bluuebunny/crossref_metadata_2025 huggingface dataset:
Building ICABL: An Inclusive Community for the Assessment of Biochemistry and Molecular Biology Learning
Authors: Moore Victoria D. G. , Cortes Kimberly , Block Kirsten F. , Engelman Shelly , Kanipes Margaret , Kennelly Peter J. , Lim Yang M. , Loertscher Jennifer , Olafimihan Tejiri , Tyler Ludmila , Upchurch‐Poole Territa L. , Dries Daniel R.
Published: May 2022

Assessment is a critical feature of effective instruction. Beyond assigning a grade to student performance, regular assessment can provide instructors, departments, and institutions with ongoing feedback on instructor, course, and program effectiveness. Despite its importance, designing assessments that are accessible to all students and measure relevant learning outcomes can be challenging for many biochemistry and molecular biology (BMB) instructors because they receive little, if any, formal training in assessment practices. Consequently, we ‐ a team of writers for the ASBMB Certification Examination ‐ have launched ICABL, an inclusive community for the assessment of BMB learning. With funding from an NSF grant (Award No. 2018204), we piloted a virtual workshop on summative assessment in Summer 2020. The 28 participants discussed best practices in summative assessment, including Bloom’s taxonomy, backward design using the ASBMB Foundational Concepts and Skills, and rubrics development as tools for informing instructors on the effectiveness of instruction. An explicit objective of the workshop was to broaden representation in the ASBMB education community. Accordingly, 68% of participants taught at minority‐serving institutions, as compared with less than 10% of current ASBMB membership. Both immediate and six‐month follow‐up surveys highlighted the efficacy of this virtual workshop in supporting participants’ professional development. In addition, we have subsequently recruited many of the participants to write and score for the ASBMB Certification Exam as one step in our goal to develop an examination team that better represents and can more effectively address the diversity of individuals in our BMB classrooms. With continued support from the NSF (Award No. 2120673), over the next three years we will be offering a series of additional workshops, both in the United States and overseas, addressing summative, formative, and alternative assessment.
Control of the Cellular Lipid Landscape by Inositol Lipids
Authors: Balla Tamas , Mandal Amrita , Kim Yeun Ju , Pemberton Joshua
Published: May 2022

Cellular organization of eukaryotic cells relies upon compartmentalization of their signaling nodes on special membrane platforms forming the various organelles. Unique lipid composition of the different organelle membranes not only defines their identity but also is critical for the proper assembly and functioning of the protein signaling complexes associated with them. Inositol phospholipids (PPIns), a class of regulatory lipids, play a critical role in defining membrane identity and forming membrane microdomains with unique signaling properties. Recent developments in lipid membrane biology revealed that phosphatidylinositol (PI) 4‐phosphate (PI4P) gradients and the PI 4‐kinases that form them drive non‐vesicular transport of several structural lipids against their concentration gradients at membrane contact sites (MCSs). These processes clearly depend on the delivery of PI from its site of synthesis in the ER to the membranes where PI4Ks convert them to PI4P. Therefore, our recent efforts have been focused on the question of how PI synthesis and PI transport systems provide the means of proper PI delivery to their other membrane destinations. In this presentation we will review our recent data on the generation and use of molecular tools to visualize and manipulate PI metabolism and delivery and demonstrate, how they can help us better understand the central role of PPIns in defining the overall lipid landscape of eukaryotic cells.
New Technologies to Visualize Gene Expression with Single Molecule Resolution Live
Authors: Stasevich Timothy
Published: May 2022

My lab is creating technology to image and quantify gene activity in real time and with single‐molecule precision in living cells. Using new mRNA and protein tags, fluorescent probes, and single‐molecule microscopy, we image DNA transcription and mRNA translation live. In this talk, I will briefly introduce our technology and describe how it can be used to amplify fluorescence from newly synthesized proteins as they are being translated from single mRNAs. I will show how we quantify these signals to determine the size, shape, subcellular localization, and mobilities of mRNA translation sites, as well as their protein synthesis dynamics. I will then highlight some of our most recent advances, including (1) single‐molecule co‐imaging of transcription and translation of CRISPR‐edited endogenous genes, (2) optogenetic tools to tether mRNA during translation, (3) open‐source software to quantify single‐mRNA translation, and (4) a palette of genetically‐encoded fluorescent probes to light up translation in multiple colors with high fidelity.
Regulation of Stress Granule Dynamicity by Valosin‐Containing Protein
Authors: Moon Stephanie
Published: May 2022

Stress granules are dynamic biomolecular condensates of proteins and non‐translating RNAs that form when translation is inhibited during stress. Stress conditions associated with disease including ER dysfunction, toxic metalloids, and inflammatory factors induce stress granule formation. Stress granules disassemble when translation resumes during the recovery from stress. Aberrant, cytotoxic stress granules are implicated in degenerative diseases of the nervous, muscular and skeletal systems. Yet, the mechanisms underlying stress granule dynamicity are not well understood. A growing body of research suggests that the ubiquitin‐proteasome system regulates the formation and disassembly of stress granules. Valosin‐containing protein (VCP) is a homohexameric AAA+ ATPase that functions as a ubiquitin‐binding protein segregase in protein quality control and general protein degradation pathways in the ubiquitin‐proteasome system. Prior research demonstrated that VCP is an important mediator of stress granule disassembly. Our recent work suggests an additional role for VCP in stress granule assembly. By imaging single mRNA molecules in living and fixed human cells, we determined that VCP and other members of the ribosome‐associated quality control pathway (i.e. listerin, nuclear export mediator factor, and the proteasome) are critical for the release of specific mRNAs from translation complexes and localization to stress granules. Overexpression of VCP alleles associated with amyotrophic lateral sclerosis and frontotemporal dementia caused increased mRNA localization to stress granules. Thus, understanding the molecular mechanisms by which VCP regulates stress granules will be important for discovering its role in disease. This research will contribute to our knowledge of how defects in stress granules and the ubiquitin‐proteasome system drive human degenerative diseases and suggest potential diagnostic and therapeutic strategies.
It's all about the students
Authors: Provost Joseph
Published: May 2022

The best moments of an instructor’s day are the times in class when everything clicks, students are sharing and are excited to be in class learning. The best time in a research mentor’s day is when a student gets a concept or designs an interesting experiment ‐ or better yet, when a student who has struggled generates exciting data and realizes they now know something that no one else in the world knows. The best time in an advisor’s day is helping a struggling student work past their fears and achieve more than they thought they could. How do we faculty get to that point where it is all about the students? Taking a scholarly approach to teaching and learning how to be a good mentor is just as important as the background research we do to write a grant and run a research laboratory. We get to be about the students by being part of larger communities like ASBMB, PKAL, CUR, daring to learn new pedagogies, studying the teaching literature – learning to be as competent in front of the students as we are with a pipette. Then when possible, we give back by creating new opportunities and mentoring other faculty. Working with the students and participating in the poster competition where it is as much a social connection as it is helping the students with their research. The community of faculty sharing our secrets, helping each other be better teachers, researchers and mentors, is what gives all of us the strength to be there for the students. I am honored for this award and hope to share some of how I’ve grown with the support of our faculty community and have given back to others – because it’s all about the students.
Snapshots of lipid synthesis and fat storage
Authors: Airola Michael
Published: May 2022

During the past thirty years, the perceived role of lipids has shifted from simple structural components of cell membranes to bioactive molecules that regulate critical cellular and pathological processes. The enzymes that generate and breakdown these bioactive lipids have emerged as novel therapeutic targets for treating the leading causes of diseases in the United States, including cancer, heart disease, and diabetes. This talk will present new insight into how key enzymes in triglyceride and phospholipid metabolism work at the molecular and structural level.
Amputation–From the Routine to the Rotationplasty
Authors: Miller Benjamin
Published: May 2022

Historically, extremity amputation was often the only surgical option for traumatic, infectious, or oncologic conditions. Despite evolution of surgical techniques and reconstructive options leading to successful limb sparing operations, amputation remains a viable and durable intervention for many conditions. The most common amputations are performed in the lower extremity and are due to complications of diabetes, peripheral vascular disease, or trauma. General principles and goals of amputations are to remove the pathology, allow for healing, maintain as much length as possible, and provide a stable base on which to weight bear. The barriers to successful outcomes include phantom pain, prosthetic cost, and short residual limbs resulting in difficulty with prosthetic wear.

Musculoskeletal oncology is a specialty that affects all ages and anatomic locations. For malignancy of the pelvis and extremities that require an ablative procedure to attain oncologic control, there can be a variety of considerations and unusual situations resulting in difficulties in rehabilitation and functional restoration. A particularly challenging scenario is a malignancy presenting in the lower extremity of a growing child; in this case, a rotationplasty can provide superior function and durability.

There have been advancements for amputees in the form of more sophisticated prostheses and rehabilitation protocols. In addition, there has been increased enthusiasm for surgical techniques reducing phantom pain or allowing for use of a myoelectric prosthesis (targeted muscle re‐innervation) and osseointegrative implants that allow for improvements in prosthetic use.
A Research‐based Approach to Pedagogy in xR ‐ What’s the Evidence?
Authors: Wainman Bruce
Published: May 2022

Three‐dimensional visualization technology such as virtual reality and other forms of extended reality (xR) are experiencing an exponential growth in interest and development. The application of xR for comprehending spatially complex, interconnected objects inherent in anatomical education is a particularly promising application of the technology. By adopting xR, educational institutions may be able address more than the challenges of pedagogy but also issues related to the resource intensiveness of acquiring, processing, and teaching with cadavers. However, there is a very long and checkered history of the use technology to augment anatomy learning. This talk will address the 150‐year history of the successes and failures of xR in anatomy education, the significant technological, physical and cognitive challenges that need to be met with the current technology and a peek at a hopeful future where those challenges are met, and xR achieves its considerable promise.
Distant‐acting Enhancers in Development and Disease
Authors: Visel Axel
Published: May 2022

Multiple converging lines of evidence from experimental and human genetic studies indicate that both common and rare sequence variants involving enhancers play major roles in Mendelian and complex human disease phenotypes. We use a combination of sequence‐based molecular approaches including chromatin mapping and synthesis‐enabled modification of enhancer sequences, large‐scale transgenic mouse studies (http://enhancer.lbl.gov), and CRISPR genome editing in the mouse model to study the in vivofunction of enhancers in developmental, evolutionary, and disease‐related processes. Using examples from our ongoing work, I will illustrate how these methods provide insight into the function and evolution of distant‐acting regulatory sequences and offer a starting point for understanding their role in human disease. I will discuss how improved in vivo mouse assays can be used to study candidate pathogenic human mutations in enhancers at scale, revealing the extent to which human genetic studies alone can be insufficient to identify truly causal mutations in enhancers. I will also show how such assays can be used to explore the sensitivity of enhancers that are extremely conserved (“ultraconserved”) in evolution to mutations, revealing a remarkable functional robustness to sequence changes that contrasts with apparent evolutionary constraints.
mRNA Modification: At the Heart of Stress Adaptation
Authors: Accornero Federica
Published: May 2022

Regulation of mRNA stability and translation is increasingly recognized as a critical control point in dynamically altering gene expression during stress or disease. In the heart, post‐transcriptional alteration of the level of select mRNAs has emerged as a driver of disease. Cardiac inflammation, hypertrophy, metabolism and electrophysiology are all aspects that are perturbed in pathophysiologic conditions and undergo regulation by RNA‐binding proteins. My talk will focus on novel molecular mechanisms responsible for controlling cardiac mRNA fate in heart disease.
Regulation of the Translatome in Cardiac Remodeling
Authors: Wang Yibin , Wang He (Iris) , Wang Frank YiFan
Published: May 2022

Gene expression is determined by both transcriptional as well as multiple layers of post‐transcriptional controls, including protein translation. One of the molecular hallmark observed in diseased hearts is augmented protein synthesis and marked reprogramming of translatome (defined as ribosomal loaded mRNAs). Furthermore, translatome reprogramming is often independent from the concurrent total transcriptome changes. However, the underlying dynamics and regulatory network remain largely unexplored. In this study, we observed a robust induction of protein synthesis associated with a rapid change of ribosome loaded mRNAs in response to hypertrophic stimulation in cultured myocytes and intact mouse hearts, associated with dynamic changes in nucleoli organization. This early onset of translatome reprogramming and subsequent protein synthesis induction requires the induction of a long non‐coding RNA (lncRNA) named Myocardial Infarction Associated Transcript (Miat). Molecular characterization reveals that Miat is specifically localized in nuclei and interacts with Nucleolin (NCL). Miat expression is essential for NCL mediated ribosome biogenesis and function. In Miat KO mouse hearts or cultured myocytes, pathological hypertrophy is significantly blunted in vitro and in vivo, along with blocked rRNA biogenesis and processing, and protein synthesis. These findings offer the first evidence that ribosome biogenesis and translatome reprogramming is an essential early molecular process of pathological hypertrophy in heart. Miat‐NCL mediated translatome reprogramming induced by pathological stimulation is a novel regulatory corner stone and post‐transcriptional check‐point for pathological hypertrophy and heart failure.
Effective and Affirming Communication with Transgender and Gender Nonconforming Patients
Authors: Cox Ryan
Published: May 2022

Bias is an important but often misunderstood factor in our learning. Too often, when faced with a novel or uncomfortable situation or set of facts, we fall back on what feels comfortable or familiar to us or adopt a stance of “neutrality.” In this instance, neutrality indicates deference to the status quo and an assumption of heteronormative and cisgender identities. Unfortunately, in medical education, this stance can effectively erase or make invisible those patients that do have unique needs, and for whom an awareness of minority identities can make the difference between affirming and competent care, or merely adequate care, or, at worst, stigmatizing care. In this roundtable discussion, we will explore methods by which clinicians and practitioners can utilize more inclusive, non‐stigmatizing, and exploratory language to create more effective, affirming, and inclusive learning environments. Critical to this practice is the knowledge and exploration of our biases and how they affect our assumptions and interactions, and what we can do to help moderate their potential influence, both in education and in practice.
Dynamics of Osteocyte Embedding and Differentiation and the Fate of Osteocytes after Bone Resorption
Authors: Dallas Sarah
Published: May 2022

The differentiation of an osteoblast into an osteocyte is a complex process involving arrest of cell motility, embedding into osteoid matrix, transformation to a dendritic morphology, interconnection of the dendrites with established osteocytes and entrapment of the new osteocyte within a mineralized matrix, all of which are accompanied by characteristic gene expression changes. To gain new insight into these processes, our laboratory has performed live‐cell and intravital imaging using transgenic reporter mice with fluorescently tagged osteocytes, osteoblasts and/or osteoclasts and GFP‐tagged collagen. This work has revealed the highly dynamic nature of the osteocyte embedding and differentiation process, it’s integration with ECM assembly and mineralization and has identified three apparent mechanisms for osteocyte embedding in collagen. Dual imaging of osteoclast resorption and osteocyte dynamics revealed that the predominant fate of osteocytes following bone resorption is to undergo cell death. These live imaging studies are providing novel insights into the properties, differentiation and function of osteocytes and the fate of osteocytes during bone resorption and enable quantitation of these cell dynamics in their natural bone environment in healthy and diseased states.
Heterotopic ossification ‐ replacement of one tissue with another
Authors: Shore Eileen M.
Published: May 2022

Our body is composed of multiple organ systems that use specific types of cells for specialized functions. Bone and cartilage cells are normally restricted to the skeleton, however in heterotopic ossification, osteogenesis and chondrogenesis occurs within tissues such as skeletal muscle, tendons, and ligaments, and leads to the formation of bone tissue outside of the skeleton. Heterotopic ossification is most commonly associated with severe trauma such as following hip arthroplasty, traumatic brain and spinal cord injury, and high impact blast injury. It is difficult to predict who will respond to these traumas by forming heterotopic bone, and there are currently no effective preventions or treatments. However, genetic forms of heterotopic ossification are providing new insights into the cellular and molecular mechanisms that control postnatal de novo formation of bone. Our investigations of a rare genetic disorder of heterotopic ossification, fibrodysplasia ossificans progressive (FOP), have not only identified key cell signaling pathways that regulate bone and cartilage cell differentiation, but also revealed the importance of the tissue microenvironment in supporting and directing aberrant cell differentiation. Our data support that heterotopic ossification involves more than mis‐directed cell differentiation, but is a multi‐step, multi‐cell type process leading to new tissue formation.
Extracellular Matrix and Skeletal Muscle Development, Homeostasis, and Disease
Authors: Henry Clarissa
Published: May 2022

Aberrant cell‐matrix interactions cause or exacerbate many musculoskeletal disorders such as myopathies, tendonitis, traumatic muscle injuries, arthritis, and sarcopenia. My lab uses the zebrafish model and primarily focuses on how signaling between muscle cells and their extracellular matrix mediates musculoskeletal development and homeostasis. I will talk about recent data regarding the impact of NAD+ signaling, proteostasis, and neuromuscular electrical stimulation on muscle cell adhesion to the matrix in the context of muscular dystrophy and dystroglycanopathies.
The Role of Annexin A6 in Triple‐negative breast cancer metabolism and disease progression
Authors: Williams Stephen D. , Sakwe Amos M.
Published: May 2022

The ability of cancer cells to alter their metabolism is one of the major mechanisms underlying therapeutic resistance in solid tumors, including the hard‐to‐treat triple‐negative breast cancer (TNBC) subtype. Here, we report the synergism between Annexin A6 (AnxA6) and Lapatinib‐resistance (Lap‐R) in the context of metabolic reprogramming. Metabolic profiling of TNBC cell lines show extensive diversity amongst the proliferative basal‐like (BSL) vs the highly motile mesenchymal‐like (MSL) molecular subtypes. In this study, we demonstrate that down regulation of AnxA6 in AnxA6‐, expressing and Lap‐R cell lines attenuated mitochondrial respiration, glycolytic flux, and cellular ATP production capacity, diminishing the overall energy phenotype of the cell. Additionally, AnxA6‐depletion altered lipid metabolism by decreasing lipid droplet accumulation, but enhancing the mitochondrial uptake and degradation of free fatty acids for ATP production via fatty acid oxidation (FAO). Our NMR‐based metabolomics revealed that AnxA6 depleted and Lap‐R TNBC cells rely on active amino acid metabolites and gluconeogenic precursors including alanine, glycine, and lactic acid to sustain their high proliferative rate. Collectively, these results demonstrate that the expression of AnxA6 underlies TNBC metabolic dependency and hence provide novel insights into the failure of EGFR‐targeted therapies as therapeutic options for TNBC patients.
Easy NanoString Gene Expression Analysis with the NanoTube
Authors: Class Caleb A. , Bristow Christopher A. , Do Kim‐Anh
Published: May 2022

The NanoString nCounter analysis system has become a popular platform for the quantification of RNA expression, and numerous statistical and bioinformatic methods have been developed to analyze the resulting data. However, many of the current methods and tools are unavailable to those without expert knowledge in computer programming. We have developed the NanoTube R package to make this analysis easier for anyone with basic R skills, as well as a simple web application (developed using R‐Shiny) for those with no programming skills. This includes all of the steps required in nCounter data analysis, from data processing and quality control to differential expression, gene set analysis, and data visualization. In addition to manufacturer‐recommended data analysis steps, functions for interoperability with other R packages for NanoString data are provided for those who prefer different analysis methods. And because this library is open‐source, it can be expanded by others with additional analysis and visualization needs.

We demonstrate the software by applying it to a publically available data set comparing the gene expression of patients with autoimmune retinopathy against healthy controls (GEO data series GSE117751). In addition to confirming the enrichment of multiple immune pathways identified in the previous analysis of the data, the EGFR signaling pathway is identified as a potential pathway of interest in autoimmune retinopathy patients. Use of this software will help researchers understand their gene expression data more quickly, and can also allow for the rapid reanalysis of public NanoString data sets to uncover new biological discoveries. The NanoTube R package and user’s guide are available on the Bioconductor software repository (https://www.bioconductor.org/packages/devel/bioc/html/NanoTube.html), while the beta version of the web application can be downloaded from GitHub (https://github.com/calebclass/Shiny‐NanoTube).
Changes in Cardiac and Vascular Contractile Protein in a Rat Model of Heart Failure with Preserved Ejection Fraction
Authors: Brozovich Frank V. , Han Young Soo , Arteaga Grace M. , Sharain Korosh , Sieck Gary C.
Published: May 2022

Heart failure is classified as heart failure with reduced ejection fraction (HFrEF) or heart failure with preserved ejection fraction (HFpEF). Although several therapies have been demonstrated to improve outcomes in patients with HFrEF, none benefit patients with HFpEF. The molecular mechanism that results in HFpEF is unknown and has been impacted by the lack of animal models, and thus, we tested the hypothesis that metabolic and hypertensive stress would result in a rat model of HFpEF. Fischer 344 rats (6mo old) were fed a high fat diet and L‐NAME in the drinking water for 4‐weeks. Invasive and noninvasive hemodynamics demonstrate that the treated animals develop HFpEF; treated animals (n=7) vs controls (n=6) have normal systolic function with LV hypertrophy (20.7±0.4mm v 27.6±2.1mm, p<0.05) and diastolic abnormalities including a decrease in both E (79.4±3.6cm/s v 64.2±3.4cm/s, p=0.003) and dP/dtmin (‐7600±1100mmHg/s v ‐5600±1400mmHg/s, p=0.015), a steeper EDPVR (0.1±1.1mmHg/μl v 3.6±1.7mmHg/μl, p=0.042) and a slower time constant of relaxation (τ; 11±1ms v 16±2ms, p=0.029), as well as edema of the liver and lungs. Compared to controls, in cardiac muscle of HFpEF rats, there is no difference (p>0.05) in the expression and/or phosphorylation of TnI or MyBPC. However, the expression of SERCA2 (p=0.021) and phosphorylation of phospholamban (p=0.046) are reduced, which would be expected to decrease the rate of SR Ca2+ reuptake and slow relaxation. In both the aorta and tertiary mesenteric vessels of HFpEF rats, the expression of nonmuscle myosin was significantly higher and the expression of the leucine zipper positive isoform of myosin light chain phosphatase was reduced, which should increase vascular tone and decrease the sensitivity of the vasculature to NO mediated vasodilatation. This study represents the first rat model of HFpEF and our results define a molecular mechanism that would contribute to both diastolic dysfunction and vascular abnormalities that characterize this disease. This rat HFpEF model could be used in future studies designed to delineate the mechanism(s) that produce the cardiac and vascular abnormalities that produce HFpEF and identify novel targets for rational drug design.
Elucidating the Role of Glycogen in Glucose Transporter 1 Deficiency Syndrome
Authors: Williams Meredith , Markussen Kia H. , Webb Madison , Mestas Annette , Pascual Juan , Gentry Matthew S.
Published: May 2022

Glucose Transporter 1 Deficiency Syndrome (G1D) is a rare genetic disorder characterized by impaired brain glucose metabolism caused by mutations in the SLC2A1 gene. Mutations in the SLC2A1 gene lead to reduced or loss of function in the glucose transporter protein type 1 (GLUT‐1), affecting its ability to deliver glucose across the blood brain barrier. This lack of glucose in the brain affects brain function and development, causing people with the disorder to suffer from seizures, learning disabilities, and struggle for independence. While decreased levels of glucose in G1D is recognized, glycogen levels in the brain have not been studied in human or animal models of the disease. We employed a mouse model of G1D syndrome characterized by a loss of function in the SLC2A1 gene to study glycogen levels. Wildtype and G1D animals of the same sex at 14 weeks old were utilized for this study. Immunohistochemical (IHC) staining with IV58B6 and an amyloglucosidase glycogen assay were used to determine regional and total glycogen levels, respectively. HALO software was employed to quantify IHC staining, and the enzymatic glycogen determination was measured with a spectrophotometric microplate reader. Through these techniques, it was determined that G1D mice had perturbed glycogen levels present in multiple regions of the brain. These data suggest that the G1D phenotype could be affected by perturbed levels of glycogen in the brain. Further research into the effect of glycogen levels in G1D may be important for determining disease modalities, defining disease progression, and understanding preclinical data and drug treatment.
Linoleic Acid Induced Angiopoietin‐Like Protein 4 Impairs Skeletal Muscle Differentiation
Authors: Son Yura , Paton Chad
Published: May 2022

Skeletal muscle differentiation is a pivotal process in embryonic development as well as regeneration/repair throughout the lifespan. It is well‐known that dietary fat intake impacts biological and physiological function in skeletal muscle, however, understanding of the contribution of nutritional factors in skeletal muscle differentiation is limited. Thus, the objective of the current study is to evaluate the effects of fatty acids on skeletal muscle differentiation in vitro. Skeletal muscle differentiation is known to be regulated by Wnt/β‐catenin signaling, and Wnt/β‐catenin signaling is inhibited by angiopoietin‐like protein 4 (ANGPTL4), a protein that is induced by free fatty acids (FFA). Therefore, the hypothesis of the current study is that FFA‐induced ANGPTL4 inhibits skeletal muscle differentiation by suppressing Wnt/β‐catenin signaling. We used C2C12 murine myoblasts and treated with various FFAs, recombinant ANGPTL4 protein, or siRNA knockdown of ANGPTL4 during cell differentiation. Among FFAs, 18:2n6 linoleic acid (LA) significantly inhibited myotube formation, showing decreased myoblast fusion. LA significantly lowered the protein expression of myogenic regulatory factors, including MyoD and MyoG and increased Pax7 during cell differentiation. We also confirmed that ANGPTL4 knockdown at day 2 and ‐6 of differentiation restored myotube formation in the presence of LA. Next, we performed an RNA‐sequencing analysis to determine which pathways were involved in the mechanism of LA‐ and ANGPTL4‐mediated inhibition of skeletal muscle differentiation at day 2 and ‐6 of differentiation. LA at day 2 and ‐6 and ANGPTL4 at day 2 downregulated Wnt/β‐catenin signaling as well as genes involved in skeletal muscle differentiation. We further found that ANGPTL4 reduced β‐catenin protein expression in nucleus, indicating that ANGPTL4 blocks β‐catenin translocation into nucleus from cytoplasm, and it was restored with ANGPTL4 knockdown. Taken together, these results demonstrate that LA‐induced ANGPTL4 inhibits skeletal muscle differentiation by suppressing Wnt/β‐catenin signaling in C2C12 myoblasts.
Assembly and Disassembly of PKA is Allosterically Controlled by Nucleotides and Metal Ions
Authors: Maillard Rodrigo A. , Zhang Yaojun , England Jeneffer , Good Lydia , Bai Lihui
Published: May 2022

Protein Kinase A (PKA) is a macromolecular assembly composed of a regulatory subunit (PKA‐R) that harbors cyclic‐nucleotide binding (CNB) domains, and a catalytic subunit (PKA‐C) that binds nucleotides and metal ions in its active site. During its phosphorylation cycle, PKA‐C experiences different conformations starting with a close conformation when bound to ATP‐Mg2+, to a dynamic equilibrium between close and open conformations with ADP‐Mg2+, and an open conformation in the absence of nucleotide and Mg2+. How the conformational changes in PKA‐C allosterically modulate interactions with the CNB domains of PKA‐R remain largely unknown. Here we use optical tweezers to selectively unfold and refold one CNB domain (termed CNB‐A) of PKA‐R in the presence of PKA‐C and different nucleotides. This approach allowed us to dissect the allosteric effects that the PKA‐C conformation exert over the folding energy landscape and conformation of CNB‐A. When PKA‐C is in an open conformation, we find that CNB‐A unfolds predominantly at low forces (9 pN) as in the apo state, indicating that CNB‐A interactions with PKA‐C require nucleotide occupancy. When PKA‐C is in a closed conformation bound to ATP‐Mg2+, CNB‐A predominately unfolds at high forces (19 pN), suggesting that CNB‐A and PKA‐C are in a single, tightly‐bound conformation. Interestingly, in the presence of ADP‐Mg2+, CNB‐A unfolds at low (9 pN), intermediate (13 pN) and high forces (19 pN), suggesting that CNB‐A can be in three conformations: apo, weakly‐ or tightly‐bound to PKA‐C. We further dissect the kinetics of interconversions between these three conformations by varying the PKA‐C concentration and the refolding and rebinding dwell time. We show that transition kinetics from the apo to the tightly‐bound conformation in the presence of ATP‐Mg2+ are much faster than in ADP‐Mg2+, which has consequences for the timing of assembly of the inactive PKA holoenzyme and dissociation of the PKA complex after cAMP‐mediate activation. Altogether, this study directly shows how nucleotides and metal ions at the active site of PKA‐C can allosterically modulate the strength and kinetics of interactions with the CNB domains, thereby highlighting the delicate regulation mechanisms for PKA to function as a proper switch in cell signaling processes.
Comparative phylogenetic analysis of the Pyruvyltransferase family
Authors: Sharma Sunita , Singh Shivani
Published: May 2022

Pyruvylation of sugars is a fairly common sugar modification, yet an extensive search of literature reveals little on successful isolations of the enzyme responsible for this sugar modification. Very recently a family of genes have been identified that appear to be involved in pyruvate transfer reactions in prokaryotes. Pyruvate moieties have been identified as one of the important modifications present on cell surfaces of bacteria. These can be utilized in in silico development of antibiotics that are able to target these moieties, and interfere with their biosynthesis, eventually leading to the arrested growth of the bacteria and death in some cases. For this, an in‐depth functional and structural characterization studies is required on the various members of the pyruvyltransferases. In this study we have performed phylogenetic analysis on the pyruvyltransferase family to trace its origin. We have been able to identify three subclasses, and surprisingly this modification is predominantly present in pathogenic bacteria, which can utilized developing a highly targeted antibacterial therapy. Various tools like MEGA and MESQUITE have been used for the analysis.
Osmotic stress and regulatory volume decrease in Crithidia fasciculataparasites
Authors: Santos Maris Leisy S. , Thakur Shivangi , Greene Amy S.
Published: May 2022

Regulatory volume decrease (RVD) describes the response to hypoosmotic stress in many cells including the parasitic flagellate Crithidia fasciculata. Hypoosmotic stress causes initial swelling, the cell expels excess liquid and returns to a normal volume. This process is not able to withstand excessive osmotic stress. We use the MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide) Assay is used to assess cell viability, while microscopy images and spectroscopic light scatter used to follow changes in cell shape and size. It was found that after being placed in 70% hypotonic (30% isotonic) solution, cells did not recover from hypoosmotic stress. We are currently developing protocols for this little‐studied parasite and investigating the mechanisms of RVD in Crithidia fasciculata including the role of the osmoregulatory contractile vacuole.
ATG9 Vesicles Are Incorporated Into Nascent Autophagosome Membranes
Authors: Olivas Taryn J. , Yu Shenliang , Wu Yumei , Luan Lin , Choi Peter , Gupta Kallol , De Camilli Pietro , Melia Thomas J.
Published: May 2022

The autophagosome is a double‐membrane organelle that traps cytoplasmic cargo and traffics it to the lysosome for degradation. How the autophagosome forms is uncertain, but a prevailing model suggests lipids are moved from the ER through the lipid transporter ATG2 to ATG9 vesicles, which then expand to comprise the growing autophagosomal membrane. However, evidence that ATG9 is ever resident within the autophagosome is scant; detection of this putative autophagosome‐resident protein is made challenging both because most ATG9 vesicles in the cell are not involved in the biogenesis at any given time and because the dilution of one or a few vesicle membranes by potentially millions of transported lipids would result in a very low density of ATG9 on the mature autophagosome. Here we develop approaches to address each of these limitations. First, we show that in genetic knockouts of ATG2, ATG9 vesicles accumulate to very high numbers at sites of aborted autophagosome formation. Focused‐ion beam scanning electron microscopy reveals that without ATG2, these putative autophagosome seed vesicles do not expand, but instead accumulate within a large vesicle cluster surrounded by ER. By fluorescence microscopy, we also detect downstream modifiers of the autophagosome membrane at these sites, including the lipid‐anchored form of the LC3 proteins, which suggests that a biochemically competent seed membrane is present. To establish whether ATG9A is found on the same membrane as LC3B, we use styrene maleic acid (SMA) copolymer nanodiscs, to isolate very small intact sections of autophagosome membrane away from all other potential contaminants. Through rigorous combinations of isolation and purification, we reveal that ATG9A and LC3B are co‐resident within the same 10 nm diameter membrane segment and likely engage in a protein‐protein complex. We then apply the same SMA‐based approach to show that even in wildtype autophagosomes, ATG9 and LC3 are co‐resident. Thus, we assert that ATG9 vesicles are the seed membrane for the autophagosome.
Development of antibacterial Type III secretion system binding antibodies
Authors: Filbrandt Peter , Piontkowsky Kaitlyn , Streu Craig
Published: May 2022

Common agricultural crops such as wild rice and tomatoes are infected by the bacterial pathogens Xanthomonas Oryzae pv. oryzae and Pseudomonas Syringae pv. syringae respectively. These species use the same cellular structure to infect their host cells called a type three secretion system or T3SS. T3SSs are composed of multiple protein subunits used to insert effector proteins into the host cell's cytoplasm. These effectors in turn begin a cascade of events that ultimately results in invasion of the host cell. Once infected, the pathogens can take over the cell’s system and ultimately kill it. Given the importance of tomatoes and rice as food sources, the spread of these infections could potentially become disastrous for the populations that depend on them. To combat this infection, biological antibiotics will be developed to bind to a critical subunit in each of the respective pathogen’s T3SS, preventing proper assembly and function, and rendering the bacteria non‐pathogenic.

A combination of yeast surface display and directed evolution are being used to find nanobodies specific to the T3SS in Xanthomonas Oryzae pv. oryzae and Pseudomonas Syringae pv. syringae. Nanobodies are inexpensive and stable antibody fragments that hold substantial potential as antimicrobials in human and agriscience applications. To do this, an antigen mimicking that of the bacterial secretion systems must be created. The antigens herein were chosen for their relative conservation and role in T3SS pathogenicity. This antigen selection strategy is expected to both increase the applicability of the method across bacterial species, and reduce the potential for the development of antibiotic resistance. This poster outlines the selection, cloning, and purification of a T3SS antigen for directed evolution of nanobodies as T3SS‐targeting antimicrobials.
Insights into the catalytic mechanism of M. tuberculosis indole‐3‐glycerol phosphate synthase
Authors: Goodey Nina M. , Cho Sarah , Maryum Bhatti , Booter Huma , Moran Cintya , Jefferson Natalie , Van Den Berg Savannah , Marin Patryjca , Peralta Ashley , Margolin Katherine , Konas David
Published: May 2022

The pathogen M. tuberculosis causes the lung disease tuberculosis (TB). Emerging TB drug resistance worldwide indicates the need for new inhibitors and targets. Recent research suggests that the tryptophan biosynthetic pathway in M. tuberculosis can be targeted to treat TB. Indole‐3‐glycerol phosphate synthase (IGPS) in M. tuberculosis(MtIGPS) is an α/β barrel enzyme that catalyzes the fourth step in the tryptophan biosynthetic pathway: the conversion of 1‐(o‐carboxyphenylamino)‐1‐deoxyribulose 5’‐phosphate (CdRP) into indole‐glycerol‐phosphate (IGP) via cyclization, decarboxylation and dehydration steps. The pH profile of MtIGPS was observed to have a bell shaped curve with pKa1 and pKa2 values or 6.7 ± 0.14 and 8.7 ± 0.12, supporting the involvement of both a general acid and a general base in MtIGPS catalysis. Mutations were introduced to three residues that had been previously proposed to serve as the catalytic base: E57, E168 and E219. The E57Q variant had a >200‐fold reduced catalytic activity compared to the wildtype and the E168Q variant had a >187‐fold increased KM. The E219 sidechain interacts with K119, which is proposed to be the catalytic acid. The E219D variant had a >233‐fold decreased catalytic activity. Combined with docking predictions, these data were consistent with E57 as the catalytic base, E168 playing a role in substrate binding, and E219 in orienting the catalytic acid K119. These data together provide an increased understanding of the structure‐function relationship in the MtIGPS active site and insights in the IGPS enzyme family.
Bile acid‐mediated activation of brown fat protects from alcohol‐induced steatosis and liver injury
Authors: Fan Mingjie , Fan Mingjie , Wang Yangmeng , Jin Lihua , Sun Zhaoli , Ding Wen‐Xing , Huang Wendong
Published: May 2022

Background & Aims

Alcohol associated liver disease (AALD) is one of the most common causes of liver injury and failure. Limited knowledge of the mechanisms underlying AALD impedes the development of efficacious therapies. Bile acid (BA) signaling was shown to participate in progression of AALD, the mechanisms of which remain poorly understood.

Approach & Results

Mice were exposed to chronic‐plus‐one‐binge ethanol feeding to induce AALD. Interestingly, ethanol feeding increased expression of the thermogenesis genes downstream of TGR5 (Takeda G protein‐coupled BA receptor 5) in brown adipose tissue (BAT) of wild‐type (WT) but not TGR5 knockout (KO) mice. TGR5 deficiency significantly diminished BAT activity and energy expenditure in mice after ethanol feeding. Alcohol increased serum BA levels in mice and humans through altering BA transportation rather than increasing hepatic BA synthesis, which activated TGR5 signaling in BAT. TGR5 deficiency did not alter alcohol‐stimulated adipose lipolysis, de novo hepatic lipogenesis, or alcohol effects on hepatic β‐oxidation of free fatty acids (FFA). However, compared to ethanol‐fed WT mice, ethanol‐fed TGR5 KO mice showed less FFA β‐oxidation in BAT, leading to increased circulation and liver uptake of FFA. Importantly, in ethanol‐fed mice housed under thermoneutral conditions, TGR5‐mediated fat burning in BAT was unchanged. Finally, administration of a specific TGR5 agonist significantly activated TGR5 signaling in BAT to increase thermogenesis, reduce serum FFA level and ameliorate hepatic steatosis and injury in AALD mice.

Conclusions

BA signaling plays a protective role in AALD by enhancing BAT thermogenesis and BAT crosstalk with the liver via TGR5 activation. Targeting TGR5 in BAT may be a promising approach for the treatment of AALD.
Acute Heat Exposure Improves Microvascular Function in Skeletal Muscle of Aged Humans
Authors: Richey Rauchelle , Hemingway Holden , Moore Amy , Olivencia‐Yurvati Albert , Romero Steven
Published: May 2022

Acute heat exposure improves microvascular function in the leg of aged adults as assessed using post‐occlusive reactive hyperemia. However, reactive hyperemia measures whole‐limb blood flow and cannot isolate perfusion among various tissues. Thus, it is unclear if the skeletal muscle circulation contributes to the improvement in microvascular function observed following acute heat exposure. We tested the hypothesis that acute hot water immersion would improve microvascular function in the vastus lateralis of aged adults. Seven aged adults (1 man, 71 ± 4 yrs) were immersed to the umbilicus for 60 min in thermoneutral (36 °C) or hot (40 °C) water. Body core temperature was measured via a telemetric pill. Two microdialysis probes were placed in the vastus lateralis ~30 min after immersion. Microdialysis was utilized to bypass the cutaneous circulation and directly assess endothelial‐dependent and endothelial‐independent microvascular function in skeletal muscle by measuring the local blood flow response to a graded infusion of acetylcholine (ACh, 27.5 and 55.0 mM) and sodium nitroprusside (SNP, 21 and 42 mM), respectively. Local blood flow was measured using the ethanol washout technique. Body core temperature increased by Δ1.1 ± 0.3 °C during hot water immersion but was relatively unchanged during thermoneutral immersion (Δ0.1 ± 0.3 °C). Baseline skeletal muscle blood flow did not differ between thermal conditions for the ACh probe (P= 0.9), nor the SNP probe (P= 0.7). The hyperemic response to 27.5 mM ACh did not differ between thermal conditions (thermoneutral immersion, Δ11.3 ± 11.5 ml/min/100g vs. hot water immersion, Δ18.6 ± 16.8 ml/min/100g; P = 0.7). However, the hyperemic response to 55.0 mM ACh was increased with prior hot water immersion (thermoneutral immersion, Δ30.7 ± 16.9 ml/min/100g vs. hot water immersion, Δ56.2 ± 19.7 ml/min/100g; P < 0.01). Similarly, the hyperemic response to 21 mM SNP did not differ between thermal conditions (thermoneutral immersion, Δ16.9 ± 16.8 ml/min/100g vs. hot water immersion, Δ18.2 ± 18.8 ml/min/100g; P= 0.9), but was increased with prior hot water immersion during the infusion of 42 mM SNP (thermoneutral immersion, Δ29.3 ± 14.4 ml/min/100g vs. hot water immersion, Δ58.5 ± 31.2 ml/min/100g; P = 0.02). These data suggest that acute heat exposure improves endothelial‐dependent and endothelial‐independent microvasculature function in skeletal muscle of aged humans. Furthermore, these data highlight the therapeutic potential of heat therapy to attenuate the hypoperfusion of skeletal muscle that occurs in aged adults during conditions that require an elevated blood supply such as exercise.