bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evidence that Epithelial Tight Junctions are Altered during Edwardsiella Infections
Authors: Aggarwal Priyanka , Leung Ka Yin , Guttman Julian Andrew 
Published: November 2023

Bacteria from the Edwardsiella genus can kill entire fish farms within days. These infections cause an array of phenotypic alterations to fish including petechial hemorrhage in the fins and skin, rectal hernia, abscesses deep within fish musculature and most strikingly, holes within (or the liquefaction of) the fish themselves. Intercellular junctions are crucial for maintaining tissue integrity, but are also common targets of bacterial pathogens. Because Edwardsiella causes alterations to various epithelia during their infectious processes, we hypothesized that tight junctions (TJs) might be targeted by these microbes. To test this hypothesis, we localized the claudin‐3 transmembrane tight junction protein in Caco‐2 cells during Edwardsiella infections and found that it was extensively re‐arranged. This work provides the first evidence of junctional alteration during Edwardsiella infections, and suggests a possible molecular modification that could lead to the observed phenotypes in diseased fish.Support or Funding InformationFunded by NSERC Discovery Grant
The presence of a variant leg muscle, flexor digitorum accessorius longus (FDAL), causing tarsal tunnel syndrome, or posterior tibial neuralgia
Authors: Swearingen Jordan V , Kennedy Patrick M , Whipp Kylen P , Miller Kyle D , Zdilla Matthew J. , Lambert H. Wayne 
Published: November 2023

The flexor digitorum accessorius longus (FDAL) muscle is a variant muscle located in the posterior compartment of the distal leg. It typically originates in the distal third of the leg and can have a variable proximal attachment to the tibia, fibula, tibia and fibula, the posterior intermuscular septum, or any of the musculature in the deep posterior compartment of the leg. The FDAL then courses posterior to the medial malleolus, enters the the tarsal tunnel (deep to the flexor retinaculum), and ends by inserting onto either the flexor digitorum longus tendon and/or the quadratus plantae muscle, which are located in the second muscular layer of the sole of the foot. The prevalence of the FDAL has been reported in 6% of asymptomatic individuals based on MRI studies or 2–12% of cadaveric studies.The presence of the FDAL has been identified as a cause of tarsal tunnel syndrome, a compression neuropathy of the tibial nerve and/or its branches that become compressed as they course deep to the flexor retinaculum. As the FDAL traverses the tarsal tunnel, it lies immediately superficial to the neurovascular bundle and can act as a space‐occupying lesion, entrapping the tibial nerve and causing numbness, pain, and paresthesias in the foot, ankle, and toes. This variant muscle is best visualized through the use of magnetic resonance imaging (MRI). While there are conservative treatments for tarsal tunnel syndrome, most often surgical intervention is needed to remove the FDAL, and the success of this operative procedure is often based on the extent of damage to the tibial nerve prior to surgery. This presentation will include a patient case and show examples of the FDAL muscle following cadaveric dissection and within MRI studies. Radiologists and surgeons should be aware of presence of the FDAL muscle when considering the etiology of tarsal tunnel syndrome.Support or Funding InformationThe research of Jordan V. Swearingen and Kyle D. Miller was supported by the West Virginia University Initiation to Research Opportunities (INTRO) Program.
The Morphological & Biomechanical Characteristics of the Ligamentum Mucosum and its Potential Role in Anterior Knee Pain
Authors: Norris Madeleine Elizabeth , Burkhart Tim , Johnson Marjorie , Sandig Martin , Smallman Thomas , Getgood Alan 
Published: November 2023

IntroductionThe ligamentum mucosum (LM), or infrapatellar plica, is a non‐isometric structure of the knee joint that traverses from the intercondylar notch of the femur to the infrapatellar fat pad. The LM is composed of dense regular connective tissue, of which four different morphologies have been identified: separate, vertical septum, split, and fenestrated. Microscopically, pathological LM appears fibrotic and calcified, which can lead to impingement, and ultimately may be implicated in the pathogenesis of anterior knee pain. The specific aims of this study are to investigate the anatomical relationship of the LM to neighboring structures, determine whether the histological characteristics of the LM further define its function and its potential role in proprioception, and lastly to perform an in vitro biomechanical analysis of the LM under normal physiological loading.MethodologyFresh‐frozen cadaveric knee specimens (n=14) were dissected to reveal the structures of the joint capsule and to identify the morphology of the ligamentum mucosum. With the knee at 90° flexion, four measurements were recorded in order to examine the important relationships within the joint capsule. The intraclass correlation coefficient was calculated to determine the consistency and reproducibility of the measurements recorded. Maintaining the proximal and distal attachments, two specimens were harvested for histological analysis using standard hematoxylin and eosin, Masson's trichrome for collagen, and mono‐clonal mouse anti‐human neurofilament protein to identify neurons and peripheral nerve endings.ResultsOverall, 64% of the knees had a LM present. With respect to morphology, 50% of the 14 specimens had a separate type, 14% had a vertical septum type, and the remaining specimens had no LM present, which is consistent with known classification systems. The histological analysis confirmed the LM to be ligamentous, thus being composed of dense regular connective tissue. Preliminary immunohistochemistry results are inconclusive at this time for evidence of peripheral nerves and nerve endings, and the biomechanical properties of LM have yet to be determined.DiscussionThe role of LM in anterior knee pain is not well documented; however, studies have shown that excising the LM can relieve idiopathic anterior knee pain. With a decrease in elasticity, pathological LM may result in patellar maltracking and instability, ultimately triggering anterior knee pain. Future biomechanical testing will utilize an Instron materials testing machine to load the ligaments under tension at a constant velocity in order to determine the force at which the specimen fails. Thus, the findings from this study can be implemented in models of anterior knee pain, more specifically models of patellar instability and maltracking, to determine the proprioceptive and pathogenic mechanisms of the ligamentum mucosum.
Using ultrasound to prevent iatrogenic injury to the dorsomedial cutaneous nerve of the hallux
Authors: Miller Kyle D , Swearingen Jordan V , Zdilla Matthew J. , Lambert H. Wayne 
Published: November 2023

IntroductionThe dorsomedial cutaneous nerve (DMCN) of the hallux is a terminal branch of the superficial fibular (peroneal) nerve which supplies the skin of the medial aspect of the great toe. This cutaneous nerve branch is particularly vulnerable to iatrogenic injury during operative procedures involving the joints of the hallux, including hallux valgus correction, hallux ridigus correction, bunionectomy, and cheilectomy. Moreover, the DMCN of the hallux can be damaged during extensor hallucis longus (EHL) tendon transfer procedures due to the fact this nerve travels superficial to EHL tendon while traveling obliquely in the dorsum of the foot from a proximal lateral location to a distal medial position.MethodsA first‐year medical student, with introductory‐level experience in ultrasonography (USG), performed all USG in the study in an attempt to identify the intersection point of where the DMCN of the hallux crosses the EHL tendon superficially. This study used an ultrasound machine (GE Venue 40 ultrasound machine) in conjunction with an L8‐L18i wide‐band high‐frequency linear array transducer to approximate this intersection. To check the accuracy of the DMCN location, a cruciate incision was made through the skin according to the midpoint markers on the L8‐18i probe.ResultsSuccessful mapping of the DMCN was gauged in binary fashion; if the DMCN was located directly beneath the center of the cruciate incision described, the method was considered successful. Of 28 feet dissected, 21 DMCN nerves (75%) were successfully located.ConclusionsThis study reports that a first‐year medical student with introductory‐level experience successfully identified the intersection of the DMCN with the EHL via USG in 75% of feet examined. Therefore, the success rate would likely improve when USG is performed by surgeons and clinicians with more experience in USG in conjunction with other examination methods, such as the four toe flexion sign. Therefore, USG may provide an inexpensive, accurate, non‐invasive means of pre‐operatively identifying the location of the DMCN of the hallux relative to the EHL tendon to prevent inadvertent nerve damage during foot surgery.Support or Funding InformationThe research of Kyle D. Miller and Jordan V. Swearingen was supported by the West Virginia University Initiation to Research Opportunities (INTRO) Program.
NURF Localizes in Gene Bodies to Regulate mRNA Processing
Authors: Landry Joseph , Alhazmi Aiman , Mack Marissa , Petencin Alexander , Nelson Humberto , Hiegel Jordan , Koparde Vishal , Scarsdale Neel , Lyons Charles , Dozmorov Mikhail 
Published: November 2023

Understanding how chromatin remodeling complexes are recruited to chromatin is important to our understanding of how they regulate gene expression. Towards this end we determined the genome wide localization and sites of putative chromatin remodeling activity for the Nucleosome Remodeling Factor (NURF) by targeting its largest and essential subunit bromodomain PHD finger containing transcription factor (BPTF). From our BPTFChIP‐Seq and FAIRE‐Seq studies in BPTF knockout ESC we observe that BPTF islocalized and regulates chromatin structure broadly throughout the genome with a preference for promoters, gene bodies and terminators. A bio informatics analysis of these data sets discovers that a majority of NURF function as aregulator of gene expression is correlated with gene bodies and terminators. Abiochemical screen for binding partners of the BPTF N‐terminal PHD finger discovers specific interactions with THOC4, a mRNA processing factor which binds to an elongating polymerase. Analysis of NURF function at the BPTF‐dependent gene Ccnd1 discovers BPTF‐dependent roles in splicing out intronDNA sequences from mRNA transcripts. These defects correlate with evidence of RNA polymerase stalling, a known mechanism for down regulating gene expression with intron retention (IR). These results present model where NURF regulated gene expression can occurs through defects in retaining intron sequences.Support or Funding InformationJeffress FoundationVirginia Academy of SciencesVirginia Commonwealth UniversityMassey Cancer Center
A Specialized Mechanism of MicroRNA‐mediated Translation in Quiescence
Authors: Vasudevan Shobha , Bukhari Syed Irfan Ahmed , Truesdell Samuel Spencer , Lee Sooncheol , Kollu Swapna , Classon Anthony , Boukhali Myriam , Jain Esha , Yanagiya Akiko , Sadyrev Ruslan , Haas Wilhelm 
Published: November 2023

Quiescence (G0) represents an assortment of reversible, cell cycle‐arrested states, associated with cancer persistence as well as development. G0 involves selective mRNA expression while decreasing canonical translation. The alternative translation mechanisms in G0 remain to be uncovered.Our data show that microRNAs, regulatory, non‐coding RNAs that target distinct mRNAs to alter gene expression, are important in G0, where they can associate with alternative translation factors to regulate specific mRNA translation. One subset of transcripts expressed in G0 includes specific mRNAs recruited by an FXR1a‐associated microRNP (microRNA‐protein complex) for translation activation in G0 mammalian cells and G0‐like immature Xenopus laevis oocytes. MicroRNPs predominantly mediate repression and downregulation; however, FXR1a‐microRNP lacks the conventional microRNP repressor, GW182, and instead, contains a specific RNA binding protein isoform, FXR1a. FXR1a promotes translation and is overexpressed and associated with poor prognosis in certain cancers. Activation is dependent on target recruitment by FXR1a‐microRNP in the nucleus, indicating compartmentalized mRNA recruitment for selective target activation. Our data reveal that microRNA‐mediated activation requires target mRNAs with unadenylated/shortened poly(A) tails. Polyadenylated mRNAs are repressed, potentially due to the role of PABP in enhancing microRNA‐mediated downregulation and in canonical translation that is impaired in these conditions. Consistently, PARN deadenylase is required for microRNA‐mediated activation. Low mTOR activity in G0 activates the cap complex inhibitor, eIF4E‐BP, and thereby, impairs canonical translation; instead, as previously shown, interaction of PARN with 5′ mRNA caps is increased in G0. The target mRNA 3′‐UTR recruits FXR1a‐microRNP, which associates with PARN and with p97, a homolog of the translation factor eIF4G, which recruits eIF3, and thereby, 40S ribosome subunits but lacks cap complex‐ and PABP‐interacting domains. These data reveal a specialized translation mechanism that is important for G0 maintenance and chemoresistance, where FXR1a‐microRNP connects specific, poly(A) shortened mRNAs to the ribosome through interactions with alternative factors, PARN and p97, in these conditions of reduced canonical translation.Support or Funding InformationCancer Research Institute, The Leukemia & Lymphoma Society, MGH funds & NIGMS (SV). Fund for Medical Discovery postdoctoral fellowship (SL).
Alternative ribosomes synthesize alternative proteins
Authors: Dow Allexa , Prisic Sladjana 
Published: November 2023

In addition to the complete set of ribosomal proteins needed to form ribosomes, many bacteria have at least one duplicated (i.e., alternative) ribosomal protein. It has been suggested that alternative ribosomal proteins (AltRP) in Bacillus subtilis and Escherichia coli serve as a replacement for zinc‐binding “primary” homologs (PrimRPs) when zinc is deficient. Therefore, it has been proposed that Prim/AltRP pairs have a role in zinc storage and release depending on zinc availability. Mycobacterium tuberculosis has four alternative ribosomal proteins organized in a single operon, whose function is not known. We hypothesize that mycobacterial AltRPs have functions other than zinc storage.In order to test if mycobacterial PrimRPs serve as a source of zinc in low zinc environment and whether or not they have some other roles, we deleted the altRP operon in M. tuberculosis. There was no growth defect in the mutant compared to the wild type when grown in zinc‐rich or zinc‐deficient medium. We have evidence that AltRPs are replacing PrimRPs in ribosomes when zinc is limited (i.e., they form “alternative” ribosomes), but this switch from PrimRPs to AltRPs did not have any measurable effect on ability of M. tuberculosis to survive under low zinc conditions. However, we observed “bulging” cell morphology in the altRP deletion mutant, which is consistent with a cell wall defect. Considering that the cell envelope defect may be caused by changes in building blocks or enzymes required for their synthesis, we used proteomics to analyze protein content in the altRP mutant vs. wild type. Indeed, proteomes of the deletion mutant (contains only “primary” ribosomes) was significantly different compared to the wild type parental strain (contains a mixture of “primary” and “alternative” ribosomes). The perturbation of the proteome caused by the lack of “alternative” ribosomes allowed us to identify a set of proteins preferably synthesized by such ribosomes. Specifically, differences in the abundance of proteins involved in cell wall synthesis or components of the cell wall may explain the difference in the cell morphology.Our data indicate that AltRPs in M. tuberculosis are not required for growth under low zinc conditions and therefore they may have only limited role in adaptation to changes in zinc availability. Importantly, our study shows that AltRPs can replace PrimRPs in ribosomes, which in turn causes a significant change in the proteome, either directly by preferentially translating a distinct set of mRNAs or indirectly, e.g., by changing the amount of repressors or proteases. Therefore, in addition to transcriptional regulation, the shift from “primary” to “alternative” ribosomes provides another layer of regulation, which may be important for adaptability and pathogenicity of M. tuberculosis.Support or Funding InformationThis research is supported by College of Natural Sciences Startup Funds
Development of Prolyl‐tRNA Synthetase from Pseudomonas aeruginosa as a Platform to Screen for Inhibitors of Protein Synthesis
Authors: Pena Noah , Hu Yanmei , Bullard James M 
Published: November 2023

IntroductionPseudomonas aeruginosa is an opportunistic pathogen and a common cause of nosocomial infections. Aminoacyl‐tRNA synthetases (aaRSs) are a class of enzymes that catalyze the covalent attachment of amino acids to their cognate tRNAs during protein biosynthesis. We describe here the enzymatic characterization and development of a screening platform based on prolyl‐tRNA synthetase (ProRS) from P. aeruginosa.ResultsProRS from P. aeruginosa was cloned and expressed in E. coli and purified to greater than 98% homogeneity. Sequence analysis shows that this protein contains the characteristic motifs of class I aminoacyl‐tRNA synthetases and when compared with E. coli exhibited approximately 70% amino acid sequence conservation. The kinetic parameters for the interaction of P. aeruginosa ProRS with its three substrates (tRNA, ATP, and methionine) were determined. Initial velocities were determined for charging of tRNA using tRNAPro concentrations between 0.8 and 3 μM. The KM and Vmax was determined to be 7 μM and 0.67 μM min−1, respectively, resulting in an observed kcat of 0.22 sec−1. This resulted in a kcat/KM value of 0.033 s−1μM−1. The ATP:PPi exchange reaction was used to monitor the interaction with ATP and proline (Pro). The KM, Vmax, observed kcat, and kcat/KM for interaction with ATP was 700, 168, 14, and 0.02 μM, respectively. Scintillation proximity assay (SPA) technology was adapted to the aminoacylation assay and then used to screen for inhibitors of activity of P. aeruginosa ProRS in a high throughput format. Using this assay, a natural product (800 compounds) and a synthetic compound (890 compounds) library was screened.ConclusionProRS identified in P. aeruginosa was cloned, expressed characterized and developed into a screening platform to identify compounds that have the potential for development as an antibacterial agent against pathogenic organisms.Support or Funding InformationResearch was funded by NIH grant 1SC3GM098173‐01A1. Partial student support was from a Departmental Grant from the Robert A. Welch Foundation (Grant No. BG‐0017), the NIH UTPA RISE program grant # 1R25GM100866‐01 and the Howard Hughes Medical Institute (HHMI) Precollege and Undergraduate Science Education Program grant #52007568.
Integrating reductive and synthetic approaches in biology using man‐made cell‐like compartments
Authors: Aoki Wataru , Komura Shun , Tamiya Eiichi , Ueda Mitsuyoshi 
Published: November 2023

We propose ‘synthetic genetics’ as a novel methodology that integrates reductive and synthetic approaches used in life science research [1]. Life science research seeks to elucidate the relationships between genotypes and phenotypes. This typically involves reductive (genetic or omics researches) and synthetic (synthetic biology) approaches. Genetic and omics research seeks to identify genes involved in a biological subsystem of interest such as transcription, translation, signal transduction, genome repair, and metabolism. This approach enables identifying individual genes in a target biological subsystem, although the entirety of the subsystem is not readily characterized. To overcome this, synthetic approaches are used, in which a known set of genes is isolated and combined to reconstruct a target biological subsystem with the aim of a complete proof for the entirety of the subsystem. Life science research combines these two methodologies. However, it is often difficult to fill in information gaps in a target subsystem on the basis of these approaches, and tremendous amounts of time and effort are required to completely understand a subsystem's functions.‘Synthetic genetics’ enables determinations of sets of genes required for the functioning of any biological subsystem [2]. This method utilizes artificial cell‐like compartments, including a randomly introduced whole gene library, strictly defined components for in vitro transcription and translation, and a reporter that fluoresces ‘only when a particular function of a target biological subsystem is active.’ The set of genes necessary for the target biological subsystem can be identified by isolating fluorescent artificial cells and multiplex next‐generation sequencing of genes included in these cells. The importance of this methodology is that screening for the set of genes involved in a subsystem and reconstructing the entire subsystem can be done simultaneously. This methodology can be applied to any biological subsystem of any species and may remarkably accelerate life science research.
Chemical Genetic Analysis Of Mast‐Cell Activation
Authors: August Avery 
Published: November 2023

Mast cells play critical roles in allergic responses, and calcium signaling controls the function of these cells, and a role for actin in regulating calcium influx into cells has been suggested. Using chemical genetic approaches, we identify the actin reorganizing protein Drebrin as a target of the immunosuppressant BTP, which inhibits calcium influx into cells and IgE‐mediated allergic responses. We also show that Drebrin−/− mice exhibit reduced IgE‐mediated allergic responses. Furthermore, Drebrin−/− mast cells exhibit defects in actin cytoskeleton organization and calcium responses downstream of the FcɛRI, and agents that relieve actin reorganization rescue mast cell FcɛRI induced degranulation. Drebrin thus represents a novel regulator of mast cell function by regulating the actin cytoskeleton.Support or Funding InformationAI108598