bluuebunny/crossref_metadata_2025 huggingface dataset:
Predicting nitroimidazole antibiotic resistance mutations in Mycobacterium tuberculosis with protein engineering
Authors: Lee Brendon , Almeida Deepak , Afriat-Jurnou Livnat , Aung Htin , Forde Brian , Harold Liam , Hards Kiel , Pidot Sacha , Ahmed Hafna , Mohamed Elaaf , Taylor Mathew , West Nicholas , Stinear Timothy , Greening Chris , Beatson Scott , Cook Gregory , Nuermberger Eric , Jackson Colin
Published: April 2020

Our inability to predict which mutations could result in antibiotic resistance has made it difficult to rapidly identify the emergence of resistance, identify pre‐existing resistant populations, and manage our use of antibiotics to effectively treat patients and prevent or slow the spread of resistance. Here we investigated the potential for resistance against the new antitubercular nitroimidazole prodrugs pretomanid and delamanid to emerge in Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). Deazaflavin‐dependent nitroreductase (Ddn) is the only identified enzyme within M. tuberculosis that activates these prodrugs, via an F420H2‐dependent reaction. We show that the native menaquinone‐reductase activity of Ddn is essential for emergence from dormancy, which suggests that for resistance to spread and pose a threat to human health, the native activity of Ddn must be at least partially retained. We tested 75 unique mutations, including all known sequence polymorphisms identified among ~15,000 sequenced M. tuberculosis genomes. Several mutations abolished pretomanid and delamanid activation in vitro, without causing complete loss of the native activity. We confirmed that a transmissible M. tuberculosis isolate from the hypervirulent Beijing family already possesses one such mutation and is resistant to pretomanid, before being exposed to the drug. Notably, delamanid was still effective against this strain, which is consistent with structural analysis that indicates delamanid and pretomanid bind to Ddn differently. We suggest that the mutations identified in this work be monitored for informed use of delamanid and pretomanid treatment and to slow the emergence of resistance.

Support or Funding Information

This work was supported by by NHMRC Project Grant APP1128929 (to C. J. J., C. G., and G. M. C.). In addition, this work was supported by an ARC DECRA Fellowship DE170100310, and NHMRC New Investigator Grant APP1139832 (to C. G.). SAB is supported by an NHMRC Career Development Fellowship GNT1090456 (to SAB). DVA and ELN acknowledge the support of the U.S. National Institutes of Health (R01‐AI111992).
Induction And Regulation of Endogenous DARC Expression in Primary Human Endothelial Cells
Authors: Khosraviani Negar , Farahani Nikki Zamani , Abramian Madlene , Wang Changsen , Lee Warren L.
Published: April 2020

The Duffy antigen receptor for chemokines (DARC, also known as atypical chemokine receptor 1) is a blood group antigen associated with susceptibility to the parasite causing malaria. Despite homology to other chemokine receptors, DARC lacks the intracellular DRY motif present on most G‐protein‐coupled receptors that is required for signaling. Human biopsy and animal studies have indicated the presence of DARC on the endothelium of post‐capillary venules where its function and regulation are unclear. The lack of literature on endothelial DARC is attributed in part to the fact that its expression disappears in cultured endothelial cells within hours of isolation from tissue. Thus, almost all studies on endothelial DARC have required in vivo approaches. However, while the in vivo work is intriguing, the ability to study endogenous endothelial DARC in vitro will be necessary to elucidate its regulation and true physiologic function. We have discovered that incubation with human whole blood for 24 hours leads to induction of DARC on cultured primary human pulmonary microvascular endothelial cells (HPMEC). DARC induced in this manner is appropriately targeted to the cell surface, as it renders endothelial cells sensitive to an extracellular leukocidin. Induction of DARC on the endothelium was sensitive to cycloheximide, implicating de novo protein synthesis, and chromatin immunoprecipitation (ChIP) assays for RNA polymerase II also confirm transcription of the gene. While exposure to whole blood for 24 hours induced endogenous endothelial DARC expression, exposure to plasma had no effect. Transwell experiments indicated that physical contact with cellular elements in blood was necessary for induction of DARC. After induction of DARC and removal of blood, we observed rapid loss of DARC protein and mRNA within 24 hours. Our preliminary experiments suggest that endothelial DARC is rapidly degraded at least in part by matrix metalloproteinases. In conclusion, DARC expression on endothelial cells is regulated by cues from the microenvironment. DARC may play an important role in the immune system and its regulation is likely to have implications for our understanding of how cultured cells maintain or lose their in situ tissue phenotype.

Support or Funding Information

This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) grant to W.L.L., and N.K. was supported by the Canadian Graduate Scholarship ‐ Master’s Program (CGS‐M).
The role of arginine vasopressin deficiency in the regulation of the kidney fibrosis
Authors: Quintanar-Stephano Andrés , Huerta-Carreón Erika Paulina , González-Blas Daniel , Herrera-Beiza Bernardo Miguel , Tinajero-Ruelas Manuel , Esparza-Ávila Verónica Alejandra
Published: April 2020

It is known that arginine vasopressin (AVP) participate in the regulation of the immune responses, including the scarring. However, the effect of how AVP regulates this process is not clear. Here we describe the impact of AVP deficiency induced by neurointermediate pituitary lobectomy (NIL) or blocking the V1a‐V2 AVP receptors with conivaptan (CON) on the kidney fibrosis (KF), induced by unilateral ureteral ligature (UUL). Groups of 5 Wistar male rats were prepared: Control Intact (CI), UUL, NIL, NIL+UUL, UUL+NIL and UUL+CON. The NIL group was operated 6 weeks before sacrifice. In the NIL+UUL group NIL was performed 3 weeks before UUL and sacrificed 3 weeks after. In the UUL+CON (3mg/kg/day/SC/3 weeks before sacrifice) the UUL was done 6 weeks before sacrifice. KF was evaluated in histological slides stained with H‐E, Masson and Sirius red. Results showed that UUL induced hydronephrosis, severe kidney fibrosis and loss of histological architecture, whose severity was related to each experimental condition: In the UUL severe fibrosis occurred in the ilium, pelvis, periglomerular, intraglomerular, intraluminal tubules and interstitial kidney areas. In comparison with the CI, NIL group showed normal kidney histology, whereas that, in comparison with the UUL the NIL+UUL group developed significant less histological alterations and less kidney fibrosis. In the UUL+NIL group (NIL surgery performed 3 weeks after UUL and sacrificed 3 weeks later), although histological changes and fibrosis were apparent, these were significantly less severe as compared with the UUL group. In comparison with the NIL+UUL group, the UUL+CON animals developed less severe fibrosis and less histopathological changes. Conclusions. Results showed that kidney fibrosis may be controlled by AVP deficiency or blocking the V1a‐V2 AVP receptors (CON) indicating a major role of AVP on the fibrotic process. Results also suggests that conivaptan may be used for kidney fibrosis treatment.

Support or Funding Information

Universidad Autónoma de Aguascalientes PIFF14‐1 and CONACYT 221262. México
Defective Autophagic Degradation in Aged D2‐mdx Diaphragms
Authors: Krishna Swathy , Quindry Tiffany , Hudson Matthew B. , Quindry John C. , Selsby Joshua T.
Published: April 2020

Duchenne muscular dystrophy (DMD) is a progressive, fatal, muscle degenerative disease caused by the absence of dystrophin, which follows an X‐linked recessive inheritance pattern. Among other cellular dysfunctions, impaired autophagy has been identified as a consequence of dystrophin deficiency in humans. Accordingly, we hypothesized that degradation of autophagosomes would be impaired in muscles of D2‐mdx mice, an emerging DMD model. To test this hypothesis, diaphragm muscles from 11 month old D2‐mdx (diseased) and DBA (healthy) mice were collected, protein extracted, and western blot analysis was performed. Data were compared with a Student’s t‐test and significance was established as p<0.05. Among the markers of upstream activation of autophagy, total AMPK was increased by 3.5‐fold, phosphorylated (p)‐AMPK was similar, and p‐AMPK/AMPK was decreased by 80% in dystrophic muscle compared to healthy muscle. Similarly, total ULK1 was increased 1.7‐fold in muscle from D2‐mdx compared to DBA, while p‐ULK1 and p‐ULK1/ULK1 were similar between groups. Total Beclin1 was increased 10‐fold and p‐Beclin1 was increased 4‐fold, resulting in a 70% reduction in p‐Beclin1/Beclin1 in dystrophic muscle compared to healthy muscle. Total mTOR, p‐mTOR, and p‐mTOR/mTOR were similar between groups. Autophagosome formation proteins ATG7, ATG16L‐1, ATG12/5 complex, LC3A/B‐I and LC3A/B‐II were increased 2–3‐fold in D2‐mdx compared to DBA, however, the ratio of LC3A/B‐II/I was similar between groups. Relative protein abundance of p62 was increased 2‐fold in D2‐mdx compared to DBA. The relative amounts of the lysosomal membrane proteins LAMP1 and LAMP2 were similar between groups, suggesting similar lysosomal content in diseased and healthy muscles. These data appear to indicate decreased autophagosome degradation in dystrophic muscle compared to healthy. Given the similar lysosome content, autophagic dysfunction in D2‐mdx mice may be due to decreased lysosome fusion or function.

Support or Funding Information

Supported in part by Ryan’s Quest, Michael’s cause and Parent Project Muscular Dystrophy (01297)
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 Information

NIH‐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

Objectives

Determine the role of adipose tissue dysfunction in programming of cardiovascular disease risk in offspring born to dams with metabolic dysfunction.

Methods

The 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).

Results

NMR 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.

Conclusions

Adipose tissue dysfunction in offspring born to metabolically adverse pregnancies stems from in utero programming of a pro‐adipogenic phenotype in preadipocytes.

Support or Funding Information

Canadian 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 Information

Funded 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 Information

NIH 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 Information

This 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 Information

Authors 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 Information

NIH INBRE Developmental Research Grant
Positive allosteric modulator of protease‐activated receptor 1 promotes skin wound healing in hairless mice
Authors: Park So-Hyeon , Seo Yohan , Namkung Wan
Published: April 2020

Protease‐activated receptor 1 (PAR1) is a G‐protein‐coupled receptor functional expressed in several skin cell types including keratinocyte and dermal fibroblast. Thrombin‐induced PAR1 activation play a crucial role in skin wound healing such as thrombosis, inflammation, proliferation and tissue repair. In the present study, we identified a novel positive allosteric modulator of PAR1, GB83, and investigated the effects of GB83 on skin wound healing. The enhancement of PAR1 activity by GB83 was measured using Fluo‐4 calcium assay. Effects of GB83 on cell proliferation and gene expression were observed using MTS assay and quantitative real‐time PCRs, respectively. SKH‐1 hairless mice were used to investigate the wound healing effect of GB83. We demonstrated that GB83 did not activate PAR1 but strongly enhanced PAR1 activation by PAR1‐AP and thrombin. GB83 significantly promoted PAR1‐mediated cell proliferation and migration in HaCaT, HDF and A2058 cells. In addition, GB83 significantly increased gene expression of TGF‐b, fibronectin and collagen type1, and promoted skin wound healing in the hairless mouse model. We have found that GB83, the first positive allosteric modulator of PAR1, significantly promotes skin wound healing in vitro and in vivo. These results suggest that GB83 may be a potential therapeutic reagent for skin wound healing.
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 Information

NIH INBRE Developmental Research Grant
Cardiomyocyte β2‐Adrenergic Receptor Plays A Protective Role in Cardiac Fibrosis
Authors: Zhu Chaoqun , Deng Bingqing , Zhang Yu , Wang Ying , Xu Bing , Xiang Yang Kevin
Published: April 2020

Background

Chronic stimulation of β adrenergic receptor (βAR) has been implicated in cardiac remodeling including hypertrophy and fibrosis associated with heart failure. Previous studies indicate a role of β2AR on inhibiting collagen production in cultured cardiac fibroblasts, however, our recent studies show that global deletion of β2AR prevent cardiac fibrosis in diabetic cardiomyopathy. Therefore, this study aims to determine the specific role of cardiomyocyte β2AR in cardiac function and the development of cardiac fibrosis in vivo.

Methods

2‐month old male β2AR flox/flox (f/f) mice (n=10) and cardiomyocyte‐specific knockout of β2AR (CKO) mice (n=10) were fed with either normal chow (NC), or high fat diet (HFD) for 6 months to induce obesity and diabetes. Echocardiography was performed using a Vevo 2100 imaging system from VisualSonics. Biochemistry and histological analyses were performed on heart tissues. Real‐time PCR was performed for mRNA expression examination.

Results

While cardiac function was normal at 2‐month old CKO mice, a significant decrease in cardiac contractility was observed in CKO mice compared to f/f mice at 8‐month old age (EF%: CKO 48.43 ± 1.314 vs. f/f 52.69 ± 0.8270, p<0.05). Moreover, HFD feeding exacerbated cardiac dysfunction in CKO mice when compared to f/f mice (EF%: CKO 42.93 ± 0.69 vs. f/f 46.79 ± 0.76, p<0.001). CKO mice also exhibited diastolic dysfunction (IVRT: CKO 20.23 ± 0.30 vs. f/f 16.83 ± 0.23, p<0.0001), which was exacerbated after HFD feeding (IVRT, CKO 24.75 ± 0.50 vs. f/f 19.94 ± 0.45, p<0.0001). The observed cardiac dysfunction was associated with severe cardiac fibrosis in 8‐month old CKO mice, and HFD feeding leads to further exacerbation in cardiac fibrosis. Cardiac fibrosis was validated with elevated protein expression of connective tissue growth factor (CTGF), and significant upregulations of mRNA expression of CTGF, α smooth muscle actin (α‐SMA), and collagen 1 in CKO HFD mice.

Conclusions

This study elucidates that cardiomyocyte β2AR is essential to maintain normal cardiac function and plays a protective role against the development of cardiac fibrosis in aging and HFD feeding in vivo.

Support or Funding Information

NIH HL113413, HL147264, VABX002900
The cardiovascular response to ambient temperature changes within the human thermoneutral zone
Authors: Ugland Nora , Lin Aihua , Bergersen Tone Kristin , Andersen Marian Berge , Holme Nathalie Linn , Zilakos Ilias , Elstad Maja
Published: April 2020

Introduction

The human temperature control within the thermoneutral zone (TNZ) depends on the regulation of skin blood flow to hands and feet (acral skin)
[1]. The acral skin blood flow is regulated by absolute level and fluctuations in arterio‐venous anastomoses’ vasculature resistance. We hypothesized that a change in ambient temperature would not affect the hemodynamic responses within the TNZ in healthy humans.

Methods

Twenty‐four young healthy human subjects were exposed to three ambient temperatures (20°C, 26°C and 32°C) while supine and lightly dressed in a climate chamber. Each temperature plateau lasted for 40 minutes. The protocol either started at 20°C or 32°C, with a subsequent increase or decrease in temperature, respectively, and the order was randomized. Beat‐by‐beat blood flux from both index fingers and the left forearm were recorded simultaneously by laser Doppler flowmetry (DRT4, Moor Instruments, Devon, UK), together with heart rate (HR, from ECG). Non‐invasive finger arterial blood pressure (Finometer, Finapres Medical System, Netherlands; calibrated at each temperature plateau) was recorded continuously from the right middle finger, providing mean arterial pressure (MAP) and estimated beat‐to‐beat cardiac stroke volume (SV). The medians and their corresponding 95% confidence intervals were calculated by Hodges‐Lehmann’s estimates. Wilcoxon signed rank sum test for paired samples tested differences between states.

Preliminary results

Increasing ambient temperature from 20°C to 32°C caused a five‐fold increase in left fingertip flux (p<0.003) while left forearm flux doubled (p<0.008). The fluctuations in the bilateral fingertip flux were maximum correlated at 26°C (>0.82) and significantly higher than the correlation at 32°C (0.73, p<0.01 as compared to 26°C). The hemodynamic variables changed significantly within the TNZ. MAP decreased from 74 (70,77) mmHg at 20°C to 64 (60,68) mmHg at 32°C (p<0.0001). SV decreased from 102 (93, 113) ml at 20°C to 97 (86, 106) ml at 32°C (p<0.0004). HR increased minimally from 20°C (56 bpm (52, 59) to 32°C (58 bpm (56, 61) (p=0.01).

Conclusions

The marked changes in acral skin blood flow within TNZ affected the central hemodynamic variables, with the vasodilation reflected in MAP decrease coinciding with an increase in ambient temperature. HR and SV showed minimal changes within TNZ.

Support or Funding Information

The Research Council of Norway (grant no: 230354)
Supracristal Plane: Is it an accurate predictor of the aortic bifurcation or the caval confluence?
Authors: Pather Nalini
Published: April 2020

An accurate knowledge of surface anatomy and anatomical planes are imperative to clinical practice. Traditionally anatomical planes are based on cadaveric studies. Radiological imaging creates an opportunity to re‐assess our current knowledge of surface anatomy. One commonly used landmark is the supracristal plane that can also be used to locate the position for needle insertion in the lumbar puncture. This study aimed to assess the location of the supracristal plane in relation to vertebral levels using Computed Tomography (CT) and to determine the structures located at this plane.

Materials and Methods

Adult CT scans (n=191, mean age, 52 years; 59% male) in the supine position were analysed to determine the vertebral level of the supracristal plane, aortic bifurcation, inferior vena cava confluence and umbilicus. The incidence of these structures at each vertebral level was correlated with sex and age. Scans (n=31) with distorting pathology and an absence of consensus amongst reporters was excluded.

Results

The supracristal plane, aortic bifurcation, IVC confluence and umbilicus were most frequently located at L4/5 intervertebral disc (47.5%), lower third of the L4 vertebra (38.1%), lower third of the L5 vertebra (30%) and at the level of the L4 vertebra (28.7%). In only 13% of patients was the aortic bifurcation located at the supracristal plane. Lumabrisation and sacralisation was present in 2 and 3 patients, respectively.

Conclusion

Modern imaging has enabled an evidence‐based prediction of the location of abdominal organs in different populations. It is imperative that this information is assimilated and incorporated in surgical anatomy education.
Effect of MED28 on epithelial‐mesenchymal transition in human colorectal cancer cells
Authors: Li Po-Chen , Hsieh Nien-Tsu , Chang Yu-Tsen , Lee Ming-Fen
Published: April 2020

Mammalian Mediator complex plays an important role in transcriptional activation by bridging RNA polymerase II and transcriptional machinery. Multiple Mediator subunits have been implicated in human diseases including cancer development. Our laboratory has previously reported that MED28, a Mediator subunit, mediates Wnt/β‐catenin signaling and promotes cell growth in human colorectal cancer (CRC) cells. The objective of the current study is to investigate the effect of MED28 on epithelial‐mesenchymal transition in CRC in addition to cell growth. Suppression of MED28 attenuated the expression of several mesenchymal biomarkers including vimentin and transcription factors Snail and Slug, but increased the expression of E‐cadherin, an epithelial biomarker; however, overexpression of MED28 reversed the reciprocal change of epithelial and mesenchymal biomarkers. The underlying molecular mechanism is currently under investigation. Our data indicate that MED28 modulates the development of epithelial‐mesenchymal transition in human CRC cells, further reinforcing the significance of MED28 in the progression of malignancy in CRC.

Support or Funding Information

This work was supported by the grant MOST 106‐2320‐B‐039‐062‐MY3 to M‐F Lee.
Natural and synthetic glycan arrays for probing interactions of the innate and adaptive immune system with zwitterionic oligosaccharides
Authors: Wilson Iain B. H. , Eckmair Barbara , Yan Shi , Hykollari Alba , Paschinger Katharina
Published: April 2020

Invertebrate glycans are often ‘foreign’ to mammals, but many invertebrates are parasites which also immunomodulate the responses of the host, while invertebrate cell lines are potential ‘cell factories’ for production of secreted glycoprotein biopharmaceuticals. However, if we wish to investigate the innate or adaptive immune response of mammals to invertebrate glycoconjugates, a methodological hurdle is that most glycan arrays primarily focus on mammalian structures and not on those from other species. We are addressing this gap in the glycobiological toolbox by preparing arrays of either natural glycans from invertebrates or synthetic glycoconjugates displaying invertebrate glyco‐epitopes. To date we have printed (i) the neutral and anionic pools of N‐glycans from honeybee royal jelly, (ii) the native and hydrofluoric acid‐treated neutral pools of N‐glycans from the canine heartworm, (iii) fractions of N‐glycans from the canine heartworm, (iii) mono‐ and disaccharides modified with zwitterionic phosphoethanolamine or phosphorylcholine and (iv) fucosylated and non‐fucosylated forms of chitobiose and LacdiNAc. These pools, fractions or compounds were probed with various lectins, pentraxins or antibodies. We show that human serum amyloid P binds glycans derived from royal jelly (the food for honeybee queens, but also used as a beauty product), while human C‐reactive protein (CRP) interacts with the glycans of the canine heartworm (a mosquito‐borne parasite). On the other hand, short phosphorylcholine‐modified glycoconjugates are recognized not only by CRP, but by IgG and IgM in the sera of some nematode‐infected animals. Not only are such arrays complementary to current resources, but they have high potential to yield new insights into the glycan‐mediated interactions of invertebrates with mammals.

Support or Funding Information

This work was supported by the Austrian Science Fund (FWF) by grants to I.B.H.W., S.Y., A.H. and K.P.
Mixed Effects Of Active Immunization Against Aß In An Alzheimer Mouse Model
Authors: Oberman Klaske , Hoogerhout Peter , Gouweleeuw Leonie , Eisel Ulrich L.M. , van Riet Elly , Schoemaker Regien G.
Published: April 2020

Since Aß plaques and cognitive function are often not related in Alzheimers disease (AD), attention has shifted towards the neurotoxic soluble oligomeric Aß as the culprit in AD. Accordingly, INTRAVAC developed a concept vaccine that is specifically directed towards these misfolded Aß molecules. A previous study indicated protection against Aß‐induced neuronal damage in vaccinated mice. The aim of this study is to investigate the effects of the vaccine in a mouse model for AD.

Methods

As model for AD, J20 mice, overexpressing the human Amyloid Precursor Protein were used. AD phenotype of J20 mice was evaluated at 6 months of age. The vaccine consisted of 3 cyclic Aß peptides, conjugated to tetanus toxoid with proper adjuvants. Mock control consisted of tetanus toxoid +adjuvants. Mice were vaccinated or mock treated at 2,3 and 4 months of age, while behavior and plaque formation were evaluated at 6, 9 and 12 months of age.

Results

AD phenotype of J20 mice was confirmed by significant decline in cognitive performance, as well as the presence of plaques. Vaccinated mice showed significant improvement of working memory at 12 months compared to mock, as tested as correct alternations in Y‐maze exploration; 59.2±1.3 vs 56.1±1.6%. However, spatial learning (AUC learning curve: 858±±29 vs758±43s) and memory (number of platform crossings: 0.47±0.19 vs 1.04±0.39), measured in the Morris Water Maze, appeared deteriorated at 12 months. Plaque load, measured as number times size, increased with age, but was even slightly higher in vaccinated compared to mock mice at 12 months;9356±1200 vs 8155±1142 pixels). Antibody response showed significant titers for the cyclic peptides, but insignificant levels for Aß.

Conclusion

Cognitive decline and plaque formation in 6 months old J20 mice provide a relevant model for AD to test effects of vaccination against oligomeric Aß. Although vaccination at young ages improved working memory at 12 months, spatial learning and memory rather deteriorated, and plaque formation was not reduced. The lack of antibody response to Aß suggests that behavioral effects are mediated by mechanisms other than Aß reduction.

Support or Funding Information

Studies are partly funded by INTRAVACC, the Netherlands