bluuebunny/crossref_metadata_2025_split huggingface dataset:
Gene expression profile analysis of immunodeficient mice after WT or mSOD1 immunoreconsitution reveals differential motoneuron death mechanisms after facial nerve axotomy
Authors: Setter Deborah Olmstead , Runge Elizabeth M , Schartz Nicole D , Sanders Virginia M , Jones Kathryn J 
Published: November 2023

When a facial nerve axotomy (FNA) is performed on recombinase activating gene‐2 knockout (RAG‐2−/−) mice lacking the adaptive arm of the immune system, there is significantly greater motoneuron (MN) death relative to wild type (WT) mice. Reconstituting RAG‐2−/− mice with whole splenocytes (WS) restores MN survival to WT levels, and CD4+ T cells specifically were found to drive immune‐mediated neuroprotection. The mSOD1 mouse model of amyotrophic lateral sclerosis (ALS), a MN disease, also exhibits significantly decreased MN survival after FNA, and this model mimics the immune dysregulation described in human ALS patients. Reconstituting RAG‐2−/− mice with mSOD1 WS fails to rescue MN survival, whereas isolated mSOD1 CD4+ T cell reconstitution confers neuroprotection. This finding suggests an inhibitory factor in mSOD1 mice blocks CD4+ T cell‐mediated neuroprotection. The objective of the current study is to characterize the mechanisms of neuroprotection in a wild‐type immune system and then identify disruptions of these processes in the mSOD1 mouse model of ALS.To accomplish this goal, gene expression profiles of the axotomized facial motor nucleus were gathered from 6 experimental groups: WT, RAG‐2−/−, RAG‐2−/− + WT WS, RAG‐2−/− + WT CD4+ T cells, RAG‐2−/− + mSOD1 WS, and RAG‐2−/− + mSOD1 CD4+ T cells. Genes associated with MN regeneration, glial activation, inflammatory cytokines, and cell death pathways were examined. Our findings revealed that relative to WT mice, MN regeneration and cell death pathways were unaffected in RAG‐2−/− mice, but glial activation and cytokine expression were significantly reduced. Adoptive transfer of WT or mSOD1 WS or CD4+ T cells into RAG‐2−/− mice restored glial activation and cytokine production to WT levels. MN‐specific Fas cell death receptor expression, which is increased in mSOD1 mice after FNA, is also elevated in RAG‐2−/− + mSOD1 WS mice. These data implicate a dysregulated glial response as a causative factor for MN death in immunodeficient mice. In contrast, promotion of the Fas/Fas ligand pathway may contribute to MN death in immunodeficient mice reconstituted with mSOD1 WS. In summary, these results suggest that peripheral immune cells can directly modulate central responses to injury. Future studies based upon these results are expected to elucidate how mSOD1 immune cells promote Fas expression, and whether inhibition of the process can preserve MN.Support or Funding InformationSupported by NIH NS40433 (KJJ and VMS)
The Ligamentum Flavum Thickens During the Advanced Stages of Hypomobility Induced Lumbar Spinal Osteoarthritis
Authors: Pulisetty Tanaya , Benckendorf Adam N. , Merlo John A. , Cramer Gregory D. , Little Joshua W. 
Published: November 2023

Low back pain (LBP) is a primary cause of global disability. The lumbar facet joint is recognized as a clinically relevant pain generator. Pain can occur through the onset and progression of facet joint degeneration consistent with spinal osteoarthritis (OA). OA is now defined as a whole joint disease involving many tissues in addition to the articular cartilage, including the joint capsule. Capsular thickening due to mechanical and inflammatory stress can present clinically as a source of LBP. We have previously examined capsular thickening that occurs during OA, by using a rat model of external spinal linkage to cause lumbar spinal hypomobility and induce degeneration in the lower lumbar facet joints (L4/5 and L5/6). We have shown that this model mimics the location, progression, and histopathological changes of the articular cartilage typically reported in human spinal OA. Additionally, we found that by 8 weeks post linkage this degeneration is associated with significant thickening of the ventral articular capsule (ligamentum flavum, LF) in the cephalad portion of the joint. These articular capsule changes are unexplored at later stages of this model when degeneration is more advanced. Here we used a morphometric approach to examine if spinal hypomobility of the bilateral L4/5 and L5/6 facet joints in rats is associated with LF thickening. We hypothesized that 12 weeks of spinal linkage would be associated with an increase in articular capsule thickness in joint regions undergoing cartilage degeneration. We tested our hypothesis by comparing LF thickness in linked and time matched control rats at 12 weeks post linkage. LF thickness measurements were performed on images obtained from a brightfield microscope (10× objective, Leica DMRB, optronics microfire camera) on formalin fixed, decalcified, paraffin embedded, Ehrlich's hematoxylin and light green stained 45 μm thick sections. Morphometric measurements were made by drawing a line through the thickest region of the LF, perpendicular to the plane of the ventral LF surface. This approach demonstrated acceptable inter‐ and intra‐rater reliability (ICC= 0.94 and 0.99). Twelve weeks of hypomobility was associated with a significant increase in LF thickness (p<0.05) compared with control animals in the cephalad and caudal regions. This agrees with our recent findings that significant degeneration is present in the superior articular process (predominantly cephalad region) and inferior articular process (predominantly caudal region) by week 12. Our findings suggest that advanced cartilage degeneration is associated with LF thickening. These findings require additional study to examine the causes of LF thickening (e.g., prolonged inflammation and fibrosis) to better understand the role of the LF in the pathophysiology of OA and OA induced LBP.Support or Funding InformationNCMIC
Diversity of microvascular endothelial cell markers in different regions of the human brain
Authors: Mbagwu Smart , Filgueira Luis 
Published: November 2023

BackgroundMicrovascular endothelial cells in the brain have various important functions, including contribution to the blood‐brain barrier and supply of nutrients and oxygen to the brain, as well coagulation control, inflammatory responses and angiogenesis. Endothelial cells have observable morphological and molecular differences in different tissues and organs. However little is known about differences of microvascular endothelial cells in the human brain. The aim of this study was to characterize qualitatively the expression of endothelial cell markers within different parts of the brain.MethodsFrozen sections from different anatomical regions (precentral and postcentral gyrus, hippocampus, rhinal and visual cortex) of human formalin fixed brains (n=3) were stained immunohistochemically for endothelial cell markers, including CD31, claudin 5, occluding, von Willebrand Factor, ZO‐1, as well as astrocyte (glial fibrillary acid protein) microglia (Iba1) markers.ResultsThe expression patterns of the markers was heterogenous in the different regions of the brain studied. CD‐31 had the most positive expression among the different biomarkers especially in the precentral cortex. ZO‐1 and and vWF had fairly positive expressions in the pre central and post central cortices compared to other regions.ConclusionThe expression pattern of the different endothelial markers is heterogenous in the microvasculature within the different anatomical regions of the cerebral cortex. This results indicate that the diversity in microvascular endothelial cells contribute to functional differences in the different brain brain regions.
Observations and practical tips on metal affinity chromatography and protein refolding techniques
Authors: Tovar Carmel N , Mullins John A , Odunuga Odutayo O 
Published: November 2023

Immobilized metal affinity chromatography (IMAC) is a specific high‐capacity technique used in large‐scale purification of proteins. In spite of the robustness of the technique, several factors still affect column binding capacity, yield and purity of proteins during IMAC. Using (His)6‐tagged proteins with comparably high levels of expression, we investigated the effect of protein size, flow‐rate, number and position of (His)6 tag on protein yield using commercial Ni2+ IMAC columns. Results suggest that regardless of manipulation of flow rate, (His)6 tag placement and number, size of protein is the major factor affecting binding and yield during IMAC purification. Small and medium sized proteins (<50 kDa) bind more tightly to the column than bigger proteins, resulting in higher yield during purification. In another study, we employed a modified gradient gel filtration technique to successfully refold a spectrum of highly aggregation‐prone enzymatic proteins back to their native states during denaturing purification. Some of the proteins chosen are well‐known to precipitate significantly during refolding. However, using this technique, precipitation was either so minimal or non‐existent. Enzymatic assays and dynamic molecular mass determination confirmed that the proteins were correctly refolded and biologically active. This strategy provides a robust approach to refolding denatured proteins that can also be used simultaneously as buffer exchange and additional purification step.Support or Funding InformationThe Welch Foundation
Modeling A Protein Story (MAPS): A Project‐based Learning Program Connecting Gene and Protein Sequence and Structure‐function Relationships with Physical Models
Authors: Munzenmaier Diane H. 
Published: November 2023

The MAPS program consists of content‐driven multimedia online modules focused on the genetics and protein structure/function/dysfunction relationships associated with a specific protein essential to grades 9–12 life sciences curricula. Teams of students, led by a teacher within a classroom, home school, or extracurricular setting, progress through hands‐on activities using models and materials such as gene and protein sequences, molecular, protein folding and protein structure models as well as use of 3D molecular visualization and design software. Following a progressive exploration of the featured protein, teams choose a “protein story” extension where they gain a deeper understanding of a related topic of interest. They then design and 3D print a physical model that helps them to tell their specific molecular story. Teams write abstracts, create posters describing their work and present their posters using their physical models at local, regional and national conferences.The MAPS program builds deep content knowledge as well as higher level integration of content through an original and unique format based on methods that have been proven to be important for student learning. Each program year begins with foundational concepts to which higher level concepts are built upon throughout the course of the program. These concepts are explored using hands‐on activities within small groups of students, guided by a teacher. Teams are encouraged to wonder, ask questions, and construct hypotheses. At the end of each unit, videos are provided to assist in pulling together and reinforcing the concepts learned through the activities before they move on to the next section. This format utilizes and encourages proven effective educational strategies such as active learning, cooperative learning, and inquiry‐based learning. Once a basic level of understanding of the protein of interest is reached, teams then choose, research and develop their unique related molecular story, design and build a physical model that represents their story, and communicate their story to the public, developing necessary presentation skills. The ability for each team to choose an extension or “protein story” that they find particularly intriguing provides additional engagement and motivation and allows for more open‐ended discovery and learning.
Promoting rural student enthusiasm for STEM by establishing a model biotechnology company in their high school
Authors: Koci Matthew , Boller Bruce , Ali Rizwana , Orders Amy 
Published: November 2023

The jobs of the 21st century require a higher level of skill, training, and practical competency in science than our high schools are currently able to provide students. This is especially true for students in rural and under‐served communities. To ensure the ability of these students to find stable and lucrative careers, we need to develop novel teaching approaches that provide better workforce development while attracting students to careers in science. This project seeks to address this issue by establishing a recombinant protein expression “mini‐biotech company” in the Bertie Early College High School, in Windsor, North Carolina. This mini‐biotech company serves as the platform to teach Essential and Common Core Standards of biology and chemistry. By going through the steps of initial process development, process scale‐up, production, and validation to ultimately produce recombinant proteins, the students get a better understanding of the concepts, technologies, inter‐relatedness of the different fields. Additionally, the students are producing proteins for active university research projects related to the industries in Bertie County. This helps the students gain a fuller appreciation for how science and biotechnology are related to the jobs in their community and better plot their own path to a science‐based career. Over the course of this project, the students developed mentoring networks with university faculty and scientists at various biotechnology companies. They have also attended local and national scientific meetings. Collectively the participants in this program gain practical work relevant experience leading to an understanding of biotechnology beyond most undergraduate students.Support or Funding InformationThis project is supported by a Student Science Enrichment Program grant from the Burroughs Welcome Fund
Genetic Engineering of Human Cells Using Lentivirus in a High School Laboratory Course
Authors: Safranek Conrad , Ross Emily , Rosenberg Martin , Cole Austin , Chandra Atharv , Pemberton Luke , Zarrinnegar Hanna , Alonzo Amanda , De Luke , Sweeney Robert 
Published: November 2023

Human cell culture, lentiviral genetic engineering, and fluorescence microscopy within a BSL‐2 laboratory provide for an unparalleled learning opportunity in molecular biology. Here we report course content that includes regulatory requirements needed to establish a BSL‐2 high school laboratory and provides detailed written and video protocols, adapted to a high school schedule, for visibly definitive positive controls in genetic engineering. This course can be fully implemented in one semester. Mechanistic descriptions and lab protocols for the following genetic engineering techniques are included in this work: lentiviral production and transduction with a GFP transgene (permanent gene delivery), with sgRNA for GFP and CAS9 payload (CRISPR; permanent gene disruption), with a GFP shRNA (permanent RNA interference), and transfection with GFP siRNA (transient RNA interference). Positive controls serve as a critical foundation for teaching fundamentals of molecular biology as well as for novel research. Students extract deeper meaning from visual representations of biological concepts, which makes GFP a particularly impactful tool in practicing positive controls. Also, students working under BSL‐2 lab conditions solidify lab safety skills that are fundamental to molecular biology. To our knowledge, there has never been published such a comprehensive guide to implementation of a BSL‐2 laboratory with associated genetic engineering protocols for visible positive controls. This information will be helpful to other secondary schools considering such programs as well as undergraduate molecular biology programs looking to incorporate these principles and techniques. All of the course content and protocols is presented and freely available at www.nuevaresearch.org.Support or Funding InformationThis work was supported by The Nueva School.
Establishing a Zebrafish Laboratory Exercise to be Used in a Course‐Based Undergraduate Research Experience (CURE) in Introductory Biology
Authors: Bruton Laura , Gruber Alex , Kalo Kadijah , Morrissette Jeffrey 
Published: November 2023

Zebrafish (Danio rerio) are biological model organisms as they are a predictive model for human diseases and development. They are also an excellent teaching tool. At Lawrence Technological University, we are establishing a population of zebrafish of all ages for the purpose of using them in a course‐based undergraduate research experience in a first‐year biology course. We have purchased a stand‐alone, portable, zebrafish “E‐Rack” from Aquaneering (San Diego, CA), which is composed of eight 1.8 L and five 2.8 L tanks. Using this system, we have been able to maintain stable water quality that has allowed us to successfully house, mate, and rear zebrafish to adulthood. We use a single pair mating procedure to obtain fertilized eggs each week that can grow to adulthood in approximately 12 weeks. We are presently designing the laboratory procedures using larval zebrafish to introduce an authentic research experience into our freshman introductory biology laboratory course. One key advantage of larval zebrafish is their transparency which allows for easy viewing of the heart and blood vessels with a low‐power light microscope. In this CURE, students will perform a cardiotoxicity assay by first lightly anesthetizing one‐month‐old larval fish. Students will then measure and record fish heart rates under the microscope. Next, they will add a variety of compounds of their own choosing to test their potential cardiotoxic effects based on observed changes to heart rate and contractility. We will have students begin with known effectors of heart rate such as nicotine and caffeine, and then move on to testing their unknowns. At the end of the course, students will produce research posters which they will present at Lawrence Tech's Research Day conference and/or other local and national conferences. We will determine the effectiveness of this new teaching strategy using the established Classroom Undergraduate Research Experience (CURE) survey administered by Dr. David Lopatto at Grinnell University.Support or Funding InformationLawrence Technological University Quest ProgramLawrence Technological University Natural Sciences Department
Mechanism of DNA Binding by Human DNA Ligase 1
Authors: Jurkiw Tom , O'Brien Pat 
Published: November 2023

Human DNA Ligase 1 (hLIG1) is an essential enzyme in the cell that seals single‐strand DNA breaks, or nicks, that occur between adjacent Okazaki Fragments during DNA replication. Although the phosphoryl transfer steps of hLIG1 have been previously characterized, many questions remain about how hLIG1 is able find and recognize nick sites. New approaches are needed to observe the rapid and transient interactions. Here we have investigated the kinetic mechanism for DNA binding using fluorescence approaches. By using a nicked DNA reporter and endogenous tryptophan fluorescence, we are able to monitor the DNA‐dependent steps of the ligation mechanism. Under the conditions used the assay appears to report on initial interactions with the nicked DNA substrate and the product release step, both of which are previously uncharacterized steps. Future experiments will extend these kinetic and thermodynamic studies to understand what features of hLIG1 and the nicked DNA substrate allow for rapid and faithful ligation of DNA breaks.Support or Funding InformationResearch was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number T32GM008353 and the Rackham Graduate School and Biological Chemistry Department at the University of Michigan.
Determination of Escherichia coli Genes Important for DNA Repair Following Alkylation
Authors: Joshi Christopher , Aiken Ariel , Nash Emma , Wong Kelly , Carlson Alyssa , Leifer Becky , Muenter Mark , Beuning Penny 
Published: November 2023

Alkylating agents, common in industrial manufacturing and chemotherapy, can damage DNA by adding alkyl groups to nucleotides. Alkylation can decrease the stability of the N‐glycosidic sugar‐base bond, making it more apt to break; alkylation can also interfere with base pairing. Either of these perturbations can lead to mutations if not remedied. DNA repair mechanisms, including direct base repair, base excision repair, and nucleotide excision repair, play important roles in correcting these lesions. Our research aims to better understand the mechanisms behind repair of alkylated DNA, first by confirming which genes are most critical. An assay was designed to screen bacterial strains harboring single deletions of more than 20 genes, selected based on prior known association to DNA repair. The survival of E. coli AB1157 strains, each with one of the 20 genes deleted, was measured upon exposure to one of two alkylating agents: styrene oxide (SO) or chloroacetaldehyde (CAA). Strains with dam deleted were sensitive to SO and CAA, suggesting that DNA adenine methylase, the protein encoded by dam, plays a critical role in DNA alkylation repair. Strains lacking recA, which plays multiple roles in DNA damage responses, were quite sensitive to both agents. Strains with mfd, mutH, or mutY deleted were not more sensitive to SO and CAA than the wild‐type strains. Next, the survival of double knockouts will be assayed, which will help determine if these genes act in the same or different DNA repair pathways.Support or Funding InformationThis work was supported by the American Cancer Society and the NU Office of the Provost.