bluuebunny/crossref_metadata_2025 huggingface dataset:
Long‐term gastrointestinal motility disruption in a mouse Gulf War Illness model treated with palmitoylethanolamide
Authors: Hernandez-Rivera Siomara , Grubišić Vladimir , Fried David , Isufi Kenny , Gulbransen Brian D
Published: April 2020

Gulf War Illness (GWI) is a chronic disorder characterized by a spectrum of six symptoms that include gastrointestinal (GI) disorders. Exposure to the anti‐nerve gas drug pyridostigmine bromide (PB) is linked with the development of GWI, but the exact mechanisms remain unclear. Our prior data show that PB exposure contributes to long‐lasting pathophysiology by driving enteric neuroinflammation, promoting immunosuppression, and altering physiological function of the colon. Here, we tested the hypothesis that palmitoylethanolamide (PEA), an endogenous anti‐inflammatory mediator, is sufficient to reduce the detrimental effects of PB in the gut and brain of mice. We tested the effects of PB in vivo by exposing male and female mice to 90 μg/mL PB for 7 days and assessing gut function using in vivo and ex vivo tests of colonic motility and barrier function for 5 months. After 2 months of treatment mice were treated with 0.07 mg/mL of PEA. Immune responses were studied using multiplex cytokine and chemokine arrays in the gut and brain. Male, but not female, mice exposed to PB gained less weight during exposure of PEA (4%, p<0.05). Regardless of sex, exposure to PEA cause a decrease in fecal pellet output (male 66.5%, p<0.01; female 66.1%, p<0.05) and fecal fluid content (male 29.3%, p<0.01; female 23.7%, p<0.05). The combination of PB and PEA treatment altered neuromuscular control and slowed colonic bead expulsion (577.7%, p<0.001) in male mice, but only PB treatment caused slower expulsion in females (777.7%, p<0.001). Colonic permeability was not altered in male mice, but females treated with PEA trended towards increased permeability (39.1%, p=0.0534). Exposure to PEA caused major shifts in the expression of pro‐inflammatory cytokines and chemokines in the colon and brain. Interestingly, immune disruption was still evident in the colon and brain of male and female animals at 2 months following exposure to PEA. Our results show that PEA alters the functional anatomy of the colon and promotes immune disruption in a sex dependent manner and provides no tangible benefit for neuroimmune disruptions driven by PB.

Support or Funding Information

Department of Defense (DOD) W81XWH1610631
The Effects of Lavender Essential Oil on Anxious Behavior and Cortisol Levels in Zebrafish
Authors: Smith Alyshia , Kasper Sherry
Published: April 2020

Stress and anxiety are issues that are increasing within the United States. Due to this increase, many have turned to essential oils for relief. Lavender essential oils are widely regarded as an effective remedy to relieve anxious behavior in stressed individuals. In this study, the effect of lavender oil on anxiety within Zebrafish was tested. When presented with a negative stimuli, zebrafish begin to dive to the bottom of the tank. In our studies, the zebrafish were placed in a novel tank, which is shown to induce a stress response, after exposure to various doses of lavender oil. There was a trend for a decrease in both anxiety and whole‐body cortisol levels in fish exposed to lavender oil compared to control. This research suggests that lavender essential oil may be an effective treatment for anxiety.
Neuronal Deletion of Ghrelin Receptor Attenuates Aging‐Associated Insulin Resistance and Cognitive Decline
Authors: Wu Chia-Shan , Ji Pengfei , Liu Yan , Sun Yuxiang
Published: April 2020

The aging process is characterized by a progressive decline of metabolic functions, insulin resistance, and cognitive impairment. We and others have reported that Growth Hormone Secretagogue Receptor (GHS‐R) plays a crucial role in metabolic regulation. GHS‐R is an endogenous G‐protein coupled receptor for the gut hormone ghrelin, and ghrelin signals the hypothalamus to increase feeding, decrease energy expenditure and reduce fat utilization. We previously reported that global GHS‐R deletion leads to a healthier metabolic profile compared to WT littermates in aging mice. We showed that ablation of GHS‐R ameliorates age‐associated metabolic impairments, reducing body weight/adiposity and improving insulin sensitivity during aging. Furthermore, we found that suppression of GHS‐R in neurons almost completely prevents diet‐induced obesity in mice (Syn1‐cre;Ghsrf/f), likely through increasing energy expenditure. Here, we further assessed metabolic and memory functions of 18–20 months‐old Syn1‐cre;Ghsrf/f and littermate Ghsrf/f control mice. Similar to young mice, old Syn1‐cre;Ghsrf/f mice showed reduced body weight and adiposity. Consistently, old Syn1‐cre;Ghsrf/f mice are more insulin sensitive than littermate controls. Interestingly, old Syn1‐cre;Ghsrf/f mice spent significantly more time exploring the novel object than the familiar object compared to controls in the novel object recognition test, at both 2 hours and 24 hours after training. These data suggest that old Syn1‐cre;Ghsrf/f mice retained short‐term and long‐term memory better than their littermate controls. In addition, old Syn1‐cre;Ghsrf/f mice exhibited significantly increased freezing behavior in both contextual and conditioned auditory stimulus tests in fear conditioning assay, indicating that old Syn1‐cre;Ghsrf/f mice retained better hippocampal‐dependent and amygdala‐dependent memory function compared to littermate controls. Overall, our data suggest that pan‐neuronal deletion of GHS‐R attenuates aging‐associated obesity, insulin resistance and memory impairment. The metabolic benefits derived from suppression of GHS‐R resemble the effects of calorie restriction, the most recognized strategy for increasing health span in multiple organisms. GHS‐R may provide a novel molecular target for improving metabolic fitness and cognitive function during aging.

Support or Funding Information

We gratefully acknowledges the support of NIH R56DK118334 (YS), NIA R01AG064869 (YS), and NIA R21AG061726 (CW).
Is the Bloom taxonomy level of student‐generated exam‐style questions predictive of exam performance?
Authors: Wisinski Jaclyn , Yu Alder
Published: April 2020

Bloom’s taxonomy provides a learning framework distinguishing low cognitive level recall and understanding from more advanced levels of application and analysis. As undergraduate level instructors, we push students to advance in cognition as they progress though a bachelor’s degree. Rote memorization and savvy test taking strategies allow many students to be successful through the first semesters of college. However, advanced learning practice must be developed to facilitate student success. Though instructors make many study strategy suggestions based on empirical evidence, many students end up “figuring it out for themselves”. Student study strategies, such as completing instructor‐provided practice questions and making/using flashcards may enhance performance, but fail to engage students in identifying what the most important concepts are and how those concepts might be tested. To encourage students to actively participate in the learning process, Genetics (BIO306) and Cell Biology (BIO315) students were asked to write question related to the lecture material several times throughout the semester. BIO306 and BIO315 instructors score student‐written questions based on Bloom’s taxonomy, with the hypothesis that students who write higher cognitive level questions will perform better on exams. End of semester course evaluations assess the students’ perception of the question writing as a study technique. Unexpectedly, the cognitive level of student‐submitted questions did not correlate with exam scores. In the next phase of the project, BIO315 and BIO306 instructors will provide students with some information about Bloom’s taxonomy and levels of cognition prior to students’ submission of questions. It is hypothesized that this intervention will encourage students to submit higher cognitive level questions in an effort to improve study skill and exam performance. This self‐quizzing strategy is being developed for 300‐level biology courses, but the results have the potential to apply to many other disciplines at other levels of education.

Support or Funding Information

University of Wisconsin ‐ LaCrosse Faculty Development Scholarship of Teaching and Learning (SoTL) grant 2019
Aseptic Conditions to Reduce Risks to Metabolic Fidelity
Authors: Henn Alicia , He Yan , Darou Shannon , Yerden Randy
Published: April 2020

Mitochondria are derived from bacteria, which puts them at risk of stress from antibiotics commonly used in cell culture. Antibiotics have been found to cause metabolic changes in cells in vitro, fundamentally altering cellular function and response. We have been culturing without antibiotics for over 8 years using the aseptic conditions in the closed Xvivo System and we have not had a single culture contaminated. With the hypothesis that routine disinfection and the conditions inside the chambers would manage microbial risks without antibiotics, we performed routine cell culture processes (cell thaw, cell passage, cell harvest) using highly permissive color‐changing TSB bacterial broth. In 4 trials, no contamination was found of any mock cultures. Monitoring of the environment inside the system with bacterial contact plates showed no evidence of contamination. Positive and negative controls showed appropriate growth. Our results showed that adding antibiotics is not necessary for aseptic conditions of cell culture, which highly reduces the metabolic risk to cells and would enhance normal cellular functions and responses in vitro.
Nucleosome sliding, histone modifications, and the epigenetic regulation of the Simian Virus 40 life cycle
Authors: Milavetz Barry , Rowbotham Kincaid , Haugen Jacob , Diaz Alexandra Rios , Balakrishnan Lata
Published: April 2020

We have recently shown that at late times in an SV40 infection a nucleosome containing modified histones is positioned over the enhancer region consistent with repression of early transcription and activation of late transcription. However, this nucleosome appears to slide toward the late region during virion formation consistent with a role in the activation of early transcription and repression of late transcription in a subsequent infection. Because the transcription factor SP1 plays a major role in regulating SV40 early and late transcription and its binding should be sensitive to nucleosome positioning, we measured the ability of HIS‐tagged SP1 to bind to various forms of SV40 chromatin in vitro and compared this to the presence of SP1 in SV40 chromatin derived in vivo. We found that HIS‐tagged SP1 bound well to chromatin from virions and very poorly to other forms of SV40 chromatin in vitro, while cellular SP1 was associated with minichromosomes isolated from infected cells and was not found in chromatin from virions. In order to determine whether the location of nucleosomes and associated histone modifications plays a role in the activation and continuation of early transcription during the initiation of an infection, we have mapped the location of RNAPII and nucleosomes containing a number of histone modifications in chromatin from virions and minichromosomes isolated 30 minutes and 2 hours post‐infection using ChIP‐Seq. During the first two hours of infection we found that RNAPII first associates with the miRNA site in the SV40 genome (at 30 minutes post‐infection) and then shifts to the early regulatory region around the enhancer (2 hours post‐infection). We next compared the location of RNAPII at the two time points to the location of nucleosomes carrying one of nine different specific histone modifications in the chromatin from virions, and minichromosomes isolated at these times. Chromatin from virions was used because it is the substrate for the activation of transcription, while chromatin from the minichromosomes would indicate any changes occurring as a consequence of transcriptional activation or extension. Based upon this rationale, we identified acetylated H3 as the most likely histone modification in SV40 predicting subsequent binding by RNAPII. We also identified acetylated H4 and the shifting of nucleosomes away from the RNAPII binding site as changes which result from activation of transcription. Together these results indicate that positioned nucleosomes carrying certain histone modifications may control accessibility of transcription factors and also serve as marks for either the binding of RNAPII during initiation of transcription or subsequent re‐initiation during active transcription.

Support or Funding Information

NIH 1R03AI127969‐01

NIH 1R03AI142011‐01
In vitro Reversal of Multidrug Resistance in Cancer
Authors: Holcomb-Webb Kasi L. , Gard Gabrielle E. , Vogel Pia D. , Wise John G.
Published: April 2020

Multi‐drug resistance (MDR) occurs when cancer cells become resistant to multiple chemotherapeutic drugs that are functionally and structurally different. MDR usually leads to failure of chemotherapy treatment and subsequently to poor patient prognosis. Cancers can become resistant to antineoplastic drugs by over‐expressing one or more ATP‐binding cassette (ABC) transporters that act as drug efflux pumps and expel xenobiotics from cells. One such protein is the Breast Cancer Resistance Protein (BCRP, or ABCG2), which is believed to confer MDR to several types of cancer. Inhibition of BCRP cellular activity is therefore thought as a way to restore sensitivity to chemotherapeutics. In previous work, we identified several potential inhibitors of BCRP using high‐throughput in silico screens. Using cell viability and cellular accumulation assays of BCRP substrates, we assessed these compounds for toxicity to cells and the ability to reverse MDR in the BCRP‐overexpressing breast cancer cell line, MCF7‐M100. Here we expand our studies of the potential BCRP inhibitors to two other BCRP‐overexpressing cell lines, the BCRP overexpressing colon cancer and non‐small cell lung cancer cell lines, S1M1‐80 and MX20, respectively. For each cell line, the level of BCRP overexpression was assessed at RNA and protein level and the MDR phenotype was confirmed using cell viability assays. The effects of our newly discovered inhibitors on reversing MDR in these two cell lines is reported.

Support or Funding Information

This work is supported by the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and private gifts from Ms. Suzy Ruff of Dallas, Texas, and Ms. Myra Williams, Ph.D., of Naples, FL.
Mutation of Schizophyllum commune Metacaspases for the Prevention of Autocatalysis
Authors: O’Reilly Kimberly Annette , Fox Kristin M.
Published: April 2020

Metacaspases are cysteine proteases found in fungi, plants, and protozoa that contain a cysteine‐histidine dyad in their active site and cleave after lysine and arginine residues. Unlike their caspase relatives, they are not found in animals, making them an ideal target for antifungals that would incur less harm to the host. Three types of metacaspases (I, II, and III) have been characterized based on their domain structure. This work focusses specifically on the five Type I metacaspases discovered in the fungus Schizophyllum commune, which have been expressed with and without their prodomains and termed MC1‐5 and MC1‐5Δpro, respectively. Many metacaspases rely on calcium for optimal proteolytic activity, and it has also been observed that some of these metacaspases undergo autoproteolysis, or self‐cleavage, during the expression and purification process. This has posed a great challenge to our lab as the autoproteolysis of these enzymes inhibits our ability to purify them in their full length form, which is necessary in order to determine a crystal structure of the protein for structure‐function analysis. In order to purify full length enzymes, we have used site‐directed mutagenesis to create mutants that lack the cleavage sites of the wild‐type enzyme. A mutant of the MC1Δpro enzyme has been created that lacks identified cleavage sites at R389 and R447. This mutant shows significantly less of the cleavage observed in the wild‐type enzyme over a 24‐hour incubation period. Future work will focus on the identification and mutation of conserved or additional cleavage sites in the other four identified metacaspases.
Interaction Networks Of A Crucial Cell Wall Enzyme In The Resistant Bacterium Acinetobacter baumannii
Authors: Tang Amy Y. , Dai Yunfei , Farinha Andrew , Geisinger Edward
Published: April 2020

Acinetobacter baumannii is a common hospital opportunistic pathogen with multidrug resistance, including resistance to last‐resort antibiotics. Many antibiotics today target the cell wall system which is critical for viability. Thus, we sought to better understand cell wall biogenesis in A. baumannii with the goal of finding novel therapeutic targets. L,D‐transpeptidases have been shown to participate in cell wall synthesis by forming unusual peptidoglycan crosslinks. A. baumannii has an L,D‐transpeptidase (Ldt1) that is essential for maintaining the characteristic rod‐shape of the bacterium. However, this transpeptidase and other aspects of cell wall synthesis remain poorly defined. We hypothesized that Ldt1 is important for cell elongation, which is mediated by a multiprotein complex known as the Rod system. To test this hypothesis and define genetic and direct‐protein interactions of Ldt1, we used transposon sequencing (Tn‐seq) and bacterial two‐hybrid techniques. Additionally, we used fluorescent D‐amino acid probes and fluorescence microscopy to visualize differences in active cell wall synthesis sites in WT A. baumannii versus ldt1 deletion mutant. The strongest negative Tn‐seq interactions were with cell division genes, consistent with the hypothesis that Ldt1 is important for the complementary Rod system. The results of the two‐hybrid assay did not show strong direct protein‐protein interaction of Ldt1 with canonical rod‐system components; however, using this method, we mapped novel interactions within the Rod system of A. baumannii. For the fluorescence microscopy, staining conditions of the D‐amino acid probe have been optimized. This study demonstrates methods of elucidating interaction networks in A. baumannii. Through increased understanding of Ldt1 and the Rod system interaction networks, novel synergistic therapeutics can be developed to combat resistant A. baumannii infections.
Role of Calcium in Proteolytic Activity of a Type I Metacaspase from Schizophyllum commune
Authors: Fox Kristin M. , Mineo Charlotte R.
Published: April 2020

Metacaspases are members of the C14 class of cysteine proteases that also includes the caspases. They are found in fungi, plants and protozoa, but not in animals. Unlike the caspases, metacaspases cleave after positively charged residues such as Lys and Arg and are activated by calcium ions. The role of metacaspases in vivo is not yet clearly defined. They have been implicated in cell death and protein quality control, and recent work in A. thaliana suggests that they are activated in response to plant wounding as part of a primitive immune system. Our work centers on understanding how the Type I metacaspases of the fungus Schizophyllum commune are activated by calcium ions. Metacaspase 1 (MC1) activity can be reduced by >90% by mutation of conserved aspartic acid residues implicated in calcium binding. A homology model for MC1 based on the structures of Type I metacaspases from S. cerevisiae and T. brucei was refined. Using the model we identified a loop near the active site that also contains the putative calcium binding site and may undergo conformational changes upon calcium binding. Mutation of a residue in the hinge region of this loop from Gly to Ala significantly reduced enzymatic activity. Kinetic solvent viscosity effects suggest that at low calcium concentrations the enzymatic reaction is limited by substrate binding, while at high calcium levels product release becomes rate‐limiting. We will combine these data to present a model for the role of calcium in Type I metacaspase activation.

Support or Funding Information

CRM was supported by a Scholarship from the Arnold and Mabel Beckman Foundation
Adipose tissue‐specific disruption of the BBSome cause insulin resistance
Authors: Guo Deng Fu , Zhao Yuying , Rahmouni Kamal
Published: April 2020

The BBSome is a protein complex composed of 8 Bardet‐Biedl syndrome (BBS) proteins including BBS1. Patients carrying mutations in any of the BBS genes display several features including obesity and type 2 diabetes mellitus. We previously demonstrated that the BBSome is required for membrane localization of the insulin receptor. We also showed that mice lacking several Bbs genes developed obesity associated with increased blood glucose, impaired glucose clearance and insulin resistance. However, the role and contribution of Bbs genes in insulin‐sensitive tissues such as adipose tissue for the development of obesity and diabetes remains unclear. To address this, we used a new conditional knockout mouse where exon 3 of the Bbs1 gene is floxed (Bbs1flox). Cre‐mediated recombination causes a frame shift resulting in a premature stop. We assessed whether deletion of the Bbs1 gene in adipocytes (by breeding Bbs1flox mice with AdiponectinCre mice) affects glucose homeostasis. Interestingly, no obesity phenotype was observed in both male and female AdipoCre/Bbs1flox mice fed either normal chow or high fat diet (45%), suggesting that development of obesity in BBS is not due to defects in adipose tissue. White fat adipose (WAT) cell size was similar between AdipoCre/Bbs1flox mice and control animals. However, mRNA and protein expression of PPARg was significantly decreased in WAT of AdipoCre/Bbs1flox mice compared to control animals (48%± 5% for mRNA and 47%± 14% for protein, respectively). In addition, fat acid synthetase (FAS) mRNA was reduced in WAT of AdipoCre/Bbs1flox mice relative to controls (34%± 10% vs control. Insulin‐induced activation of AKT was significantly decreased in the WAT, but not in the liver and skeletal muscle of AdipoCre/Bbs1flox mice versus control animals. Finally, fasting glucose level was similar between AdipoCre/Bbs1flox mice and control animals, and glucose tolerance test revealed no difference between AdipoCre/Bbs1flox mice and controls. However, insulin tolerance test uncovered impaired insulin‐induced glucose clearance in AdipoCre/Bbs1flox mice. These findings demonstrate the importance of the adipocyte BBSome for insulin sensitivity and glucose homeostasis.
Interrogating the Mechanism of Eukaryotic Initiation Factor 3 (eIF3) through Molecular Dissection
Authors: Yoo Kyungyoon , Aitken Colin Echeverría
Published: April 2020

Eukaryotic translation initiation is the rate‐limiting and most regulated step of protein synthesis. It begins with the formation of a pre‐initiation complex (PIC) on the small (40S) ribosomal subunit. The PIC is then recruited to the 5′ end of an mRNA, from which it scans in the 5′ to 3′ direction to identify the start (AUG) codon. This convoluted pathway is mediated by multiple protein factors called eukaryotic initiation factors (eIFs).

eIF3, the largest and the most complex of these protein factors, is comprised of five essential subunits (eIF3a/b/c/g/i). Previous genetic and biochemical studies have shown that eIF3 mediates PIC formation, is essential for mRNA recruitment, and participates in scanning for the start codon. eIF3 has also been shown to play a critical role in the stabilization of mRNA in the exit channel and contributes to the rate of recruitment at the entry channel. This latter role of eIF3 may involve its interaction with the 40S latch elements Rps2/3 and h16/h18. In fact, mutations of Rps3 exacerbate 40S dependence on eIF3 for stabilizing mRNA binding by the PIC. Using an in‐vitro reconstituted system, we are interrogating the interaction between eIF3 and the 40S latch by combining Rps2/3 mutations and mutations within elements of eIF3 thought to interact the latch.

To further dissect the role of the eIF3 entry and exit‐channel arms, we are reconstituting yeast eIF3 by recombinant expression in bacteria. Previous work has relied on eIF3 purified from yeast cells. This new approach will alleviate the limitations previously imposed by the essential nature of the eIF3 subunits. Our initial work will examine the activity of reconstituted eIF3 in mRNA recruitment and PIC stabilization. Then, we will focus on a set of mutations targeted at interrogating the many functions of eIF3: truncation of a region of eIF3c thought to promote accurate start‐codon selection; isolation of the eIF3a/c heterodimer that stabilizes mRNA at the ribosomal exit channel; isolation of a eIF3b‐g‐i complex found near the mRNA entry channel; and truncation of an RNA binding domain of eIF3b. The effects of these mutations on component events during translation initiation will shine new light on the role of eIF3 and its subunits.
A Novel Data Analysis Pipeline for Bottoms‐Up Mass Spectrometry Proteomic Analysis of Primary Tissues
Authors: Silverwood Tyler , Radosavljevic Adriana , Charntikov Sergios , Chu Feixia
Published: April 2020

Modern mass spectrometry‐based proteomic studies require reliable, and robust data analysis for a comprehensive and quantitative profile of histone post‐translational modifications (PTM). Studies involving primary tissue samples face additional challenges in comparative sample analysis, due to various protein concentrations, multiple modification sites and modification states. A novel data analysis pipeline was developed in order to streamline the analysis process and normalize samples of varying concentration. This is relevant in the proteomic analysis of addiction, where various modification isoforms can correlate with different addiction phenotypes. A self‐administration model addiction study of methamphetamine (METH) addiction was used to develop this pipeline.

Following nuclear protein isolation, proteins underwent tryptic digestion and histone H3 samples were derivatized with propionic anhydride. Identification, validation, and label‐free quantification of histone modifications was performed. Protein loading of different samples was normalized, using high abundance, proteotypic unmodified peptides. To localize changes in specific site and state, peptide isoforms for each site‐ and state‐specific modification were binned. Using this approach, we were able to identify histone modifications that underwent statistically significant changes in METH self‐administrated rat brain samples. In addition, we were able to identify histone modifications that are indicative of METH controllability in tested individual subjects. This data analysis pipeline has led to robust analysis of histone PTMs. Information on changes in site‐ and state‐specific histone modifications can be integrated with RNA‐seq data to elucidate driving chromatin pathways in the formation and maintenance of pathophysiological epigenetic states.

Support or Funding Information

This project was supported in part by the University of New Hampshire Hamel Center for Undergraduate Research.
Do Undergraduate Choices Predict Medical Gross Anatomy Performance?
Authors: Veazey Kathryn , Robertson Ellen
Published: April 2020

A 2019 study conducted by Robertson et al. suggested that the amount of prior anatomy experience does not predict the performance of first‐year medical students in medical gross anatomy at the University of Mississippi Medical Center (UMMC). In recent years, it has not been possible for UMMC to accept non‐state residents to its medical program. Thus, nearly seventy‐five percent of first‐year medical students matriculate from one of five major in‐state institutions. At current writing, four of the five schools directly communicate with the UMMC admissions program regarding the relationship between their courses and UMMC’s specific institutional mission. First‐year students enter UMMC with a wide range of previous undergraduate and graduate degrees. The undergraduate choices investigated were which institution(s) students attended, what type and how many degree(s) they attained, and whether they took an anatomy course prior to attending UMMC. We hypothesized 1) that undergraduate degree does not predict performance in a medical gross anatomy course, 2) individuals with science majors are more likely to have prior anatomy experience, 3) the collaboration of the previous institution with UMMC does not predict performance in a medical gross anatomy course, and 4) students from Mississippi College who have prior anatomy experience will perform better in the medical gross course at UMMC. Mississippi College implements a gross anatomy curriculum that is nearly identical to the course taught at UMMC. Data was collected from 280 first‐year medical students at UMMC in the fall of 2016 and fall of 2017. Using a self‐reported survey, data was collected on the number and type of prior degrees and prior anatomy experience. The course director provided student performance data in the medical gross anatomy course. The office of admissions of the medical school confirmed the prior degrees and previous academic institutions of the students. Quantitative analysis was used to determine the validity of the research hypotheses. The results of this study could provide additional guidance to undergraduate students during the degree selection process prior to applying to medical school.
Role of MAB21L2 in Ocular Disease
Authors: Klestinski Keith , Malloy Joseph , Patel Siddharth , Colella Benedetto , Homberg Logan , Khan Taha , McLaughlin Andrew , Panichikudiyil Abraham , Sajanlal Ahmed Ali , Scaffidi Isaiah , Warner Luke , Kaiser Carl
Published: April 2020

MAB21L2 is a protein linked to fetal eye development. The MAB21L2 encoding gene is found on the fourth human chromosome and is found in all vertebrate species. Exome sequencing in human cousins affected by coloboma, microcornea, cataracts, and skeletal dysplasia identified an p.Arg51Gly variant in MAB21L2. Sanger sequencing confirmed that five affected family members were heterozygous for the wild‐type p.Arg51Gly variant, while unaffected family members were wild‐type, thus identifying this variant as a cause of coloboma, a disease characterized by missing tissue in one or more structures of the eye in humans. Studies using zebrafish models confirms the role of this highly conserved gene in ocular development. The affected amino acid (Arg 51) is conserved across all family members and vertebrate species.

The crystal structure for MAB21L2 has yet to be successfully solved. The MUHS SMART team submitted four amino acid sequences from the MAB21L2 gene to I‐TASSER, a bioinformatics method for predicting three‐dimensional protein structure, in order to study the effect of the p.Arg51Gly variant on protein structure and function. The first submissions contained human and zebrafish wild‐type amino acid sequences, while the second contained the human and zebrafish p.Arg51Gly variant. The predicted protein structures were 3‐D printed in order to visualize any structural differences between the wild‐type and variant proteins. Through JMOL we focused on the residue 51 mutation site. The mutation site was found on the exterior of the protein. Modeling results showed that in the mutant type residue 51 was glycine rather than the arginine which is typical of the wild type. About 48% of the protein is made up of alpha‐helices and 15% of the protein is made up of beta‐sheets. Research conducted by the Semina Lab of the Medical College of Wisconsin through Yeast Two Hybrid assays found that a protein top2b interacted with wild‐type MAB21L2 but the interaction appeared to be weakened in the mutant. Other proteins typically involved with misfolded proteins that could lead to degradation (hspa8 and hspa5) both interacted with mutant but not wild‐type MAB21L2.

Future research into the effects of the protein mutation may provide a useful step towards better understanding of ocular development and ultimately finding cures for various ocular diseases. Possible treatment of this protein mutation may involve gene‐editing technology like CRISPR‐Cas9 to replace the mutated amino acid.
Interactions between chemical and environmental factors and bacterial community composition in a Great Lakes watershed
Authors: Cooper Carolyn , DeLoach Idgena , McMorris John , Slater Adam , Brokus Sarah , Wade Randall , Best Aaron , Krueger Brent , Pikaart Michael
Published: April 2020

Urbanization and agricultural activity have degraded the water quality of the Great Lakes and their tributaries. These impacts include nutrient and fecal coliform loading and high sediment levels. In this presentation, we report on three years of continuous weekly monitoring of the Macatawa watershed in Ottawa and Allegan Counties, Michigan, a drowned river mouth entering Lake Michigan. Five lake locations and seven stream sites have been analyzed for microbiological content by community 16S rRNA sequencing, fecal indicator bacteria presence, and Escherichia coli whole‐genome sequencing. Along with these biological indicators, we profile chemical and physical parameters including dissolved oxygen, nutrients, temperature, and pH. We find continued high phosphorus, total suspended solids, and E. coli levels, exceeding total maximum daily load targets, or other benchmarks, for the watershed. Patterns in microbial communities show variation influenced by season and geographic location; for example, total E. coli increases in the stream tributaries in summer but decreases in the lake fed by those streams relative to winter. Furthermore, overall diversity of microbial communities increases in late fall and winter. These data provide a baseline for monitoring remediation efforts in the Macatawa Watershed. We suggest these will serve as a comparison for hypereutrophic watersheds around the nation and to aid decisions about remediations and public access to these recreational waters.

Support or Funding Information

NSF RUI 1616737
Rats are Protected from Chronic Pressure Overload Compared with Mice Through Angiogenesis, but not Myogenesis.
Authors: Nishimura Koichi , Oydanich Marko , Zhang Jie , Babici Denis , Fraidenraich Diego , Galindo Cristi , Vatner Dorothy E , Vatner Stephen
Published: April 2020

It is generally assumed that rats and mice respond to cardiovascular stress similarly. The first goal of this investigation was to compare the effects of chronic pressure overload, induced by 4 weeks of transverse aortic constriction (TAC), in Sprague Dawley rats and C57/BL/6J mice. TAC, after 1 day, induced similar left ventricular (LV) pressure gradients in both rats and mice (113 ± 5.4 vs. 103 ± 11.5 mmHg), and after 4weeks a similar amount of LV hypertrophy (LV/tibial length increase: 51 ± 6.0 vs. 51 ± 5.7). After 4 weeks of TAC, LV systolic and diastolic function were preserved in rats, whereas LV ejection fraction decreased in mice (19 ± 2%), along with increases in LV end diastolic pressure (159 ± 54%), LV systolic wall stress (90 ± 12%), and LV diastolic wall stress (102 ± 41%). The second goal was to determine whether angiogenesis or myogenesis was the mechanism protecting against chronic pressure overload. Immunofluorescent Ki67 staining of capillaries revealed greater increases in angiogenesis with TAC in rats (10 ± 2 positive cells/field) than in mice (6 ± 1 positive cells/field). Myocardial blood flow fell by 55 ± 15% in mice but was preserved in rats. Coronary reserve, the increase in blood flow to the heart with adenosine, was preserved in rats, whereas it fell by 28% in mice with TAC. The next goal was to determine if myogenesis, through generation of stem cells, mediated these differences. This was accomplished by staining the myocardium with c‐kit. More c‐kit positive cells were found in rats with TAC compared to mice with TAC (0.30 ± 0.03 vs. 0.07 ± 0.02 cells/mm2). Surprisingly, these c‐kit positive cells were located not in myocytes, but in the interstitial space and were all negative for Troponin I. However, some of these c‐kit positive cells were positive for CD31, i.e., endothelial cells, which could be involved in mediation of the angiogenesis. Thus, rats tolerate severe pressure overload better than mice, with mechanisms involving increased angiogenesis, but not myogenesis.
Anatomy across the pond; how anatomy is taught and in how many hours in the United Kingdom.
Authors: Smith Claire , Freeman Samuel , Heylings David , Davies Ceri
Published: April 2020

It has been suggested that a deficit in junior clinicians anatomical knowledge is causing harm to patients, evidenced by a rise in surgical negligence legal claims resulting from a lack of knowledge of underlying anatomy. This study set out to examine the state of anatomy in the UK and Ireland in 2019 and how it has changed over the past two decades. The study used questions previously published by Heylings (2002), with some additions to reflect recent innovations in anatomy teaching. The project was awarded ethical approval (ER/BSMS3867/8) and a survey comprising 53 questions was hosted on the University of Sussex Qualtrics platform. Heads of Anatomy in the UK and Ireland (n=40) were asked to complete the survey and a 100% response rate was obtained. The results showed that anatomy is a defined ‘group’ or department in 62% of institutions. For medical teaching, 38% of institutions follow a systems‐based curriculum and 35% a hybrid curriculum. The number of medical student being taught in a cohort ranged from 71–450. The time dedicated to gross anatomy teaching ranged from 34–145h. Only one institution teaches anatomy by dissection only, 12 using prosection only, and 17 use a combination of the two. Five institutions do not use human cadaveric material. In 2018, five new medical schools were created in the UK, each following an existing institution’s curriculum. At the time of the survey, were yet to enroll students and therefore, were not included in the study. In comparison to data gathered therein 1999 (Heylings, 2002) when 160h was the mean teaching time for traditional regional anatomy courses and 116h for system based courses, the results of the current study reveal a considerable reduction in anatomy teaching hours. Is this reduction responsible for the increase in surgical negligence claims?
Early Maturation of the Hypoxic Ventilatory Response in Hyperoxic Rat Pups: Evidence for Trait‐Specific Heterokairy
Authors: Pinette Michaela , Bavis Ryan W.
Published: April 2020

Newborn mammals exhibit a biphasic hypoxic ventilatory response (HVR) in which an initial increase in ventilation is followed by a decline back toward baseline levels. The magnitude of the secondary decline diminishes with age, but rat pups reared in moderate hyperoxia make this transition earlier (Bavis et al., J. Appl. Physiol. 109(3): 796–803, 2010). This is consistent with heterokairy, a form of developmental plasticity in which environmental factors alter the rate and/or timing of developmental events. The present study investigated whether this effect is specific to the HVR or if hyperoxia instead accelerates overall development. Rat pups reared in 60% O2 (Hyperoxia) exhibited a less biphasic HVR at both P4 and P10‐11 compared to pups reared in 21% O2 (Control). However, growth curves and the average ages at which pups attained key developmental milestones (i.e., fur development at P5, incisor eruption at P9, and eye opening at P15) were similar between treatment groups. We also studied the relationship between metabolic rate and ambient temperature as another indicator of physiological maturity. The lower critical temperature (LCT) decreased with age, but the LCT was the same for Control and Hyperoxia rats at both P4 (~32°C) and P10‐11 (~30°C). The ability to increase metabolic rate at low ambient temperatures also increased with age, but this thermogenic capacity tended to be reduced in Hyperoxia rats at both P4 and P10‐11 (i.e., lower rates of metabolic CO2 production at temperatures below the LCT). We conclude that hyperoxia accelerates the rate at which the HVR matures without affecting the maturation of other morphological and physiological traits.
Optical tweezers and multimodality imaging: a platform for dynamic single‐molecule analysis
Authors: Raja Ali , Hadizadeh Nastaran , Candelli Andrea
Published: April 2020

Utilization of single molecule technologies offer researchers the unique opportunity to perform direct real‐time observations and measurements for a plethora of biological mechanisms. Here, we present our efforts to facilitate biologically meaningful discoveries using fully integrated optical tweezers with single‐molecule fluorescence microscopy technology. Correlative fluorescence and optical tweezer microscopy (C‐trap™) is the only technology that truly integrates optical tweezers, confocal/STED microscopy and an advanced microfluidics. The C‐Trap ™ enables live, simultaneous and correlative visualization and manipulation of molecular interactions with sub‐piconewton (pN) force resolution and a kilo‐Hz (to mega‐Hz) temporal resolution, such that even the quickest cellular events can be captured. This technology can be utilized as an easy‐to‐use and stable instrument with the capacity to elucidate biologically meaningful mechanisms. Here we present applications detailing protein (un)folding and conformational changes; DNA‐protein interactions and genome modifications; effects of mechanical stress on DNA/RNA structure; motility of cytoskeletal molecular motors; and protein droplet and aggregation dynamics. These experiments demonstrate technological advances in hybrid single‐molecule methods and the potential for their use in future scientific breakthroughs.
PPP6C Regulation of ERK Signaling in Melanoma
Authors: Cho Eunice , Lou Hua Jane , Turk Benjamin E
Published: April 2020

Melanoma, the deadliest form of skin cancer, is characterized by aberrant hyperactivation of the ERK mitogen‐activated protein kinase signaling pathway. Inhibitors of BRAF and MEK1/2, both members of this pathway, have shown clinical efficacy and are used in combination to treat BRAFV600E melanoma, the most common melanoma genomic subtype. However, not all BRAFV600E melanomas respond to these inhibitors, and those that do respond eventually acquire resistance. To better understand mechanisms of drug susceptibility in BRAFV600E melanoma, we performed a loss‐of‐function screen to identify kinases and phosphatases that modulate sensitivity to two clinical MEK1/2 inhibitors. In this screen, we identified PPP6C, the catalytic subunit of protein phosphatase 6 (PP6), as a factor promoting sensitivity to MEK1/2 inhibition. PPP6C is a potential tumor suppressor mutated in 7–12% of melanomas, and has roles in cell cycle regulation and DNA damage repair. shRNA‐mediated knockdown and CRISPR/Cas9‐mediated knockout of PPP6C in BRAF mutant melanoma cells both lead to hyperactivation of MEK1/2 and ERK1/2, resulting in decreased sensitivity to pathway inhibitors. This finding was confirmed in a panel of BRAFV600E melanoma cell lines. Through in vitro biochemical and cell‐based studies, we have established that PP6 directly dephosphorylates MEK1/2. As with complete loss of PPP6C, cells expressing most recurrent cancer‐associated PPP6C mutants similarly have hyperactive ERK signaling. Our studies support a novel negative regulatory role for PPP6C in ERK signaling that contributes to its activity as a tumor suppressor and promotes pathway inhibitor sensitivity in BRAFV600E melanoma.

Support or Funding Information

Funding was provided by the Gruber Foundation, T32 GM007324, and F31 CA220999.
Rapid effects of aldosterone and phosphatase inhibition on water and urea permeabilities
Authors: Wang Yanhua , Ma Fuying , Klein Janet D. , Sands Jeff M.
Published: April 2020

Aldosterone promotes renal sodium uptake, thereby increasing water reabsorption. But our previous data showed that aldosterone decreases UT‐A1 urea transporter expression, suggesting aldosterone could inhibit water reabsorption. In this study, we investigated the rapid effect of aldosterone on vasopressin‐stimulated osmotic water permeability in rat inner medullary collecting ducts (IMCDs). In IMCDs from male Sprague Dawley rats weighing 50–75g, we saw that aldosterone significantly decreased osmotic water permeability from 152 ± 21 to 122 ± 17 μm/s (45 min, n=4, p<0.01). Given the rapid nature of the response, we further investigated whether the effect of aldosterone might be through phosphatase‐mediated modulation of transporter phosphorylation. Inhibition of PP1/PP2a phosphatases by calyculin increased basal osmotic water permeability from 58 ± 8 to 92 ± 14 μm/s (n=4, p<0.01). In the presence of vasopressin, stimulation by calyculin remained significant in both male and female rats 77 ± 10 to 119 ± 15 μm/s (30 min, n=6, p<0.01). Addition of aldosterone to the bath in the presence of calyculin reversed the increase in osmotic water permeability from 119 ± 15 to 89 ± 10 μm/s (45 min, p<0.01). Inhibition of pp2B with tacrolimus also stimulated water permeability (88 ± 13 to 108 ± 19 μm/s), which was reversed by addition of aldosterone (81 ± 10 μm/s). Urea permeability was also increased by both calyculin (25 ± 7 × 10−5 to 32 ± 7 × 10−5 cm/s; n=3, p<0.05) and tacrolimus (15 ± 1 × 10−5 to 18 ± 1 × 10− 5 cm/s; n=4, p<0.05). Aldosterone decreased pser256‐ and increased pser261‐AQP2. Inhibition of PP2B by tacrolimus increased pser256‐ and decreased pser261‐AQP2. Calyculin increases pser256‐AQP2 and this was reversed by aldosterone. Treatment of inner medullary tissue with 50 nM calyculin or 62 nM tacrolimus resulted in significantly increased UT‐A1 phosphorylation. We conclude that aldosterone is able to rapidly inhibit water and urea reabsorption, possibly by altering phosphatase activity and changing the phosphorylation states of AQP2 and UT‐A1.

Support or Funding Information

AHA 18CDA34060053

NIH/NIDDK

RO1‐DK41707
Investigating Polycystin‐2‐mediated mitophagy as a stress response through its role in NAFLD
Authors: Brill Allison L. , Kuo Ivana Y. , Ehrlich Barbara E.
Published: April 2020

Non‐alcoholic fatty liver disease (NAFLD), the most common chronic liver disease worldwide, is characterized by aberrant dysregulation of cellular energy mechanisms, including defective autophagy and mitochondrial function. Inhibiting general autophagy in models of NAFLD has produced contradictory results, resulting in both protection against and causation of hepatic steatosis and insulin resistance. However, the specific role of autophagy to selectively degrade mitochondria—the process known as mitophagy—has not been studied in the context of NAFLD development. The main goals of this project are to establish the function of dysregulated mitophagy in the development of NAFLD, and to determine how Polycystin‐2 (PC2), a Ca2+‐modulating protein on the endoplasmic reticulum (ER) membrane, works to promote hyper‐activation of mitophagy through its interaction with Bcl‐2 associated athanogene 2 (BAG2). My interest in PC2 and BAG2 is based on published and preliminary data demonstrating 1) increased PC2 and BAG2 expression in human and murine livers with NAFLD, 2) BAG2 as an interacting partner of PC2 that promotes PTEN‐induced putative kinase 1 (PINK1)‐mediated mitophagy following PC2 up‐regulation, and 3) protection against insulin resistance and hepatic steatosis in PC2 haploinsufficient mice fed a high fat diet (HFD). In this work, I examine how PC2 is up‐regulated under stress to modulate mitophagy and mitochondrial function, as well as how PC2‐null hepatocytes respond to HFD‐induced NAFLD, and determine how observed mitochondrial morphological and functional alterations that occur in hepatocytes following the onset of NAFLD are altered by PC2 and BAG2. These results will confirm PC2 as a stress response protein capable of regulating mitophagy, and will help resolve the role of dysregulated autophagy in NAFLD development.

Support or Funding Information

Funding provided by T32GM007324 and F31DK118836.