bluuebunny/crossref_metadata_2025 huggingface dataset:
Networks of Exchanging Antibiotic Resistomes in Human and Environmental Microbiota
Authors: Dantas Gautam
Published: June 2021

While the most acute effects of increasing antibiotic resistance is observed in clinical settings, the evolution and transmission dynamics of resistance gene dissemination is an ecological problem. Indeed, steady use and abuse of antibiotics over the past century in food animals, humans, and the environment has provided substantial selective pressure for enrichment of resistance genotypes in each of their associated microbiomes. An over‐reliance on culture‐based methods, the standard in the study of clinical resistance, has vastly underestimated these reservoirs of resistance genes (or ‘resistomes’). To address this issue, we have recently developed high‐throughput metagenomic functional selections, aided by next‐generation sequencing, to characterize resistomes encoded by the microbiota of healthy human adults and children as well as diverse soils. Hundreds of resistance genes we identify from specific taxa in these different microbial communities are identical to resistance genes found in major human pathogens, indicating recent genetic exchange between these microbes. We also find thousands of functionally validated resistance genes which are genetically novel, but flanked by genes involved in horizontal gene transfer, including transposases and integrases. Together, these findings highlight the substantial antibiotic resistome encoded by microbes from diverse environments, which is available for exchange with pathogens, with the potential to severely exacerbate the problems with clinical resistance.
Coxsackieviral Infection Causes Cytoplasmic Aggregation and Cleavage of TAR DNA Binding Protein‐43
Authors: Fung Gabriel , Shi Junyan , Deng Eric , Zhang Jingchun , Hong Andy , Luo Honglin
Published: June 2021

Coxsackievirus B3 (CVB3) is a positive single‐stranded enterovirus that infects the heart. CVB3 infection can cause myocarditis and lead to dilated cardiomyopathy (DCM). In North America, DCM accounts for approximately 20% of heart failure and sudden death in children and young adults. Recent findings have shown similar molecular pathologies between cardiomyopathies and neurodegenerative diseases. In neurodegenerative diseases such as frontotemporal dementia and amyotrophic lateral sclerosis, insoluble‐cytoplasmic TAR DNA binding protein‐43 (TDP‐43)‐aggregates are a common biomarker contributing directly to pathological disease progression. The importance of insoluble protein aggregates in viral pathogenesis has also been recognized, resembling that of neurodegenerative diseases. However, the significance of TDP‐43 in CVB3‐induced cardiomyopathy remains unstudied.

In this study, we investigated the interplay between CVB3 and TDP‐43 using A/J mice, a strain susceptible to virus‐induced cardiomyopathy, and HeLa cell culture models. Our studies demonstrated that CVB3 infection leads to redistribution of nuclear TDP‐43 into cytoplasmic aggregates in a viral protease 2A dependent manner. We also found that viral protease 3C, actively cleaves TDP‐43 at amino acid 327 into two respective cleavage fragments. The respective N‐terminal cleavage fragment inhibited native TDP43 activity in the nucleus as a transcriptional regulator. We conclude that TDP‐43 pathologies in CVB3‐induced cardiomyopathy may be similar to those previously found in neurodegenerative diseases.

This work was supported by the Canadian Institutes of Health Research (HL)
Gene 33 mediates Cr(VI)‐induced DNA damage response
Authors: Park Soyoung , Li Cen , Zhao Hong , Darzynkiewicz Zbigniew , Xu Dazhong
Published: June 2021

Hexavalent chromium [Cr(VI)] is a known human lung carcinogen. But, the molecular players that mediate its carcinogenesis are poorly defined. Gene 33 (Mig‐6, ERRFI1) is an adaptor protein implicated in lung cancer. It regulates multiple signaling pathways mediating cell proliferation, migration, apoptosis, and senescence. In this study, we found that Cr(VI) triggers a typical DNA damage response in BEAS‐2B lung epithelial cells, manifested as elevation of γH2AX and P‐p53(S15) levels, in a dose‐dependent fashion. The Cr(VI)‐induced DNA damage response appeared mainly in the S and G2/M phases of the cell cycle. However, the dose‐dependent increase in the γH2AX level was more pronounced in G1 and S phases than in the G2/M phase. In contrast, P‐p53(S15) signals changed more uniformly in all phases of the cell cycle. Cr(VI) also induced the DNA damage response in A549 lung carcinoma cells. Gene 33 knockdown with siRNA significantly inhibited Cr(VI)‐induced γH2AX signals in A549. More interestingly, inhibition of the γH2AX signal by Gene 33 knockdown mainly occurred in G1 and G2/M phases of the cell cycle. In contrast, P‐p53(S15) signals were dramatically elevated by Gene 33 knockdown. In addition, the level of Gene 33 protein was suppressed by Cr(VI) treatments. Moreover, ectopic overexpression of Gene 33 elevates the basal γH2AX level while having limited effect on the P‐p53(S15) signal in A549 cells. Surprisingly, Gene 33 containing a point mutation disrupting its interaction with EGFR elicited much stronger increases in basal γH2AX and P‐p53(S15) levels. Our results revealed a novel and apparently complex function of Gene 33 in mediating the DNA damage response and a potential involvement of this protein in Cr(VI)‐mediated carcinogenesis.
Measuring Brain Activity in Fourth Grade Students Through Biopac Science Lab
Authors: Hamilton Shannon , Brown Carson , Evans LeVisa , Majors Katharyn , Thomas Destini , Gurovich Alvaro
Published: June 2021

Recently the American Physiology Society (APS) has been working to increase learning and making science fun for elementary school students. Physiology Understanding Week (PhUn) is the APS initiative to use interactive experiments to engage school‐age children with science and physiology. In this project we used a Biopac MP40 system to measure brain activity of fourth grade students at Dixie Bee Elementary School. The activity was divided in 5 different sessions, one per room, in two different days, reaching 116 elementary school students. Each room was divided in small 4‐student groups with the following general activity set‐up: 1) brief presentation about brain physiology using a brain model, 2) measuring brain activity during 3 different conditions, 3) analyzing collected data, and 4) discussing obtained results. Three electrodes were placed behind the student's ear, 2 students per group. Brain activity was collected during 4 different activities: 1) resting with eyes closed, 2) doing a math problem, 3) writing a sentence in cursive, and 4) playing catch with tennis ball. Our goal was to illustrate how brain activity changes while performing different tasks. The other 2 students controlled the computer and observe the changes in brain activity while they recorded the results. The students completed charts with the values of alpha, beta, and delta waves for each of the activities performed. This activity allowed the students to use applied technology while learning about the functions of the brain. This interactive method enables students to be more involved and interested in science; it also captures their attention and makes learning ‘PhUn’.

Supported by IU AHEC 14/15 #PIU081 and APS PhUn week program
Core Competencies in Animal Physiology
Authors: Cliff William , Hull Kerry , Blatch Sydella , Halpin Patricia , Beason‐Abmayr Beth
Published: June 2021

The past decade has seen greater emphasis placed on competency‐based education in the biomedical sciences, as evidenced in the calls to reform by AAAS (Vision and Change) and AAMC/HHMI (Scientific Foundations for Future Physicians). A working group of animal physiology instructors, established at the 2014 APS Institute of Teaching and Learning (ITL), formulated a list of ten core competencies required of students to fully master animal physiology. These competencies address core concepts such as body size, life history, phenotypic plasticity, trade offs, adaptations to common environmental challenges, adaptations to extreme environments, and evolutionary constraints. While relevant to human physiology, these concepts are most clearly understood from the perspective of animal physiology. The core concepts were unpacked into constituent ideas and learning objectives specific to each core competency were developed. Comparison to the foundational chapters of several widely‐used textbooks in animal physiology showed substantial consonance but also revealed that coverage of some concepts was scant or lacking. Further refinement of the competencies will continue through consultation with a wider group of instructors in animal physiology. Formulation of the core competencies in animal physiology provides faculty with demonstrable and measurable outcomes for student learning and sets the stage for developing assessment tools designed to evaluate student understanding of the core concepts in animal physiology.
Assessing Organizational Changes to a First Year Medical Gross Anatomy Dissection Course
Authors: Darling Ryan , Notebaert Andrew , Lu Yuefeng , Conway Marianne , Maklad Adel , Sinning Allan
Published: June 2021

With expanding undergraduate medical student class sizes, it is becoming increasingly important to develop and assess efficient policies and procedures to maintain an effective learning environment. We divide our students into two laboratory groups that rotate weekly, resulting in each student only participating in half of the complete cadaveric dissection. This year, our faculty instituted a new laboratory design with the goal of increasing accountability, professionalism, and performance on assessments by designating a rotating team leader (TL) within each dissecting group (DG). The TL was responsible for 1) creating and implementing a plan to carry out the weekly goals of the dissector, 2) organizing peer‐teaching for the three students in the non‐dissecting group (NDG), and 3) keeping a “patient chart” to aid in the weekly handoff between groups. Each week, the students were quizzed orally and graded on their dissection and patient chart. These grades were shared across all six members of the table, even for the NDG. Preliminary analysis of individual quiz grades during the first half of the course revealed a significant one‐way ANOVA, F(2, 444) = 4.82, p = 0.01. The average TL grade (out of four) was 3.69 ± 0.41, while the DG average was 3.64 ± 0.31, and the NDG average was 3.57 ± 0.29. This suggests that being a TL aided in learning the material, while not being part of the dissection potentially hindered learning. Data will be updated and analyzed in more detail upon completion of the course, including an analysis of peer assessments.
Continued Development of the Role of Teaching Assistants in Mount Sinai's “Structures” Course
Authors: Maffucci Patrick , Laitman Benjamin , Laitman Jeffrey
Published: June 2021

The Structures course at Mount Sinai is a combination of three courses taken by first year medical students: Anatomy, Histology, and Embryology. Functioning as a single unit, this course allows integration of these areas to enhance student learning and offer a unique perspective for medical students. Last year, we reported our efforts to reshape how Teaching Assistants (TAs) function within our course. This year, with the second iteration of Structures and as part of a continued effort to increase the role of student TAs, TAs became responsible for a broader range of activities. Notably, table conferences, a substitute for practical exams in Anatomy normally conducted by faculty members, were transitioned entirely to Senior TAs (4th year or scholarly year medical students, or PhD phase MD/PhD students). Second year medical student TAs became responsible for organizing and teaching a number of review sessions, including weekly review sessions, midterm/final examination reviews, and in‐lab review sessions before each table conference. Course evaluations have been completed and are being analyzed. Feedback on TAs and their role in the course was overwhelmingly positive. In addition, focus groups consisting of first year students are being organized to solicit feedback and suggestions. Of equal importance, TAs have responded favorably to increased involvement in the course and have cited the value of increased opportunities to teach and of strengthening their understanding of topics taught. Overall, we have found that an increase in the role and responsibilities of TAs in medical education have been extremely positive for both students and TAs alike.
Developing Lifelong Learning Skills through Self‐Directed Learning in the Gross Anatomy Laboratory using Narrated Digital Slideshows
Authors: Dunham Stacey
Published: June 2021

The Liaison Committee on Medical Education has identified self‐directed learning as a critical process in developing lifelong learning skills. Gross anatomy laboratories present an opportunity to incorporate self‐directed learning. The goal of this study was to develop a technology based, self‐directed learning opportunity that could be assessed by instructors. It was conducted in a medical gross anatomy course of about 40 students divided into nine groups, each working with an individual donor. Alternating dissections were used and only half of each group was present for each dissection. At the dissection students were tasked with creating a checklist of structures and required to take digital photographs on an iPad. Outside class, checklists and images were uploaded to a digital application, VoiceThread. VoiceThread allowed students to create narrated slideshows of the dissection, highlighting structures or issues of significance. All students had access to all slideshows through the online learning management system. Groups were graded both for completion of their narrated slideshows, and for their comments on other students' slideshows. VoiceThread slideshows were viewed by the researcher and scored using a rubric to analyze their content, accuracy, and presentation style. Students were also surveyed about their use of VoiceThread and its impact in the gross anatomy laboratory. Early results show that students were able to use VoiceThread in a self‐directed capacity. Students also indicated narrated videos and checklists provided an incentive for lab preparation and were an additional learning tool. This study provides an example of how instructors can incorporate and document self‐directed learning opportunities in the gross anatomy laboratory.
Variations in Branching of the Profunda Femoris Artery
Authors: Hurley Mary , Stevens Todd , Estaris Sharine , Duen Kelvin , Olivieri Marion
Published: June 2021

The profunda femoris artery is a major branch of the femoral artery in the femoral triangle. The medial and lateral femoral circumflex arteries are typically branches of the profunda femoris artery within a few centimeters of its branching from the femoral. Netter's atlas shows the profunda femoris branching off the femoral artery closer to the inguinal ligament than is shown by Grant's atlas

In addition, Netter shows the medial and lateral circumflex femoral arteries branching opposite of each other close to the beginning of the profunda femoris, while Grant shows the lateral circumflex artery branching from the profundus femoris much more distal to the branching of the medial circumflex. After approval from D'Youville College IRB, thirteen cadavers were examined for variations in the branching of these arteries. Besides typical branching, in 6 of the cadavers the medial circumflex branched off the femoral artery, and in one, both medial and lateral circumflex were branches of the femoral artery.
Examining Coronary Arterial Variations in an Urban Sample: Implications to Cardiovascular Disease
Authors: Zhang Zhihang , Mastriano Daniel , Tan Jun Yuan , Hasan Mohammed , Lazar Jason , Pagano Anthony , Marquez Samuel
Published: June 2021

Cardiovascular disease causes more deaths worldwide than any other medical affliction usually targeting older adults for which one of the antecedents of the disease – atherosclerosis, begins early in life thus making primary prevention efforts necessary from childhood. This study examined angiograms from a cross‐sectional random sample of patients (n=25) in order to document the cardiac vascular pattern, grading the size and dominance of the coronary arterial branching pattern to determine if one can predict which coronary artery branch may be more predisposed to disease processes. An additional mixed‐sex urban population sample from Downstate anatomy laboratory (n=61) was included to compare and contrast the angiogram data. The four branches of the coronary arteries (sinoatrial nodal; posterior interventricular [PIV]; anterior interventricular [AIV] and left circumflex) were examined and similarly graded. Results showed 79% of the cadaveric hearts to be right‐dominant, 15% co‐dominant with dual origin of the PIV artery, and 7% being left dominant. Angiogram data revealed similar results with 80% showing right dominance. While the sinoatrial nodal artery was graded normal 80% of the time, the PIV and AIV arteries were frequently identified as hypertrophied in both sample sets suggesting that these vessels are the most common sites for blockage.
Establishing an Anatomical Proximity Between the Coronary Circumflex Artery and the Mitral Valve Annulus: Implications for Mitral Valve Surgery
Authors: Winkler Eliot , Malakouti‐Nejad Bayan , Johnson Marjorie , Catrip Jorge , Nagendran Jeevan , Chu Michael
Published: June 2021

The coronary circumflex artery (CX) lies in the left atrioventricular groove, running intimately parallel to the mitral valve annulus. Thus, unintentional damage to the CX can occur via a fixation suture during mitral valve surgery, typically reported in hearts with a left dominant or co‐dominant circulation. This study aims to elucidate the anatomical relationship between the CX and annulus, and evaluate damage to the CX following three different mitral valve surgical repairs. It is predicted that left dominant or co‐dominant hearts should exhibit the closest anatomical relationship between the CX and annulus and experience a greater incidence of damage despite the chosen mitral valve surgery. Using fixed (n=18) and fresh‐frozen (n=9) cadaveric hearts, coronary circulation was dissected and dominance determined. After opening the left atrial wall, a clock face was overlaid on the mitral valve (12:00 position at the A2 leaflet midline) and the distance between the annulus and the CX was measured at each hour. The CX was closest to the annulus in fresh‐frozen, left dominant hearts. All left dominant and co‐dominant hearts had CXs that hugged the posterior length of the annulus, diving deep at 3:00 (before the posterior commissure), a finding previously unreported. Preliminary results from the ongoing surgical repairs advocate for cautionary suture placement in areas formerly considered to be at low risk for damage.
The cardiac potassium channel HERG is blocked by calcium at a site near the outer mouth of the channel
Authors: Nguyen Andrew , Wong Alice , Oberoi Angad , Le Thuyvy , Miller Alan
Published: June 2021

A reduction in the number of HERG channels has been implicated in Long QT Syndrome, which can degenerate into the lethal arrhythmia Torsades de Pointes. Patients can present with abnormal serum electrolyte levels due to a variety of conditions including gastrointestinal dysfunction, renal and endocrine disorders, diuretic use, alcoholism, and aging. The aim of this study is to determine if extracellular calcium blocks the pore of the HERG channel by acting at a site in the outer vestibule of the channel.

Divalent block was measured using two‐electrode voltage clamping of Xenopus oocytes expressing either wild‐type HERG or the HERG mutants S631A, and G628CS631C. S631 is thought to be located in the outer pore of the HERG channel.

Changing extracellular potassium from 0 mM to 20 mM resulted in a greater decrease in WT HERG current due to an increase in extracellular calcium, as well as a number of other divalent ions (Mg2+, Co2+ and Mn2+). In addition, block of WT HERG by calcium was voltage dependent, with increased block at negative voltages. Finally, there was little or no block of the HERG double mutant G628CS631C by calcium at all voltages tested, whereas there was increased block of the HERG mutant S631A by calcium at negative voltages.

3 results suggest that calcium blocks HERG by physically occluding the pore of the channel: 1) the dependence of block on extracellular potassium. 2) the lack of block of G628CS631C 3) the altered voltage dependence of block of S631A. This study has implications for an increased risk of cardiac arrhythmias with hypokalemia as well as for patients with acute promyelocitic leukemia treated with arsenic, a trivalent ion that might block HERG at the same site as calcium.

Funded by Touro University and NIHR 15‐1R15HL079971
PrdxI Encode Peroxiredoxin is Required for Vascular Development in Zebrafish
Authors: Wu Changyi , Syue Hai‐hong
Published: June 2021

Genetic signaling and redox homeostasis are required for proper growth of blood vessels. Using zebrafish as a model organism have been intensively identified many molecules that control vasculature. Some antioxidant genes have been shown important for vascular growth. However, there is still limited information about oxidative stress and vascular development during embryogenesis. Here, we report a novel function of prdx1 that play critical roles in vascular growth during zebrafish development

Prdx1 encodes a cytosolic peroxidase responsible for the remove of hydrogen peroxide and is highly conserved from yeast to mammal. Our in situ hybridization data showed the expression pattern of prdx1 is in developing vessels. Knockdown of prdx1 by morpholino injection impairs the vascular growth of ISV (intersegmental vessel) and CVP (cardinal vein plexus). We further showed the reduction of ISV is due to a decrease of cell proliferation and migration, but not cell death. Consistent with the vascular defects in prdx1MO, we found a decreased expression of vascular markers, flt4,mrc1, stabilin and ephrinb2. Loss of prdx1 results in vascular defects suggests the antioxidant function is important. Thus, we test if oxidative stress could cause vascular defects. Our data showed H2O2 treatment impaired CVP formation and synergetic effects are showed while H2O2‐treated in prdx1MO. We also found the expression of main antioxidant genes (SOD1, SOD2, catalase and prdx2) are decreased while loss of prdx1. Finally, prdx1 likely regulates hemangioblast fate decision because the increased expression of blood marker gata1 concurrent with the decreased expression of endothelial markers in prdx1 MO. Together, we showed a novel function of prdx1 that play critical roles in vascular growth during zebrafish development.
Sex‐Specific Calcium Handling Underlying Sex‐Specific Cardiomyocyte Death in Heart Failure
Authors: Kararigas Georgios , Molina Cristina , Summer Holger , Baczko Istvan , Golz Stefan , Regitz‐Zagrosek Vera
Published: June 2021

Heart failure manifests differently between men and women and there are major sex differences in cardiomyocyte death. We aimed at studying the impact of sex on global gene expression in left ventricles of male (n = 5) and female (n = 5) patients with end‐stage dilated cardiomyopathy (DCM) compared with samples of men (n = 10) and women (n = 8) with no cardiovascular disorder. Pathway analysis (adjusted P < 0.05) revealed induction of ECM‐receptor interaction and inflammatory pathways in male hearts, while the oxidative phosphorylation and proteasome pathways were repressed. In contrast, the Wnt and Hedgehog signaling pathways were induced in female hearts, while the mTOR signaling pathway was repressed. The Ca2+ pathway was induced in both male and female hearts, but male hearts had a significantly higher induction. In isolated human ventricular myocytes (HVMs) from DCM patients, we found by patch‐clamp a 2‐fold increase in L‐type Ca2+ current density in male vs. female HVMs (n = 6‐8 cells/group), while there were no sex differences in sarcoplasmic reticulum Ca2+ load. Cytochrome c cytosolic release was significantly higher in male than female hearts (P < 0.05), while female hearts had significantly higher BCL2 protein levels than male hearts (P < 0.05). In conclusion, there are major sex differences in Ca2+ handling in the failing heart, which may be part of the molecular factors underlying sex differences in heart failure.
Developmental Regulation of Epicardial Cell Adhesion and Coronary Vasculogenesis
Authors: Hatcher Cathy , Tanner Brendan , Abdurahman Dina , Nelson Kolby , Laidlaw William , Gorski Grzegorz
Published: June 2021

Mutations in the gene encoding the TBX5 transcription factor cause Holt‐Oram syndrome (HOS). Individuals with HOS exhibit structural congenital heart defects that affect formation of the pericardium and coronary vessels. These structures descend from cells that migrate out of the transitory proepicardium during early cardiogenesis. Our previous analyses revealed that Tbx5 is expressed in the proepicardium, visceral pericardium/epicardium and coronary vessels. Analyses of mice with proepicardial‐specific deletion of Tbx5 (Tbx5epi‐/‐) revealed that Tbx5 contributes to epicardial cell adhesion to the myocardium and epicardial‐derived cell migration into the myocardium to form coronary vessels. Genes encoding cell adhesion and extracellular matrix (ECM) proteins were decreased in Tbx5epi‐/‐mouse hearts. These cell adhesion and ECM proteins have established roles in extracardiac tissues, but their functional roles in cardiovascular development remain unknown. Our current study aims to identify cardiovascular developmental regulation of these genes. We demonstrate that Tbx5 regulates transcription of genes encoding these cell adhesion and ECM proteins. Our analyses also reveal that proteins encoded by these Tbx5‐dependent genes are detected as early as embryonic day 11.5 in the developing heart. Cardiac expression of these proteins correlates with formation of the epicardium and coronary vessels. Further studies will determine the requisite functional contributions of the cell adhesion and ECM proteins to establishment and maintenance of the epicardium and underlying coronary vessels. (Support: PCOM Center for Chronic Disorders of Aging)
Selective Inhibition of Osteosarcoma Cell Functions Induced By Curcumin‐Loaded Self‐assembled Arginine‐Rich‐RGD Nanospheres
Authors: Chang Run , Sun Linlin , Webster Thomas
Published: June 2021

Objectives

To develop a treatment against osteosarcoma with higher selectivity towards osteosarcoma cells and lower cytotoxicity towards normal osteoblast cells.

Statement of methods

Curcumin was found to have significant selectivity against osteosarcoma cells in vitro, and the ampiphilic peptide C18GR7RGDS was used as a carrier in aqueous solution. This peptide contains an arginine‐rich and an arginine‐glycine‐aspartic acid (RGD) structure, which may lead to efficient cell penetration and targeting for cancerous cells since RGD sequence can target to the overexpressed ανβ3 integrin. Using a method of co‐dissolution with acetic acid and dialysis tubing, the solubility of curcumin was enhanced and formed a homogeneous solution with the help of amphiphilic nanoparticles (APNPs). The cytotoxicity and cellular uptake of these materials on both osteosarcoma and normal healthy osteoblast cell lines were evaluated by MTT assays and confocal fluorescent microscopy

Results

It was demonstrated for the first time that APNPs formed nanospheres with diameters of 10‐20 nm in water and phosphate buffer saline (PBS), and opened up when the pH value was reduced to 4. The successful encapsulation of curcumin in APNPs was confirmed by Fourier transform infrared (FT‐IR) and X‐ray diffraction (XRD) analysis. The curcumin‐loaded APNPs exhibited a significant selective internalization against the MG‐63 osteosarcoma cell line

image

Conclusions

APNPs can encapsulate hydrophobic curcumin in their hydrophobic cores, and the curcumin‐loaded APNPs could be an innovative candidate for the selective inhibition against osteosarcoma.
MEASUREMENT OF LEVEL OF BRANCHED CHAIN AMINO ACIDS IN NUTRITIONAL SUPPLEMENTS
Authors: Lamela Daniel , Sachandani Kunal , Hubble Kyle , Keenan Kelly
Published: June 2021

The goal of this project was to develop a laboratory experience suitable for college students using nutritional supplements. An active ingredient in protein based nutritional supplements are the branched chain amino acids (BCAA) which have been linked to a quicker recovery for muscle. BCAA consist of three amino acids: leucine, isoleucine and dehydrogenase. These supplements are usually made from whey protein in milk. The objective in this study was to measure the level of BCAAs in a variety of supplements and milks using the leucine dehydrogenase enzyme assay. Supplements were prepared and were first hydrolyzed to release the amino acids. BCAAs were measured using the leucine dehydrogenase enzyme assay and arginine was measured with a colorimetric test. In order to validate the method, the complete hydrolysis of protein was demonstrated using SDS‐PAGE as well as the Lowry assay. Proteins of known amino acid composition were hydrolyzed and the levels of BCAA and arginine were measured and compared to the predicted values based on the sequence of the protein.
YZGD, a Two‐Domain Enzyme, as a Teaching Tool in a Biochemistry Laboratory
Authors: O'Handley Suzanne
Published: June 2021

YZGD is a two domain enzyme that is both a member of the HAD superfamily with pyridoxal phosphatase activity and a member of the Nudix Hydrolase superfamily with comparable activity on CDP alcohols, ADP sugars and ADP coenzymes, TDP sugars, and some activity on UDP and GDP sugars. Due to its two‐domain nature, I have found it to be a useful enzyme to use in the teaching laboratory. We have created a mutant (YZGDD12N) that has no pyridoxal phosphatase activity but full Nudix activity and a mutant (YZGDE324Q) that has no Nudix activity but full pyridoxal phosphatase activity, demonstrating the independent nature of enzyme domains. We also have cloned both a his‐tagged YZGD and a non‐his‐tagged YZGD, thus demonstrating the power of affinity chromatography in enzyme purification. The students express the various YZGDs (mutants, wildtype, and his‐tagged), purify these enzymes, and characterize them (Bradford assays, gel electrophoresis, and enzyme assays). We then work as a group to compare and contrast the results that each student gets; it is this comparison that especially shows the students the power of site‐directed mutagenesis and affinity chromatography purification. In addition, the students do a protein structure ‐ function project where they use the structural database SCOP, the visualization program CN3D, BLAST searches and CLUSTAL alignments, and the scientific literature on any HAD or Nudix superfamily member that has been structurally determined.
Utilizing Ligation Independent Cloning in an Undergraduate Biochemistry Laboratory Course
Authors: Hazzard James , Forte Brittany , Nelp Micah , Young Anthony , Hausrath Andrew
Published: June 2021

As a continuation of the transformation of our laboratory course to a more research focused experience we tested whether the Ligation Independent Cloning (LIC) technique would be suitable for students with little to no prior experience in molecular biology methodologies. To produce an expression system for for E. coli alkaline phosphatase (PhoA) we cloned its gene into the pETHSUL vector kindly donated by Dr. Patrick Loll. The construct expresses a fusion protein where a His‐tagged SUMO protein is fused to PhoA. The sumo moiety can be proteolytically released from PhoA following affinity column purification. We compare the ease of building the pETHSUL‐PhoA construct and enzyme activity data for the purified enzyme with an expression system developed previously in our class using a classical double restriction enzyme sub‐cloning into a pET29a vector that incorporates a His‐tag at the C‐terminus of PhoA.
A Theoretical Study of the Factors that Contribute to the Conformational Energy of Six‐Membered Rings
Authors: Abdulsalam Maryam , Balilli Rabih , Baello Rofe , Booth Robert , Suresh Sandeep , Kasner Marc
Published: June 2021

Various studies have shown that 2‐hydroxyltetrahydro‐2H‐pyran has a negative conformational energy and is said to manifest an anomeric effect. The cause of the anomeric effect is not well understood and by computationally studying the energies and geometries of a series of compounds in a series of solvent systems, the contributions of steric factors, of hyperconjugation effects, of stereoelectronic interactions and solvent interactions to the relative stability of axial and equatorial conformers were investigated. A series of monosubstituted cyclohexanes with minimal electronic interactions were studied followed by a series of monosubstituted tetrahydro‐2H‐pyrans. The relaxed scans of energy as a function of the orientation of the ring substituent of the tetrahydro‐2H‐pyran series show the combination of the electronic effects and the steric effects and comparison to the scans of the cyclohexane series to the allowed separation of the electronic effects from the steric effects. Natural Bond Orbital analysis of the full sequence of both sets of compounds is used to establish the extent of any hyperconjugative effects. To study the solute‐solvent interactions, the energy and geometry studies were repeated using a continuum solvent model with water (ε = ~80), acetone (ε = ~20) and cyclohexane (ε = ~2) selected as the solvent.
Evidence‐Based Design of a Fragile Site in Budding Yeast
Authors: Fernando Shanika , Arlt Martin , Glover Thomas , Wilson Thomas
Published: June 2021

Common fragile sites (CFSs) are seen as breaks on metaphase chromosomes when cells are grown under replication stress. Our mammalian cell study demonstrates that CFSs overlap copy number variant (CNV) hotspots characteristic of human diseases including autism, schizophrenia, and cancer. These at‐risk regions are characterized by late replication and large transcription units, supporting hypotheses that CFSs result from a replication‐transcription conflict. However, mammalian systems fall short of explaining what the repair mechanisms and intermediates are at these sites and whether transcription unit length is correlative or causative of prolonged replication. We have used budding yeast in an attempt to prospectively design a fragile site/CNV hotspot to elucidate the roles of transcription and repair in the formation and resolution of chromosome breaks. We examined genome‐wide replication data for expansive replicons that are free of Ty elements, repetitive sequences, and essential genes. We selected loci on chromosomes XII and XV as candidates for higher genomic instability under replication stress, especially when replication and transcription are manipulated. A URA3‐CAN1 reporter was used to ablate a late‐firing origin and detect locus‐specific deletion rates using fluctuation analysis. We are delineating the effects of these large replicons lacking late‐firing origins and hydroxyurea, a replication inhibitor and inducer of CNVs in mammalian cells, on CNV formation in yeast. This yeast model will provide insight into the mechanisms that create CNV breakpoint junctions with a goal of establishing a high‐throughput assay for agents that induce CNV formation. Source of Funds: R25 GM086262, R21 ES022311.
Saccharomyces cerevisiae as a Tool to Examine Rpa2 N‐Terminal Phosphorylation Across Common Eukarya
Authors: Wilson Timothy , Ghospurkar Padmaja , Senger Barbara , Haring Stuart
Published: June 2021

Background

Replication Protein A (RPA) is a heterotrimeric complex that serves critical roles in eukaryotic genome duplication and maintenance. In human cells, the N‐terminus of the 32‐kDa subunit of RPA (Rpa2) is hyper‐phosphorylated by multiple kinases in response to DNA damage. We demonstrated previously in the budding yeast Saccharomyces cerevisiae that Rpa2 N‐terminal phosphorylation does not occur under genotoxic conditions, yet this domain is vital for the damage response.

Study Objective: Determine why there is a difference between yeast and human Rpa2 phosphorylation in response to genomic insult and its importance for the DNA damage response.

Methods: We utilized chimeric Rpa2 proteins, yeast Rpa2 antibodies, and human Rpa2 phospho‐specific antibodies to examine the potential for human and other eukaryotic Rpa2 N‐termini to be phosphorylated and their contribution to the DNA damage response in yeast.

Results and

Conclusions

We determined that the human Rpa2 N‐terminal domain is phosphorylated in yeast. Furthermore, hyper‐phosphorylation of chimeric Rpa2 in yeast occurs in a manner analogous to that of Rpa2 in human cells. We also determined the yeast kinases responsible for phosphorylating specific residues within the human N‐terminus of chimeric Rpa2. Finally, we identified other eukaryotic Rpa2 N‐termini that have the potential to be phosphorylated. Identification and characterization of phosphorylation of eukaryotic Rpa2 N‐termini in yeast allow for an understanding of why yeast Rpa2 phosphorylation does not occur as well as insight into the evolutionary significance of higher eukaryotic Rpa2 phosphorylation.

Funding: This research was supported by NSF‐CAREER 1253723 to SJH.
Re‐replication Origins in Sciara DNA Puffs Revealed by New and Old Genomic Technologies including Nanopore Sequencing
Authors: Urban John , Bashir Ali , Sebra Robert , Foulk Michael , Howison Mark , Gerbi Susan
Published: June 2021

Each one of the numerous origins of DNA replication (ORIs) in metazoan genomes is typically regulated to fire no more than once in any given S‐phase. “Intrachromosomal DNA amplification” occurs when an ORI re‐fires leading to extra copies of that locus. This event is rare, aberrant, and deadly in most metazoans, which makes it difficult to study. However, the fungus fly Sciara coprophila has several “DNA puffs” in the salivary gland genome where multiple rounds of DNA re‐replication occur during normal larval development, offering a unique opportunity to investigate how particular ORIs can re‐fire while the other ORIs in the genome do not. Previously, we studied the amplification ORI at locus II/9A and showed that a steroid hormone, ecdysone, plays a role in its repeated firing. Expanding our studies to the other DNA puffs is a first step in further understanding the mechanism of DNA re‐replication in Sciara. However, the other DNA puff sequences were unknown as was the Sciara genome sequence. To obtain new DNA puff sequences we first assembled the genome using many genomics technologies, including Illumina, PacBio, BioNano Genomics Irys, and ultra‐long reads from Oxford Nanopore minION. With the assembled genome we used Illumina sequencing of salivary gland genomes before, during and after amplification followed by copy number analysis to identify new DNA puffs. In addition, we have sequenced the salivary transcriptomes before, during, and after amplification to search for candidate genes that are involved in regulating DNA re‐replication in Sciara. [JMU supported by NSF GRFP (DGE‐1058262) NSF EPSCoR (grant# 1004057) fellowships]
Interactome Dynamics of the Transcription Elongation Complex Revealed by Quantitative Proteomics
Authors: Harlen Kevin , Churchman Stirling
Published: June 2021

At each stage of transcription, RNA polymerase is regulated by a host of dynamically recruited factors. Many stages are associated with the phosphorylation state of the C‐terminal domain of the largest Pol II subunit, which consists of the peptide repeat Y1S2P3T4S5P6S7. Factors that co‐purify with a particular phosphoisoform of the CTD likely act at the associated stage of transcription and are recruited either through direct phospho‐CTD binding, other Pol II subunits and regulatory factors, the nascent RNA or the DNA template. To probe the dynamic composition of the transcription elongation complex, the interactome of each CTD phosphoisoform in S. cerevisiae was determined using a sequential immunoprecipitaiton strategy followed by label‐free mass spectrometry. Our data identify an average of 80 statistically significant Pol II interactors per phoshoisoform, many of which display a preference for purifying with specific isoforms. For example, Spt4, a transcription elongation factor, is highly enriched in the Ser5P dataset, suggesting that it acts in the 5′ region of genes, where Ser5 phosphorylation is most abundant. Indeed, our analysis of global Pol II density in spt4 deletion mutants by native elongating transcript sequencing (NET‐seq) revealed strong transcriptional defects in the 5′ region. Other factors, such as the termination factor Rai1, interacted with all isoforms. NET‐seq analysis of a yeast strain lacking rai1 revealed major transcriptional defects across gene bodies and in termination. This defect was seen in both sense and anti‐sense transcripts, indicating that Rai1 has a previously unappreciated general role in regulating transcription. Taken together these data provide a network of Pol II interacting factors that function to regulate transcription.
Polyisoprenylated Cysteinyl Amides Induce Apoptosis and Inhibit Proliferation and Migration of Human K‐Ras Mutant Cancer cells
Authors: Nkembo Augustine , Salako Olufisayo , Poku Rosemary , Aguilar Byron , Flores‐rozas Hernan , Lamango Nazarius
Published: June 2021

Activating K‐Ras is present in 90, 50 and 25% of pancreatic, colorectal and lung cancers, respectively, making them refractive to chemotherapies. Tumors with Ras mutations are challenging to drug, leading to poor prognoses. We hypothesize that polyisoprenylated cysteinyl amides inhibitors (PCAIs) can be effectively used to target and disrupt Ras signaling resulting in potentially very effective new therapies. To test this, MIAPaCa‐2, Panc10‐05, Caco‐2 and A549 human cancer cells were treated with PCAIs for their effects on cell viability, proliferation, apoptosis and migration. The PCAIs inhibited MIAPaCa‐2, Panc10.05, Caco‐2 and A549 cell viability and proliferation with EC50 values ranging from 2.0 to 6.0, 1.8 to 3.3, 0.2 to 4.0 and 0.4 to 10 µM, respectively. Ethidium bromide/acridine orange analysis suggests an apoptotic cell death mechanism while annexin v/propidium iodide flow cytometry analysis revealed cell arrest at the G0/G1 phase. Submicromolar concentrations of PCAIs significantly inhibited MIAPaCa‐2 cell migration in the wound‐healing assay. The effect on migration appeared to be linked to the disruption of filamentous actin organization as observed with phalloidin staining and transfections with a plasmid expressing an RFP‐tagged F‐actin marker protein. The potency of PCAIs in K‐Ras mutant cells is likely due to the disruption of signaling by Ras and related monomeric G‐proteins such as RhoA/B. PCAIs might therefore represent attractive therapeutic molecules for cancers with activating Ras mutations and hyperactive G‐proteins.

Supported by the NIMHD of the NIH under Award Number G12 MD007582
Regulation of Ubiquitin Independent Proteasomal Degradation of Intrinsically Disordered Proteins
Authors: Shaul Yosef
Published: June 2021

Intrinsically disordered proteins (IDPs) are labile proteins and undergo degradation by ubiquitin dependent and independent mechanisms. Previously we reported on the 20S proteasomal degradation pathway of the IDPs that is regulated by NQO1, a 20S associated protein, and by Nannies. Nannies are IDP interacting proteins functioning to inhibit IDPs proteasomal degradation. However the question of whether and how the IDPs are targeted to the 20S proteasome remained open. To address this question, we adopted the BiFC cell based technique for visualizing protein interactions with 20S proteasome. We found that IDPs interact with a subset of 20S proteasome alpha subunits. The results suggest that the 20S proteasome has two hub sites for interaction with IDPs in the alpha ring. We focused on the subunit PSMA3 and showed that its C terminus interacts with multiple IDPs. Thus, it implies that IDPs are targeted to the 20S proteasome prior to their degradation. In addition, lately we have identified a new type of 26S proteasomes whereby no ATP but NADH regulates its assembly. We have evidence for this new type of NADH‐26S proteasomes preferentially involved in IDPs degradation. Finally we will report on a new proteomic approach in identifying the selective 20S proteasomes substrates.

Asher, G., Reuven, N. and Shaul, Y. (2006). 20S proteasomes and protein degradation by default. BioEssay. 28(8):844‐849.

Tsvetkov, P., Reuven, N. and Shaul Y. (2009). The nanny model for IDPs. Nat Chem Biol. 5(11):778‐81

Tsvetkov P, Myers N, Eliav R, Adamovich Y, Hagai T, Adler J, Navon A, and Shaul Y. (2014). NADH Binds and Stabilizes the 26S Proteasomes Independent of ATP. J Biol Chem. Apr 18;289(16):11272‐81.
Selective RNA Sequestration Mediated by a Heat‐Sensing Disordered Protein Region
Authors: Drummond D Allan , Pilipenko Evgeny , Riback Joshua , Scott Jamie , Rojek Alexandra , Budnik Bogdan , Wallace Edward , Katanski Christopher
Published: June 2021

Heat‐shocked eukaryotic cells attenuate protein synthesis, mount a transcriptional response, and assemble proteins and RNAs into dynamic cytosolic granules. The constituents of these granules, the mechanism of their formation, their function, and even the mechanism by which eukaryotic cells sense temperature remain unclear. We have developed quantitative mass spectrometric methods that reveal more than a hundred proteins which reversibly assemble in response to heat in budding yeast. Assembly is in some cases protein‐autonomous: we report purified proteins self‐assembling in vitro with kinetics matching their in vivo assembly, a process encoded in eukaryote‐specific domains, including low‐complexity, intrinsically disordered regions (IDRs). We report the thermally induced, IDR‐mediated assembly of an RNA‐binding protein under physiological conditions that is selective for the type of RNA bound, a phenomenon with potential regulatory consequences. Evolutionary analysis of these domains has been challenging because they align poorly between species, a possible consequence of their unusual functional constraints. We introduce novel methods for analyzing liquid/gel‐forming sequences, informed by these constraints, which reveal novel signatures of selection and coevolution between proteins interacting through these domains. Together, our results illuminate a corner of a vast cellular regulatory system based on assembly and disassembly which evolves according to unfamiliar yet sensible rules.
Computational Analysis of the Interaction between Kinesin Light Chain and Amyloid Precursor Protein
Authors: Shah Soleil , Taylor Cooper , Miller Billy , Parish Carol
Published: June 2021

Disruption of binding between the cargo binding domain of kinesin‐1 light chain (KLC) and amyloid precursor protein (APP) has been implicated in a variety of diseases, including familial Alzheimer's. KLC serves to transport cargo, such as APP, across a microtubule; however, when this function is disrupted, APP releases and disrupts neuronal function. In this project, computational molecular dynamics (MD) was conducted on apo KLC, apo APP, and docked KLC‐APP structures using the Amber MD package to understand the dynamics and interactions between the structures in atomistic detail. KLC remained stable in the apo state and helped to stabilize APP when docked. Preliminary per‐residue free energy decomposition was utilized to determine the key residues involved in KLC‐APP binding. MD was also conducted on an extended tetratricopeptide repeat (TPR) structure to observe fast‐folding dynamics and produce the correct alpha‐helical formation. TPR units, in tandem, form KLC's cargo‐binding domain. Within 700 ns, TPR was able to fold to mostly resemble the consensus crystal structure (CTPR) to within an RMSD of 2.13 Å.