bluuebunny/crossref_metadata_2025_split huggingface dataset:
Knockdown of Letm1 Protein Affects Mitochondrial Calcium Homeostasis and Cell Viability in Trypanosoma cruzi
Authors: dos SANTOS Guilherme Rodrigo , Leite Ana CR , Lander Noelia , Chiurillo Miguel A , Docampo Roberto , Vercesi Anibal E 
Published: April 2020

Calcium ion (Ca2+) is an important second messenger in the life cycle of the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease. It is well established that mitochondria have a central role in intracellular Ca2+ homeostasis. While the mechanisms of mitochondrial Ca2+ release in T. cruzi are not completely understood, it has been proposed that Letm1 (Leucine Zipper and EF‐Hand Containing Transmembrane Protein 1), a protein localized to the inner mitochondrial membrane, mediates the extrusion of Ca2+ from mitochondria via a Ca2+/H+ exchange mechanism. Using the CRISPR/Cas9 system we generated TcLetm1 knockdown (TcLetm1‐KD) cell lines. By electroporation of the parasite in presence of pTREX‐Letm1 plasmid, we also obtained a parasite line overexpressing Letm1 protein (OE‐Letm1). Using control parasites for knockdown (transfected with a scrambled sgRNA ‐ SCR) or overexpressing Letm1 protein (pTREX‐n) we have performed cell biology and biochemical experiments to assess the function of Letm1 in T. cruzi. We found that, although overexpression of Letm1 has no effect in epimastigotes proliferation, TcLetm1‐KD epimastigotes have a significantly lower proliferation rate. The ongoing biochemical experiments suggest that the protein Letm1 is, indeed, involved in mitochondrial Ca2+ homeostasis. Using digitonin permeabilized cells, we observed that, while overexpression of Letm1 increases the rates of both net mitochondrial Ca2+ uptake and release, TcLetm1‐KD has the opposite effect. Additionally, we found that TcLetm1‐KD parasites showed an increase in basal mitochondrial calcium concentration. In cell respiration experiments, TcLetm1‐KD cells showed lower oxygen consumption rates than control cells and increased ADP:ATP ratio. Knockdown and overexpression of Letm1 were achieved in T. cruzi for the first time and these cells are being used to study the activity of TcLetm1 as a mitochondrial Ca2+/H+ exchanger.Support or Funding InformationFAPESP, CNPq, and CAPES.
Effects of Environmental Acclimation versus Transport Stress on Barrier Recovery in a Pig Model of Intestinal Ischemia and Repair
Authors: Ziegler Amanda L. , Blikslager Anthony T. 
Published: April 2020

The pig is a powerful model for intestinal barrier studies, and it is important to carefully plan animal care and handling for optimal study design as psychological and physiological stressors significantly impact mucosal barrier function. Here, we report the effects of a period of environmental acclimation versus acute transport stress on mucosal barrier repair after intestinal ischemic injury. Jejunal ischemia was induced for 30‐minutes in 8–10‐week‐old pigs which had been transported and allowed to acclimate to a biomedical research housing environment for 3‐days prior to injury or been transported immediately prior to injury. Jejunal mucosa was then allowed to recover ex vivo in Ussing chambers while transepithelial electrical resistance (TEER) and mannitol flux were measured, and epithelial integrity was assessed by histomorphometry. In uninjured mucosa, there was no difference in basal TEER (P=0.8801) or epithelial integrity on histology between groups (P=0.5686), however, acclimated pigs had increased flux as compared to transported pigs during the first hour of ex vivo incubation (P=0.0186). Ischemia‐injured mucosa of acclimated pigs had less robust TEER recovery ex vivo (P=0.0101) as compared to transported pigs, but an increased initial flux in this group was significantly reduced during recovery (P=0.0025). Ischemia induced greater epithelial loss in transported pigs as compared to acclimated pigs (68.5% versus 87.8% epithelial coverage, P<0.0001), yet both groups restored control levels of epithelial coverage after 120‐minutes ex vivo recovery. These results indicate that acute transport stress on the day of experimental intestinal injury modeling may increase mucosal susceptibility to epithelial loss, but also prime the tissue for a more robust barrier repair response. Brief environmental acclimation, on the other hand, appears to increase intestinal permeability to molecular flux in the absence of injury, while possibly having a protective effect on epithelial loss during injury. These are important considerations for appropriate study design when utilizing highly translational pig models for intestinal barrier research.Support or Funding InformationNIH K01 OD 028207, NIH P30 DK 034987, NIH‐NICHD R01 HD095876, USDA‐NIFA VMCG‐0065, USDA‐NIFA 1007263.
Regulation of ENaC by aldosterone and potassium
Authors: Leite Dellova Deise C. A. , Saha Bidisha , Takagi Enzo , Shabbir Waheed , Pearce David 
Published: April 2020

In the cortical collecting duct (CCD), aldosterone regulates the transcription of serum and glucocorticoid‐regulated kinase 1 (SGK1) and stimulates the activity of epithelial sodium channel (ENaC). In addition, an aldosterone‐independent mechanism contributes to regulation of ENaC. We have shown that K+ itself acts through the master kinase mTOR within its type 2 complex (mTORC2) to activate SGK1, which stimulates ENaC to enhance K+ excretion in principal cells. However, the potential interconnections of these mechanisms have not been determined. Here, we have explored the aldosterone effect in association with K+ effect on ENaC activity regulation in the distal nephron. mpkCCD cells were grown on type I collagen–coated filters and subjected to aldosterone treatment and changes in extracellular [K+]. Amiloride‐sensitive short‐circuit current (ISC), which reflects ENaC activity, was measured by voltohmmeter (Millipore Millicell ERS). Following ISC measurements, cells were harvested and processed for immunoblot analysis. Exposure of mpkCCD cells to 1 μM aldosterone for 3 h increased ISC about 1.9‐fold compared to serum and hormone restricted cells. Elevation of basolateral [K+] to 3, 5 and 7 mM, after aldosterone treatment, promoted an additional increase in ISC of 1.5, 1.6 and 1.7‐fold, respectively. SGK1 HM phosphorylation increased proportionately to the increase in ENaC activity. The effect of K+ on ENaC activity and SGK1 HM phosphorylation is rapid and mediated by basolateral Kir 4.1 K+ channel activity. Overall, our data suggest that the effects of aldosterone and elevation of basolateral [K+] on ENaC activity and SGK1 phosphorylation were additive. Therefore, it is possible that basolateral [K+] acts in concert with aldosterone to regulate ENaC activity in CCD cells. However, in this cell culture model the effects are not synergistic suggesting that they do not proceed through convergent signaling pathways.Support or Funding InformationThis work was supported by NIH R01‐DK56695, Manning Foundation and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001.
Using a lipidomics approach to analyze the role of sphingomyelin in aging in C. elegans
Authors: McIntyre Grace E. , Radeny Joycelyn , Wang Lou , Chan Jason 
Published: April 2020

Research breakthroughs on aging and diseases have helped extend life expectancy in the United States over the last 25 years. However, as individuals continue to age, they lose the ability to perform motor and cognitive functions and their risk of developing chronic diseases increases. Here, we used a lipidomic approach to identify important lipid mediators of aging in C. elegans. This study focuses on the bioactive lipid sphingomyelin (SM), whose abundance depends on metabolic enzymes including acid sphingomyelinase (ASM). In C. elegans, one ASM ortholog is coded by the asm‐3 gene, and loss of asm‐3 extends lifespan in C. elegans. To explore longitudinal changes in lipids between wildtype (wt) and mutant asm‐3 C. elegans during aging, we collected six samples of wt and asm‐3 animals, at ages 1, 5, and 10 days, for lipid analysis. Approximately 700 different lipids were abundant enough in concentration for analysis. We found that 10‐day old asm‐3 mutants have increased levels of two Omega‐3 fatty acids, docosahexaenoic acid (DHA/22:6) and docosapentaenoic acid (DPA/22:5); both are linked to increased lifespans and decreased mortality when present in higher concentrations. Previous studies show that SM accumulates later in life causing aging to accelerate, but less is known regarding changes in specific types of sphingomyelin. Therefore, we also sought to examine how chain length and saturation levels of SM change with age by comparing the SM of 1 and 10 day old wt worms. Future work plans to correlate lipid composition to physiological changes in old animals. Together, we hope that we can better understand the intrinsic biochemical lipid processes associated with late life conditions to better prevent illness care for our aging populations.Support or Funding InformationResearch reported in this publication was supported by the National Institute On Aging of the National Institutes of Health under Award Number R15AG052933. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
The Intracranial baroreflex is attenuated in hypertension
Authors: Ramchandra Rohit , Guild Sarah-Jane , George Bindu , Vari Sydney 
Published: April 2020

BackgroundWe have previously shown that there is a novel control pathway between physiological levels of intracranial pressure (ICP) and blood pressure via the sympathetic nervous system. Increases in ICP of up to 20 mmHg over a 30‐minute infusion period were matched by increases in mean arterial pressure such that cerebral perfusion pressure remained constant. However, whether this ‘intracranial baroreflex’ is altered in hypertension is not known. Hence, in the present study we tested the hypothesis that the intracranial baroreflex is attenuated in a group of hypertensive animals.MethodsExperiments were conducted in two groups of adult ewes which either underwent unilateral renal artery clipping for three weeks (n=6) or sham surgery (n=6). Three weeks later, the mean arterial pressure and renal blood flow response to intracerebroventricular infusion of saline was determined.ResultsIntracerebroventricular infusion of saline produced a ramped increase in ICP of up to 20 mmHg over a 30‐minute infusion period (baseline 4.3±1.1 mmHg) in the normotensive group of animals. The ICP increase was matched by an increase mean arterial pressure in the normal animals such that cerebral perfusion pressure remained constant. Direct recordings of renal blood flow indicated that renal blood flow did not change but renal vascular conductance decreased with increasing ICP. In the hypertensive group, while intracerebroventricular infusion of saline produced similar ramped increases in ICP, the increase in mean arterial pressure was significantly attenuated such that cerebral perfusion pressure decreased. Administration of the ganglionic blocker, hexamethonium, prior to the infusion of saline abolished the increase in blood pressure in both groups.DiscussionIn conclusion, we have shown that the ‘intracranial baroreflex’ ensures increases in mean arterial pressure during physiological increases in intracranial pressure to maintain cerebral perfusion. This reflex is attenuated in hypertension.Support or Funding InformationHealth Research Council of New Zealand
The Role of Proteoglycans in Glomerular Physiology and Pathophysiology
Authors: Ebefors Kerstin , Khramova Alina , Nyström Jenny 
Published: April 2020

BackgroundDiabetic kidney disease (DKD) is the leading cause of renal failure in the world. Diabetes is associated with damage to the endothelial glycocalyx (eGCX), the layer of proteoglycans (PGs) and other negatively charged molecules covering the glomerular endothelial capillaries. The eGCX restrain the flow of charged molecules, such as albumin, over the filtration barrier in the kidney. Loss of the glomerular eGCX leads to proteinuria without other visible damage to the filtration barrier, but the detailed structure and composition is still largely unknown. The aim of this study was to gain new knowledge about the composition and role of the EGCX in health and DKD.MethodsThe negatively charged PGs in the eGCX was eluted from rat kidneys using 1 M NaCl solution (high salt, HS). In addition 1 M mannitol was used as osmotic control (HO) and 0.15 M NaCl to mimic physiological salt concentrations (NS). Solutions were introduced intra‐arterially to rat kidneys under anesthesia in vivo. Venous effluent was analyzed using mass spectrometry. Fractional clearance of albumin and GFR was measured. Electron microscopy (EM) was used to investigate morphological changes. Expression of PGs and PG related genes in glomeruli from patients with DKD was investigated using deep sequencing data from a Swedish DKD cohort (DKD, n=19, controls; pre‐transplant biopsies, n=20).ResultsWe identified 15 PGs in the eluate from rats. PGs were found in highest yields in the HS samples. EM demonstrated that the eGCX thickness was significantly reduced in the HS rats −28% (p<0.05) compared to NS. Rats perfused with HS had increased fractional clearance of albumin and reduced GFR, compared to NS and HO, 10 minutes after perfusion. In glomeruli from patients with DKD 12 PGs were found to be significantly regulated, and 4 of these PGs were also identified in the eGCX eluates from rats. There was an overall decrease in expression of enzymes responsible for building up PG side chains, as well as an increase in proteins involved in PG degradation.ConclusionThe eGCX itself represents a dynamic structure with significant molecular turnover. In our study, we identified several PGs novel to the glomerular eGCX. We show that loss of eGCX leads to proteinuria and reduced GFR, strongly supporting that the eGCX is important for preventing proteinuria. In glomeruli from patients with DKD we found significant alterations in the gene expression of PGs and the enzymes regulating their composition. Further investigation is needed to clarify in detail when and how these alterations appear in DKD.
Role of CO2 in the Expression of TRPV1 in Vascular Smooth Muscle Cells
Authors: Nishimura Naoki , Sugimoto Naotoshi , Nakamura Taichi , Wada Taizo 
Published: April 2020

ObjectivesCarbon dioxide (CO2) is known to affect thermal sensation and vasodilatation in human skin immersed in CO2‐rich water. However, the role that CO2 plays in these mechanisms is unclear. Transient receptor potential vanilloid 1 (TRPV1) is a member of a non‐selective cationic channel family expressed in human sensory neurons. Activation of TRPV1 is induced by heat and chemical agonists such as H+. This study aimed to clarify the role of CO2 in regulating TRPV1 expression in vascular smooth muscle cells.MethodsA7r5 rat smooth muscle cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 incubator. The cells were exposed to 20% CO2 for 24 hours. The expression of TRPV1 and TRPV4 were subsequently evaluated by real time PCR.ResultsThe expression levels of TRPV1, but not TRPV4, were significantly increased ( P =0.013) in the vascular smooth muscle cells following exposure to 20% CO2 for 24 hours.ConclusionsThese results suggest that exposure to CO2 may regulate the expression of TRP family proteins in cultured vascular smooth muscle cells.Support or Funding Informationnone
Open Heart Surgery with Cardiopulmonary Bypass using Dexmedetomidine‐based Monitored Anesthesia Care without Endotracheal intubation Improves Postoperative Recovery
Authors: Chen Wenting , Yuan Lan , Wang Yongqiang , Tang Wei , Guo Feng , Xia Zhengyuan , Song Jiangang 
Published: April 2020

BackgroundStudies have demonstrated the feasibility of performing awake cardiac surgery (ACS) in non‐intubated patients with consciousness and spontaneous breathing using epidural anesthesia. However, major concerns about severe complications of epidural anesthesia, such as epidural hematoma, have seriously hampered the development of ACS. Monitored anesthesia care (MAC) is a planned procedure during which the patients undergo local anesthesia together with sedation and analgesia. Dexmedetomidine (Dex), a highly selective α‐2 agonist, can facilitate fast recovery in patient undergoing cardiac surgery. We aimed to evaluate the feasibility and safety of Dex‐based MAC without intubation and the potential of achieving enhanced recovery after surgery (ERAS).MethodsWith patients’ consent, 11 patients scheduled to undergo cardiac surgeries under cardiopulmonary bypass (CPB) were arranged for MAC. Surgery was performed with a conventional median sternotomy. MAC composed of continuous Dex infusion (0.2 – 0.5 g/kg/hour) supplemented with low‐dose remifentanil/sufentanil, regional anesthesia and electroacupuncture was applied to satisfy the need for analgesia and sedation during cardiac surgery while avoid significant respiratory depression. 11 patients who routinely underwent general anesthesia (GA) that matched for age, gender and types of operation were used as controls. Intraoperative respiration and hemodynamics, postoperative vasoactive‐inotropic score (VIS), serum troponin I, complications and recovery after surgery were evaluated.Results11 cardiac surgeries including mitral valve replacements, valvoplasty, valve replacement plus valvoplasty, resection of atrial myxoma, and repair of atrial septal defect, were uneventfully accomplished under MAC. None of them required conversion to GA. The whole process of surgery was accompanied by adequate spontaneous breathing, stable hemodynamics, satisfactory surgical conditions, good patient satisfaction and no anesthesia awareness. Compared with GA group, intraoperative dosage of opioids and general anesthetics, VIS at 6 hours after entry into intensive care unit (ICU), serum cardiac troponin I concentrations, the time to drink and duration of ICU stay were significantly reduced in the MAC group (all p<0.05).ConclusionThe minimally invasive Dex‐based MAC for heart surgery with CPB is safe and feasible. Moreover, it shows the potential of cardioprotection and enhanced recovery after surgery.Support or Funding InformationStudy supported in part by a NSFC grant(81774108)
Perceptions of Pain Treatment in Pediatric Patients With Functional Gastrointestinal Disorders
Authors: Hale Amy E. , Smith Allison M. , Christiana Julie Snyder , Burch Elizabeth , Schechter Neil L. , Beinvogl Beate C. , Paul Fiona , Henaghan Amelia S. , Logan Deirdre , Nurko Samuel 
Published: April 2020

Objectives:
Individual understanding of and expectations for chronic pain treatment can influence treatment adherence and thus success, but little is known about these critical factors in parents and children presenting with pain-predominant functional gastrointestinal disorders. The aim of this study was to identify parent and patient understanding of pain-predominant functional gastrointestinal disorders, expectations for treatment, and interventions utilized before presenting to a multidisciplinary clinic.

Materials and Methods:
This was a prospective study of patients evaluated in a Multidisciplinary Functional Abdominal Pain Program. Before the clinic visit, parents and patients completed questionnaires regarding their understanding of chronic pain, perceptions of abdominal pain contributors, expectations regarding treatment, and identification of previous interventions utilized.

Results:
Participants were knowledgeable regarding the biology of chronic pain. Perceptions of contributors to abdominal pain included a sensitive stomach, general stress, and nerves/worry. Most had attempted to treat their pain with medication, exercise or physical therapy, or a psychological treatment. Participants reported that receiving a definite diagnosis would be the most helpful intervention, followed by psychological treatment.

Discussion:
Participants were knowledgeable regarding chronic pain, but still indicated that receiving a definite diagnosis would be the most helpful intervention. Most had tried multiple interventions and did not believe that further medication, testing, or surgery would solve their pain. Instead, parents presenting at this Functional Abdominal Pain Program appeared most hopeful about the benefits of multidisciplinary treatment approaches including psychological interventions, a focus on activity and functioning, and complementary and alternative medicine interventions.
Wnt Signaling Pathway Proteins in Scar, Hypertrophic Scar, and Keloid: Evidence for a Continuum?
Authors: Chaudet Kristine M. , Goyal Amrita , Veprauskas Katy R. , Nazarian Rosalynn M. 
Published: April 2020

Abstract:
Hypertrophic scars and keloids are fibroproliferative lesions characterized by excessive collagen deposition. It is unclear whether these entities represent distinct disorders or share a common pathogenesis and the molecular underpinnings of these lesions are poorly understood. Accumulating evidence suggests that the Wnt signaling pathway is a key regulator of wound healing. In this study, tissue microarray was used to evaluate the protein expression profile for Wnt3a, phosphorylated glycogen synthase kinase 3 alpha (pGSK-3α), WNT1-inducible-signaling pathway protein 1 (WISP1), and WISP2 in normal skin, scars, hypertrophic scars, and keloids. Analysis revealed significantly increased fibroblast expression of pGSK-3α in scars (27.2%), hypertrophic scars (30.4%), and keloids (57.3%) compared with normal skin (16.4%) (all differences statistically significant; P < 0.01). Analysis of WISP2 showed 94% of fibroblasts in normal skin expressing WISP2 and significantly decreased expression in scars (46.8%), hypertrophic scars (27.0%), and keloids (61.3%) (all differences statistically significant; P < 0.01). The parallel patterns of expression of pGSK-3α and WISP2 in scars and hypertrophic scars and significantly increased expression in keloids may support the notion that keloids are a truly distinct fibrosing disorder and may provide further evidence for targeting the Wnt signaling pathway in the treatment of keloids.