bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of Sucrose Synthase in the Metabolism of Indican
Authors: Inoue Shintaro , Morita Rihito , Minami Yoshiko 
Published: April 2020

Indigo,a blue dye, is obtained from the cultivated plant species Polygonum tinctorium. It is derived from indican (indoxyl β‐D‐glucoside), one of the secondary metabolites stored in the vacuole of the leaf cells. In the cells, indican is biosynthesized by the catalysis of indoxyl b‐D‐glucoside synthase (IGS) that uses indoxyl and UDP‐glucose as its substrates. Indoxyl is possibly synthesized from indole by the catalysis of a monooxygenase, such as cytochrome P450 (CYP), whereas, sucrose synthase (SUS) or UDP‐glucose pyrophosphatase catalyzes the synthesis of UDP‐glucose. In the metabolism of indican, except IGS, the related proteins have not been identified to date; therefore, this study aimed to identify the specific proteins involved in the metabolism of the compound indican. Indoxyl is usually a very unstable substrate and is easily oxidized to indigo; due to its instability, it must be allowed to complete its indican synthesis reaction as fast as possible. In the plant secondary metabolism, the ER membrane‐associated CYPs that hydroxylate metabolites are well known. Furthermore, these CYPs may function as a scaffold for a temporary complex, known as metabolon, on the ER membrane. We assumed that the IGS also associates with some ER membrane proteins, because a part of IGS localizes on the microsome. We then performed protein–protein interaction analysis (coimmunoprecipitation and pull‐down assay) and detected some candidate proteins. Some proteins including CYPs and SUS were identified by the MS/MS analysis. We then examined the involvement of SUS in the metabolism of indican by performing protein–protein interaction analysis and cell fractionation using recombinant SUS.The full‐length cDNA of SUS gene obtained using the RACE‐PCR encodes a soluble cytosol protein composed of 806 amino acids. The recombinant SUS expressed in Escherichia coli was used to perform the experiments, as shown below. Firstly, we examined the intracellular localization of SUS using the cytosol and microsomal fractions prepared from the crude extract of leaf cells. Similar to IGS, SUS was also detected in both the fractions. The interaction between IGS and SUS was examined by coimmunoprecipitation using specific antibodies. We found that both proteins interacted with one another, suggesting that SUS might be a supply source for UDP‐glucose in the reaction involving the synthesis of indican. We are going to further discuss the data required to elucidate the entire indican metabolic pathway and to confirm the presence of its metabolon.
Notch‐1 signaling through MCL‐1 modulates macrophage polarization and immune defense against Mycobacterium avium paratuberculosis infection in autoimmune disease
Authors: Keewan Esra’a , Naser Saleh A. 
Published: April 2020

We previously reported that IL‐6 plays a major role in the survival of Mycobacterium avium paratuberculosis (MAP) in infected macrophages. MAP has been associated with Crohn’s disease (CD) and Rheumatoid Arthritis (RA) pathogenesis. The role of Notch‐1 signaling in macrophages during intracellular infection such as MAP is not clearly understood. We hypothesis that IL‐6 expression in macrophages is induced by Notch‐1 signaling which inhibits apoptosis through Myeloid cell leukemia sequence‐1 (MCL‐1), which ultimately induce persistence of chronic infection. In this study, we measured the in vitro effect of MAP infection on Notch‐1 signaling and downstream influence on IL‐6 and MCL‐1. Specifically, we evaluated the role of Notch‐1 signaling in apoptosis and consequent MAP viability in infected macrophages and its effect on macrophage polarization and inflammatory response. Overall, the data showed that MAP infection upregulated Notch‐1, IL‐6, and MCL‐1 by (1.2, 1.8 and 4.2 fold expression, respectively) in activated THP‐1 compared to uninfected macrophages. Interestingly, treatment of MAP‐infected macrophages with IL‐6 showed upregulation of Notch‐1 and MCL‐1, which suggest a role for Notch‐1 and IL‐6 in the upregulation of MCL‐1 and possibly regulation of apoptosis in MAP infected macrophages. Targeting Notch signaling by a gamma‐secretase inhibitor resulted in decreased expression of IL‐6 and MCL‐1 (1.38 ± 0.32 and 0.63 ± 0.034, respectively) in infected macrophages compared to untreated macrophages (2.7 ± 0.25 and 2.0 ± 0.21, respectively). Moreover, gamma‐secretase inhibitor decreased MAP viability by 1.3 log CFU/ml. On the other hand, MAP modulates the macrophage polarization toward M1 macrophage polarization (M1/M2=2.20) compared uninfected macrophages (M1/M2=1.27), whereas the Notch inhibitor promotes M2 and diminish M1 macrophage polarization (M1/M2=0.76). Collectively, targeting Notch‐1 signaling will regulates the inflammatory response and enhances bacterial clearance in MAP infected macrophages. The study provides significant data that support the mechanistic role of Notch‐1 signaling in chronic infection in autoimmune disease.Support or Funding InformationThis work was funded, in part, by the Florida Legislative Grant.
Cutaneous microvessel circulation and vasomotion responses to local skin heating in women with HIV.
Authors: Wang Dan , Ma Yuchi , Guendsechadze Shirin Claudia Nouri , Yang Xiaoru , Sun Philena , Kalakurthy Samantha , Carter Gabriela H. , Kummar Princy , Kassaye Seble , Wilcox Christopher S. 
Published: April 2020

BackgroundPatients infected with the human immunodeficiency virus (HIV) who are well treated by ART still suffer from many complications of microvascular dysfunction. Our previous ex vivo study reported that subcutaneous microvessels from women living with HIV had severe endothelial dysfunction. However, it is unknown whether the microvascular defect is more widespread. We tested the hypothesis that microvascular endothelial, neural and myogenic functions are impaired in women living with HIV compared to control women (control).MethodsWomen with HIV (n = 35) were compared to matched controls (n = 24). HIV infection was virally suppressed on antiretroviral therapy and both groups were enrolled in the DC Women’s Interagency HIV Study (DC – WIHS). Cutaneous blood flow (CBF) was measured by laser Doppler flowmetry (LDF) during an increase of local skin temperature from 33°C to 42°C. Vasomotion responses were analyzed using a wavelet transform to calculate the power of the frequency intervals associated with endothelial (0.0095 ‐0.012Hz), neural (0.021‐0.05Hz) and myogenic (0.051 ‐0.15Hz) activities of the Laser‐Doppler signal. Data are present as mean ± SEM.ResultCBF was increased in both groups with LH at 42°C, but the increases of CBF to heat was lower in HIV infected women than controls (13.9 ± 1.1 vs 15.6 ± 1.2 fold, p<0.05). Vasomotion activities were similar between groups at baseline (33°C) and increased in both groups with LH at 42°C, but the increase was less in HIV compared to controls (13.9 ± 1.1 vs 15.6 ± 1.2 fold change, P <0.05). Moreover, compared to the control group, HIV had lower wavelet amplitude during LH in endothelial (100±3.8 vs 149± 15 PU/Hz/min, P<0.05), neural (92 ± 5 vs 132 ±15 PU/Hz/min, P<0.05) and myogenic (46±3 vs 60 ±7 PU/Hz/min, P=NS) frequency intervals.ConclusionWomen living with HIV have impaired microvascular dysfunction with a complex derangement of microvascular circulatory regulation involving the endothelial, neural and myogenic components. Thus, fully preventing microvascular disease in HIV will require correction of widespread microvascular defects.Support or Funding InformationNational Heart, Lung, and Blood Institute (NHLBI), R01 HL134511
Mitomycin C Induces Immunogenic Cell Death Biomarkers In Human Bladder Tumors
Authors: Sun Yajing , Lu Ze , Chao Fangching , Taylor John A. , Au Jessie L.-S. 
Published: April 2020

IntroductionStandard‐of‐care for non‐muscle invasive bladder cancer is complete transurethral tumor resection followed by intravesical therapy with BCG, mitomycin C (MMC), gemcitabine, or other agents. Tumor recurrence is common and occurs in 40% to 80% of patients, with 10–20% progressing to muscle‐invasive disease. Earlier studies have shown selected cytotoxic agents induce immunogenic cell death (ICD) that promotes tumor response to immunotherapy and thereby offers the opportunity of developing synergistic combinations of ICD‐inducing cytotoxics with immune checkpoint blockers. The current qualifications for an ICD‐inducing cytotoxic include induction of DAMPs (danger‐associated molecular patterns) in vitro and vaccination with cytotoxic‐treated tumor cells in immunocompetent mice. Under these criteria, MMC is considered a poor ICD inducer. The present study tested the hypothesis that MMC can induce ICD in a more clinically relevant model, i.e., 3D histocultures of bladder tumors obtained from patients.MethodsWe evaluated the ability of MMC to induce two ICD biomarkers (i.e., ecto‐CRT (calreticulin) and release of HMGB1 from nucleus) and/or dendritic cell (DC) maturation in 3 bladder cancer cell lines with different differentiation status (RT4, T24, and TCCSUP), and 21 MMC‐treated human bladder tumor histocultures including 11 archived paraffin‐embedded tumors and 10 freshly obtained surgical tumor specimens. The methods included immunocytochemical staining flow/immune‐cytometry, immunoblotting, and immunohistochemical staining and quantitative imaging. Co‐cultures of human peripheral blood DC with MMC‐treated tumor cells (48 h) were used to detect DC maturation markers (CD83 and HLA‐DR).ResultsOver 85% of human bladder tumors (7/10 fresh tumors and 11/11 archived tumors) showed ecto‐CRT and/or HMGB1 release upon treatments with MMC at clinically achievable concentrations (1 and 3 μM). Induction of ICD markers by MMC was time‐dependent (peaked at 18 h) and increased with tumor grade (TCCSUP (grade 4) >T24 (grade 3)>RT4 (grade 1) (3 experiments, P<0.01, 2‐way ANOVA). HMGB1 release was increased in three cell lines with a dose‐dependent manner after 48 h MMC treatment (2 experiments, p value<0.05, 2‐way ANOVA). MMC further induced DC maturation in TCCSUP cells but not RT4 cells.ConclusionOur results indicate MMC induced ICD biomarkers and/or DC maturation in cultured human tumor cells and histocultures of human bladder tumors; the MMC effects were dependent on time, tumor grade and culture conditions.Support or Funding InformationSupported in part by Mosier Endowed Chair in Pharmaceutical Sciences at University of Oklahoma, Albert Charitable Trust, 1R01CA163015 from National Cancer Institute, and University of Kansas Cancer Center P30‐CA168524‐03.
Expression, purification and initial characterization of LysE membrane exporter from Mycobacterium tuberculosis: Towards comprehensive functional and structural study
Authors: Georgieva Elka R. , Fanouraki Christina , Borbat Peter P. 
Published: April 2020

Mycobacterium tuberculosis (Mtb) causes the very severe disease tuberculosis. To fight Mtb infection, detailed understanding of the pathogen molecular mechanisms in the host is needed. To this end, comprehensive knowledge about Mtb membrane transport system, which is currently not well researched, is a high priority. Our focus is on the Mtb‐encoded L‐lysine exporter (LysE), which removes the excess of metabolically produced basic amino acids from the bacterial cytosol. LysE is a target for inhibition, since its deficiency leads to toxic levels of L‐lysine and suppression of bacterial growth. The most studied H37Rv strain of Mtb encodes two LysEs, but one of then, the Rv1986 protein, is considered to be the primary exporter. Also, Rv1986 expression is increased during the early stages of Mtb infection under conditions of hypoxia, thus, the protein could be a vaccine target. Therefore, we selected to study this protein. To do so, we developed custom protocols for its cloning, expression, purification, and initial characterization. To overcome the low expression levels in E. coli, we fused LysE at its C‐terminus to a combination of FLAG and 8xHistidine tags. Through these tags, we employed a dual‐affinity purification. We purified the protein in lipodiscs made of native E. coli membranes and in detergent. We obtained highly pure protein as confirmed by SDS‐PAGE and Western blotting. Both techniques visualized multiple bands at LysE suggesting protein oligomerization. We further created and purified a single cysteine mutant N175C for spin labeling. Pulse EPR study on it also points to multimeric protein organization. These studies as well as electron microscopy on LysE‐nanogold particle complexes are ongoing. To the best of our knowledge, this is the first highly‐pure LysE produced, which will facilitate further thorough investigations of the functional mechanisms of this protein.Support or Funding InformationNIAID R03 AI137735
Getting the message: expansion of a bacterial three‐hybrid assay to mRNA‐protein interactions
Authors: Berry Katherine E. , LeBlanc Hannah , Nguyen Linh 
Published: April 2020

Non‐coding small RNAs (sRNAs) contribute to bacterial biofilm formation and virulence, but there is much we do not understand about their molecular mechanisms. Hfq is the best‐studied bacterial RNA chaperone protein and has become the paradigmatic example for how RNA‐binding proteins facilitate mRNA regulation by sRNAs. Here we present progress on establishing and broadening the capabilities of a recently developed bacterial three‐hybrid (B3H) assay for studying interactions between Hfq and its RNA targets. While Hfq interactions with both mRNAs and sRNAs are critical for its function, only sRNA‐Hfq interactions have been accessible in the B3H assay to date. In order to expand the assay to mRNA‐Hfq interactions, we first demonstrated the assay’s capacity to detect interactions between synthetic mRNA mimics and Escherichia coli Hfq. Furthermore, we have developed constructs that support detection of interactions between mRNA 5′UTRs and two E. coli RNA‐chaperone proteins: Hfq and ProQ. This development points to the potential of the B3H assay to detect a wider variety of RNA‐protein interactions than previously possible, and will allow us to dissect the molecular mechanisms of these interactions. Current efforts aim to understand the relationship between the translational status of an mRNA fragment and its ability to bind protein partners in the B3H assay. In addition, we are working to understand the relationship between the in vivo B3H data on RNA‐protein interactions and the binding energetics of these interactions, which are typically measured in vitro. The results of these efforts will provide a deeper understanding of the implications of B3H data for RNA‐protein binding and regulatory mechanisms. By expanding both the capabilities of this bacterial three‐hybrid assay and our understanding of the data it provides, we aim to broaden its utility in studying bacterial gene regulation and RNA‐protein interactions.Support or Funding InformationThis work was supported by National Institutes of Health [R15GM135878], Mount Holyoke College and the Henry Luce foundation.
KMT2D Haploinsufficiency in Kabuki Syndrome Impairs Differentiation of Neural Crest Cells
Authors: Lavery William J. , Kotliar Michael , Chang Ching F. , Brugmann Samantha A. , Barski Artem , Lindsley Andrew W. 
Published: April 2020

ObjectiveKabuki Syndrome (KS), caused by haploinsufficiency in lysine methyltransferase KMT2D, is a multi‐system disease characterized by distinct facial dysmorphisms, mild‐to‐moderate intellectual disability, renal and cardiac abnormalities, short stature and humoral immune deficiency. Many of the tissues in which these abnormalities manifest are derived from or developmentally modulated by the neural crest cell (NCC) lineage. This study’s objective was to determine which features of the KS phenotype can be directly linked to genetic and epigenetic dysregulation in KMT2D haploinsufficient NCCs.MethodsPeripheral blood mononuclear cells from a human donor haploinsufficient for KMT2D (KS patient, KMT2D+/−) or wild type (WT) control were used to generate induced pluripotent stem (iPS) cells. WT and KS NCCs were generated from WT and KS iPS cells, respectively. Whole transcriptome RNA sequencing (RNAseq) was performed on iPS cells (WT and KS) and NCCs (WT and KS) to characterize differential gene expression. Chromatin immunoprecipitation sequencing (ChIPseq) of histone 3 lysine 4 trimethylation (H3K4Me3) and histone 3 lysine 27 acetylation (H3K27Ac) was performed on NCCs (WT and KS) to characterize differential histone modifications at gene regulatory elements. Analysis of RNAseq and ChIPseq data was performed using BioWardrobe platform.ResultsGene set enrichment analysis of transcriptome data indicates that, compared to WT NCCs, KMT2D haploinsufficient NCCs have ectopic expression of several genes critical to normal NCC organization, morphogenesis and tissue differentiation, including NCC transcriptional repressor Msh homeobox 2 (MSX2). Analysis also identified several genes with decreased expression in KMT2D+/− NCCs, including transcription factor AP‐2 alpha (TFAP2A), which serves as a master regulator of NCC lineage progression. ChIPseq data indicate differential enrichment of H3K4Me3 and H3K27Ac histone modifications at the TFAP2A locus in WT compared to KS NCCs, which suggests that TFAP2A may have a direct role in the impaired differentiation of NCCs in KS. Further studies to explore the roles of TFAP2A and other genes identified by these data are underway.ConclusionThese results show that KMT2D haploinsufficiency impairs differentiation of the NCC lineage cells through epigenetic and transcriptomic dysregulation, thus supporting the classification of KS as a neurocristopathy.Support or Funding InformationNIH T32 Training Grant 5T32AI060515‐14 (WJL), R35 DE027557 (SB), Center for Pediatric Genomics Cincinnati Research Foundation (AWL).
Withdrawn: A Controllable Protein Translation Strategy for Exploring Cellular Signal Transduction Based on Reading through Premature Termination Codons
Authors: Cheng Xiyao , Huang Yongqi , Su Zhengding 
Published: April 2020

Withdrawal: Xiyao Cheng, Yongqi Huang, and Zhengding Su. A Controllable Protein Translation Strategy for Exploring Cellular Signal Transduction Based on Reading through Premature Termination Codons. The FASEB Journal. 34:s1. doi: 10.1096/fasebj.2020.34.s1.09195.The above abstract, published online on April 19, 2020, in Wiley Online Library (wileyonlinelibrary.com), has been withdrawn by agreement between the authors, FASEB, and Wiley Periodicals, Inc. The abstract has been withdrawn at the request of the authors.
Characterizing the role of an α1,6‐mannanase needed for Neurospora crassa cell wall biogenesis
Authors: Patel Pavan , Free Stephen 
Published: April 2020

Cell wall components such as glucans, chitins and mannans are needed for fungal virulence. The dfg‐5 gene encodes a GH‐76 α‐ 1,6 mannanase that is needed to cleave the α‐1,6‐mannose backbone of fungal N‐linked oligosaccharide‐associated galactomannans. The processed galactomannan is then used as substrate by GH‐72 lichenin transferases, that cross‐link it into the cell wall. However the mechanism of how DFG‐5 processes the galactomannan structure remains unclear. Here, we characterize the DFG‐5 protein. A pair of adjacent aspartic acid (D) or glutamic acid (E) residues are normally found at the active sites of GH‐76 family glycosylhydrolases. We used site directed mutagenesis to mutate 7 conserved aspartate and glutamate residues in DFG‐5 protein. Complementation analysis demonstrates that D76, D116, D117, and E130 mutations renders DFG‐5 inactive. 3D Modeling of DFG5 protein strongly suggests that D116 and D117 define the active site based on homology with other characterized glycosylhydrolases. Modeling of DFG‐5 mannanase shows it has a second groove containing D76 and E130. To further characterize DFG‐5, we generated a HIS6 tagged version of the protein and demonstrated that it complemented a dfg‐5 gene deletion. A number of glycoproteins co‐purify with the HIS6‐tagged DFG‐5 and these co‐purifying glycoproteins require galactomannan for DFG‐5 binding. Three known cell wall glycoproteins co‐purify with DFG‐5 mannanse.Support or Funding InformationFunding for the study is provided by the UB Foundation.
When the Efferent Becomes Afferent ‐ the Cellular Response of Sympathetic Neurons to Sensory Stimuli
Authors: Davis Harvey , Musa Ayesha , Li Dan , Herring Neil , Paterson David 
Published: April 2020

Responding to the so called “inflammatory soup” has often been regarded as the sole domain of sensory afferent neurons. We tested the hypothesis that efferent sympathetic neurons can also directly respond to traditionally afferent stimuli. We used single cell RNA‐sequencing (scRNAseq) to explore transcript expression of membrane G protein and ion channel coupled receptors in the sympathetic neuronal population within the stellate ganglia of 5–6‐week‐old Wistar rats. Agonist responses were then evaluated using Fura‐2 AM calcium imaging and perforated patch clamp recordings in isolated cultured neurons.ScRNAseq identified transcript expression of transient receptor potential (TRP) channels (TRPV2>TRM7>TRPC1>>TRM4). Certain TRP channel activators (10 mM camphor n=18, 10 mM cannabidiol n=16, and 100 mM 2‐APB n=15) caused large baseline calcium influxes, which were blocked by the TRP channel inhibitor ruthenium red (RuR, 10 mM; Camphor n=7; Cannabidiol n=24; 2‐APB n=11). An accompanying depolarization of the resting membrane potential and spontaneous firing was also observed, which was reduced by RuR. Other TRP channel activators (100 mM menthol n=15, 1 mM capsaicin n=16, 500 mM AITC n=9, 1 mM vanillin n=15, 300 nM JT010 n=15, 100 mM cavracol n=13, 10 mM icillin n=10, 50 mM naltriben n=15, pH=6.5 n=16 and 100 ng/ml IGF‐1 n=12) failed to produce significant responses.ScRNAseq also identified transcript expression of receptors for inflammatory mediators, for example histamine (HRH3) and prostaglandin E2 (PTGER2 and PTGER3). Stimulation of these receptors (100 mM histamine n=11 and 1 mM prostaglandin E2 n=8) had no effect on baseline [Ca2+]I, but were able to reduce Ca2+ influx in response to KCl depolarization (50 mM) or nicotine (100 mM).Overall, these data are important when interpreting stellate ganglia sympathetic neurons as purely efferent, with evidence that neuronal excitability can be directly influenced by sensory signaling pathways.Support or Funding InformationThis project was supported by the Wellcome trust and the British Heart Foundation