bluuebunny/crossref_metadata_2025_split huggingface dataset:
Microbiome and Respiratory Diseases: A Study Using a Novel Non‐invasive Saliva Sample Collection Method
Authors: Suarez-Martinez Edu B. , Rivera-Mariani Felix , Ocasio Marinel , Arroyo Gerardo 
Published: April 2020

Microbiome studies are contributing to decipher the role of the microbiota in a spectrum of human disorders. However, current sample collection methods often limit the feasibility of Bacteriome approaches to study respiratory diseases such as allergic rhinitis and asthma. Most are invasive such as bronco‐alveolar lavage, lung brushing, and lung biopsy, and other as the nasopharyngeal swabbing causes inflammatory reactions. Non‐invasive methods, such as induced sputum sampling, had shown comparable results of representative microbiota to those with lung brushing. However, most population‐based studies used the non‐invasive oral washing method (OWM). Studies showed that OMW is the least comparable to any invasive sampling. Usually, oral wash bacteriome outcomes show non‐informative respiratory disease‐related data because the identified taxa are mostly oral, periodontal, and nasopharyngeal commensals or area‐specific pathological microbiota. The objective of this study was to evaluate the salivary Bacteriome in participants with and without allergic rhinitis using a novel saliva collection method. This method considers the contribution of the mucociliary clearance (MCC) process as part of a comprehensive bacteria‐representative model to study respiratory tract diseases. Protocols were approved by the Ponce Health Sciences University IRB #171205‐ES. As part of the method, participants washed their mouths at night. Then, the following morning before mouth washing, they collected the saliva from deep in the throat and inner mouth. DNA from saliva samples was isolated, and 16S amplicons of 300bp were sequenced using the MiSeq platform. Analyses for allergic rhinitis and microbiome associations included Operational Taxonomic Units (OTUs), Chao1, Shannon, Simpson, Weighted Unifrac, and NMDS PERMANOVA. We identified seven bacterial taxonomic levels, including for species. The alfa diversity showed differences in allergic and non‐allergic participants employing OTUs and Shannon using the Mann‐Whitney U test (p< 0.05). More importantly, NMDS PERMANOVA effectively categorized allergic and non‐allergic individuals (p< 0.05). We identified bacteria taxa not previously reported using the OMW. Based on these results, we concluded that the novel non‐invasive saliva collection method provided robustly, representative, and informative bacteriome results that can be adopted to study other respiratory diseases such as asthma and those with MCC‐related illnesses.Support or Funding InformationNIH‐NIGMS #2R25GM096955;Puerto Rico Science Technology and Research Trust 2017‐00173
Programming of a Pro‐Adipogenic Phenotype in Offspring Born to Metabolically Compromised Pregnancies
Authors: Mikolajczak Anna , Sallam Nada , John Cini , Singh Radha , Easson Sarah , Thompson Jennifer 
Published: April 2020

ObjectivesDetermine the role of adipose tissue dysfunction in programming of cardiovascular disease risk in offspring born to dams with metabolic dysfunction.MethodsThe female mouse heterozygous for leptin receptor deficiency (Hetdb) was used as a model of maternal metabolic dysfunction. These females exhibit higher adiposity, dyslipidemia and hyperinsulinemia during pregnancy. Our data show that this model reproduces programming of early‐onset cardiovascular disease risk reported in human offspring born to obese or diabetic mothers. We sought to test the hypothesis that programming of cardiovascular disease risk is attributable to perturbed adipose tissue development leading to later‐life adipose tissue dysfunction. Wild type (Wt) offspring from both Wt and Hetdb pregnancies were studied as neonates and adults. At 7 weeks of age, offspring were placed on either a control or high fat/sugar (HF/HS) diet. Adipogenic potential was examined in preadipocytes isolated from the stromal vascular fraction (SVF) of inguinal subcutaneous adipose tissue (iSAT) in both neonatal and adult Wt offspring. After induction of differentiation, Oil Red O staining was used to measure lipid droplet accumulation and qPCR was used to measure adipogenic markers. Flow cytometry was utilized to quantify adipogenic progenitors in SVF. Adipokine secretion was measured with ELISA. Several indices of adipose tissue function were measured in adult offspring, including adipose tissue insulin resistance (Adipo‐IR).ResultsNMR whole‐body fat mass was 1.3 fold higher (p < 0.01) and plasma resistin levels were 1.7 fold higher (p < 0.01) in Wt neonates born to Hetdb pregnancies. The iSAT of Wt neonates and adults born to Hetdb vs. Wt dams exhibited a shift in the cell size distribution from smaller adipocytes to larger adipocytes (p < 0.05), indicating that adipogenesis was accelerated in the iSAT of Hetdb neonates. The percentage of CD31‐CD45‐CD29+CD34+ viable SVF cells was higher in neonates from the Hetdb vs. Wt pregnancy. Preadipocytes isolated from Hetdb offspring exhibited a pro‐adipogenic phenotype in early life that persisted into adulthood. After HF/HS feeding, preadipocytes isolated from Wt female (p < 0.05) and male (p < 0.05) adult offspring born to Hetdb pregnancies accumulated more lipid during differentiation. Heightened adipogenesis in adult offspring was also accompanied by impaired insulin‐stimulated inhibition of lipolysis.ConclusionsAdipose tissue dysfunction in offspring born to metabolically adverse pregnancies stems from in utero programming of a pro‐adipogenic phenotype in preadipocytes.Support or Funding InformationCanadian Institutes of Health Research, Alberta Children’s Hospital Research Institute, Heart & Stroke Foundation of Canada and Libin Cardiovascular Institute of Alberta
Endothelin Receptor A and B Co‐operate to Mediate GPER‐induced Natriuresis
Authors: Gohar Eman Y. , Almultaq Rawan , Pollock David M. 
Published: April 2020

G protein‐coupled estrogen receptors (GPER) are membrane‐associated receptors that mediate rapid estrogenic signaling. We recently found that activation of GPER in the renal medulla of female rats, but not males, evokes endothelin‐dependent natriuresis. Endothelin‐1 (ET‐1) inhibits tubular reabsorption of Na+ via stimulating ET receptor B (ETB). Evidence points that ET receptor A (ETA) may contribute to natriuresis in female rats only. The current study was designed to identify whether GPER‐induced natriuresis is mediated via activation of renal medullary ETA and/or ETBreceptors in female rats. We determined the effect of stimulating GPER in the renal medulla on urine flow and urinary Na+ excretion in anesthetized female ETB deficient rats (ETB def) and transgenic (TG) controls. We infused saline to the renal medullary interstitium during an equilibration period of 60–80 min at a flow rate of 500 μl/h, followed by vehicle, then the GPER selective agonist, G1 (5 pmol/kg/min). Infusion of G1 to the renal medulla increased urine flow and Na+excretion in TG control animals (from 4.77 ± 1.19 to 7.36 ± 2.41 μl/min and from 0.47 ± 0.09 to 0.91 ± 0.27 μmol/min, respectively, n=7, p<0.05). Similarly, medullary GPER activation evoked a diuretic and natriuretic response in ETB def rats (urine flow: from 4.07 ± 0.47 to 6.52 ± 1.01 μl/min and Na+ excretion: from 0.29 ± 0.06 to 0.64 ± 0.16 μmol/min, respectively, n=8, p<0.05). G1 infusion did not change blood pressure or urinary K+ excretion in ETB def or TG control animals. Mean arterial blood pressure was higher in ETB def rats, compared to TG controls (117 ± 6 vs. 99 ± 3 mmHg, n=7–8, p<0.05). We also determined the physiological response to medullary GPER activation in anesthetized female Sprague Dawley rats after individual blockade of ETA or ETB receptors, achieved by an intravenous bolus injection of ABT‐627 (5mg/kg) or A‐192621 (10 mg/kg), respectively, or vehicle. Neither selective antagonism of ETA nor ETB antagonism impacted the diuretic or natriuretic actions of G1. However, simultaneous blockade of ETA and ETB receptors abolished G1‐evoked diuresis and natriuresis. Blood pressure and urinary K+ excretion remained unchanged during G1 infusion in the presence or absence of ET receptor antagonists. Thus, using pharmacological and genetic approaches, our study reveals that ETA and ETB receptors in the renal medulla work co‐operatively to mediate the diuretic and natriuretic response to GPER activation.Support or Funding InformationFunded by AHA 18CDA34110010 and K99DK119413 to EYG and P01 HL136267 to DMP.
Mid‐cervical spinal contusion injury with compression causes extensive neurodegeneration in medullary and spinal neural networks
Authors: Seven Yasin Baris , Allen Latoya L. , Zwick Amanda , Mitchell Gordon S. 
Published: April 2020

Cervical spinal cord injury (cSCI) is a multifaceted disorder which disrupts respiratory neural pathways and causes neurotoxicity, tissue ischemia, inflammation and extensive neurodegeneration, which may impair breathing capacity and airway defense (cough). Neurodegeneration after cSCI extends beyond the injury epicenter (where the most severe effects are observed), affecting multiple neuromotor networks. Following the initial trauma, a suite of sub‐acute mechanisms contributes to secondary degeneration, including: ischemia, excitotoxicity and inflammation. These features activate both neurodegenerative and compensatory signaling cascades. Most contusion models of cSCI utilize a single impact with a specified magnitude to the exposed cervical spinal cord. However, compression from fractured vertebrae, edema and other factors associated with the initial impact can subsequently impair local circulation. Thus, it is important to model cSCI with experimental injuries incorporating both impact and compression. Here, we determined the extent of neurodegeneration in the medulla and cervical spinal cord following cSCI incorporating both impact and compression. We hypothesized that cervical spinal contusion with compression (cSCC) injury causes extensive neurodegeneration in the medulla and cervical spinal cord, far from the site of impact. cSCC was performed at C4 by adding brief compression with dwell time following the initial impact. We demonstrate: 1) high mortality (~50% compared to ~15% without compression); 2) more severe limb paralysis; 3) delays in regaining of the righting reflex; and 4) ventilatory deficits when challenged with 7% CO2 + 10.5% O2. cSCC injury at C4 causes profound neurodegeneration extending rostral to the brainstem visualized as reduced medullary NeuN immunoreactivity in the sub‐acute time‐points (~4h), degenerating neural tracts rostral and caudal to injury, abnormal interneuron shape and nucleus location rostral to the injury, and neuronal loss caudal to injury. In addition, cSCC induces neurodegeneration in descending raphe serotonergic axons, undermining the capacity for serotonergic plasticity. These data support the idea that spinal cord injury extends beyond the injury epicenter highlighting potential therapeutic targets at different locations of the CNS following cSCI.Support or Funding InformationNIH R01 HL147554 and HL69064, NIH SPARC (OT2OD023854), NIH T32 HD043730 (LLA) and the UF McKnight Brain Institute.
Mapping the Minimal Promoter Sequence for Human Prolidase (Peptidase D; PEPD‐ gene)
Authors: Ismail Farah Adel , Aganga Ireti Eni , Lanaghan Zeljka Adel Miletic , Balasubramanian Muthukumar Eni , Dash Chandravanu Eni , Pandhare Jui Eni 
Published: April 2020

Collagen, the most abundant protein in humans, is the primary component of the extracellular matrix. Collagen is also a critical reservoir of proline that, along with hydroxyproline, comprise one‐fourth of the amino acid composition of collagen. During collagen turnover, sequential action of matrix metalloproteinases and peptidases convert collagen into dipeptides. In the subsequent final rate‐limiting step, prolidase (PEPD) hydrolyzes dipeptides containing C‐terminal proline or hydroxyproline into the constituent amino acids, which are then recycled towards collagen synthesis. Due to its critical role in collagen turnover and protein metabolism, prolidase is a key player in several physiological and pathological processes, including wound healing, cell proliferation, inflammation, and carcinogenesis. Consequently, alterations in prolidase expression can profoundly affect normal physiological processes and cause pathological changes. The goal of this study is to narrow down such knowledge gaps, especially, in our understanding of the regulation of PEPD transcription. First, towards determining a functionally active promoter region of PEPD (PEPDp), we cloned a 1587 base pair (bp) DNA region—spanning 1537 bp upstream, and 50 bp downstream, of the transcription start site (TSS)—of PEPD gene into a firefly luciferase reporter plasmid. We confirmed that the PEPDp is functionally active by demonstrating its ability to robustly drive the luciferase expression in the HEK293T cell line. Second, we sought to determine the minimal region of PEPDp that is sufficient and necessary to drive gene expression. We subcloned two progressively shorter deletion fragments of PEPDp—537 bp and 387 bp long into the firefly luciferase reporter system and then compared their promoter activity with that of the full‐length PEPDp. Our findings thus indicate that the lack of any cancellation in the promoter activity of the PEPDp deletion fragments points out that the functionally active region of PEPDp spans ~350 bp upstream and 50 bp downstream of the transcriptional start site (TSS), thereby advancing our knowledge of the molecular mechanisms regulating prolidase expression.Support or Funding InformationThis work is partly supported by National Institutes of Health (NIH) Grants DA037779 and MD007586 (to Jui Pandhare) and DA024558, DA30896, DA033892, DA021471, AI22960 and MD007586 (to Chandravanu Dash). Ireti Eni‐aganga is supported by RISE Grant. The work is also supported by the RCMI Grant G12MD007586, the Vanderbilt CTSA Grant UL1RR024975, the Meharry Translational Research Center (MeTRC) CTSA grant (U54 RR026140 from NCRR/NIH, the U54 Grant MD007593 from NIMHD/NIH, and the Tennessee Center for AIDS Research (P30 AI110527).
Modelling a Novel Superoxide Dismutase 1 Mutation in Motor Neurons: An Insight into Molecular Pathomechanism of Amyotrophic Lateral Sclerosis
Authors: Verma Sagar , Vats Abhishek , Gourie-Devi Mandaville , Khurana Shiffali , Ganguly Nirmal Kumar , Chakraborti Pradip , Taneja Vibha 
Published: April 2020

Amyotrophic lateral sclerosis (ALS) is a fatal, adult onset neurological disorder characterized by progressive degeneration of upper and lower motor neurons. ALS is a multifactorial disease with a complex etiology and misfolded protein aggregates are considered as the major neuropathological hallmark. Several genetic mutations are associated with familial and sporadic forms of ALS. We have recently reported the first mutation in Superoxide Dismutase 1 (SOD1) in a family with ALS from India. The purpose of this study is to explore the functional consequences of L84F SOD1 mutation using in‐vitro cell model. We observed that this novel triallelic mutation (L84F) caused metal chelation and destabilized the native SOD1 dimer. Cells expressing mutant SOD1 showed increased protein carbonylation and were more sensitive to oxidative stress. Further, L84F SOD1 mutant readily formed detergent‐resistant aggregates in motor neuronal cell line. Modulating protein homeostasis pathways led to enhanced aggregation of mutant SOD1. Interestingly, the cells expressing mutant SOD1 exhibited significant decrease in neuronal differentiation compared to wild type. Our data shows that altered differentiation in mutant SOD1 cells is probably due to hampered autophagy and aberrant deposition of misfolded protein. We also observed that treatment with flavonoids including curcumin, luteolin abolished mutant SOD1 aggregates by inducing autophagy.ALS pathology also involves disassembly of neuromuscular junction (NMJ) which leads to muscle atrophy. However, the series of events that leads to skeletal muscle denervation is poorly understood. To examine the effect of mutant SOD1 on NMJ, we have established an in‐vitro NMJ model by co‐culturing skeletal muscle and motor neuronal cells. We are currently using this model to delineate the neuron‐ and muscle‐ specific cues that affects the structural and functional integrity of NMJ. To conclude, L84F SOD1 mutant exhibits characteristics of ALS such as aggregation of misfolded protein and deregulated proteasome machinery. Further targeting protein homeostasis pathways may aid in developing novel therapeutics for ALS.Support or Funding InformationAuthors acknowledge Indian Council of Medical Research (ICMR) (Project Code: 1.3.2.183.025) and Research Development Program (RDP) at Sir Ganga Ram Hospital for funding the research project. SV acknowledges Council of Scientific and Industrial Research (CSIR) and AV, SK acknowledges Indian Council of Medical Research (ICMR) for fellowships.
Regulation of Extracellular Matrix Gene Expression by Desmosomal Cadherins
Authors: Alexander Hunter , Conway Daniel , Dubash Adi D. 
Published: April 2020

Desmosomes are protein complexes crucial for maintaining cell‐cell adhesion and integrity of tissues. These complexes are made up of proteins from three families: transmembranecadherins(Desmoglein and Desmocollin) linkadjacentcellsin the extracellular space, armadilloproteins (Plakophilin and Plakoglobin)stabilizethe intracellular plaque, and the cytolinker Desmoplakin (DP) connects the plaque to the intermediate filament network. Desmosomal proteins have also been shown to coordinate gene expression pathways required for processes such as proliferation, differentiation and cell migration. In particular, several lines of evidence have linked desmosomal proteins to gene expression of extracellular matrix (ECM) proteins. Loss of Plakophilin‐2 or Desmoplakin causes increases in expression of fibronectin and collagen, while in contrast, loss of plakoglobin results in a significant decrease in expression of fibronectin. These data indicate that individual components of the desmosomal complex control ECM gene expression via distinct cellular signaling networks. In our study, we sought to investigate the role of desmosomal cadherins in ECM gene expression, via use of A431 cells lacking either Desmoglein‐2 or Desmocollin‐2, generated via CRISPR‐mediated knock‐out (Dsg2 KO and Dsc2 KO, kindly provided by Dr. Daniel Conway, VCU). Compared to control cells, Dsg2 KO cells demonstrated a dramatic (~10‐fold) increase in expression of Fibronectin (Fn1), and relatively minor changes in expression of Collagen 1 (Col1a1) and Collagen 2 (Col2a1). In contrast, no major changes in expression of Fn1 was observed in Dsc2 KO cells. We show that loss of Dsg2 KO (but not Dsc2 KO) also caused a significant increase in expression of the pro‐fibrotic signaling molecule transforming growth factor beta 2 (Tgfb2), but not Tgfb1 or Tgfb3. Increased expression of Fn1, Col1a1, Col2a1 and Tgfb2 was also observed upon siRNA‐mediated knockdown of Dsg2 in A431 cells, verifying that these changes are not clone‐specific or due to off‐target CRISPR effects. While expression of other desmosomal proteins is unchanged in Dsg2 KO cells (mRNA and protein levels), the integrity of the junctional complex was expectedly perturbed, as observed via a significant increase in Triton‐solubility of Plakophilin‐2, Plakophilin‐3, Plakoglobin and Desmoplakin. Nevertheless, siRNA‐mediated knockdown of these proteins could not rescue Fn1 or Tgfb2 increases in Dsg2 KO cells, indicating that changes are not due to mislocalization of other desmosomal components. Taken together, our study highlights a novel role for Dsg2 in mediating ECM gene expression, adding significant insight into the mechanisms by which desmosomal cadherins control the adhesive behavior of cancer cells.Support or Funding InformationNIH INBRE Developmental Research Grant
The MAL/SRF Pathway Regulates Desmosome Protein Expression and Localization in Cancer Cells
Authors: Godwin Ashley , Eldredge Luke , Dubash Adi D. 
Published: April 2020

The desmosome is a cell‐cell adhesion complex which facilitates the mechanical stability of tissues and cell‐cell communication. Desmosome function depends upon a tripartite organizational structure wherein transmembrane cadherins (Desmoglein and Desmocollin) link adjacent cells in the extracellular space, armadillo proteins (Plakophilin and Plakoglobin) stabilizethe intracellular plaque, and the cytolinker Desmoplakin (DP) connects the plaque to the intermediate filament network. In addition to their central role in maintaining cell‐cell junction integrity, desmosomal cadherins also coordinate biological processes such as proliferation, apoptosis, differentiation and cell migration. Many studies have reported diverse mechanisms by which cancer progression alters DSM gene expression, but the signaling pathways that govern the expression and localization of DSM constituent proteins remain elusive. Recent work has identified the transcription factor serum response factor (SRF) as a regulator of mRNA levels and localization of DSM proteins such as the cadherin Desmoglein‐1. In our study, we treated several cancer cell lines with siRNA to SRF and its cofactor, MAL, to broadly investigate the role of these transcription factors in regulating DSM gene expression and protein localization. Our results demonstrate that abrogation of MAL/SRF signaling through RNAi results in a decrease in localization of DP to cell‐cell borders, along with perturbation of other desmosomal and adherens junction markers. Importantly, we also demonstrate that MAL/SRF abrogation causes a reduction in mRNA levels (by quantitative PCR) and protein levels (by western blot) of DP, indicating the importance of these transcription factors for controlling the expression of DP. Our data therefore highlight a novel link between MAL/SRF signaling and DP expression, and contribute to a growing understanding of the variety of signaling pathways involved in mediating DSM gene expression.Support or Funding InformationNIH INBRE Developmental Research Grant