bluuebunny/crossref_metadata_2025 huggingface dataset:
Vitamin D deficiency contribute to the development of preeclampsia through increased sFlt‐1 production and impaired vascular function
Authors: Yang Weiwei , Li Qinghua , Yin Lingxuan , Si Yanru , Gao Zhiqin , Meng Yuhan , Cao Bin , Wang Yanling
Published: April 2020

Preeclampsia (PE) is a pregnancy‐specific syndrome that affects 5~8% pregnancies and is a leading cause of maternal and neonatal morbidity and mortality. Although the underlying mechanism remains unclear, PE is widely accepted to be induced, in part, by uteroplacental ischemia, which leads to increased antiangiogenic factors, such as sFlt‐1, which antagonizes the angiogenic factors, causing widespread vascular dysfunction. Vitamin D deficiency (VDD) is a widespread public health issue, and increasing studies suggested that VDD is a possible risk factor for the development of PE; however, the underlying mechanism that contributes to the increased risk of preeclampsia in vitamin D deficient patients is not clear. We tested the hypothesis that vitamin D deficiency contributes to the development of PE in a placental ischemic rat model by increasing sFlt‐1 production and impairing vascular function. We first induced VDD in Sprague‐Dawley rats (DYET 119273 vs 110842, Dyets Inc., USA) prior to RUPP (Reduced Uterine Perfusion Pressure) surgery on gestational day (GD) 14. Blood pressure was monitored via carotid catheter. On GD19, uterine arteries were harvested to determine vascular reactivity on a wire myograph. We found that RUPP surgery significantly increased mean arterial blood pressure as expected(Ctrl+Sham, n=12, 102±10 mmHg; Ctrl+RUPP, n=12, 117±11 mmHg; P<0.01), and this was significantly enhanced by vitamin D deficiency (VDD+RUPP, n=12, 136±11 mmHg; P<0.01). Plasma sFlt‐1 concentrations were significantly increased in VDD rats (n=12, 117±15 pg/mL) compared to the control group(n=12, 49±17 pg/mL)(P<0.05), and VEGF concentrations were significantly decreased(Ctrl, n=12, 1899±371 pg/mL; VDD, n=6, 1071±249 pg/mL, P<0.05). VDD induced endothelial dysfunction in uterine arteries (% relaxation, control, n=6, 95±7%; VDD, n=6, 37±11%, P<0.05). The circulating biomarkers for endothelial dysfunction also significantly increased. The E‐Selectin increased from 5166±1217 pg/mL to 5756±1053 pg/mL (P<0.05). The ICAM‐1 levels increased from 3.15±1.03 ng/mL to 3.50±0.94 ng/mL (P<0.05). The VCAM‐1 level increased from 44±13 ng/mL to 53±11 ng/mL (p<0.05). These results suggest that vitamin D deficiency contributes to the onset of preeclampsia by increased sFlt‐1 production and impaired vascular function.

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).
Repeatability of Cerebral Vasomotor Reactivity to Hypercapnia in Humans
Authors: Nandadeva Damsara , Patik Jordan C. , Martin Zachary T. , Fadel Paul J. , Brothers R. Matthew
Published: April 2020

Impaired cerebrovascular function is a risk factor for the development of various neurocognitive and cerebrovascular diseases including cognitive dysfunction, dementia, Alzheimer’s disease, and stroke. Since hypercapnia elicits a robust cerebral vasodilator response, cerebrovascular function is commonly assessed as an increase in cerebral blood flow to elevations in arterial carbon dioxide (i.e., cerebral vasomotor reactivity ‐ CVMR). Hypercapnia induced by a rebreathing protocol is one such technique used to assess cerebrovascular function. Although this technique is used to evaluate CVMR within and between subjects before and after interventions, to our knowledge, the repeatability of CVMR assessed by rebreathing has not been comprehensively investigated. Moreover, the National Institute of Health’s recent guidelines emphasize the need for rigor and reproducibility of data collection techniques. Accordingly, this study was designed to determine the within‐day repeatability of CVMR response to rebreathing‐induced hypercapnia. Eleven young healthy males (age = 24 ± 3 years, BMI = 24.3 ± 3.8 kgm−2, mean ± SEM) reported to the laboratory following an overnight fast and performed 3 trials of rebreathing‐induced hypercapnia each separated by approximately 2 hours. Heart rate (ECG), respiration (Pneumotrace), beat‐to‐beat blood pressure (Finometer), middle cerebral artery mean blood velocity (MCAv ‐ transcranial Doppler) and breath‐by‐breath end‐tidal carbon dioxide concentration (PETCO2 ‐ capnograph) were continuously measured. Cerebral vascular conductance index (CVCi) was calculated as MCAv divided by mean arterial pressure. CVMR was assessed as the slope of the linear regression between the increase in %MCAv and %CVCi across the range of change in PETCO2 (ΔPETCO2). The increase in %MCAv and %CVCi was also assessed at a ΔPETCO2 of 15 mmHg which was the highest common change in magnitude of hypercapnia achieved across all rebreathing trials among all participants. Repeatability between the 3 trials was assessed using intra class correlation coefficient (ICC). The slopes of %CVCi vs. ΔPETCO2 (3.9 ± 0.4, 3.8 ± 0.3 and 3.6 ± 0.3 %mmHg−1 for 1st, 2nd and 3rd trial respectively, p = 0.82) was not different and showed very good repeatability (ICC = 0.86, p < 0.001) between the 3 rebreathing‐induced hypercapnia trials. Likewise, the % increase in CVCi at ΔPETCO2 of 15 mmHg was not different (40 ± 4, 38 ± 4 and 36 ± 4% for 1st, 2nd and 3rd trial respectively, p = 0.72) and also demonstrated very good repeatability between trials (ICC = 0.86, p < 0.001). Similar results were obtained when MCAv was used as the dependent variable. These results suggest that within‐day CVMR assessed by rebreathing‐induced hypercapnia is highly repeatable and therefore can be used as a reliable index of cerebrovascular function.

Support or Funding Information

Supported by UTA College of Nursing and Health Innovation
Group A Streptococcus May Suppress V‐ATPase Activity To Inhibit THP‐1 Macrophage Phagolysosomal Acidification
Authors: Lee Jimin , Nishioka Scott , Snipper Joshua , Okumura Cheryl Y.M.
Published: April 2020

Infection with Group A Streptococcus (GAS), a human‐specific bacterial pathogen, results in a multitude of diseases ranging from strep throat to toxic shock, and can lead to serious post‐infection sequelae such as rheumatic heart disease. During infection, macrophages engulf bacteria within the phagosome, which then fuses with the lysosome. Subsequent acidification of the phagolysosome allows for degradation of bacteria. Our lab and others have shown that during GAS infection, phagolysosomes are not acidified, allowing GAS to persist. The overall objective of this study was to determine how GAS inhibits phagolysosomal acidification. Our lab has previously shown that secretion of the pore‐forming toxin Streptolysin O (SLO) causes phagolysosomal leakage of relatively large proteins. In our current work, we demonstrate that during infection using a mutant strain lacking SLO or using heat‐killed bacteria that GAS‐infected phagosomes are still not acidified, indicating that lack of acidification is not due to mere leakage. Phagolysosomes undergo acidification using the enzyme complex vacuolar ATPase (VATPase), which needs to properly assemble cytosolic and membrane components to function. To assess whether V‐ATPase assembly was affected, infected cells were fractionated and recruitment of cytosolic V‐ATPase subunits to the phagosome membrane was monitored by Western blot. Our preliminary results indicate that V‐ATPase assembly is not inhibited, suggesting that GAS may instead prevent acidification by inhibiting V‐ATPase activity. We are currently conducting further research to investigate this potential inhibitory mechanism of GAS. Restoring the ability of lysosomes to become acidified and activate proteolytic enzymes may serve as a means to enhance macrophage function, both bactericidal capacity and antigen presentation capability, to elicit effective long‐lasting immunity.

Support or Funding Information

American Heart Association (17GRNT3410851), Occidental College Undergraduate Research Center
A context dependent requirement for Fig4 catalytic function in yeast
Authors: Reeves Hannah , Khan Imran , Muñoz-Rivera Alejandra , Habib Alim , Weisman Lois , Strunk Bethany S.
Published: April 2020

PI lipid phosphatases modulate signaling cascades through localized dephosphorylation of specific phosphoinositides (PI) on subcellular membranes. Additionally, some of these phosphatases regulate cellular processes through lipid phosphatase independent functions. Notably, Fig4, a PI 5‐phosphatase proposed to dephosphorylate PI3,5P2, promotes the synthesis of its substrate independent of its catalytic activity. Fig4 activates its opposing kinase, Fab1, to facilitate dynamic elevation of PI3,5P2 by associating with the Fab1‐Vac14‐Fig4 complex. While Fig4 is critical for development, its catalytic function is not, as catalytically impaired Fig4 rescues neonatal defects in a Fig4 knock‐out mouse. Nevertheless, mice expressing catalytically impaired Fig4 alone display late on‐set neurological abnormalities and reduced life spans indicating that Fig4 catalytic activity is critical for long‐term homeostasis. Strikingly, we show that catalytic impairment of yeast Fig4 is more detrimental to yeast growth on rapamycin and heat stress than total loss of Fig4. Toxicity of catalytically impaired Fig4 can be rescued by co‐expression of wild‐type Fig4 implying that this toxicity results from loss of catalytic function. Moreover, yeast expressing hyperactive Fab1 show similar growth defects on rapamycin and heat stress in the absence of Fig4, which suggests that elevated levels of PI3,5P2 are responsible for this phenotype. These studies provide a new model for investigating the physiological implications of loss of Fig4 catalytic activity.

Support or Funding Information

NIH R00 GM120511‐04
Inhibition of Androgen Receptor Decreases Fat Metabolism by Decreasing Carnitine Palmitoyltransferase I Levels in Skeletal Muscles of Trained Mice
Authors: Kim Jisu , Hwang Hyejung , Park Hun-young , Jung Wonsang , Kim Sung-Woo , Lim Kiwon
Published: April 2020

Purpose

Androgen hormone levels are strongly associated with obesity in adult mammals, especially with advanced age. We investigated androgen receptor inhibition on fat metabolism and long‐chain fatty acid (LCFA) transport proteins in skeletal muscle during exercise.

Methods

Male ICR mice were randomly divided into three groups: CON (control), EX (exercise), and EXIN (exercise + androgen receptor inhibition). EX and EXIN groups were trained on a treadmill five times a week. After four weeks, the fat metabolism of each group was measured using open‐circuit calorimetry during one hour of exercise. After the metabolism measurement, the expression levels of LCFA transport proteins (FAT/CD36, CPTI) were analyzed in skeletal muscle.

Results

Weight gain and final body weight were significantly lower in the EX group than in either the CON or EXIN groups. Conversely, food intake was significantly higher in the EX group than it was in the CON and EXIN groups. The total weight (CON; 2.07 ± 0.6, EX; 1.64 ± 0.2, EXIN; 1.95 ± 0.2) of the abdominal adipose tissue were significantly lower in the EX group than in the CON and EXIN groups (P < 0.05). However, there was no different between the CON and EXIN group. Oxygen uptake and fat oxidation during exercise tended to be lower (12%) in the EXIN group than in the EX group. Total fat oxidation in the EXIN group was significantly lower during the initial 20‐min (P < 0.003) and 40‐min (P < 0.041) phases compared to that in the EX group. In addition, the level of FAT/CD36 protein in the EX and EXIN groups was approximately double that in the CON group (P < 0.001, P < 0.001). CPTI expression in the EX group was higher than that in the EX group (P < 0.0069) as well as in the CON group.

Conclusion

Exercise training increases the expression of LCFA transport proteins (FAT/CD36, CPTI). However, blocking androgen receptors decreases the expression of CPTI in the skeletal muscle, which reduces fat metabolism. Thus, reducing sex hormones or suppressing the sensitivity of AR receptors can inhibit energy efficiency and fat metabolism by suppressing CPTI.

Support or Funding Information

This work was supported by the Ministry of Education of the Republic of Korea and the National Research Foundation of Korea (NRF‐2015S1A5B5A01016088).
Needs and Opportunities for Ethics and Responsible Conduct of Research (E/RCR) integration in Course‐based Undergraduate Research Experiences: the ENCOUR Fellowship Program
Authors: Diaz-Martinez Laura A. , D'Arcy Christina E. , Olimpo Jeffrey T.
Published: April 2020

Course‐based undergraduate research experiences (CUREs) have been shown to enhance students’ development of cognitive and non‐cognitive skills in science, technology, engineering and mathematics (STEM) fields resulting in the proliferation of CURE offerings in college course catalogues. These courses often constitute the initial research experience for undergraduate students in the biological sciences, thereby establishing students’ perceptions of what it is to be a researcher. Though ensuring the integrity of the scientific enterprise through the ethical and responsible conduct of research (E/RCR) is central to the scientific process, documented efforts to integrate E/RCR education in the context of CUREs is lacking. In an effort to address this information gap, we conducted a mixed methods study to evaluate E/RCR implementation efforts within biological sciences CUREs at the national level. Results from these efforts indicate a general awareness from CURE stakeholders of the importance of E/RCR education in CUREs. The majority (65.15%) of individuals surveyed reported having to resolve E/RCR issues within their CUREs and agree that E/RCR education should be included. Although a great majority (96.96%) of respondents reported addressing at least one E/RCR topic in their CUREs, the responses evidenced a lack of planned and intentional integration of E/RCR education into such learning environments. Prominent issues identified by our study participants as barriers for the effective integration of E/RCR education into their CUREs were a perceived lack of time (92% of respondents), resources (85% of respondents), and appropriate training for instructors. Based on these results, the Ethics Network for Course‐based Opportunities in Undergraduate Research (ENCOUR) has developed a fellowship program that aims to serve as a national professional development model for instructors facilitating E/RCR education in biology CUREs. ENCOUR fellows will participate in a year‐long program designed to enhance their E/RCR knowledge and to guide them through the development and assessment of educational resources for effective integration of E/RCR topics into their CUREs.

Support or Funding Information

This work was supported by the National Science Foundation Awards 1727867 and 1919312
Endogenous β‐endorphin Plays a Pivotal Role in Angiotensin II‐mediated Pressor Response
Authors: Badr Safy , Rezq Samar , Hassan Rasha
Published: April 2020

Background

Endorphins are endogenous opioid neuropeptides that are mainly produced from the pituitary gland in response to pain and stress. A number of mediators were reported to stimulate β‐endorphin production including interleukin 1 beta (IL‐1β) and corticotropin‐releasing factor (CRF). Angiotensin II (Ang II) can stimulate β‐endorphin production from anterior pituitary gland, but the exact molecular mechanisms involved in this effect, as well as the role of the released β‐endorphin in Ang II‐mediated pressor response, remain elusive. Our study aimed to fill this knowledge gap.

Methods

Male rats (6 per group) were either injected with IL‐1β receptor blocker (IL‐1R antagonist, 100 μg /kg, s.c.), the CRF receptor blocker, astressin (100 μg /kg, Intranasal) or a combination of both prior to Ang II injection (200 μg/kg, ip). Other groups of rats were given naloxone (1.6 mg/kg, s.c.) or telmisartan (10 mg/kg, oral) prior to Ang II injection. Serum and hippocampal β‐endorphin levels, brain IL‐1β and CRF levels were detected. Moreover, markers of oxidative stress including malondialdehyde (MDA) and superoxide dismutase (SOD), the inflammatory marker, C‐reactive protein (CRP) and sympathoexcitation (c‐Fos, L‐glutamate, and phosphorylated MAPK) markers were measured in the hippocampus of different groups.

Results

Ang II increased serum and hippocampal β‐endorphin levels by 37.95% and 28.03%, respectively. This response was attenuated when astressin or/and IL‐1R antagonist were given before Ang II injection. Moreover, Ang II induced a pressor response, increased hippocampal IL‐1β (1.4‐fold), CRF (7‐fold), MDA (1.5‐fold) and CRP (3.3‐fold) while deceased SOD (by 31.49%) levels. Also, Ang II injection increased c‐Fos (9.9‐fold), glutamate (3.1‐fold) and phosphorylated‐MAPK (5.6‐fold) levels. The previous effects were abolished when telmisartan was given prior to Ang II injection, while naloxone aggravated Ang II‐mediated pressor response and most of the biochemical changes.

Conclusion

These findings suggest that Ang II can induce β‐endorphin release by increasing the levels of both IL‐1β and CRF and that the released endorphin mitigates Ang II‐mediated sympathoexcitatory response. This study reveals new insights on the intermediary role of β‐endorphin in Ang II‐mediated sympathoexcitation and highlights it as a possible target for treating hypertension.
Investigation into the Metabolic Limitations of Alpha Conotoxins as Drugs in the Gastrointestinal Tract
Authors: Gusman Emma , Sandbach Arika , Espiritu Michael
Published: April 2020

Peptides isolated from cone snails, known as conotoxins, have potential in treating neuropathic pain. Peptides often lack efficacy as drugs due to poor pharmacokinetic profiles despite displaying high potency and selectivity. Conotoxins present significant sequence diversity, but little information exists about their pharmacokinetic properties. We hypothesize that this diversity will correlate with dynamic pharmacokinetic profiles, with some peptides exhibiting increased stability. In this study, we aimed to gain a basic understanding of the stability of the conotoxins α‐IMI and α‐GI in the stomach and intestines. Both have different primary sequences despite serving as antagonists of the acetylcholine receptor, which is a potential target in cancer, chemotherapy‐induced allodynia and possibly pain associated with Crohn’s disease. In order to study these peptides, novel mass spectrometry (LC‐MS/MS) methods were developed incorporating multiple reaction monitoring scans for each peptide in simulated gastric and intestinal fluids. The quantitation range of our method was 0.5–5 μg/mL, which is 20 times more sensitive than traditional methods. To compare gastrointestinal metabolic stability, each peptide was incubated in simulated gastric fluid and intestinal fluid and their stability was measured using our LC‐MS/MS method at times ranging from 0–90 minutes. Both peptides displayed a high amount of stability in gastric fluid, as no significant loss of either peptide was determined after 90 minutes. In intestinal fluid IMI degraded rapidly (t1/2= ~6 minutes), whereas α‐GI displayed far more stability in the same environment (t1/2= ~257 minutes). α‐GI incorporates a prominent helix in its structure which is likely responsible for its increased stabilization in comparison to IMI. Considering the differences in the metabolic stability of these peptides, further study of conotoxin sequences may result in the identification of stable molecular scaffolds. In the future, we plan to pinpoint what particular features are responsible for metabolic stability or susceptibility across more conotoxins, such as differences in sequence, structure and rigidity and plan to use this information to produce analogs with increased stability within the gastrointestinal tract.

Support or Funding Information

Source of Research Support: This work was supported by the Pacific University Research Incentive Grant Program and an ASPET Summer Undergraduate Research Fellowship (SURF) award at Pacific University.
Spinal trigeminal subnucleus pars muralis: An interface for apneic reflex
Authors: Perez Amalia Callado , Kleinfeld David , Deschênes Martin
Published: April 2020

Noxious stimulation of the nasal cavity elicits protective reflexes for the upper respiratory tract, including sneezing, mucus secretion, apnea and cardiovascular reflexes. Yet, no studies have shown the full circuit responsible for the apneic response upon noxious stimulation of the nasal mucosa. Here, we show that injection of cholera toxin beta subunit into the nasal mucosa leads to terminal labeling in the transition zone between the interpolaris and caudalis subnuclei of the trigeminal sensory complex, i.e. subnucleus muralis (SpVm). We also provide physiological evidence that ammonia vapor delivered into the nasal cavity induces an apneic reaction and activates some SpVm cells. Subnucleus muralis cells also respond to mechanical stimulation of the narial pad and the nasal mucosa. Yet, none of the muralis cells responded to vibrissa deflection. Electrolytic lesion of SpVm or unilateral transection of the anterior ethmoidal nerve prevent apnea induced by ammonia delivery to the nasal epithelium, demonstrating their crucial role in the apneic reflex. We also found direct projections from SpVm to the respiratory centers, particularly the preBötzinger complex (preBötC). Inhibition of inspiratory preBötC cells and increased firing of expiratory preBötC cells are associated with ammonia‐induced apnea.

Anatomical and physiological data presented here support the hypothesis that nasal induced apnea may be mediated by direct projections from nasal innervated Subnucleus muralis neurons to the PreBötC complex.
Control of Vasoconstriction by Myoendothelial Ca2+ Signaling Through Gap Junctions
Authors: Saavedra Andrea , Espinoza Hilda , Burboa Pia , Figueroa Xavier
Published: April 2020

In resistance arteries, smooth muscle cells (SMCs) and endothelial cells (ECs) are communicated through gap junctions (i.e. myoendothelial gap junctions), which provide a signaling pathway for control of vasomotor tone. In this context, the Ca2+ signaling associated to smooth muscle contraction might also be transmitted via myoendothelial gap junctions and activate a regulatory feedback mechanism (myoendothelial feedback) by the production of endothelium‐dependent vasodilators. It is thought that IP3 is the main Ca2+‐related signaling transmitted from smooth muscle cells to endothelial cells, but the direct contribution of Ca2+ to the myoendothelial feedback is not clear. We investigated the temporal pattern of vasoconstriction in mesenteric resistance arteries, and their temporal relationship with the increase of Ca2+ in SMC and EC. We used intact mesenteric resistance arteries of Sprague dawley male rats (≤ 10 mm in length) to measure the vasoconstriction and changes in [Ca2+]i of smooth muscle cells and endothelial cells observed in response to phenylephrine (PE) or KCl using the fluorescent indicators of Ca2+, Fluo‐4 and X‐Rhod‐1.Changes in [Ca2+]i were also evaluated in primary cultures of mesenteric endothelial cells. All procedures were approved by the local Bioethical Committee (ID:180806005). The vasoconstriction induced by PE and KCl was not only associated with an increase of [Ca2+]i in smooth muscle cells, but also in endothelial cells, which was prevented by the blockade of L‐type voltage‐dependent Ca2+ channels with 10 μM nifedipine or the inhibition of gap junctions with 50 μM 18‐b‐Glycyrrhetenic acid. Although the stimulation with PE or KCl did not evoke an increment of [Ca2+]i in primary cultures of endothelial cells, the response induced by these vasoconstrictors was larger after blocking nitric oxide (NO) production with NG‐nitro‐L‐arginine (100 μM). These results suggest that the direct diffusion of Ca2+ contributes to the NO‐mediated component of the myoendothelial feedback.

Support or Funding Information

Conicyt 21171840
Characterization and Comparison of Human IAPP to Animal IAPPs
Authors: Torres Jaris , Lamba Arleen , Nogaj Luiza A.
Published: April 2020

Islet Amyloid Polypeptides (IAPPs), are insoluble amyloids or small oligomers that are notorious for aggregating into deposits that destroy pancreatic β‐islet cells, the primary producers of insulin. The aggregation of the 37‐amino acid polypeptide human islet amyloid polypeptide (hIAPP) seems to play a role in this aggregation and cell death process. IAPP aggregation has been associated with the development of type II diabetes, a disease that affects 30.3 million people of all ages—or 9.4% of the U.S. population, and is an epidemic that has devastated the lives of many. MTT assays were performed to assess cell viability of HeLa cells that were treated with the IAPPs of various animals. Subsequently, LDH Cytotoxicity assays were conducted to test the viability of the cellular membrane upon addition of the same IAPPs. This research explores the relationship between the IAPP sequences, their propensity to aggregate, and their effect on mammalian cell viability. To do this we compared aggregation potential and cellular toxicity of full‐length IAPP from several diabetic and nondiabetic organisms whose aggregation propensities had not yet been determined for full‐length IAPP.
Evaluating the Dose Dependent Response to DTT Treatment Resulting in HSF Activation in Drosophila cells
Authors: Shirk Austin , Choi Annette , Hrizo Stacy , Buckley Martin
Published: April 2020

Proteins drive many of the biological processes in cells. To do this, they fold into complex 3D structures that are integral for their functionality. Notably, various environmental and chemical stressors can disrupt protein folding and thus disable the functions of proteins, threatening the livelihood of cells. To mitigate this stress, organisms initiate the highly‐conserved heat shock stress response. In eukaryotes, the master heat shock activator heat shock factor (HSF) is rapidly recruited to the Hsp70 heat shock protein genes and triggers the recruitment of additional co‐activator proteins that facilitate gene expression. This leads to the production of heat shock proteins that function as molecular chaperones to promote refolding of proteins, prevent aggregation and increase protein degradation pathways. Notably, activation of the heat shock response pathway, can be visualized by measuring GFP‐tagged HSF binding to the heat shock protein genes in living Drosophila salivary gland nuclei. Our lab is currently using this technique to identify novel compounds that induce the heat shock response pathway. Our pioneering experiments have shown that diamide and hydrogen peroxide, two chemicals known to cause protein misfolding and activation of the heat shock response pathway, trigger the recruitment of GFP‐HSF to the Hsp70 loci in living salivary gland cells to a similar level as HS stress. In addition, to our surprise, high levels of Dithiothreitol (DTT, 100 mM), a chemical known to cause protein misfolding and activation of the unfolded protein response pathway (UPR), results in the recruitment of GFP‐HSF to the Hsp70 gene loci. Here we describe experiments to further explore a possible dose dependent activation of HSF by DTT.

Support or Funding Information

Student Faculty Research Grant for Slippery Rock University
Catalytic Characterization of Human Malate Dehydrogenase 1 (MDH1)
Authors: Huynh Ngoc H. , Hoang An , Sohl Christal D.
Published: April 2020

Lung cancer is the leading cause of cancer death. Non‐small cell lung cancer (NSCLC) makes up 85% of all types of lung cancer, and includes squamous cancer, adenocarcinomas, and large cell cancer. Malate dehydrogenase (MDH) exists as two forms, MDH1 (cytosolic) and MDH2 (mitochondrial). Both forms are elevated in tumor cells, but only high expression of MDH1 is associated with poor prognosis. Patients with amplified MDH1 have a 50% reduction rate of survival. MDH1 appears to support proliferation of tumor cells by fueling glycolysis with the regeneration of nicotinamide adenine dinucleotide (NAD+). However, studies of the kinetics of human MDH1 is limited. Our goal is to characterize the catalytic features and regulation of MDH1. We hypothesized that MDH1 activity is inhibited in the presence of reactive oxygen species (ROS). Human MDH1 was expressed heterogenously from Escherichia coli and purified using immobilized cobalt affinity chromatography. The enzyme catalytic activity was determined in the optimal conditions of salt, pH, temperature, and buffer. In the assay, MDH1 activity was calculated from the oxidation rate of NADH to NAD+ at 340 nm using a spectrophotometer after incubating MDH1 with oxaloacetate and NADH under steady‐state conditions. To test our hypothesis under oxidative environment, we repeated this assay using oxidized glutathione. The enzyme activity was 6‐fold lower than that measured in reducing conditions. In the absence of the enzyme, oxidized glutathione does not convert NADH to NAD+, which implied the enzyme’s conformation was affected and hence the observed loss in enzyme activity. In the future, we will employ site‐directed mutagenesis to gain an insight whether our observed inhibition was due to the sensitivity of cysteine residues to oxidation.

Support or Funding Information

This work was funded by a Research Scholar Grant, RSG‐19‐075‐01‐TBE, from the American Cancer Society (C.D.S.), National Institutes of Health R00 CA187594 (C.D.S.), U54CA132384 (SDSU) & U54CA132379 (UC San Diego), MARC 5T34GM008303 (SDSU), and IMSD 5R25GM058906 (SDSU), as well as the California Metabolic Research Foundation (SDSU).
The Molecular Magic of “Meatless” Meats: Structural and Sequence Similarities between Soy Leghemoglobin and Bovine Globins
Authors: Shelton Katelyn , Najera Karina , Ajredini Sara , Navarro Jazhel , Frangias Themistocles
Published: April 2020

Plant‐based proteins are becoming more prevalent in the modern day due to humanity’s fight against climate change. How do scientists produce plant‐protein that tastes similar to animal‐protein? When comparing bovine hemoglobin (2qss), soy leghemoglobin (2GDM) and bovine myoglobin (2qss), the proteins differ by location, number of heme proteins, oxygen affinity and backbone alignment. Hemoglobin is found in red blood cells of animals. More specifically, myoglobin is found in the red blood cells of muscle tissue in animals. Leghemoglobin, however, is the byproduct of the symbiotic relationship between root nodules on legumes and nitrogen‐fixing bacteria that lives on them. However, all of these proteins are linked by the common molecule, heme. Plant‐based “meat” manufacturers suggest that this heme group, that is embedded in each of these globins, is responsible for this “meaty” taste. Additionally, the presence of umami‐inducing amino acids was investigated and glutamic acid surface prevalence related to flavor. Using the suite of structural analysis applications in the Protein Data Bank, such as FATCAT and fast2BLAST, the structural similarities between bovine hemoglobin, especially the heme itself, bovine myglobin and soy leghemoglobin can be illustrated, both quantitatively and qualitatively (e.g. 3D‐printed models).
Epigallocatechin‐3‐gallate inhibits tumor angiogenesis via endoglin/Smad1 pathway in human umbilical vein endothelium cells
Authors: Sheu Ming-Jyh , Chen Chiao-Yun
Published: April 2020

Endoglin, a transforming growth factor‐β (TGF‐β) co‐receptor, is highly expressed in endothelial cells and various solid tumors, including lung cancer. It may be involved in endothelial cell (EC) differentiation, migration, and proliferation. TRC105, an endoglin‐neutralizing antibody, increased VEGF signaling in ECs. Besides, anti‐VEGF treatment increased the endoglin level. Therefore, we hypothesized that a combination of both endoglin and VEGF inhibitors may increase and maintain anti‐angiogenetic activity in human umbilical vein endothelial cells (HUVECs). Structures of all 2000 compounds from the TCM database were used for screening to find the potential compounds. Discovery Studio (version 4.5) was used for the virtual screen of various compounds targeting the endoglin. Among them, epigallocatechin‐3‐gallate (EGCG) showed its possibility to bind with endoglin. The sulforhodamine B (SRB) and western blotting assay were used to determine the cell density and measurement of the cellular protein content. Our results showed that EGCG demonstrated the strongest binding affinity to endoglin, and inhibited the endogin protein expression in HUVECs. The main contribution of the study indicated that EGCG significantly decreased the semaxanib‐induced overexpression of endoglin. Therefore, a combination treatment including EGCG will probably solve the drug resistance to anti‐VEGF treatments.

Support or Funding Information

China Medical Universityand Asia University (CMU106‐ASIA‐14) and Show Chwan Memorial Hospital, Changhua, Taiwan (RD107042)
Usual and Unexpected Variations in the Veins of the Posterior Abdomen: A Case Study
Authors: Stewart Ian F.
Published: April 2020

Two venous system components comprise the drainage of the posterior abdominal cavity. One component drains blood from the visceral derivatives of the urogenital ridge (e.g., kidneys, gonads) directly to the inferior vena cava (IVC). The other venous system collects blood from the body wall and drains into the azygos system through the subcostal vein, ascending lumbar vein, and lower posterior intercostal veins. Veins, and especially the azygos system, are notoriously variable. Much of this variability arises from the complex embryological development of the caval venous system in the thoracic and abdominopelvic cavities; but the formation of venous anastomoses representing multiple routes of drainage from a single region also contribute to the variability of veins.

During routine dissection of the abdomen of a male cadaver (Caucasian, 67 years), the right testicular vein demonstrated multiple anatomical variations. An anomalous vein arising from several small tributaries at the lateral end of the 11th and 12th ribs was discovered during the dissection. This vein crossed the posterior abdominal wall inferomedially to drain into the right testicular vein. Further exploratory dissection revealed direct communication between the anomalous vein and the right 11th posterior intercostal vein, which could be traced from the origination of the anomalous vein to the connection with the azygos vein at the appropriate vertebral level. The anomalous vein represents an unusual route for collateral venous circulation between the azygos system and the caval system in the abdomen via the right 11th posterior intercostal vein. Additionally, the right testicular vein drained into the right renal vein instead of directly draining into the IVC. No other notable variations were observed in the abdominal vasculature. The left testicular vein, left renal vein, left posterior intercostal veins, and left subcostal vein all demonstrated typical merging patterns. Accompanying renal, testicular, and posterior intercostal arteries on the right side also appeared to have normal branching patterns.
Inhibition of Spinal Inflammation Restores Compensatory Plasticity Induced by Recurrent Reductions in Respiratory Neural Activity in Offspring Exposed to Gestational Intermittent Hypoxia
Authors: Mickelson Carly R. , Gumnit Maia G. , Meza Armand L. , Radcliff Abigail B. , Watters Jyoti J. , Baker Tracy L.
Published: April 2020

Sleep disordered breathing (SDB) during pregnancy is increasing in parallel with the obesity epidemic, yet the long‐term effects on the adult offspring are unknown. In our animal model of SDB during pregnancy, pregnant rats are exposed to chronic intermittent hypoxia (8 hrs/day, 2 min 10.5% O2 separated by 2 min of 21% O2) or intermittent normoxia from gestational days 10–21. Preliminary data show that adult male (but not female) offspring exposed to gestational intermittent hypoxia (GIH) show deficits in respiratory control that manifest as increased spontaneous central apneas during presumptive sleep and impaired compensatory responses to recurrent reductions in respiratory neural activity, a form of plasticity known as inactivity‐induced inspiratory motor facilitation (iMF). Preliminary data also show that microglia isolated from male GIH offspring show primed inflammatory responses in respiratory control regions. Since neuroinflammation impairs respiratory plasticity, we tested the hypothesis that inhibiting NF‐kappaB and STAT3 inflammatory transcription factor activation restores the capacity to elicit iMF in GIH offspring. Phrenic inspiratory output was measured in urethane‐anesthetized, vagotomized, mechanically ventilated GIH or gestational intermittent normoxia (GNX) offspring. Vehicle or 2‐[(aminocarbonyl)amino]‐5 ‐(4‐fluorophenyl)‐3‐ thiophenecarboxamide (TPCA‐1) was delivered intrathecally over the phrenic motor pool ~1 hour before exposure to recurrent reductions in neural activity (5, ~1 min neural apnea episodes, separated by 5 min). As expected, recurrent neural apnea elicited a significant increase in phrenic inspiratory output in vehicle‐ and TPCA‐treated GNX offspring (54±17% and 62±12% baseline, n=6, 5, respectively, p<0.05) when compared to time controls not receiving activity deprivation (11±4% baseline, n=4). By contrast, recurrent neural apnea did not elicit iMF in vehicle‐treated GIH offspring (25±3%, n=7, p>0.05). However, in GIH offspring receiving intrathecal TPCA‐1, recurrent neural apnea induced a significant increase in phrenic inspiratory output (75+/−10% baseline, n=7, p<0.05), indicating that NF‐kappaB and STAT3 inflammatory transcription factor inhibition rescues the capacity to elicit iMF in GIH offspring. These data indicate that increased neuroinflammation in male GIH offspring impairs the capacity for recurrent reductions in respiratory neural activity to trigger compensatory forms of neuroplasticity.

Support or Funding Information

Supported by R01HL142752 (JJW and TLB)
Development of a hands‐on learning activity on potential DNA damage and skin cancer for a nursing biochemistry class
Authors: Paliwal Vipin , Masoud Nazieh , Nickel Anne-Marie
Published: April 2020

Skin cancer (basal cell carcinoma) is one of the most common forms of cancer in the US (about 4.3 million cases/year). Prolonged exposure to sunlight is one of the major causes of skin cancer due to mutations in the DNA. Ultraviolet‐B (UV‐B) radiations in the region of 290–320 nm of the solar spectrum are absorbed by basal keratinocyte cells resulting in mutated DNA leading to cancer. We developed a two‐part active learning experience for the nursing biochemistry class. Part I: In this activity, the relative intensity of UV radiation at 308 nm in the absence and presence of sunscreen was measured using a UVX Digital Radiometer‐UVX31 Sensor by first‐year nursing students in a biochemistry course at the Milwaukee School of Engineering (MSOE). A class of 20 students brought in their own commercially available sunscreens to class. Students tested the sunscreens’ ability to block the UVB light. A thin layer of sunscreen was applied to a plastic wrap sheet and placed between the UV source and the sensor. Students compared the intensity of the UV light (in mW/cm2) transmitted through an uncoated and coated plastic wrap. They then observed that different commercial sunscreens have different abilities to block UV light.

DNA damage by UVB causes mutations due to the development of cyclobutane‐pyridine photodimers commonly recognized as thymine‐thymine (T‐T) dimers. Part II: Students made connections between the real‐life classroom activity performed in Part I and a three‐dimensional physical model of DNA segment (crystal structure source entries: ndb UD0053 or PDB 1SM5) showing the formation of the T‐T dimer using a hand‐held model and images. The model was constructed by 3D‐printing. Students were challenged to locate the T‐T dimers in the model and discuss the possible impact of the change in the DNA structure.

The activity motivated the nursing biochemistry students and enhanced their learning experience with DNA mutations by directly relating it to everyday products such as sunscreens and a 3‐D model showing the biochemical DNA damage which could lead to skin cancer. Additionally, the two‐part activity enhanced one‐on‐one student‐faculty interactions in the classroom. In the future we plan to conduct an in‐depth quantitative educational study to understand the benefits of this active learning exercise as compared to traditional classroom instruction.

Support or Funding Information

Summer professional development grant, Milwaukee School of Engineering, Milwaukee, WI. Center for BioMolecular Modeling (CBM), Milwaukee School of Engineering, Milwaukee, WI.
The Commander protein complex and its role in intracellular membrane trafficking
Authors: Collins Brett
Published: April 2020

Membrane compartmentalisation is a defining feature of all eukaryotic cells and we have evolved sophisticated protein machineries to control the flow of transmembrane molecules and lipids between organelles. One of the most dynamic membrane bound compartments is the early endosome, which controls the sorting of transmembrane cargos as they transit through the secretory and endocytic system. Our lab is deciphering the molecular mechanisms that control this membrane sorting using a combination of structural and cellular biology. ‘Retromer’ is one of the best characterised endosomal transport protein machines, and is mutated in Parkinson’s Disease and recently we and others identified a ‘Retromer‐like’ protein complex named ‘Retriever’. Retriever forms a large macromolecular assembly with other proteins including the so‐called COMMD and CCDC complexes and a member of the sorting nexin protein family called SNX17. The mega‐complex has been dubbed ‘Commander’, and plays an important role in endosomal trafficking of many receptors including integrins, lipoprotein receptors, ATP7A copper transporters, ENaC epithelial sodium channels, and amyloid precursor protein and other receptors. In this seminar I will discuss our work towards an understanding of the molecular basis for how Commander is assembled, interacts with transmembrane cargos, and regulates their recycling or degradation.
A Combination Therapy of Cisplatin and Lovastatin Reduces Ras Activation in an A375 Melanoma Cell Line
Authors: Shock Nicole Kuss , Byrd Shere K.
Published: April 2020

In many cancers mutations in the Ras protein lead to cancer growth and metastasis. In a mutated form, Ras allows cells to avoid a normally programed death called apoptosis. Ras is a GTP‐binding protein and attaches to the cell membrane through a prenylated tail. When attached, it will activate and signal for the promotion of cell survival. The prenylated tail and membrane insertion will keep Ras in its activated state. Typical antineoplastic agents are highly toxic therapies, but by using them in combination with alternative therapies such as lovastatin, proliferating cells may be held in check with reduced side effects. The purpose of these experiments was to examine Ras prenylation and activation in a melanoma cell line, following treatment with a combination of lovastatin and cisplatin. Flow cytometry using annexin‐V apoptosis assay was performed to determine drug toxicities for each drug and in combination. Melanoma cells were treated for 48 with 10 uM lovastatin, then 1.5 ug/ml cisplatin was added for an additional 24 hr. Membrane localization of Ras was determined using immunohistochemistry followed by analysis with ImageJ software. The results show that Ras localization to the membrane in cells undergoing the combined treatment was reduced 4‐fold relative to cisplatin alone (p<0.05). These results indicate that statin therapy combined with traditional cancer drugs such as cisplatin may be more effective at slowing cancer growth with lower toxicities, than with cisplatin alone.

Support or Funding Information

Supported by the NIH Common Fund, through the Office of Strategic Coordination, Office of the NIH Director with the linked awards: TL4GM118992, RL5GM118990, & UL1GM118991 and a Fort Lewis College Undergraduate Research Grant.
Probing the Function of Perilipin 5b
Authors: Dalton Rebekah C. , Tansey John T.
Published: April 2020

Many modern health issues arise from aberrant lipid metabolism, among these are type II diabetes and non‐alcoholic fatty liver disease. One commonality these diseases share is neutral lipid metabolism and storage. Regulation of neutral lipid metabolism is of vital importance in the body, and among the proteins responsible for such regulation are the perilipins. Perilipins are a family of five conserved proteins that are found on the surface of lipid storage droplets and play a central role in the regulation of cellular neutral lipid metabolism. Perilipin 5 specifically, is expressed in tissues with a high capacity for fatty acid oxidation such as cardiac muscle, oxidative muscle, and fasting liver. Using western blotting and reverse transcriptase PCR, we have identified a splice variant of perilipin 5 we have termed perilipin 5b. This shortened form of the protein retains the amino terminal 35 kDa of the protein but lacks the putative 4‐helix bundle found in the C‐terminus. Based on the reports in the literature, this protein would lack the carboxy terminal domains reported to be necessary for interactions with lipases and other proteins but would retain serine 155 that is requisite for PKA phosphorylation and translocation to the nucleus. We have constructed a carboxy terminal truncation of perilipin 5 and are expressing it in CHO cells to further define the function of this protein. Immunofluoresence microscopy of CHO cells expressing perilipin 5b shows localization on lipid storage droplets and on puncta in the cytosol. Stimulation of the PKA signaling pathway in these cells leads to the phosphorylation of perilipin 5 and a shift to the nucleus. Collectively these data indicate that perilipin 5b plays a role in lipid storage, but the function of this truncation is still unknown.
Mgr2 regulates membrane insertion of marginal stop‐transfer signals in the mitochondrial inner membrane
Authors: Lee Seoeun , Kim Hyun
Published: April 2020

The TIM23 complex mediates membrane insertion of presequence‐containing mitochondrial proteins via a stop‐transfer mechanism. Stop‐transfer signals consists of a hydrophobic transmembrane segments and flanking charges. Mgr2 functions as a lateral gatekeeper of the TIM23 complex. However, it remains elusive which features of stop‐transfer signals are discriminated by Mgr2. To determine the effects of Mgr2 on the TIM23‐mediated stop‐transfer pathway, we measured membrane insertion of model transmembrane segments of varied hydrophobicity and flanking charges in Mgr2 deletion or overexpression yeast strains. We found that upon deletion of Mgr2 the threshold hydrophobicity for membrane insertion or the requirement for matrix‐facing positive charges are reduced. These results imply that Mgr2‐mediated gatekeeper function is important to control membrane sorting of marginal stop‐transfer signals.

Support or Funding Information

This research was funded by grants from National Research Foundation of Korea (NRF‐2016R1A2B2013459) and Promising‐Pioneering Research Program through Seoul Nation University to HK. Work in the laboratory of MvdL is supported by the Deutsche Forschungsgemeinschaft (SFB 894, IRTG 1830). Work in the laboratory of RI is supported by the CNRS ATIP program.