bluuebunny/crossref_metadata_2025 huggingface dataset:
Examining the Research Experiences of Undergraduate Biochemistry Students: A Case Study Approach
Authors: Johnson Sara L. , Bodner George M.
Published: November 2023

Undergraduate research is a high‐impact educational practice that provides students with opportunities to gain a variety of advantages. In the past 20 years, a growing body of researchers have sought to identify both the positive and negative outcomes that result from students' participation in undergraduate research. This body of work has confirmed the important role an undergraduate research experience can play in a student's education. Unfortunately, not all students who participate in undergraduate research realize these benefits. The outcome of a student's participation in undergraduate research can vary, not only between research groups but at the individual level. Many factors play a role in the experience students will have when engaging in undergraduate research. Our research focuses on the discursive elements of undergraduate students' research experiences in biochemistry. Using a case‐study approach, we investigate the norms of discourse of and between undergraduate students and their mentors while working in the laboratory. We use video‐recorded observations of student‐mentor pairs working together to identify discursive norms. We have been employing a stimulated recall interview protocol to explore how participants establish and negotiate norms during a research experience, focusing on the invisible processes by which undergraduate students learn to do research. Our approach allows us to examine the complex processes involved in a student's acculturation into the community of practice of biochemistry researchers. Using this approach, we have detected differences between the discursive norms of students and their mentors. We have also observed variation in the discourse that is constructed between distinct student‐mentor pairs. This diversity suggests that, to continue improving undergraduate research as an educational practice, research efforts should expand beyond investigation of programmatic outcomes into the fine‐grain details of students' experiences. By characterizing these discursive processes, we intend to inform and equip the future efforts of those interested in the research, design, and evaluation of undergraduate research programs. Additionally, research advisors interested in improving the experiences of their own undergraduate students may benefit from the practical mentoring strategies we will suggest based on our findings.
Rheostat or Toggle: Examining the role of non‐conserved mutations to LURE students into research
Authors: Ayella Allan Klara , Moriah Beck R
Published: November 2023

There is a growing movement to involve undergraduate students in authentic research experiences. A variety of studies have indicated the strength of this approach in developing scientific aptitude, confidence, critical thinking skills and likelihood to become career scientists. Course‐based undergraduate research experiences (CUREs) provide the opportunity to lure students into legitimate research at both primarily undergraduate and large research institutions. Here, we describe how to use a CURE‐based biochemistry lab course to explore the role of conserved and non‐conserved amino‐acid sites in structure and function of lactate dehydrogenase enzyme (LDH). Recent research suggests that substitutions at non‐conserved positions behave more like rheostats in modulating protein function, whereas substitutions at conserved positions result in “toggle switching.” In the first phase of this CURE, students used bioinformatics tools to carry out protein sequence alignments and 3D structural explorations to identify potential sites to mutate in the LDH gene. Site‐directed mutagenesis was then used to generate amino acid substitutions at non‐conserved sites in the LDH protein. Students confirmed mutations to the DNA sequence in protein expression vectors before carrying out protein purification of both wildtype and mutant LDH proteins. In the final phase, students assessed protein function using a variety of techniques. All students were required to determine LDH kinetic parameters (Km and Kcat). In addition, students were prompted to explore other enzyme structural or functional changes through alternative assays or broader biophysical and structural studies of their choice. For example, students initiated studies included the use of circular dichroism to determine structural changes and/or comparisons of thermal stability, examination of coenzyme binding effects and conformational changes via fluorescence spectroscopy. Formal assessments were carried out to determine gains in scientific aptitude, confidence and critical thinking. A culminating poster session allowed students to disseminate their research findings beyond the classroom and receive critical feedback from faculty and students from a broader realm. Attitudinal changes resulting from this experience were monitored by administering the CURE assessment tool developed by Lopata et. al before and after the course. Informal attitudinal assessments indicated that participating in this new curriculum increased student interest in attending graduate school and pursuing careers in biochemical research. Student confidence in their ability to conduct independent research also increased. We feel that because the outcome of each experiment was not known beforehand, students were able to primarily focus on the experimental design and data analysis hence increasing their scientific aptitude, confidence and critical thinking skills.

Support or Funding Information

This research was funded by Wichita State University and McPherson College.
Successes and Teachable Moments During a Partnership to Create a Research‐like Experience in a 2 Year College Organic Chemistry II Laboratory.
Authors: Jaco Nathan , Lutz Christopher , Grant Jennifer
Published: November 2023

As part of the NSF ATE grant, MALDI‐based Research‐like Experiences in a 2YC/4YC Collaboration with a Renewable Fuels Industry Partner (DUE 1400885), the authors examined how best to combine state of the art training in research‐like experiences that included use of major research instrumentation embedded in an organic chemistry course. In this case a research‐like experience utilizing MALDI‐TOF mass spectrometery was conducted, an experience that also utilized Process Oriented‐Guided Inquiry Learning (POGIL) techniques to serve students at 2‐year colleges. This unique research experience fulminated into sharing of data, methods and feedback between the students and executives of a local biodiesel fuels company, Ever Cat Fuels of Anoka Minnesota.

This presentation examines the structure and effectiveness of the artifacts used, as well as the outcomes generated as the research experience was implemented, and the feasibility of creating a modular curriculum that allows student to participate in research‐like experiences using a variety of different major research instruments. At the basis of the curriculum, we created a suite of materials that include a faculty training manual, a student manual, a paced set of Process Oriented Guided Inquiry Learning materials, and two videos. The first video has garnered an average of 1,500 view per month, representing a viewership from 59 countries.

Materials used to deliver this research‐like experience will be delineated, and both the successes achieved and the lessons learned during implementation will be summarized. In particular we will highlight our efforts to make fatty acid identification more facile using triglyceride mass tables developed using Excel software. Triglyceride data used was based on the most highly abundant fatty acids present in similar materials shown with GC‐MS analysis. The tables have been constantly modified to benefit the user, by including possible cation adducts and a creating a search feature. Finally, several teaching scripts were developed introducing the biochemistry of Triglycerides and how to properly use the Excel tables in analyzing seed oils. Information from these scripts were then used create a student manual instructing the user on the process of setting up MALDI‐TOF analysis of triglycerides in natural oils.

As this is the final year of this project, the authors are soliciting feedback on the quality and utility of these educational resources.

Support or Funding Information

This work was supported by the NSF Advanced Technology Education grant “MALDI‐based Research‐like Experiences in a 2YC/4YC Collaboration with a Renewable Fuels Industry Partner” (DUE 1400885).
The Council on Undergraduate Research (CUR): What's in it for Post‐Docs and New Faculty? Advancing your research career with/through the Council on Undergraduate Research
Authors: Wolyniak Michael , Wimmers Larry
Published: November 2023

This poster is intended for post‐docs and new faculty to identify the Council on Undergraduate Research (CUR) as a professional society that supports and promotes high‐quality undergraduate student‐faculty collaborative research and scholarship. What is CUR: The Council on Undergraduate Research, is a national organization of individual and institutional members representing over 900 colleges and universities. CUR achieves its vision through efforts of its membership organized into 13 disciplinary divisions including Biology. In an effort to raise the visibility of this professional organization, The Biology Division of CUR has recently initiated outreach efforts aimed at post‐docs to raise awareness about building a successful and fulfilling research program with primarily undergraduate students. Our workshops and topics have included topics such as ‐ (1) Examining the academic environment at primarily undergraduate institutions, and others that do not have doctoral program, (2) Preparing oneself to be competitive for faculty positions at these institutions, and (3) Balancing research and teaching responsibilities as a beginning investigator. Conferences: A biennial conference features workshops as well as presentations by faculty and administrators, and other stakeholders with an interest in promoting undergraduate research. A yearly forum called CUR Dialogues brings together representatives of funding agencies and foundations, to discuss funding opportunities to support undergraduate research. NCUR is a conference for students which is held each year at an academic campus. In addition to providing a forum for undergraduate presentations, this venue assists under‐represented students in the development of networks and support structures. Undergraduate Students are selected to participate in a yearly forum called Posters on the Hill, which raises awareness with legislators about the educational significance of undergraduate research. Other CUR Activities: Faculty development opportunities are offered through Institutes and Workshops (Institutionalizing Undergraduate Research, Initiating and Sustaining Undergraduate Research Programs, Proposal Writing, and Beginning a Research Program); Publishing books and articles related to creating, managing, and evaluating undergraduate research programs, including CUR Quarterly, the organization's journal; Assisting administrators and faculty members in improving and assessing the research environment at their institutions; Providing information on the importance of undergraduate research to state legislatures, private foundations, government agencies, and the U.S. Congress.
Year‐long Research Experiences in Drug Discovery May Lead to Positive Outcomes for Transfer Students
Authors: Beckham Josh , Metola Pedro , Strong Lisa
Published: November 2023

Conceptual understanding of biochemistry and success in science can be challenging for transfer students when transitioning into a degree plan from another institution. These goals may be more readily achieved through a curriculum tied to research. We have begun to test this hypothesis through establishing a pilot CURE (course based undergraduate research experience) called the Accelerated Research Initiative (ARI) built upon the successful framework of the Freshman Research Initiative (FRI) in the College of Natural Sciences at The University of Texas at Austin. The ARI caters to upper‐division undergraduates science majors (juniors and seniors). Several of the students in a starting class of 16 have transferred into the university from other 4 year institutions or from community colleges. The student‐researchers are guided by a non‐tenure track professor, several undergraduate TAs and peers who have prior research experience. For each project, the overall goal is to identify small molecule inhibitors of an enzyme from an infectious disease organism (bacterial or protozoan). Computational molecular docking software is used to make predictions about which compounds may bind. They then seek to test these in biochemical assays against the enzyme they have made through recombinant DNA cloning and protein expression. In order to gain insight into the students' perceptions of the experience so that the curriculum could be tailored in subsequent semesters, surveys and focus groups were used. The questions and discussion focused on gaining insight about the contexts, activities, and outcomes cited in other CUREs (Corwin et. al. 2015), such as: content and skills, self‐efficacy, persistence in science, ownership, communication, science identity, and mentoring. When compared to responses from transfer students who did not participate in a CURE, these perceptions of the course may help identify which aspects are most meaningful to achieving science success.

Support or Funding Information

Howard Hughes Medical Institute (HHMI)
Stabilization of Dsg2 binding enhances GJ function and ameliorates arrhythmia caused by PG deficiency
Authors: Erber Bernd Markus , Schinner Camilla , Schlipp Angela , Waschke Jens
Published: November 2023

Arrhythmogenic cardiomyopathy (AC) is a genetic disease of the cardiomyocyte intercalated disc which causes cardiac arrhythmia and sudden cardiac death. Mutations primarily affect desmosomal proteins including the cadherin‐type adhesion molecule desmoglein 2 (Dsg2) and the plaque protein plakoglobin (Pg), which are known to be required for mechanical cohesion of cardiomyocytes, and thereby impair gap junction (GJ)‐mediated propagation of excitation by unknown mechanisms. Therefore, therapy is symptomatic at present. We provide evidence that stabilization of Dsg2‐binding by a tandem peptide (TP) enhances GJ signal transduction in cardiomyocytes. TP was designed to cross‐link Dsg2 molecules separate from the hydrophobic binding pocket of the extracellular domain 1. TP stabilized Dsg2 interaction on the surface of living cardiomyocytes as revealed by atomic force microscopy (AFM) and rescued cardiomyocyte cohesion when either tryptophan was used to block the binding pocket or Dsg2 and Pg were depleted by siRNA. In parallel, disturbed localization of connexin 43 (Cx43) and impaired GJ‐mediated excitation propagation were also abolished by TP, partly via a mechanism involving PKC phosphorylation of Cx43. In hearts from a cardiomyocyte‐specific Pg‐deletion mouse model for AC, TP reduced arrhythmia immediately upon perfusion. These results indicate that stabilization of Dsg2 binding may represent a new approach to treat arrhythmia in AC.

Support or Funding Information

Förderung für Forschung und Lehre (FöFoLe)
CD43 syalomucin contributes to cardiac inflammation and fibrosis in non‐ischemic heart failure
Authors: Velázquez Francisco , Salvador Ane , Nevers Tania , Ngwenyama Njabulo , Aronovitz Mark , Blanton Robert , Alcaide Pilar
Published: November 2023

Left ventricular (LV) dysfunction and Heart Failure (HF), are one of the leading causes of hospitalizations and death in the developed world. HF is a very complex syndrome involving multiple factors that lead to LV remodeling and impairment of cardiac function. T cells have been found infiltrated in the LV of patients with ischemic and non‐ischemic HF and also increased in the systemic circulation, and this correlates with LV dysfunction. Moreover, studies in T cell deficient mice demonstrate a pathogenic role for T cells in non‐ischemic HF induced by LV pressure overload. We have previously demonstrated that intra‐myocardial E‐selectin and intracellular adhesion molecule‐1 (ICAM‐1) are upregulated in mice with HF, and suggested that these adhesion molecules may regulate T cell recruitment to the heart during pressure overload induced HF. Syalomucin CD43 is highly expressed on leukocytes, including T cells, and its highly glycosylated form expressed in activated T cells functions as an essential E‐selectin ligand that regulates specific T cell subset recruitment to sites of inflammation. However, the role of CD43 in cardiovascular disease is largely unexplored and its role in leukocyte recruitment in HF remains unknown. We hypothesized that CD43 regulates T cell recruitment to the LV during pressure overload induced HF. To test this, we used the mouse model of thoracic aortic constriction (TAC) in WT and CD43−/− mice and monitored them for 4 weeks. We observed that the expression of the highly glycosylated form of CD43 was induced in T cells isolated from cardiac lymph nodes from TAC WT mice as compared to Sham mice. CD43−/− mice had a higher survival rate and reduced interstitial and perivascular fibrosis as compared to WT mice. Echocardiography measurements determined that the ejection fraction and fractional shortening were both preserved in CD43−/− mice in response to TAC as compared to WT TAC mice, indicating preserved systolic function in CD43−/− mice. Immunohistochemistry showed that CD43−/− mice had decreased LV leukocyte infiltration, more specifically, decreased CD4+ T cells and CD11b+ monocytes as compared to WT mice in response to TAC. Our results demonstrate that CD43 plays a crucial role in leukocyte recruitment to the heart and in the pathology associated with non‐ischemic heart failure. Future studies will determine the molecular mechanisms by which CD43 functions in leukocyte trafficking and disease progression in non‐ischemic heart failure.

Support or Funding Information

HL123658‐01S1
Resolvin D1 Programs Myocardial Infarction‐induced Cardiorenal Syndrome in Heart Failure Pathology
Authors: Halade Ganesh V. , Kain Vasundhara , Serhan Charles N. , Ingle Kevin A.
Published: November 2023

Resolvins are innate immune responsive lipid mediators produced after myocardial infarction (MI) to resolve inflammation. MI‐induced bidirectional interaction between progressive left ventricle remodeling and kidney dysfunction is known to advance cardiorenal syndrome (CRS). Whether resolvins limits MI‐induced cardiorenal dysfunction is unclear. Thus, to define the role of resolvin D1 (RvD1) in post‐MI CRS, we tested the hypothesis using 8 to 12 weeks old male C57BL/6 mice that were subjected to coronary artery ligation surgery. RvD1 (3ug/kg/day) was injected 3 hour post‐MI for day (d)1 or continued till d5. No‐MI naïve and saline‐injected MI mice were used as controls. RvD1‐injected group promoted neutrophils (CD11b+/Ly6G+; 2.0±0.3%) egress from infarcted LV compared with MI‐control group (4.2±2.0%) at d5 post‐MI. Further, RvD1 injected mice showed higher reparative macrophages (F4/80+/Ly6Clow/CD206+; 20.0±1.0%) phenotype in the infarcted LV compared with MI‐control group (14.4±2.1%) at d5 post‐MI measured by flow cytometry. MI‐induced LV remote hypertrophy was limited in RvD1‐injected mice compared with MI‐control mice confirmed using wheat germ agglutinin staining. Post‐MI nephrin expression was diffused to glomerular membrane at d5 in MI‐control mice indicating renal injury impacting kidney glomerulus structure but to a lesser extent in RvD1‐injected mice validated using immunofluorescence staining. Administration of RvD1 attenuated MI‐induced renal inflammation decreasing neutrophil gelatinase‐associated lipocalin (2.0±0.1 fold p<0.05) gene and protein expression in the kidney at d1 post‐MI (2.2±0.1 fold; p<0.5) and d5 compared to MI‐control. In conclusion, RvD1 not only clears MI‐induced inflammation by increasing resolving leukocytes but also is renoprotective in nature in heart failure pathology.

Support or Funding Information

HL132989 and AT006704 to GVH
Muscle Ring Finger‐1 Knockout (MuRF1−/−) Mice are Resistant to LPS‐induced Cardiac Dysfunction Due to Decreased NF‐κB Activity
Authors: Parry Traci L. , Brown David I. , Mwiza Jean Marie , Willis Monte S.
Published: November 2023

Sepsis is associated with a high mortality (up to 40%) in hospitalized patients. Cardiac dysfunction plays a critical role in the outcome of severe sepsis, and sepsis‐targeted cardiac therapies are lacking. Fortunately, recent studies have identified the role of Toll‐like receptors, cytokines, and NF‐kappaB signaling in sepsis‐associated heart failure. We recently identified that the muscle‐specific ubiquitin ligase MuRF1 (Muscle Ring Finger1) inhibits peroxisome proliferator‐activated receptor alpha (PPARalpha) in cardiomyocytes by mediating its nuclear export in a ubiquitin‐dependent manner. We found that MuRF1 interacts with PPARalpha to mediate a multi‐monoubiquitination using three lysines surrounding a newly identified nuclear export sequence. Interestingly, MuRF1−/− mice have a five‐fold increase in cardiac PPARalpha activity. Since studies have shown that PPARalpha competitively inhibits NF‐kappaB and downstream inflammation, we hypothesized that MuRF1−/− mice would exhibit cardiomyocyte anti‐inflammatory responses through its competitive increase in PPARalpha activity. To test this, we performed echocardiography on MuRF1−/− and wildtype mice at baseline and 4, 24, 48, and 72 hours post‐lipopolysaccharide injection (2 mg/kg of LPS). LPS interacts with Toll‐like receptor 4 on cardiomyocytes and inflammatory cells, releasing cardiodepressant cytokines (e.g. TNFa, IL‐1, IL‐6) that activate NF‐kappaB. No mice died during the course of the 72 hour study and no cardiac differences were observed between groups at baseline. Four hours after LPS, wildtype mice exhibited significantly worse systolic function compared to MuRF1−/− mice (Fractional Shortening (FS)%=34.5+1.5 vs. 42.0+4.5). Wildtype LPS treated hearts exhibited significant wall thinning, while MuRF1−/− wall thickness did not change from baseline. MuRF1−/− LPS‐induced cardiac dysfunction was completely resolved by 72 hours (FS%=57.5+1.4), whereas wildtype hearts still exhibited residual dysfunction (FS%=48.4+1.9). Analysis by immunoblot (from hearts harvested 72 hours post LPS treatment) showed (active) P‐Ikappa Balpha was decreased in MuRF1−/− mice, indicating NF‐kB signaling was decreased in these mice. Similarly, inflammatory cytokine markers IL‐1alpha and IL‐1beta were decreased in MuRF1−/− mice, indicating MuRF1−/− mice had less inflammation. These data are in step with our hypothesis, demonstrating that MuRF1's regulation of PPARalpha may affect the heart's ability to respond to NF‐kappaB (due to enhanced nuclear PPARalpha activity inhibiting NF‐kappaB signaling). Therapeutic inhibition of the MuRF1‐PPARalpha interaction provides a muscle‐specific therapy that may prove beneficial in NF‐kappaB‐mediated cardiac dysfunction seen in sepsis, heart failure, and myocardial infarction.
Full‐thickness Engineered Skin Tissue with 3D Biomimetic Micro‐topography
Authors: Yu Jonelle , Korkmaz Emrullah , LeDuc Philip , Ozdoganlar Burak
Published: November 2023

Many native tissues possess three‐dimensional (3D) micro‐topographies that have evolved to be more biologically functional. As one of the functional features, dermal papillae, 3D undulated interface between epidermis and dermis in skin is centered on increased interface area, resulting in, for instance, reinforced mechanical contact, enhanced thermal conductivity, and improved biomolecular transportation. To reserve the micro‐topographies in engineered skin tissues would bring tremendous advances to patient‐specific skin transplantation (for higher graft take rate and shorter recovery time) and organ‐on‐a‐chip development (for more accurate disease models and drug screening). To accomplish this, various approaches have been developed in academic researches. However, those approaches only achieved the topographies as 2D rectangular channels, 2D sine wave channels, or 3D taper pillars, which have not mimicked precisely the natural topographies of dermal papillae.

Here we developed 3D undulated topographies at multiple micro‐scales on hydrogel spongy scaffolds for mimicking dermal papillae by using coupled 3‐axis micro‐milling and micro‐molding approaches. We micro‐milled master molds with 3D sine wave matrix, where the structure possesses continuous curvature in all directions, to mimick the undulated topographies in native skin. We afterwards accomplished hydrogel porous scaffolds with the 3D undulated surface, which was transferred from master molds by micro‐molding and lyophilization. We finally examined cell‐scaffold interfacial response by culturing fibroblasts on the undulated surfaces. As a result, the hydrogel porous scaffolds replicated the precise master molds without obvious distortion or shrinkage. The cells grew and migrated along the micro‐patterned scaffolds over 7 days with very few dead cells. Our biological engineering approach applied to tissue scaffolds paves the way to fabricate more 3D structurally realistic artificial tissues in areas such as organ repair and organ‐on‐a‐chip applications, which are applicable to biological functions such as cell‐cell interactions and cell signaling.
Bisphenol‐A and 17‐β Estradiol Alter Colon Microbial Metabolites and Exacerbate Dextran Sodium Sulfate‐Induced Colitis in Mice
Authors: DeLuca Jennifer A.A. , Menon Rani , Riordan Rebekah , Allred Kimberly F. , Jayaraman Arul , Allred Clinton D.
Published: November 2023

Inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis (UC), differ in their responses to estrogen status in female patients. Previous studies investigating the role of 17‐β estradiol (E2) in animal models of both CD and UC have shown anti‐inflammatory or pro‐inflammatory responses, respectively. E2 has been reported to worsen disease activity scores in the Dextran Sodium Sulfate (DSS)‐induced colitis model. Additionally, data from DSS models suggest changes in intestinal microbes play a role in inflammation initiation and exacerbation of colitis. Bisphenol‐A (BPA) has been shown to exert estrogenic activity in the colon and alter intestinal function at relatively low doses, but the role that xenoestrogens such as BPA play in colonic inflammation is poorly described. We investigated the ability of E2 and BPA to exacerbate colonic inflammation and alter microbial metabolites in an acute DSS‐induced colitis model in vivo. Treatment with DSS and E2 resulted in 100% mortality, while DSS and BPA co‐treatment resulted in 67% mortality. Body weight decreased significantly in E2 mice, indicating worsened disease severity. BPA treatment also resulted in reduced body weight. Rectal bleeding and fecal consistency scores were most severe in E2 treated mice, but BPA treatment also worsened these scores. DSS treatment significantly decreased colon length and increased colon inflammation score. Inflammation score was worsened in BPA treated animals in the presence and absence of DSS. Alterations in the levels of tryptophan‐derived metabolites were determined using LC‐MS metabolomics and identified unique patterns of metabolites associated with BPA exposure. To further assess the effects of BPA on non‐transformed colonocytes, cell number, cell cycle, and apoptosis were measured in vitro in young adult mouse colonocytes (YAMCs). BPA at concentrations of 1 μM and 10 nM reduced cell number similarly to 1 nM E2. Apoptosis was increased in E2 treated cells, but not in BPA treated cells. Collectively, these data suggest BPA can alter cellular function of nonmalignant colonocytes and exacerbate inflammation in vivo which may be associated with changes in microbiota‐derived metabolites.

Support or Funding Information

Funding was provided by the Center for Translational Environmental Health Research through National Institute of Environmental Health Sciences grant number P30ES023512.
Genetic Variations at 15 Forensically Relevant Microsatellite Loci (STRs) in the Three Major Ethno‐Linguistic Population Groups in Nigeria
Authors: Agbo Benedict Usman , Ebuehi Osaretin Albert Taiwo , de Osuntoki Akinniyi A
Published: November 2023

This study is to identify genetic markers that will show distinct genetic variability among the three major ethno‐linguistic population groups in Nigeria that can help establish ethnic origin of forensic evidential materials. Samples from 315 unrelated individuals representing the three populations; ‘Hausa’, ‘Ibo’ and ‘Yoruba’ were analyzed using 15 Microsatellite loci (STRs) from Applied Biosystems. Analyses of inter‐ and intra‐population diversity were carried out using average heterozygosity, allele range (base pair), most frequent allele per a locus, Fixation index (Fst), Forensic and Paternity parameters. Multi‐locus genotype frequencies were utilized for testing conformity with Hardy‐Weinberg equilibrium (HW). 7 loci was found to conform to Hardy Weinberg equilibrium after X2 analysis. However, Bonferoni corrections show all loci to be in HW equilibrium, equally, exact test showed all loci to be in conformity with HW. Forensic and paternity parameters across the loci showed adequate discriminatory power for forensic identification of individuals as well as sufficient exclusionary power for parentage analysis. Average heterozygosity Fixation Index and Nei's standard genetic distance gave the least distance between the Ibo and Yoruba ethic groups. Allele frequency distribution comparisons at all loci analyzed show substantially no significant difference at 5% level of confidence between the Nigerian population and the FBI African American population allele frequencies. Ethnically blinded test from twenty samples among the three ethnic groups show a high degree of certainty in ethnic origin inferences from unknown samples. The study show Genetic variations between the three populations with marked phylogenetic relationships.

Support or Funding Information

University of Lagos, Lagos Nigeria
Residues of Escherichia coli thioredoxin critical for interaction with phage T7 DNA polymerase to increase processivity
Authors: Lee Seung‐Joo , Tran Ngoc , Richardson Charles C
Published: November 2023

Bacteriophage T7 uses the thioredoxin of its host, Escherichia coli, to greatly enhance processivity of its DNA polymerase. The structure and amino acid sequence of thioredoxins of all organisms are highly conserved. Therefore, thioredoxins from other organisms may interact with T7 DNA polymerase to support phage growth. This hypothesis was examined using human thioredoxin whose x‐ray crystallographic structure overlaps completely with that of the Escherichia coli protein. However, human thioredoxin cannot support T7 phage growth. It does not form a complex with T7 DNA polymerase as determined by isothermal calorimetry and surface plasmon resonance, and thus does not stimulate the polymerase activity. Homologous scanning analysis using this non‐functional homolog reveals that the N‐terminal 20 and the C‐terminal 12 amino acid residues of Escherichia coli thioredoxin can be substituted for its human counterpart without significantly losing ability to support the phage growth. Further investigation using site‐directed mutagenesis identifies leucine‐95 as a critical element that may contribute to hydrophobic interaction with the thioredoxin‐binding loop of the polymerase. The results suggest that specific interactions at the junction of thioredoxin and the DNA polymerase rather than the overall structure are important to enhance the processivity of T7 DNA polymerase.
Dynamics of the E. coli beta clamp and its influence on DNA loading
Authors: Koleva Bilyana , Baez Joe , Conway Jaime , Wu Amy , Beuning Penny
Published: November 2023

Sliding clamps are found in all domains of life and are essential for efficient DNA replication, which is required every time a cell divides, as well as for coordinating traffic on DNA. The Escherichia coli (E. coli) beta‐clamp is a ring‐shaped protein consisting of two identical monomers encoded by the dnaN gene that associates with DNA polymerase III and facilitates the high degree of processivity required for DNA replication. Sliding clamp proteins open and are loaded onto specific DNA structures by clamp loaders in the presence of ATP. We aim to gain insights into the contributions of different domains to the function of beta using a linked‐beta‐clamp that constrains the homodimer at one of two interfaces. Usually the homo‐dimeric structure of the beta‐clamp allows it to act as a molecular tool belt, binding more than one protein simultaneously at specific protein interaction sites on the clamp. Thus, a better understanding of the sliding clamps' role in the process of DNA damage tolerance will be gathered by investigating whether one interface or binding site is adequate for DNA loading or for other protein interactions. To create this construct, the linker length and sequence, as well as expression conditions, were optimized. The linked‐beta proteins were characterized through a thermal denaturation assay, and showed comparable thermal stability to wild‐type beta. In an ATPase assay, similar amounts of inorganic phosphate were detected in reactions containing either linked‐beta or wild‐type beta, indicating that the clamp loader can interact with both versions of the beta clamp. Currently, we are investigating whether linked and wild‐type beta improve the processivity of a polymerase in a primer extension assay. We are now constructing and characterizing variants of wild‐type beta and linked‐beta to test the importance of residues at only one of the interfaces or partner protein binding sites.
Chromatin accessibility of the dosage compensated Drosophila male X‐chromosome is established by a context‐specific role for the CLAMP zinc finger protein
Authors: Larschan Erica , Urban Jennifer , Kuzu Guray
Published: November 2023

Most transcription factors (TFs) bind to thousands of sites throughout the genome to differentially regulate subsets of target genes. However, it is often difficult to study how the same TF mediates different modes of transcriptional control because it is not known how these context‐specific functions are differentiated. Identification of the Drosophila CLAMP zinc finger protein provides a new opportunity to understand how the same TF can function in different contexts because it has at least two distinct functions: 1) CLAMP specifically targets MSL complex‐dependent dosage compensation to the male X‐chromosome; 2) CLAMP binds to the promoters of thousands of genes on all chromosomes and is essential for the viability of both males and females. To define the mechanisms by which CLAMP performs its context‐specific functions, we have precisely measured chromatin accessibility, performed nascent RNA sequencing, and proteomics. We determined that CLAMP establishes the open chromatin environment across the entire dosage compensated X‐chromosome, not the MSL complex as was previously hypothesized. Moreover, CLAMP recruits the ISWI chromatin remodeler to several key sites on the X‐chromosome that are critical for MSL complex recruitment. At promoters, differential occupancy of CLAMP and the similar GA‐binding GAGA factor (GAF) modulates gene expression patterns. Overall, we have provided new insights into how a single transcription factor can function in context‐specific ways to promote differential gene expression.
Role of the ELL complex in transcriptional regulation in S. pombe
Authors: Gopalan Sneha , Gibbon Dana , Seidel Chris , Zhang Ying , Florens Laurence , Washburn Michael , Conaway Joan , Conaway Ronald
Published: November 2023

The ELL family of transcription elongation factors can suppress the transient pausing of Pol II at each step of nucleotide addition during transcription, thereby stimulating the overall rate of transcription. ELL‐associated factors (EAFs) bind directly to ELL and are positive regulators of ELL elongation activity. ELL and EAF have been implicated in the release of RNA pol II from promoter proximal pausing by an unknown mechanism. Orthologs of ELL and EAF were not identified in fungi until recently. Previous data from our lab identified genes in S. pombe similar to ELL and EAF, which have no obvious orthologs in S. cerevisiae. SpELL and SpEAF also interact with each other to form a heterodimer and can positively regulate transcription by RNA pol II. We identified proteins interacting with SpELL and SpEAF by immunoprecipitation followed by Multidimensional Protein Identification Technology (MudPIT). Proteomic analyses revealed an uncharacterized sequence orphan protein that associates strongly with both ELL and EAF. Biochemical analysis using recombinant proteins confirmed that this protein binds directly to ELL and forms a ternary complex with ELL and EAF. ChIP‐seq experiments suggest that all three proteins co‐localize at genes with high RNA pol II occupancy. Synthetic Genetic Array (SGA), a high‐throughput technique for exploring genetic interactions, was performed on the deletion mutants lacking the genes encoding ELL, EAF and the novel ELL‐interacting protein. Preliminary data analysis suggests that these genes may interact genetically with overlapping set of genes.

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Characterization of the SUN Domain Protein Mps3 Function in Gene Expression
Authors: Antoniacci Lisa , Lukasak Rebecca
Published: November 2023

The yeast nuclear envelope protein Mps3 functions in several aspects of chromosome metabolism such as sister chromatid cohesion, telomere clustering, and DNA damage repair. In addition, Mps3 physically and functionally interacts with the histone variant Htz1. Htz1 was previously identified as a histone variant that prevents DNA from forming silent heterochromatin as a way to regulate transcription. Because of the involvement of both of these genes in chromosome metabolism, it is hypothesized that they may function in regulating gene expression. In order to assess genome expression wild type yeast, mps3‐3, mps3‐5, and an htz1Δ strain were examined for genome wide expression levels in both asynchronously and synchronized growing cells. The data generated was analyzed for both up‐regulation and down‐regulation of gene expression in comparison to the wild type strain. Identification of expression differences between the wild type and mutant strains would suggest a function for Mps3 and Htz1 in gene expression.

Support or Funding Information

Shierling Endowed Chair Marywood University
Fine‐tuning of FACT by the ubiquitin proteasome system in regulation of transcriptional elongation
Authors: Bhaumik Sukesh R. , Sen Rwik , Ferdoush Jannatul , Kaja Amala
Published: November 2023

FACT (facilitates chromatin transcription), an evolutionarily conserved histone chaperone involved in transcription and other DNA transactions, is upregulated in cancers, and its downregulation is associated with cellular death. However, it is not clearly understood how FACT is fine‐tuned for normal cellular functions. We find that the FACT subunit Spt16 is ubiquitylated by San1 (an E3 ubiquitin ligase) and degraded by the 26S proteasome. Enhanced abundance of Spt16 in the absence of San1 impairs transcriptional elongation. Likewise, decreased abundance of Spt16 also reduces transcription. Thus, an optimal level of Spt16 is required for efficient transcriptional elongation, which is maintained by San1 via ubiquitylation and proteasomal degradation. Consistently, San1 associates with the coding sequences of active genes to regulate Spt16's abundance. Further, we find that enhanced abundance of Spt16 in the absence of San1 impairs chromatin reassembly at the coding sequence, similarly to the results seen following inactivation of Spt16. Efficient chromatin reassembly enhances the fidelity of transcriptional elongation. Taken together, our results demonstrate for the first time a fine‐tuning of FACT by an ubiquitin proteasome system in promoting chromatin reassembly in the wake of elongating RNA polymerase II and transcriptional elongation, thus revealing novel regulatory mechanisms of gene expression.

Support or Funding Information

National Institutes of Health (2R15GM088798‐02) and American Heart Association (15GRNT25700298).
L3MBTL2 is Required for Spermatogenesis and the Maintenance of Male Fertility
Authors: Meng Chenling
Published: November 2023

L3MBTL2 is a member of the MBT‐domain proteins, which are involved in transcriptional repression and implicated in chromatin compaction. L3MBTL2 is found to regulate cell proliferation and differentiation in mouse embryonic stem cells and early embryonic development. Our previous study has shown that L3mbtl2 is highly expressed in the testis, but its role in spermatogenesis remains unclear. In the present study, we show that L3MBTL2 is highly expressed in pachytene spermatocytes. Germ cell‐specific knockout of L3mbtl2 in the testis led to mice with smaller testes, increased abnormal spermatozoa and reduced sperm counts, and the mice ceased to produce progeny at a much earlier age. Mechanistically, L3mbtl2 deficiency resulted in inappropriate retention of γH2AX on autosomes, and defective crossing‐over and synapsis during the pachytene stage of meiosis I, and more germ cell apoptosis and degeneration. Acetylation of H4, H2B and H3 in germ cells, and the subsequent protamine 1 deposition and chromatin condensation in sperm were reduced in L3mbtl2 deficient mice as compared with wild type mice. These results suggest that L3mbtl2 plays important roles in meiosis and germ cell survival, and in chromatin remodeling during spermiogenesis.
Role of chromatin remodeling and spacing factor 1 in histone H2A ubiquitination mediated gene silencing
Authors: Wang Hengbin , Zhang Zhuo , Jones Amanda E , Renfrow Matthew B , Liu Chuan Fa , An Woojin , Luo Jianjun , Wu Wei , Kang Yue , Tong Yufeng
Published: November 2023

Posttranslational modification of histones plays important regulatory roles in chromatin‐based nuclear processes. Histone H2A ubiquitination (H2Aub) is a predominate modification and has been primarily linked to gene silencing; however, the mechanism by which H2Aub achieves the gene silencing function remains unclear. Here we report the identification of RSF1 (remodeling and spacing factor 1), a subunit of the RSF (remodeling and spacing factor) complex, as a novel H2Aub binding protein which mediates the repressive function of H2Aub on gene expression. RSF1 preferentially associates with H2Aub‐enriched nucleosomes through a previously uncharacterized region designated as the ubiquitinated H2A binding (UAB) motif. RSF1 specifically interacts with H2Aub nucleosomes and the UAB motif is required for RSF1 to interact with H2Aub nucleosomes in vitro and in vivo. Genes regulated by RSF1 overlap significantly with genes regulated by RNF2 or Ring1B, the catalytic subunit of Polycomb repressive complex 1 (PRC1), in both human and mouse cells. Strikingly, virtually all overlapped genes show changes in the same direction. RSF1 binds to gene promoter regions, and over 85% of H2Aub enriched genes, including the classic PRC1‐target Hox genes, are co‐bound by RSF1. Reduction H2Aub levels by Ring1B knockout resulted in a significant reduction of RSF1 binding. RSF1 knockout did not affect RNF2/Ring1B or H2Aub levels but resulted in re‐expression of H2Aub target genes, accompanied by a reduction in the spacing and stability of H2Aub‐containing nucleosomes. Finally, the direct role of RSF1 in H2Aub‐mediated gene silencing was demonstrated by chromatin based in vitro transcription assay. Therefore, this study identifies RSF1 as a novel H2Aub binding protein and reveals that H2Aub represses gene expression by maintaining a regular spaced, stable nucleosome array at promoter regions through RSF1.

Support or Funding Information

Hengbin Wang is a Leukemia and Lymphoma Scholar.
Determination of Altered Bromodomain Function of Cancer‐Associated Missense Mutations in Polybromo‐1
Authors: Goetz Christopher , Dykhuizen Emily , Smith Brian
Published: November 2023

Bromodomains are chromatin interaction modules of approximately 110 amino acids that bind acetyl‐lysine residues on histones and other nuclear proteins. Bromodomains target transcriptional proteins and their associated protein complexes to specific gene loci on chromatin, and thus influence transcriptional activity of selected genes. Humans encode 61 bromodomains distributed among 46 bromodomain‐containing proteins, one of these proteins, Polybromo‐1 (PBRM1), contains six tandem bromodomains and is a subunit of the Polybromo‐1‐BRG‐Associated‐Factors (PBAF) chromatin remodeling complex. Missense mutations in PBRM1 cluster in the bromodomains and are present in many cancers. In particular, PBRM1 is mutated in approximately 40% of the sequenced cases of Renal Clear Cell Carcinoma, the most aggressive and lethal subtype of renal cell carcinomas. However, the functional consequences of these missense PBRM1 bromodomain mutations was unknown. Here, we compiled 178 missense mutations located within the PBRM1 bromodomains through analysis of cancer databases including Cancer Driver Mutation, COSMIC, and IntOgen. To determine the consequences these missense mutations have on PBRM1 bromodomain function, we developed a high‐throughput ThermoFluor assay using an acetyl‐lysine mimetic, 1‐methyl‐2‐quinolinone, which mimics the acetyl‐lysine: bromodomain interaction through formation of a hydrogen bond between its N‐methyl amide moiety and a conserved asparagine residue found in the acetyl‐lysine binding pocket of bromodomains. The ThermoFluor assay simultaneously quantifies both bromodomain thermal stability and the ability of the bromodomains to bind acetyl‐lysine residues by measuring the shift in melting temperature of mutant and wild‐type bromodomains in the presence or absence of the acetyl‐lysine mimic ligand. The thermal shift data strongly correlates with dissociation constants measured by isothermal titration calorimetry (ITC), rendering the ThermoFluor assay a rapid and quantitative way to assess bromodomain function for a large number of mutations. Our preliminary data shows that cancer‐associated PBRM1 bromodomain mutants exhibit altered stability (to varying degrees) in the presence of the acetyl‐lysine mimetic in comparison with the wild‐type PBRM1 bromodomains. This research provides insights into function of the PBRM1 bromodomains under normal conditions and during cancer development and may lead to novel methods to selectively target missense mutations in PBRM1 as potential therapeutics.

Support or Funding Information

American Heart Association Scientist Development Grant 15SDG25830057 Medical College of Wisconsin Research Affairs Committee American Cancer Society Institutional Research Grants #14‐247‐29‐IRG and #86‐004‐26‐IRG Advancing a Healthier Wisconsin Endowment
Roles of PARP‐1‐dependent poly(ADP‐ribosyl)ation in regulation of the catalytic activity of human ALC1 chromatin remodeling enzyme
Authors: Ooi Soon‐Keat
Published: November 2023

ALC1 is a macrodomain‐containing SNF2‐family ATPase with apparent roles in DNA repair and transcription and has been implicated in the pathogenesis of various cancers. ALC1 displays robust catalytic activity only when its macrodomain interacts with the poly(ADP‐ribose) (PAR) on nucleosome‐bound PARP‐1. However, little is known about the mechanism by which PARylated PARP‐1 regulates ALC1 activity. We seek to define the features of PARP‐1 required for ALC1 activation. PARP‐1 is a multi‐domain enzyme that catalyzes most PAR synthesis in cells. PARP‐1 includes DNA‐binding zinc finger domains (Zn1, Zn2 and Zn3), BRCA1 C‐terminal domain (BRCT), tryptophan‐glycine‐arginine‐rich domain (WGR) and catalytic domain (CAT). As studies have found that Zn2 and BRCT domains are dispensable for PARP‐1's PARylation activity, we have tested PARP‐1 mutants lacking certain domains for their abilities to activate ALC1. Our results thus far show that Zn2 and BRCT domains of PARP‐1 are also dispensable for ALC1 nucleosome remodeling activity.