bluuebunny/crossref_metadata_2025 huggingface dataset:
Exploring a 13‐nucleotide Motif Capable of Driving Cap‐independent Translation
Authors: Kharbanda Pankhuri , Flores Megan A. , Juba Amber N. , Wellensiek Brian P.
Published: April 2020

In the classical model of eukaryotic translation, a 7‐methylguanosine cap at the 5’ end of the messenger RNA (mRNA) acts as the recognition signal to recruit the ribosome to the RNA strand. The ribosome then scans the mRNA for the translation initiation codon, AUG. However, under stressful circumstances—such as apoptosis or viral infection—the recognition signal can be lost. Despite this, there is increasing evidence that translation still seems to proceed under certain circumstances. This cap‐independent mode of translation has been seen to occur through various regulatory elements along the mRNA. In an attempt to identify sequences that drive cap‐independent translation, the human genome was surveyed using mRNA display. The results of this survey led to the identification of many translation enhancing elements (TEEs) scattered throughout the genome. Further characterization of these TEEs provided evidence that they are able to recruit the ribosome to internal mRNA sequences, therefore bypassing the cap structure. A combination of sequence function and phylogenetic analysis led to the isolation of a short 13 nucleotide motif within several TEEs that is capable of driving cap‐independent translation. Further evaluation of the 13mer suggests that the functional motif maybe larger than originally thought, and the sequence context surrounding the 13mer can influence its effectiveness. When exploring the sequence around the 13mer, protein translation appeared to be the most efficient when the upstream nucleotides, preceding the start site, followed a triplicate pattern. To further investigate this phenomenon, we have explored exact start codon usage, performed cryptic promoter analysis, and conducted mutational mapping of the sequence the 13mer was originally discovered within. The results of our study suggest that the ribosomal binding site is larger than 13 nucleotides and translation initiation likely occurs at a position other than the original ribosomal binding site.

Support or Funding Information

Midwestern University Intramural funds
The Them1 START Domain Regulates Nuclear Trafficking in Brown Adipocytes
Authors: Baqai Mahnoor , Li Yue , Tillman Matthew , Ortlund Eric A. , Cohen David E. , Hagen Susan J.
Published: April 2020

Thioesterase superfamily member 1 (Them1) is highly expressed in brown adipose tissue, where it limits energy expenditure. Them1 is a fatty acyl‐CoA thioesterase with tandem hotdog fold thioesterase domains, an N‐terminal set of phosphorylation sites at S15, 18, and 25, and a C‐terminal lipid‐binding steroidogenic acute regulatory protein‐related lipid transfer (START) domain, which modulates thioesterase activity. Cell fractionation studies have demonstrated that Them1 thioesterase activity is localized in microsomes and nuclei, suggesting that it is targeted to specific intracellular compartments. Our aim was to determine the mechanism by which Them1 traffics to the nucleus and its function therein.

Methods

Identification of a putative nuclear localization signal was determined using cNLS mapper (
http://nls‐mapper.iab.keio.ac.jp/cgi‐bin/NLS_Mapper_form.cgi). Full‐length, mutant, or truncated THEM1 constructs were cloned and linked to EGFP. Plasmids or adenoviral vectors were transfected into a differentiated brown adipocyte cell line (MUCAR cells) that does not express Them1. The localization of Them1 was visualized by confocal microscopy via EFGP and mRNA expression was determined using qPCR. Confocal z‐stacks were used to produce 3‐D images with Volocity software. Them1 nuclear localization was evaluated in the presence or absence of the nuclear export inhibitor leptomycin B. Cyclohexamide was used to block new protein synthesis.

Results

Phosphomimic mutations that simulated Them1 phosphorylation at S15 resulted in a diffuse Them1 localization. Stimulation of cells with norepinephrine resulted in Them1 phosphorylation and its reorganization from discrete puncta to a diffuse localization. In the diffuse state, Them1 was transported into the nucleus. Treatment with cyclohexamide suggested that the nuclear Them1 pool was not newly synthesized. We identified a nuclear localization signal in a hinge region located between the 2nd thioesterase domain and the START domain. Deletion of either the nuclear localization signal or the START domain in full length Them 1 blocked nuclear targeting. Conversely, mutant Them1 containing only the START domain was targeted to the nucleus. Entry into the nucleus was balanced by trafficking out of the nucleus, as evidenced by exit blockade using leptomycin B. Nuclear Them1 regulated mRNA expression levels for a number of genes that regulate lipid metabolism in brown adipocytes.

Conclusions

Our results demonstrate that trafficking of Them1 to the nucleus occurs via a nuclear localization signal within the START domain. We speculate that nuclear Them1 regulates the transcription of lipid metabolic enzymes by a mechanism that is distinct from its acyl‐CoA thioesterase activity.

Support or Funding Information

This work was supported by NIH DK103046 to DEC, SJH and EAO.
White Matter Hyperintensities and Carotid Arterial Stiffness in Amnestic Mild Cognitive Impairment
Authors: Curtis Bryon Matthew , Tomoto Tsubasa , Chiles Collin , Austin Courtney , Zhang Rong
Published: April 2020

Introduction

White matter hyperintensities (WMH) is a risk factor of cognitive impairment and Alzheimer’s disease (AD). Arterial stiffening is associated with increased arterial pressure and cerebral blood flow (CBF) pulsatility which may lead to WMH pathogenesis. However, the relations among WMH, arterial stiffness, and pulsatile change in arterial pressure and CBF remain unclear. Therefore, this study investigated the associations among WMH volume, carotid arterial stiffness, carotid arterial pressure, and CBF pulsatility in patients with amnestic mild cognitive impairment (MCI), a prodromal stage of AD.

Methods

Fifty‐four amnestic MCI patients and twenty‐four cognitively normal subjects (CN) underwent measurements of WMH volume using T2‐weighted‐fluid‐attenuated inversion recovery imaging. Carotid β‐stiffness index and arterial pressure at the common carotid artery (CCA) were measured using ultrasound and applanation tonometry. CBF pulsatility index (PI) was measured from the middle cerebral artery using transcranial Doppler.

Results

WMH volume, PI, carotid β‐stiffness index, carotid systolic blood pressure (cSBP) and pulse pressure (cPP) did not differ between groups. Greater WMH volume was correlated with higher CBF PI in both groups (MCI: r=0.373, NC: r=0.463, p<0.05), whereas it was correlated positively with carotid β‐stiffness index, cSBP, and cPP only in the MCI group (MCI: r=0.499, r=0.432, r=0.468, all p<0.01). In multiple linear regression analysis, cSBP was an independent determinant of WMH (β=0.293, p=0.005) with adjustment for age, sex, body mass index, and MCI status.

Conclusion

These findings collectively suggest that carotid arterial stiffness, carotid SBP, and pulse pressure may play an important role in WMH pathogenesis in MCI.

Support or Funding Information

This work was funded by the National Institutes of Health ((R01AG033106, R01HL102457, P30AG012300)
Using Human Stem Cells to Analyze the Effects of the Immune System on Chronic Kidney Disease
Authors: D'Agostino Victoria Anne , Dunkley Truman , Toth Charles
Published: April 2020

Recent studies have shown that there exists a correlation between Chronic Kidney Disease (CKD) and macrophage‐induced inflammation. Utilizing human induced pluripotent stem cells (hiPSC), the inflammatory process in kidney disease could be recapitulated and the mechanisms behind it better understood. Kidney organoids and macrophages were grown from two hiPSC cell lines, BJFF.6 and WTC11, following previously described procedures. An inflammatory response was induced by the addition the pro‐inflammatory mediator Poly (I:C) and Lipopolysaccharide (LPS). The role of the microbiome in kidney disease was examined by the introduction of the tryptophan metabolite, indole‐3‐sulfate (I3S). The difference in gene expression in the kidney and macrophage co‐cultures for two inflammatory markers, TNFα and IL‐6, were analyzed using qPCR. ELISA analysis was also performed, which showed that there was an overall increase in inflammation in the co‐cultured kidney organoids, which was reduced in the presence of 100 uM I3S. This demonstrates that microbiome metabolites can influence an inflammatory response. Examining the role of the microbiome in an inflammatory disease model can be used to further study kidney inflammation and detect potential targets for treatment of CKD.

Support or Funding Information

Providence College Undergraduate Research Grant, and a gift from Dr. John Mullen
Resveratrol Attenuates Doxorubicin Cytotoxicity and Mitochondrial Dysfunction and Human Proximal Tubular Epithelial Cells
Authors: Valentovic Monica A. , Brown Kathleen C. , Dial Mason , McGuffey Rachel
Published: April 2020

Doxorubicin (DOX, Adriamycin), is a cancer chemotherapy employed in the treatment regimen for breast, Hodgkin and Non‐Hodgkin lymphomas, small cell lung cancer and acute lymphoid leukemia. The U.S. FDA issued a Black Box warning for Cardiomyopathy in patients treated with doxorubicin. Other adverse effects include renal impairment. The cellular mechanisms for doxorubicin mediated renal cytotoxicity are not known. Resveratrol (RES) is a phytoalexin found in many fruits including grapes, blueberries, cranberries, dark chocolate and nuts. Antioxidant and anticancer properties have been associated with RES in cell culture and in human clinical studies in cancer patients and in vivo rodent models. Our studies tested the hypothesis that RES protection for DOX renal cytotoxicity, was mediated by maintaining mitochondrial function. All studies were conducted in human noncancerous renal proximal tubular epithelial cells (HK‐2). HK‐2 cells were plated and equilibrated for 48 h. Cells were next pre‐incubated for 1 h with 0 (DMSO) or 5 or 7.5 uM RES followed by a 24 h co‐incubation with 0–5 uM DOX. All results were obtained from 3 independent experiments. Western analysis for protein carbonylation and 4‐hydroxynooneal (4‐HNE) were used as an indicator of oxidative stress. Mitochondrial function was assessed using a Seahorse analyzer system. Prior to examining mitochondrial function, cell number and substrate concentrations were optimized based on the manufacturer’s recommendations. Mitochondrial Stress Test was conducted following the treatments described above and each well was normalized by protein. Viability was assessed using MTT leakage and tyrpan blue exclusion cell counts. RES did not alter cell viability as indicated by comparable MTT values between DMSO and RES groups (p>0.05). DOX was cytotoxic to HK‐2 cells within 24 h. Pretreatment with RES provided protection from DOX to HK‐2 cells. Basal mitochondrial respiration assessed as oxygen consumption rate (OCR) was diminished by 4 uM DOX. Mitophagy mediated by DOX and the effect of RES compared protein expression of LC3BI, LC3BII and the ratio of LC3BII/I. In summary, RES attenuated DOX renal cytotoxicity by maintaining mitochondrial function. Further studies are needed to probe changes in specific mitochondrial proteins mediated by DOX.

Support or Funding Information

(Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence; M.D. and R.M. supported by WV NASA Undergraduate Research Fellowship: M.D. Supported by MUSOM Summer Research Fellowship).
Faculty Perceptions of the Implementation of Flipped Teaching in Undergraduate STEM Courses Across Two Institutions
Authors: Givens Adriana , Bracey Georgia , Fickas Julie , Bartels Lynn , Locke Sharon , Gopalan Chaya
Published: April 2020

The flipped classroom is an active learning and teaching environment that has been shown to improve student engagement and thus student performance. In order to expand the use of flipped teaching (FT) in STEM classrooms, we recruited faculty members from two institutions, six faculty from a local community college and an additional six from a regional state university. We were particularly interested in how faculty perceived the implementation of FT, barriers in the implementation of FT, and the differences in FT implementation at a four‐year university and a two‐year community college. All 12 faculty members participated in six faculty development FT workshops, that were spread over an entire semester. While undergoing training, the participants were expected to be preparing for their implementation of FT during the following semester. Pre‐implementation survey data from both institutions showed that 6 out of 12 faculty had a strongly favorable attitude toward FT and 4 out of 12 strongly agreed that they possessed the knowledge to use FT. When implementation of FT was considered, 4 out of 6 community college faculty and 2 out of 6 university faculty strongly agreed that they could successfully implement FT. One common barrier contributing to the resistance in the implementation of FT as identified by the participants at both institutions in the pre‐implementation survey was the lack of time to implement. Additional barriers identified specifically by the university faculty included lacking the knowledge to implement FT as well as anticipating potential resistance from students. Post‐implementation survey data showed that university faculty attitudes toward FT increased from 3 out of 6 to 5 out of 6 while community college faculty’s attitudes remained at 3 out of 6. There was an increase in knowledge of using FT from 2 out of 6 to 6 out of 6 by university faculty and from 2 out of 6 to 5 out of 6 by community college faculty. Additionally, faculty perceptions of successful implementation increased from 2 out of 6 to 5 out of 6 by university faculty and remained unchanged at 4 out of 6 among community college faculty. Multiple barriers were identified through post‐implementation survey by faculty from both institutions which included lacking the time to implement, limited knowledge related to implementation, grading, and class sizes. An additional barrier identified specifically by the university faculty was anticipating potential resistance from students. Addressing these barriers is critical in allowing the use of active learning methodologies such as FT.

Support or Funding Information

National Science Foundation
CRF Release in the Nucleus Accumbens Promotes Arousal in Anticipation of Cocaine Availability: Implications for Stress‐Induced Drug‐Seeking
Authors: Leonard Michael , Covington Herb , Miczek Klaus
Published: April 2020

Dopamine signaling within the nucleus accumbens core (NAcC) is critical to the generation of motivated behaviors in both appetitive and aversive contexts. Within the accumbens, various neuropeptides influence motivational states through the modulation of dopamine release dynamics. Here, microdialysis studies show that the stress peptide corticotropin releasing factor (CRF) is released in the NAcC when rats are exposed to environmental contexts that predict either an aversive outcome (i.e. social defeat) or drug‐reward opportunity (i.e. cocaine availability), and moreover, intra‐NAcC infusion of exogenous CRF elicits a robust increase in extracellular dopamine. The present studies further interrogate accumbens‐specific CRF actions as a candidate mechanism for maladaptive arousal evoked by drug‐predictive stimuli – a hallmark feature of cocaine dependence. In order to assess the role of NAcC‐CRF on instrumental responding directed towards cocaine procurement, male Long‐Evans rats were trained to self‐administer cocaine under a heterogeneous chained schedule of reinforcement (FIFR) in order to dissociate anticipatory (‘drug‐seeking’) from consummatory (‘drug‐taking’) behavior. Completion of a fixed‐interval (10 min) was followed by a period (10 min) of continuously reinforced responding (0.32mg/kg cocaine; FR1) on another lever. Microdialysis conducted during this procedure revealed that extracellular dopamine levels within the NAcC gradually ‘ramp up’ across the fixed‐interval in parallel with lever pressing activity, as drug availability approaches. Similarly, dialysate levels of CRF were specifically and progressively elevated during the fixed‐interval component preceding drug‐access, but were unaffected by drug‐taking. Infusion of CRF into the NAc produced a rapid elevated tonic dopamine levels that was accompanied by enhanced responding during the fixed‐interval component of the procedure, but notably did not affect subsequent cocaine intake. Conversely, pharmacological blockade of CRF‐R2, dampened extracellular dopamine levels in the NAc and suppressed lever pressing behavior during the fixed‐interval. Taken together, these data suggest that in response to appetitive or aversive stimuli, CRF may be a critical mediator of motivated processes that are classically associated with dopamine transmission. To this end, stress‐evoked CRF release in the NAc may represent a candidate mechanism by which stress triggers aberrant cocaine‐seeking.

Support or Funding Information

NIDA R01‐DA031734 to KAM
Dicer as a Repressor of Antiviral Responses in Mouse Embryonic Stem Cells
Authors: Gurung Chandan , Sapkota Krishna , Huang Faqing , Guo Yan-Lin
Published: April 2020

Recent studies have demonstrated that pluripotent stem cells (PSCs), including human and mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have underdeveloped antiviral innate immunity. Specifically, they fail to express IFN in response to viral stimuli. This finding has led to the hypothesis that the lack of IFN response is an intrinsic property of PSCs. It has also inspired the investigation of the underlying molecular mechanisms. In an effort to conduct a holistic analysis of antiviral responses in ESCs, we have developed an assay that can simultaneously measure RNA‐mediated gene expression and RNA‐induced antiviral responses. In this system, GFP is expressed from functionalized synthetic GFP‐mRNA and is used to measure the cellular translation activity, whereas dsRNA and 5’‐triphosphate‐ssRNA act as viral RNA analogs. Transfection of ESCs with these synthetic RNAs can potently activate all common antiviral signaling pathways, including RNA interference (RNAi), dsRNA activated protein kinase (PKR), OAS‐RNase L, and IFN responses. Our data revealed that RNAi activity was detected in ESCs, but not in Dicer knockout ESCs (D‐/‐ESCs) as expected since Dicer is a key enzyme in miRNA and siRNA processing. The antiviral responses mediated by PKR, OAS‐RNase L, and IFN were either not detected or detected at low levels in ESCs, but they increased dramatically in D‐/‐ESCs, as indicated by increased activity of PKR (measured by low level GFP expression and PKR phosphorylation) and RNase L (determined by RNA degradation). Furthermore, the components of the IFN signaling pathway are expressed at higher basal levels in D‐/‐ESCs than in normal ESCs and are further up‐regulated by RNA transfection. The most notable effect of RNA transfection into D‐/‐ESCs is the induction of cell death and decreased cell proliferation, which is at least partly attributed to PKR activation and increased expression of the cell cycle inhibitors p21 and p19. Our findings suggest that ESCs are exquisitely sensitive to cellular damages caused by antiviral response and that Dicer acts as a repressor of antiviral responses in ESCs under normal conditions.

Support or Funding Information

This work was in part supported by the National Institute of General Medical Sciences (R15GM128196‐01). We thank Mississippi‐IDeA Network of Biomedical Research Excellence for the use of the imaging facility (funded by the National Institute of General Medical Sciences P20 GM103476‐11).
Enhanced Mitochondrial Turnover in Aged Human Right Atrial Tissue
Authors: Oliveira Ashley N. , Karmanova Lioubov , Murugavel Shamini , Yanagawa Bobby , Hood David A.
Published: April 2020

Age is one of the greatest risk factors for cardiovascular disease, which is the leading cause of death in the developed world. Therefore, the maintenance of cardiac health throughout the lifespan is of the upmost importance. Aging is associated with a plethora of biochemical and metabolic alterations, many of which converge on the mitochondrion. As a highly oxidative tissue, mitochondria supply roughly 95% of all ATP required in the myocardium. This reliance on mitochondria underscores the importance of maintaining the quality of these organelles, especially with age, however little is known about how age effects quality control mechanisms in the human heart. We hypothesized that the removal of mitochondria through mitochondrial specific autophagy, or mitophagy, would be impaired in aged human cardiac tissue. To evaluate mitochondrial quality control, samples of right atria were collected from young (≤50 years) and aged (≥70 years) patients undergoing coronary artery bypass surgery (CABG). Tissue was collected prior to and immediately following surgery, providing a useful model of ischemia‐reperfusion injury. Patients were matched for hyperention and dyslipidemia, as well as prescribed medications such as statins, and ACE inhibitors. Exclusion criteria included smoking, a prior stroke or myocardial infarction, or other comorbidities (e.g. diabetes, cancer). Mitochondrial content as indicated by various markers such as COX IV, VDAC, and UQCRC2 was reduced in the aged samples, however a modest 1.2‐fold increase in Citrate Synthase was observed, suggesting age‐associated compositional changes. The upstream marker of autophagy Beclin‐1 and the transcriptional regulators TFEB and TFE3 did not change with age, suggesting that aging does not affect the drive for autophagy. Despite this, constituents of the autophagosome p62 and LC3‐II were reduced by 20% and 30%, respectively, in the aged samples, indicative of increased autophagy flux. The mitophagy marker Parkin displayed a trend to decrease with age, suggesting a decline in the signaling for mitophagy. The lysosomal marker Cathepsin D was unaffected by age in cardiac muscle, but the lysosomal calcium channel Mucolipin‐1 increased in aged atrial muscle by 2‐fold. Analysis of post‐CABG samples indicated marked elevations in HSP70 and Caspase‐3 protein compared to pre‐CABG values. This was only evident in the atria of aged individuals. Our data suggest that increased mitophagy flux could account for the reductions in mitochondrial markers in aged atria, and that CABG can induce a stress response leading to apoptosis signaling that is more prominent with age.

Support or Funding Information

Supported by NSERC Canada.
Characterizing the Action of Arginine Vasopressin at Hypoglossal Motoneurons In Neonatal Mice
Authors: Bolte Kristen , Revill Ann L.
Published: April 2020

Vasopressin, traditionally considered a hormone, also acts as an excitatory neurotransmitter. The effects of vasopressin have been characterized in brainstem motoneurons, including hypoglossal motoneurons (XII MNs). The XII motor nucleus receives inspiratory rhythm from the preBötzinger Complex where it is relayed to the muscles of the tongue via the XII nerve. With some variation across postnatal maturation, the vasopressin V1a receptor gene is expressed from birth to adulthood in the XII motor nucleus, and is enriched in the XII nucleus in adults compared to other cranial motoneuron populations. Previous research demonstrated that vasopressin, acting via V1a receptors on preBötzinger Complex, can stimulate breathing in adult rats by increasing breathing frequency as well as diaphragm and genioglossus (primary tongue protruder) muscle activity. Whether vasopressin can also act directly at XII MNs to potentiate inspiratory bursting has not been well characterized. Thus, the aim of this research was to characterize vasopressin effects on XII MN inspiratory bursting behavior. We generated rhythmic, transverse medullary slices (600μM) from CD1 mice of either sex (postnatal days 0–5), and recorded inspiratory activity from XII nerve rootlets using a suction electrode. We then bath applied arginine vasopressin (0.01μM and 0.1μM, AVP, V1a receptor agonist), or locally injected AVP (0.1μM and 1.0μM). In doing so, we were able to assess the effects of arginine vasopressin on XII inspiratory burst amplitude. Bath applied AVP increased inspiratory burst amplitude by 43.4%±12.5% (n=9) when applied at 0.01μM, and 56.4%±18.1% at 0.1μM. When locally applied (n=7), AVP at 0.1μM increased inspiratory burst amplitude 38.8%±6.2%, and AVP at 1.0μM caused a 26.5%±6.3% increase in inspiratory burst amplitude with a notable increase in tonic activity. In a separate set of experiments (n=8) to test whether AVP acts via V1a receptors, local AVP increased inspiratory burst amplitude by 40.2%±9.3%. Local pre‐application of the V1a receptor antagonist ((d(CH2)51Tyr(Me)2Arg8) Vasopressin, 500nM) for 60 s attenuated the response to AVP (6.7%±14.7% decrease in inspiratory burst amplitude), but the AVP response was recovered after 15 minutes with an increase in XII inspiratory burst amplitude of 22.3%±4.1%. Overall, our results support that AVP can increase XII inspiratory burst amplitude via V1a receptor activation. Further research will need to determine whether AVP plays a role in maintaining airway patency in vivo.

Support or Funding Information

Kenneth A. Suarez Research Fellowship, Midwestern University
Proteome and MicroRNAs in Human Milk: Term‐Dependent Changes in Exosome Composition
Authors: Vahkal Brett , Ferretti Emanuela , Altosaar Illimar , Beaulieu Jean-François
Published: April 2020

Infants born prematurely are a vulnerable population. Human milk is considered the best source of nutrition for infants as it contains not only nutrients, but also a variety of bioactive components to help infants adjust to life outside the womb. Among these components are exosomes, which are lipid bilayer vesicles that contain messenger RNA (mRNA), microRNA (miR) and proteins. Human milk exosomes contain various immunomodulatory components that may provide protection and promote microbial tolerance in the GI tract of newborns. However, mothers birth infants at very different times. Could preterm birth signal mother’s mammary gland to manufacture magical milk? We propose that term and preterm human milk convey different exosome constituents to the infant gut. We hypothesized that term and preterm human milk have distinct protein and miR content, and the exosomal isolates have unique immunomodulatory effects on intestinal cells. To investigate this, exosomes were isolated from human milk after term and preterm births. The exosomal miRs were characterized using RNA sequencing. Exosomal proteome enabled determination of abundant protein classes, narrowing of protein profile and discovery of essential proteins in preterm versus term milk. To investigate downstream pathways of either exosomes or specific exosomal‐contained proteins, we used the Caco 2/15 intestinal cell line to measure levels of immunomodulatory proteins following treatments with milk derived exosomes or proteins. These results provide novel insights for adaptations that exist in human term and preterm milk on both the proteome and expressional level. Ultimately, this research can guide the development for therapeutics in infant gastro‐intestinal diseases, such as necrotizing enterocolitis, and elucidate unique proteins that are released into human milk for downstream action(s) in the developing infant. Therapeutic benefits of preterm milk constituents are being explored.

Support or Funding Information

Canadian Institutes of Health Research
Mechanism of Action of Moringa oleifera (Moringa) in the Treatment of Diabetes Mellitus
Authors: Obih John-Clifford Agbasi , Obih Patience Ogechi , Arome Okai Samuel
Published: April 2020

Diabetes mellitus is a metabolic disease characterized by impairment of carbohydrate, protein and fat metabolism which results from defect in the secretion of insulin, insulin utilization or both. According to CDC, 30.3 million people have diabetes in the USA out of which 23.1 million people have been diagnosed and 7.2 million people have not yet been diagnosed. Approximately 84 million American adults have prediabetes. The prevalence of diabetes is increasing both in developed and developing countries. Standard drugs are available in the market for the treatment of diabetes. However, many people with diabetes are turning to medicinal herbs for treatment. The assumption of those who use medicinal herbs is that the herbs are edible and do not carry attendant side effects as the drugs in the market. Moringa oleifera is among the medicinal herbs used locally for diabetes. It is widely cultivated in India. Research has shown that Moringa oleifera is an edible plant that has multiple benefits in diabetes. In many parts of Africa, moringa is widely used for self‐medication in diabetes and hypertension. Its mechanism of action as an antidiabetic has not been well elucidated. This study was undertaken to examine if moringa affects carbohydrate metabolism like acarbose that reduces hyperglycemia by inhibiting alpha‐glucosidase. The alpha‐glucosidase inhibition study of moringa aqueous leaf extract was performed in vitro in a 96 well‐plate using Saccharomyces cerevisiae as the source of alpha‐glucosidase. The assay was analyzed in a multiskan spectrophotometer and the absorbance was read at 400 nm. A parallel test was run with acarbose. Acarbose, gave a dose‐dependent inhibition of alpha‐glucosidase and moringa showed also activity of a similar mechanism, but not as potent as acarbose. It has been reported that herbal medicines have multiple mechanisms of action. More studies need to be carried out to identify other possible mechanisms of action of Moringa oleifera as an antidiabetic.

Support or Funding Information

COE
Alterations in Phospholipase A2‐Mediated Pathways in Smokers: A Potential Mediator of Skin Cancer Development
Authors: McHowat Jane , Cosby Courtney , Lozano Juan Carlos , Kispert Shannon E. , Missall Tricia
Published: April 2020

Cigarette smoking is a major preventable cause of disease that maintains a disproportionate prevalence in populations distinguished by socioeconomic status, race, and gender. Much remains unknown about the biochemical effects of smoking on the development of skin cancers including squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). However, a recent meta‐analysis study demonstrated that cigarette smoking is associated with a 52% increase in the risk for SCC, but does not appear to modify the risk for BCC. Our previous studies have demonstrated that cigarette smoking is associated with changes in phospholipase A2 (PLA2)‐mediated pathways that are implicated in cancer development and progression. More specifically, PLA2 activation leads to increased platelet‐activating factor (PAF) and prostaglandin E2 (PGE2) synthesis, metabolites that are increased in cancer cells and mediate tumor growth, survival and invasion. To assess the effects of cigarette smoking in non‐melanoma skin cancer, we analyzed PAF and PGE2 expression in skin biopsies from six smokers and six non‐smokers with SCC, and six smokers and six non‐smokers with BCC using immunohistochemistry. PAF was not detectable in BCC patient samples. SCC patient samples revealed positive PAF expression with nuclear localization in all patient samples, but there was no significant difference between expression in samples of smokers when compared to nonsmokers. PGE2 expression was detected in BCC patient samples, with no difference between smokers and nonsmokers. PGE2 expression was greater in SCC when compared to BCC, with a 50% increase in expression in SCC samples from smokers compared to nonsmokers. Increased PGE2 accumulation was accompanied by a 25% increase in COX‐2 expression in SCC samples from smokers compared to nonsmokers. Increased COX‐2 and PGE2 production can stimulate the production of vascular endothelial growth factor (VEGF), accelerating tumor growth, survival and invasion. We detected a 40% increase in VEGF expression in SCC samples from smokers when compared to nonsmokers. COX‐2 and VEGF expression remained unchanged between BCC samples taken from smokers or nonsmokers. Taken together, our data suggest that increased PGE2 accumulation may contribute to the increased risk of SCC in smokers.
Dissecting Amino Acid Osmolytes: Is a Zwitterion Required for Increasing Protein Thermal Stability?
Authors: Canepa Jacob , Wilkinson Steven
Published: April 2020

Amino acids are an important class of osmolytes that demonstrate the ability to stabilize certain proteins under acute thermal stress. However, little consensus exists on what structural aspects produce this phenomenon. It has been proposed that the stabilizing nature of amino acids emerges from their zwitterionic nature at neutral pH, but little evidence has supported this view, especially in regards to the breadth of its applicability to proteins of varying structure and composition. In this report, we structurally dissect the amino acids L‐alanine and β‐alanine to determine functional group contributions to protein‐stabilizing activity on two structurally dissimilar proteins: human C‐reactive protein (CRP) and human myoglobin. The stabilizing effects of the osmolytes (and their structural derivatives) were quantified by Differential Scanning Fluorimetry (DSF) in three metrics: protein melting temperature (Tm), standard free energy change (ΔGo), and “m”‐values. Our results indicate that the zwitterionic theory holds for myoglobin, as removal of either the carboxylate or amino moiety from the amino acid abrogated all thermal stabilizing effects. Furthermore, substitution of the carboxylate group with a hydroxyl group did not restore protein‐stabilizing activity, arguing for the zwitterionic requirement for amino acid‐mediated thermal stabilization. However, the results with CRP were significantly different from those observed with myoglobin. In the case of CRP, the amino acid carboxylate proved to be the dominant stabilizing group, as compounds lacking the amino group conferred greater thermal stability than the complete amino acids. These results affirm that the zwitterionic theory is limited in its ability to describe amino acid‐mediated effects on protein thermal stability and suggests that the nature of the interactions between amino acid osmolytes and proteins is highly dependent on protein structure. These results may have significant implications for the development of chemistries to stabilize diverse protein targets in biospecimens for diagnostic applications.

Support or Funding Information

California State University Program for Education and Research in Biotechnology (CSUPERB)

Chevron Biotechnology Applied Research Endowment

Bill and Linda Frost Fund
Mature Adipocytes and FGF2 Influence Angiogenic Processes
Authors: Lowke Makenzie Taylor , Cortez Xiomara , Garcia Michelle Renee
Published: April 2020

The process of wound healing requires the transportation of substrates needed for the rebuilding of tissue through appropriate vessel formation; angiogenesis. Adipose tissue extract is often used to facilitate the wound healing process, which is attributed to its potent angiogenic properties. Most angiogenic process are regulated through growth factors such as vascular endothelial growth factor (VEGF), angiopoietin, and fibroblast growth factor (FGF). Fibroblast growth factor has been utilized to facilitate wound healing in the biomedical community, which is one of the growth factors synthesized and secreted by adipocytes. Therefore, it was hypothesized that mature adipocytes augment angiogenic processes in dispersed adipose tissue in the presence of FGF2 (basic). Adipose tissue was collected from 4 prepubertal female pigs (gilts) and enzymatically dispersed (collagenase type II). Cells were separated into 2 groups 1) mature lipid‐filled adipocytes (MA) and 2) all other cell types (OC) i.e., vascular endothelial cells (VEC), preadipocytes, and fibroblasts. The OC were incubated for 24 hr in cell culture media with 10% fetal bovine serum (FBS) to promote attachment and angiogenic processes. After the first 24 hr incubation period, media was removed and replaced with 2% FBS and MA were added with or without FGF2 (10−11 M) or rabbit polyclonal anti‐FGF2 (1:100 dilution) and incubated for an additional 24 hr. The MIXED procedure of SAS was utilized to determine the effect of treatment on in vitro angiogenic cellular processes. The addition of MA alone augmented cellular angiogenic processes including elongation, sprouting, formation of nascent tubes, and vessel tube formation (P < 0.05). However, vessel tube formation in the FGF2 treatment group was lower than MA alone (P < 0.05), which was ameliorated, in part, with FGF2 antibody treatment. Mature adipocytes appear to augment angiogenic processes; however, FGF2 may diminish or impair the progression of vessel tube formation.

Support or Funding Information

USDA LEADERS 2 Award #2017‐38422‐27298
Peroxidase activity along soil depth of a metal contaminated brownfield in New Jersey
Authors: Dyson Eshariah N. , Idrovo Cesar , Hagmann Diane , Krumins Jenifer , Goodey Nina
Published: April 2020

The peroxidase enzyme is a member of the oxidoreductase family of enzymes. It has been referred to as a remediation enzyme, as more peroxidase activity is observed when soils are experiencing oxidative stress. At Liberty State Park, a New Jersey brownfield, there is a section of the park that is closed to the public because of high levels of metal contamination. Despite the presence of metals in the soil, much of the area supports plant life. The goal of this study is to determine how the peroxidase activity and metals are distributed along soil depth and to compare results from a forested and barren area of the site. To do this, we collected three 10 cm deep soil cores from each of the two sites. Using the cores, we examine the peroxidase activity and metal concentrations of each 2 cm cross section. We found a similar trend in distribution along depth for metal concentration and peroxidase activity in the forested site. The barren site had a measurable peroxidase activity with a high metal concentration found in the top 2 cm cross section. These studies can serve as tools to understand the relationship between peroxidase activity, metal load, and soil depth and determine how these factors effect plant growth.

Support or Funding Information

NSF Grant CBET #1603741
Phosphorylation of Serine 263 in the Human Choline Transporter 1 alters the transporter’s function
Authors: Rizvi Midhat Ali , Arutyunov Denis , Caluseriu Oana , Leslie Elaine , Cordat Emmanuelle
Published: April 2020

Disruptions of the neuromuscular junction result in congenital myasthenic syndromes (CMS). CMS is characterized by akinesia, apneas or even death. CMS type 20 is caused by mutations in the SLC5A7 gene which encodes for the choline transporter (CHT1). The 13 transmembrane domain CHT1 protein is thought to be phosphorylated at several sites but the role of phosphorylation remains unknown. We have recently reported the missense recessive substitution c.C788>T in SLC5A7 in a family with a history of CMS using whole exome sequencing. This mutation encodes a p.S263F substitution in CHT1 and results in a complete loss of transport function despite a normal plasma membrane localization. To assess whether 1) the p.S263F substitution alters CHT1 phosphorylation state, and 2) a change in phosphorylation results in altered transport function, three substitutions were introduced into CHT1: p.S263A, and phosphomimetic p.S263D and p.S263E. Upon expression in HEK293 cells, we found that p.S263A mutant retained a high level of transport activity while the p.S263D and p.S263E mutants were non‐functional, suggesting that S263 phosphorylation inhibits choline transport. Immobilized metal affinity electrophoresis supports that wild‐type CHT1 may be more phosphorylated than p.S263F CHT1 mutant. These results support the essential role of Serine 263 residue in CHT1 choline transport possibly via altering its phosphorylation status.

Support or Funding Information

Women and Children’s Health Research Institute, Stollery Children’s Hospital Foundation, Canadian Institute of Health Research & The University of Alberta
IMPLICATING RIBONUCLEASES IN MAMALIAN TAILED MIRTRON BIOGENESIS
Authors: Zia Farid , Flynt Alex
Published: April 2020

MicroRNAs are pervasive small noncoding ~22 nt gene regulators that collectively control gene expression by destabilizing mRNA through repressing translation. Since they are broadly involved in cell differentiation and maintenance, they are linked to most diseases including the major causes of mortality, heart disease and cancer. Several thousand miRNA genes have been reported in mammals that typically generated thorough a canonical pathway involving sequential cleavage by Drosha and Dicer, however, there are many non‐canonical miRNAs that mature through alternative pathways. Mirtrons are the most abundant class of atypical miRNA that bypass Drosha cleavage step and instead are produced by splicing. Following lariat debranching, the host intron RNA can be exported to cytoplasm for Dicer processing. There are 3 classes of mirtrons: conventional, 5′‐tailed, and 3′‐tailed. In tailed mirtrons, splicing defines only pre‐miRNA hairpin end. While a mechanism has been reported for 3′‐tailed mirtrons, processing of 5′‐tailed varieties remains a mystery. Here, we studied the biogenesis of human and mouse 5′‐tailed mirtrons to implicate RNases in tail processing. Through a mutational approach we have identified endocnucleolytic cleavage of precursors to be important to processing of 5′‐tailed mirtrons. This is a novel mechanism that contrasts with what has been shown for 3′ tailed mirtrons. Our findings shed light on the most substantial variety of mirtrons encoded by the human genome.

Support or Funding Information

This work was supported by the National Institute of Health and also Mississippi INBRE program (R15GM120716‐01).
Tissue Factor Pathway Inhibitor (TFPI) release by bovine, ovine and porcine unfractionated heparins in primates. Comparative studies at mass and potency adjusted intravenous dosing
Authors: Kouta Ahmed , Bontekoe Emily , Duff Richard , Jeske Walter , Hoppensteadt Debra , Yao Yiming , Fareed Jawed
Published: April 2020

Background

Unfractionated heparin (UFH) is a highly sulfated glycosaminoglycan (GAG) that consists of repeating disaccharide units, containing iduronic acid (or glucuronic acid) and glucosamine, exhibiting variable degrees of sulfation. UFHs release tissue factor pathway inhibitor (TFPI) which inhibits extrinsic pathway of coagulation by rapidly inactivating of Factor Xa and the Factor VIIa/TF complex. Bovine mucosal heparins (BMH) and ovine mucosal heparin (OMH) are currently being developed for re‐introduction for both medical and surgical indications. In contrast to the porcine mucosal heparins (PMH), the active pharmaceutical ingredient (API) BMH exhibits a somewhat weaker anti‐coagulant activity as cross‐referenced against PMH. In this study, we determined the TFPI antigen release level by various dosages of UFHs using ELISA method. Also, we projected that potency adjusted BMH at equivalent dosage has the same TFPI release profile compared to OMH and PMH.

Materials & Methods

BMH, OMH and PMH were obtained from various commercial sources. Potencies of each heparin were determined by an amidolytic anti‐Xa assay in relation to the USP heparin standard. Individual groups of primates (n=4) were administered 0.5 mg/kg or (50 or 100) U/kg dosage of bovine, ovine or porcine UFH via intravenous route. Blood samples were collected at baseline, 15, 30, 60 and 120 minutes post‐administration. Tissue Factor Pathway Inhibitor (TFPI) antigen levels were measured using a commercially available sandwich ELISA methods. All results were compiled as mean ± SD.

Results

At a gravimetric level (0.5 mg/kg), BMH exhibited a lower TFPI level than OMH & PMH at all time points with a maximum value of (200 ng/ml) at 15 mins post drug administration. OMH and PMH showed comparable TFPI levels at all time points with maximum values of (250 ng/ml) and (265 ng/ml) respectively at 15 mins post drug administration. However, USP cross‐referenced anti‐Xa potency adjusted based dosing showed comparable TFPI antigen levels for all three UFHs at all time points with a maximum value of 250 ng/ml at 15 mins post drug administration. At 60 mins post drug administration, potency adjusted BMH showed slightly higher TFPI level of (200 ng/ml) compared to both OMH & PMH.

Conclusion

Tissue factor pathway inhibitor (TFPI) levels at 15 minutes after IV administration of 0.5 mg/kg heparins to non‐human primates showed that both PMH and OMH release comparable TFPI antigen levels which were significantly higher than BMH. Potency equated dosing resulted in comparable TFPI release profiles for all three heparins. Therefore such dosing may provide uniform levels of anticoagulation for the parenteral indications for UFHs. These observations suggest that potency equated bovine heparin has comparable release of TFPI and therefore is biosimilar to the OMH and PMH.
“My Vagina Did What?”: Evaluating a 3D Mobile App of Enterocele Anatomy in Post‐Hysterectomy Women
Authors: Park Yuna K. , Corral Janet , Lee Lisa M.J. , Muffly Tyler M.
Published: April 2020

Pelvic organ prolapses are becoming more prevalent, with the number of diagnoses projected to increase by 46% in the United States by 2050. One type of prolapse is enterocele, a condition in which a peritoneal sac containing the small intestines herniates into the vaginal wall. Secondary to weakened pelvic floor muscles, enterocele is strongly associated with hysterectomy, with 60% of women diagnosed with enterocele having a past history of hysterectomy. Healthcare providers discover prolapse during a physical exam when the vaginal wall bulges outside the vaginal opening. Despite the increasing prevalence and complicated anatomy of enterocele, there are few resources to educate women on the anatomy of a prolapse. Knowledge gaps cause patients stress and shame surrounding their condition, limiting their ability to confide in others and seek medical advice. Thus, supplementing counseling with patient education resources on enterocele anatomy may increase patient understanding and improve communication between patient and provider. Effective counseling may decrease patient anxiety by addressing patient fears, relieving humiliation, and putting an end to self‐blame. The first aim of this project was to develop an interactive three‐dimensional (3D) mobile application (app) to assist healthcare providers in educating post‐hysterectomy women on the anatomy of enterocele. The second aim was to assess the educational value of the mobile app as a visual aid during counseling and its ability to increase patient understanding and decrease anxiety. The 3D model utilized in the app was segmented using 3D Slicer from a de‐identified CT urogram and pelvic MRI of a female diagnosed with enterocele post‐hysterectomy. Artifacts were smoothed in ZBrushCore 2018, and the model was animated in Autodesk Maya 2019. The model was imported into a game engine, Unreal Engine 4, and packaged into a mobile app, which was iteratively designed to highlight relevant structures of pelvic anatomy and allow the model to be enlarged and rotated. The mobile app was revised based on surveys from healthcare providers on the app’s usability and educational value. To test the efficacy of the app, patients were randomized into two groups: (1) a control group with standard counseling and (2) an experimental group with counseling supplemented by the mobile app. Patients completed pre‐ and post‐intervention surveys assessing anatomical understanding and anxiety regarding their diagnosis. Preliminary data suggest the mobile app has a high potential to assist healthcare providers with educating patients. Complete analysis of patient and clinician data will be presented. Women’s perspectives and issues are traditionally underrepresented in medicine. By developing a mobile app to increase awareness, we intend to normalize the conversation and overcome the social stigma of female pelvic organ prolapse.