bluuebunny/crossref_metadata_2025_split huggingface dataset:
DNA methylation contributes to chronic hypoxia‐induced downregulation of large‐conductance Ca2+‐activated K+ channels in uterine arteries in pregnancy (889.3)
Authors: Hu Xiang‐Qun , Zhu Ronghui , Zhang Lubo 
Published: June 2021

Uterine blood flow increases dramatically during pregnancy in part due to an upregulation of the large‐conductance Ca2+‐activated K+ (BKCa) channel in the uterine artery. However, this hemodynamic change is complicated by chronic hypoxia. We hypothesized that DNA methylation may play a role in chronic hypoxia‐induced downregulation of BKCa channels. Uterine arteries were isolated from nonpregnant (NPUA) and near‐term (~140 days) pregnant (PUA) sheep and incubated under 21% or 10.5% O2 for 48 hours at 37°C. Exposure to chronic hypoxia significantly reduced the BKCa current density in PUA (p < 0.05), but not in NPUA. 5‐aza‐2'‐deoxycytidine (5‐aza), a DNA methylation inhibitor, blocked the hypoxia‐mediated effect in PUA. Accordingly, chronic hypoxia decreased a BKCa activator NS 1619‐induced relaxations of PUA, which was abrogated by 5‐aza. Treatment of NPUA ex vivo under 21% O2 with physiological concentrations of estrogen and progesterone (E2β/P4) resulted in increased BKCa current density; and chronic hypoxia suppressed this upregulation. However, 5‐aza reversed the impact of chronic hypoxia and restored the ability of E2β/P4 in upregulating the BKCa current density. Taken together, our findings suggest that an epigenetic modification of DNA methylation plays an important role in chronic hypoxia‐induced downregulation of BKCa channel function in uterine arteries in pregnancy.Grant Funding Source: HL089012, HL110125
IGF‐1 acutely increases activity of basolateral ClC‐K2‐like Cl‐ channels in the distal nephron (892.12)
Authors: Zaika Oleg , Mamenko Mykola , Boukelmoune Nabila , Pochynyuk Oleh 
Published: June 2021

Insulin growth factor 1 (IGF‐1) is known to promote anti‐natriuresis by stimulating ENaC‐mediated sodium reabsorption in the distal renal tubule. However, it remains unclear whether IGF‐1 also augments Cl‐ transport at this place. Using patch clamp electrophysiology in freshly isolated mouse distal nephrons we detected highly abundant single channel conductance of approximately 14 pS in a cell‐attached configuration. Decreased Cl‐ concentration in the pipette resulted in a rightward shift of I‐V relation, indicating Cl‐ selectivity of the recorded channel. The single channel properties of the recorded channel are comparable with ClC‐K2 that was reported to be functionally expressed predominantly in the intercalated cells of distal nephron. In tandem with apically localized pendrin, ClC‐K2 is thought to contribute to distal nephron Cl‐ absorption. We found that acute application of 100 nM IGF‐1 significantly increases open probability of the ClC‐K2‐like channel in a reversible manner. Pretreatment of distal nephrons with a selective PI3‐kinase inhibitor LY292002 abolished activation of the channel by IGF‐1. We propose that, concomitantly with its stimulatory effects on sodium transport, IGF‐1 also favors distal nephron Cl‐ absorption by increasing activity of basolateral ClC‐K2‐like channels in a PI3‐K‐dependent manner.Grant Funding Source: DK095029
Low basolateral Na+ pump activity in macula densa cells may be necessary to generate tubuloglomerular feedback responses (892.32)
Authors: Nadal‐Quiros Monica , Nieves‐Gonzalez Aniel , Moore Leon , Marcano Mariano 
Published: June 2021

Macula densa (MD) cells sense changes in tubular fluid [Cl–] and this initiates a chain of events within the juxtaglomerular apparatus (i.e., the tubuloglomerular feedback (TGF) response) that regulates GFR. It is known that MD cells have much lower basolateral Na+/K+ATPase activity than neighboring cortical thick ascending limb (cTAL) cells. Also, an increase in luminal [NaCl] leads to the depolarization of the MD cell basolateral membrane, an event thought to be an essential step in the TGF response. To study the role of the Na+ pump in MD cells, we created a mathematical model that includes kinetic and thermodynamic models of the transport proteins and channels in MD cells. We examined the influence of luminal [NaCl] on basolateral membrane potential difference (PD). For comparison, similar computations were made with a model of a cTAL cell. When the luminal NaCl was varied from 20 to 150 mM, the basolateral membrane of the MD cell depolarized from –71 to –47 mV. For the cTAL cell, the basolateral membrane potential remained near –96 mV. These results suggest that the low expression of the basolateral Na+ pump may be appropriate in MD cells because, in these sensory cells, cytosolic [Na+] tracks luminal [Na+]. In a reabsorbing cTAL cell, the high basolateral Na+ pump expression stabilizes cytosolic [Na+] and basolateral PD.Grant Funding Source: Supported in part by MBRS‐RISE grant 2R25GM061151‐12 and by NIH‐NIGMS grant SC1GM084744.
Regulation of phosphate transport by dietary Pi in the small intestine of rats (908.2)
Authors: Candeal Eduardo , Caldas Yupanqui , Levi Moshe , Sorribas Victor 
Published: June 2021

Pi homeostasis is tightly controlled by the harmonized action of the intestine, kidney and bone. To date, three Na‐dependent Pi transporters have been found in the small intestine: NaPi2b, Pit‐1 and Pit‐2, but the coordinated modulation of all three by dietary Pi is unclear.Male Wistar rats were fed ad libitum 0.1% vs. 1.2% Pi diets, for either 1, 5 or 10 days. Brush border membrane vesicles (BBMV) and RNA were obtained from duodenum (D), jejunum (J) and ileum (I).0.1% Pi diet increased Na‐dependent Pi transport in D and J BBMV at 1, 5 and 10 days, as well as the expression of NaPi2b, but only in J for Pit‐2. Pit‐1 expression was increased only at 10 days in D and J. In the I, Pi transport was very modest and adaptation to 0.1% Pi diet was not observed, however, NaPi2b and Pit‐2 expressions were increased at all three times. The changes in NaPi2b and Pit‐1 protein expressions were confirmed by immunohistochemistry, but the changes in Pit‐2 were not noticeable.Pi transport was always higher at pH 6.0 than 8.5 but inhibited by phosphonoformic acid. This suggests the existence of a novel Na‐dependent transporter, different from NaPi2b, Pit‐1 and Pit‐2, and regulated within 24 h by dietary Pi.
22q11.21 Deletion associated with sporadic Tetralogy of Fallot in Han Chinese (912.3)
Authors: He Guo‐Wei , Maslen Cheryl L. , Bai Xiao‐Yan , Liu Xiao‐Cheng , Liu Zhi‐Gang , Yang Qin 
Published: June 2021

Tetralogy of Fallot (TOF) occurs in one in 3,000 live births and accounts for 10% of all serious congenital heart disease in the U.S. and the incidence is substantially higher in China. We investigated the genetic basis of TOF in Han Chinese by using Affymetrix genome‐wide human SNP array 6.0 in DNA extracted from blood of patients and controls. Copy number was calculated based on probe hybridization signal intensity data relative to the signal of disease‐free normal controls. To determine CNV status on each sample we performed real‐time PCR. TaqManTM probes designed by the manufacturer (Applied Biosystems, Foster City, CA) were used to target each of the specific region. The RNase P was chosen as the reference gene. Deletion of 22q11.21 was seen in 8/60 (13.3%) cases. However, the deletion was seen in different patterns in those patients, shown by different starting and ending loci and involving genes. Parents of TOF probands with a disease‐associated CNV provided with ability to determine if any of the CNVs were inherited or de novo. The CNVs were de novo mutations for all these cases. Thus, 22q11.21 deletion is associated with sporadic tetralogy of Fallot in Han Chinese involving a large number of genes and the related individual genes warrant further detailed investigations.Supported by NSFC 1170148, 2010CB529500, 2012‐BH110004, 2011BHKZ001, 2012BWKZ 008, and CUHK4774/12, China.
Rectus capitis posterior minor and its relationship to the spinal dura mater: a histological investigation (913.3)
Authors: Venne Gabriel , Reifel Conrad , Pang Stephen , Ellis Randy 
Published: June 2021

The rectus capitis posterior minor (RCPm) is a deep suboccipital muscle connecting the tubercule of the posterior arch of the atlas to the inferior nuchal line of the occipital bone. Although controversial, a number of dissection‐based or imaging‐based reports suggested a direct connection between the RCPm and the dura mater. Detailed histologic investigation is required to establish any link between RCPm and the dura mater.Left and right RCPms, their bony attachments and adjacent spinal dura mater, were resected from 6 embalmed and 2 unfixed cadavers. Tissue samples were fixed with 4% paraformaldehyde in phosphate buffered saline and decalcified. Both left and right RCPms were subdivided into medial and lateral parts and processed for histological examination. Serial histological sections were prepared in order to include maximum surface area; Masson’s trichrome stain was used to visualize muscle and connective tissue connections.Histological analysis suggested an indirect link between RCPm and the dura mater. This investigation also found tissue distributions previously unreported, i.e.: periosteum of the occipital bone blends into the spinal dura mater on the medial aspect; tissue between the RCPm and dura mater has a structural organisation similar to a bursa or fat pad and this structure is reinforced by traversing collagen bundles taking origin at the peri‐ and/or endomysium of the RCPm.This histological investigation clarifies some anatomical features of the suboccipital region and indicates a secondary function of the RCPm, which may include posterior traction of the dura mater during neck hyperextension.Grant Funding Source: Natural Sciences and Engineering Research Council of Canada (NSERC)
EU‐OPENSCREEN: novel chemical tool compounds for molecular biologists (972.3)
Authors: Stechmann Bahne 
Published: June 2021

EU‐OPENSCREEN (www.eu‐openscreen.eu) is an academic European open‐access research infrastructure on the European roadmap with the aim to develop novel research ‘tool compounds’ (i.e. chemical inhibitors or activators of biological targets) for all areas of the Life Sciences (incl. molecular, cell, plant and microbiology; chemistry; pharmacology, early drug discovery etc.). EU‐OPENSCREEN supports all stages of a tool development project, incl. assay miniaturization, high‐throughput screening and chemical optimization of ‘hit’ compounds.EU‐OPENSCREEN offers to external researchers access to its shared resources, incl. the latest screening technologies, chemistry services and a unique compound collection. Applications of biologists are welcome who have developed a robust assay and are interested in collaboratively developing a chemical ‘tool compound’ for their target‐of‐interest. Selected assays are screened against 100,000‐200,000 compounds, incl. confirmatory and counter screening, IC/EC50 determination, SAR (structure‐activity relationships) and QC of confirmed hit compounds.Currently in its conceptualization phase, EU‐OPENSCREEN will start operations in late 2015, but it can already look back on a growing number of transnational activities: joint screening projects, exchange of local compound libraries and development of new design principles for its compound collection.
Affinity extraction of proteins from acidic environments (979.2)
Authors: Teichert Kristian , Stocks Bradley , Engen John 
Published: June 2021

Affinity extraction of proteins from acidic environments Affinity purification is widely used to isolate an analyte of interest from a complex mixture, such as a cell lysate. The process involves immobilization of one ligand followed by incubation with a sample containing the binding partner. While most affinity purifications are performed at near‐physiological pH, there are situations where performing affinity extraction at low pH would be advantageous, including isolating proteins from acidic environments such as the stomach or acidic hot springs. The affinity pairs most often used for protein purification (e.g., antibody:antigen, GST‐tags:glutathione or immobilized metals:6xHis tags) have limited or abolished binding at low pH. Here we employed a published double mutant of streptavidin which is known to form a tight complex with biotin at low pH. A vector designed to incorporate an N‐terminal biotinylation sequence onto a protein was used to direct in vivo biotinylation of a selected lysine residue in several target proteins. This strategy has advantages over non‐specific in vitro, post‐purification biotinylation wherein multiple labels are added randomly and thereby cause disruption of key protein‐protein interactions. We demonstrate the utility of the mutant streptavidin:biotin system for extracting proteins of interest by characterizing the binding kinetics of this affinity pair in various solutions, pH’s and temperatures. Support: NIH R01‐GM101135Grant Funding Source: Supported by NIH R01‐GM101135
Protein‐protein interactions between CIB1 and novel integrin binding partners: structural and thermodynamic basis of CIB1 promiscuity (979.5)
Authors: Freeman Thomas , Black Justin , Bray Holly , Dagliyan Onur , Wu Yi , Tripathy Ashutosh , Dokholyan Nikolay , Leisner Tina , Parise Leslie 
Published: June 2021

The cytoplasmic protein CIB1 (calcium‐and‐integrin‐binding protein 1) has been shown to promote cancer cell growth by affecting the RAS/RAF/MEK/ERK and the PI3K/AKT pathways, and was previously identified as an αIIb binding partner that inhibits agonist‐induced activation of the platelet‐specific integrin, αIIbβ3. To better understand the nature of the protein‐protein interactions between CIB1 and other proteins, we characterized the interaction between CIB1 and several α‐integrins. A sequence alignment of all α‐integrin CTs revealed that key residues in the CIB1 binding site on αIIb are well‐conserved, and was used to delineate a consensus binding site (I/L‐x‐x‐x‐L/M‐W/Y‐K‐x‐G‐F‐F). A hydrophobic binding pocket on CIB1 was shown to be the major binding site for multiple α‐integrins by all‐atom replica exchange discrete molecular dynamics, competitive binding assays, and mutational analysis. We also demonstrated that CIB1 can bind to other whole integrin complexes in vivo using co‐immunopreceipitation assays. Isothermal titration calorimetry measurements indicated that this binding is driven by hydrophobic interactions and depends on residues in the CIB1 consensus binding site. These new mechanistic details of CIB1‐integrin binding imply that CIB1 could bind to all integrin complexes and act as a broad regulator of integrin function. Moreover, this work provides new insight into the nature of the protein‐protein interactions between CIB1 and its binding partners.Grant Funding Source: Supported byNHLBI 5R01HL092544 and NIGMS 5K12GM000678
Systems‐level analysis of xylose fermentation by experimentally‐evolved Saccharomyces cerevisiae (981.4)
Authors: Sato Trey , Parreiras Lucas , Ong Irene , Avanasi Ragothaman , Zhang Yaoping , Higbee Alan , Keating David , Coon Joshua , Gasch Audrey , Landick Robert 
Published: June 2021

Experimental directed evolution and systems‐level analysis are powerful tools that can provide insights into the genetic regulation of biological and industrial processes. The fermentation of renewable plant biomass into cost‐effective biofuels is one such applied process. For the yeast biocatalyst, Saccharomyces cerevisiae, a major barrier to cellulosic ethanol production is its inability to rapidly ferment xylose from hemicellulose. To identify genetic factors regulating xylose fermentation, we performed directed evolution of an engineered S. cerevisiae strain on xylose and generated two evolved strains; GLBRCY127 was isolated by aerobic selection and a second was isolated from subsequent evolution of GLBRCY127 anaerobically. Metabolomic analyses between evolved and parent strains during the consumption of xylose from lab and cellulosic media suggested enhanced flux through the Pentose Phosphate Pathway and altered levels of TCA cycle intermediates. Genome sequencing and RNAseq analysis identified mutations in three genes that regulate mitochondrial function and signal transduction pathways. Importantly, deletion of these genes conferred xylose fermentation by the unevolved parental strain. This work identifies unexpected metabolic and physiological processes that limit cellulosic ethanol production and, when genetically modified, may help to realize a viable biofuels industry.Grant Funding Source: Supported by DOE BER Office of Science DE‐FC02‐07ER64494.
Quantitative lc‐ms/ms analysis of cytoplasmic bacterial cell wall biosynthesis intermediates: application to vancomycin resistance VRE (982.2)
Authors: Vemula Harika , Putty Sandeep , Bobba Sudheer , Gutheil William 
Published: June 2021

Vancomycin resistance in vancomycin‐resistant enterococci (VRE) is due to an alternative cell wall biosynthesis pathway in which D‐Ala‐D‐Ala is replaced, most commonly by D‐Ala‐D‐Lac. Methods for quantitative analysis of these intermediates has been lacking, which has impeded progress in characterizing the details of this resistance, and in developing new agent to counter this resistance.Methods: LC‐MS/MS assays for both the alanine branch and the UDP‐linked cytoplasmic peptidoglycan intermediates have been developed. Alanine branch intermediates used Marfey’s reagent derivatization to enable analysis, and analysis of the UDP‐linked intermediates used an ion‐pairing approach. For the development of the UDP‐linked intermediate assays, these intermediate were purified from VRE using preparative ion‐ pairing HPLC. To demonstrate this assay, VRE was grown to log phase and treated with increasing concentrations of vancomycin.Results: Analysis of alanine branch and UDP‐linked intermediates demonstrates the utility of LC‐MS/MS‐based methods for the study of the of the metabolite level changes in VRE in response to vancomycin.
Insulin signaling and copper homeostasis are functionally linked in 3T3‐L1 adipocytes (992.2)
Authors: Yang Haojun , Dhawan Neha , Ivy Kristin , Kaplan Jack , Lutsenko Svetlana 
Published: June 2021

Copper is an essential element for human growth and development. Abnormal copper metabolism is associated with severe and potentially lethal disorders. Previous studies of copper misbalance in the animal model of Wilson’s disease have shown that copper levels influence cholesterol and triglyceride content in tissues. To understand the mechanisms connecting copper and lipid metabolism, we characterized copper homeostasis in 3T3‐L1 adipocytes upon differentiation and insulin signaling. We show that during adipogenesis copper uptake is upregulated 2‐3 fold. Protein levels of the copper‐transporting ATP7A also increase, presumably to facilitate copper delivery to semicarbazide‐sensitive amine oxidase (SSAO), a copper‐containing enzyme that exerts an insulin‐like effect and is highly upregulated in differentiated adipocytes. Both copper depletion and copper elevation inhibit SSAO’s activity, suggesting a fine‐tuned regulation of this enzyme by the metal. In turn, SSAO’s substrates and product regulate ATP7A localization in cells triggering trafficking form the trans‐Golgi network to vesicles. In response to insulin, copper uptake remains unchanged, whereas ATP7A traffics to the plasma membrane and vesicles indicating insulin‐dependent redistribution of intracellular copper pools. Altogether, our data revealed a novel metabolic connection between copper homeostasis and insulin signaling in differentiated adipocytes.* Equal contributionGrant Funding Source: NIH R01 GM101502
The effect of toxic heavy metals, Hg and Cd on the mRNA expression levels of the novel gene CIA7 of Chlamydomonas reinhardtii (995.4)
Authors: Sanchez Alvaro , Ynalvez Ruby 
Published: June 2021

Heavy metal pollution of the environment is a growing concern to biological researchers due to its potentially harmful effects. Bioremediation of these toxic metals by species of algae has been researched as a possible alternative. Chlamydomonas reinhardtii contain a novel gene: CIA7of unclear function. Due to its possession of conserved cysteine amino‐acid residues, this gene is hypothesized to play a role in the binding and tolerance of heavy metals.This study aims to qualitatively assess the levels of mRNA expression obtained from two strains of C. reinhardtii upon exposure to sub‐lethal levels of Hg and Cd. The two strains referred to are a wild‐type (D66) and a mutant strain (cia7) of C. reinhardtii. The mutant was isolated from inorganic carbon acquisition mutants. Upregulation of the CIA7 gene following a toxic metal treatment will provide a basis for CIA7’s role in heavy metal tolerance.To perform the expression analysis of the CIA7 gene, D66 and cia7 algal strains were exposed to sub‐lethal concentrations of Hg and Cd in liquid media. A separate control treatment with no heavy metal content was also performed. After an exposure time of three hours, total RNA was extracted and purified. The entire RNA content of the algae was then converted into its complementary DNA (cDNA). Amplification of the CIA7 gene was performed using the polymerase chain reaction and the band intensities were compared using Quantity One analysis software. The comparison in expression levels between both algal strains and between metal treatments will help determine whether CIA7 has the potential for abatement of toxic heavy metals.Grant Funding Source: Supported by: Norman Hackerman Advanced Research Program
Mcm10 Interacts with Mrc1 and May Play a Possible Role in DNA Damage Checkpoint Pathway (LB123)
Authors: Hendrix Chance , Fultz Brandy , Das‐Bradoo Sapna 
Published: June 2021

To ensure genome integrity, cells must replicate with absolute fidelity. However, cells are constantly under stress that causes DNA damage or block replication. To circumvent these problems, cells have a quality control system termed DNA damage checkpoint. In humans, defects in this checkpoint cause genome instability and a strong predisposition to cancer. A recent study has identified minichromosome maintenance protein 10 (Mcm10) as one of the few replication proteins that prevent DNA damage. However, whether Mcm10 has an active role in preventing DNA damage is unknown. We have observed that Mcm10 and mediator of replication checkpoint 1 (Mrc1) interact in S. cerevisiae whole cell extracts. Moreover, a robust interaction was observed between Mcm10 and Mrc1 by yeast two‐hybrid assays. Our data suggests Mcm10 interacts through the conserved C‐terminus of Mrc1. Interestingly, it is the N‐terminus of Mcm10 that binds to Mrc1. We are currently conducting experiments to further elucidate this interaction. These studies promise to identify a novel role of Mcm10 in DNA damage response pathway and maintenance of genomic integrity.Grant Funding Source: Supported by the National Institute of General Medical Sciences of the National Institutes of Health through Grant Number 8P20GM103447 to SDB.