bluuebunny/crossref_metadata_2025 huggingface dataset:
The lysine demethylase KDM6A regulates pancreatic β cells function
Authors: Zhang Bo , Wang Shirley , Sun Zhongjie
Published: May 2022

Background and Objective

Diabetes mellitus refers to a group of metabolic disorders characterized by impaired glucose homeostasis. Both type 1 diabetes (T1D) and type 2 diabetes (T2D) are characterized by insulin insufficiency. β cell failure in diabetes are often due to posttranslational modifications of histones and transcription factors. Lysine demethylase 6A (KDM6A) belongs to the KDM6 family of histone H3 lysine 27 (H3K27) demethylases which is important for development of various organs. The objective of this study is to investigate whether KDM6A regulates pancreatic β cell function using targeted β cell‐specific deletion of KDM6Agene.

Methods and Results

Briefly, inducible insulin 1‐cre and KDM6A‐loxp mice were bed to generate cre‐loxp mice. Injection of tamoxifen induced deletion of KDM6A gene in β cells which led to increased H3K27me3 levels. KDM6A‐KO mice exhibited reduced baseline insulin levels compared with wild‐type mice. Interestingly, despite low baseline insulin levels, KDM6A‐KO mice demonstrated enhanced glucose tolerance. Insulin release was increased in KDM6A‐KO mice vs WT mice at 15 min following glucose challenge. Furthermore, ex vivo glucose‐induced insulin secretion was increased in cultured islets isolated from KDM6A‐KO mice. In cultured Min6 β cells, knockdown of KDM6A using KDM6A shRNA largely enhanced glucose‐induced insulin release. When challenged with high‐fat diet, KDM6A‐KO mice displayed similar weight gain vs wild‐type mice. However, the insulin sensitivity test showed that KDM6A‐KO enhanced peripheral insulin sensitivity as evidenced by the delayed recovery of glucose level in KDM6A‐KO mice fed with high‐fat diet.

Conclusion

Our results suggest a novel role of KDM6A in the regulation of pancreatic β cell function. KDM6A may be a potential therapeutic target for β cell dysfunction.
The Role of ATF5 in Mitochondrial Maintenance, Biogenesis and UPRmt Signaling Following Acute Exercise in Skeletal Muscle
Authors: Slavin Mikhaela , Kumari Rita , Hood David
Published: May 2022

Maintenance of the mitochondrial protein folding environment is essential for organellar and cellular homeostasis. Over 99% of mitochondrial proteins require import into mitochondria, followed by their folding and intraorganellar sorting. Mitochondrial stress can result in the accretion of misfolded proteins, establishing a requirement for mitochondrial protein quality control (MQC) strategies. The Mitochondrial Unfolded Protein Response (UPRmt) is a compartment‐specific MQC mechanism that increases the expression of protective enzymes by Activating Transcription Factor 5 (ATF5) to restore mitochondrial function. Contractile activity during acute exercise is a stressor that has the potential to temporarily disrupt organellar protein homeostasis. However, the roles of ATF5 and the UPRmtin basal mitochondrial maintenance and exercise‐induced UPRmt signaling in skeletal muscle are not known. To investigate this, we subjected WT and whole‐body ATF5 KO mice to a bout of acute exercise and collected skeletal muscle tissues immediately after. ATF5 KO animals exhibited 2‐fold increases in phosphorylated JNK protein levels, indicative of enhanced stress signaling. Interestingly, in KO muscle, PGC‐1a protein was enhanced by 50% and 40% in nuclear and cytosolic compartments, respectively, suggesting an increased drive toward mitochondrial biogenesis in the absence of ATF5. Muscle from these animals also displayed a more abundant, but dysfunctional, mitochondrial pool, with a 20% increase in mitochondrial content, 30‐40% reductions in respiration, and a 20% increase in ROS emissions, corresponding with no changes in exercise performance. The UPRmt proteins mtHSP70 and LONP were upregulated 20‐30% in KO muscle, while ATF4 mRNA was upregulated 2.5‐3.7‐fold, along with an 8% increase in its nuclear localization. Furthermore, KO muscle showed an impaired UPRmt mRNA response to acute exercise, suggesting a regulatory role for ATF5 in the maintenance of a high‐quality mitochondrial pool, and in mediating the transcription of UPRmt genes during exercise.
Investigating Susceptibility of ꞵ‐catenin‐mutated Hepatocellular Carcinoma to Checkpoint Inhibitors
Authors: Delgado Evan R. , Tao Junyan , Bell Aaron W. , McGaughey Jackson , Ganesh Jai , Poddar Minakshi , Singh Sucha , Monga Satdarshan P.
Published: May 2022

Hepatocellular Carcinoma (HCC) is the 5th most common cause of cancer‐related death in the United States with an estimated 32,000 annual deaths. The standard of care for HCC as established by the IMBrave150 trial is a combination of the PD‐L1 inhibitor, Atezolizumab, and an antiangiogenic, Bevacizumab. While this combination extends overall survival to 19.2 months in unresectable HCC, only 52% of patients survive to 18 months. Furthermore, objective response rates are around 32% and hence clarifying underlying mechanisms regulating the immune response in HCC oncogenesis might strengthen immunotherapy efficacy. Especially important is to identify response rates of these agents in specific molecular subclasses of HCC may help in better patient selection. The oncogene ꞵ‐catenin is consistently mutated and activated among 30% of HCC patients. The Wnt/ꞵ‐catenin pathway has been approached as a target for precision medicine in HCC. We recently modeled HCCs with ꞵ‐catenin mutations in mice up to 69% genetic similarity. Here, we combine hydrodynamic tail vein injections with the Sleeping Beauty Transposase which is a non‐viral method of expressing genes of interest. Our model co‐expresses an S45Y point mutant of ꞵ‐catenin with the tyrosine kinase receptor MET (B+M). This combination represents ~10% of patient HCCs and provides a clinically relevant tool for the investigation of personalized HCC therapeutics.

A recent study demonstrated HCCs driven by a Δ90‐truncation mutant of ꞵ‐catenin in combination with the Myc oncogene are shown to lack sufficient immune cell surveillance. These HCCs have poor cytotoxic T cell and dendritic cell recruitment while responding poorly to checkpoint inhibitors. Since activating point mutations in CTNNB1 gene are common in patient HCCs, we asked whether poor immune surveillance extends to HCC models harboring point mutations. We first checked and found the presence of CD45+, F4/80+, and CD11b+ cells both 2 and 4 weeks after B+M tumor induction. This indicates the presence of both adaptive and innate immune cells in the B+M tumor microenvironment. We next asked whether checkpoint inhibition will effectively reduce tumor burden in the B+M model. Therefore, we treated B+M mice with either IgG or anti‐PD1 monoclonal antibodies 4 weeks after tumor induction. Tissues were harvested 3.5 weeks after treatment. We found a modest 20% reduction in liver weight to body weight ratio, a measure of tumor burden, in B+M mice treated with anti‐PD1 compared to IgG controls. Our results demonstrate that HCCs with missense ꞵ‐catenin mutations do respond albeit marginally to checkpoint inhibitors. In the future, combination of checkpoint inhibitors with other inhibitors will be tested in these models to determine their efficacy in treating ꞵ‐catenin‐mutated HCCs.
A New Method to Analyse Myocardial Substrate Metabolism and Mitochondrial Function in Intact Cardiac Tissue Slices
Authors: Heinen Andre , Bottermann Katharina , Spychala Andre , Eliacik Asena , Amin Ehsan , Moussavi‐Torshizi Seyed‐Erfan , Klöcker Nikolaj , Gödecke Axel
Published: May 2022

The progression of cardiac diseases is often accompanied by disturbances in substrate metabolism. Extracellular flux analysis has become a standard tool to investigate metabolic alterations in cell lines. However, the enzymatic digestion of the heart to isolate adult cardiomyocytes as well as the cultivation procedure that is required for cell attachment to the cell culture plates might affect metabolism. Therefore, we developed a flux analyser‐based method to measure substrate metabolism of intact cardiac tissue slices. Furthermore, we tested this method in a proof‐of‐principle approach in remote myocardium after myocardial infarction.

To yield cardiac tissue pieces of comparable size, mouse cardiac tissue was sliced (150 µm) using a vibratome, and tissue pieces (diameter 1.9 mm) of these slices were punched out. Using “islet capture plates” in a Seahorse XFe 24 analyser, oxygen consumption rates (OCR) were measured at baseline and after FCCP‐induced uncoupling in palmitate, glucose (Glc) and glutamine (Gln) enriched medium. To determine long‐chain fatty acid metabolism, CPT1 was inhibited by etomoxir, and Glc/Gln metabolism by inhibition of mitochondrial pyruvate carrier (MPC) and glutaminase (Gls) with UK5099/BPTES. Optical mapping of membrane potential was used to assess action potentials in tissue slices as indicator of cellular integrity. Finally, the developed method was used to analyse substrate metabolism in the remote myocardium at day 3 after myocardial ischemia and reperfusion (n=7) in comparison to sham mice (n=8). Data are mean±SD; unpaired two‐sample t‐test.

Basal OCR was 53±8 pmol/min, and FCCP increased OCR to 92±18 pmol/min. Both etomoxir and UK5099/BPTES reduced OCR indicating that both palmitate and Glc/Gln are metabolised. Optical mapping of tissue slices showed regular action potential characteristics and propagation. After myocardial infarction, CPT1 inhibition caused a smaller reduction of uncoupled mitochondrial OCR in I/R animals compared to sham (40±13 vs. 52±4%, p<0.05). This effect was caused by an increased metabolism of Glc/Glu (37±13 vs. 24±4 pmol/min, p<0.05), whilst the effect of CPT1 was not different.

Here, we describe a new method to analyse cardiac metabolism using cardiac tissue slices that metabolise fatty acids as well as glucose, and show high functional integrity. Therefore, this method has the potential to expand the methodological alternatives to investigate cardiac substrate metabolism. In a proof‐of‐principle approach, the analysis of cardiac substrate metabolism of the remote myocardium after I/R showed an augmented glucose/glutamine metabolism.
LPS transport during fat absorption is inhibited in caveolin‐1 knockout murine jejunum
Authors: Nishii Shin , Akiba Yasutada , Mizoguchi Akinori , Kaunitz Jonathan D.
Published: May 2022

Lipopolysaccharides (LPS) entry from the intestinal lumen to the circulation is associated with systemic inflammation. We have found that LPS is transcellularly transported to portal vein during long‐chain fatty acid (LCFA) exposure via CD36‐ and lipid raft‐mediated pathways in rats, suggesting the involvement of caveolae‐mediated endocytosis. We thus examined LPS transport in caveolin‐1 (Cav1) knockout (KO) murine jejunum.

FITC‐LPS was applied to the mucosal bath of Ussing chambered muscle‐stripped jejunal mucosa‐submucosa preparations of Cav1 KO and wild type (WT) mice. Serosal appearance of FITC‐LPS was measured with or without luminal application of oleic acid (OA, 30 mM) with taurocholic acid (TCA, 0.1 mM), or medium‐chain fatty acid sodium caprate (C10, 10 mM).

Luminal application of OA/TCA increased FITC‐LPS m‐to‐s transport in WT jejunum, inhibited by the CD36 inhibitor sulfosuccinimidyl oleate (SSO, 0.1 mM) or the lipid raft inhibitor methyl‐β‐cyclodextrin (MβCD, 0.1 mM), not by the clathrin inhibitor chlorpromazine (0.1 mM) or Pitstop2 (30 µM), suggesting that LCFA‐induced LPS transport is mediated by caveolae‐mediated endocytosis, not by clathrin‐mediated endocytosis. In contrast, OA/TCA‐induced FITC‐LPS transport was abolished in Cav1 KO jejunum. Nevertheless, luminal application of C10 increased FITC‐LPS transport both in WT and Cav1 KO jejuna without transepithelial electrical resistance changes, suggesting that C10 enhances transcellular LPS transport via caveolin‐independent endocytosis in the jejunum.

These results suggest that LPS transport during LCFA exposure is mediated by Cav1‐mediated endocytosis, whereas MCFA‐induced LPS transport is likely via clathrin‐mediated endocytosis. Modulation of epithelial endocytosis may be a new therapeutic target for LPS‐associated diseases mediated by dietary fatty acids, including metabolic syndrome.
Hypoxia Inducible Factor is partially involved in mitochondrial and metabolic responses to hypoxia in rats DEMAREST Maud 1, MARCOUILLER François 1, SOLIZ Jorge 1, JOSEPH Vincent 11 Institut universitaire de cardiologie et de pneumologie, Université Laval, Québec
Authors: Demarest Maud M. , Marcouiller François , Soliz Jorge , Joseph Vincent
Published: May 2022

High altitude environments present many constraints for mammals, the most well‐known being the lack of oxygen or hypoxia. Nevertheless, several ecological reports show that mice (Mus musculus) unlike rats (Rattus norvegicus) are present above 4000m1 although they are not native to this environment. The associated general hypothesis is that mice are pre‐adapted to high altitude environments. There are also differences in mitochondrial2, ventilatory and metabolic3 responses to hypoxia between these two species. One of the mechanisms involved in many of these responses is the hypoxia‐inducible factor HIF, which helps maintain the cell's oxygen homeostasis. What precisely are the different roles of HIF in mitochondrial, ventilatory, and metabolic responses to hypoxia in rats and mice, two species more or less adapted to high‐altitude environments? Does the pharmacological stabilization of HIF in normoxia mimic the responses observed in hypoxia? Conversely, does blocking HIF in hypoxia suppress these same responses? We measured the ventilatory and metabolic responses (whole body plethysmography) of rats (SD) and mice (FVB) under the following conditions: (i) 6h normoxia (21% O2); (ii) 6h hypoxia (10% O2); (iii) 6h normoxia with injection of deferoxamine, a HIF stabilizer (200mg/kg) and (iv) 6h hypoxia with injection of 2‐methoxyestradiol, a HIF blocker (5mg/kg). In addition, we evaluated mitochondrial respiration in homogenates of liver and cerebral cortex, two organs sensitive to hypoxia. Our preliminary results show that hypoxia increases ventilation and reduces metabolism and complex I efficiency in the cerebral cortex. Furthermore, pharmacological stabilization of HIF has no effect on the ventilatory response, but seems to mimic some mitochondrial and metabolic responses to hypoxia, whereas blocking HIF has no effect on these same responses. If in mice blocking HIF in hypoxia reduces the amplitude of the observed responses, our data will demonstrate fundamental differences in the mechanisms of responses to hypoxia between these two species that would strongly support our general hypothesis.
Anomalous Obturator Arteries with Multiple Pelvic Branches: A Urogynecological Approach
Authors: Acevedo Arroyo Alexandra N. , Santiago Negrón Camille L. , Fonseca Salgado Carlos , Jiménez Dietsch Sofía , Quiñones Rodríguez Jailenne I.
Published: May 2022

The obturator artery (OA) is known to often present multiple anatomical variations. These can be due to atypical origin, variable anastomosis, or abnormal course within the pelvis. We report a rare arterial variation in a Puerto Rican female cadaver that showed two abnormal obturator arteries with multiple pelvic branches. The OA emerged from the anterior branch of the internal iliac artery, which normally runs anteroinferior along the lateral wall of the pelvis to the upper part of the obturator foramen where it exits the pelvis. Within the pelvis, the atypical obturator artery provided two anatomically variant branches: abnormal obturator artery I (AOAI) gave rise to three branches, while abnormal obturator artery II (AOAII) gave rise to two branches. Identification of these accessory arteries and anastomoses is important for surgical interventions. Therefore, knowledge of the anatomical variations in this region must be taken in multiple pelvic, gynecological, and urological procedures to avoid iatrogenic injury.
Depletion of gut microbiota suppresses spontaneous ileitis in mice driven by mitochondrial dysfunction induced by Phb1 deficiency
Authors: Alula Kibrom , Theiss Arianne
Published: May 2022

Introduction

Dysbiosis in gut microbiota is associated with inflammatory bowel diseases (IBD), such as Crohn’s disease. Prohibitin 1 (PHB1) is a mitochondrial protein important in optimal mitochondrial respiration and is decreased in biopsies from IBD patients. Intestinal epithelial mitochondrial dysfunction is emerging as an underlying contributor to inflammation in IBD. Previous studies from our laboratory have demonstrated that mice with Phb1‐deficiency in the intestinal epithelium (Phb1iΔIEC) manifest mitochondrial dysfunction, Paneth cell defects, and spontaneous inflammation in the ileum with a penetrance of 87% at 12 weeks after inducing Phb1 deletion. This inflammation is associated with a reduction in the abundance of short chain acid (SCFA)‐producing bacteria, such as Blautia, Roseburia, Ruminococcus, and Coprococccus. However, little is known whether gut dysbiosis is a driver or consequence of ileitis during intestinal epithelial mitochondrial dysfunction. In the present study, we hypothesized that the gut microbiota is necessary to induce intestinal inflammation during epithelial cell mitochondrial dysfunction.

Methods

Phb1iΔIEC mice and control Phb1fl/fllittermates were co‐housed and treated with broad spectrum antibiotics (ABX) (1 g/L ampicillin sodium salt, 1 g/L neomycin sulfate, 1 g/L metronidazole, and 500 mg/L vancomycin) in autoclaved drinking water for 4 weeks beginning 8 weeks after the induction of Phb1 deletion. Ileal stools were collected for 16S rRNA sequencing and quantification of SCFAs by liquid chromatography‐mass spectrometry. Ileum was collected for histological (inflammation) scoring after H&E staining and for immunofluorescent staining of lysozyme and Muc2 to quantitate Paneth cell defects (lysozyme allocation patterns, increase of lysozyme+/Muc2+ staining, and crypts devoid of lysozyme+ cells). Spleen weight was also measured at the time of sacrifice.

Results

Ileal stool from Phb1iΔIEC mice exhibited a significant reduction in the SCFA butyrate compared to Phb1fl/fl littermates. 16S rRNA sequencing showed that ABX treatment abolished the ileal bacterial population in both Phb1fl/fland Phb1iΔIEC mice. ABX treatment ameliorated enlarged spleen weights, histological inflammation, and Paneth cell defects characteristic of Phb1iΔIEC mice to measurements noted in Phb1fl/fl mice.

Conclusions

These results suggest that mitochondrial dysfunction in IECs on its own is not sufficient to cause inflammation, but triggers disease when coupled with gut microbiota interaction. This is especially relevant to IBD given the prevalence of epithelial mitochondrial dysfunction in these patients.
Micromolar Concentrations of Methionine Aminopeptidase Inhibitor has Little Cytotoxic Effect on Cancerous and Non‐Cancerous Mammalian Cells
Authors: Croes Alex , Clymer Jennifer , Loveless Jasmine , Nguyen Calvin , Tawfiq Hassan , Romo Caroline , Patel Nikita , Flores Robert , Gomez Justin , Alvarez Melissa , Olaleye Omonike , Rosell Rosemarie
Published: May 2022

Tuberculosis (TB) is a disease of the lung caused by the bacterium, Mycobacterium tuberculosis (Mtb), and is the second leading cause of mortality from an infectious agent after SARS‐CoV‐2. Mtb is an opportunistic pathogen that benefits from a compromised immune system and is particularly deadly in HIV patients. New therapies targeting TB are necessary due to an increase in multi‐drug resistant strains. Our collaborators at Texas Southern University identified novel bacterial methionine aminopeptidase (MtMetAP1) inhibitors. Genetic knockout studies support the lethality of the deletion of MtMetAP1 in Mtb. Further studies support the existence of an ortholog in Homo sapiens (HsMetAP1) that performs similar essential functions. Although these novel inhibitors are known to be effective against Mtb, their cytotoxicity in mammalian cells has yet to be established. We hypothesize that there will be low levels of cytotoxicity in cancerous and primary lung cell lines at varying concentrations of the MetAP novel inhibitor, UST‐001. Using flow cytometry via a MUSE® Cell Count and Viability Kit, the cytotoxicity of UST‐001 was assayed in H1299 cancer cells. The H1299 cells were exposed to concentrations of UST‐001 ranging from 0.1μM to 750μM for 48 hours at 37℃and 5% CO2. Three negative controls were included in each replicate: DMSO (carrier), pure media, and Isoniazid (INH). INH is a common clinically effective TB treatment. The viability of H1299 cells was still over 60% at the highest inhibitor concentration tested. Trypan Blue Assays were performed on the same H1299 cells at each concentration and the controls, which corroborated flow cytometry results. In establishment of a dose‐response curve, our data showed that at UST‐001 concentrations below 100 μM, induced little to no cell death and cell viability of 90% or higher. Since cancerous cells have higher mitotic rates and more repair pathways, we hypothesize that we will be able to establish an IC50 curve in non‐cancerous Mlg‐2908 lung fibroblast cells at higher concentrations. Current flow cytometry testing on Mlg‐2908 cells, using the same protocol as the H1299 cells, shows cell viability of 89% or higher in 0.1 μM to 10 μM UST‐001 concentrations. Since cancerous cells have higher mitotic rates and more repair pathways, we hypothesize that we will be able to establish an IC50 curve in Mlg‐2908 cells at higher concentrations. Further studies using a Muse Annexin V apoptosis assay will focus on the UST‐001 mechanism of cell death. Our ultimate goal is to characterize the toxicity of this drug in mammalian systems, with hopes of potentially identifying a new class of antibiotics and pharmaceutical targets.
Single‐Nucleotide Polymorphisms Inside MicroRNA Target Sites: Influence in Hepatocellular Carcinoma Risk
Authors: Woo John I.
Published: May 2022

Hepatocellular carcinoma (HCC) is the fifth most common cancer and is responsible for over 700,000 deaths worldwide each year. Although diagnostic measures and surgical interventions have improved in recent years, the absence of early‐stage symptoms, lack of adequate biomarkers, and the acquired resistance to chemotherapeutic agents still poses a problem. Previous studies have demonstrated that non‐coding nucleic acids that regulate gene expression, known as micro‐RNAs (miRNA), can be associated with the development of cancer. The main purpose of this investigation was to identify single nucleotide polymorphisms (SNPs) inside miRNA target sites that could influence HCC tumor susceptibility. First, we used the National Center for Biotechnology Information’s Gene Expression Omnibus (NCBI GEO) to identify genes that were highly expressed in HCC. Then HCC patient raw read data were downloaded from the Sequence Read Archive database, mapped, sorted, and then analyzed. Our analyses identified SNPs in 30 miRNAs located on AKR1B10 that could be associated with the risk of HCC.
PGE receptor EP4 signaling increased hyaluronan‐vesican complex in abdominal aortic aneurysm
Authors: Hiromi Taro , Iida Saki , Nakamura Takashi , Yokoyama Utako
Published: May 2022

Chronic inflammation and dysregulation of extracellular matrices are hallmarks of abdominal aortic aneurysm (AAA) pathophysiology. We have demonstrated that PGE receptor EP4 signaling in vascular smooth muscle cells (VSMCs) has a pivotal role in AAA formation via immune infiltrates and impairment of elastic fiber repair. However, EP4 signaling‐mediated changes in extracellular matrices have not been fully understood, and expressions of the chondroitin sulfate proteoglycan versican are controversial. Versican has multiple binding partners (e.g., hyaluronan and fibrillin‐1) and modulates VSMC phenotypes. We, therefore, aimed to clarify an expression change in versican in AAA.

We administrated angiotensin‐II (AngII) to VSMC‐specific EP4 overexpression mice (EP4‐Tg) to induce AAA. The expression level of versican V0/V1 was gradually increased in the tunica media of EP4‐Tg mice 4 days after AngII administration, but not in Non‐Tg mice. This increase in versican V0/V1 became prominent 14 days after AngII administration in EP4‐Tg mice. Protein expression of versican V0/V1 was increased in the tunica media of human AAA tissues. EP4 stimulation in VSMCs derived from EP4‐Tg mice increased levels of versican mRNAs (20.0±1.4‐fold, n=5, p<0.01) and versican V0/V1 proteins (4.1±0.7‐fold, n=5, p<0.01), while administration of EP1/3 or EP2 agonist did not. The EP4‐mediated increase in versican mRNAs was completely suppressed by Actinomycin D. Versican V0/V1 are cleaved by a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS‐1) and ADAMTS‐5. The expression levels of ADAMTS‐1, ADAMTS‐5, and cleaved versican protein expressions were not changed in EP4‐Tg VSMCs. The expression level of Hyaluronan synthase1 (Has1) mRNA was also increased in EP4‐Tg VSMCs (66.9±10.1‐fold, n=5, p<0.01). Western blotting revealed that versican V0/V1 proteins of approximately 200‐400 kDa in size in the supernatants of EP4‐Tg VSMCs stimulated with an EP4 agonist were increased by hyaluronan digestion with hyaluronidase (6.3±1.5‐fold, n=6, p<0.01). Transmission electron microscopic analysis demonstrated that elastin contractile units between elastic laminae and VSMCs were disrupted in EP4‐Tg mice administrated with 10 days of AngII, but not in Non‐Tg mice.

These data suggested that EP4 signaling in VSMCs increased hyaluronan‐versican V0/V1 complex. Versican V0/V1 proteins were increased in the early stage of a mouse AAA model. It has been suggested that versican has inhibitory roles in elastic fiber formation. EP4‐mediated hyaluronan‐versican V0/V1 complex may exacerbate AAA via disruption of elastin contractile units.
A new β2 integrin activation reporter mouse reveals localized intra‐ and extra‐vascular neutrophil integrin activation in vivo
Authors: Wen Lai , Marki Alex , Wang Zhihao , Orecchioni Marco , Makings Jeffrey , Kim Kenneth , Kiosses William B. , Mikulski Zbigniew , Ley Klaus
Published: May 2022

β2 integrins (LFA‐1, Mac‐1, CD11c‐CD18, and CD11d‐CD18) are leukocyte‐specific adhesion receptors that play critical roles in leukocyte recruitment, as well as other immunological processes such as phagocytosis and immunological synapse formation between T cells and antigen presenting cells. Adhesion of leukocytes to other cells such as endothelial cells are regulated by integrin affinity changes for their ligands (“activation”). For human β2 integrins, activation reporter antibodies including mAb24 and KIM127 can be used to study integrin activation. No such activation epitopes are known in mouse β2 integrins. Because of the lack of mouse β2 integrin activation reporter antibodies, nothing is known about β2 integrin activation in vivo. Here, we generated a humanized β2 integrin knockin mouse strain by targeting the human β2 integrin coding sequence into the mouse Itgb2 locus. We show that this enables imaging of β2 integrin activation using the KIM127 (extension conformation) and mAb24 (high affinity) reporter antibodies. Human β2 pairs with the mouse integrin α chains, yielding normal expression of the β2 integrins LFA‐1, Mac‐1 and CD11c‐CD18 in all major leukocyte populations. Using a CXCL1‐induced acute inflammation model, we uncovered the dynamics and subcellular localization of β2 integrin activation in arresting neutrophils in vivo in venules of the mouse cremaster muscle. Activated integrins in arresting neutrophils in vivo are concentrated at the interface of neutrophils and the endothelium at the rear side of neutrophils facing against the blood flow. In a high‐dose lipopolysaccharide (LPS) model, we found that β2 integrins are activated in association with elevated neutrophil adhesion in lung and liver. Thus, these mice, for the first time, enable studies into β2 integrin activation in vivo.
Diacerein attenuates 1,2‐dimethylhydrazine induced colon cancer progression in rats: Modulation of IL‐6/STAT3/ lncRNA HOTAIR pathway
Authors: El‐Sherbiny Mohamed , Eisa Nada H. , Said Eman M. , Khodir Ahmed E. , Elsherbiny Nehal , Aloufi Abdulrahman M. , Aldobikhi Yousef A.
Published: May 2022

Diacerein (DIA) is an FDA approved anti‐inflammatory drug. Over the past few years, several studies reported its anti‐proliferative action against different types of cancers. However, the anti‐tumor effect against colon cancer remains obscure. Colon cancer is one of the top solid tumors diagnosed around the world. The current study aims to investigate DIA‐associated anti‐tumor effect In vivo and invitro and to further elaborate the potential underling molecular mechanism. In vitro, human Caco‐2 colon cancer cell lines were treated with serial dilution of DIA and were evaluated for cell viability and apoptotic percentages via MTT and Annexin V assays, respectively. In vivo, subcutaneous injection of 1,2‐dimethylhydrazine (DMH) (20 mg/kg for 15 weeks) was used to induce colon carcinogenesis. DIA oral administration at dose of 50 mg/kg/day for additional of 8 weeks was tested. This therapeutic regimen showed significant anti‐tumor effect and improvement of colonic tissue structure as evident by histopathological examination. Mechanistically, colon tissue samples were collected for ELISA and immunohistochemical analyses. Data showed that DIA treatment reduced colonic inflammatory status as evident by reduction of TLR4, NF‐κB and TNF‐α protein levels. In addition, DIA treatment reduced angiogenesis levels as evident by downregulation of VEGF tissue expression levels. Furthermore, DIA treatment modulated Wnt/β‐catenin pathway, induced caspase‐3 expression and interrupted IL‐6/STAT3/lncRNA HOTAIR axis. Altogether, this study provides evidence that DIA treatment might be beneficial in controlling colon cancer progression via multi pathways targeting mechanism.
The potential role of LCZ696 in ameliorating diabetic cardiomyopathy by inhibiting the myocardial inflammation, ER stress, and apoptosis through the RAGE/NF‐κB and PERK/CHOP signaling Pathways
Authors: Al Rejaie Salim , Belali Osamah
Published: May 2022

The development of diabetic cardiomyopathy (DCM) is multifactorial that leads to myocardium structural and functional disruption resulting excessive activity of the renin‐angiotensin system (RAS) and finally heart failure. LCZ696 newly established as chronic heart failure drug which combines a neprilysin inhibitor (sacubitril) and an angiotensin II receptor blocker (valsartan). Present study is designed to find the effect of LCZ696 on DCM in rats. Diabetes was induced by feeding rats with a high‐fat diet for 6 weeks following a single injection of STZ (30mg/kg). Diabetic rats were randomly divided in three group (n=8), LCZ696 and valsartan treatment was started two weeks after the DM induction and continued for 8 consecutive weeks. Blood pressure of individual rats was recorded using tail‐cuff instruments. At end of the treatment echocardiography was done. In serum glucose, insulin, creatine kinase‐MB and lactate dehydrogenase (LDH). In cardiac tissues, pro‐inflammatory cytokines and advanced glycation end‐products (AGEs) formation estimated. Western blot and RT‐PCR were used to determine the apoptotic genes, endoplasmic reticulum stress (ER) parameters. LCZ696 and valsartan ameliorated the DCM progression by inhibiting of AGEs, its receptor (RAGE) at mRNA, pro‐apoptotic gene markers (BAX and caspase‐3) associated with induction of the antiapoptotic gene (Bcl‐2) at mRNA and protein expressions, NF‐κB at the mRNA and protein levels associated with restoring the elevation of pro‐inflammatory cytokines such as TNF‐α, IL‐6, and IL‐1β at the activity level. Furthermore, LCZ696 and valsartan helps in restoring the induction of ER stress parameters (GRP78, PERK, eIF2a, ATF4, and CHOP) at the mRNA and protein levels. LCZ696 and valsartan improved diastolic dysfunction and attenuated histological abnormalities. LCZ696 and valsartan attenuated DCM by inhibiting the myocardial inflammation, ER stress, and apoptosis through the RAGE/NF‐κB and PERK/CHOP signaling cascades. Collectively, present results revealed LCZ696 has more strong protective effect against DCM than valsartan.
Microbial multidimensional signature assessment reveals microbial SNVs as the superior non‐invasive biomarkers for early detection of colorectal cancer
Authors: Gao Wenxing , Sun Ruicong , Zhu Lixin , Gao Sheng , Feng Zhongsheng , Wu Dingfeng , Gao Xiang , Zhao Rui , Yao Ye , Liu Zhanjiu , Zhu Ruixin , Jiao Na
Published: May 2022

Background

The early detection of colorectal cancer (CRC), especially at precancerous adenoma stage, significantly reduces its incidence. Gut microbiome has become a promising non‐invasive tool for CRC screening, whereas the potential of microbial multidimensional signatures remains poorly understood. Here we performed a cross‐cohort analysis to evaluate the capability of microbial multidimensional biomarkers for adenoma detection.

Design

Whole metagenome sequencing data of four public datasets including 183 adenoma patients and 439 healthy controls were reprocessed consistently to obtain taxonomic‐, functional‐ and single‐nucleotide variants (SNVs)‐profiling. With MMUPHin, differential multidimensional signatures were identified after adjusting confounders, based on which random forest (RF) models were constructed and then optimized by recursive feature elimination. Finally, internal validation and external validation with in‐house dataset (10 controls and 6 adenomas) and five resampled datasets were conducted to further assess the robustness of the best biomarker panel.

Results

The integrated analysis identified 103 multi‐kingdom differential species between adenoma and control group, of which 15 optimal species were selected to construct a RF model achieving an AUC of 0.75. Meanwhile, the model constructed with 31 optimal biomarkers out of 386 differential KO genes reached an AUC of 0.74. Notably, the diagnostic model with 75 SNVs from 10 species showed superior accuracy (AUC = 0.85) with high specificity to adenoma. Co‐abundance analysis revealed intensive bacterial‐fungal associations in line with functional abnormalities related to microbial quorum sensing, purine and butanoate metabolism.

Conclusion

Microbial SNV biomarkers outperform other biomarkers and display high specificity to adenoma, which may serve as a novel non‐invasive tool for early detection of CRC. Furthermore, multidimensional signatures provide potential therapeutic targets for adenoma.
Inverse Neurovascular Coupling Contributes to Vasopressin Neuronal Activation in Response to Systemic Homeostatic Challenge
Authors: Roy Ranjan , Althammer Ferdinand , Brown Colin , Stern Javier
Published: May 2022

Neurovascular coupling (NVC), the process that links neuronal activity to cerebral blood flow changes, has been mainly studied in superficial brain areas, namely the neocortex. Still, whether the conventional, rapid and spatially restricted NVC response can be generalized to deeper, and functionally diverse brain regions remains unknown. The hypothalamic supraoptic nucleus (SON) is a deep brain region with unique cytoarchitectural properties including a high vascularization. Paradoxically, the dense vascular network is not required to maintain its metabolic demand. This mismatch in metabolic supply and demand suggests that the SON vasculature may serve additional functions beyond the delivery of energy substrates. To address this important gap in our knowledge, we set up the objective to implement a novel experimental approach to enable in vivo, real‐time two‐photon imaging from the ventral surface of the brain to monitor SON NVC responses to a systemic homeostatic challenge. Using this approach in anaesthetized male and female transgenic rats expressing GFP driven by the vasopressin (VP) promoter (VP‐GFP rats), we found that a systemic hypertonic saline (HTS) infusion evoked a gradual and linear increase in the firing rate of VP neurons (p < 0.0001; n = 4). Rather than resulting in a classical vasodilatory NVC, the increased VP neuronal activity was accompanied by a significant intraparenchymal arteriole (but not venule) vasoconstriction (one‐way RM ANOVA; p < 0.0001; n = 13 vessels from 5 rats). This “inverse” NVC was prevented when a VP V1a receptor antagonist was locally delivered within the SON (one‐way RM ANOVA; p < 0.0001; n = 4 vessels from 4 rats). The HTS infusion also significantly decreased RBC velocity in SON capillaries (one‐way RM ANOVA; p < 0.0001; n = 17 vessels from 6 rats) indicative of a decreased SON blood flow. The salt‐induced inverse NVC response resulted in a rapid and progressive decrease in SON pO2 (one‐way RM ANOVA; p < 0.002; n = 6 rats), along with an increased expression of the hypoxia inducible factor (HIF)‐1a mRNA (p < 0.001; one‐sample t test n = 6 rats). Finally, we show that a hypoxic SON microenvironment significantly increased the firing activity of VP neurons in ex vivo slices (p < 0.02; paired t test; n = 14 cells from 10 rats). Taken together, we show that the salt‐induced inverse NVC in the SON results in a hypoxia‐mediated positive feedback mechanism, which we propose contributes to sustain the activity of VP neurons in response to a systemic homeostatic challenge.
The gut‐liver axis in the liver’s disease: the pathophysiological basic of dysbiosis and hepatopathy’s pharmacorrection
Authors: Ponamarev Vladimir , Popova Olga
Published: May 2022

The decreasing of hepatopathy’s spread in the animal industry can be achieved by determining the correlation between dysbiosis and hepatopathies, assessing metabolic aspects on diseases, and forming optimal protocols for their diagnostic and treatment. The study aimed to research the efficiency of pharmacorrection’s protocol in case hepatopathy of cows. The studies were carried out on farms of the Pskov region (Russia). Holstein high‐productive cows were used aged from 3 to 4 years. The farm’s management was included the indoor housing of cows and the twice‐a‐day feeding. The ration was included the earlage, the medic haylage and dry forage, the presser, the molasses, and the mixed cattle feed. Cows with clinical signs of hepatosis and their biochemical findings were formed into 2 groups (n=10 by each). The results of the biochemical analysis were showed hypoproteinemia and hypoalbuminemia. The concentration of total protein was 53.28±1.16 g/l. The albumins level was 25.42±0.86 g/l. The BUN level was decreased in 61 % of blood samples and was equal to 3.81±0.23 mmol/l. The animals have had a different protocol of hepatopathy’s pharmacorrection. The protocol of 1st group cows (n=10) was included the complex with herbal and sorbents (reptile thymum, chamaemelon, inula summus, absinthium, juniperi oleum, origanum, perlite, vermiculite, polyphepan) as a supplementation to the ration (4 % per the daily rate during 14th days) and the hepatoprotective agent “Hepaton‐vet” (the compound: lac Carduus, ursodeoxycholic acid) at a dose of 100 ml per cow during 21 days. The protocol of 2d group cows (n=10) included only the hepatoprotective agent “Hepaton‐vet” for 21 days. We were used the software program “Statistica 10.0” with the Mann‐Whitney test as a statistical analysis of results. We have observed that the cows of the 1st group had a higher daily milk production (28.42±4.2 kg) than the cows of the 2d group (25.32±2.3 kg). The difference between groups was 12.2 %. The fat content of milk in the cows of the 1st group was 4.07±0.9 %, 2d group – 3.8±0.4 %. The milk protein content in the cows of the 1st group was up by 2.57±0.3 % compared with the value of the 2d group. So, we can assume that combining the hepatoprotective agent with proven efficiency and the complex with herbal and sorbents is the perspective treatment approach of the liver’s disease and the modulation of the gut‐liver axis.
Oxidative Crosstalk with Venular Capillary Mitochondria Mediates Barrier Failure in Acute Lung Injury
Authors: Hough Rebecca F. , Emin Memet T. , Jin Guangchun , Bhattacharya Jahar
Published: May 2022

OBJECTIVE

. To elucidate mechanisms underlying alveolar‐capillary crosstalk in the context of lipopolysaccharide (LPS)‐induced Acute Lung Injury (ALI), which causes The Acute Respiratory Distress Syndrome (ARDS).

HYPOTHESIS

Oxidative crosstalk between alveolar epithelium and adjacent endothelium causes mitochondrial injury and barrier failure in Acute Lung Injury

METHODS

. We instilled LPS (5mg/kg) by the intranasal (i.n.) route in anesthetized mice. We evaluated microvascular endothelia of isolated blood‐perfused mouse lungs by confocal microscopy, and ALI by bronchoalveolar lavage (BAL) 48h later. For microscopy, we gave the potentiometric mitochondrial dye MTDR, and the reactive oxygen species (ROS) indicator DCF by intravascular infusion. We determined the role of endothelial uncoupling protein 2 (UCP2) by (1) UCP2 knockdown in endothelia by vascular siRNA injection, and (2) endothelial‐specific knockout of UCP2. We transfected alveolar epithelium by i.n. instillation of catalase‐expressing plasmid, or depleted neutrophils by intraperitoneal injection of anti‐Gr1 antibody, 24 hours before i.n. LPS. We quantified barrier permeability through BAL protein content.

RESULTS

. Confocal imaging indicated that baseline fluorescence intensity of both endothelial MTDR and DCF was steady, indicating that endothelial mitochondrial potential and endothelial ROS were stable. However, 48 hours after LPS instillation, MTDR fluorescence decreased in venular capillaries more than in alveolar capillaries (n=4, p<0.05), indicating endothelial mitochondrial depolarization. Simultaneously, DCF fluorescence increased in venular capillaries more than in alveolar capillaries (n=4, p<0.05), indicating an increase in endothelial ROS. LPS instillation increased BAL protein (n=4, p<0.05). Knockdown of endothelial UCP2 expression, or expression of transfected catalase in the alveolar epithelium blocked LPS‐induced endothelial mitochondrial depolarization and BAL protein increase (p<0.05). Endothelial‐specific knockout of UCP2, but not neutrophil depletion, blocked LPS‐induced BAL protein increase (p<0.05).

CONCLUSIONS

. We show here for the first time that the hyper‐permeable responses of LPS‐induced ALI result from mitochondrial depolarization in venular capillaries. Transfer of H2O2 from alveoli, not neutrophils activated UCP2, causing proton influx into the mitochondrial matrix. Our findings reveal a novel mechanism of LPS‐induced ALI, implicating mitochondria‐oxidant coupling in barrier failure. Thus, UCP2 presents a potential therapeutic target for ARDS.
You gotta work, BASIL! Reimagining an established CURE to provide high‐quality digital learning experiences that are intentionally equitable, inclusive and accessible for all students
Authors: Sikora Arthur K. , Hall Bonnie , Mills Steven , Roberts Rebecca , Craig Paul
Published: May 2022

In recent years, Course‐based Undergraduate Research Experiences (CUREs) have become increasingly valuable models to cultivate student interest in research, especially when few other research opportunities at an institution exist. Since the start of the COVID‐19 pandemic, there has been the need to operate in an online environment while maintaining high standards. Biochemistry Authentic Student Inquiry Lab (BASIL) students hypothesize and test functions of enzymes from the Protein Data Bank with no known function, utilizing a combination of wet‐lab and computational approaches. Here we describe how this CURE was adapted to an online format, simulating the lab environment using a low stakes iterative assessment. Using Google forms, students answer questions about experimental background, procedure and lab safety that correlate with published BASIL experiments. Using a mix of multiple choice, free response, and video/ image‐based questions, students engage with the material at a deeper level despite not being physically present in the lab. These forms can function as self‐contained experiments or pre‐lab/post‐lab assignments to enhance the in‐lab experience. The BASIL consortium is dedicated to developing high‐quality teaching and learning experiences to reach and engage the modern learner. This CURE is flexible and has been found to improve the levels of personal comprehension and knowledge of STEM concepts and research design in students. These online modules provide another way for learners to reap the benefit of research‐based courses in an ever‐changing educational landscape. Built to equitably and inclusively reach and engage all students, these tools integrate intentional opportunities for community‐building and interaction only possible in the digital environment. Novel strategies developed to accommodate all students will help to enhance exposure for undergraduate students to vital STEM research experiences and promote sustainability for institutions.
Analysis of Attitudinal Student Learning Benefits from a Biochemistry CURE Adapted for the Online Format
Authors: Kapil Amika , Gonzalez Luis , Pathak Niraj , De Santanu , Sikora Arthur
Published: May 2022

Due to the ongoing COVID‐19 pandemic, institutions across the world have had to adjust their teaching methods. This shift to emergency remote instruction resulted in modifications to existing curricula, especially in the experimental science laboratory. There is a need to better understand student benefits of CUREs in this new environment. Using the Participant Perception Indicator (PPI) survey, we measured changes in students’ knowledge, experience, and confidence (KEC) growth over the course of a fully online biochemistry course. The results and analysis of the survey data gave light to three significant findings. Our analysis indicated the following: (1) Students learned more through bioinformatic experiments and related concepts than about their wet‐lab counterparts. During this time, students were not able to perform the wet‐lab techniques in person, relying instead on the virtual experience to learn these skills. (2) Students reported greater gains in learning outcomes KEC compared to wet‐labs and computational techniques. The utilization of learning outcomes can only partially fill the gap left by the loss of physical interaction in experimental procedures. (3) Finally, students reported their experience and confidence gains lagged behind their knowledge of technique‐based skills. Clearly, students are not as confident in their understanding of techniques when unable to perform them in the physical laboratory. Thus, even though students have extensive knowledge and understanding of the structure, protocols, and purpose of the experiments and techniques, student responses indicated that their experience and confidence is not on par with their knowledge acquired through fully online education.