bluuebunny/crossref_metadata_2025 huggingface dataset:
A Horseshoe Kidney with Convoluted Vasculature: Gross and Histological Anatomy
Authors: Cook Ryan L. , Herrold Christina B. , Auten Matthew W. , Grise Joy , Santos Rosaysela , Zdilla Matthew J. , Lambert H. Wayne
Published: April 2020

This report documents the incidental finding of a horseshoe kidney identified during routine dissection in a gross anatomy course. The anatomic examination demonstrated malrotated bilateral kidneys, which were fused at the inferior poles. Histologic examination revealed no significant pathologic change; however, the fused portion was shown to have an intact cortex and collecting system. Furthermore, the renal vasculature was anomalous with multiple accessory and convoluted arterial vessels. An aortic aneurysm was also noted just superior to the renal fusion. Operative procedures and diagnostic imaging performed in the region of such an anatomical variation may be complicated by unanticipated vascular patterns. Therefore, special attention should be paid to vasculature in the setting of a horseshoe kidney.

Support or Funding Information

T. Walley Williams Summer Anatomy Research Fellowship;

WV Research Challenge Fund [HEPC.dsr.17.06]
Crystal structures and Directed Evolution of the α‐L rhamnosidase RHA‐P from Novosphingobium sp. PP1Y
Authors: Terry Brendan , Driza Danicah Vinh , Izzo Vivianna , Sazinsky Matthew Howard
Published: April 2020

Flavonoid natural products are well‐known for their beneficial antimicrobial, antitumor, and anti‐inflammatory properties. However, some of these natural products often are rhamnosylated, which severely limits their bioavailability. The lack of endogenous rhamnosidases in the human GI tract not only prevents many of these glycosylated compounds from being of value in functional foods but also limits the modification of natural product libraries being tested for drug discovery. RHA‐P is a catalytically efficient, thermostable α‐L‐rhamnosidase from the marine bacterium Novosphingobium sp. PP1Y that selectively hydrolyzes α‐1,6 and α‐1,2 glycosidic linkages between a terminal rhamnose and a flavonoid moiety. Due to its promiscuity, relative thermostability compared to other known rhamnosidases, and catalytic efficiency even in significant concentrations of organic solvents, RHA‐P shows potential for biocatalytic applications. In a recent work, we reported the 2.2. Å resolution crystal structure of RHA‐P and, in part through in silico docking experiments, offered insight into the substrate preference of RHA‐P for glycosylated aryl compounds such as hesperidin, naringin, rutin, and quercitrin. This work reports the 3.0 Å resolution crystal structure of a E506Q RHA‐P mutant soaked with naringin and applies the structural data to‐date to the semi‐rational directed evolution of highly thermostable mutants of RHA‐P with retained activity on flavonoid glycosides.

Support or Funding Information

This research was supported by FARB 2014 ORSA 132082 (VI), the Beckman Scholars program (BT), and the Pomona College Chemistry Department (BT and DD). Use of the Stanford Synchroton Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE‐AC02‐76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393).
Long non‐coding RNAs regulate NF‐κB activation, inflammation, and glucose metabolism
Authors: Mandal Subhrangsu S. , Obaid Monira , Chini Avisankar
Published: April 2020

Inflammation plays a central role in immune response and the inflammatory response is closely linked with metabolism. Our recent study demonstrates that lncRNA HOTAIR plays key roles in NF‐kB activation, cytokine expression, and inflammation. Here, we investigated if HOTAIR plays any role in regulation of glucose metabolism in immune cells during inflammation. Our study demonstrate that lncRNA HOTAIR regulates the expression of glucose transporter isoform 1 (Glut1) which controls the glucose uptake into macrophages. SiRNA‐mediated knockdown of HOTAIR suppressed the LPS‐induced expression of Glut1 and glucose update in macrophages suggesting key roles of HOTAIR in LPS‐induced Glut1 expression and glucose metabolism. HOTAIR induces NF‐kB activation which in turn increases Glut1 expression and glucose metabolism in response to LPS. Beyond HOTAIR, we also discovered several novel lncRNAs (termed as LinfRNA:
Long‐noncoding
inflammation associated
RNA) which are closely associated with inflammation and metabolism. Overall, our study demonstrated that HOTAIR and LinfRNAs play key roles in NF‐kB activation and inflammation and regulate metabolic programming in immune cells potentially to meet the energy needs at the priming of immune response to pathogenic infection or other stressors.

Support or Funding Information

National Institute of Health grant # 1R15 ES019129‐01
Impaired Bmp signaling in Foxl1+ telocyte influence the maintenance of the intestinal stem cell niche
Authors: Perreault Nathalie , Servant Raphaelle , Pomerleau Veronique , Jones Christine , Boudreau Francois
Published: April 2020

New components of the intestinal stem cell niche are constantly being discovered. Recently, the subepithelial Foxl1+telocytes (TCFoxl1+) were found to play a key role in maintaining and regulating intestinal crypt homeostasis. TCFoxl1+ produce crucial niche components such as WNT2b, WNT5a and RSPO3 as well as the Bone Morphogenic Proteins (BMP) inhibitors Gremlin 1 and 2. Previous work in our laboratory showed that epithelial BMP signaling is involved in terminal differentiation and maturation of cells from the secretory lineage. Our recent work showed that the loss of mesenchymal BMP signaling leads to the development of a pathological microenvironment inducing colonic and gastric polyposis in older mice, drawing attention to the impact of a pathogenic microenvironment in polyposis.

By genetic means, we propose to address how BMP signaling in TCFoxl1+ modulates the intestinal stem cell niche. Using the Cre/loxP system, we generated mice with a deletion of BmpR1a in all gastrointestinal subepithelial TCFoxl1+. Histological analysis and terminal differentiation assessment were performed with cellular staining and immunofluorescence (H&E, Alcian Blue and Lysozyme). Niche components were analysed by RT‐qPCR from crypt epithelium or total ileal RNA extracts. H&E staining on 9 months‐old BmpR1aΔFoxl1+ and control mice ileum demonstrated perturbed architecture in the BmpR1aΔFoxl1+ such as fused and enlarged villi compared to control mice. Alcian Blue staining showed an increase of goblet cells in the mutant mice. Electron microscopy showed that Paneth cells from mutant mice have larger vesicles than controls. The RT‐qPCR analysis showed a decrease in WNT signaling targets in the crypts epithelium cells. Indeed, LGR5 is decreased by 50% and mTERT by 34% in the mutant mice, whereas the stem cell marker BMI1 is not modulated. Maturity of Paneth cells is impaired in the mutant mice as shown by a 41% decrease in SOX9 expression. This lack in Paneth cell maturity is also reflected by a 60% decrease in WNT3 expression in BmpR1aΔFoxl1+ mice, an important stem cell niche factor. Secretory cell determination transcription factor ATOH1 is decreased by 35% in the mutant mice. Analysis from total ileal extract revealed a decrease of WNT2b, a key niche factor from the mesenchymal cells.

Thus, our results show that altering Bmp signaling in subepithelial TCFoxl1+ modulates crucial niche factors influencing intestinal stem cells homeostasis and their subsequent determination.

Support or Funding Information

Canadian Institutes of Health Research‐Institute of Nutrition, Metabolism and Diabetes
Ezh2 loss is a haploinsufficient mediator of cell lineage in prostate cancer and has divergent effect on androgen receptor expression
Authors: Wadosky Kristine M. , Wang Yanqing , Goodrich David
Published: April 2020

Patients with advanced, metastatic, and/or treatment‐resistant prostate cancer (PCa) can develop aggressive tumor variants with neuroendocrine (NE) features. These PCa variants are found in 20–30% of patient autopsies relapsing from androgen deprivation therapy (ADT), can lack androgen receptor (AR), and have altered histology and clinical course. A recent comprehensive study of patients with advanced PCa shows that alteration in tumor suppressor gene RB1 is the strongest genomic predictor of survival. We have previously shown that loss of Rb1 promotes NE lineage transformation in genetically engineered mouse models (GEMMs) of PCa. Rb1 deficiency also induced pervasive changes in gene expression characteristic of NE variants in human PCa. These changes included increased expression of histone methyltransferase Ezh2. These data led us to hypothesize that Ezh2 is a driver of NEPC transformation in Rb1‐deficient PCa. To test this hypothesis, we developed new GEMMs of Rb1‐deficient PCa lacking Ezh2. Data show that overall survival of PBCre4:Ptenf/f:Rb1f/f:Ezh2f/+ (DKOE/+) mice with loss of one Ezh2 allele is significantly increased compared to PBCre4:Ptenf/f:Rb1f/f (DKO) (42 weeks vs. 38 weeks). To our surprise, overall survival of PBCre4:Ptenf/f:Rb1f/f:Ezh2f/f (DKOE/E) was decreased compared to DKO (33 weeks vs. 38 weeks). Histological analysis and expression of endstage primary tumors from DKOE/E and DKOE/+ diverge from the original DKO model in regards to AR expression. While DKO tumors are ARlow‐NEpos, DKOE/E tumors are ARneg‐NEhigh and DKOE/+ tumors are ARhigh‐NEhigh, which has been confirmed by RNA sequencing. Interestingly, ARhigh‐NEhigh tumors in DKOE/+ is similar to the “amphicrine” histological PCa variant that has recently been reported in patients. Together, these data suggest that Ezh2’s effect on PCa progression in the absence of Rb1 is haploinsufficient. Future work is required to investigate of the exact mechanism of Ezh2’s contribution to NE lineage, AR expression, and AR activity in advanced PCa.

Support or Funding Information

This work was supported by the National Cancer Institute funding sources R01CA207757, R01CA207757‐03S1, R01CA234162 to D.G. and 3P30CA016056 to Roswell Park.
Endoplasmic Reticulum Stress Induction of Yeast Bax Inhibitor (BXI1) Gene Expression Does Not Entirely Require IRE1
Authors: Finn John Patterson , Parker Wesley , Cardona Andrew , Pineda Edy , Austriaco Nicanor O.P.
Published: April 2020

Bax Inhibitor (BI‐1/TMBIM6) is a regulator protein within the Unfolded Protein Response (UPR) that influences rates of apoptosis within cells. Studies have shown that BI‐1 is upregulated in human cancer cells like prostate and breast, therefore it is important to study the factors that control BI‐1 within a cell. In this paper, the Saccharomyces cerevisiae homolog of BI‐1, BXI1, is studied to better understand the signaling pathway that influences concentrations of BXI1, specifically the Inositol Requiring Enzyme 1 (IRE1) pathway. Ire1p is an Endoplasmic Reticulum (ER) bound transmembrane ligand receptor which detects the UPR and activates downstream targets which play further roles in regaining homeostasis. Here, our lab used two genetically modified S. cerevisiae strains, both with Green Fluorescent Protein (GFP) bound to BXI1 (Bxi1p‐GFP) and one lacking genetic information to code for IRE1 (ΔIre1) to test the effect of the IRE1 pathway on the expression of BXI1. When cells were cultured in media containing either tunicamycin or DDT, we observed an increase in the expression of BXI1 that was not entirely dependent on the IRE1 pathway. This suggests that there may be other signaling pathways that influence the expression of BXI1.

Support or Funding Information

Our laboratory is supported by grant NIGMS R15 GM110578, awarded to N. Austriaco.
Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by the Nuclear Bile Acid Receptor, Farnesoid X Receptor
Authors: Smyth Jessica S. , Bussayajirapong Anchalin , Lajczak-McGinley Natalia K. , Foulke-Abel Jennifer , Donowitz Mark , Keely Stephen J.
Published: April 2020

Introduction and Aims

The intestinal epithelium functions to transport nutrients, fluid and electrolytes, while at the same time acting as a barrier to the entry of harmful substances. CFTR is a transmembrane Cl− channel important in regulating intestinal fluid transport and is implicated in the pathogenesis of a number of intestinal diseases. Bile acids, classically known for their roles in lipid digestion, are now also recognised as important enteric hormones that regulate many aspects of epithelial function. Indeed, it is thought that bile acid metabolism is a primary mechanism by which the microbiota communicates with its human host. Here, we set out to investigate the role of the nuclear bile acid receptor, farnesoid x receptor (FXR), in regulating epithelial CFTR expression.

Methods

T84 human colonic epithelial cells were cultured as polarised monolayers on permeable supports and treated bilaterally with the FXR agonist, GW4064 (5 μM), over a range of times. Levels of FGF19, an index of FXR activation, CFTR, FXR, FOXA1, and miR‐494 were measured by qRT‐PCR, western blotting, or ELISA.

Results

Treatment of the cells with GW4064 significantly increased FGF19 mRNA expression by 1235 ± 237.3 fold (n = 8; p < 0.05) and protein by 719.6 ± 92.0 pg/mL (n = 5; p < 0.01) after 48 hrs. Moreover, GW4064 downregulated CFTR mRNA to 0.47 ± 0.1 fold after 12 hrs (n = 8; p < 0.01) and protein levels to 0.36 ± 0.1 fold after 48 hrs, compared to vehicle‐treated controls (n = 4; p < 0.05). Transcriptomic analysis confirmed FXR‐induced downregulation of CFTR in primary human colonic enteroids. Studies in Ussing chambers showed that GW4064 treatment for 48 hrs inhibited Cl− secretory responses to the Ca2+‐dependent agonist carbachol (CCh; 100 μM) and the cAMP‐dependent agonist, forskolin (10 μM) by 79.9 ± 7.5 % (n=4; p <0.01) and 74.2 ± 8.9 % (n=4; p < 0.01), respectively. Expression of miR‐494, which is known to target CFTR, was not increased by GW4064 treatment in T84 cells. However, mRNA expression of FOXA1, a transcription factor that regulates CFTR, was inhibited by 33.2 ± 5.2% after 3 hrs (n = 4; p <0.05).

Conclusion

The nuclear bile acid receptor, FXR, regulates colonic epithelial CFTR expression. Such actions are likely to be important in the setting of microbial regulation of host intestinal physiology in health and disease. As such, FXR represents an excellent target for development of new drugs to treat intestinal diseases associated with dysregulated CFTR function.

Support or Funding Information

This work was funded by a Science Foundation Ireland Principal Investigator Award to SJK
ALTERATION OF MITOCHONDRIAL MEMBRANE POTENTIAL (ΔΨM): POTENTIAL ROLE IN THE MECHANISM OF MEHP‐INDUCED INHIBITION OF STEROID BIOSYNTHESIS IN MA‐10 LEYDIG CELLS
Authors: Traore Kassim , Li Yunbo , Papadopoulos Vasilios , Zirkin Barry
Published: April 2020

INTRODUCTION AND OBJECTIVES

Mono‐(2‐ethylhexyl) phthalate (MEHP) is the active metabolite of di‐(2‐ethylhexyl) phthalate (DEHP), a plasticizer with endocrine disruptor activity that is widely used in the manufacturing industry. MEHP inhibits luteinizing hormone (LH)‐stimulated steroid biosynthesis in Leydig cells by molecular mechanisms that remain unclear. In the present study, using MA‐10 mouse tumor Leydig cells, we examined changes in mitochondrial function caused by MEHP exposures in relationship to possible link to alteration of progesterone biosynthesis.

METHODS

The effect of MEHP on LH‐stimulated progesterone production was assessed after treating MA‐10 cells with (0–300 μM) MEHP for 24 hrs. Progesterone production was quantified by enzyme‐linked immunosorbent assay (ELISA). To measure oxygen consumption, cells were treated with MEHP (0–300 μM) for 24 hrs, and oxygen consumption was monitored using a Clarke‐type oxygen electrode. Mitochondrial membrane potential (ΔΨm) was assessed using the JC‐1 dye mitochondrial membrane potential indicator. Mitochondria‐derived superoxide was measured using the MitoSOX™ red mitochondrial superoxide indicator.

RESULTS

Exposures of MA‐10 cells to 0–300 μM MEHP caused inhibition LH‐stimulated progesterone biosynthesis in a dose‐dependent manner. The decrease in the Leydig ability to produce progesterone correlated with a loss in the Leydig cell mitochondrial membrane potential (ΔΨm), and was linked to perturbation of the capacity to energize the mitochondrial membrane. MEHP exposure was suggested to cause a disruption in the electron flow through the electron transport chain, evidenced by a decrease in the ability to consumption molecular oxygen and an increase in the generation of superoxide in the mitochondria.

CONCLUSIONS

Together, these observations suggest that MEHP inhibits steroid biosynthesis in MA‐10 cells via a mechanism involving a loss of the mitochondrial membrane potential (ΔΨm) and an increase in the oxidative stress level, two crucial changes known to affect mitochondrial function and cause inhibition of STAR‐mediated cholesterol transport and steroid biosynthesis.

Support or Funding Information

This material is based upon work supported by NIH grant SC2GM099578 and by the North Carolina Biotechnology Center, NCBC grant #2019‐IDG‐1017.
Cytosolic glutathione‐S‐transferase A1 (GSTA1) plays a primary role in the metabolic clearance of the aromatase inhibitor, Exemestane
Authors: Teslenko Irina L. , Chen Gang , Watson Christy , Lazarus Philip
Published: April 2020

Exemestane (EXE) is used to treat estrogen receptor positive (ER+) breast cancer in postmenopausal women by inhibiting the enzyme aromatase in the final step of estrogen biosynthesis. Variability exists in both overall patient response and adverse events, which may be attributed to inter‐individual variation in EXE metabolism. EXE is metabolized primarily to the active metabolite, 17β‐dihydro‐EXE (17β‐DHE), and preliminary studies have demonstrated that cysteine conjugates of EXE and 17β‐DHE comprise 77% of total EXE metabolites in the urine of patients. Cysteine conjugation is a multi‐step process, the first of which is glutathione (GSH) conjugation, catalyzed by the glutathione‐S‐transferase (GST) family of the enzymes. The goal of the present study was to identify the hepatic GSTs active in the metabolism of EXE and 17β‐DHE. In a screening of the Human Protein Atlas, 12 cytosolic GSTs were found to be hepatically expressed and were initially screened by incubating 0.5 μg of pure recombinant GST enzyme with EXE or 17β‐DHE (125 μM) and reduced glutathione (GSH). The resulting GS‐conjugates of EXE and 17β‐DHE were detected and quantified using UPLC‐MS/MS. Results from the conjugation assays indicated that the cytosolic enzyme GSTA1 is highly active against both EXE and 17β‐DHE, while GSTM3 and GSTM1 are only moderately active against EXE. No other recombinant cytosolic GST enzymes tested (GSTA2, GSTA4, GSTK1, GSTM2, GSTM4, GSTO1, GSTP1, GSTZ1 and GSTT1) exhibited activity against either EXE or 17β‐DHE. KM values for human liver cytosol (HLC) and purified recombinant GSTA1 were similar for both EXE (45 μM vs. 77 μM, respectively) and 17β‐DHE (54 μM and 30 μM, respectively) as substrate, suggesting that GSTA1 may be the main driver for EXE and 17β‐DHE glutathione conjugation in human liver cytosol. Using EXE as a substrate, Vmax value for GSTA1 was >7‐fold higher than that observed for GSTM3 (9.8 nmol/min/mg vs. 1.3 nmol/min/mg, respectively). The EXE intrinsic clearance (ClINT) for GSTA1 (130 nl/min/mg) was >10‐fold higher than that observed for GSTM3 (11 nl/min/mg). These data suggest that GSTA1 may be an important component in the metabolic pathway contributing to the elimination of EXE in breast cancer patients.

Support or Funding Information

This work is supported by grants from NIH, National Cancer Institute (grant R01‐CA164366; to P. Lazarus) and the Health Sciences and Services Authority of Spokane, Washington (grant WSU002292 to College of Pharmacy and Pharmaceutical Sciences, Washington State University)
Analysis of Assessments for Biochemistry Students of Varying Chemistry Backgrounds in Thermodynamics and Kinetics of Chemical Reactions as well as Research Skills
Authors: Keenan Kelly
Published: April 2020

Threshold concepts represent abstract topics in a subject that students must grasp in order to advance understanding in the subject. In Biochemistry, they include several topics related to thermodynamics and kinetics of chemical reactions including, free energy, thermodynamics, and steady state. Many of these topics are covered in the introductory chemistry sequence, which is taught traditionally as a year of general chemistry followed by organic. Chemistry courses at Stockton University are presented in a different order: general I, organic I and II and general II. In the Biochemistry classes at Stockton, students have varied chemistry backgrounds. A Previous assessment was designed to test student’s knowledge in several threshold concepts as a pre and post‐test and correlate it with amount of chemistry classes previously taken. Students scored the lowest marks in questions related to free energy and thermodynamics: the initial scores were lowest and the lowest amount of improvement was observed as well. This was true regardless of number of previous chemistry courses taken. The goal of this project was to investigate the reason for the low scores. A new assessment was used and tested on 170 Biochemistry students as a pre ‐test at the beginning of the semester and a post‐test at the end. In order to separate knowledge of the concept from knowledge of definitions and symbols, questions were designed to assess both. Half of the questions required students to correctly identify symbols and definitions for the terms related to steady state, thermodynamics and kinetics. The other required students to predict the outcomes if a change occurs without requiring the use of symbols to represent the key terms. Results are presented based on both areas. In addition to questions on these topics, students are also assessed for some of the outcomes listed in the skills in the ASBMB Foundational Concepts. The skills assessed required the students to interpret the results of an enzyme assay in which various mistakes were made as well as comment on experimental design and best way to express results. In addition to these higher level questions, there were also ones on how to prepare solutions. The scores are also related to the amount of Chemistry and Biology classes previously taken.
S‐Nitrosoglutathione Reductase Inhibition Attenuates Long Term Effects of Neonatal Hyperoxia on Airway Hyperreactivity
Authors: Sopi Ramadan B. , MacFarlane Peter M. , Gaston Benjamin , Martin Richard J. , Raffay Thomas M.
Published: April 2020

Bronchopulmonary dysplasia survivors display long‐term obstructive lung disease with airway hyperreactivity (AHR). While often treated with asthma medications, patient responses are variable. We have previously shown that the endogenous smooth muscle relaxant molecule, S‐nitrosoglutathione (GSNO), is degraded in neonatal hyperoxia by upregulated GSNO reductase (GSNOR) in the murine lung (Raffay, et al. Molecular Pharmacology 2016; 90(4):418‐26). We hypothesize that inhibition of GSNOR is a novel approach to attenuate neonatal hyperoxia‐induced AHR in room air recovered mice. Newborn C57BL/6 mice were randomized on the first day of life and assigned to room air (21% O2) or hyperoxic (60% O2) groups for three weeks to induce bronchopulmonary dysplasia AHR. Animals were then recovered in room air until six weeks of age. AHR was assessed in vitro using precision‐cut living lung slice preparations in response to increasing doses of bath‐applied methacholine (MCh, 0.25–64 μM). Lung slices were pre‐incubated with or without a GSNOR inhibitor (100 μM N6022) or a β2 adrenergic receptor agonist (10 or 100 μM levalbuterol). AHR is reported as percent change in airway lumen area from baseline (± SEM). 2–3 airways were imaged and averaged per animal per condition. Neonatal hyperoxia significantly increased airway contractile responses to MCh in room air recovered mice with a mean maximal effect (Emax) of 71.9 ± 2.7% change from baseline compared to the room air control Emax of 39.8 ± 5.2% (p<0.01). GSNOR inhibition significantly attenuated hyperoxic effects on AHR (32.2 ± 2.0%, p<0.001). Pre‐incubation of hyperoxic slices with either a GSNOR inhibitor or a β2 adrenergic receptor agonist (100 μM) also attenuated the response to the maximal dose of MCh (64 μM) compared to vehicle (both p<0.01). These studies show that neonatal hyperoxia‐exposed mice display longer‐term in vitro AHR to MCh and that inhibition of GSNOR reverses hyperoxia‐induced AHR comparable to a β2 adrenergic receptor agonist. We speculate that GSNO‐based therapies may serve as novel treatments for AHR in bronchopulmonary dysplasia survivors that do not respond to β2 adrenergic receptor agonists.

Support or Funding Information

Supported by NIH 5K08HL133459‐03 and ATS Research Foundation Unrestricted Critical Care Grant
Evaluation of Lysis and Recovery of Genomic DNA from Microbiota to Maximize Efficiency for DNA Sequencing
Authors: Proctor Caleb McKinley , Okparanta Akelachi , Ryan Gabriella , Easparro Brandon , Nash Rodney
Published: April 2020

Microbes, specifically microbes found in mixed populations in guts, soil, feces, and other tissues, have been the object of much research for commensal relationships for decades. Many of these microbes are ubiquitous across many species or biomes but are present very in low quantities due to the inherent challenges of microorganism survival in environmental conditions. While, in some cases, only present in low quantity, soil microorganisms are a critical component of our ecosystem. Soil microbiome studies aim to characterize the microbial repertoire and to correlate the microbial population with soil and ecological functions. Given the time involved and complexity of culture‐based enrichments, along with the inability to grow some species in culture, many modern studies involve direct cell lysis and DNA recovery from soil followed by PCR or genetic sequencing for target enrichment, detection, and analysis. In order to evaluate the organisms, present in such low quantities, methods must be developed to lyse all organisms present in soil of various sizes and toughness’. In this study we evaluate the lysis and recovery of a microbiological standard using commercially available kits and genetic sequencing to show an ideal bead milling matrix for microbiota in soil.

Support or Funding Information

Funding for this project was provided by Omni International
Bacterial Expression of Chimeric Escherichia coli and Trypanosoma brucei DNA Methyltransferases
Authors: Taber Cassandra C. , Militello Kevin T.
Published: April 2020

Our laboratory is interested in DNA and RNA methylation in E. coli and T. brucei as little is known about this form of epigenetic regulation in microorganisms. One methyltransferase being studied at this time is a putative DNA methyltransferase (TbDmt) from Trypanosoma brucei. The exact function of TbDmt is unknown but the protein strongly resembles bacterial DNA methyltransferases such as DNA cytosine methyltransferase (EcDcm) from E. coli. To test our hypothesis that TbDmt is a DNA methyltransferase, we expressed TbDmt in bacteria and created two chimeric protein sequences switching the DNA binding domain and enzymatic domain of EcDcm and TbDmt. Exchanging the DNA binding domain and enzymatic domain of TbDmt with a known methyltransferase may help us discover the function of the enzyme and, if it is a methyltransferase, what DNA sequence is targeted for methylation. Plasmids were made containing the sequences for EcDcm, TbDmt, and both chimeric proteins where the genes are adjacent to the lac operator. E. coli were transformed with the plasmids and expression was induced with IPTG. All four proteins were produced at 20°C, but the proteins with the TbDmt enzymatic domain were less soluble than those with the EcDcm enzymatic domain. The plasmids were re‐isolated after three hours of growth in the presence of IPTG. The plasmids were then digested with eight restriction enzymes blocked by methylation. Each digestion was run on an agarose gel with DNA from uninduced cells and unmethylated phage lambda DNA as controls. EcDcm methylated at its expected site, 5′CCWGG3′, as determined by resistance to PspGI. In addition, there is partial resistance to HpaII in cells containing EcDcm indicating methylation at 5′CCGG3′ or related sequences. The proteins were denatured using guanidium HCl, isolated and analyzed by SDS‐PAGE with Coomassie blue staining. EcDcm and the chimeric protein with the EcDcm enzymatic domain and TbDmt DNA binding domain were successfully purified and were then purified using partial denaturing conditions. These proteins will be tested for methyltransferase activity. TbDMT and the chimera with the TbDMT enzymatic domain were not able to be purified using the guanidium HCl denaturing method so stronger denaturants will be tested in attempt to purify the proteins. Further work will be done in purifying the proteins using both denaturing and nondenaturing conditions to produce functional enzymes for use in methylation assays. This work will contribute to the limited knowledge of methyltransferases in bacteria and protists.

Support or Funding Information

NIH grant 1R15AI133428‐01 to KTM
Assessment of the Lateral Ventricle as a Means to Identify Pre‐Dementia
Authors: Ritz Brianna Kathryn , Zdilla Matthew
Published: April 2020

Dementia is a component of several neurological diseases including Alzheimer Disease. Unfortunately, predictive factors regarding dementia are elusive and early diagnosis remains challenging; yet, early diagnosis is a key factor in decreasing the severity of the disease. The authors hypothesized that ventricular expansion characteristic of hydrocephalus ex vacuo may be reflective of dementia diagnosis. Therefore, this study assessed the anatomy of the lateral ventricles via MRI in an unremarkable population (n=733), a pre‐dementia population (i.e., a population who was considered unremarkable at the time of MRI but subsequently developed dementia) (n=82), and a population of individuals with diagnosed dementia (n=235). Significant differences in total left‐ and right‐sided lateral ventricle volumes were found between unremarkable and pre‐dementia populations (LEFT: t=10.332, Mean difference=10,627mm3, 95% CI of Mean = (8,607.7mm3, 12,645mm3; p<0.0001. RIGHT: t=9.89, Mean difference=9,700mm3, 95% CI of Mean = 7775mm3, 11,625mm3; p<0.0001.). Also, significant differences in left‐sided and right‐sided inferolateral (i.e., inferior horn) ventricle volumes were found between unremarkable and pre‐dementia populations (LEFT: t=16.966, Mean difference=798mm3, 95% CI of Mean = 706mm3, 891mm3; p<0.0001. RIGHT: t=17.552, Mean difference=778mm3, 95% CI of Mean = 691mm3, 864mm3; p<0.0001). Moreover, significant differences were identified in ventricular volumes between the unremarkable population and the dementia population; however, no significant differences were found in the ventricular volumes between the pre‐dementia and dementia population. Therefore, an anatomical assessment of the lateral ventricles and their inferior horns may aid in the early identification of individuals who are at risk of having diagnosed dementia.

Support or Funding Information

Funding: WV Research Challenge Fund [HEPC.dsr.17.06] and NASA West Virginia Space Grant Consortium, Training Grant #NNX15AI01H
The structure of LRRK2 and the conformational changes that are associated with LRRK2 activation and Parkinson’s disease pathogenesis
Authors: Weng Jui-Hung , Auto Philip , Lu Tsan-Wen , Taylor Susan
Published: April 2020

Parkinson’s Disease (PD), a major neurodegenerative disorder, is characterized by chronic and progressive loss of dopaminergic neurons ultimately leading to death. Leucine‐rich repeat kinase 2 (LRRK2) is one of the greatest known genetic contributors to PD. LRRK2 is a large multi‐domain protein that contains a kinase domain as well as a Ras/GTPase (ROC) domain, followed by a COR domain. LRRK2 also acts as scaffold for the assembly of signaling complexes with its armadillo, ankyrin, leucine‐rich and WD40 domains at the N‐ and C‐termini. Studies have shown that most of the common familial mutations resulted in the formation of fibrous structures docked on microtubules in cells. However, how the fibrous structure is associated with LRRK2 activation is still unclear. Little is known about how LRRK2 activity is regulated, as well as the pathogenic mechanisms that drive PD. In this study, we use hydrogen‐deuterium exchange mass spectrometry (HDX‐MS) to measure the solvent exposed regions of LRRK2. Purified truncated LRRK2 (RCKW), which includes ROC, COR, kinase and WD40 domain, was used to map conformational changes associated with ATP, GTP and kinase inhibitor binding. Mutations in the kinase domain, including two familial mutations (G2019S and I2020T), are characterized as well. This information will help us to understand the protein interfaces within and between LRRK2 oligomers involved in LRRK2 activity. Also, it will help to understand the conformation that drives the activation of LRRK2 and pathological docking onto microtubules.
Lipid Nephrotoxicity and Increased Risk of Nephrolithiasis in Model of Estrogen Deficiency
Authors: Fiebiger Bryce J. , Van Erdewyk Jonathan I. , Bilal Khan , Babich Victor , Barnes Maria , Di Sole Francesca
Published: April 2020

Menopause is a period during which women tend to gain body fat. Estrogen deficiency is proposed to impact body fat composition and energy balance in these women by influencing the levels of leptin, an adipocyte‐derived hormone that acts primarily on the central nervous system. Specifically, lipid nephrotoxicity increases the risk for kidney disease and nephrolithiasis due to renal lipid accumulation and overly acidic urinary pH. Mechanisms underlying these kidney disturbances and extent of their effects when women become estrogen deficient (i.e. post‐menopausal) are not well‐defined. Utilizing an estrogen deficient (i.e. ovariectomized) rodent model, we hypothesize that central leptin receptor activation regulates renal function by augmenting the accumulation of renal lipids and urinary pH acidification. We measured the glomerular filtration rate (GFR) by creatinine clearance and protein level of the neutrophil gelatinase‐associated lipocalin (NGAL), an early biomarker of kidney injury, by immunohistochemistry. An increase in creatinine clearance (from 1.3 ml/min to 2.3 ml/min) and protein levels of NGAL (over 90% increase in both glomeruli and tubules) was measured in ovariectomized adult female rats when compared with their sham‐operated controls. Treatment with leptin receptor antagonist (LAN‐6, 3μg/day), delivered for four weeks into the lateral ventricle by an osmotic pump, significantly attenuated these effects. Furthermore, we determined that perirenal fat accumulation was markedly higher in the same estrogen deficiency model when compared with the sham‐operated control. Perirenal fat accumulation has been associated with lipid accumulation in renal tissues and excess of intrarenal lipid is increasingly recognized to contribute to kidney injury through lipotoxicity. A significant accumulation of renal lipid, tested using oil red O staining, was detected in the renal tubules of the ovariectomized animals when compared with their controls. We also found significant acidification of urinary pH and decreased protein expression of a key player in urinary pH regulation, Na/H exchanger‐3 (NHE3), in ovariectomized animals when compared with their controls. Renal lipid accumulation, urinary pH acidification and decrease in NHE3 expression were all partially reversed by blocking activation of central leptin receptors. We propose that: 1. An increase in GFR combined with kidney injury presupposes kidney dysfunction, 2. infiltration of renal lipid leads to renal injury, through lipotoxicity, 3. urinary pH acidification and reduction of NHE3 expression increases the risk of nephrolithiasis. Taken together, these findings suggest that leptin plays a key role in the development of menopausal‐associated kidney disease.
GATA‐4 interacts with the UCS domain of the Myosin Chaperone, Striated Muscle UNC‐45
Authors: Odunuga Odutayo , Anderson Morgan , Duncan Drake
Published: April 2020

Striated muscle UNC‐45 (SM UNC‐45 or UNC‐45B) protein acts as a chaperone for cardiac and skeletal muscle myosins; regulating their folding, assembly into thick filaments, interaction with other sarcomeric proteins, and degradation. GATA‐4 is a transcription factor that regulates the expression of several cardiac muscle genes, including myosin. A previous study by Chen et al (2012) demonstrated for the first time that SM UNC‐45 (UNC‐45B) physically interacted with GATA‐4 and promoted its transcriptional activity in an overexpression experiment. The aim of this study was to identify the specific domain in UNC‐45B that interacted with GATA‐4. Pull‐down assays showed that GATA‐4 bound to the UCS domain of UNC‐45B. The major revelation from these studies is that UNC‐45B has the potential to exert both short‐term (protein level) and long‐term (gene level) controls over myosin and therefore muscle structure and function. Experiments are ongoing to determine whether UNC‐45B acts as a chaperone on GATA‐4 protein.

Support or Funding Information

Robert A. Welch Foundation Grant #AN‐0008
Determining the Kinetics of Desferroxiamine D from Streptomyces coelicolor using Isothermal Titration Calorimetry
Authors: Goncuian Eliana , Hoffmann Katherine
Published: April 2020

The long‐term objective for this research is to create a new class of antibiotics that targets the non‐ribosomal peptide synthetase independent siderophore (NIS) pathway in bacteria. The focus of my current research is to quantify the first and second order kinetic constants and obtain the kinetics curve of our model system, Desferroxiamine D (DesD), from Streptomyces coelicolor. DesD reacts with ATP as a cofactor, and either the small (hydroxysuccinylcadaverine, HSC), medium or large (desferrioxamine G, or dfoG) substrates in an iterative manner. We have so far collected activity curves with ATP and dfoG limiting, and are pursuing HSC curves. To do this we used Isothermal Titration Calorimetry (ITC) to collect data and GraphPad Prism 8.2 along with Malvern MicroCal Peaq ITC software to fit the kinetics curves and determine the kinetics constants. We successfully gathered and analyzed ITC data for both the catalytic (wild type, Wt) and non‐catalytic (mutant, R306Q) forms of DesD and determined the optimal buffer conditions for solubility. We also gathered data regarding the activity of our Wt DesD enzyme in order to determine the lifespan and the time of optimal catalytic activity. We have found that our enzyme is still active within 96 hours where it was previously thought to have a 36–48 hour catalytic lifespan.

In conducting these experiments, we observed that an industry standard amount of magnesium chloride (MgCl2) in our solution caused an oscillating signal during equilibration of the ITC prior to enzyme injection. We hypothesized that the data reflects metal chelation of the Mg by our substrate (dfoG). We plan on conducting a binding experiment using the ITC in order to quantify the substrate (dfoG) and MgCl2 interaction, and ultimately recommend a new standard amount of MgCl2 to the field for future experiments. Other future directions include determining the kinetic constants of Wt DesD interacting with its small substrate HSC (which must be synthesized and purified,) and determining the catalytic role of other residues in the active site by site‐directed mutagenesis and ITC. This experimental assay and data will be the foundation for future drug design targeting DesD.

Support or Funding Information

This research was supported in part by the National Science Foundation (NSF‐RUI grant 1716986 to K.M.H.), and by the John Stauffer Charitable Trust.
Bile Acids Directly Bind ENaC and Allosterically Regulate its Activity
Authors: Wang Xue-Ping , Ertem Merve , McKernan Abagail S , Lei Xiaoguang , Kashlan Ossama B
Published: April 2020

The epithelial sodium channel (ENaC) mediates Na+ transport in several epithelia, including the aldosterone‐sensitive distal nephron, distal colon, and biliary epithelium. ENaC is a heterotrimer composed of α, β, and γ subunits. Numerous extracellular ligands regulate the activity of the channel. Bile acids are abundant in the biliary tree and intestinal tract, and can be elevated in the urine of patients with advanced liver disease. Our previous work showed that bile acids that regulate ENaC activity have specific chemical moieties, but are not necessarily membrane permeable. We hypothesized that bile acids directly bind ENaC. To test this, we used light‐sensitive bile acid probes derived from cholic acid and deoxycholic acid (p‐CA and p‐DCA). We incubated Fisher Rat Thyroid cells expressing V5‐tagged ENaC subunits with p‐CA and p‐DCA. After brief exposure to light, crosslinked proteins were conjugated to biotin, precipitated with streptavidin, subjected to SDS‐PAGE/immunoblotting with detection by anti‐V5. We found that both p‐DCA and p‐CA crosslinked to α, β, and γ ENaC subunits. In competition experiments with excess unlabeled bile acids, both DCA and CA decreased crosslinking by the corresponding bile acid. DCA competition vs. p‐DCA was more effective than CA competition vs. p‐CA. These data show that these bile acids directly and specifically bind ENaC subunits. Using Xenopus oocytes, we observed that taurocholic acid (t‐CA) activation of ENaC currents is voltage‐dependent. Modeling of the voltage‐dependent data suggests at least two binding sites, consistent with the crosslinking data. We also determined whether t‐CA regulated currents of αβγ, α‐only, αβ‐, and αγ‐channels. We found that α‐only channels were greatly activated by t‐CA, followed by αβγ and αβ channels. Our data suggest that the a subunit is sufficient for bile acid regulation. Our results suggest that bile acids regulate ENaC through direct ligand‐channel interactions.
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 Information

MMR 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

Introduction

Externally 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.

Hypothesis

We hypothesized that all three drugs would inhibit cell proliferation.

Aims

Our 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.

Methods

PI‐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 Discussion

All 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 Information

Summer research stipend to medical students at MUCOMDepartmental faculty research support to faculties at MUCOM