bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Amplification of ITS‐2 and Cox1 Regions of Paramphistomum cervi in Three Breeds of Nigerian Cattle
Authors: Jeremiah Olalekan Taiwo , Wahab Yunus Abiola , Fagbohun Olusegun Adesina
Published: April 2020

Rumen fluke infections (paramphistomoses) in cattle are increasingly being reported. Serious issues with the parasite genus Paramphistomum are reported in sub‐tropical and tropical regions, with cases of paramphistomosis causing morbidity and mortality in ruminants.

Routine laboratory diagnosis involves conventional methods, such as optical microscopy used for the morphological and histological identification of the parasites. However, the occasional difficulty of identifying parasite structures may decrease the sensitivity of such methods.

Molecular technology demonstrates the presence of the parasites based on their antigenic components or Deoxyribonucleic Acid (DNA) segments. Polymerase Chain Reaction (PCR) makes it possible to perform selective amplification from complex genomes. Therefore, we describe the molecular amplification of the internal transcribed spacer 2 (ITS 2) region of the ribosomal DNA (rDNA) and Cycloxygenase region of the mitochondrial DNA (mDNA) encoding transfer Ribonucleic Acid (RNA) (tRNA‐Thr/Cox1) of adult Paramphistomum cervi worm samples by Polymerase Chain Reaction (PCR) using species specific primers: [5′‐ TGTGTCGATGAAGAGCGCAG‐3′ and 5′‐ TGGTTAGTTTCTTTTCCTCCGC‐3′] (5′‐ TGGAGAGTTTGGCGTCTTTTT‐3′ and 5′‐ CCATCTTCCACCTCATCTGG‐3′) respectively. Adult Paramphistomum cervi worm samples were obtained from the rumen of twenty bulls and twenty cows of White Fulani (n=18) Sokoto Gudali (n=13) and Red Bororo (n=9) breeds in a municipal abattoir in Ibadan city of Nigeria.

The amplification of the rDNA and mDNA of the parasite at the two spacer regions yielded 500bp (ITS2=100%) and 880bp (Cox1=91.7%) bands on agarose gel electrophoresis. The results suggest Paramphistomum cervi as common rumen flukes of cattle breeds in the study location. In addition, Polymerase Chain Reaction can be used as a rapid confirmatory diagnostic tool for bovine paramphistomosis.
Developing Medical Students as Future Educators Through a Teaching Distinction Track
Authors: Pizzimenti Marc A. , Bernat Carrie K. , Ruden Billie , Pettit Jeffery E.
Published: April 2020

Preparing undergraduate medical students to develop skills related to teaching and facilitation of learning requires a planned approach across multiple years. Students at the Carver College of Medicine (CCOM) may elect to enroll in the Teaching Distinction Track (TDT) where they focus on exploring multiple forms of education‐based experiences. These experiences are in addition to those required for the general medical curriculum. Students commit to participating in the TDT in their M2‐year by establishing a mentor and developing a learning contract. The learning contract outlines planned activities, opportunities for feedback, coursework, participation on education‐based committees, and the development of an education‐based project. The requirements for the TDT are a minimum of 60‐h of relevant teaching experience that may include tutoring, teaching science concepts, creating new educational materials, serving as a small‐group facilitator, assisting in physical examination skills development, and participation on committees related to medical education. Students are also required to develop a teaching portfolio and must successfully complete a 4‐week teaching elective. During their final semester at the CCOM each student gives a presentation on how the TDT has impacted their studies, showcasing some of their work. Included in the presentation are a synopsis of their teaching philosophy, specific teaching skills in which they have seen growth, examples of the educational materials they have developed, and the project they have undertaken. Since it was developed in 2010, approximately 128 students have graduated with a teaching distinction.
The Oxygen Mirror‐J‐Curve: Mild Hyperoxia and Mild Hypoxia Induce Beneficial Adaptations
Authors: Lovrenčić Luka , Kovac Zdenko
Published: April 2020

Background

Mirror‐J‐response, named based on the shape of dose response curve, claims that physiological agents of living organism (including humans) when they deviate mildly from the reference range produce beneficial effects (e.g. glucose, calcium and osmolality, etc). A higher degree of deviations of those same agents produces harmful effects.

Hypothesis

Both mild hyperoxia and mild hypoxia may induce a positive adaptive response. Could the mirror‐J be substantiated for oxygen?

Methods

Objective of this study was to search for a support of hypothesis in the published data.

Results

Beneficial effects of mild hypoxia have been shown both on cells and whole organisms. Cellular response to hypoxia consists of gene transcription modification and metabolic adaptations. These effects translate to adaptations of whole organisms. Animal studies show improved outcomes in both healthy subjects and disease models when challenged with hypoxia. Studies on healthy human subjects were mainly focused on physical activity performance, but have also shown decrease in inflammatory cytokines and glucose resistance. Studies on patients mostly focused on cardiovascular and neurologic diseases and show improvement in cardiorespiratory fitness similar to physical exercise.

Mild hyperoxia animal studies demonstrated that ischemia improved tolerance following hyperoxia in myocardium, brain and kidney. Human studies focus primarily on exercise performance in healthy subjects and cardiovascular and neurological pathology, same as hypoxia research. Mild hyperoxia during exercise promoted fat oxidation, reduced sugar oxidation, and delayed time to exhaustion. Hyperoxia preconditioning reduced risk of heart surgery. Also, following traumatic brain injury, periods of hyperoxia can normalize oxidative metabolism markers in brain long after return to normoxia.

Conclusion/Discussion

Hypoxia and hyperoxia are in antithesis, but their effects are surprisingly similar. Both were shown to precondition various tissues to ischemia and to improve cardiorespiratory fitness. These similarities invoke a usage of a similar mechanism of action, as suggested for disarrangements in calcium, glucose and osmolality. Indeed, it was shown that both, mild hypoxia and mild hyperoxia induce production of reactive oxygen species which induce cellular adaptive response. Furthermore, studies have shown that both hypoxia and hyperoxia activate hypoxia inducible factor and promote erythropoietin production. Taken together, this data supports single unified cellular response to tipping oxygen balance. Those responses follow the mirror‐J nonlinearity. These adaptive responses appear to function on cellular level and level of organism as a whole and may improve functioning in normoxia.
The Effect of Different Magnesium Sources on Renal Magnesium Transporters in Rats Fed a High Fat Diet
Authors: Sahin Kazim , Yazgan Kubra , Tuzcu Mehmet , Orhan Cemal , Sahin Nurhan , Ojalvo Sara Perez , Sylla Sarah , Komorowski James R.
Published: April 2020

Magnesium (Mg), which plays a role in many biochemical functions of the body, is the second most common cation in the cell. A high‐fat diet (HFD) intake causes hypomagnesemia, which is an electrolyte disorder. In this study, the molecular effects of Mg forms [magnesium oxide (MgO); magnesium picolinate (MgPic)] on renal Mg transporters (TRPM6, TRPM7, EGF, claudin‐16, claudin‐19, NCC) were investigated in HFD rats. For this purpose, 42 male Sprague‐Dawley (8 weeks old) rats were divided into 6 groups 1) Control; 2) MgO; 3) MgPic; 4) HFD; 5) HFD+MgO; 6) HFD+MgPic. Mg was supplemented at 500 mg of elemental Mg/kg diet. No effect on serum creatinine and urea levels of both Mg forms was detected (P >0.05). In HFD rats, kidney Mg levels were lower and kidney MDA levels were higher than other groups. However, the kidney Mg levels were increased and the kidney MDA levels were decreased by the Mg supplementation (P<0.001). The highest increase/decrease was obtained in the MgPic group. In addition, TRPM6, TRPM7, NCC, EGF, claudin16 and claudin19 Mg transporters were negatively affected by the HFD intake. However, it has been determined that Mg forms, especially MgPic, improved the levels of these proteins (P<0.001). As a result, MgPic may prevent oxidative stress and regulate Mg transporters in kidney including TRPM6, TRPM7, NCC, claudin16, and claudin19 in HFD rats

Support or Funding Information

Nutrition21, NY Firat Universitry ‐VF.18.02Turkish Academy of Science (KS)
Fetal Exposure to the Synthetic‐Progesterone Levonorgestrel (LNG) targets the brain resulting in hyperactive behavior using the Zebrafish (Danio rerio) as a model
Authors: Fuentes Austin K. , Ho-Shing Benjamin K. , Zamora Emily , Hinojosa Gabriella , Dearth Robert K.
Published: April 2020

Anxiety‐related disorders are a rising health concern in adolescent and young adults. Evidence suggests that disrupting normal neuronal development (neurogenesis) in the fetal brain could play a role in the manifestation of mental health disorders later in life. Growing literature suggests a role in hormone receptor (HR)‐regulated brain development, particularly in the hypothalamus, the neuroendocrine section of the brain and part of the limbic center. Yet, this link has not been thoroughly investigated. Levonorgestrel (LNG), a synthetic steroid used in contraceptive pills in adults, may also pose a threat to the developing brain by targeting HRs. Furthermore, this endocrine disrupting chemical (EDC) has been found at physiologically relevant levels in potable water sources worldwide providing an alternative route of exposure for unknowing individuals. Therefore, we investigated the effects embryonic exposure to LNG had on neurogenesis and hyperactivity using the zebrafish (Danio rerio) as neurobiological model. First, computational modeling using Quantitative structure‐activity relationships (QSAR), determined the predicted bioavailability, physiological half‐life and overall potential efficacy of LNG as a neuroendocrine disruptor. Next, Zebrafish were bred and fertilized embryos were collected and individually placed in 96‐well plates. Embryos were exposed to LNG levels (at doses ranging from 5–1000ng/L) or a vehicle control starting at 3 hours post fertilization (hpf) to 5 days pf (dpf), the time encompassing neurogenesis. There were no observable differences in fertilization ratios, hatching or morphology between LNG treated groups and vehicle controls. On 5 dpf, all treated and controls were assayed for changes in locomotor activity (i.e. hyperactivity busts). Larval movement were analyzed using computational locomotor activity software. Interestingly, the 5ng dose of LNG significantly (p<0.05) increased hyperactive behavior compared to vehicle controls. HR expression and neuronal marker α‐HuC were used to document neurogenesis. Our data suggests that the observed LNG‐induced hyperactivity represents anxiety‐like behavior as a manifestation of aberrant neurogenesis. This is the first study to show that exposure to low environmentally relevant levels of LNG causes hyperactivity and thus, potentially alters HR regulated neuronal development increasing the risk of other mental issues later in life.

Support or Funding Information

This project was supported in part by UTRGV College of Science (COS) Research Enhancement Seed Grants Program to RKD, the UTRGV Presidential Graduate Research Fellowship to AF and GH, the UTRGV COS Dean's Fellowship to EZ, and the UTRGV Engaged Scholars Award to BHS.
Contributions of Creatine Kinase to Mucosal Immune Responses in Colitis
Authors: Lanis Jordi M. , Lee J. Scott , Cartwright Ian , Hall Carrie , Colgan Sean P.
Published: April 2020

Intestinal epithelial cells facilitate nutrient transport as well as protect against invasion of luminal bacteria and microbial products. Disruption of the intestinal epithelial barrier is a hallmark of IBD. The regulation of intestinal epithelial barrier is made possible by junctional complexes including tight junctions and adherens junctions that are supported by the actin cytoskeleton. Maintenance of the actin cytoskeleton is an energy demanding process and intestinal epithelial cells shuttle energy to the locations of the cellular junctions through the creatine‐ATP energy circuit. Within the cell, creatine and its associated enzyme(s) creatine kinase(s) (CK) facilitate the shuttling of high energy phosphates in the form of phosphocreatine between sites of ATP generation. Work in our lab has shown that CK contributes to apical junction assembly in vitro and that supplementation with creatine in murine colitis models is protective. Further studies have shown that mice lacking the brain and mitochondrial isoforms of CK (CK dKO) are significantly more susceptible to DSS‐induced colitis as measured by multiple disease parameters, including disease activity, weight loss, intestinal permeability and by histological assessment. In addition to impaired barrier function, mice lacking CK showed defective inflammatory responses underscored by nearly non‐existent levels of colonic IFNγ. Flow cytometry from splenocytes and MLNs of CK dKO mice showed that T cells produced less IFNγ upon stimulation in vitro, and this finding was confirmed in human PBMC treated with the CK inhibitor cyclocreatine. These results implicate a CK‐dependent regulation of IFNγ in protective innate immune responses during mucosal inflammation, and ongoing studies are focused on elucidating the mechanism of CK activity on T cell signaling.

Support or Funding Information

Crohn's and Colitis Foundation RFA #546183
Transient silencing of transforming growth factor‐β1 expression restores nitric oxide generation in diabetic hematopoietic stem/progenitor cells: Role of thrombospondin‐1.
Authors: Jahan Jesmin , Miguel Duran , Quiroz Julio , Gracia Charles , Bartelmez Stephen , Jarajapu Yagna
Published: April 2020

Adult hematopoietic stem/progenitor cells (HSPCs) participate in the cardiovascular repair by stimulating regeneration and angiogenic functions of endothelium. Long‐term diabetes is associated with impaired vasoprotective functions of HSPCs. Transforming growth factor β (TGFβ1) is pleiotropic regulator of HSPC functions. Previous studies have shown that transient silencing of TGFβ1 expression improves in vivo migratory functions of diabetic HSPCs partly by restoring nitric oxide (NO) generation. This study tested the hypothesis that restoration of NO by TGFβ1‐silencing is mediated by thrombospondin‐1 (TSP1)/CD47 pathway. TSP1 is known to attenuate NO generation via inhibition of endothelial nitric oxide synthase (eNOS). HSPCs were isolated from peripheral blood samples obtained from either male or female healthy (n=25) or diabetic (both type 1 and type 2) (n=27) individuals (50–80 years of age) by immunomagnetic enrichment. TGFβ1 expression was transiently blocked by using TGFβ1‐antisense delivered in the form of phosphorodiamidate morpholino oligomer (PMO‐TGFβ1). TGFβ1 and TSP1 gene expressions were determined in HSPCs treated with either PMO‐control or PMO‐TGFβ1. NO generation induced by stromal‐derived factor‐1α (SDF) was determined by DAF‐FM flow cytometry. Diabetic cells have higher expression of TGFβ1 or TSP1 compared to that observed in cells derived from healthy individuals (n=10). Treatment with TGFβ1‐PMO decreased the expression of TGFβ1, which was associated with decreased TSP1 expression. CD47 expression is similar in healthy or diabetic individuals. NO generation by SDF is attenuated in diabetic compared to healthy cells (P<0.01, n=5). PMO‐TGFβ1‐treated diabetic cells showed increased generation of NO in response to SDF compared to PMO‐control‐treatment (P<0.01, n=5). Future studies will determine the effect of TSP1 on SDF/NO generation in cells with or without CD47‐siRNA, to further support the hypothesis. Collectively, these studies indicate the involvement of TGFβ1/TSP1/CD47/eNOS pathway in diabetic impairment of NO generation however involvement of other pathways cannot be ruled out.

Support or Funding Information

This study is partly supported by funding from NIH National Institute of Aging (AG056681).
Respiratory Dysfunction in a novel mouse model of Duchenne Muscular Dystrophy.
Authors: McCall Angela L. , Bailey Aidan M. , Pucci Logan A. , Dhindsa Justin S. , Strickland Laura M. , Robinson-Hamm Jacqueline , Gersbach Charles A. , ElMallah Mai K.
Published: April 2020

Duchenne muscular dystrophy (DMD) is the most common genetic disease in adolescent males. It is caused by a loss of dystrophin protein, necessary for skeletal and cardiac muscle fiber membrane integrity, linking the sarcolemma to the extracellular matrix. Without dystrophin, muscle fibers are unable to maintain structure, with each contraction and relaxation leading to progressive muscle weakness. Weakness of the diaphragm results in respiratory failure for patients in early adulthood. For over 30 years, the mdx mouse which lacks dystrophin expression has served as a relevant model for DMD. However, the genotype of the mdx mouse does not reflect that of the patient population.

Recently, a new mouse model harboring a human mutation in the dystrophin gene (DMD) was introduced into the mdx mouse (hDMD/Δ52;mdx) to better recapitulate a common genotype in patients. Since respiratory pathology is a significant cause of morbidity and mortality in DMD, this study sought to characterize the respiratory pathology in a novel DMD model – the hDMD/Δ52;mdx ‐ and further investigate the mdx mouse, using C57BL/6J mice as wildtype controls. The hypothesis driving this work is that the hDMD/Δ52;mdx mouse has greater respiratory deficiency compared to the mdx mouse. Whole body plethysmography (WBP) was used to assess respiration at baseline and during a challenge with hypercapnic and hypoxic conditions (FiCO2: 0.07, FiO2: 0.10; nitrogen balance). While exposed to normoxic air (FiO2: 0.21; nitrogen balance) the hDMD/Δ52;mdx was indistinguishable from either control at each time point. However, during the respiratory challenge the hDMD/Δ52;mdx mice had reduced frequency and minute ventilation by 6 months, which continued to decline at 12 months compared to the C57B/6J mice. Post mortem immunohistochemical studies of the neuromuscular junctions within the diaphragm showed that hDMD/Δ52;mdx and mdx mice had reduced overlap between presynaptic and postsynaptic endplates. Myofiber atrophy, fibrosis, and myofiber regeneration were also observed in the diaphragms of both mouse models. In conclusion, the hDMD/Δ52;mdx exhibits moderate respiratory pathology, and serves as a relevant animal model to study the impact of novel therapies on respiratory function.

Support or Funding Information

Duke Department of Pediatrics Derfner Grant; R01 HD099486‐01; 1R21NS098131‐01
Arterial Stiffening and Calcification Associated with Abnormal Lysosomal Positioning and Exosome Secretion in Mice Lacking Mucolipin 1 Gene
Authors: Bhat Owais M. , Yuan Xinxu , Camus Sarah , Salloum Fadi , Li Pin-Lan
Published: April 2020

Arterial calcification is a highly regulated cellular process that is characterized by the pathological mineral deposition leading to reduced arterial compliance. The aim of the present study was to investigate whether mucolipin 1 gene contributes to lysosomal positioning and exosome secretion which may results in phenotype change in SMCs during arterial medial calcification (AMC). In mucolipin 1 gene knock out (KO) mice, a high dose of Vit D (500,000 IU/kg/day) resulted in increased aortic calcification compared to their wild type littermates, which was accompanied by downregulation of SM22‐a and upregulation of RUNX2 and osteopontin in the arotic media, indicating phenotypic switch to osteogenic. Furthermore, we observed decreased colocalization of lysosome marker (Lamp‐1) with lysosome coupling marker (Rab 7 and ALG‐2) in the arterial wall of mucolipin 1 KO mice as compared to their wild type littermates. In addition, mucolipin 1 KO mice showed increased expression of exosome markers, CD63 and annexin‐II (AnX2), in the arterial medial wall, which was associated with reduced co‐localization of lysosome marker (Lamp‐1) with multivesicular body (MVB) marker (VPS16). This suggests reduction of the lysosome‐MVB interactions. Furthermore, we found significantly increased exosomes in the plasma of mucolipin 1 KO mice as compared to the wild type littermates. Functionally, in mucolipin 1 KO mice pulse wave velocity (PWV) significantly increased and Vit D treatment further enhance such stiffening. All these data indicate that mucolipin‐1 gene deletion leads to abnormal lysosome positioning and exosome secretion in the arterial wall, which may contribute to the arterial stiffness during the development of AMC.

Support or Funding Information

(Supported by NIH grants HL057244, HL075316 and DK120491)
Nicotinamide mononucleotide (NMN) treatment attenuates oxidative stress and rescues angiogenic capacity in aged cerebromicrovascular endothelial cells: a potential mechanism for the prevention of vascular cognitive impairment.
Authors: Kiss Tamas , Tarantini Stefano , Yabluchanskiy Andriy , Csipo Tamas , Balasubramanian Priya , Lipecz Agnes , Reglodi Dora , Farkas Eszter , Zhang Xin , Bari Ferenc , Csiszar Anna , Ungvari Zoltan
Published: April 2020

Age‐related impairment of angiogenesis likely has a critical role in cerebromicrovascular rarefaction and development of vascular cognitive impairment and dementia (VCID) in the elderly. Recently, we demonstrated that aging is associated with NAD+ depletion in the vasculature and that administration of NAD+ precursors exerts potent anti‐aging vascular effects, rescuing endothelium‐mediated vasodilation in the cerebral circulation and improving cerebral blood supply. The present study was designed to elucidate how treatment with nicotinamide mononucleotide (NMN), a key NAD+ intermediate, impacts age‐related impairment of endothelial angiogenic processes. Using cerebromicrovascular endothelial cells (CMVECs) isolated from young and aged F344xBN rats, we demonstrated that compared with young cells, aged CMVECs exhibit impaired proliferation, cellular migration (measured by a wound‐healing assay using electric cell‐substrate impedance sensing [ECIS] technology), impaired ability to form capillary‐like structures, and increased oxidative stress. NMN treatment in aged CMVECs significantly improved angiogenic processes and attenuated H2O2 production. We also found that pre‐treatment with EX‐527, a pharmacological inhibitor of SIRT1, prevented NMN‐mediated restoration of angiogenic processes in aged CMVECs. Collectively, we find that normal cellular NAD+ levels are essential for normal endothelial angiogenic processes, suggesting that age‐related cellular NAD+ depletion and consequential SIRT1 dysregulation may be a potentially reversible mechanism underlying impaired angiogenesis and cerebromicrovascular rarefaction in aging. We recommend that pro‐angiogenic effects of NAD+ boosters should be considered in both preclinical and clinical studies.

Support or Funding Information

This work was supported by grants the Oklahoma Center for the Advancement of Science and Technology, the Oklahoma Shared Clinical and Translational Resources (OSCTR) program funded by the National Institute of General Medical Sciences (GM104938), the NIA‐supported Geroscience Training Program in Oklahoma (T32AG052363), the Oklahoma Nathan Shock Center (P30AG050911), the Cellular and Molecular GeroScience CoBRE (1P20GM125528, sub#5337), and the NIGMS supported Center of Biomedical Research Excellence (CoBRE).
Transport of Degraded Ferric Enterobactin by Fiu
Authors: Yang Taihao , Newton Salete M. , Klebba Phillip
Published: April 2020

Under iron deficient conditions, Gram‐negative bacteria like Escherichia coli secrete enterobactin, a catecholate siderophore that complexes and solubilizes iron in the extracellular environment. Ferric enterobactin (FeEnt) is the most avid natural or synthetic iron complex. It may be glucosylated (FeGEnt), and it is also subject to both chemical degradation and oxidation (FeEnt*). Neverthless, bacterial outer membrane transporters acquire FeEnt, FeGEnt and FeEnt*. For example, the E. coli Fiu protein is known to catalyze the uptake of monomeric catecholate iron complexes, and using siderophore nutrition tests and radioisotopic iron accumulation experiments, we found that Fiu preferentially recognizes and transports FeEnt*. To further analyze the specificity of the FeEnt* uptake process, we created Cys substitutions in the external surface loops of Fiu and labeled them with fluorescein maleimide (FM). During binding of ferric siderophores, the surface loops undergo conformational motion that quenches attached fluorophores, allowing quantitative determinations of affinity and specificity. We measured the affinity of Fiu‐FM for different ferric catecholate compounds: FeEnt*, FeEnt, and FeGEnt produced Kd values of 0.46, 20, and 0.5 μM, respectively. Thus, Fiu manifests specificity for the degradation products of FeEnt, but also mediates the uptake of FeEnt and FeGEnt, identifying it as an important component of bacterial iron acquisition systems that are essential to bacterial survival.

Support or Funding Information

National Institutes of Health
Mitochondria Amplify TRPV4‐dependent Calcium Dynamics within the Pulmonary Microvascular Endothelium
Authors: Knighten Jennifer , Phillips Vantel , Taylor Mark , Townsley Mary , Francis Christopher Michael
Published: April 2020

Acute respiratory distress syndrome (ARDS) is an inflammatory lung disease common in critically ill patients marked by diffuse alveolar damage and increased pulmonary endothelial barrier permeability. A large body of evidence supports the role of mitochondrial dysfunction in ARDS, which has been shown to mediate endothelial permeability. For example, in cultured rat pulmonary microvascular endothelial cells (PMVECs), the mitochondrial complex I inhibitor rotenone increases endothelial permeability and in isolated perfused lung studies, rotenone increases the pulmonary vascular filtration coefficient. In both cell culture and isolated perfused lungs, the amphipathic quinone coenzyme Q1 (CoQ1) rescues rotenone‐dependent increases in endothelial permeability by bypassing complex I to restore mitochondrial bioenergetics.

The transient receptor potential vanilloid protein 4 (TRPV4), a mechanically gated calcium channel, has also been implicated in mediating endothelial permeability by increasing intracellular calcium in response to high pressure ventilation. The phorbol ester 4α‐phorbol‐12,13‐didecanoate (4αPDD) provides a pharmacological means of TRPV4 activation and stimulates endothelial permeability responses in both cell culture and animal models of lung injury. Given that mitochondria actively participate in calcium buffering, we hypothesized that TRPV4‐dependent calcium signals may be compartmentalized by mitochondria. To test this hypothesis, we used confocal microscopy to measure calcium signals in PMVECs in response to TRPV4 activation via 4αPDD (5 μM) after treatment with vehicle, rotenone (0.5 μM), or rotenone in combination with CoQ1 (10 μM), and developed a sensitive, automated technique for intracellular calcium signal transient analysis.

Using our novel analysis approach, we found that rotenone significantly decreased the number of calcium signal transients stimulated by 4αPDD over a 15 minute interval (mean ± SEM: 22 ± 4 rotenone vs 96 ± 14 vehicle, p <0.01), and that CoQ1 in combination with rotenone reversed this effect (60 ± 15, p = n.s. vs vehicle). Rotenone also dampened the amplitude of calcium responses to TRPV4, an effect that was partially restored by CoQ1. These findings support the hypothesis that mitochondria regulate TRPV4‐mediated calcium dynamics by amplifying calcium signals, and that mitochondrial dysfunction impairs this phenomenon. This suggests that mitochondrial regulation of endothelial calcium signaling is essential to the maintenance of barrier integrity and provides a possible therapeutic target in ARDS.

Support or Funding Information

NIH K25HL136869 and NIH P01 HL066299‐17
An Essential Fringe‐Like Protein in the Plasmodium falciparum Asexual Life Cycle
Authors: Villegas Alejandra , Fierro Manuel Antonio , Muralidharan Vasant
Published: April 2020

Malaria is a deadly disease caused by the apicomplexan parasite Plasmodium. Plasmodium asexual replication occurs in the red blood cell (RBC) and is what causes clinical symptoms of disease. In the RBC, Plasmodium moves through 3 developmental stages ending with schizogony and creating 16–32 daughter merozoites. At the end of schizogony, merozoites egress the host cell by breaking out of two membranes, the parasitophorous vacuole membrane and the RBC membrane. Schizogony and successful egress allows for exponential Plasmodium replication and RBC infection. Our lab recently identified a B3GLCT‐like protein in the Plasmodium ER (PfB3ER) that interacts with an essential ER chaperone and whose expression profile is similar to that of proteins required for egress. B3GLCT‐like proteins are known to act as glucosyltransferases and work in concert with protein O‐fucosyltransferases (POFUT2) to modify thrombospondin‐like repeats in mammalian cells. Surprisingly, the P. falciparum POFUT2 has been shown to be non‐essential while PfB3ER is predicted to be essential in the asexual stages. Based on PfB3ER homology to the mammalian B3GLCT and its predicted expression profile, we hypothesize that the O‐glycosylation function of PfB3ER is required for efficient egress of P. falciparum from RBCs. To test this, we have employed CRISPR/Cas9 gene editing to HA‐tag PfB3ER and create conditional mutants utilizing the TetR‐DOZI‐aptamer system. We show that PfB3ER localizes to the ER, unlike previous published results that suggest that PfB3ER is exported to the host RBC, and that PfB3ER is primarily expressed during schizogony. Further, we show that PfB3ER is essential for the asexual replication of P. falciparum. Future studies will focus on the specific stage of the intraerythrocytic lifecycle that are affected by PfB3ER knockdown and test the requirement of its glycosyltransferase activity. Together, these data have uncovered an essential function for a putative glycosyltransferase in the intraerythrocytic lifecycle of P. falciparum.

Support or Funding Information

R01 AI130139/AI/NIAID NIH HHS/United States
Embryonic Temperature Shifts Increase Adult Size in Drosophila melanogaster
Authors: Marks Christopher P.
Published: April 2020

The developmental history of organisms is critical in determining phenotypic outcomes. Shifts in environmental factors can constrain or redirect ontogeny in ways that traditional models of plasticity have failed to predict. In this study we examined the impact of shifting between high and low thermal regimes during the embryonic stage in Drosophila melanogaster. We found that extreme temperatures (both high and low) resulted in larger flies at enclosure than the temperature during subsequent development would have predicted. Thus, embryonic temperature poses a critical factor that can have lasting effects on morphology and fitness.
cAMP Biosensors with Restricted Subcellular Localization Reveal Persistent Signaling Changes Following Antidepressant Withdrawal
Authors: Senese Nicolas B. , Rasenick Mark M.
Published: April 2020

Many patients experience a constellation of side effects following termination of antidepressant therapy. Collectively these effects are known as the antidepressant discontinuation syndrome. All currently prescribed antidepressant medications can cause this syndrome, regardless of primary mechanism of action. This suggests that some shared mechanism, independent of these drugs’ known binding targets, may contribute to the discontinuation syndrome.

Lipid rafts are membrane microdomains with distinct signaling characteristics and protein expression compared to other membrane regions. Previous work has shown that chronic, but not acute, treatment with traditional antidepressants induces translocation of Gs out of lipid raft microdomains. In contrast, rapid acting antidepressants such as ketamine cause Gs translocation following acute exposure. This promotes Gs signaling in general, likely due to increased coupling to downstream effectors such as adenylyl cyclase.

Using a lipid raft restricted cAMP biosensor we show that perturbations of Gs signaling persist for over 24 hr after antidepressant withdrawal in an in vitro model system. These signaling changes are variable between different subcellular microdomains, and between different cell types. In general, signaling changes in lipid rafts are more persistent than changes observed in non‐raft membrane regions, or in the cytoplasm. Effects are compared between c6 glioma, SK‐N‐SH neuronal, and HEK‐293 kidney derived cell lines.

We also show that changes in lipid raft cAMP signaling are reflected by persistent reductions in lipid raft Gs expression following drug withdrawal. Protein expression is quantified following membrane isolation and sequential detergent extraction to separate lipid raft from non‐raft membranes.

The observed antidepressant effects occur in model systems lacking the primary target for these drugs (e.g. SERT and escitalopram), and as such may represent a novel mechanism by which antidepressants regulate cellular activity. Understanding these residual antidepressant effects may aid in the development of novel antidepressants without an associated discontinuation syndrome, and in the discovery of new strategies to mitigate these persistent effects following drug withdrawal.

Support or Funding Information

Supported by: VA Merit BX001149, NIH T32 MH067631 and NIH R01 AT009169
Neuregulin‐4 Limits Pro‐Inflammatory Cytokine Production in Macrophages
Authors: Schumacher Michael A. , Dennis Isabella , Robinson Cache , Frey Mark R.
Published: April 2020

Background

Pro‐inflammatory macrophages are critical mediators of intestinal inflammation. Over‐aggressive responses from these cells, including elevated cytokine release and prolonged persistence in tissue, may contribute to inflammatory bowel disease. We have previously found that the ErbB4 receptor tyrosine kinase is induced on macrophages by pro‐inflammatory stimulation, and that treatment with the ErbB4‐specific ligand neuregulin‐4 (NRG4) reduces macrophage numbers through apoptosis and ameliorates colitis. This suggests that macrophage‐expressed ErbB4 limits colonic inflammation, but the impact of NRG4/ErbB4 on cytokine regulation in these cells remains untested.
We hypothesized that ErbB4 inhibits pro‐inflammatory cytokine production in macrophages.

Methods

To test the role of ErbB4 in regulating cytokine expression, we generated bone‐marrow derived macrophages from mice lacking ErbB4 in cells of myeloid lineage (LysMCre/ErbB4FF) or sex‐matched littermate controls, and activated with IFN□/LPS (pro‐inflammatory M1) or IL‐4 (anti‐inflammatory M2). To test the role of NRG4, we treated activated cells with recombinant ligand at 100 ng/ml for 24 hours, harvesting cells before the onset of NRG4‐induced apoptosis to compare functional effects on cytokine expression. RNA was collected and analyzed for cytokines by qPCR.

Results

Pro‐inflammatory M1 activation of macrophages induced NRG4 expression 11‐fold (p=0.01). M1 macrophages from LysMCre/ErbB4FF mice expressed elevated levels of TNF (32% higher, p=0.02) and CXCL1 (69% higher, p=0.01) versus M1 macrophages from littermate controls. No differences in expression were seen in anti‐inflammatory M2 macrophages. NRG4 treatment led to reduced TNF (31% lower, p=0.005) and IL‐1β (16% lower, p=0.01) in M1 macrophages from wild‐type mice.

Summary/Conclusion

NRG4 reduces pro‐inflammatory cytokine production in macrophages through its receptor, ErbB4. This work suggests that ErbB4 signaling in macrophages drives an anti‐inflammatory feedback loop to limit colonic inflammation.

Support or Funding Information

NIH 2R01DK095004‐06A1 (Frey); Crohn's and Colitis Foundation (Frey and Schumacher)
Novel roles of PFKFB3 in mediating endothelial dysfunction in obesity
Authors: Batori Robert Karoly , Bordan Zsuzsanna , do Nascimento Thiago Bruder , Padgett Caleb , Huo Yuqing , Stepp David , de Chantemele Eric Belin , Fulton David
Published: April 2020

Obesity is a major and growing health problem in the US with over 93 million afflicted. Although, obesity itself is not considered a disease, it is a strong risk factor for cardiovascular disease, in large part due to an altered metabolic state. The relationship between metabolic dysfunction and impaired cardiovascular health remain incompletely understood.

One of the earliest consequences of cardiovascular dysfunction in obesity is the loss of endothelial function and impaired nitric oxide (NO) signaling. NO is best known as the mediator of endothelium‐dependent relaxation, but it is also vital for maintaining blood vessel homeostasis through inhibition of leukocyte adhesion and smooth muscle cell proliferation. In blood vessels, NO is synthesized by endothelial nitric oxide synthase (eNOS) and dysregulation of eNOS has been shown to occur through changes in its expression and post‐translational phosphorylation. The biological actions of NO can be further compromised by rapid inactivation by reactive oxygen species (ROS) such as superoxide that are generated by NADPH oxidases (NOX) as well as numerous other enzyme systems.

Our laboratory has previously shown that an imbalance between NO and superoxide‐generating pathways is responsible for the loss of endothelial function in obesity. However, the signals coordinating the loss of NO and increased superoxide with altered metabolism remain poorly defined.

In our studies we have found that expression of the glycolytic enzyme 6‐phosphofructo‐2‐kinase/fructose‐2,6‐biphosphatase 3 (PFKFB3) is significantly increased in endothelial cells from obese animals. PFKFB3 is responsible for fructose‐2,6‐bisphosphate generation in vascular cells which is a potent allosteric activator of phosphofructokinase‐1 (PFK‐1), one of the rate‐limiting enzymes of glycolysis. We have also found that adenovirus‐mediated expression of PFKFB3 within the endothelium of mouse blood vessels is accompanied by the loss of endothelium‐dependent relaxation.

In addition to its effects on blood vessels, overexpression of PFKFB3 in cultured cells resulted in decreased NO production as a result of increased eNOS phosphorylation on the well‐known inhibitory PKC phosphorylation site, threonine 495 (T495). Furthermore, PFKFB3 overexpression blunted Akt‐S473 phosphorylation, reducing stimulus‐dependent phosphorylation of eNOS at S1177. In addition to its negative effect on NO signaling overexpression of PFKFB3 increased the activity and mRNA level of NOX1, a major contributor to endothelial dysfunction in obesity.

These results demonstrate a novel functional relationship between endothelial cell metabolism, nitric oxide and ROS production that may contribute to endothelial dysfunction in obesity.

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1R01HL147159‐01
Determining the Role of Obesity and Muscarinic Signaling in Asthma
Authors: Bruggink Stephanie , Kentch Kyle , Kronenfeld Jason , Renquist Benjamin
Published: April 2020

In the U.S., one in thirteen individuals have been diagnosed with asthma. Obesity increases the incidence of asthma by 56%. The degree of obesity is associated with the severity of reduction in lung volume and lung volume can be restored with weight loss. Obesity also increases the risk of hospitalization by 460%. Although asthma and altered lung function is commonly assessed with spirometry in humans, plethysmography is recommended by the American Thoracic Society for assessing lung volume. The most common and effective method for assessing lung function in mice, forced ventilation, is terminal and limited to assessing lower airway function, preventing repeated measures. We aimed to develop a head‐out, variable pressure plethysmography system that would allow for repeated sensitive assessment of lung function and volume in lean and diet‐induced obese mice while mimicking the measures in human clinical practice. Preventing air leak from the chamber is vital for recording accurate measurements in a variable pressure plethysmography system. To eliminate air leak, we created an inflatable cuff that encompassed the mouse’s neck. This allowed us to repeatedly and accurately assess airway function in mice. To validate the head‐out, variable pressure plethysmography system, we used a series of models with altered muscarinic signaling. First, we assessed the response to a nebulized bronchoconstrictor, methacholine, establishing that methacholine resulted in the expected decreased breaths/minute, increased expiratory and inspiratory time, and decreased the rate of air flow at 50% of exhalation (EF50). To assess chronic muscarinic signaling, we used an adeno‐associated viral delivery to induce expression of a mutated constitutively active muscarinic 3 receptor (Q409L M3R) in airway smooth muscle, creating a model of chronic bronchoconstriction. In mice that expressed the Q490L M3R, we similarly found a decrease in breaths/minute and an increase in expiratory and inspiratory time at tidal breathing. Finally, we observed a decrease in breaths per minute at tidal breathing in muscarinic 3 receptor knock‐out mice. In our model of diet‐induced obesity we showed that obesity increased breaths per minute and decreased expiratory time at tidal breathing relative to that observed in lean mice. We have developed a reliable tool to repeatedly assess lung function in mice using the same clinically relevant measure applied in human asthmatics. Future research will apply this tool to better understand the mechanism by which obesity alters lung function and tidal breathing volume in mice.

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ABRC ADHS14‐082986

ABRC ADHS17‐00002043

T32 HL 007249
Early Life Stress Mediates Enhanced Aortic Vasorelaxation in Sv129 Mice
Authors: Kellum Cailin E. , Allan J. Miller , McPherson Kasi C. , Neder Thomas H. , Seifert Michael E. , Feig Daniel I. , Pollock Jennifer S.
Published: April 2020

Early life stress (ELS) is an under‐appreciated risk factor for cardiovascular disease (CVD). In humans, adverse childhood experiences (ACEs) or ELS is recognized as inducing behavioral or emotional stress. We reported that young adults exposed to ACEs display enhanced vascular stiffness, increased peripheral resistance, and elevated plasma ET‐1. We hypothesized that vascular endothelial‐derived ET‐1 is associated with vascular dysfunction after exposure to ELS. We hypothesized that endothelial‐derived ET‐1 is linked to vascular dysfunction in ELS. To test this hypothesis, we utilized vascular endothelial‐specific ET‐1 knockout (VEETKO) and flox control mice exposed to normal rearing (NR) or maternal separation with early weaning (MSEW). Briefly, MSEW involves maternal separation for 4h/day (postnatal (PD) 2 to 5), 8h/day (PD6 to 16), and weaned at PD17. NR litters were weaned at PD21. Male and female adult NR and MSEW, flox control (n=3–5) and VEETKO (n=5) mice were used to study acetylcholine (Ach)‐ and sodium nitroprusside (SNP)‐mediated aortic vasorelaxation. Interestingly, aorta from MSEW flox mice showed significantly greater maximal Ach‐mediated vasorelaxation compared to NR flox mice (Flox‐control 74.7% SEM 4.15, Flox‐MSEW 93.7% SEM 1.63, VEETKO‐control 78.0% SEM 6.81, VEETKO‐MSEW 86.9% SEM 2.05; p=0.0023). EC50 responses to Ach were not significantly different (Flox‐NR −6.22 SEM 0.330, Flox‐MSEW −6.88 SEM 0.124, VEETKO‐NR −6.70 SEM 0.144, VEETKO‐MSEW −6.88 SEM 0.268; p=0.066). All responses to SNP were similar. The VEETKO and flox control mice are on a Sv129 background. We previously reported that C57Bl/6J mice exposed to MSEW displayed endothelial dysfunction when compared to NR. Thus, these data suggest that exposure to MSEW has a different mechanistic response to the MSEW protocol with different strains of mice. Future studies will include behavioral studies to determine whether the MSEW protocol in Sv129 mice is distinct from the pro‐anxiety response reported in C57Bl/6J mice.

Support or Funding Information

Funding K01‐DK‐105038 KAH, RO1 HL136936 JSP and DMP

PROmoTE R25 DK 112731
Delayed delivery of metformin promotes functional recovery in a model of neonatal stroke
Authors: Livingston Jessica , Gilbert Emily , Syeda Tasfia , Christie Taryn , Morshead Cindi
Published: April 2020

Hypoxia‐ischemia (HI) is one of the leading causes of neonatal death and long‐term disability. Currently, the only standard treatment for HI is therapeutic hypothermia, which affords only modest benefit. The drug metformin has been shown to have pleiotropic effects in the brain, including activation of endogenous neural precursor pools. Importantly, administration of metformin leads to improved functional improvements following neonatal HI. Our previous work has demonstrated that metformin treatment beginning 24 hours after HI injury on post‐natal day (P8), and lasting for 1 week leads to attenuation of motor deficits at P22 and activation of endogenous neural precursor cells (NPCs) in the subventricular zone (SVZ). NPCs expanded in number, migrated into the parenchyma and differentiated into neurons and glia. Improved cognitive outcomes in young adults was also observed with extended metformin treatment in HI injured mice. Given these promising outcomes, and to improve the clinical application of the therapeutic intervention, we asked if delayed metformin treatment would be equally effective at improving functional outcomes. Male and female C57/BL6 mice were subjected to HI (or a sham procedure) at P8. Metformin (or vehicle) treatment began on P15 and lasted 4 weeks. Motor and cognitive functions were assessed across time using various behavioural assays (cylinder, foot fault, and puzzle box). Tissue was collected at P28 and 63 and analyzed for changes in inflammation, proliferation and differentiation. We found that delaying metformin treatment was effective at promoting motor and cognitive recovery. Treatment led to a decrease in inflammation after injury, as seen by a reduction in microglia, and an increase in oligodendrocytes in the motor cortex. Hence, our findings demonstrate that a clinically relevant delayed metformin treatment paradigm has therapeutic potential following neonatal HI.

Support or Funding Information

CIHR, Medicine by Design