bluuebunny/crossref_metadata_2025_split huggingface dataset:
Podocyte proteome analysis reveals stress responses in glomerular sclerosis
Authors: Koehler Sybille , Brinkkoetter Paul , Rinschen Markus M. 
Published: April 2020

Podocyte injury is a major cause of chronic kidney disease. However, understanding podocyte‐specific responses at a systems level is difficult when glomerular cellular composition is altered by loss of podocytes or increase of extracellular matrix. We developed and applied proteome analysis to very small sample amounts to podocyte fractions isolated from single mice after induction of glomerular disease and to single glomeruli from patients with Focal segmental glomerulosclerosis (FSGS). Transcriptome and proteome analysis of glomeruli from patients with FSGS revealed an underrepresentation of podocyte‐specific genes and proteins in late‐stage disease. To circumvent this problem, we utilized two commonly used mouse models of chemically‐induced FSGS and performed proteomic analysis of pure podocyte fractions in early disease states. We observed perturbations of propagating pathways of glomerular disease such as Jak‐Stat signaling, central carbon metabolism such as TCA cycle enzymes, and mechanical stress response. Multi‐layered priorization approaches identified a co‐regulation of proteinuria with the mechanosensor protein Filamin‐B. Podocytes in human biopsies, single glomeruli from proteinuric rats and podocytes undergoing mechanical stress, showed increased expression of Filamin‐B, suggesting a role in detrimental stress responses. A nephrocyte‐specific depletion of Cher, the Filamin homolog in Drosophila, showed an altered filtration capacity while nephrocyte morphology was unaffected.This study identified conserved mechanisms of the podocyte stress response by analyzing ultrasensitive proteomes from human glomerular FSGS tissue and purified native mouse podocytes during early disease stages. The pipeline put forward here enables systematic comparisons of large‐scale proteomics data and phenotype to protein correlation.Support or Funding InformationMMR was supported by the DFG (Ri2811/1 and Ri2811/2).
The cytotoxic role of the inhibition of PI‐3‐kinase, and acto‐myosin cross‐bridge formation in human metastatic breast cancer cells.
Authors: Thirumeni Rajashree , Bhetwal Bhupal Prasad 
Published: April 2020

IntroductionExternally added survival/growth factors are essential for the survival of healthy cells, however cancer cells are believed to be self‐sufficient in supporting their auto‐proliferation without the need of exogenously added growth factors. The Phosphatidyl‐Inositol‐3‐kinase (PI‐3‐kinase) pathway is crucial in cell survival. Also, cancer cells exhibit enhanced potential to metastasize via increased acto‐myosin cross bridges formation which requires increased Ca2+ influx via cell surface voltage‐gated Ca2+ channels leading to activation of myosin light chain kinase (MLCK). The MLCK then phosphorylates myosin and forms acto‐myosin cross‐bridges. The role of MLCK, PI‐3‐kinase, and calcium channel in cancer cell growth has not been extensively tested. In this study, we tested the individual effects of blocking cell survival pathway (PI‐3 kinase), voltage‐gated Ca2+ channels and MLCK on MCF‐7 cell (a human metastatic breast cancer cell) survival using pharmacological inhibitors.HypothesisWe hypothesized that all three drugs would inhibit cell proliferation.AimsOur aim was to firstly test if these drugs had any effect at all on cancer cell growth. Secondly, it was to test each drug at different concentrations and see which concentration had the most effect on cell death.MethodsPI‐3‐kianse inhibitor (Wortmannin); MLCK inhibitor (ML‐7), and lastly, a voltage‐gated calcium channel inhibitor (nifedipine) were used. 1% Bitter melon extract (BME) was also tested as this was found to inhibit MCF‐cell growth in our lab by earlier investigators. Cells were thawed, mixed with a medium that contained all essential nutrients for cell growth (DMEM + fetal bovine serum), and then were added onto 96‐well plates and allowed to grow for 48 hours. We incorporated control plates (no drug), plates with drugs at different concentrations, a plate with the compound the drug was originally dissolved in (DMSO or ethanol), and a plate with 1% BME (bitter melon extract). The plates were analyzed under a microscope and cell viability was measured using MTT assays. Images of each plate were taken and correlated with the MTT assay results. Each experiment was repeated at least 4 times in duplicate or triplicate to plot graphs and draw statistical inferences.Results and DiscussionAll three drugs independently inhibited cell growth in a dose‐dependent manner. The observable differences between the different drugs were the concentrations at which they were effective. The results imply that Ca2+ influx and its downstream pathway leading to acto‐myosin interaction is essential for the MCF‐7 cell attachment to the plate and hence its survival.Support or Funding InformationSummer research stipend to medical students at MUCOMDepartmental faculty research support to faculties at MUCOM
The Effect of a Histone Deacetylase Inhibitor on PD‐L1, HLA‐ABC, HLA‐E, and HLA‐G on Human Breast Cancer Cell Lines
Authors: Toufexis Alec George , Reddy Nikhil Cheruku , O’Donnell Robert W. 
Published: April 2020

Increased expression of human leukocyte antigen (HLA) allows tumor cells to be more easily detected by the immune system. In previous research, epigenetic modifiers including the histone deacetylase inhibitor 3 (HDAC3), RGFP966, has been shown to decrease PD‐L1 expression. PD‐L1 expression leads to inhibition of T cell cytotoxicity, and decreasing it therefore can enhance the immune response. We hope to elucidate whether or not HLA‐ABC expression is similarly decreased upon exposure to the HDAC3 inhibitor which would be detrimental to immune detection. Two breast cancer cell lines that are known to express HLA‐ABC are MCF‐7 and MDA‐MB‐231. Our initial results show that HLA‐ABC is expressed on the MDA‐MB‐231 and MCF‐7 cell lines, while PD‐L1 is only expressed by MDA‐MB‐231 cell line as demonstrated by flow cytometry. However, in subsequent experiments testing the effect of the drug RGFP966, there were inconsistent results regarding the change in expression of HLA‐ABC which requires further investigation. In addition, we also wish to look at other HLA proteins, such as HLA‐E and HLA‐G, of which expression which has been shown to be detrimental to immune detection. Future experiments are planned to optimize the doses of RGFP966 to maximize HLA expression in these breast cancer cell lines while limiting the expression of PD‐L1 and HLA‐E and HLA‐G.Support or Funding InformationGeneseo FoundationCarl Savino ‘53 and Olga Gerych‐Savino ‘53 EndowmentDavid Wolf, DO
The Size of the Inferior Horn of the Lateral Ventricle is Negatively Correlated with the Thickness of the Entorhinal Cortex
Authors: Ritz Brianna Kathryn , Zdilla Matthew 
Published: April 2020

The hippocampus and entorhinal cortex are implicated in the progression dementia. With structural changes in the parenchyma of the hippocampus and entorhinal cortex, will the inferolateral aspect of the lateral ventricle expand to fill the void? This study sought to determine the relationship between the volume of the lateral ventricle / inferolateral aspect of the lateral ventricle and the volume of the hippocampus and entorhinal cortex, as well as the thickness of the entorhinal cortex. The methods included analysis of Freesurfer data collected from 2100 hemispheres from 1050 brain MRIs. Imaging included scans from 733 unremarkable individuals, 82 pre‐dementia individuals, and 235 individuals with dementia. Pearson’s correlation coefficients were calculated to assess correlation. The inferolateral aspect of the lateral ventricle was significantly negatively correlated with hippocampus volume, in general; although, the correlation was small. The strongest statistically significant correlations existed between the inferolateral ventricular volume and the entorhinal thickness. Analysis of the total population revealed a moderate negative correlation (r=−0.51, p<0.0001), as did analysis of the pre‐dementia and dementia populations (r=−0.45, p<0.000 and r=−0.51, p<0.0001, respectively). However, in the unremarkable population, the correlation was very small albeit significant (r=−0.26, p<0.001). Therefore, the inferolateral ventricular size increases as entorhinal thickness decreases, in general, but especially in pre‐dementia and dementia populations. The results of this study suggest a relationship between the inferior horn of the lateral ventricle and key regions of the brain that are implicated in dementia and, therefore, have clinical value in the assessment of dementia.Support or Funding InformationFunding: WV Research Challenge Fund [HEPC.dsr.17.06] and NASA West Virginia Space Grant Consortium, Training Grant #NNX15AI01H
Regulation of SPRTN Metalloprotease in DNA‐Protein Crosslink Repair
Authors: Perry Megan , Kollala Sai , Halbkat Halle , Su Grace , Kreiling Natasha , Ghosal Gargi 
Published: April 2020

DNA‐protein crosslinks (DPCs) are generated when proteins become covalently trapped on the DNA upon exposure to agents such as endogenous reactive aldehydes or exogenous chemotherapy drugs. Failure to repair DPCs causes cells to acquire oncogenic mutations; conversely, specific crosslinking of replication enzymes can target highly proliferative cancer cells and cause fork collapse and cell death. Despite the potency of DPC‐inducing chemotherapies, cancer cells often become resistant to these treatments likely due to robust DNA damage repair pathways. Previously, we and others identified SPRTN metalloprotease as a critical DNA damage response protein. SPRTN cleaves proteins cross‐linked to DNA and facilitates DPC repair, but little is known about the molecular mechanisms that regulate SPRTN protease activity. SPRTN is mono‐ubiquitinated, and we are currently investigating the functional significance of SPRTN ubiquitination. Using mass spectrometry and co‐immunoprecipitation, we have identified USP11 as an interacting partner of SPRTN. USP11 shRNA knockdown cells are hypersensitive to DPC‐inducing agents and have delayed repair of DPCs. Continuing work is focused on the molecular function of USP11 in DPC repair. Our studies provide insight into the regulatory processes underlying the DPC repair pathway and identify repair enzymes that can be therapeutically targeted in cancer cells to prevent chemoresistance to standard DPC‐inducing therapies.Support or Funding InformationThis work is supported by K22, NCI Award CA188181‐01A1 to Gargi Ghosal and University of Nebraska Presidential Graduate Fellowship to Megan Perry.
Novel Salinomycin Analogs Show Improved Selectivity Towards Breast Cancer Stem Cells
Authors: Urbaniak Alicja , Reed Megan R. , Antoszczak Michał , Sulik Michał , Huczyñski Adam , Eoff Robert L. , MacNicol Melanie C. , MacNicol Angus M. , Chambers Timothy C. 
Published: April 2020

Breast cancer tumor metastasis is the leading cause of mortality among women. Cancer stem cells (CSCs) are the tumor‐initiating cells which drive initiation, progression, metastasis, and tumor reoccurrence. Their resistance to conventional chemo‐ and radiotherapies and their ability to survive such treatment enables tumor reestablishment. Thus, therapeutic strategies targeting CSCs hold great potential for novel advances in cancer treatment. Salinomycin (SAL) is a naturally occurring polyether ionophore antibiotic with anti‐cancer activity towards various types of tumor cells, but its toxicity towards normal cells limits broad application. A library of 21 novel SAL analogs was screened to identify compounds with improved selectivity especially against breast cancer stem cells. SAL analogs were either single modified with ester or amide in the C1 position or double‐modified C20‐oxo derivatives. Eight single‐ and two double‐modified derivatives were more potent (IC50 range of 1.13 ± 0.19 to 3.93 ± 0.39 μM) towards the breast cancer cell line MDA‐MB‐231 compared to SAL (IC50 of 5.81 ± 1.50). These derivatives induced DNA fragmentation suggestive of apoptotic cell death. Further, most of these derivatives had improved selectivity toward MDA‐MB‐231 cells compared to SAL when also tested against normal breast epithelial MCF10A cells. In addition, a butyl ester derivative of SAL, chosen based on increased potency and selectivity versus SAL, significantly reduced the colony‐forming efficiency of MDA‐MB‐231 cells in association with selective loss of the CD44+/CD24−/low stem‐cell‐like subpopulation. Select derivatives were next screened against the NCI‐60 Human Tumor Cell Line Panel. A double‐modified analog of C20‐oxo‐SAL with hydroxamic acid was found to be more potent than SAL towards all 6 breast cancer cell lines in the panel as well as other tumor types especially leukemia. Studies are underway to test the analogs in three dimensional culture using breast cancer organoids derived from both cell lines and patients. The present findings highlight the therapeutic potential of SAL analogs towards breast cancer and breast cancer stem cells and support further research and clinical development of these compounds.Support or Funding InformationThe present study was funded by grants (to AMM and TCC) from the Arkansas Breast Cancer Research Program. Testing was performed by the Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute,
http://dtp.cancer.gov.
Brain connectivity and activity during Alzheimer’s disease progression in a mouse model by manganese‐‐enhanced MRI in living brain correlated with post‐mortem histopathology.
Authors: Bearer Elaine L. , Medina Christopher S. , Uselman Taylor , Jacobs Russell E. 
Published: April 2020

Alzheimer’s disease (AD) arises from inherited mutations in genes associated with plaque formation: amyloid precursor protein (APP), tau, and presenilin, or sporadically without these mutations. Studies of mice expressing these familial Alzheimer’s disease (FAD)‐associated mutations has led to new insights into the biological processes in sporadic AD. We study a mouse model in which expression of the FAD protein, APPSwInd, can be suppressed after plaque formation. This mouse thus corresponds to sporadic Alzheimer’s disease, where plaques are present in the absence of mutated protein. We use manganese‐enhanced magnetic resonance imaging (MEMRI), an emerging technology, coupled with histopathology of serial sections to investigate how plaques affect connectivity, neural activity degeneration throughout the whole brain as the mouse ages. MEMRI can be used in two ways: to determine axonal transport dynamics and to map neural activity in living mice. Stereotactic intracranial injection of Mn2+ traces transport in hippocampus occurring over 24h in the awake behaving mouse. Systemic Mn2+ enters active neurons in the brain of freely moving mice over 24h and is imaged retrospectively. Amyloid precursor protein (APP) is the precursor to A□eta plaques. The cytoplasmic domain of APP mediates cargo‐motor attachments for axonal transport. Our results show that in APP‐KO mice, axonal transport on Mn2+ is decreased demonstrating the APP plays a role in Mn2+ transport. In old transgenic mice APPSwInd, with both over‐expression of mutated protein and plaques, axonal transport is altered, as detected by time‐lapse manganese‐enhanced magnetic resonance imaging (MEMRI) of the brain in living mice. Time‐lapse MR images were captured before and successive time points after stereotactic injection of Mn2+ (3–5nL) into CA3 of the hippocampus. Images of multiple individuals from the two groups (with plaques and no expression, or without plaques) were aligned and processed with our automated computational pipeline, and voxel‐wise statistical parametric mapping (SPM) performed. Whole brain neural activity was decreased in 3yr old mice with plaque and expressing APPSwInd compared to non‐expressing littermates, particularly in the hippocampus. Brains were harvested after MEMRI for biochemistry, or fixed, embedded and serially sectioned for histopathology. Histopathology detected mouse p‐tau–positive dystrophic neurites, decreases in the volume of septal nuclei, decrease in dentate gyrus width and cell death. Thus, plaque alone depresses transport in the important hippocampal to basal forebrain memory circuit, and decreases neural activity in the aging mouse.Support or Funding InformationSupported by NINDS RO1 NS062184 and The Harvey Family Endowment.
Amelioration of Podocytopathy by Smpd1 Gene Deletion in Podocyte‐Specific Asah1 Gene Knockout Mice
Authors: Li Guangbi , Huang Dandan , Camus Sarah , Li Ningjun , Li Pin-Lan 
Published: April 2020

Podocyte‐specific deletion of Asah1 (AC gene code in mouse) gene has been found to produce podocytopathy and nephrotic syndrome in mice (Asah1fl/fl/PodoCre mice). However, the mechanism underlying the pathogenesis of podocytopathy in these mice remains unknown. To test whether ceramide accumulation contributes to podocyte injury in Asah1fl/fl/PodoCre mice, Asah1fl/fl/PodoCre mice were mated with Smpd1−/− mice (Smpd1 is mouse gene coding acid sphingomyelinase), which produced mice lacking both Smpd1 and Asah1 gene in podocytes, namely, Smpd1−/−/Asah1fl/fl/PodoCre mice. Using LC‐MS/MS, ceramide accumulation was detected in isolated glomeruli of Asah1fl/fl/PodoCre mice, and Smpd1 gene knockout blocked glomerular ceramide accumulation Asah1fl/fl/PodoCre mice. Correspondingly, severe proteinuria and albuminuria in Asah1fl/fl/PodoCre mice were ameliorated by Smpd1 gene deletion. By immunofluorescence staining, we found reduced glomerular podocin and increased glomerular desmin in Asah1fl/fl/PodoCre mice, which was confirmed using Western blot analysis in isolated glomeruli from these mice. All these podocyte injuries in Asah1fl/fl/PodoCre mice were substantially blocked by cross breeding with Smpd1 gene knockout mice. Under transmission electron microscope, Asah1fl/fl/PodoCre mice were found to have foot process effacement and microvillus formation in podocytes, which were also reversed by deletion of Smpd1 gene. These results suggest that AC deficiency induces ceramide accumulation in mouse glomeruli which is critical to the development of podocytopathy and nephrotic syndrome. Blockade of ceramide production via ASM may attenuate ceramide accumulation and consequent podocytopathy due to AC gene deficiency.Support or Funding InformationThis study was supported by NIH grants DK54927 and DK102539.
Carotid body‐specific shRNA knockdown of PKCɛ blunts TRPV1‐dependent asthmatic bronchoconstriction
Authors: Wilson Richard J.A. , Roy Arijit , Jendzjowsky Nicholas 
Published: April 2020

RationaleAggravated airway bronchoconstriction and mucus production in response to allergen and/or bradykinin (i.e., airway hyperresponsiveness) is a primary defining symptom of asthma. Recently, we discovered a critical role for the carotid bodies: inhaled allergen causes an increase in arterial plasma concentrations of asthmakines which excite the carotid bodies via a transient receptor potential vanilloid 1 channel (TRPV1) dependent mechanism. Elevated carotid body excitation in turn increases efferent vagal activity, resulting in heightened bronchoconstriction. While our data demonstrate that the carotid bodies are strongly excited by multiple Th‐2 cytokines, in the ovalbumin primed and sensitized Brown Norway rat model of asthma, the primary asthmakine is likely lysophosphatidic acid (LPA). However, the cellular link between LPA receptors and TRPV1 has not been identified. Here we show that the effect of LPA on the carotid bodies is likely mediated by PKCɛ‐dependent phosphorylation of TRPV1.MethodsLentivirus vectors containing shRNA to knockdown PKCɛ or scrambled sequence were injected bilaterally into the carotid bodies of asthmatic rats (n=7, each). The responses of carotid bodies to LPA (5μM), and their subsequent responses to TRPV1 blockade (AMG9810, 10μM) were tested using the en bloc perfused carotid body preparation. The effects of carotid body‐specific PKCɛ knockdown on changes in lung mechanics in response to inhaled bradykinin were tested using the Flexivent system.ResultsCarotid bodies from asthmatic rats injected with PKCɛ knockdown sequence mounted smaller responses to LPA than those injected with scrambled sequence. No difference in LPA response was detected between groups following TRPV1 blockade, suggesting the primary target for PKCɛ is TRPV1. Asthmatic rats with carotid bodies injected with PKCɛ knockdown sequence had reduced lung resistance in response to bradykinin at 1, 10 and 20 minutes. Similar results were obtained using a PKCɛ inhibitory peptide.ConclusionWe show for the first time that carotid body‐mediated bronchoconstriction in asthmatic rats occurs through LPA receptor activation of PKCɛ and subsequent TRPV1‐mediated carotid sinus nerve excitation. Targeting carotid body LPA receptors, PKCɛ and/or TRPV1 may provide a new therapeutic target to reduce asthmatic airway hyperresponsiveness.Support or Funding InformationCIHR, Alberta Innovates, Alberta Lung Association and the Parker B. Francis Foundation.
Probiotic‐generated product protects against alcoholic liver disease through increasing intestinal AhR‐IL22‐Reg3 and Nrf2 signaling
Authors: Gu Zelin , Liu Yunhuan , Li Fengyuan , Jiang Mengwei , Zhang Lihua , McClain Craig , Feng Wenke 
Published: April 2020

BackgroundProbiotics are known to modulate intestinal barrier integrity against alcohol‐induced endotoxin leakiness and bacterial translocation. Our previous studies showed that Lactobacillus rhamnosus GG (LGG) and its culture supernatant protected against alcoholic liver disease (ALD) through increasing intestinal mucus and epithelial tight junction protein expression resulting in a reduction of circulation LPS level. The precise mechanisms underlying this protective effect are still not clear. Recent studies demonstrated the critical role of intestinal aryl hydrocarbon receptor (AhR) in the regulation of RORγt+ innate lymphoid cell (ILC3) function to produce IL22, which modulates intestinal microbiota homeostasis and barrier function through increasing the expression of epithelial antimicrobial peptide regenerating islet‐derived protein 3γ (Reg3γ) and Reg3β.MethodsWe isolated a product (LGGp) from LGG and examined its role in the modulation of ALD. C57B6 mice were subjected the Lieber DeCarli liquid diet containing 5% alcohol for 10 days, and one bolus of alcohol (5 g/kg) was gavaged on the last day 9 hours before tissue collecting. Control mice were fed isocaloric maltose dextrin. LGGp was supplemented on day 7 once a day for three days.ResultsThis three‐day administration of LGGp reversed alcohol‐induced hepatic steatosis and injury, demonstrated by reduced Oil red O staining of liver section, decreased serum ALT and AST levels and reduced hepatic apoptosis. The protective effects were associated with decreased circulating LPS concentration and hepatic bacterial load, indicating an increased gut barrier function. Further analysis showed that LGGp activated AhR luciferase activity and increased Cyp1a1 (AhR target) enzymatic activity. LGGp treatment increased intestinal mRNA expression of IL22, Cyp1a1, Reg3g and Reg3b, which play a critical role in modulating intestinal immune response to bacteria invasion and translocation. In addition, LGGp treatment significantly increased intestinal epithelial nuclear factor erythroid 2‐related factor 2 (Nrf2) protein expression, which were associated with increased protein expression of epithelial tight junctions ZO‐1, occludin, claudin‐1. Furthermore, LGGp inhibited LPS‐induced pro‐inflammatory factors TNFα and IL1β in mRNA and protein levels in Raw264.7 macrophage cells.ConclusionsOur results demonstrate that LGGp treatment protects against ALD through activation of intestinal AhR‐mediated upregulation of IL22 and Reg3 and through Nrf2‐mediated upregulation of intestinal tight junction proteins that lead to the decreased LPS release and bacterial translocation and reversal of ALD.Support or Funding InformationSupported by grants from NIH.