bluuebunny/crossref_metadata_2025 huggingface dataset:
The effects of varying DNA flap length from 20 to 50 deoxynucleotides on the mechanism of single‐strand annealing in Saccharomyces cerevisiae
Authors: Odango Jane , Fregoso Fred , Camberos Juan , Fischhaber Paula
Published: April 2020

Mutagens constantly threaten the integrity of genetic material by causing damage in the form of double‐strand breaks (DSBs). Such damage is typically repaired through various modes of homologous recombination (HR). This project focuses on a form of HR known as single‐strand annealing (SSA), which is triggered when a DSB is located between two DNA repeats. This pathway generates 3' overhanging single‐stranded DNA “flaps” which are cleaved by the Rad1‐Rad10 endonuclease, aided by the mediator protein Saw1 in S. cerevisiae. Recent literature has demonstrated that Saw1‐dependent recruitment of Rad1‐Rad10 to damage sites, and SSA repair proficiency, are both a function of flap length. In this study, we set out to elucidate the precise flap length threshold requiring Saw1 for optimal SSA repair in vivo through fluorescence microscopy and real‐time quantitative PCR (qPCR).

In order to investigate the importance of DNA flap length on Rad1‐Rad10 and Saw1 recruitment, we designed yeast strains generating 20, 30, or 50 nt flaps during SSA. A DSB was induced at a fluorescently labeled site in the yeast genome (DSB‐RFP), allowing us to monitor the recruitment of Rad10‐YFP via fluorescence microscopy. The percentages of cells containing colocalized foci in strains either wild‐type or mutant in Saw1 were quantified, revealing that Saw1‐mediated recruitment of the Rad1‐Rad10 complex increased with flap length. Surprisingly, we found that Saw1 was actively recruited to the DSB site across all flap length substrates via experiments with a Saw1‐CFP strain. Analyses via qPCR were also carried out to quantify SSA repair products formed in WT and saw1Δstrains. The data show diminished repair product formation in the absence of Saw1 as flap lengths increased. Together, these results suggest that Saw1 is required for Rad1‐Rad10 recruitment and efficient SSA repair in longer flap substrates with ~20–30 nt representing the key size triggering Saw1‐dependence for repair. Current efforts are underway to determine the Saw1‐dependence of proficient SSA repair under conditions in which background induction is not amplified by qPCR.

Support or Funding Information

The authors thank our Department and the NIH SC3 program for funding.
The EBP50 and VDAC1 Interaction Mediates Cytoprotective Signaling on Mitochondria during Oxidative Stress
Authors: Jou Tzuu-Shuh , Chang Pei-Yu
Published: April 2020

Mitochondria, the organelles controlling ATP production and cellular metabolism, play a vital role in a number of cellular stress responses by communicating with other cellular compartments, for example, mitochondria‐associated membranes (MAMs), the tightly membrane contact site between mitochondria and endoplasmic reticulum (ER). We recently found a scaffold protein, Ezrin/radixin/moesin (ERM)‐binding phosphoprotein 50 protein (EBP50), plays a cytoprotective role on mitochondriam. Knockdown of EBP50 elicited severe mitochondrial morphological change fro fusion to fission and augmented apoptotic rate upon H2O2‐induced stress and could be rescued by overexpressing siRNA resistant construct of EBP50 in Madin‐Darby Canine Kidney (MDCK) cells. Furthermore, PDZ1 domain of EBP50 interacted with one of pivotal MAMs proteins, voltage‐dependent anion channel 1 (VDAC1), which is localized on outer mitochondrial membrane (OMM). The N‐terminus of VDAC1 regulates its oligomerization and channel activity. Amine‐terminal deletion (aa 1‐26) of VDAC1 diminished EBP50‐VDAC1 interaction while carboxyl‐terminal deletion (aa 240–283) retained the intact association. Moreover, the central component of the translocase of outer membrane (TOM) Tom20 also participated in EBP50‐VDAC1 interaction.

Our study paves the way for unraveling the detailed mechanisms and provides molecular insights into how mitochondria are protected from damaging during oxidative stress response.
Controlled Cortical Impact Increases the Relative Abundance of Chondroitin 6‐Sulfate in the Lateral Posterior Thalamus
Authors: Logsdon Aric , Alonge Kimberly , Wight Thomas , Guttman Miklos , Banks William
Published: April 2020

The brain is limited in its capacity to repair and reorganize neural networks following traumatic brain injury (TBI). Sulfated chondroitin sulfate (CS)‐glycosaminoglycan (GAG) extracellular matrix chains are known to influence formation and maintenance of neural networks, and maladaptive changes to the CS‐GAG abundance or sulfation pattern are associated with neurocircuit dysfunction. Recent reports indicate that TBI can alter the sulfation pattern of CS‐GAG chains, however, little is known about the anatomical and functional relevance of these changes. In this study, adult male C57BL/6 mice were subjected to a TBI via a controlled cortical impact (CCI) to the somatosensory cortex (SSC), where an established thalamocoritcal connection between the SSC and the lateral posterior thalamus (LPT) exists for sensorimotor function. At 7 and 28 days following CCI, immunofluorescence analysis of the ipsilateral, but not the contralateral LPT revealed a significant increase in the 6S‐CS sulfated GAG marker CS‐56. To analyze the relative changes in all CS‐GAG sulfation, CS disaccharides were released from both ipsilateral and contralateral SSC and LPT regions and quantified using a recently optimized LC‐MS/MS + MRM technique, which can differentiate between the relative abundance of five specific CS isomers (Δ4S‐, Δ6S‐, Δ4S6S‐, Δ2S6S‐, Δ0S‐CS) and dermatan sulfate (Δ2S4S‐DS). Here, we show that both the ipsilateral SSC and LPT exhibited higher percentages of Δ6S‐CS disaccharides compared to the contralateral brain regions, as well as the sham control brain regions. To determine whether the increase in 6S‐CS abundance in the SSC and LPT associate with sensorimotor function, CCI mice were subjected to the rotarod task to measure sensorimotor performance. Mice exposed to CCI exhibited a decrease in their latency to fall from the rotarod, which inversely correlated to the percentage of Δ6S‐CS. Collectively, these data suggest that CCI alters the CS‐GAG sulfation pattern within the thalamocortical circuitry, and these changes may contribute to persistent sensorimotor dysfunction. Future studies will investigate novel therapeutic interventions targeting CS‐GAG chains to potentially improve outcomes from TBI.

Support or Funding Information

R01GM127579 to M.G.R01AG046619 to W.B.F32DK12266201 to K.A.
Exploring the Effects of a V104F/V477F Double Mutation on CYP2B6 Activity Towards Efavirenz
Authors: Prewett Alisa , Riegsecker Ben , Angle Ethan , Cox Philip
Published: April 2020

Background

The Cytochromes P450 2B6 (CYP2B6) and 2A6 (CYP2A6) participate in the metabolism of the anti‐HIV drug Efavirenz (EFV). CYP2B6 monohydroxylation of EFV results in the formation of 8‐hydroxyefavirenz (8‐OHEFV) whereas CYP2A6 monohydroxylation of EFV forms 7‐hydroxyefavirenz (7‐OHEFV). To date, little is known about the structural characteristics of these enzymes that lead to differential EFV metabolite formation. In order to better understand such characteristics, we sought to mutate CYP2B6 towards CYP2A6 in hopes of uncovering amino acids critical for the formation of 8‐OHEFV. Specifically, we decided to focus on two phenylalanine residues in the CYP2A6 active site that are valines at the analogous positions in CYP2B6.

Methods

The valine residues at positions 104 and 477 of CYP2B6 were mutated to phenylalanines using site directed mutagenesis, creating a V104F/V477F double mutant. Wild type (WT) and mutant CYP2B6 expression was accomplished in COS7 cells and, following treatment with EFV, metabolites were extracted and detected using a novel gas chromatography‐mass spectrometry (GC‐MS) method after chemical derivatization using BSTFA + 10% TMCS. Protein expression was assessed via western blotting.

Results

We observed little to no difference in the protein expression of WT and mutant CYP2B6 proteins. 8‐OHEFV production in the COS7 system was detected for wild type and mutant CYP2B6, but no 7‐OHEFV production was observed. Interestingly, GC‐MS analysis of 8‐OHEFV formation by the V104F/V477F mutant indicates a marked increase in 8‐OHEFV production, suggesting the combination of these mutations might enhance 8‐OHEFV formation by CYP2B6.

Future Goals

Future experiments will seek to characterize each single mutant and determine kinetic parameters for 8‐OHEFV formation by all three mutant CYP2B6 proteins.

Support or Funding Information

Funding for this work was generously provided by the Department of Biology and Chemistry at Azusa Pacific University and the Faculty Research Council of Azusa Pacific University
Changes in the gut microbiota of wild mice according to environment
Authors: Cho Seongbeom , Kim Junhyung , Guk Jae-Ho , Song Hyokeun , Suh Jun Gyo , Seong Je Kyung
Published: April 2020

Background

The gut microbiota is closely linked with the health and disease status of hosts. This could be affected by multifactorial factors, among which environmental factors have a great effect on the gut microbiota, which is the cause of different phenotypes in mice. Here, we investigated the changes in the gut microbiota of wild mice according to habitat and diet.

Methods and Results

Forty‐nine Mus musculus were captured from fields that were separated by rivers, roads, and mountains in Korea. Sixteen representative wild mice were transferred to laboratory animal facilities and their gut microbiota was analyzed at 2–4 weeks intervals up to 20 weeks. At the phylum level, 16 phyla were identified; among them, four phyla (Firmicutes, Bacteroidetes, Epsilonbacteraeota, and Proteobacteria) comprised the core gut microbiota and accounted for about 98% of the total microbiota. In alpha diversity, the number of observed OTUs and richness significantly reduced, while the evenness significantly increased. In beta diversity, the distribution of the gut microbiota initially differed depending on the habitat of wild mice. However, as wild mice adapted to laboratory animal facilities, the gut microbiota clustered together in the PCoA plot and the volatility of beta diversity also showed distinct differences. Furthermore, the relative abundance of Staphylococcus (p < 0.01) and Alistipes (p < 0.05) tended to increase at the genus level.

Conclusions

Due to the variety of wild environments in which wild mice are exposed, wild mice have a greater diversity and distribution of the gut microbiota than mice exposed to constant environments. As wild mice adapted to a constant environment, the gut microbial diversity decreased and the distribution of the gut microbiota became constant. This study provides a new approach, gut microbiota analysis, in the assessment of environmental exposure in wild mice, which is limited based on phenotyping and genotyping.

Support or Funding Information

This research was supported by the National Research Foundation of Korea (NRF‐2018R1A2B6002396) and the Korea Mouse Phenotyping Project (2016M3A9D5A01952417) of the Ministry of Science, ICT and Future Planning.
Genetic Association of ACE Polymorphism with Sarcopenia and Effects of Genetic Regulation of ACE Activity on Muscle Function
Authors: Kim Chulhyun , Kim Hyeoijin , Song Keon-Hyung , Kwon Insu , Ambegaonkar Jatin
Published: April 2020

The purpose of this study was to investigate associations between skeletal muscle mass and ACE polymorphism, or sarcopenia‐related candidate gene, and to examine the effects of genetical angiotensin‐converting enzyme (ACE) activity on physical performance and respiratory muscle function in Korean adults. For this study, 1,464 Korean adults aged 18~79 were recruited. The anthropometrics were 1.69±0.06m (height), 63.1±5.5kg (weight), 29.4±6.5% (percent body fat), and 24.5±3.0kg/m2 (body mass index). Fat‐free mass, physical performance, gait speed (Timed Up‐and‐Go), pulmonary function (maximal inspiration and expiration pressure) and fatiguing maximum voluntary isometric contraction (40% MVIC) of ankle plantar flexor muscles with dynamic Electromyography were tested. Data of body composition and muscle functions were analyzed using one‐way ANOVA and post‐hoc Scheffe test for continuos variables and the Chi‐square test and host‐hoc Bonfferioni correction for countable variables. Blood samples for the whole blood and serum were obtained and assayed for circulating ACE activity and the ACE I/D genotype. The distribution of ACE polymorphism throughout the groups was analyzed using the Chi‐square test and host‐hoc Bonferroni correction for genetic distribution and multiple logistic regression analysis for the odds ratios (ORs) with 95% confidence intervals (CIs). We found that the distribution of ACE Indel genotype for sarcopenia was significantly different from normal population who have normal or high fat‐free mass index. Especially, we found a significantly higher (p<.05) distribution of DD genotype in sarcopenia participants (27%) than the incidence (12.5%) in normal population. The D allele of ACE genotypes in older adults indicated significantly slower gait speed than I allele of them in the Timed Up‐and‐go test (p<.05), and significantly lower levels for maximal inspiration and expiration pressure (p<.05). The II genotype in older adults indicate the highest tolerance of muscle fatigue during muscle contraction (p<.05). In conclusion, ACE Indel polymorphism and level of ACE activity is associated with sarcopenia in older adults and would genetically regulate muscle function in part. Our findings suggest that genetical concentrations of angiotensin‐converting enzyme should play a role in determining low muscle mass, slow walking speed, respiratory muscle function, and fatigue after muscle contraction in individuals. Therefore, examining ACE Indel polymorphism and ACE activity level could be used to predict sarcopenia and muscle function, and subsequently this information can be applicable to prevention and treatment to the aging muscle.

Support or Funding Information

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (358‐2011‐1‐G00025).
Effects of Artesunate with Paclitaxel on the Proliferation and Morphology of Androgen‐sensitive (LNCaP), Androgen‐insensitive (PC‐3) Human Prostate Cancer Cell Lines and Normal Epithelial Prostate cell line (RWPE‐1)
Authors: Adedeji Dolapo , Fabian Juan , Adedeji Adetayo , Payne Gloria
Published: April 2020

Background

Prostate cancer is one of the diseases worldwide that causes cancer‐related deaths in men and is the second most common cancer affecting thousands of men each year in the United States behind only lung cancer. Artesunate (ART) is part of the artemisinin drugs for the treatment of malaria. Paclitaxel (PTX) is an anticancer agent that belongs to the Taxane class and is used for the treatment of metastatic hormone‐refractory prostate cancer.

Purpose

The aim of this study was to examine the effects of combining ART with PTX on human prostate cancer cell lines ‐ LnCAP and PC‐3, normal epithelial prostate cell line RWPE‐1 and observe any morphological change using Scanning Electron Microscope (SEM).

Method

In vitro anti‐proliferative 3‐(4,5‐Dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium (MTS) assay was used to assess the effects of ART with PTX on human prostate cancer cell line (LnCaP and PC‐3) and normal prostate cell line (RWPE‐1) after 72‐ and 120 – hour exposure at a concentration range from 1 nm – 100 μM. For the SEM images, cell lines were cultured with thin films of gold, titanium and zinc oxide deposited by magnetron sputtering on silicon substrate at room temperature. Images of samples with and without the drugs were acquired.

Results

The average cell death observed for both LnCAP and PC‐3 cell lines was 40% and 73% after 72‐ and 120‐ hours incubation with ART and PTX at different combination ratios of 1:1; 1:2 and 2:1 for ART: PTX and 1:1; 1:2 and 2:1 for PTX: ART with concentrations ranging from 1 nm – 100 μM. For normal epithelial prostate cell line, RWPE‐1, the average cell death observed ranged from 0.35% – 23% using the same combination ratios. Cell adhesion was detected on silicon substrates and on thin films of gold, titanium and zinc oxide. Morphological changes were observed when samples were treated with and without drugs. These SEM images were associated with cell lines undergoing apoptosis when compared with control (images with no drugs).

Conclusion

Although further observations are needed, it appeared that combination of ART with PTX displayed cytotoxicity regardless of whether the prostate cancer cell line is androgen‐sensitive or androgen‐insensitive after 72 and 120 ‐ hour exposure with little or no effect on normal epithelial prostate cell line. Therefore, combination of both drugs may aid in reducing cardio‐toxicity exerted by PTX alone thereby reducing the prostate cancer mortality rate in men.

Support or Funding Information

Work support by ECSU Mini‐grant and in part by NSF Award# 1818774
BACTERIAL CELLULOSE MEMBRANES WITH PLASMA‐MODIFIED SURFACES FOR TISSUE ENGINEERING APPLICATIONS
Authors: Benevenuto Luíz Guilherme Dercore , Cruz Sandra Andrea , Alves Eduardo Donato , Achcar Jorge Alberto , da Silva Barud Hernane , Montrezor Luís Henrique
Published: April 2020

Bacterial cellulose (BC) is a promising biomaterial notable for its features such as strength, hydrophilicity, and biocompatibility, which enable it to be used as implants and scaffolds in tissue engineering. However, its topography does not present the ideal characteristics for cell attachment and development, so surface modifications are required in order to improve the cellular response. The objective of this study was to analyze the effects of oxygen plasma treatment applied to bacterial cellulose (BC) for 2.5 and 5 minutes, together with its effects on cell behavior during 21 days of culture. Osteo‐1 cells were cultured in the absence and presence of BC, at an initial density (day 0) of 1000 cells per 200 μL of osteogenic medium (DMEM + FBS + ascorbic acid + dexamethasone + β‐glycerophosphate), in 48‐well culture plates, for 7, 14, and 21 days, with exchange of the medium every 72 h. Cell counting was performed with a Bio‐Rad TC20™ cell counter, viability was analyzed using the MTT test, and mineralization was quantified by the Alizarin Red test. Alkaline phosphatase (AP) in the culture medium was measured using a commercial LabTest kit. Statistical analysis of the data was performed using ANOVA and Fisher’s test for multiple comparisons. Alkaline phosphatase results: Higher AP was observed for the group with application of the plasma to the BC for 5 minutes, while no statistically significant differences were found among the groups without treatment, treatment for 2.5 minutes, or in the absence of BC, as well as between the days of culture. Culture time effects: Higher cell counts were obtained for the control group without BC and the group with untreated BC. The highest cell counts were observed at 14 and 21 days of culture, with no statistically significant differences among the means. Mineralization and cell viability: The highest responses for mineralization and viability were observed at 7 days of culture, followed by decreases at 14 and 21 days. The results were indicative of changes on the bacterial cellulose surface caused by the oxygen plasma treatment, which influenced the cell features. The findings indicated that plasma treatment for up to 5 minutes changed the topography of the surface, leading to better conditions for cell differentiation and development from Osteo‐1 to osteoblast, as indicated by the higher AP concentration for this group. The treatment for 5 minutes led to enhanced cellular responses on the bacterial cellulose, contributing to better cell development.

Support or Funding Information

Funding

FAPESP (grant #2018/14840‐4).
Heparinox, a generic version of low molecular weight heparin enoxaparin, is bioequivalent to the branded version
Authors: Qneibi Dalia , Ramacciotti Eduardo , Kouta Ahmed , Hoppensteadt Debra , Daravath Bharathi , Jeske Walter , Iqbal Omer , Fareed Jawed
Published: April 2020

Introduction

Enoxaparin is the most widely used low molecular weight heparin. Several generic versions of enoxaparin have also become available for clinical use in the indications for which this product is approved. The regulatory bodies require the generic products to conform with the product specifications in terms of biological and structural equivalence. Heparinox (Cristália, São Paulo, Brazil) represents a new generic version of enoxaparin which has become available in the South American continent recently. The purpose of this investigation is to compare the branded enoxaparin (Sanofi Aventis, Paris, France) with heparinox using validated biologic and physico‐chemical assays to demonstrate the similarities of this low molecular weight heparin with its branded version.

Materials and methods

Heparinox brand of enoxaparin was obtained in commercial syringes containing 20 and 40 mg doses. Branded enoxaparin was commercially obtained in syringes containing 100 mg. The molecular weight profiling was carried out by gel permeation chromatography using a narrow range calibrator method. The anticoagulant activities were measured in whole blood and plasma based assays. The antiprotease activities were measured using anti‐Xa and IIa chromogenic substrate methods. USP compliant assays were used to calculate potency relative to the NIBSC low molecular weight heparin standard (NIBSC, London, England). Protamine titration studies were carried out to compare the extent of neutralization of the generic and branded products.

Results

The molecular weight of 2 batches of heparinox was comparable to that of the branded enoxaparin (4.1 kDa vs. 4.1 kDa). In the ACT, APTT and thrombin time assays the concentration curves for heparinox and branded enoxaparin were superimposable. Similarly, the antiprotease activities of the branded and generic products were comparable. There was no difference in the USP potency of the heparinox (96 anti‐Xa units/mg, 32 anti‐IIa units/mg) in comparison to the branded product (97 anti‐Xa units/mg, 32 anti‐IIa units/mg). The protamine titration studies revealed similar neutralization profiles for both the generic and branded products.

Conclusions

These studies demonstrate that the newly introduced generic low molecular weight heparinox exhibits similar molecular weight and functional profiles in comparison to the branded enoxaparin. Additional structural studies using NMR and oligosaccharide analysis, and pharmacological profiling in terms of antithrombotic, bleeding and PK/PD parameters are currently in progress.
Gender Differential Upregulation of 15‐Hydroxyprostaglandin Dehydrogenase Associates with Aspirin Decreasing Intracranial Aneurysm (IA) Rupture in Mice
Authors: Lu Yongjun , Ishii Daizo , Hasan David M.
Published: April 2020

Previously we showed 1) that aspirin decreased the risk of IA rupture significantly more in male than in female in the International Study of Unruptured Intracranial Aneurysms (ISUIA) and 2) the presence of sex‐differential plasma level of 15‐hydroxyprostaglandin dehydrogenase (15‐PGDH) in the lumen of human IAs. These observations were confirmed in our animal experiments using established mouse model of IAs. However, the aspirin dose used in these experiments was high (25 mg/kg/d).

In the present study, we used a lower dose of aspirin 5 mg/kg/d (comparable to 325 mg for human) in our mouse‐model experiments to examine the protective effect of aspirin at lower doses and to test whether such sex differential response still exists in response to lower dose of aspirin.

Aneurysms were induced with hypertension using Angiotensin II infusion (1000ng/kg/min for 21 days) and elastase (35m Units, single injection into mice basal cisterns). When grouping males and females together (n=30), aspirin decreased significantly the incidence of aneurysm when compared to control (60% vs 87%, p=0.0164), and the incidence of aneurysm rupture (43% vs 68%, p=0.0480). When analyzing the incidence of aneurysm rupture by gender (n=15), the incidence of aneurysm formation was significantly lower in male versus female mice on aspirin (40% vs 80%, p=0.0302), and the incidence of aneurysm rupture was lower in male mice (33% vs 53%, p=0.2311).

qPCR analysis from individual mouse cerebral arteries showed significantly higher wall tissue of 15‐PGDH mRNA in male than in female mice at baseline (before administering aspirin) (1.69±0.12 vs 1.10±0.14, p=0.027). Administering aspirin upregulated this expression and maintained the sex difference (2.37±0.30 vs 1.25±0.12, p=0.012).

These observations further validate the previous findings that aspirin protect against IA rupture significantly more in males than in females, potentially via 15‐PGDH pathway, which may provide a new promising therapeutic target for decreasing risk of aneurysm ruptures.

Support or Funding Information

NIH, 1R01NS107293‐01A1
Protective Effects of Magnesium Biotinate on Propionic Acid‐Induced Autistic Features in Rats
Authors: Komorowski James , Ojalvo Sara Perez , Orhan Cemal , Erten Fusun , Sahna Engin , Kılıc Ertugrul , Sahin Kazim
Published: April 2020

Objective

This study investigated the protective effects of magnesium biotinate (MgB) on propionic acid (PPA)‐induced autistic features in rats.

Background

Biotin is involved in energy metabolism and various immune and nerve functions. MgB is a novel biotin complex that has enhanced absorption. Compared to D‐Biotin, MgB has superior tissue uptake and anti‐inflammatory effects. Neurological problems can occur in biotin‐deficient individuals. Supplementation with MgB may prevent or reduce neurodevelopmental disorders such as those seen in autism spectrum disorder.

Design/Methods

Thirty‐five male Wistar rats (3 weeks old) were randomized into the following five groups; 1: Control (received saline), 2: PPA alone, 3: PPA + 10 mg human equivalent dose (HED) MgB, 4: PPA + 100 mg HED MgB, 5: PPA + 500 mg HED MgB. Animals were fed a standard diet, with or without MgB, for 35 days and on day 30, rats were administered PPA (500 mg/kg BW/day) by subcutaneous injection for 5 days. Neurobehavioral tests were then performed for two days. Serum and tissue samples were then collected for analysis.

Results

Social interaction deficits and signs of brain toxicity, including higher brain levels of inflammatory and oxidative stress markers, were observed in PPA alone rats. Brain levels of inflammatory markers were lower in all MgB groups compared to PPA alone (p<0.05). Oxidative stress markers were lower and antioxidant markers were higher in the 100 mg and 500 mg HED MgB groups compared to PPA alone (p<0.05). These effects were most significant in the 500 mg HED MgB group. Sociability and social preference scores were greater in the 100 and 500 mg HED MgB treatment groups compared to PPA alone (p<0.05). The 500 mg HED MgB dose improved social preference score to the extent that it did not differ from normal controls.

Conclusions

Pre‐treatment with MgB significantly reduced brain toxicity and improved autistic features induced by PPA.

Support or Funding Information

This study was conducted at Firat University and funded by JDS Therapeutics, LLC.
Evaluation of Tumor Necrosis Factor Alpha (TNF‐α) in Sleep‐Deprived Menopausal‐Induced Rats and the Impact on Bone Health
Authors: Ellsworth Nicole Lynn , Frisch Frank
Published: April 2020

Post‐menopausal osteoporosis as a consequence of estrogen depletion is a growing concern for women in the United States. As more women take on executive positions and experience sleep deprivation, there is the potential for up regulation of pro‐inflammatory cytokines, such as tumor necrosis factor alpha. It follows that the homeostatic imbalance of osteoclastic and osteoblastic activity leads to a greater risk of disease. Bisphosphonates generally, and Zoledronate specifically works by decreasing the number of osteoclasts. This current study investigated the impact of Zoledronate on the concentrations of tumor necrosis factor alpha‐type (TNF‐α) in 32 ovariectomized Wistar rats that were ovariectomized to simulate post‐menopausal estrogen‐deficiency. After one week of surgical recovery, rats were separated into 4 groups. The control group (C, n=4) was given an intraperitoneal (IP) injection of 0.45 mL of 0.9% saline, were housed in standard conditions permitting 12 hours light and 12 hours dark. The Zoledronate group (Z, n=8) were given an IP injection of 50 ug/kg of 10% Zoledronate and housed in standard conditions with a 12 hour light/dark cycle. The sleep‐deprived group (SD, n=8) were given the same IP injection as the rats in C, but were housed in chambers that did not permit sleep for 18 hours, then moved to sleep chambers for 6 hours. The sleep‐deprived Zoledronate group experienced the same sleep deprivation as the SD group, but received an IP injection of 50ug/kg body weight of 10% Zoledronate. Throughout the five‐week period of sleep deprivation cycles, the concentrations of TNF‐α were collected and examined. It was originally hypothesized that the sleep‐deprived group of rats would have the highest concentration of TNF‐α due to the biological stress associated with insomnia. However, TNF‐α levels were significantly higher in the Zoledronate group than both the control and sleep‐deprived groups, as well as the sleep‐deprived with Zoledronate groups (p<0.01). We ascribe this to bisphosphonate induced transient fever seen in Zoledronate usage in previous studies (Dicuonzo, 2003). It is also suspected that the low concentrations of TNF‐α in the sleep‐deprived groups are seen due to the short time frame of this experiment along with a challenged immune system in the animals. With a longer period of sleep deprivation, it is possible that the hypothesized cytokine levels would be reached.

Support or Funding Information

This study was supported in part by Novartis, Basel, Switzerland
Heparan Sulfate Deficiency Enhances Wound Healing
Authors: Riley Raquel , Phan Anne Q. , Zurich Raymond , Li Jing Amelia , Li Sophie Lu , Trinh Erica , Lee Erica , Padala Vikram , Liang Iris , Wilson Andrew , Esko Jeffrey D.
Published: April 2020

Heparan sulfate has many biological activities, in particular positive effects on growth factor signaling and cell adhesion. Thus, mutations in heparan sulfate assembly typically manifest in loss‐of‐function, leading to developmental defects or altered physiology. Here, we demonstrate that genetic and pharmacological reduction of heparan sulfate enhances wound healing, a gain‐of‐function phenotype. In one model, 8‐week‐old mice were shaved and subjected to a full thickness 6‐mm diameter punch biopsy through the dorsal skin. Imaging of the wound over two weeks showed that wound closure occurred more rapidly in mice bearing a defect in Ext gene function, which encodes the copolymerase responsible for the assembly of the backbone of the chain. Enhanced wound closure also occurred after tamoxifen‐induced deficiency of heparan sulfate in skin (Rosa26‐CreER; Ext1flox/flox mice) or in keratinocytes (K14‐CreER; Ext1flox/fflox). Wildtype mice treated orally with xyloside also showed significant enhancement in dermal wound repair. We conclude that manipulation of the levels of heparan sulfate in vivo increases the capacity for wound repair, suggesting a novel approach for enhancing the regenerative property of tissues.
Identification of Predictors Underlying the Attitude of Medical Students towards Medical Biochemistry in their Pre‐clinical Years and its Influence on Students’ Learning Outcome
Authors: Sengupta Anamika
Published: April 2020

Background

The importance of positive attitude in the learning process of students has been acknowledged in numerous educational studies (Reiss 2004; Berg 2005; Banya 2005; Trumper 2006). The frequently observed negative attitude of medical students towards Biochemistry among pre‐clinical students in US medical schools served as the motivation for this study. It aims at identifying predictors underlying the attitude of 150 participating medical students (1st, 2nd, and 3rd semesters at Ross University School of Medicine) towards medical biochemistry (a basic science subject that they are exposed to in their pre‐clinical years) and explores the relationship between the possible predictors, attitudes and learning outcomes.

Methods

A 7‐item questionnaire paper survey with questions (related to students’ demography (age, sex, marital status, first language), their familiarity with the subject matter, and a 1–7 Likert scale rating of their attitude & confidence levels towards biochemistry was distributed in class at the start of the semester. MCAT scores and GPAs were obtained from the University database. Correlation and Multiple Regression models were used for data analysis.

Results

Preliminary results indicated students’ attitude towards Biochemistry was positively correlated with their levels of confidence for the subject. Multiple Regression analysis identified Confidence and Age as predictors of attitude. However, MCAT Biology scores and familiarity with the subject matter were the best predictors of students’ confidence levels. Learning outcomes were directly influenced by gender and first language, none of which predicted their attitude towards biochemistry.

Conclusion

Although students’ prior exposure to the subject matter determines their approach to medical biochemistry in the pre‐clinical years (in addition to their MCAT scores and ages), the ultimate performance outcomes in the subject are notably higher in males with English as a first language.
Comparisons of catalytic efficiency during the reduction of lignocellulosic substrates between free enzyme versus enzyme bound to mobile enzyme sequestration platforms (mESP)
Authors: Fatema Nour , Moore Blakely , Wangnai Chinnapong , Ceballos Ruben Michael
Published: April 2020

Plants represent a vast, renewable source of biomass. Plant biomass serves as a major substrate for sustainable biofuel production. However, to overcome the food‐versus‐fuel dilemma, effective use of plant residues for cellulosic (versus corn‐ or sugarcane‐based) ethanol must be realized. Effective deconstruction of lignocellulosic matrix and efficiently accessing and reducing cellulose to fermentable sugars (i.e., glucose) would overcome current constraints that prevent cellulosic ethanol from becoming an economically viable alternative for liquid/transportation fuel. Cellulosic feedstocks consist of cellulose (20–50%), hemicellulose (15–35%), and lignin (5–30%). In previous work, we demonstrated that the use of thermotolerant and acidotolerant engineered mobile enzyme sequestration platforms (mESP) could enhance lignocellulosic deconstruction of two acid pretreated (and alkaline pretreated) feedstocks. Specifically, acid‐pretreated wheat straw and corn pericarp were subjected to a suite of cellulases, an enzyme cocktail, under two conditions: (a) pretreated substrate plus enzymes free in solution (experimental control); and, (b) pretreated substrate plus enzymes bound to a prototype mESP. The mESP‐bound enzymes showed enhanced ability to convert complex carbohydrates to glucose (i.e., sugar reduction) when compared to the free enzyme in solution control (at equimolar enzyme concentrations). Although the mESP was known to be thermotolerant and acidotolerant, enhancement of sugar reduction efficiency was also observed with alkaline pretreated substrate. Still, these preliminary experiments only demonstrated a 2‐ to 3‐fold increase in enzymatic efficiency over controls. Indeed, with some enzyme ‘cocktails’, sugar reduction efficiency actually decreased when employing mESPs. It was suggested that steric interference between enzymes due to the close proximity of enzyme attachment points on the platform and the use of enzymes with different target points (exo‐versus endo‐) on the substrate (i.e., cellulose) may limit the magnitude of enhancement that can be achieved. After re‐engineering the mESP system to separate attachment points and strategic development of free enzyme cocktails and enzyme composition on charged mESPs, this study demonstrates that next‐generation mESPs can improve catalytic efficiency more than 3‐fold. In addition, alternative substrates have been tested (e.g., rice straw) with similar results. These results and implications for mESP technology are discussed in detail.

Support or Funding Information

U.S. National Science Foundation Molecular and Cellular Biosciences (MCB) grant (Award No. 1818346; PI‐Ceballos); U.S. National Science Foundation Research Coordination Network (RCN) grant (Award No. 1624171; PI‐Ceballos)
Assessment of the Alveolar Bones’ Unique Structure and Impact on Periodontology
Authors: Shah Kushal , Lacin Nursima , Sun Lu , Naveh Gili
Published: April 2020

The composition and structure of bones is important to the function of the periodontal ligament (PDL) in particular and to tooth movement overall. One bone crucial to the arrangement of teeth is the alveolar process, which connects teeth to the jawbone through intramembranous bone formation housing the cementum. Comprising of both cortical bone and cancellous bone, the alveolar process is unique in function as it is able to endure tension during mastication by working with the PDL

Seeing that alveolar bone can endure tension and compression forces provokes question towards how the composition and structure influences this bone.

Using immunohistochemistry (IHC) staining, collagen II, IX, and XI, have all been found in the alveolar bone; these are a set of tissue specific collagens that are found at low levels in intramembranous bone. Identifying this type of collagen in the alveolar bone is unusual as it is mainly found in cartilage and the vitreous humor in the eye.

These observations will provide insights into the connection between collagen in the alveolar process and basic structure‐function relations of the tooth‐PDL‐bone system, while establishing a better understanding of tooth movement reactivity and the development of treatments to periodontal diseases.

Support or Funding Information

Harvard School of Dental Medicine AFM project
Identifying species‐specific Hfq‐RNA Interactions using a bacterial three‐hybrid assay
Authors: Tran Anh M. , Chambers Kelly M. , Berry Katherine E.
Published: April 2020

Bacteria use non‐coding small RNAs (sRNAs) to regulate expression of target mRNAs, often with the help of protein chaperones. About half of bacterial species possess a well‐characterized globally‐acting RNA‐binding protein, a hexameric protein called Hfq, which contributes to virulence in many pathogenic species. While much is known about the mechanism of RNA interactions by Hfq proteins in certain well‐studied model bacteria, there are significant gaps in our understanding of how conserved the RNA‐binding behavior of Hfq homologs from diverse bacterial organisms. Here, we use a recently developed bacterial three‐hybrid (B3H) assay to further define the interactions between Hfq and its RNA substrates. This assay connects the interaction between a DNA‐bound RNA and a RNA polymerase (RNAP)‐bound protein Hfq to the expression of a reporter gene, providing a genetic route to the analysis of RNA interactions from a heterologously expressed RNA‐binding protein. This provides a facile route to study the behavior of diverse RNA‐binding proteins in an innocuous laboratory strain of E. coli. Here we present data collected using the B3H assay to compare the RNA interactions of Hfq from several bacterial species, including Listeria monocytogenes, Caulobacter crescentus, and Escherichia coli. We have observed distinct RNA‐binding profiles between these Hfq proteins. Sequence alignments of these three proteins led to a hypothesis that charged residues along the rim surface of Hfq may underlie the differences in binding preferences for different sRNAs. Data will be presented that test this model using both chimeric and site‐directed mutants. Our long‐term goal is to gain a better understanding of what structural elements of an RNA‐binding protein such as Hfq determine its RNA binding and function in driving bacterial gene regulation.

Support or Funding Information

This work was supported by National Institutes of Health [R15GM135878], Mount Holyoke College and the Henry Luce foundation.
Astrocytic modulation of IKA, NMDA and AMPA currents in NTS neurons of rats is affected by sustained hypoxia
Authors: Machado Benedito H. , Bonagamba Leni G.H. , Accorsi-Mendonça Daniela
Published: April 2020

Short term sustained hypoxia (SH, FiO2 0.1, 24 h) produces cardiovascular and respiratory changes mainly due to peripheral chemoreflex activation. SH alters the electrophysiological properties of neurons in the NTS, where the first synapses of chemoreflex afferents are located. In the present study we evaluated alterations produced by SH on neuronal excitability and excitatory synaptic transmission in the NTS neurons integral to chemoreflex pathways. After SH protocol, brainstem slices were obtained for electrophysiological recordings using whole cell patch–clamp and fluoracetate (FAC) was used to produce metabolic inhibition of astrocytes. SH increased the action potential discharge in response to positive injected current into NTS neurons (p=0.0048). The presence of A‐type potassium current (IKA) was observed and its peak amplitude was larger in neurons from control [Vm = 10 mV, 1428 ± 600 pA (n=10)] than in SH rats [Vm = 10 mV, 334 ± 76 pA (n=10)]. Inhibition of astrocytes with FAC decreased the IKA amplitude in neurons from control rats [1227 ± 129 pA vs 605 ± 210 pA (n=7)], but not in SH rats [475 ± 110 pA vs 427 ± 114 pA (n=6)]. With respect to excitatory neurotransmission, SH increased AMPA/kainate [−183 ± 122 pA (n=10) vs −353 ± 101 pA (n=10)] and NMDA currents amplitude [61 ± 10 pA (n=7) vs 102 ± 37 pA (n=10)]. Astrocytic modulation on AMPA [aCSF: −353 ± 101 pA vs aCSF + FAC: −369 ± 76 pA, (n=10)] and NMDA currents [aCSF: 102 ± 37 pA vs aCSF + FAC: 108 ± 32 pA, (n=10)] was blunted by SH. In addition, SH increased AMPA current density [control: −6 ± 3.5 pA/pF (n=6) vs SH: −20 ± 12 pA/pF (n=7)]. The data shows that astrocyte modulation of synaptic transmission in NTS neurons integral to the chemoreflex pathways is reduced by SH, which may contribute to the observed increase in the chemoreflex responsiveness on this experimental model.

Support or Funding Information

FAPESP, CAPES and CNPq
Intermittent but not Chronic Access to Western Diet Selectively Induced Binge‐like Eating and Risk‐taking Behavior in Rats.
Authors: Shaw Cemilia , Patterson Sasha , Shah Krishna , Longley Megan , Sirohi Sunil
Published: April 2020

Binge eating disorder (BED), the most common eating disorder in the US, has a lifetime prevalence of 2.8% and has been associated with many other psychological and metabolic disorders. Several preclinical models developed to study binge‐eating disorder and related pathologies use stress, food restriction, or limited access conditions to induce binge‐like intake. Using limited access model, Corwin and colleague, demonstrated that limited‐short (2 hr Mon, Wed and Fri) access to vegetable shortening induced a binge‐compensate feeding pattern which was not present in limited‐regular (2 hr every day) access conditions. Similar findings were reported when a nutritionally complete high‐fat diet was utilized either every day (HFD‐ED) or every‐third day (HFD‐3D). Using this model, we have recently shown that both HFD‐ED and HFD‐3D groups of rats undergo similar binge‐compensate eating patterns and display compulsive feeding/risk‐taking behavior, suggesting the frequency instead of presence/absence of binge‐like feeding episodes as a major difference between HFD‐3D and HFD‐ED. It was also unclear if the intermittent availability or the HFD itself triggered compulsive feeding. Therefore, in the present study, we evaluated the impact of ad libitum chow and western diet (WD; rich in both sugar and fat) consumed in a binge manner in relation to the same diet consumed in a non‐binge manner. Male Long Evans rats received chronic (every day) or intermittent (Mon, Wed, and Fri) 24 hr access to Western Diet for six weeks. The control group received normal rodents chow. All the animals had ad libitum access to chow and water throughout the experiment. We observed that intermittent access‐group significantly overconsumed on WD‐access days and under‐consumed on chow‐access days as compared to both chow controls and chronic access group. Chronic access group, on the other hand, significantly overconsumed every day as compared to chow controls but not in a binge/compensate manner. Only intermittent access‐group showed elevated food intake than their baseline during the first two hours of WD‐access days. Finally, only the intermittent access‐group showed risk‐taking behavior when tested in the light/dark box. These data demonstrate selective induction of risk‐taking behavior following binge‐like eating but not when the same diet was consumed in a non‐binge manner.

Support or Funding Information

Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number TL4GM118968 and RL5GM118966. This publication was also made possible by funding, in part, by NIMHD‐RCMI # G12MD007595‐10, NIGMS‐BUILD # UL1GM118967 and NIGMS‐SCORE # 1SC3GM127173‐01A1 to SS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Selection of Mutations that Affect Herpesvirus Entry into Cells
Authors: Hippler Daniel P , Fan Qing , Longnecker Richard , Connolly Sarah A
Published: April 2020

During virus entry into cells, herpesviruses rely on the coordinated action of multiple viral glycoproteins. For herpes simplex virus type 1 (HSV1), four glycoproteins (gD, gH, gL, and gB) are required to fuse the viral and host membranes. gB functions as a fusion protein and is conserved in the herpesvirus family. It mediates viral entry by undergoing a conformational change that results in fusion viral and host membrane. This conformational change is triggered by both gD, the receptor‐binding protein, and gHgL, a heterodimer that modulates fusion. Capturing a physical interaction among these glycoproteins has been challenging, most likely because the interactions are low affinity and/or transient. The goal of this study is to characterize the interactions between gB and other herpesvirus proteins using natural selection. We previously generated a fusion‐deficient gB mutant. By serially passaging HSV1 carrying this gB mutant, we are selecting for mutations that restore fusion function. To shift the selective pressure away from second‐site mutations within gB, we generated a virus that is deleted for the gB gene but carries the mutant gB protein in its membrane. After six rounds of passage, the virus showed enhanced replication and the viral genome was submitted for next‐generation sequencing. The sequence results will be analyzed to determine the locations of novel mutations. The impact of these mutations on virus entry will be examined by mapping them to existing structures and analyzing mutant function in cell‐cell fusion assays.

Support or Funding Information

NIH 1R01AI148478‐01