bluuebunny/crossref_metadata_2025 huggingface dataset:
Loss of β‐cell Gαz protects against high‐fat diet induced glucose intolerance by preserving incretin responsiveness and enhancing insulin secretion
Authors: Carbajal Kathryn , Schaid Michael , Green Cara , Laundre Erin , Harrington Jeffrey , Kelly Grant , Reuter Austin , Fenske Rachel , Wehner Molly , Lamming Dudley , Kimple Michelle
Published: April 2020

Cyclic‐adenosine monophosphate (cAMP) is an important secondary messenger in the insulin‐secreting pancreatic β‐cells. Intracellular production of cAMP is augmented by stimulatory heterotrimeric G proteins (Gs) and blocked by inhibitory heterotrimeric G proteins (Gi). Pancreatic expression of the unique Gi family member, Gαz, is restricted to the islet, which contains β‐cells and other endocrine cell types, including glucagon/GLP‐1‐secreting alpha cells and somatostatin‐secreting delta cells. We have previously shown that full body Gαz‐null mice are protected from hyperglycemia in several diabetes model systems. Here, we use a high fat diet (HFD) model of obesity and insulin resistance to explore the impact of loss of beta‐cell Gαz on the type 2 diabetes (T2D) phenotype of male C57BL/6J mice. We hypothesized that loss of beta‐cell Gαz in the context of HFD feeding would enhance insulin secretion, protecting mice from HFD‐induced glucose intolerance. To generate beta‐cell specific Gαz‐null and control mice, we bred Gαz flox/flox mice with rat insulin promoter RIP‐Creherr mice. At 12 weeks of age, transgenic Gαz‐null mice were fed a diet containing 45 kcal% fat or a 10 kcal% fat control diet for 26 weeks. At study end, mice were metabolically phenotyped and sacrificed for whole pancreas and islet phenotyping. Beta‐cell‐specific Gαz loss protected mice from HFD induced glucose intolerance due to a selective effect on islet insulin secretion, particularly in concert with a stable agonist of the Gs‐coupled GLP‐1 receptor. This protective effect was independent of changes in Gαz‐coupled EP3 receptor signaling, as neither EP3 expression nor production of its endogenous ligand, PGE2, were enhanced in islets from HFD‐fed mice. This protective effect was also beta‐cell autonomous, as both full‐body and beta‐cell‐specific Gαz‐null mice had enhanced GLP‐1‐positive alpha‐cells, albeit with no changes in active GLP‐1 secretion. Finally, gene microarray results confirm that Gαz loss and HFD feeding synergize to differentially regulate islet gene expression, specifically up‐regulating secretion‐centric genes, many of which are related to cAMP signaling. This study presents a novel inhibitory mechanism of receptor‐independent G‐protein signaling that may hold promise for developing beta‐cell targeted diabetes therapeutics.
After exertional heat stroke, is cooling to <40°C sufficient? Revisiting an established protocol in a mouse model
Authors: Alzahrani Jamal Mohammed , Garcia Christian K. , Murray Kevin O. , Robinson Gerard P. , Sheikh Laila A. , Laitano Orlando , Clanton Thomas L.
Published: April 2020

Exertional Heat Stroke (EHS) is a life‐threatening illness that can lead to multisystem tissue damage and increased mortality. To attenuate these consequences, immediate cooling to <40°C is the current recommendation. It is believed that the rate of cooling is a key factor in preventing tissue injury; however, much is unknown. In this study, we have used a model which allows us to test the effect of different cooling rates on recovery from EHS.

METHODS

Thirteen female mice were randomly assigned to one of two groups, a slow‐cooling group (SC) and the rapid‐cooling group (RC). Both groups underwent emitter implantation surgery which allowed us to monitor core temperature (Tc) in real time. After two weeks of recovery, mice were introduced to free running wheels inside their cages for another two weeks. After that, mice received forced running wheel training for one week. On the day of EHS exposure, mice were placed inside a chamber, kept at 37.5°C/40% humidity. Mice ran at closely controlled speeds until they reached symptom limitation (i.e. loss of consciousness at ~42.3°C). The RC group was immediately taken outside the chamber and allowed to recover at room temperature ~23°C. However, the SC group was kept inside the running wheel enclosure until consciousness (~9 min) and then allowed to recover for another 2 hours inside their home cage at 37.5°C.

RESULTS

The total thermal area averaged higher in the SC group, but did not reach statistical significance (P=0.1291). The time mice spent >39.5°C to the maximum Tc (Tc,max), and from Tc,max to 39.5°C was not statistically different. However, the SC group exhibited greater mortality than the RC (P=0.012). In the SC group, the time spent to cool from Tc,max to 37.5°C was statistically significant (P<0.0025). Also, the time from Tc,max to the minimum core temperature was statistically significant (P<0.0001).

CONCLUSIONS

In conclusion, this model helped us to distinguish the effect of different cooling rates on mortality. The data suggests that quickly cooling to only 40°C may be insufficient to avoid heat injury or death, but cooling to 37.5°C may improve outcomes.

Support or Funding Information

Supported by the BK and Betty Stevens Endowment and a Fellowship from King Saud University, Saudi Arabia
Stimulator of Interferon Genes (STING) Regulates Endothelial Healing and Reendothelialization Following Angioplasty‐Mediated Vascular Injury
Authors: Anastasiou Marina , Carrillo-Salinas Francisco Javier , Smolgovsky Sasha , Aronovitz Mark , Kaur Kuljeet , Alcaide Pilar
Published: April 2020

Angioplasty and stent implantation are common clinical interventions to improve blood flow in Peripheral Artery Disease, which is rising in prevalence in America. However, these interventions result in vascular endothelial damage, leading to the activation of signaling pathways that can have a major effect on short‐ and long‐term vessel health. We hypothesized that endothelial cell‐intrinsic Stimulator of Interferon Genes (STING), a crucial regulator of immune responses triggered by pathogen or self‐derived DNA, regulates reendothelialization and healing in response to vascular injury. To study whether STING‐mediated reendothelialization was an endothelial cell specific effect, in vitro wound healing assay was performed on WT and STING deficient primary mouse heart endothelial cells (MHEC) using live imaging and quantification with Cytation and IncuCyte. We found that, while WT MHEC completely covered the scratch area within 24 hours of initial scratch, only 75.59% ± 5.8 of the initial wound was healed in STING deficient MHEC monolayers. Furthermore, our in vivo experiments using the wire injury‐mediated experimental model of angioplasty in mice followed by Evans Blue Dye staining to distinguish the areas with denuded endothelium, demonstrate a decrease in reendothelialization in injured carotid arteries in STING deficient mice as compared to WT mice five days post injury (43.57% ± 9.79 area reendothelialized vs 95.16% ± 2.77, respectively). Seven days post wire injury, WT carotid arteries were completely healed, in contrast to those of STING deficient mice that remained unhealed (67.74% ± 5.80 of reendothelialized vessel area). Additionally, multi‐parameter flow cytometry analysis of the enzymatically digested arteries from the seven days post wire injury group demonstrated an increase in myeloid cell infiltration in WT mice, which was not observed in either the injured STING deficient arteries or in the uninjured contralateral WT carotid arteries, indicating an early localized and differential immune response likely affecting long term vascular remodeling. Our results support that endothelial STING contributes to endothelial healing in vitro and in vivo, and that myeloid cell infiltration in the carotid arteries is required for reendothelialization and may be responsible for lack of healing in STING deficient mice. Ongoing experiments aim to definitively quantify temporal recruitment of immune cells over the course of reendothelialization and chronic vascular remodeling following wire injury and the mechanisms involved in short‐ and long‐term vascular health.

Support or Funding Information

This work was supported by the NIH‐RO1‐HL123658
Experimentally evolved resistance to ionizing radiation in Escherichia coli after 100 cycles of selection
Authors: Lancaster Illissa Jasmine , Liu Jessica , Trimarco Joseph , Bruckbauer Steven , Cox Michael M.
Published: April 2020

Ionizing radiation (IR) causes damage to proteins, RNA, and DNA molecules within a cell, primarily through generation of reactive oxygen species (ROS). Building on previous work, we have now experimentally evolved four lineages of Escherichia coli (IR9, IR10, IR11, IR12) to be extremely IR resistant. Starting with the first round of selection using a dose of 750 Gy to kill 99% of the Founder strain, at round 130 of selection the populations now require a dose of nearly 4,000 Gy to maintain similar lethality. As of this selection cycle, these populations are near the IR resistance of the radioresistant bacterium Deinococcus radiodurans. Each of these populations have evolved separately to counteract sources of DNA damage in exchange with fitness tradeoffs in the absence of IR. All four populations display clonal interference, and three of the populations contain over 1,000 polymorphisms. We previously explored variants of DNA repair enzymes that contribute to IR resistance in the four populations and recently discovered an additional DNA repair protein variant (RecJ). Along with this RecJ variant, we have also uncovered novel pathways to IR resistance through variants of proteins in ATP synthesis (AtpA), iron‐sulfur cluster biogenesis (SufD) and cadaverine synthesis (CadA). Although these variants describe IR resistance in lineage IR9, other mechanisms in the other three evolved lineages remain to be explored.
Inhibition of Notch signalling using fucose analogs
Authors: Hao Huilin , Liu Zilei , Wu Peng , Haltiwanger Robert
Published: April 2020

Notch is an evolutionarily conserved cell surface receptor for short range cell‐cell communication. Notch signalling has critical roles in many cell‐fate related events, such as cell differentiation, proliferation and survival. Dysfunction of Notch signalling is associated with various congenital disorders and a wide range of human malignancies. Notch is an important drug target for cancer therapy. However, the initial efforts to inhibit Notch activity with γ‐secretase inhibitors (GSIs) have failed because of severe gastrointestinal toxicity. Novel Notch‐targeting strategies are urgently needed. One unique characteristic of the Notch receptor is the heavy O‐glycosylation of its extracellular domain (ECD). The O‐linked glycans, especially O‐fucose added by POFUT1 (Protein O‐Fucosyltransferase 1), are necessary for proper Notch function, such as protein stability, trafficking and interaction with ligands. We hypothesize that disrupting Notch O‐fucosylation can be a potential approach for Notch inhibition. We have demonstrated that L‐fucose analogs with carbon 6 modifications are potent Notch inhibitors, especially 6‐alkynyl and 6‐alkenyl fucose. The analogs are efficiently incorporated to Notch EGF repeats by POFUT1, and inhibit Notch signalling at sub‐micromolar concentrations. Interestingly, these analogs inhibit Delta but not Jagged‐mediated Notch activity, suggesting they are the first small molecule, ligand‐specific Notch inhibitors, and can be a powerful tool for Notch study. Notch controls the early differentiation of T cells in thymus. Blocking Notch signalling with the analogs showed a complete inhibition of T cell differentiation with Delta ligands in an in vitro OP9 cell culture system. We have preliminary results showing the analogs also showed inhibitory effects in vivo using fetal thymus organ culture (FTOC). Many cancers have mutations in Notch pathway components that make Notch hyperactive. We found the analogs strongly inhibit the constitutively active signalling of NOTCH1 mutants identified from acute lymphoblastic leukemia (T‐ALL) patients. We are now examining the anti‐tumor ability of the analogs in various cancer cell lines where Notch or Notch pathway players contribute to tumorigenesis. The 6‐alkynyl fucose analog can also act as a probe for EGF repeat O‐fucosylation, and help to discover novel POFUT1 substrates. Additional fucose analogs are being synthesized and evaluated to find more effective inhibitors or analogs with distinct activities (e.g. Notch activation, Jagged‐specific or ligand‐nonspecific inhibition).

Support or Funding Information

This work was supported by NIH grant GM061126.
Acute Systemic Inhibition of Inflammation Augments Endothelium‐dependent Dilation in Women with a History of Preeclamptic Pregnancy
Authors: Stanhewicz Anna E. , Dillon Gabrielle A. , Serviente Corinna F. , Alexander Lacy M.
Published: April 2020

Women who have had preeclampsia demonstrate microvascular endothelial‐dysfunction, mediated in part by reduced nitric oxide (NO)‐dependent dilation. Preeclamptic pregnancies are associated with elevated inflammation, and inhibition of inflammation attenuates endothelial damage in animal models of preeclampsia. However, it is unclear if systemic inhibition of inflammation improves endothelial function in women after a preeclamptic pregnancy. Using the cutaneous microcirculation as a model, we hypothesized that acute systemic inhibition of inflammation (oral salsalate; 1500mg/twice daily/4 days) would improve endothelium‐ and NO‐dependent vasodilation in women with a history of preeclampsia (PrEC) but not in women with a history of uncomplicated pregnancy (HC). Eleven HC (30±1yrs, 9±2 months post‐partum) and 8 PrEC (29±3yrs, 6±1 months post‐partum) participated in a double‐blind placebo‐controlled study. Following each treatment, 2 intradermal microdialysis fibers were placed in the skin of the ventral forearm for graded infusion of acetylcholine (ACh, 10−7–102 mM) or ACh+15mM L‐NAME (NO synthase antagonist). Red cell flux was measured over each site by laser‐Doppler flowmetry (LDF). Cutaneous vascular conductance was calculated (CVC=LDF/mean arterial pressure) and normalized to maximum (%CVCmax; 28mM SNP + local heat 43°C). ACh‐induced (75±3 vs. 92±4%CVCmax; p=0.002) and NO‐dependent (18±8 vs. 32±4%; p=0.04) vasodilation were attenuated in PrEC compared to HC following placebo. Salsalate augmented ACh‐induced (92±3%CVCmax; p=0.001) and NO‐dependent (38±4%; p=0.04) dilation in PrEC compared to placebo but had no effect in HC (all p>0.05). Women who have had preeclampsia demonstrate attenuated microvascular endothelial function compared to women with a history of uncomplicated pregnancy. Salsalate treatment augmented endothelium‐dependent vasodilation via NO‐mediated pathways in these women, suggesting that inflammatory signaling plays a role in mediating persistent endothelial‐dysfunction in women with a history of preeclampsia. ClinicalTrials.gov registration # NCT03482440

Support or Funding Information

NIH K99 HL138133 (Stanhewicz); NIH R01 HL093238 (Alexander); NIH T32 AG049676 (Serviente)
Neuroprotective Effects of Estrogen on Beta‐Amyloid Induced Neurotoxicity in Murine Hypothalamic Cells
Authors: Persaud Nikita Sudarshana , Fletcher Jada , Hindieh Jennifer , Chakraborty Sanjoy , Chakraborty Tandra R.
Published: April 2020

Alzheimer’s disease (AD) is a neurodegenerative affliction marked by significant cognitive decline and eventual dementia. The two most notable pathological hallmarks of AD include beta‐amyloid plaques, formed from the extracellular aggregation and deposition of beta‐amyloid fibrils, and the intracellular accumulation of hyperphosphorylated tau as neurofibrillary tangles in the brain. Previous research has suggested a possible neuroprotective property of estrogen, the female sex hormone, in AD. Despite the implications of a hypothalamic role in the early pathophysiology of AD, such as disturbances in sleep and emotional affect, scant literature exists outside of cortical and hippocampal AD research. This study is designed to replicate AD models using a control cell line, SH‐SY5Y human neuroblastoma cells, and an experimental cell line, N38 murine hypothalamic cells in culture, by incubating these cells with oligomerized human beta‐amyloid 42 (Aβ42). Each cell line is then treated with estrogen in order to observe the hormone’s efficacy as a neuroprotective agent. Cell viability, cytotoxicity and proliferation were measured in order to determine estrogen’s neuroprotective potential, using a variety of bioassays such as MTT and LDH. Preliminary results show lower mitochondrial activity and higher rates of cell lysis when treated with physiologically relevant concentrations of estrogen and low beta‐amyloid concentrations in the SH‐SY5Y cell line. In addition, the low concentration of Aβ42 (10μmol) appeared to have neuroproliferative effects on the SH‐SY5Y cells as opposed to its expected neurotoxicity. Conversely, N38 cells treated with estrogen at dosages higher than physiologically relevant concentrations, were found to have higher mitochondrial activity than cells treated with only Aβ42 after 24 hours, indicating a possible neuroprotective effect of estrogen at high concentrations on the hypothalamic cell line. Cell lysis and cytotoxicity of Aβ42 in N38 cells will be tested in order to further elucidate the pathology of the hypothalamus in AD, as well as estrogen’s potential neuroprotective effect on this unexplored brain region.

Support or Funding Information

Biology Department, Adelphi University, Garden City, New York. Department of Biological Sciences, New York City College of Technology/CUNY, New York, NY.
Deciphering the Mechanisms of Action of Altertoxin II with Genome‐Wide CRISPR/Cas9 Knockout Screening
Authors: Robles Andrew J. , Dai Wentao , Vaseva Angelina , Risinger April L. , Cichewicz Robert H. , Mooberry Susan L. , Houghton Peter J.
Published: April 2020

Ewing sarcomas (ES) are bone and soft tissue tumors that affect children and adolescents. These cancers are characterized by chromosomal translocations that result in expression of an oncogenic fusion protein (typically EWSR1‐FLI1). Despite advances in cure rates for many childhood malignancies, advanced, metastatic or relapsed ES remain a challenge. We identified the fungal metabolite altertoxin II (ATXII) as having highly selective activity against six ES cell lines compared to a panel of pediatric and adult cancer cells. On average, ATXII is 94‐fold (range 16‐ to 400‐fold) more potent against ES cells compared to rhabdomyosarcoma (RMS) cells, indicating high selectivity for ES. Mechanism of action studies indicated that ATXII selectively induces DNA damage in ES cells. However, the high degree of selectivity for ES suggests other mechanisms may be involved. To determine if ES sensitivity to ATXII is a dominant or recessive phenotype, we hybridized sensitive EW8 cells (ES) with resistant Rh30 cells (RMS) and determined the sensitivity of these hybrids to ATXII. Five single‐cell subclones derived from these hybrids were resistant to ATXII, suggesting that sensitivity to ATXII is a recessive phenotype in ES cells. Based on these data, we further investigated the mechanism of action of ATXII to identify which cellular pathways confer resistance. A genome‐wide CRISPR‐Cas9 knockout screen was conducted in resistant Rh30 cells using the GeCKOv2 library. After selection with puromycin, the transduced cells were treated for three weeks with ATXII or vehicle and genomic DNA was isolated every 4 days. These samples were subsequently prepared for next‐generation sequencing to identify sgRNAs that are depleted in the ATXII‐treated samples compared to vehicle‐treated control samples. Sequencing of these samples has recently been completed and initial bioinformatic analysis was conducted. Preliminary results have identified multiple differentially expressed sgRNAs in ATII‐treated Rh30 cells compared to vehicle‐treated cells, suggesting loss of these genes influences growth in the presence of ATXII. Overall, these studies have the potential to identify new molecular targets for the development of ES therapies.

Support or Funding Information

Funded by P01CA165995 (PJH), R01GM107490 (RHC, SLM), T32CA148724 (Cancer Biology Training Program) and RP170345 (CPRIT Training Program)
Comprehensive substrate specificity map of the mycobacterial serine hydrolase, LipN
Authors: Johnson R. Jeremy , Schemenauer Daniel , Pool Emily , Hoops Geoffrey
Published: April 2020

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), uses a battery of lipases to scavenge host cell lipids and maintain an infection in its host. With their central roles in infection, lipases have become a viable target for drug development, but many of the lipases in M. tuberculosis remain poorly characterized. Our goal was to determine the substrate specificity, biochemical properties, and potential structural information of LipN, a proposed mycobacterial lipase. Initially, wild type LipN was heterologously expressed, purified to homogeneity, and its substrate specificity characterized against a library of 35 fluorogenic ester substrates. Wild type LipN demonstrated the highest catalytic efficiency against small carbon esters, including methyl ether, ethyl ether, and oxazole esters. LipN’s preference for short (three carbons or less) esters with polar substituents suggests its physiological role as an esterase rather than a lipase and a selectivity for polar metabolites. Substitutional mutagenesis across the modeled active site and binding pocket of LipN allowed identification of the essential catalytic serine and histidine residues with the catalytic aspartate playing a less essential role in catalysis. The rest of the binding pocket showcased a range of polar residues required for full catalytic activity and two conserved glycines as the oxyanion hole. Herein, we showed that LipN likely catalyzes the ester hydrolysis of small, polar metabolites potentially aiding in the acquisition of additional energy sources from a host.
Further Optimization of Heterologous Glucoraphanin Biosynthesis Through Co‐Expression Analysis
Authors: Pignolet Roxanna , Endelman Benjamin , Barnum Collin , Shih Patrick M.
Published: April 2020

It is estimated that 38.4% of adults in the United States will be diagnosed with cancer (National Cancer Institute). With this high rate of incidence, the exploration of new options for cancer prevention and treatment is of high importance. Plant secondary metabolites are strong candidates for study due to their potential to be used in anti‐cancer dietary supplements. Isothiocyanates are products of the glucoraphanin biosynthesis pathway that have been demonstrated to display potent anti‐cancer and anti‐inflammatory properties. Previous studies have identified the genes in the core glucoraphanin biosynthesis pathway (CGBP); however, heterologous expression of the CGBP in tobacco has produced glucoraphanin at only 0.35 μmol g‐1 dry weight (Mikkelsen et al. 2010). In order to increase our titer, we used co‐expression analysis to identify a total of forty‐two gene candidates for optimizing the CGBP. We tested these candidates for their effect on glucoraphanin biosynthesis by transiently expressing the CGBP with candidate genes in tobacco. LC‐MS analysis of the transformed leaf tissue for glucoraphanin content revealed that six of the forty‐two genes increased glucoraphanin production. We also found several genes that produced significantly less glucoraphanin per gram dry weight when combined with the CGBP than the CGBP alone. The increases in heterologous glucoraphanin production implicate the six genes as important factors in the glucoraphanin biosynthetic pathway with the potential to raise concentrations closer to recommended supplement quantities.

Support or Funding Information

This project is funded by NIH Grant R00AT009573
Role of myeloid cell CYP19A1 in lung airway tissue repair following naphthalene induced epithelial injury in a mouse model
Authors: Han Weiguo , kovalchuk Nataliia , Ding Liang , Liu Pengfei , Yang Weizhu , Zhang Qing-Yu , Van Winkle Laura , Ding Xinxin
Published: April 2020

CYP19A1 (the aromatase) is a key enzyme in the biosynthesis of estrogens, which have important regulatory functions, including roles as a major mitogenic stimulus for cellular proliferation. In this study, we are testing the hypothesis that macrophage CYP19A1 can increase estrogen production and/or regulate the function of macrophages at sites of tissue injury, thereby influencing the balance between normal tissue repair and pathogenic outcomes related to abnormal epithelial repair. A myeloid cell (including macrophage) specific Cyp19a1‐null mouse model was generated and characterized. Wild‐type and Cyp19a1‐null mice were treated with naphthalene (200 mg/kg, IP) to induce acute lung injury, or with corn oil as a vehicle control. Mouse lung was examined at 24 hour, 72 hours, or 14 days after the treatment, for extent of acute airway injury, post‐injury cellular proliferation, and tissue repair, respectively. At 24 hours, a genotype difference in the extent of naphthalene‐induced airway injury was not detected. At 72 hours, the fraction of airway epithelial cells that are positive for the cell proliferation marker Ki67 was higher in naphthalene‐treated wild‐type mice (40%–70%) than in naphthalene‐treated Cyp19a1‐null mice (20–30%); whereas the fraction was much lower in corn oil treated control mice (3–5%). At 14 days, the bronchiolar epithelium was completely recovered in wild‐type mice, but only partially (<30%) in the Cyp19a1‐null mice, as indicated by the abundance of Club cells (positive for Clara cell secretary protein) in the airway epithelium. Thus, the loss of macrophage CYP19A1 did not impact the degree of naphthalene‐induced lung injury; but it impeded the post‐injury cellular proliferation and tissue repair in the airways.

Support or Funding Information

Supported in part by NIH grant ES020867
Do Medical School Textbooks Accurately Represent Public Health Concerns? Addressing Discrepancies between References in Textbooks and the Actual Prevalence of Five Congenital Disorders
Authors: Williams Avery A. , Dobson Seth D. , Muldoon Kathleen M.
Published: April 2020

The pre‐clinical years of medical school are designed to give future physicians the foundational knowledge essential to practicing medicine. Characteristically, the curriculum explores vital topics such as anatomy, physiology, biochemistry, pharmacology, immunology, and other basic sciences. In addition to learning about the “typical” function in a healthy patient, medical students are also taught “pathological” conditions they may see in their clinical settings to discuss treatment, prevention, and prognosis. Due to lecture hour time restraints, it is not possible to speak in depth about every relevant condition; therefore, courses usually assign textbooks to provide additional information to students outside the classroom. We sought to explore the relationship between the frequency with which certain congenital differences were identified in textbooks used in medical school courses, and the prevalence of those conditions within patient populations in the United States. A sample of allopathic and osteopathic schools were surveyed to compile a list of 121 textbooks used in years one and two of medical school. These books were examined to evaluate the references to five congenital conditions with teratogenic and/or genetic origins: congenital cytomegalovirus (cCMV), Zika virus, fetal alcohol syndrome, spina bifida, and Down syndrome. Frequency of mentions was defined as the presence or absence of the name of each condition determined by both automated and manual searches of the texts. Prevalence rates were taken from the Centers for Disease Control and Prevention (CDC). Our results indicate that the frequency of mentions of the selected conditions do not accurately reflect the prevalence of these conditions in the United States as identified by the CDC. Most surprisingly, congenital cytomegalovirus was one of the least mentioned conditions in the texts, despite cCMV having the highest relative prevalence. Our results indicate that the foundational education that first and second year medical students receive through textbooks may not accurately represent their future patient populations. It is important to increase the representation of common, preventable teratogens in the basic science years of medical education in order to augment general physician awareness, which may translate to better patient education of common prevention techniques and health outcomes.
Epigenetic regulation of phenylethanolamine N‐methyltransferase: implications for adrenaline biosynthesis
Authors: Khurana Sandhya , Tharmalingam Sujeenthar , Murray Alyssa , Tai T.C.
Published: April 2020

Adrenaline, a neurohormone and neurotransmitter, plays an extensive role in the physiological response to stress, and the sympathetic regulation of blood pressure. Phenylethanolamine N‐methyltransferase (PNMT), the terminal enzyme in the catecholamine biosynthetic pathway, directly responsible for adrenaline synthesis, is elevated in hypertensive animals; genetic linkage studies have associated the PNMT gene to the development of hypertension. Epigenetic changes are heritable changes in gene expression that are a consequence of the modification of the DNA (methylation at CpG sites) or histones (e.g. by methylation, acetylation) that facilitate packaging the DNA into nucleosomes. Changes in the epigenome have been associated with incidences of cancer, metabolic disorders and cardiovascular pathologies. In this study, alteration in PNMT expression modifiable by epigenetic regulation was examined using the rat adrenal pheochromocytoma derived PC12 cells. In vitromethylation of a PNMT promoter driven luciferase construct, using CpG methylases, consequently lead to a radical decrease in promoter activation, even in presence of dexamethasone (Dex) or Forskolin (Fsk), otherwise potent activators of PNMT expression. Further, the influence of a DNA methylation inhibitor 5‐aza‐2′‐deoxycytidine (5aza2DC), and histone deacetylase inhibitor valproic acid (VPA) was examined. Transcript analysis of endogenous PNMT, and PNMT promoter driven luciferase assays, both revealed that PNMT transcription is elevated in presence of these epigenetic modifiers, and these compounds can interact with the activation by Dex or Fsk to modulate PNMT expression. The extent of CpG methylation at the promoter of PNMT using bisulphite‐sequencing, and transcription factor binding sites that are sensitive to epigenetic modification using site directed mutagenesis, are currently being examined. The data suggests that PNMT regulation is sensitive to epigenetic modification which can have repercussions for adrenaline biosynthesis, and consequently on its role as a neurotransmitter.

Support or Funding Information

CIHR
Spectroscopic and Biochemical Studies on Metallo‐β‐lactamase IMP‐1
Authors: Zhang Huan
Published: April 2020

β‐Lactamases are a primarily cause for clinical resistance to β‐lactam antibiotics, such as penicillins, cephamycins, carbapenems, and cephalosporins. Therefore, there are considerable efforts to discover inhibitors of β‐lactamases, which can be given in combination with existing β‐lactams as a treatment for antibiotic resistant bacterial infections. According to previous studies, several types of metallo‐β‐lactamases inhibitors have been discovered by organic chemists: metal‐ion binding inhibitors, covalent inhibitors, allosteric inhibitors, and the inhibitors with uncharacterized mechanisms. The blaIMP gene was initially found in clinical imipenem‐resistant Serratia marcescens and Pseudomonas aeruginosa isolates in Japan. The IMP enzymes deliver broad substrate specificities, and comparing to penicillins they have greater binding affinities for carbapenems and cephalosporins. IMP‐1, the first variant identified, is the most studied IMP variant, and there have been many reported inhibitors of IMP‐1. However, most of these studies reported IC50 and/or MIC values, and most of the inhibitors have not been characterized for mechanism of inhibition. This lack of understanding of mechanism of inhibition could potentially be reducing our efforts to find effective inhibitors of IMP variants and other metallo‐β‐lactamases. We used spectroscopic and biochemical studies to probe the mechanism of inhibition for several known metallo‐β‐lactamase inhibitors with IMP‐1. Equilibrium dialysis was used to incubate IMP‐1 with different inhibitors, and metal analyses were used to determine the zinc content in the resulting samples. This technique allows for us to determine whether an inhibitor strips zinc from IMP‐1. Native mass spectrometry (Orbitrap) of IMP‐1 samples containing inhibitors were used to confirm the equilibrium dialysis results and also to determine whether an IMP‐1/zinc/inhibitor ternary complex forms. Native mass spectrometry can be used to determine whether inhibitor binds to the zinc‐containing form of IMP‐1, but this technique cannot determine whether the inhibitor(s) bind to the metal ions in the active site. Therefore, we utilize UV‐VIS spectrophotometry with cobalt‐substituted analogs of IMP‐1. This technique can be used to probe whether the inhibitor binds to one or both of the metal ions. We also probe inhibitor binding to the cobalt‐containing enzyme by using Electron Paramagnetic Resonance (EPR) spectroscopy. We have discovered that EPR can be used to determine whether inhibitors bind to both metal ions. By using these techniques, we hope to identify inhibitors that form ternary complexes, rather than those that strip metal ions from the metallo‐β‐lactamases. We believe that the inhibitors that form ternary complexes are the best candidates for rational re‐design efforts.

Support or Funding Information

National Institutes of Health (NIH) and Miami University
The Development of 3D Printable Stretch Bioreactors
Authors: Howell Andrew P. , Fischer Kristin
Published: April 2020

Introduction

Cyclic stretch bioreactors for research and medical field applications have proven beneficial in enhancing muscle tissue growth when compared to static cultures. However, the cost for many commercially available cyclic stretch bioreactors carry large price tags and have limited customizability. Three‐dimensional (3D) modeling combined with 3D printing allows for highly customizable cyclic stretch bioreactors to be designed and printed at a lower cost while potentially maintaining the high degree of precision seen in commercially available bioreactors. The importance of this work is to reduce the cost of cyclic stretch bioreactors to increase availability for use in research and medical fields.

Materials and Methods

The 3D modeling software, TinkerCAD, and a Flashforge Creator Pro 3D printer with FlashPrint operating software were used for this project. Poly(‐lactic acid) (PLA) filament purchased from Flashforge was used to print several ready‐for‐assembly pieces. O‐rings and glass were purchased from McMaster‐Carr. For lid fabrication, an O‐ring was placed in a molded channel in the body of the bioreactor and a glass cover was affixed. The chamber was sealed and subjected to an air tight test as well as a water tight test. The chamber was submerged for an hour in water first and then dried prior to filling the chamber with water, inverting the chamber for one hour, and checking for leaks.

Results and Discussion

A one‐chamber‐four‐well system was chosen as it allowed for multiple scaffolds to be stretched at once, minimized the potential sealing failure points, and maximized the utility of the chamber’s small footprint. Models for a one‐chamber‐four‐well system were successfully printed using PLA filament. A notable feature of the model is an integrated channel for an O‐Ring in conjunction with a glass lid to allow for sealing of the chamber to ensure an air and water tight internal environment. The chamber successfully maintained all seals for the air tight test, but during the water tight test some water seeped into the chamber frame. This seepage into the frame could lead to contamination and loss of samples.

Conclusions

Construction for a cyclic stretch bioreactor, using 3D printing, is an attractive option for lowering cost while increasing customizability. The production of a water and air tight one‐chamber‐four‐well system can allow for multiple replicates per study and can allow for easy replication of the chamber itself by printing the number of chambers required for the study. Further investigation into the development of 3D printable cyclic stretch bioreactors is a worthwhile effort with future work for this bioreactor including addressing sealing or friction issues, the creation of an adaptable computer control unit with accompanying motor system, and cellularized scaffold testing.

Support or Funding Information

‐The Hampden‐Sydney Honors Research Council and The Virginia Academy of Science
Class I HDAC Differentially Regulates Blood Pressure and Renal Injury in Npr1 Gene‐targeted Male and Female Mutant Mice
Authors: Kumar Prerna , Bloodworth Meaghan , Gogulamudi Venkateswara R. , Pandey Kailash N.
Published: April 2020

Atrial and brain natriuretic peptides (ANP, BNP) activate guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA), which plays a critical role in the regulation of blood pressure (BP) and fluid volume homeostasis, and inhibits the cell proliferation and fibrosis. Mice lacking functional Npr1 gene (coding for GC‐A/NPRA) exhibit pathophysiology of hypertension, kidney disease, and heart failure; however, the mechanisms regulating Npr1 expression are not well understood. The objective of the present study was to gain insight into the effect of class I‐specific histone deacetylase (HDAC) inhibitor, mocetinostat (MGCD0103) on Npr1 gene expression and regulation of BP and renal injury in gene‐targeted mutant mice. We utilized 16‐ to 20‐weeks old male and female Npr1 gene‐knockout haplotype (1‐copy; Npr1+/−), wild‐type (2‐copy; Npr1+/+), and gene‐duplicated heterozygous (3‐copy; Npr1++/+) mice. All mice genotypes were injected intraperitoneally with MGCD0103 (2 mg/kg) at an alternate day for 2‐weeks. The Western blot results showed that MGCD0103 significantly increased the renal NPRA protein levels in male and female mice compared with vehicle‐treated (olive oil) controls. After MGCD treatment, the renal GC‐A activity was significantly increased in both male and female mice. Male mice exhibited significantly higher systolic BP (SBP) than female mice and treatment with MGCD0103 decreased SBP in both 1‐copy male (106 ± 0.6 vs. vehicle‐treated, 129 ± 1.9; mmHg) and female (97 ± 2.2 vs. vehicle‐treated, 110 ± 2.1 mmHg, p < 0.001) mice. Renal HDAC activity was considerably higher (p < 0.05) in male mice than female mice; however, treatment with MGCD0103 attenuated (p < 0.05) HDAC activity by 40% in male and ~50% in female animals. Significantly higher urinary albumin to creatinine ratio was detected in male mice compared with female mice, which was attenuated by MGCD0103 treatment in male mice. The present results demonstrate that class I HDAC inhibitor, MGCD0103 upregulates NPRA expression in vivo, lowers SBP, and repairs renal injury differentially in male vs. female mice. These findings will have important implications for treatment of hypertension and renal injury in humans in a gender‐related manner.

Support or Funding Information

This work was supported by NIH grant HL062147.
Impaired Autoinhibition of Protein Kinase Cγ in Spinocerebellar Ataxia Type 14
Authors: Pilo Caila A. , Kunkel Maya T. , Van An-Angela N. , Kobori Evan K. , Longman Cheryl , Gorrie George , Taylor Susan S. , Newton Alexandra C.
Published: April 2020

Spinocerebellar ataxia type 14 (SCA14) is a neurodegenerative disease caused by germline mutations in the diacylglycerol (DG)/Ca2+‐regulated protein kinase C gamma (PKCγ), leading to Purkinje cell degeneration and progressive cerebellar dysfunction. Curiously, the majority of the approximately 50 missense mutations identified in PKCγ cluster to the DG‐sensing C1A and C1B domains. Here, we use a genetically‐encoded FRET‐based C Kinase Activity Reporter (CKAR) to show that ataxia‐associated PKCγ mutants have higher basal activity in cells, and thus are less autoinhibited, than wild‐type enzyme. However, whereas reduced autoinhibition generally renders PKC sensitive to degradation, we show that mutations in the C1B domain allow translocation to membranes but protect PKCγ from phorbol ester‐induced down‐regulation. Indeed, deletion of the C1B domain prevents PKCγ down‐regulation with phorbol esters, potent ligands for the C1 domains. Strikingly, the degree of impaired autoinhibition correlates inversely with age of disease onset. Patients with the most severe mutation we examined (V138E) present with symptoms as young children, whereas symptoms in patients with the least severe mutation examined (D115Y) manifested in their 40s. To understand the structural basis of mutations outside the C1 domains, we generated a model of PKCγ using homology modeling and molecular docking. Mutations outside the C1 domains occur in regions also predicted to disrupt autoinhibition, including the pseudosubstrate, a predicted interface between the kinase and C1B domains, and the C‐terminal tail of PKCγ. Taken together, our data support a model in which SCA14 mutations enhance PKCγ activity without compromising stability. Furthermore, because many of the genetic causes of the 40+ types of SCA alter Ca2+ homeostasis, deregulated PKCγ activity may be a common cause for the disease. This raises the possibility that inhibition of PKCγ will be a potentially viable therapeutic target for SCA.

Support or Funding Information

NIH T32 GM007752
Elucidation of HIV‐1 5' Leader Secondary Structure Through Novel Long Range Nuclear Magnetic Resonance (NMR) Techniques
Authors: Walker Claudia , Kan Elisabeth , Davis Faith , Catazaro Jonathan , Summers Michael
Published: April 2020

The human immunodeficiency virus (HIV‐1) is a global pandemic that has infected approximately 37 million individuals. The high mutation rate in the HIV‐1 genome renders current treatments that target regions of the replication cycle often insufficient. Thus, structural elucidation of the 5' leader within the HIV‐1 RNA is pertinent due to the high conservation of this packaging signal, making it paramount to viral replication and a promising target for antiretroviral therapy.

Structural analysis of RNA constructs below 60 nucleotides has been effective by Nuclear Magnetic Resonance (NMR) spectroscopy. However, the size of the 5' leader – approximately 300 nucleotides – has stifled structural studies by NMR due to excessive signal broadening and long instrument acquisition times. To circumvent the latter complication, site‐specific lanthanide labeling of a reporter protein with a high binding affinity to a cognate RNA loop is proposed. It is well‐known that paramagnetic lanthanide ions induce measurable pseudocontact shifts (PCS) in NMR spectra. However, binding the metal ion tag directly to the RNA is deleterious. Consequently, the spliceosomal U1A reporter protein is utilized as an intermediary between the metal ion tag and the RNA. Prior to applying the technique to the 5' leader, a 46 nucleotide Moloney Murine Leukemia Virus (MMLV) derived RNA construct was used as a model for PCS observation and NMR experiment optimization. Through the use of Heteronuclear Multiple‐Quantum Correlation experiments, PCS data was obtained in conjunction with residual dipolar couplings, resolving the quaternary structure of U1A: MMLV. Furthermore, the experiment illustrated an increase in distance measurements from 5Å to 30Å in the presence of paramagnetic lanthanides. Our novel method is currently being applied toward the elucidation of the secondary structure of the 5' leader with the U1A binding loop strategically placed in regions of interest in an effort to enhance our understanding of HIV‐1 biology and to facilitate the design of novel therapeutics in the future.

Support or Funding Information

This investigation was sponsored by NIH/NIGMS MARC U*STAR T34 HHS 00026 National Research Service Award to UMBC. This research was also supported in part by a grant to UMBC from the Howard Hughes Medical Institute through the HHMI Adaptation Project. We would like to thank the MARC U*STAR program and UMBC Summer Biomedical Training Program for the strong support.
DECREASING OF ERYTHROCYTES MEMBRANES PLASMALOGENS LEVEL AT THE DIABETES MELLITUS
Authors: Shamansurova Zulaykho , Saatov Talat , Akhrarova Nilufar
Published: April 2020

Introduction. Plasmalogens are important part of glycerophospholipids and plays a role in the tissue development and metabolism. Changing their level shown in coronary heart diseases, peroxysomes disorders, Diabetes Mellitus (DM).

Aim. Study the role of plasmalogens in the development of diabetes complication, especially in diabetic nephropathy (DN) presented interest in our study.

Material and methods. In 12 healthy subjects (HS) and in 49 patients with DM2 in age 39 – 65 with disease duration from 2 month to 25 years the level of fasting blood glucose (FBG) and HbA1c, plasma triglycerides (TG) content, plasmalogens in blood serum (SP) and erythrocytes membrane (EP) were observed. The stage of DN detected by urine protein excretion level, glomerular filtration rate (GFR), blood urine nitrogen (BUN) and creatinine level. Patients according to GFR and BUN level were selected into the main 3 groups: without proteinuria (BPU), with proteinuria and normal BUN (PU), with proteinuria and higher BUN (CKD).

Results. FBG and HbA1c level were significantly higher in all DM patients than HS, by 2.1, p<0.01 and in 1.7, p<0.05 respectively and was comparable between DN groups. TG level were significantly higher by 1.7 times in DM group and shown relationship with severity of DN. SP and EP level were significantly lower in DM group and showed strong linkage between GFR and BUN level and suggest about involvement of plasmalogens into development of DN.

Conclusion. In patients with DM SP and EP level significantly decreased, had a strong linkage with GFR, BUN and can reflect the severity of DN.