‐rich lipid rafts (LR). A detergent‐free cell fractionation protocol was used to show that SR‐A was 3‐fold enriched in LR relative to non‐raft membranes isolated from primary macrophages, THP‐1 macrophages, and stably transfected HEK cells. An association between SR‐A‐mediated cell adhesion and LR was indicated by truncating the cytoplasmic tail of SR‐A (SR‐AΔ1–49), which abolished receptor internalization but did not affect SR‐A‐mediated adhesion or localization in LR. Further, disrupting LR function by depleting membrane cholesterol with methyl‐beta‐cyclodextrin (MβCD) inhibited cell adhesion mediated by SR‐A, but not that mediated by SR‐AΔ1–49. This suggests the presence of a LR‐dependent regulatory element in the cytoplasmic domain of SR‐A. In contrast to its effect on cell adhesion, MβCD treatment did not significantly affect the SR‐A‐mediated uptake of modified lipoprotein. Thus, localization of SR‐A in LR, perhaps with specific signaling proteins, is a mechanism that differentially regulates SR‐A function. Support: NIH‐HLRO1–076682 and AHA‐11PRE‐7950020.
Atorvastatin‐induced cardiac function impairment
Authors: Qiao Congzhen , Lauver Dale Adam , Lucchesi Benedict
Published: June 2021

Statins, known as 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase inhibitors, are reported to reduce low‐density lipoprotein cholesterol levels in blood. In spite of the dramatic cholesterol‐lowering effect, statin‐induced myalgia and fatigue have been widely reported. The inhibition of HMG‐CoA reductase by statins prevents the synthesis of mevalonate, a precursor for the synthesis of cholesterol as well as coenzyme Q10 (CoQ10). It has been well documented that CoQ10 plays a critical role in the synthesis of mitochondrial adenosine‐5′‐triphosphate (ATP), the depletion of which can cause a reduction of intracellular ATP, leading to myalgia. Given the high‐energy demand that cardiac muscle requires, we are interested in statin's off‐target effect on the skeletal and cardiac muscle in rabbits. Several clinical studies have shown that there is a link between the reduction of CoQ10 and cardiomyopathy. In addition, endomyocardial biopsies have shown a decrease of myocardial ATP in a failing heart, suggesting that the reduction of ATP may exacerbate heart failure and contribute to heart remodeling. However, few publications have reported the adverse cardiac effects of statins most likely due to the depletion of ATP. To address this issue, we demonstrated atorvastatin's effect on rabbit cardiac function by employing Langendorff heart model as well as morphological changes detected by transmission electronic microscopy. Our preliminary data suggest that atorvastatin impairs cardiac function in 7 and 28 days treatment compared to placebos. In addition, the disruption of the mitochondrial ultrastructure in 28 days atorvastatin‐treated group indicates a long‐term deleterious cardiac effect of atorvastatin. Hence, we believe that statin‐induced CoQ10 deficiency leads to the depletion of ATP, which impairs heart function and may contributes to the pathogenesis of heart failure.

The source of research support: the University of Michigan Cardiovascular Research Fund
The Protective Effect of 11‐Keto‐β‐Boswellic Acid a selective 5‐LOX Inhibitor on Myocardial ischemia Reperfusion Injury in Rats
Authors: Nassar Noha N , El Shazly Shimaa M , El Motteleb Dalia M Abd
Published: June 2021

Myocardial ischemia induces 5‐lipoxygenase (LOX) translocation and leukotriene production in the heart. Leukotrienes increase inflammatory responses and could thereby aggravate ischemic injury. This study aimed to investigate whether 11‐Keto‐β‐boswellic (a selective 5‐LOX inhibitor) in three different dose levels exert protective effect on myocardial ischemia reperfusion injury in an in vivo rat heart model. In the current study, forty rats were randomly assigned into 5 groups. GP1: sham operated, Gp 2: ischemic reperfusion (I/R) model while Gps 3,4,5 received 11‐ Keto‐boswellic acid in doses 250, 500, 1000 mg/kg respectively via an oral gavage for 7 days, then subjected to I/R which was induced by ligation of left anterior descending coronary artery for 45 minutes followed by 4 hours reperfusion. At the end of the experiments hearts were excised for analysis. There were significant elevation in the expression of nuclear factor (erythroid‐derived 2)‐like 2, heneoxygenease‐1 and restoration of glutathione peroxidase in a dose dependent manner. Furthermore, there were significant and dose dependent reduction in elevated myeloperoxidase, reduction in the expression of nuclear factor kappa B, tumor necrosis factor ‐α, 5‐LOX, cyclooxygenase‐2 and intercellular adhesion molecule 1 in the heart tissues. We concluded that 11‐Keto‐boswellic acid exerts a protective effect in myocardial I/R injury through mechanisms related to enhancement of antioxidant capacity and prevention of inflammatory cascades.
Sestrin2 is cardioprotective against ischemia/reperfusion injury by promoting LKB1‐mediated AMPK activation
Authors: Morrison‐Nozik Alexander , Tong Chao , Lee Jun Hee , Budanov Andrei , Karin Michael , Li Ji
Published: June 2021

The mechanism of AMP‐activated protein kinase (AMPK) activation in the ischemic heart remains poorly understood. Recent evidence implicates the role of Sestrin2 (Sesn2) in the AMPK signaling pathway and we hypothesize that Sesn2 plays an influential role during myocardial ischemia in order to promote AMPK activation. Sesn2 protein was found to be expressed in adult cardiomyocytes. Interestingly, Sesn2 was found to accumulate in the heart during ischemic conditions. Cardiac phenotype analysis of WT and Sesn2 KO mice were not significantly different. When subjected to in vivo ischemia/reperfusion (I/R), myocardial infarction was significantly greater in Sesn2 KO versus WT hearts. Similarly, Langendorff perfused hearts indicated exacerbated cardiac function after I/R injury in Sesn2 KO versus WT as evidenced by decreased LVDP, dP/dt, and rate‐pressure products. Moreover, ischemic AMPK activation was reduced in Sesn2 KO hearts. Immunoprecipitation of Sesn2 demonstrated an association with AMPK. Importantly, immunoprecipitation of LKB1 from WT hearts demonstrated AMPK association during ischemia while LKB1‐AMPK interaction in Sesn2 KO hearts was nearly abolished. The results demonstrate an important cardioprotective role of Sesn2 against I/R injury by acting as a LKB1‐AMPK scaffold.
Mechanism of Central Atypical Cannabinoid Receptor GPR18‐Mediated Hypotension in Conscious Rats
Authors: Penumarti Anusha , Abdel‐Rahman Abdel A
Published: June 2021

Background and objectives

Vascular GPR18 activation causes vasodilation, and our preliminary findings are the first to show: (i) GPR18 expression in tyrosine hydroxylase (TH) immunoreactive neurons in the rostral ventrolateral medulla (RVLM); (ii) RVLM GPR18 activation (Abn CBD) and blockade (O‐1918) dose‐dependently reduced and increased BP, respectively, in conscious male Sprague Dawley rats. Here we tested the hypothesis that NO generation, via ERK1/2 activation, coupled with reduced reactive oxygen species (ROS) generation in RVLM underlies GPR18‐mediated hypotension. We complemented the integrative studies with signaling studies in differentiated PC12 cells (catecholamine expressing cells with neuronal phenotype) to elucidate the molecular mechanisms implicated in GPR18 signaling.

Key results: Ex vivo studies, on RVLM neuronal tissues, collected during the hypotensive response elicited by intra‐RVLM GPR18 activation in conscious rats revealed increases in ERK phosphorylation and NO level and reduced ROS in RVLM while GPR18 blockade elicited opposite BP and molecular responses. In PC12 cells, GPR18 is associated with lipid rafts, and its activation replicated the molecular responses obtained in the RVLM.

Conclusion

GPR18 activation in the RVLM lowers BP by reducing oxidative stress and increasing NO, via ERK1/2 phosphorylation. Similar molecular findings in PC12 cells support the use of this model system in ongoing GPR18 signaling studies. Specifically, pharmacologic/genetic manipulation studies will elucidate the role of the lipid rafts and their major protein, caveolin, in GPR18 signaling.
TEMPORAL EFFECTS OF CHRONIC ANGIOTENSIN II INFUSION ON BLOOD PRESSURE AND VASCULAR FUNCTION
Authors: Gomolak Jessica R , Faulkner Jessica L , Didion Sean P
Published: June 2021

While Angiotensin II (Ang II) infusion is commonly used as a model of Ang II‐dependent hypertension, little is known regarding the temporal effects of Ang II infusion on blood pressure or endothelial function. The goal of this study was to compare and contrast the effects of Ang II (1000 ng/kg/min via osmotic minipump) infused for 14 days and 28 days on systolic blood pressure (SBP) and endothelial function. Ang II infusion for 14 days significantly increased SBP relative to control mice (eg, SBP, 143±4 vs.115±2 mmHg, respectively). SBP was also increased in mice infused with Ang II for 28 days and was significantly greater (p<0.02) than that observed in mice infused with Ang II for 14 days (eg, SBP, 158±5 vs.143±4 mmHg, respectively). In terms of endothelial function, responses to acetylcholine (ACh) were markedly reduced (p<0.05) in carotid arteries from 14 day Ang II‐infused mice compared to control mice (eg, 100 μM ACh produced 57±7 vs. 97±2% relaxation, respectively). Ang II infusion for 28 days produced a similar degree of endothelial dysfunction to that produced by 14 day Ang II infusion (eg, 100 μM ACh produced 58±7 vs. 57±7% relaxation, respectively). Ang II infusion for 14 and 28 days produced a temporal increase in blood pressure, but a similar degree of endothelial dysfunction. Supported by NIH HL‐089884 & HL‐107632.
Celecoxib Offsets The Negative Renal Influences of Cyclosporine Via COX‐2/Endothelin ETB Receptor crosstalk
Authors: El‐Gowelli Hanan M , Helmy Maged W , Abdel‐Halim Rabab M , El‐Mas Mahmoud M
Published: June 2021

We tested the hypotheses that (i) celecoxib, a selective COX‐2 inhibitor, counterbalances renal derangements caused by cyclosporine A (CSA) in male rats, and (ii) the endothelin ETB receptor mediates the CSA‐celecoxib interactions. Ten‐day treatment with CSA (20 mg/kg/day) significantly increased biochemical indices of renal function (serum urea and creatinine) and inflammatory cytokines (renal transforming growth factor‐β and interleukin‐2). CSA also caused renal tubular atrophy and congestion in renal arterioles along with perivascular and interstitial fibrosis. These detrimental renal effects of CSA were dramatically reduced in rats treated concurrently with celecoxib (10 mg/kg/day). Moreover, immunohistochemical analyses showed that glomerular and tubular protein expressions of COX‐2 and ETB were reduced by CSA and restored to near‐control values in rats treated simultaneously with celecoxib. The adverse biochemical, inflammatory, and histopathological profiles of CSA were replicated in rats treated with the endothelin ETB receptor antagonist BQ788 (0.1 mg/kg/day, 10 days). The BQ788 effects, like those of CSA, were alleviated in rats treated concurrently with celecoxib. Together, facilitation of the interplay between COX‐2 and endothelin ETB receptors is the cellular mechanism by which celecoxib ameliorates the nephrotoxic manifestations of CSA in male rats.
Discovery, characterization, and optimization of highly selective D2 dopaminergic antagonists
Authors: Free R. Benjamin , Xiao Jingbo , Doyle Trevor , Conroy Jennie , Titus Steve , Bryant‐Genevier Melanie , Southall Noel , Hu Xin , Ferrer Marc , Javitch Jonathan A , Marugan Juan J , Sibley David R
Published: June 2021

The D2 dopamine receptor (DAR) is central in the etiology and/or therapy of many neuropsychiatric disorders. Unfortunately, truly specific drugs for this receptor have been difficult to obtain, primarily due to high conservation of the orthosteric binding site within DAR subtypes and other G protein‐coupled receptors (GPCRs). In order to develop novel molecular scaffolds for the D2 DAR, we used high throughput screening to interrogate a small molecule library and identify hit ligands with distinct functional characteristics, mechanisms of action, and selectivity among DAR subtypes. Here we present the discovery of a novel series of small molecule selective D2 DAR antagonists found in this campaign. One such hit was optimized via medicinal chemistry efforts, and a lead compound was identified with high D2 selectivity versus D1, D3, D4 and D5 DARs. Interestingly the binding of this compound to the D2 DAR demonstrated near complete dependence on Na+. Furthermore, the lead compound showed favorable ADME and in vivo pharmacokinetic (PK) properties. In a profiling screen of a large panel of GPCRs the compound showed relatively low cross reactivity. This probe could be a useful pharmacological tool for studying structure activity relationships with the help of computational docking and simulations, as well as an ideal scaffold for the further development of even more highly selective D2 DAR compounds.