bluuebunny/crossref_metadata_2025 huggingface dataset:
Neuronostatin Induces C‐FOS Response and Gluconeogenic Enzymes in HEPG2 Cells: A Potential Role of Neuronostatin in the Liver
Authors: Mahmood Ammar , Grote Stephen , Samson Willis , Yosten Gina
Published: April 2020

Hepatic glucose production (HGP) and output are regulated by a complex system of hormones, receptors, signaling mechanisms, glycolytic and gluconeogenic enzymes, and many overlapping transcription factors. The proteins involved in HGP are attractive targets for antidiabetic therapy, yet better pharmacological interventions are still needed for treatment. Therefore, it is critical to identify and characterize new targets within HGP regulation. We identified a novel peptide, neuronostatin, encoded in the somatostatin prohormone that exerts effects in the central nervous system, the heart, and the pancreas. Neuronostatin is produced in the delta cells of the pancreatic islets and plays an important role in maintaining glucose homeostasis through enhancing glucagon secretion from the α cell and reducing insulin release from the β cell. In male rats, a bolus injection of neuronostatin significantly increased blood glucose over 30 minutes after administration. Likewise, the peptide delayed glucose clearance and diminished the insulin response to hyperglycemia in a glucose tolerance test. Our group also identified the orphan GPCR, GPR107, as a cognate receptor for the peptide. GPR107 is expressed in numerous tissues, including the liver.

In searching for the role of neuronostatin in hepatic physiology, we observed that hepatocellular carcinoma cell line HEPG2 expressed GPR107. Thus, we hypothesized that neuronostatin would elicit a biologic effect on the liver using HEPG2 cell line as in‐vitro model. We found that treatment of HEPG2 cells with neuronostatin significantly elevated cFos mRNA expression, indicating that HEPG2 cells respond to the peptide. In addition, neuronostatin induced expression of gluconeogenic enzymes, such as phosphoenolpyruvate carboxykinase and glucose‐6‐phosphatase. These results indicate potential effects of neuronostatin on HGP, so we sought to determine if the effects of neuronostatin on blood glucose were due to increased glucagon secretion or due to a direct effect in the liver. In rats treated with a glucagon receptor antagonist, neuronostatin increased blood glucose, supporting a glucagon‐independent action on HGP. Thus, our results suggest that neuronostatin could play a physiologically relevant role in the liver and that GPR107 may be a new target in hepatic glucose production.

Support or Funding Information

National Institute of Health
Pulmonary microvascular dysfunction develops in rodent models of HFpEF
Authors: Czikora Istvan , Davila Alec C. , Tian Yanna , Belanger Kasey , O'Connor Paul , Sullivan Jennifer C. , Bagi Zsolt
Published: April 2020

Pulmonary complications are common in patients with diastolic heart failure, also known as heart failure with preserved ejection fraction (HFpEF). We tested the hypothesis that pulmonary microvascular dysfunction develops in HFpEF. We employed two different rodent models of HFpEF, uninephrectomized and DOCA (UNX‐DOCA) salt treated rats as well as uninephrectomized aldosterone (UNX‐Aldo) infused mice. Left ventricle (LV) diastolic function was assessed by transthoracic echocardiography (E/A ratio and deceleration time). Microvascular vasodilator function in rats was examined in isolated small pulmonary arteries (3rd or 4th order) using wire myography, whereas pulmonary vascular resistance (PVR) was measured in isolated perfused lungs in mice. Our results showed that LV diastolic dysfunction and increased pulmonary edema formation was accompanied by a reduced acetylcholine‐induced pulmonary artery relaxation and increased PVR in UNX‐DOCA rats and UNX‐Aldo mice, respectively, when compared to corresponding controls. Responses to the direct nitric oxide donor, sodium nitroprusside were maintained and found to be similar in the magnitude in the different groups. We also found that these functional changes were accompanied by a reduced artery lumen diameter and increased wall thickness in UNX‐DOCA rats and UNX‐Aldo mice. We concluded that both functional and morphological changes develop in the pulmonary microcirculation that could contribute to pulmonary complications, such as edema formation and pulmonary artery hypertension in HFpEF.

Support or Funding Information

1R01AG054651‐01

17SDG33680024
The female menstrual cycle: impact on cardiovascular, ventilatory and neuromuscular responses to whole body exercise
Authors: Georgescu Vincent P. , Thurston Taylor S. , Jarrett Catherine L. , Weavil Josh C. , Richardson Russell S. , Amann Markus
Published: April 2020

BACKGROUND

The female menstrual cycle (MC) is characterized by varying levels of endogenous sex hormones. Previous investigations have indicated that fluctuating levels of estrogen and progesterone may influence vascular function, ventilatory chemosensitivity, and exercise‐induced fatigue throughout the MC. However, a comprehensive analysis of the role of these sex hormones on determining cardiovascular, ventilatory, and neuromuscular responses during whole body exercise is currently lacking. Therefore, the purpose of this study was to investigate cardiovascular, ventilatory and neuromuscular responses to submaximal cycling exercise of eumenorrheic females throughout the MC.

METHODS

Females were studied during three MC phases: early follicular (EF; days 1–4), late follicular (LF; days 8–12), and mid luteal (ML; days 8–10 post LH peak). Prior to exercise, venous blood was collected each visit to characterize endogenous hormone concentrations. Subjects cycled at 50 W, 75 W, 100 W, and 80% Wpeak (129 ± 12 W) for 4 min per stage. Heart rate (HR), mean arterial pressure (MAP), femoral artery blood flow (QL, Doppler ultrasound), minute ventilation (VE), oxygen consumption (VO2), carbon dioxide production (VCO2), and estimated arterial hemoglobin saturation (SpO2) were quantified throughout all cycling trials. Preto post‐exercise changes in potentiated quadriceps twitches (ΔQtw) were used to quantify peripheral locomotor muscle fatigue.

RESULTS

HR, MAP, and QL were not different at any intensity throughout the MC (P > 0.4). VE, VO2, VCO2 were also not different throughout the MC (P > 0.2). Furthermore, neither hemoglobin content, nor SpO2 and arterial oxygen content changed significantly across the MC (P > 0.1). Consequently, leg O2 delivery during exercise at all intensities were similar during the MC. Finally, exercise‐induced peripheral fatigue was not different throughout the MC (ΔQtw: ~47%; P = 0.2).

CONCLUSION

Fluctuating endogenous female sex hormones do not significantly influence central or peripheral hemodynamics or the ventilatory response to submaximal whole‐body exercise. Similarly, the exercise‐induced development of peripheral fatigue also remains unaltered across the MC during submaximal cycling exercise of the same duration and intensity. Although depending on the research question, these data suggest that controlling for MC phase may not be necessary when examining cardioventailtory or neuromuscular responses to whole body exercise in females, and should encourage the participation of more females in these types of studies.

Support or Funding Information

National Heart, Lung, and Blood Institute (HL‐116579)
Understanding the Cellular Functions Polyamines Play in Proliferation, Replication, and Survival of Leishmania donovani
Authors: Perdeh Jasmine , Love Quintin , Leizorek Yvette , Berioso Brandon , Le Linh , Roberts Sigrid
Published: April 2020

Leishmaniasis, a neglected tropical disease, affects an estimated billion people worldwide. A better understanding of parasite biology is vital for the development of much needed new therapeutic strategies. Our research addresses this critical need by focusing on the role of polyamines for growth and survival of Leishmania parasites. The main polyamine biosynthetic enzymes in Leishmania are ornithine decarboxylase (ODC) and spermidine synthase (SPDSYN), which sequentially convert ornithine to the polyamines, putrescine and spermidine. With generated gene deletion mutants, Δodc and Δspdsyn, we have demonstrated in vitro that the metabolite putrescine is essential beyond its function as the precursor for spermidine formation. Additionally, we observed that putrescine‐depleted Δodc parasites immediately ceased proliferation and died within two weeks. In contrast, putrescine‐rich Δspdsyn mutants showed an intermediate growth phenotype and entered a quiescent‐like state with cell death occurring after six weeks. Furthermore, putrescine‐depleted cells were unable to synthesize DNA and stopped replicating. We also observed two hallmarks of programmed cell death in the putrescine‐depleted cells: cell‐rounding and phosphatidylserine exposure. To reduce the variability associated with working with two cell lines and to further understand the functions polyamines play, we are in the process of generating a double gene deletion mutant, ΔodcΔspdsyn.

Support or Funding Information

Pacific University School of Pharmacy Research Incentive Grant
Student Design of a Proteopedia Page to Reinforce Foundational Biochemistry Concepts and Molecular Visualization Skills
Authors: Hall Bonnie L.
Published: April 2020

In our curriculum, we have implemented a variety of strategies to promote mastery of ASBMB core concepts in biochemistry, and more recently of molecular visualization skills. For the past two years we have utilized student design of a Proteopedia page in our Biochemistry course as a way to gain proficiency in both of these areas. Working in small groups, students are assigned a journal article that characterizes the function of a protein. Each article contains one or more solved protein structures along with a variety of other biochemical data. A series of scaffolded assignments lead the students through an overview of the article, more in‐depth analysis of the protein’s function, individual design of a draft Proteopedia page, and a final revision of that page. Using the Jmol tools in Proteopedia and the original PDB file, students construct and annotate novel images to highlight key points of the article. They identify critical molecular interactions, explore topology and connectivity, generate alternate renderings, and emphasize relationships between molecular structure and function. As students complete these activities, they are accessing, comprehending and communicating science topics. After completing their Proteopedia pages, students report a better understanding of foundational concepts and molecular visualization. They also appreciate this visually‐based project, rather than a written or oral analysis of the journal article. The Proteopedia project also helps the students see the relevance of their growing biochemistry knowledge to both current literature and to solving real‐world problems.
Dietary Fatty Acid Composition Influences Growth Performance and Hepatic Metabolism in Neonatal Pigs
Authors: Gerrard Sam , Seymour Kacie , Yonke Joseph , El-Kadi Samer
Published: April 2020

Medium‐chain fatty acids (MCFA) are often included in neonatal milk formulas as a more readily oxidized source of energy compared with long‐chain fatty acids (LCFA). This rapid oxidation of MCFA is thought to enhance growth. Thus, the objectives of this study were to evaluate the effect of energy supplementation and fat source on growth and body composition, and determine the rate of Krebs Cycle substrate utilization in neonatal pigs. Eleven three‐day‐old pigs were allocated to one of three sow milk replacer formulas: a control (CONT) and diets rich in either LCFA or MCFA. The CONT formula provided 80%, whereas the MCFA and LCFA formulas provided 120%, of the metabolizable energy requirements of neonatal pigs. Pigs were fed for 20 days and body composition measured by DXA before initiation and on day 20 of feeding. At the end of the study, pigs were euthanized and muscles and organs collected and weighed. Livers were digested with collagenase and isolated hepatocytes were incubated in media containing all amino acids, glucose, and short‐chain fatty acids in the presence of [U13C] labeled alanine, glucose, propionate, and glutamate for 90 min. The enrichment of Krebs Cycle intermediates was determined using gas chromatography/mass spectrometry. Body weight of pigs in the LCFA group was greater than those in the MCFA and CONT groups on days 18 and 20. In addition, percent fat measured by DXA was greater for MCFA (8%) and LCFA (7%) than those in the CONT (4%) group, and this increase in fat deposition occurred at the expense of lean tissue (P < 0.05). Longissimus dorsi and heart weights as percent of body weight were similar for all groups; however, soleus weight was less for MCFA compared with those in the CONT and LCFA groups. In addition, liver and kidney weights as a percentage of body weight were greater for pigs in the MCFA group compared with those in the CONT group with LCFA being intermediate (P< 0.05). There was no effect of formula feeding on glucose and alanine contribution to lactate synthesis in isolated hepatocytes, but this contribution was greater for glucose (36%) than for alanine (15%) (P<0.05). Conversely, the contribution of alanine (35%) carbon to pyruvate was greater than the contribution of glucose (7%) (P<0.05). Furthermore, in hepatocytes of pigs fed CONT formula 39% of pyruvate carbon was derived from alanine and 31–34% for pigs in the LCFA and MCFA groups (P<0.05). Lastly, glutamate contributed 64% of α‐ketoglutarate carbon in hepatocytes of CONT and MCFA and only 40% for LCFA pigs. These data suggest that dietary MCFA increase body fat without increasing lean tissue accretion compared with CONT. In addition, anaplerosis from glutamate was greater for pigs fed MCFA and CONT than for those fed LCFA.
Elevated miR‐125b inhibits trophoblast invasion by directly targeting KCNA1 in preeclampsia
Authors: Li Qinghua , Yin Lingxuan , Si Yanru , Meng Yuhan , Yang Weiwei
Published: April 2020

Preeclampsia (PE) is a pregnancy‐specific syndrome of human pregnancy associated with poor trophoblast invasion, which attributed to 63 000 maternal and 500 000 infant deaths annually worldwide. The incidence of PE has increased by 40% over the last several decades. Elevated miR‐125b in the placenta has been reported to be linked with the risk of PE. Our bioinformatic analysis showed that voltage‐gated potassium channel subfamily A member 1 (KCNA1) is a predicted target for miR‐125b. However, roles of miR‐125b in the pathogenesis of PE is not fully explored. Here we test the hypothesis that miR‐125b is involved in the development of PE by targeting KCNA1 in human trophoblast cells. We found that expression of miR‐125b was upregulated on first trimester in patients who later developed preeclampsia(p=0.0009<0.01), while KCNA1 levels were significantly decreased and negatively correlated with miR‐125b levels in the PE placentas (r2=0.6099, p<0.01). We validated that KCNA1 is a direct target of miR‐125b using luciferase assay and western blotting(p<0.05). We then conducted transwell invasion assay and found that miR‐125b significantly inhibited trophoblast cell invasion by 58.67±3.48% (compared to mock, p<0.05), knockout of KCNA1 significantly attenuated the invasion‐inhibitory effect of miR‐125b (p<0.05) while overexpression of KCNA1 reversed the effect of miR‐125b (p<0.05). In conclusion, these data suggest that elevated miR‐125b impaired trophoblast invasion via directly targeting KCNA1 in the pathology of PE. The findings highlight the function of KCNA1 in human placental cells and provide new insight of how miR‐125b is involved in the pathogenesis of preeclampsia.

Support or Funding Information

This research was supported by the National Natural Science Foundation of China (81601318) and Shandong Province Health and Medical Science and Technology Program (2016WS0668), Weifang Medical University (No. 2017BSQD11).
Acute Cervical Spinal Cord Injury Alters Swallow Motor Patterns
Authors: Iceman Kimberly , Strecker Courtney , Greene Clint , Howland Dena , Pitts Teresa
Published: April 2020

Cervical spinal cord injuries (cSCIs) can cause swallow disorder (dysphagia), potentially resulting in aspiration and pneumonia. Dysphagia is characterized by disruption of the swallow motor pattern, which involves sequential activation of muscles that propels the bolus into the esophagus while avoiding the airway. Normal swallow includes diaphragm activity that generates negative pressure, drawing the bolus into the esophagus. Centers in the brainstem control the swallow patterns. Thus, despite reports of dysphagia in some patients with cSCI, little consideration has been given to the potential contributions of spinal circuitry to swallow. We aim to characterize swallow dysfunction in cats with cSCI. Using EMG to record breathing patterns, water‐induced swallow in pentobarbital‐anesthetized spontaneously breathing cats is captured before and after acute high cervical hemisections (at C2 or C3). EMGs are recorded from the mylohyoid, thyrohyoid, thyroarytenoid, thyropharyngeus, cricopharyngeus (upper esophageal sphincter), and diaphragm muscles, and trans‐diaphragmatic pressure is measured by monitoring esophageal and stomach pressures. After hemisection, swallow is dramatically altered, as indicated by a dysfunctional swallow motor pattern. Coordinated, sequential muscle activation necessary for proper movement of a bolus through the pharynx is lost, esophageal pressure is positive rather than negative due to reduced activation of the diaphragm, and upper airway EMG activity increases. These alterations in behavior‐specific muscle recruitment patterns are profoundly dysphagic, indicating that disruption of spinal circuits in the high cervical spinal region can produce major dysfunction in swallow motor patterns. This work may inform screening and treatment protocols of the cSCI patient population, in which dysphagia is likely to be underdiagnosed.

Support or Funding Information

NIH grants HL 111215, NS 110169 and OT20D001983, the Craig H. Neilsen Foundation Pilot Research Grant 546714, Kentucky Spinal Cord and Head Injury Research Trust, the Commonwealth of Kentucky Challenge for Excellence, VA RR&D B9249S and the Rebecca F. Hammond Endowment.
Crystalizing Structured RNAs Using a Surface‐Entropy‐Reduced Fab as a Crystallization Chaperone
Authors: Roman Christina Alexandra , Koirala Deepak , Piccirilli Joseph
Published: April 2020

Our lab studies the molecular mechanisms of regulatory RNAs elements by determining their atomic resolution structure using x‐ray crystallography. However, RNA crystallization presents many challenges. One strategy to meet these challenges is the use of a protein chaperone to assist RNA crystallization. Antibody fragments (Fabs) provide one type of crystallization chaperone, and we have used these successfully for RNA crystallization in two ways. In one approach, a Fab‐binding‐RNA epitope is grafted into the RNA of interest to create a binding site for the cognate Fab crystallization chaperone. To improve this method, I performed surface entropy reduction on the Fab framework to make a version of our crystallization chaperone that is more suited to well‐ordered crystal packing. Using structural analysis software as a guide, I made twelve surface‐entropy‐reduced (SER) mutant versions of the Fab framework, in which patches of bulky, flexible residues are mutated to alanine. To test the performance of these mutants, I carried out crystallization trials of their complexes with the yjdF RNA, a novel bacterial riboswitch that binds to aza‐aromatic compounds. The wild type Fab (wt BL3‐6) and many of the mutants were able to crystalize with the riboswitch and gave diffraction datasets with 3.5 – 3 angstrom resolution range, and the structure is currently being solved. Using the mutants, I have also crystalized a second RNA target, PEMV2, which is a viral cap‐independent‐translation‐element. This RNA crystalized with wild type but could only diffract to 2.75 angstrom resolution with the aid of a mutant. I am in the process of solving this structure which will give valuable hints to the mechanism of action of this class of viral cap‐independent‐translation‐elements. As I refine this crystallization chaperone technology, I will also apply it to a notoriously difficult crystallization target, domain V of the human polio virus IRES.

Support or Funding Information

NIH, HHMI, The University of Chicago
ETB Receptor Function is Modulated by Estradiol in Young Women
Authors: Shoemaker Leena N. , Haigh Katherine M. , Kuczmarski Andrew V. , McGinty Shane J. , Welti Laura M. , Edwards David G. , Feinberg Ronald , Wenner Megan M.
Published: April 2020

It is well known that vascular endothelial function declines in women after menopause. Our laboratory has recently shown that impaired endothelial function in postmenopausal women is due in part to a loss of endothelin‐B (ETB) receptor mediated dilation. However, it is unclear whether these changes in ETB function are due to aging or menopause‐related declines in ovarian hormones. Therefore, the purpose of this study was to test the hypothesis that short‐term estradiol (E2) administration would enhance the contribution of ETB receptor mediated dilation during local heating in young women. Twelve young women (24 ± 4 years, 24 ± 3 kg/m2, mean arterial blood pressure: 84 ± 6 mm Hg) participated in this study. Endogenous sex hormone production was suppressed with daily administration of a gonadotropin‐releasing hormone antagonist (GnRHant; Ganirelix) for 10 days. E2 (0.1 mg/day, Vivelle dot patch) was added back on days 4–10. We measured cutaneous vasodilation to local heating (42°C) on day 4 (GnRHant) and day 10 (GnRHant+E2) via laser Doppler flowmetry coupled with intradermal microdialysis for the perfusion of lactated Ringer’s (control) and ETB receptor antagonist (BQ‐788, 300 nM). Cutaneous vascular conductance (CVC) was calculated as cutaneous red blood cell flux/mean arterial blood pressure, and expressed as a percent of maximal vasodilation (CVC %max) achieved with local heating to 43°C and perfusion of sodium nitroprusside (28 mM). E2 administration improved cutaneous vasodilation by 8 ± 10% (control site; P = 0.02) compared to GnRHant. ETB receptor blockade increased cutaneous vasodilation to local heating during hormone suppression with GnRHant (control: 84 ± 7 vs. BQ‐788: 89 ± 5 CVC %max, P = 0.02). However, ETB receptor blockade tended to reduce cutaneous vasodilation to local heating during short duration E2 administration (control: 90 ± 7 vs. BQ‐788: 85 ± 8 CVC %max, P = 0.08). These data indicate that suppression of endogenous ovarian hormone production alters ETB receptor responses in young women, mimicking the response profile in postmenopausal women observed in our previous data. Furthermore, these data suggest that E2 modulates ETB receptor function. Additional data are needed to determine ETB receptor function and sensitivity to E2 in postmenopausal women.

Support or Funding Information

Research supported by AHA Award 16SDG30700015 (MMW), NIH Grant U54‐GM104941 (PI: Binder‐Macleod), and P20 GM113125 (DGE).
Cathepsin‐B is a Mediator of Barrier Dysfunction and Hyperpermeability in Blood‐Brain Barrier Endothelial Cells
Authors: Peng Jasmine , Lambertis Amira , Hunter Felicia , Childs Ed , Tharakan Binu
Published: April 2020

Cathepsin B belongs to the papain‐like family of cysteine proteases and is an upstream mediator of the NLRP3 inflammasome pathway that leads to interleukin 1β (IL‐1β) secretion. Interleukin 1β is a potent inducer of tight junction disruption and hyperpermeability in blood‐brain barrier (BBB) endothelial cells. However, the involvement of cathepsin B in BBB dysfunction and hyperpermeability is not clearly known. Understanding this pathway is critical to preserving BBB integrity and protection against brain edema. In order to study this relationship, rat brain microvascular endothelial cells were transfected with active cathepsin B. Tight junction organization and changes in permeability were evaluated based on immunofluorescence, immunoblot analysis of zonula occudens‐1 (ZO‐1) and Transwell monolayer permeability assay using FITC‐dextran (10‐kDa). Cathepsin B activity and cell viability were evaluated fluorometrically. F‐actin stress fiber formation was studied using rhodamine phalloidin staining. Active cathepsin B induced ZO‐1 dislocation/tight junction disorganization, F‐actin stress fiber formation and monolayer hyperpermeability significantly. These effects were inhibited by pretreatment with NLRP3 inhibitors isoliquiritigenin and MCC950. Active cathepsin B had no significant effect on ZO‐1 protein expression or cell viability. Furthermore, cathepsin B inhibitor E‐64d attenuated hydrogen peroxide‐induced cathepsin B activity and monolayer hyperpermeability significantly. These results suggest that cathepsin B is a mediator of BBB dysfunction/hyperpermeability. Cathepsin B inhibition has protective effects against barrier dysfunction and hyperpermeability in BBB endothelial cells. One of the mechanisms by which cathepsin B induces barrier dysfunction is via the NLRP3 inflammasome pathway.
Effects of Acclimatization on Cognitive Function at High Altitude
Authors: Oeung Britney , Heinrich Erica C. , Puvvula Nikhil , Pham Kathy , Frost Shyleen , Brena Rebbecca , DeYoung Pamela N. , Orr Jeremy E. , Malhotra Atul
Published: April 2020

Acute high‐altitude exposure can lead to impaired neurocognitive function. However, it remains unclear if the duration of exposure impacts the degree of impairment or if acclimatization improves performance over time. We hypothesized that measures of sustained attention and reaction time would be impaired by acute high‐altitude exposure and would improve over three days of acclimatization. 14 participants (n=9 men; n=5 women) were included in the study. Participants completed a 30 minute cognitive function test battery (Cognition by Joggle Research) at sea level and each morning over three days at 3800 m elevation. Half of the participant group completed baseline sea‐level cognitive function testing before ascent to high altitude and half completed baseline tests at least 2 days after returning to sea level. The cognitive test battery evaluated sensory motor speed, visual learning, working memory and spatial working memory, abstraction, spatial orientation, complex scanning and visual tracking, risk decision making, and vigilant attention. Mean reaction time on the Psychomotor Vigilance Task (PVT) was higher at high‐altitude compared to sea level (SL: 199 ± 27 ms; Day 1 Alt: 224 ± 33 ms; p=0.005) and did not improve over three days of acclimatization. However, mean reaction time on the Line Orientation Task (LOT) was lower at high‐altitude compared to sea level (SL: 6076 ± 1831 ms; Day 1 Alt: 5173 ± 988 ms; p=0.02) and remained lower throughout acclimatization. The number of correct responses on the LOT did not change significantly upon ascent. Reaction times on the Digit Symbol Substitution Task (DSST) decreased (R=‐0.33, p=0.04) and the number of correct responses increased (R=0.32, p=0.04) throughout acclimatization. Mean reaction time on the Balloon Analog Risk Task (BART) decreased throughout acclimatization (R=‐0.35, p=0.02). These data suggest that while some cognitive domains including complex scanning and visual tracking (DSST) improve with acclimatization, behavioral alertness and sustained attention (PVT) remain impaired. Furthermore, spatial orientation measured by the LOT was improved at high altitude, consistent with studies showing no change, or improvements in visuospatial perception, under mild stress. Further work is required to determine the mechanisms underlying the effects of acclimatization on cognitive function, the domains in which improvement occurs, the time course of these changes, and whether interventions can impact these processes.

Support or Funding Information

This work was funded by the University of California, Riverside, School of Medicine.
Influenza A Virus Escape Mutations and their Effects on Binding Affinity
Authors: Burke Lucas S. , Kalyanaraman Karshana , Flores Stephanie , Khong Trang , Selover Danielle , Rusch-Salazar Laura
Published: April 2020

The Influenza A virus (IAV) causes a common and potentially fatal infection. The Center for Disease Control reports that 3–11% of the U.S. population is affected annually by IAV. The 2017–2018 burden of influenza led to over 950,000 patient hospitalizations and almost 80,000 deaths. The surface of IAV contains two proteins essential for infection: hemagglutinin (HA) and neuraminidase (NA). HA binds to a receptor on an epithelial host cell surface, and helps IAV penetrate the cell and release the viral RNA genome. As newly formed IAV particles leave the infected cell, NA assists in viral release and driving the infection of neighboring cells. Due to frequent, natural mutations in the IAV genome, annual IAV vaccines are engineered but are not always effective. The Schiffer Lab propagated IAV in vitro in the presence of an antibody to identify mutations of IAV that can escape immune detection. A critical asparagine located in the binding pocket of HA (N203HA) facilitates a hydrogen bond to sialic acid on LSTc (a human receptor analog). The N203VHA escape mutation disrupts the virus‐ligand interaction; however, an allosteric mutation on NA (E329KNA) can compensate for the HA mutation. Our 3‐D printed model of the HA active site visually depicts the binding of the N203 residue with the LSTc. This model further illustrates that viruses can use allosteric mutations to circumvent the immune system. Physically modeling the active site of HA allows students to investigate how substrates fit into active sites and visualize mutations within proteins.

Support or Funding Information

The CREST Program is funded through DUE‐IUSE #1725940 awarded to Milwaukee School of Engineering Center for BioMolecular Modeling.
NF‐κB activation in Osteoarthritis Occurs Independent of Primary Cilia Associated Proteins HSP27 and IKK Activity
Authors: Rose Brandon James , Kooyman David L. , Porter James , Woolstenhulme Jonny , Hanson Romney , Woodbury Reagan , Marchant Drew
Published: April 2020

Abnormal or nonfunctioning primary cilia are known to be deleterious in cartilage homeostasis, promoting both abnormal cartilage modeling and early onset osteoarthritis. Two proteins specifically regulated by the primary cilia are involved in modulating transcriptional factor nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) activity; heat shock protein 27 (HSP27) aids in activating NF‐κB by degrading inhibitor of kappa‐B kinase alpha (IκBα), IκB kinase (IKK) phosphorylates and inactivates IκBα, freeing NF‐κB to translocate to the nucleus and initiate transcription of inflammatory genes. We hypothesized, therefore, that HSP27 and IKK would be upregulated in osteoarthritic tissue, which would allude to inflammatory mismanagement by the primary cilia as a possible mechanism. The destabilization of the medial meniscus technique (DMM) was successful in inducing osteoarthritis, as manifest by increased Mankin scores compared to rats undergoing the sham surgery (P = 0.009) and increased prevalence of cyst‐like lesions (P = 0.01). Surprisingly, we found no statistically significant difference in HSP27 (P = 0.768) or IKK (P = 0.214), though a statistically significant increase in NF‐κB expression was observed (P = 0.0000877). We therefore conclude that ciliary intervention in osteoarthritis occurs independent of this mechanism, and propose that future research regarding the role of primary cilia in osteoarthritis center on indirect activation of inflammatory mechanisms.
Small‐Molecule Sigma1 Modulator Suppresses PD‐L1‐Mediated Cancer Cell Adaptive Immune Resistance in Response to Interferon‐Gamma
Authors: Haas Derick A. , Kim Felix J.
Published: April 2020

Background

Sigma1 (also known as sigma‐1 receptor) is a unique ligand‐regulated integral membrane protein enriched in the endoplasmic reticulum (ER) of cancer cells. Sigma1 acts as a multi‐functional scaffolding or chaperone protein to maintain protein and lipid homeostasis in the secretory pathway of these cells. Programmed Death‐Ligand 1 (PD‐L1) is a type I integral membrane glycoprotein that is processed through the secretory pathway. Upon reaching the cell surface, PD‐L1 acts as an inhibitory immune checkpoint molecule by binding to its cognate receptor, programmed death‐1 (PD‐1), on activated T cells, resulting in T cell inactivation, thus suppressing the anti‐tumor immune response. PD‐L1 protein expression and transport to the cell surface can be upregulated by interferon‐gamma (IFN‐γ). This mechanism is known as adaptive immune resistance, whereby cancer cells change their phenotype in response to a proinflammatory immune response. PD‐L1 is also incorporated onto cancer cell‐derived extracellular vesicles called exosomes, which can inactivate T cells within the tumor microenvironment and at distant sites. We previously reported that Sigma1 associates with PD‐ L1 and that small molecule Sigma1 inhibitors (Sigma1i) can trigger lysosomal degradation of PD‐L1.

Objective

The purpose of our present study is to examine the role of Sigma1 in the PD‐L1 life cycle, including interferon gamma (IFN‐γ) signal transduction, upregulation of PD‐L1 mRNA, post‐translational modification of PD‐L1, transport to the cell surface, and incorporation onto exosomes.

Methods

Six cancer cell lines from multiple cancer types were assessed for PD‐L1 protein expression by immunoblot and PD‐L1 mRNA by qRT‐PCR. PD‐L1 surface expression was assessed by flow cytometry and biotinylation assays. Exosomes were isolated by ultracentrifugation and concentrations and particle size profiles were determined using nanoparticle tracking. We used Sigma1i to examine the role of Sigma1 in the PD‐L1 life cycle.

Results

We found that Sigma1i blocks IFN‐γ induced PD‐L1 at a post‐translational step. Cotreatment of Sigma1i with IFN‐γ results in accumulation of Endoglycosidase H (EndoH) sensitive PD‐L1 in the ER and thus prevents cell‐surface expression of PD‐L1. Furthermore, Sigma1i blocked IFN‐γ‐induced incorporation of PD‐L1 on cancer cell exosomes.

Conclusion

We have found that Sigma1i causes nascent PD‐L1 to be retained in the ER, preventing it from progressing through the secretory pathway, reaching the cell surface, and from being packaged onto exosomes. By reducing cell surface PD‐L1 and exosomal PD‐L1, Sigma1 lation could provide a novel approach to enhancing anti‐tumor immunity.
The Incidence and Anatomical Characteristic of Anterior and Posterior Meniscofemoral Ligaments. A Meta‐Analysis.
Authors: Paziewski Mateusz Paweł , Pękala Przemysław Andrzej , Pękala Jakub Radosław , Mann Mitch , Pasieka Paweł Melchior , Tomaszewski Krzysztof Andrzej , Walocha Jerzy Andrzej
Published: April 2020

Introduction

The meniscofemoral ligament consists of two divisions ‐ anterior (aMFL) and posterior (pMFL). MFL plays a role as a knee stabilizator and supports the posterior cruciate ligament (PCL). The main purpose of this study was to summarize the anatomic features of the meniscofemoral ligament in the field of arthroscopic surgery, to update current clinical guidelines.

Materials and methods

A comprehensive search was conducted through the largest medical databases. Two reviewers were involved in search and data extraction. Both AQUA tool and PRISMA guidelines were strictly followed during the research.

Results

The study enrolled 61 articles into the analysis. The prevalence of aMFL was 56.2% (95%CI: 46.7–65.4%) of 4366 knees. It was more common in males than in females (37.2% vs. 29.1%) and occured more often in right than in left knees (53.4% vs 46.6%). Mean length and thickness of aMFL equaled 23.00 mm (95%CI 22.8–23.5mm) and 1.7mm (95%CI 1.6–1.8mm) respectively. The incidence of pMFL, was 7 1.0% (95%CI 64.3–77.2%) out of 5056 lower limbs. The pMFL was more likely to appear among females than males (81.1% vs. 76.7%) and in right legs (56.1% vs. 43.9%). Mean length and thickness was 27.4mm (95%CI 27.1–27.7mm) and 2.5mm (95%CI 2.4–2.5mm) respectively. Both aMFL and pMFL occurred together in 33.4% (95%CI 22.1–38.6%), but both were absent in 7.2% (95%CI 2.7–11.8) of cases.

Conclusions

Anterior and posterior meniscofemoral ligaments are very common structures of knee joint. Clinicians should take it under attention during arthroscopic surgery to avoid its damage. MFLs high prevalence develop a high demand to expound its function and relevance in knee surgery.
Gastrin release in a mucosal organ culture model exposed to a simulated digest of an equine dietary supplement
Authors: MacNicol Jennifer L. , Pearson Wendy
Published: April 2020

Horses are prone to GI diseases including colic, colitis, and gastric ulcers. Equine dietary supplements are commonly used as a means of supporting optimal equine gastrointestinal health. G’s Formula (manufactured by G’s Organic Solutions INC., BC, CAN) is an equine dietary supplement composed of dried cabbage, carrot, oat flour, and hemp meal, formulated to promote optimal GI health in horses. The objective of the current study was to investigate the impact of a simulated gastric digest of GF on gastrin (Gt) release in an in vitro mucosal organ culture model. Porcine stomachs were collected from a local abattoir (Reist and Weber, ON, CAN). A section of mucosa from the pyloric antrum was isolated and the smooth muscle removed. A 4mm punch biopsy was used to divide the mucosa, and 2 biopsies were placed into each well of a 24‐well culture dish. Each well was filled with 1.5 ml of media supplemented with glutamine. Media was changed at 24h, at which time 100 ul of either a simulated gastric digest of GF (GF), a simulated gastric digest blank (BL), or PBS (CO) was added into sample wells. Media and digests were refreshed at 48h, at which time wells were either stimulated with 10−5M carbachol (GF‐s, n=12; BL‐s, n=12; CO‐s, n=11) or sham stimulated with PBS (GF‐n, n=9; BL‐n, n=13; CO‐n, n=10). This process was repeated at 60h, and the experiment was terminated at 72h. Gt release in the media was assessed using a porcine Gt ELISA (MyBioSource INC., SD, USA) and standardized /g tissue. Data was analyzed using a repeated measures ANOVA in SAS 9.4 using PROC GLIMMIX (SAS Institute, NC, USA). There was a difference between the treatments GF and BL (16534±797 vs 14072±723 pg/ml/g, p=0.03) and a trend towards a difference between GF and CO (14581±794 pg/ml/g, p=0.09). At 60h GF‐n was higher than CO‐n (17475±1784 vs 12164±1688 pg/ml/g) and GF‐s was higher than BL‐s (16903±1543 vs 12434±1544 pg/ml/g). GF‐s was higher at 72h compared to 48h (18289±1543 vs 14051±1543, p=0.03). In conclusion, a simulated gastric digest of GF added into an in vitro organ culture of porcine pyloric antrum appears to enhance tissue Gt release.

Support or Funding Information

This project was funded by G’s Organic Solutions INC.

J.L. MacNicol was funded by NSERC.
Recombinant Expression and Purification of Wild‐type JHA15 from Aedes aegypti
Authors: Cancino Leah Serena Hagemann , Rascón Alberto A.
Published: April 2020

The Aedes aegypti mosquito serves as a major vector of the Dengue, yellow fever, and zika viruses, which can be found in tropical regions around the world, as well in certain regions of the United States. The primary method of disease transmission is through the bite of an infected female mosquito taking a blood meal from an uninfected human host, which is a process necessary for the gonotrophic cycle. Once the blood meal is consumed, midgut proteases degrade blood meal proteins into amino acids and peptides that are then used in egg development. The common proteases found in the midgut, which are expressed after a blood meal, consist of chymotrypsins, trypsins, and exo‐peptidases. The focus of this study is on the JHA15 protease, which is believed to exhibit chymotrypsin‐like activity and is constantly expressed immediately after a blood meal. However, the exact role in this process has not been fully elucidated. Therefore, we are working towards understanding the catalytic and specific activity of the protease in vitro, which will help determine the specific role in the mosquito. To initiate these studies, JHA15 with the wild type pro‐peptide region was cloned into the pET28a vector. Once the gene was cloned, soluble protease expression was achieved utilizing Shuffle T7 competent Escherichia coli cells (NEB) grown in TB media at 10°C. The protease was purified with a Ni+2‐chelating column and purity analyzed through SDS‐PAGE. Future research will focus on activation and enzyme kinetics of the protease to determine if it is indeed involved in blood meal digestion.
Reduced sympathetic baroreflex sensitivity in individuals with multiple sclerosis during the Valsalva maneuver
Authors: Trotter Claire E. , Huang Mu , Keller David M. , Davis Scott L.
Published: April 2020

Multiple sclerosis (MS) is a neurodegenerative autoimmune disease characterized by demyelination in central nervous system leading to potential impairments in the autonomic control of cardiovascular function. We have previously demonstrated individuals with MS exhibit a diminished ability to increase blood pressure in response to a simulated hypotensive stimulus (carotid neck pressure) compared with healthy controls likely due to impaired sympathetic modulation of the vasculature. The aim of the current investigation was to test the hypothesis that individuals with MS have a blunted muscle sympathetic nerve response (MSNA) to drops in arterial blood pressure induced via a Valsalva maneuver compared to matched healthy controls. Four patients with relapsing‐remitting MS (2 females/2 males, EDSS < 4) and 4 sex‐, age‐ and mass‐matched healthy controls were instrumented for MSNA (peroneal nerve), arterial blood pressure (Finometer), and heart rate. Participants performed a Valsalva maneuver (VM) at 40 mmHg for a 15 s after a normal inspiration. Sympathetic baroreflex function was assessed by relating all sympathetic burst occurring during the 15 s straining period of the VM to the maximum fall in DBP measured from highest values (Phase I) to the nadir prior to the end of the VM (Phase III). Drops in diastolic pressure during the Valsalva were similar between groups (MS: −31 ± 10 mmHg: CON: −25 ± 15 mmHg; P = 0.30). Individuals with MS produced less total MSNA bursts during the 15 s VM compared to healthy controls (MS: 8.3 ± 4.1 bursts/15 s; CON: 15.0 ± 4.1 bursts/15 s; P = 0.003). Sympathetic baroreflex sensitivity was reduced in individuals with MS (MS: −0.32 ± 0.25 bursts/15 s/mmHg) vs. CON: −0.77 ± 0.43 bursts/15 s/mmHg; P = 0.048). Individuals with MS have a reduced ability to increase MSNA in response to decreases in blood pressure during the VM confirming previous speculations of abnormal sympathetic baroreflex control of arterial blood pressure in this clinical population.

Support or Funding Information

NIH R15HL117224; National MS Society RG4696A3
Observations on the Correlation of Basic Science Content in Year 1 With the Clinical Science Content in Year 2 Within a Modified System Based Integrated Spiraled Medical Curriculum
Authors: Osborne David L. , Funk Cindy K. , Ketchum Robert J.
Published: April 2020

In August 2016, Instruction began at the Burrell College of Osteopathic Medicine. Curriculum planning commenced in 2015 and has been constant in preparation of teach subsequent year. The class that began as the inaugural class (class of 2020) will graduate in May of 2020. Now, the first year curriculum is being presented in its fourth iteration and the second year curriculum is presented in its third iteration. Input from students, faculty and administration has been incorporated as the curriculum has evolved into its present form. This presentation will highlight the observations and inputs that have contributed to the growth of the curriculum from planning to present form. The original iteration of the curriculum was modeled after the two pass integrated curriculum that was developed at Texas College of Osteopathic Medicine and employed at Rocky Vista College of Osteopathic Medicine. Faculty designed the basic order of systems for presentation and the weekly design of the sessions in each system over the fall and spring of 2015–2016. The number of faculty involved continually grew as the faculty were hired during this period. The basic science faculty designed the systems courses in the Year 1 curriculum. Clinical faculty designed the Clinical Skills and Osteopathic Manipulative Medicine components of both years 1 and 2. The clinical faculty also designed the systems courses of Year 2 of the curriculum. As a result, the first year of the curriculum is based in the general science that serves as a basis for understanding medicine while the second year of the curriculum has a distinctly clinical focus related to diagnosis and basic treatment of diseases. Connection of the content between year 1 and year 2 is the area of biggest concern related to the curriculum. Planning and execution do not always align and leads to many gaps that are seen in the knowledge base of the students. Communication between the faculties teaching each year is vital but is often less than optimal. Bloating in each year of the curriculum is the result, but often exacerbates problems as opposed to resolving issues. Administration and faculty opinions often differ as to the cause of the problem, as well as, how to correct the situation. Administrative organization, curriculum order, length of semester, and faculty turnover have all be looked at to address various issues. We will address what has been done to facilitate improvements in the program as the first four years have progressed.
Impact of increases in heat stress frequency associated with climate change on lifespan in different thermally adapted populations of an inter‐tidal marine invertebrate
Authors: Smith Autumn , Sun Patrick
Published: April 2020

Climate change is expected to change temperature regimes though multiple avenues such as increasing maximal environmental temperatures as well as frequency of daily maximum temperatures, which has been relatively understudied to the former. This study examines the chronic effect of heat stress due to higher frequencies of current daily maximum temperature on lifespan of two populations the intertidal marine copepod Tigriopus californicus with vastly different thermal tolerances. This study applied different heat stress regimes to the two populations and monitored their survival for the entirety of their lifespan. Our results show that frequency of heat stress impacts lifespan.