bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of Mono‐Lecture and Dual‐Lecture Modes in the First Year Medical Education
Authors: Chen Jian , Daley William P , Yang Gongchao , Cui Dongmei
Published: November 2023

Contemporary basic medical science education at medical schools is more clinically oriented both in preparing students for the medical board examination and the following clinical rotations. Major issues facing medical educators in the basic science curriculum for first medical students is how to properly integrate clinical knowledge to facilitate their learning, and consolidate useful basic scientific knowledge for future clinical practice. We investigate whether dual‐lecture mode is a useful tool, comparing with the mono‐lecture mode in the first year medical course. Our survey results indicated about 70% of students strongly agree or agree that a dual ‐ lecture would be more effective than the traditional mono‐lecture mode. The dual‐lecture mode may better integrate basic science knowledge and clinical information during the first year medical education, and serve as a bridge for students to become active learners.
Determination of Escherichia coli Genes Involved in Resistance to DNA Alkylating Agents
Authors: Bellou Sirine , Muenter Mark , Travers Meghan , Leifer Becky , Baig Samir , Kramer Caitlin , Qi Macee , Joshi Christopher , Carlson Alyssa , Conway Charles , Beuning Penny
Published: November 2023

DNA repair is a vital aspect of cellular life that restores DNA from damage often caused by environmental aggressions, such as chemical mutagens. Alkylating agents, a common family of DNA damaging agents that form adducts on DNA, are frequently used for chemotherapy as well as for industrial purposes, thus the importance to deepen our understanding of their mechanisms of damage and the cellular responses to these agents. In order to identify genes that confer resistance to alkylating agents, our research focuses on the ability of twenty‐seven genes in Escherichia coli to confer resistance to chloroacetaldehyde (CAA) and styrene oxide (SO). An assay has been designed to assess survival of the strains, each of which possess a deletion of a specific gene, upon exposure to different concentrations of the alkylating agents relative to the wild‐type strain. Strains in which recJ or nfo were deleted were sensitive to SO, whereas strains lacking recA were sensitive to both SO and CAA. The genes deleted in the strains that show drastic killing were selected for complementation, which would confirm the role of each specific gene in the repair mechanism.

Support or Funding Information

American Cancer Society
Functional Significance of Meiotic Proteins in the DNA Repair System of Bdelloid Rotifers
Authors: Dickinson Lauren E , McReynolds Jeanita C , Schurko Andrew M
Published: November 2023

Bdelloid rotifers are microinvertebrates that repair DNA double‐strand breaks (DSBs) with an efficiency unparalleled in other eukaryotes. These animals inhabit transient aquatic environments and survive frequent desiccation by entering a state of anhydrobiosis. Their DNA repair system is necessary for repairing the DSBs induced by extreme desiccation. Bdelloids have long been considered ancient asexuals that have survived without males and sex for millions of years. Evidence for canonical meiosis and sexual reproduction in bdelloids is absent, although some form of atypical genetic exchange might occur in some populations. Despite the long‐standing absence of meiosis in bdelloids, their genomes contain four genes that encode proteins (HOP1, SPO11, MSH4, and MSH5) known to be specific to meiosis in other eukaryotes. Since bdelloids lack meiosis yet possess a noteworthy DNA repair system, the objective is to investigate the potential role of meiotic proteins during DNA repair. We used real‐time PCR to quantify meiotic gene expression in the bdelloid Adineta vaga following exposure to 280 Gray of ionizing radiation (which induces DSBs). Genes encoding meiotic proteins were upregulated, relative to non‐irradiated A. vaga. Western blots using custom antibodies for HOP1, SPO11, MSH4, and MSH5 from A. vaga revealed that these proteins are produced in bdelloids. To characterize binding partners of these meiotic proteins during DNA repair, we generated 6×histidine‐tagged copies of HOP1, SPO11, MSH4, and MSH5 from A. vaga. Pull‐down assays using the histidine‐tagged proteins were done to identify the proteins that interact with HOP1, SPO11, MSH4, and MSH5 in irradiated and non‐irradiated bdelloids. These findings will shed light on the functional significance of these meiotic proteins in bdelloids. Overall, this study represents the first genetic and proteomic data to address whether meiotic genes in bdelloids have evolved a novel function in DNA repair, which could justify the maintenance of these genes in an ameiotic lineage. Data supporting a role for “meiotic proteins” in DNA repair would be the first evidence of a non‐meiotic function for these genes and could be used to further our understanding of DNA repair in eukaryotes.

Support or Funding Information

This work was supported by grants from the NCRR (5P20RR016460) & NIGMS (8P20GM103429) from the NIH to AMS, and a Summer Undergraduate Research Fellowship from the Arkansas Department of Higher Education to LED.
DNA Repair Proteins XLF and XRCC4 Interact With Telomeric Proteins TRF1 And RAP1
Authors: Castle Eric Anthony , Zaibaq Pilar , Pham Quoc‐Khanh , Aviles Yicenia , Hou Jianyi , Vu Kimberly Thien , Fahmy Nicole , Guirette Melanie , Ruff Aaron , Jain Renu , Jabbur James , Ribes‐Zamora Albert
Published: November 2023

Non‐homologous end‐joining (NHEJ) is the main repair pathway for processing double‐strand DNA breaks in Eukarya. In mammalian cells, NHEJ is mediated by a complex of proteins comprised of the Ku subunits (Ku70 and Ku86), Ligase IV, XRCC4, Artemis, DNA‐PKcs and XLF. Paradoxically, several of these NHEJ proteins are also found at telomeres, despite the fact that one of the main functions of the complex structure at telomeres is to prevent NHEJ proteins from recognizing the natural ends of chromosomes as DSBs. For instance, Ku is present at telomeres through interactions with resident telomeric proteins like TRF1 and TRF2, as is the case for DNA‐PKcs. Currently, the telomeric status of several NHEJ proteins is still unknown. Using protein fragment complementation assays, we have found interactions between two NHEJ proteins, XLF and XRCC4, with TRF1 and RAP1, two proteins present at telomeres. Co‐localization studies with TRF2 indicate that TRF1 interactions with XLF and XRCC4 occur at telomeres, while XLF‐RAP1 and XRCC4‐RAP1 interactions occur throughout the nucleus. Mutations that disrupt XRCC4‐XLF filament formation did not impair XRCC4 or XLF interaction with TRF1. Similarly, XRCC4 mutations in known phosphorylation sites for ATM and DNA‐PKcs did not impair XRCC4‐TRF1 interaction nor did they impair XRCC4 localization at telomeres. Overall, these results suggest that XRCC4 and XLF can localize at telomeres through direct interaction with telomeric resident proteins.

Support or Funding Information

Supported by the UST/HCCS STEM Scholars Program (P031C110128‐12) from the US Department of Education and by the University of St Thomas Cullen Smith Chair of Biology
Distinct Kinetic Phases Associated With Specific Transcription Factors Differentially Regulate IL1B and TNF Genes
Authors: Pulugulla Sree H , Adamik Juraj , Grillini Andrea N , Galson Deborah L , Auron Philip E
Published: November 2023

Interleukin 1b (IL‐1β) and Tumor Necrosis Factor α (TNFα) are pro‐inflammatory cytokines that are normally expressed and released by monocytes in response to infection and injury. These cytokines are important mediators of inflammatory responses and intracellular communication in the body against infection. However, chronic overexpression of these cytokines are involved in many inflammatory diseases such as sepsis, osteo‐ and rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, gout and asthma. To understand the transcriptional kinetics of these genes in THP‐1 monocytes, we first examined the 24‐hour mRNA kinetic profile following the Lipopolysaccharide (LPS) or Phorbol 12‐Myristate 13‐Acetate (PMA) stimulation. IL1B gene showed two distinct profiles: a rapid‐vigorous initial phase (0–4 hours) followed by decreased continuous expression (4–24 hours), whereas the TNF gene showed only a rapid‐transient initial induction (0–4 hours) followed by a complete shut‐down in the later stage (4–24 hours). In contrast, PMA generated only a delayed but sustained lower level of expression (7–24 hours) for IL1B, with only a minute effect on TNF. Next, in order to understand the mechanisms involved in these distinct modes of IL1B and TNF gene regulation, we performed experiments comparing the kinetic mRNA expression patterns of these genes, in the presence of various transcription factors inhibitors that are known to play a role in the transcription of IL1B and TNF. For IL1B, rapid‐vigorous initial phase expression was dependent on both NF‐κB and C/EBPβ transcription factors, whereas the late phase continuous expression required C/EBPβ and HIF1α, and was independent of NF‐κB. For TNF, early phase expression was almost exclusively dependent on NF‐κB, with minimal involvement of C/EBPβ. TNF gene was completely shutdown in late expression phase with no dependence on NF‐κB, C/EBPβ or HiF1α. This indicates a role for HIF1α in late sustained expression of IL1B replacing NF‐κB. Also, either LPS or PMA increased the transcription of continuously expressed Hif1a gene in a NF‐κB dependent manner. Finally, when the THP‐1 cells were re‐stimulated with LPS after 24‐hour initial LPS stimulation, the IL1B mRNA profile was similar to the initial LPS stimulation profile, with a rapid‐transient expression followed by a decreased continuous expression. In contrast, the TNF gene was completely refractive to LPS re‐stimulation (Endotoxin tolerance). Also, our results demonstrated that extracellular succinate in the media was important for IL1B gene re‐stimulation.
Analysis of nuclease hypersensitive‐equivalent sites in SV40 by Next Generation Sequencing
Authors: Milavetz Barry , Balakrishnan Lata , Woods Benjamin , Christensen Kendra
Published: November 2023

Chromatin fragments obtained by sonication or Micrococcal nuclease digestion of SV40 minichromosomes or disrupted virions were analyzed by Next Generation Sequencing (NGS) to determine the location of regions of the SV40 genome differentially sensitive to either treatment. Both treatments yielded samples which upon analysis allowed us to identify regions of hypersensitivity and moderate sensitivity. With sonication all of the samples contained regions of hypersensitivity centered on nucleotides 141 and 221 and regions of moderate sensitivity surrounding these sites which included the 21 base pair repeat regions and enhancers. Following Micrococcal nuclease treatment Hypersensitive sites were found in minichromosomes centered on nucleotides 30 and 92 and in chromatin from disrupted virions centered at nucleotide 106. Micrococcal nuclease also generated a pattern of peaks and valleys based upon the number of reads at each nucleotide consistent with the location of nucleosomes. While the pattern was very similar throughout most of the SV40 genome in minichromosomes and chromatin from disrupted virions, the pattern differed significantly in the regulatory region. In minichromosomes we observed a nucleosome‐free region extending from approximately nucleotide 5000 to nucleotide 250 encompassing most of the early and late transcription regulatory sequences and the origin of replication. In chromatin from disrupted virions we observed a very small open region which at most included only the enhancers.

Support or Funding Information

This work was supported by grants from NIH AI094441 (to BM) and NIH GM098328 (to LB)
Target Discrimination in the Nonsense‐Mediated Decay Pathway Requires ATPase‐dependent Release of Upf1
Authors: Lee Suzanne Rebecca , Pratt Gabriel , Martinez Fernando , Yeo Gene W , Lykke‐Andersen Jens
Published: November 2023

RNA binding proteins (RBPs) play key roles in gene expression, involved in every aspect of RNA metabolism. RBPs are generally thought to identify their target RNAs through affinity for specific RNA elements or bound proteins. However, this mechanism of target recognition has limited utility for RBPs functioning in RNA quality control pathways, which target aberrant RNAs that have the potential to vary greatly in sequence and associated proteins. Here we present several lines of evidence suggesting that in the nonsense‐mediated decay (NMD) quality control pathway, target discrimination involves the selective release of the RNA helicase Upf1 from non‐target mRNAs, rather than Upf1‐target specific affinity. By examining Upf1 association with reporter mRNAs and the mRNA transcriptome, we found that Upf1 mutations preventing ATP hydrolysis eliminate target discrimination, elevating the association of NMD factor complexes with both target and non‐target mRNAs. Conversely, Upf1 mutants with enhanced ATPase activity are severely compromised in association with both targets and non‐targets. Moreover, ATP hydrolysis‐dependent release of Upf1 from non‐target mRNA is faster than release from target mRNAs in cell extracts. Intriguingly, known modulators of NMD depend on the ATPase activity of Upf1 to limit Upf1‐non‐target association. Together, our findings suggest that mRNP‐regulated ATP hydrolysis‐dependent release is a previously unappreciated mechanism by which a regulatory RBP can achieve target specificity. This mechanism may be particularly advantageous for RNA quality control pathways, which need to continuously survey the RNA population for those that deviate from the norm.

Support or Funding Information

This study was supported by a Helen HayWhitney Postdoctoral Fellowship to S.R.L., NIH grant R01

GM099717 to J.L.‐A.; NIH grants NS075449, HG004659, HG007005, to G.W.Y.; ; and a National Science Foundation Predoctoral Fellowship to G.A.P. F.J.M. is supported in part by a University of California, San Diego, Genetics training grant from the National Institute of General Medical Sciences (T32 GM008666).
The Production of ExtraCellular Vesicles Is Increased after Heat Shock
Authors: De Maio Antonio , Hawisher Dennis , Lara Bernardo , Cauvi David M.
Published: November 2023

When organisms are exposed to extreme conditions or stresses homeostasis is compromised. The natural cellular response to changes in homeostasis is mediated by the expression of a family of proteins coined heat shock proteins (hsp). These proteins are involved in the stabilization of basic cellular processes to preserve cell viability and to guaranty the return to homeostasis. In addition, they protect cells from subsequent insults in a time‐dependent fashion. The bulk of hsp function occurred within the cytosol and subcellular compartments. However, hsp have also been found outside cells acting as signaling molecules directed at activating a systemic response to stress. Extracellular hsp are released after cell death by necrosis in a soluble form. Hsp are also exported by an active mechanism independent of cell death, in which hsp are associated with extracellular vesicles (ECV). Indeed, hsp have been detected in numerous preparations of ECV from different sources. We investigated the release of ECV after heat shock (HS) in human hepatoblastoma cells (HepG2). We observed a rapid production of ECV within a few hours after the insult. There were no differences in the size of vesicles derived from HS and control cells. However, HS cells produced between 2–3 folds more ECV than non‐stressed cells. Analysis of the biochemical composition of ECV after HS revealed the presence of Hsp70, the major inducible form of hsp. Stress ECV were highly enriched in other cellular components, including tubulin and actin, in comparison with vesicles derived from stressed cells. These observations suggest that the mechanism of ECV release is different between stress and normal conditions. Finally, we propose that stress ECV induce a systemic response directed at avoiding the propagation of the insult, which has been named the “stress observation system” (SOS).
Mortalin Caught in Action: Structures of the Nucleotide Binding Domain in the Apo, ADP‐bound and AMP+PPi‐bound states
Authors: Moseng Mitchell , Mignery Matthew , Larney Paige , Emerson Corey , Nix Jay C , Page Richard C
Published: November 2023

The heat shock protein 70, Hsp70, chaperone family plays key roles in cellular homeostasis and stress response. Hsp70s not only regulate the folding of nascent proteins, but also prevent aggregation, promote disaggregation, and refold misfolded proteins. The mitochondrial heat shock protein Mortalin functions in a variety of processes such as apoptosis, cellular stress response, mitochondrial protein import and quality control, Fe‐S cluster biogenesis, mitochondrial homeostasis, and regulation of p53, an important tumor suppressor. Mortalin is implicated in neurodegenerative diseases, and is known to play an anti‐apoptotic role in cancer cells. The Page laboratory has elucidated crystal structures of the nucleotide binding domain of human Mortalin in the apo, ADP‐bound, and AMP‐bound states at 2.8 Å, 2.78 Å, and 1.75 Å resolutions, respectively. The crystal structure of Mortalin‐NBD with the modified ADP homolog N6P‐ADP bound has also been determined at 1.49 Å. The overall structures and active site organizations are similar, with a few key differences in side chain positions. Our data identifies specific residues in the nucleotide binding pocket, which are not conserved in other Hsp70‐family members, that lead to lower nucleotide affinity and slower turnover relative to cytosolic Hsp70. Our structural data will also contribute to the understanding of disease‐associated Mortalin mutations and to improved Mortalin‐targeted compounds with the aim of influencing the function of Mortalin. Regulation of Mortalin has been proposed as a potential avenue for the treatment of cancer and neurodegenerative diseases.
Compulsive Grooming and Hyperactivity in Nicastrin Conditional Knockout Mice
Authors: Dries Daniel R. , Zhu Julia , Birnbaum Shari G. , Yu Gang
Published: November 2023

The gamma‐secretase complex is a proteolytic machine of four subunits and is involved in the generation of the pathogenic and neurotoxic beta‐amyloid peptides found in the brains of Alzheimer's patients. Germline deletion of any of the four gamma‐secretase components results in embryonic lethality, necessitating tissue‐ and/or age‐specific deletion. Conditional deletion of presenilin (the catalytic subunit) or nicastrin (the substrate receptor subunit) from neurons in the mouse forebrain results in mild cognitive defects and neuronal loss. Here, we asked whether gamma‐secretase activity in other brain cell types (i.e. glia) also contributes to behavioral phenotypes. To this end, we have developed a conditional knockout mouse in which nicastrin has been specifically eliminated from oligodendrocytes, the myelinating cells of the brain. These mice are hyperactive and display reduced anxiety. Adult mice appear do not display gross brain pathology, although adult mice do have hypomyelination in the central nervous system. The work presented suggests that elimination of gamma‐secretase activity from oligodendrocytes may result in altered neuronal circuitry to elicit behaviors consistent with obsessive‐compulsive disorder and attention‐deficit‐hyperactivity disorder, as well as some of the non‐cognitive symptoms of Alzheimer's disease.

Support or Funding Information

This work was supported by NIH grants F32 AG031625, R01 AG023104, R01 AG029547, the Welch Foundation (I‐1566), the Hartwell Foundation, and the Alzheimer's Association.
Liquid‐Liquid Phase Separation of hnRNP A2 and Implications for Structure and Disease Propagation
Authors: Chabata Charlene Vongai , Ryan Veronica , Fawzi Nicolas Lux
Published: November 2023

A family of RNA‐binding proteins characterized by the presence of putatively disordered domains containing high glutamine, asparagine, and aromatic content have recently been linked to the formation of intracellular puncta and neurodegenerative disease‐associated aggregates. One member of this family, heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2), is known to aggregate and contribute to the pathogenesis of multisystem proteinopathy (MSP) and inclusion body myositis (IBM). HnRNPs dynamically associate into large protein‐RNA complexes, termed ribonucleoprotein (RNP) granules, which have important roles in RNA processing, transport, and sequestration. These granules may act as nucleation sites for aggregates in neurodegenerative diseases. The precise mechanism by which hnRNPA2 exerts this toxic influence is unknown, therefore we performed structural studies on hnRNP A2 to determine how granules nucleate aggregates. Here, we used differential interference contrast microscopy and fluorescence recovery after photo bleaching to show that the isolated disordered domain of hnRNPA2 and its disease mutant D290V form a liquid‐liquid phase separated state mimicking RNP granules in vitro. We used the same methods to investigate how the full length protein changed the dynamics of the liquid‐liquid phase separation. Furthermore, using solution NMR, we determined the structure of monomeric wild‐type and D290V hnRNPA2. These results lay the foundation for observing liquid‐liquid phase separated states of disorder proteins with atomistic resolution.
Global Ubiquitylome Profiling for the Identification of Drug Targets in Cancer
Authors: Udeshi Namrata D , Kronke Jan , Fink Emma , Svinkina Tanya , Schenone Monica , Ebert Benjamin , Carr Steven A
Published: November 2023

Mechanisms of action for drugs targeting ubiquitin E3 ligases that show efficacy in disease treatment have been extremely difficult to establish. Through advances in proper utilization of ubiquitin enrichment reagents and instrumentation, we now routinely characterize unprecedented numbers of ubiquitylation sites in disease relevant samples to reveal cancer‐relevant substrates. Lenalidomide is a highly effective drug for treatment of multiple myeloma but the precise mechanism of action has not been established. Previous work found that thalidomide, an analogue of lenalidomide, binds and modulates the activity of the CRBN‐DDB1 E3 ligase. We hypothesized that the pleiotropic effects of lenalidomide may be caused by altered ubiquitination of targets. Quantitative proteomics using SILAC revealed that transcription factors IKZF1 and IKZF3 have altered abundance and ubiquitination status upon lenalidomide treatment in multiple myeloma cells. Protein interaction analyses showed that IKZF1 and IKZF3 bind to CRBN and that binding is enhanced in the presence of lenalidomide. Multiple myeloma cells were found to be dependent on IKZF1 and IKZF3 expression. Knockdown of these proteins inhibited cell growth while expression of the lenalidomide‐resistant IKZF3 conferred lenalidomide resistance of MM1S cells. Here we present findings that reveal that lenalidomide modulates the CRBN‐DDB1 ligase to increase ubiquitination of two transcription factors that would otherwise be considered “undruggable.” Our findings reveal a novel mechanism of action for a therapeutic agent; alteration of ligase activity leading to selective degradation of specific targets. This work will further discuss the mechanism of action for Lenalidomide in MDS 5q‐.

Support or Funding Information

This work was supported by funding from the NIH (R01HL082945, P01 CA108631), Leukemia and Lymphoma Society, Evans Foundation, Starr Cancer Consortium, SPARC consortium, National Human Genome Research Institute (RL1‐HG004671) and by the German Research Foundation (DFG, Kr‐3886/1‐1).
Measurements of Endogenous Opioid Peptides in the Rat Brain Using Online‐Preservation Microdialysis with Mass Spectrometry
Authors: Robles Dagoberto , Laude Nicholas D. , Kramer Catherine , Lemister Eric , Meske Diana , Navratilova Edita , Heien Michael , Porreca Frank
Published: November 2023

In the mammalian central nervous system, opioid receptors and their endogenous ligands are involved in mediating reinforcing effects of natural rewards such as food. Endogenous opioid system also plays an important role in pain and analgesia. Human neuroimaging studies have demonstrated that endogenous opioid peptides (EOPs) are released in cortical and subcortical regions of the brain during appetitive food consumption, as well as following an acute pain stimulus. Dynamic monitoring of EOPs in laboratory animals, however, has been limited due to the low concentration of EOPs that are released and their high susceptibility to degradation. To address these issues, on‐line preservation microdialysis with nano liquid chromatography and tandem mass‐spectrometry (nano LC‐M) has been developed to facilitate measurements of EOPs and prevent peptide degradation. By using this method, in vivo ultra‐trace measurements of leucine and methionine enkephalins was successfully achieved in conscious and freely moving rats. This approach serves as a powerful tool to quantitatively monitor endogenous opioids in cortical and subcortical regions of the brain in behaving rats.

Support or Funding Information: Department of Pharmacology, Arizona Health Sciences Center, University of Arizona, Tucson, AZ, 85721, USA Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721, USA
Characterization of Vitamin D and Serotonin Pathway Variations in Patients with Irritable Bowel Syndrome
Authors: Dussik Christopher M , Grozić Aleksandra , Hockley Maryam , Zhang Lin , Park Jin , Wang Jie , Nickerson Cheryl , Yale Steven H , Foxx‐Orenstein Amy , Sandrin Todd , Jurutka Peter
Published: November 2023

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder associated with chronic abdominal pain along with recurrent diarrhea, constipation, or both that afflicts millions of people worldwide. Currently, the etiological foundation of the disorder remains enigmatic. However, there is mounting evidence suggesting that serotonin may play a critical role in the pathophysiology of IBS. DNA microarrays analyzing the transcriptomes of 11 IBS patients versus 13 asymptomatic control patients have identified a series of 858 differentially‐expressed genes that may characterize the IBS pathological state. Gene ontology (GO) analysis of this subset of genes revealed that nodes featuring serotonin‐related pathways displayed the highest number of functions relative to other GO groups. Quantitative real‐time polymerase chain reaction (qRT‐PCR) analysis of IBS patient‐derived RNA displayed reduced expression of the rate‐limiting enzyme of the serotonin biosynthetic pathway, tryptophan hydroxylase 1 (TPH1), relative to a pool of RNA comprised of eight control patients. Further, this analysis revealed potential compensatory regulation of several other genes integral to the maintenance of serotonin metabolism. In addition, concentrations of serum 25‐hydroxyvitamin D in both the IBS and control sampled populations indicated a trend towards decreased vitamin D levels within the IBS cohort, regardless of IBS subtype. Finally, vitamin D was shown to be effective in regulating the expression of several key serotonin metabolism genes, alluding to a potential link between vitamin D and serotonin pathways modulating IBS. These findings suggest that the pathogenesis of IBS may be associated with abnormal serotonin metabolism and/or vitamin D deficiency, and provide novel insight into possible treatment regimes.

Support or Funding Information

Wisconsin Network for Health Research
Large scale structural rearrangement provides dual control over the catalytic and membrane binding activity of a bacterial serine hydrolase
Authors: Johnson R. Jeremy , Smith Mackenzie , Hart Whitney
Published: November 2023

The serine hydrolase FTT258 from the highly pathogenic bacterium Francisella tularensis exists in distinct open and closed conformations. Based on comparison to the homologous human acyl protein thioesterase, this large structural rearrangement was proposed to provide connected control over the catalytic and membrane binding activity of FTT258. Using comprehensive mutagenesis and differential kinetic and membrane‐binding measurements, we determined the contribution of a key flexible loop to controlling the structural rearrangement and biological activity of FTT258. For the catalytic activity, a centrally located tryptophan residue (Trp66) was deemed essential, with the resulting alanine variant showing complete ablation of enzyme activity. Other amino acids localized near this essential tryptophan residue, including Met63, Arg64, Tyr67, and Asp68, also significantly decreased the hydrolase activity, indicating a critical target area for controlling the enzyme activity and structural rearrangement of FTT258. Combinatorial variants containing the Trp66 substitution with other important residues showed enzymatic activity similar to the Trp66 variant alone, suggesting a controlling role for this residue. Liposome‐binding experiments with the tryptophan variants did not however show significant changes in membrane binding activity. Instead, removal of a proximal positively‐charged arginine residue (Arg64) significantly decreased the membrane binding activity of FTT258. Together, the conformational change in FTT258 dually controlled the catalytic and membrane binding activity of FTT258, but through distinct subsections of a key flexible loop.

Support or Funding Information

Funding provided by a Butler University Holcomb research award and a Senior Research Grant from the Indiana Academy of Sciences.
Dynamic Equilibria Studies of FTT258, a Serine Hydrolase from Francisella tularensis
Authors: Rabin Perry , Johnson R. Jeremy
Published: November 2023

Francisella tularensis is a highly infectious bacterium and the causative agent of tularemia. In this project, we investigated two different structural forms of FTT258, a serine hydrolase from F. tularensis and a potential novel drug target. Specifically, the goal of my project was to determine the role of the active site of FTT258 in controlling the equilibria between its inactive and active forms. Substitutional analysis combined with size exclusion chromatography was used to analyze the importance of amino acids in the active site and in a flexible loop region of FTT258 to controlling this equilibria. Although amino acids within the flexible loop were found to not control the structural equilibria of FTT258, deletion of the catalytic serine active site disrupted the transition between the inactive and active forms of FTT258. Furthermore, the structural composition of FTT258 was found to be highly concentration dependent. These experiments confirmed the essential role of the active site and catalytic amino acids in controlling the structural shifts in FTT258. Understanding this regulation will allow for new methodology in purifying the different forms of FTT258 and analyzing the potential of FTT258 as a novel drug target.

Support or Funding Information

Funding provided by a Butler University Holcomb research award and a Senior Research Grant from the Indiana Academy of Sciences.
DISSOCIATION CONSTANTS FOR REVERSIBLE HOMODIMERS OF RECOMBINANT MONOMERIC HUMAN ACETYLCHOLINESTERASE
Authors: RADIC ZORAN
Published: November 2023

Size exclusion chromatography profiles of highly purified monomeric recombinant human acetylcholinesterase (hAChE; EC 3.1.1.7) at micro molar concentration range reveal elution volumes smaller than expected for a monomeric globular protein comparable in size to the bovine serum albumin. Dilution of hAChE to the nano molar concentration range results in the elution peak shifts towards larger volumes indicating formation of hAChE monomers. This observation is consistent with small angle X‐ray scattering (SAXS) experiments on hAChE that indicate formation of reversible homodimers of the micro molar monomeric recombinant enzyme in solution (Blumenthal et al., EB2016 abstract).

FPLC chromatography of a series of concentrations of recombinant monomeric hAChE in the range of 0.00001 – 1 mg/ml (150 pM‐15uM) was performed to assess concentration dependent elution shifts and determine dimer dissociation constants in 50 mM Tris/HCl, 100 mM NaCl, pH 7.4 at 4 ºC. Peaks of enzyme activity in FPLC elution fractions were detected using Ellman spectrophotometric activity assay. Wild type hAChE constructs with and without N‐terminal FLAG tag sequence as well as Tyr337Ala/Phe338Ala double mutant hAChE not containing FLAG tag were assayed. Size of the hAChE active center gorge in the choline binding domain was increased in this double mutant. Corresponding dimer dissociation constants were determined to be in the range of 1 – 2 uM for both wild type and mutant hAChE, consistent with hAChE oligomerisation interface residing outside of the active center gorge opening as indicated by crystallographic dimers of hAChE.

Support or Funding Information

This work was supported by the Counter ACT Program, National Institutes of Health Office of the Director, Grant 1U01NS083451 from NINDS.
OVERLAY INDEPENDENT COMPARISON OF ACETYLCHOLINESTERASE 3D STRUCTURES REVEALS LIGAND SPECIFIC BACKBONE CHANGES UPON REVERSIBLE AND COVALENT BINDING
Authors: RADIC ZORAN , Rohrer Jacqueline , Sidhom Morgana , Han Jingting
Published: November 2023

We developed a simple overlay independent comparison of protein 3D structure alpha carbon backbones and applied it to PDB deposited structures of acetylcholinesterase (AChE; EC 3.1.1.7) in order to identify backbone domains amenable to ligand binding associated conformational flexibility. Distance of each alpha carbon in the structure to a frame of reference point was determined first. We then calculated differences in corresponding distances between two different PDB structures and plotted as a function of amino acid number, or color coded and mapped onto ribbon or surface representation of an AChE structure. To reduce the influence of crystal packing we averaged corresponding distances determined in individual chains of crystallographic dimers before comparing different structures. We were able to observe systematic, previously undetected, conformational changes upon binding of tight and weak reversible ligands as well as covalent conjugates, phosphonylated and aged. Analogous structures of AChE from different species, human mouse and fish were compared revealing consistent conformational changes. Finally, our simplified comparative 3D structure analysis was contrasted to the sophisticated error‐scaled difference distance matrix analysis.

Modest to moderate fluctuations of AChE backbone conformation upon complex or adduct formation may remain undetected in classical overlay analysis. Small, but systematic conformational changes in protein structures determined at low, 100 K temperature conditions, may prove as an important indicator of larger conformational changes at physiological conditions relevant for structure based design of modern therapeutics.
Structural, Biochemical Characterization, and Evolutionary Origin of Alcohol/Aldehyde Dehydrogenases (ADHE) in Entamoeba
Authors: Leitao Joshua , Espinosa Avelina
Published: November 2023

Bifunctional alcohol/aldehyde dehydrogenase (ADHE) enzymes are essential for anaerobic respiration in protists and bacteria (i.e. group‐A‐Streptococcus, Entamoeba histolytica, Pasteurella multocida, Escherichia coli, Clostridium perfingens, Clostridium difficile). Structural and biochemical analysis of ADHE enzymes would contribute to the understanding of their evolution in prokaryotic and eukaryotic lineages. We have previously characterized the bifunctional Entamoeba histolytica alcohol/aldehyde dehydrogenase (EhADH2). Since EhADH2 is required for amoebic survival, it is an ideal target for the search of antiamoebic alternatives. Here we elucidate the ADHE structure and function of six glycolytic proteins from six Entamoeba varieties (isolated, cloned and expressed from E. dispar, E. invadens‐IP‐1, E. invadens VK, E. moshkovskii Snake, E. moshkovskii Laredo, and E. terrapinae). Alcohol and aldehyde dehydrogenase activities from each ADHE enzyme were analyzed. Considering that the evolutionary origin of ADHE enzymes appears to be from diverse bacterial lineages, the purpose of this study is to examine the kinetic properties and efficiency of Entamoeba ADHEs. Concurrently, inhibitory studies using pyrazoline derivatives on Entamoeba growth by targeting its EhADH2 enzyme are being pursued in our laboratory as a novel anti‐amoebic strategy.

Support or Funding Information

American Society for Microbiology (ASM) Undergraduate Research Fellowship
Biodiesel Production via Genetically Altered Lipase from Proteus mirabilis Transesterification in Methyl Acetate
Authors: McDonnell Alaina Nicole , Altier Jake , Carey Demetrius , Witherow D. Scott
Published: November 2023

Lipase catalyzed biodiesel production presents a potential methodology that exceeds the traditionally utilized acid/base protocols. Advantages include economic favorability and convenience due to more efficient catalysis, which leads to less time, starting material, and energy required for production. A lipase has been previously genetically engineered for methanol tolerance and was shown to be an effective catalysis for transesterification reactions utilizing methanol to produce fatty acid methyl esters, or biodiesel, and glycerol. An alternate acyl acceptor to methanol, methyl acetate, produces a fuel stabilizer, triacetin, as opposed to glycerol when used in the transesterification. This methodology has not been previously utilized with the characterization of these genetically engineered lipases, which are also described as dieselzymes. Dieselzyme mutations have been expressed in E.coli using a pET28 bacterial expression vector and purified utilizing immobilized metal ion affinity chromatography. Kinetic optimization was performed for three mutants using p‐nitrophenyl palmitate as substrate at varying pHs and in methyl acetate versus methanol reactions. Further investigation of the dieselzymes includes use of synthetic triglycerides as substrate to continue kinetic optimization.

Support or Funding Information

University of Tampa Department of Biochemistry, Chemistry and Physics Summer Fellowship; University of Tampa Honors Program Fellowship; University of Tampa David Delo Research Professor Grant; University of Tampa Dana Foundation Grant
Divorced women who are in informal unions are most vulnerable to food insecurity
Authors: Dallmann Diana , DALLMANN Marquis Grace S , Colecraft Esi K , Clark Shelley , Aryeetey Richmond
Published: November 2023

Divorced women in Africa experience economic disadvantages. This analysis uses baseline data from the Nutrition Links project to determine if marital experience of women is associated with food insecurity (FI). 1094 Households (HH) with an infant (< 12 months) were selected from 137 villages in the Upper Manya Krobo district in Ghana. FI was assessed using the Latin‐American and Caribbean Food Security Scale (ELCSA). HH where caregivers were the mothers of the index child and where the ELCSA questionnaire was completed are included in this analysis (n=1065). Internal validity of ELCSA was confirmed using the Rasch model. The relative severity of the items was according to the theoretical framework underlying the ELCSA. Additionally, no substantial DIF CONTRASTS were found when comparing groups by primary education (DIF <0.52 logit), and all items had INFIT values within acceptable range (0.7 to 1.3 logit). Principal Components Analysis was performed with HH assets to create a score as a proxy of wealth. Women's age and age at first pregnancy were 27.1±0.2 and 19.2±0.1 years, respectively. FI affected 57% of HH (27% mild, 18% moderate, and 12% severe). FI was associated with HH wealth score, women's education level, and age at first pregnancy (p<0.05) but not with age. Sixty percent of women in severely food insecure HH had not finished primary school compared to 44% in food secure HH (p=0.01). Age at first pregnancy and wealth score decreased with severity of FI (p<0.01). Marital experience was also associated with FI. Women who were in an informal union (i.e.: cohabiting or in a union), either divorced or never married, had higher odds for being food insecure (OR: 1.68 and 2.27, respectively) compared to women who were in their first marriage (p<0.01). After adjusting for education level, age, age at first pregnancy, and wealth, only divorced women in informal unions still had higher odds for being food insecure when compared to those in their first marriage (OR: 1.59, p<0.05). These results are consistent with previous publications, implying that poorer never‐married and divorced women may enter into informal relationships out of economic necessity. They also suggest that much more attention must be placed in better understanding marital history of women and the social aspects that are linking these informal unions to FI. Considering that divorce is not uncommon, and that informal unions are increasing in Ghana, a large number of women and their children could be experiencing FI. Finally, this study highlights the importance of identifying divorced women, specially divorced women in new informal unions as a vulnerable population for interventions against FI.
Antiseptic mouthwash use and effect on resting metabolic rate and blood pressure among healthy females: a randomized, double‐blind, crossover study
Authors: Sundqvist Michaela Linn , Lundberg Jon , Weitzberg Eddie
Published: November 2023

BACKGROUND

Inorganic nitrate, from dietary or endogenous sources can be metabolized in vivo to generate bio‐active nitrogen oxides including nitric oxide (NO) with robust NO‐like cardiovascular and metabolic effects. The bio‐activation of nitrate involves its active accumulation in saliva followed by a sequential reduction to nitrite, nitric oxide, and other reactive nitrogen species. Commensal oral bacteria have a key role in nitrate bio‐activation since they reduce salivary nitrate to the more reactive nitrite anion.

A recent study demonstrates that resting metabolic rate (RMR) decreases significantly after dietary supplementation with nitrate. This effect is likely caused by bio‐active nitrogen oxides directly interacting with mitochondria to affect proton leak and other parameters. It is unclear however if endogenous nitrate, derived mainly from the oxidation of NO in blood and tissues, also has this effect. We recently showed that blocking oral bacterial conversion of nitrate to nitrite with an antiseptic mouthwash, leads to a reduction in salivary and plasma nitrite levels together with a concomitant increase in blood pressure. Such effect indicates that endogenous nitrate is sufficient to have physiological effects after reduction to nitrite and then NO.

OBJECTIVE

Here we studied the effects of an antiseptic mouthwash on nitrate and nitrite kinetics and RMR in humans.

DESIGN

In a randomized, double‐blind, crossover study, we measured the RMR using indirect calorimetry in 17 healthy young females after a 3‐d intervention with a chlorhexidine‐containing antiseptic mouthwash. The subjects were instructed to use antiseptic mouthwash or placebo 3 times a day after meals. We measured 24h ambulatory blood pressure (ABP) and collected saliva‐ and blood samples in the end of the intervention period.

RESULTS

Saliva nitrate levels were significantly increased after mouthwash compared to placebo (mouthwash: 1443 ± 262 μmol/L; placebo: 652 ± 199 μmol/L, p = 0.002), but nitrate and nitrite levels in plasma showed no significant changes. There was no significant increase in RMR after mouthwash compared to placebo (p = 0.982) and saliva nitrate did not correlate with RMR. The 3‐d use of antiseptic mouthwash did not significantly change ABP.

CONCLUSIONS

Differently from a recent study, the use of an antiseptic mouthwash did not affect circulating nitrite levels in this group of healthy young females, despite markedly reducing salivary conversion of nitrate to nitrite. Similarly, RMR was not affected by this intervention. Further studies are needed to clarify the role of endogenous nitrate, derived from NOS activity, in regulation of NO homeostasis in blood and tissues.
Anthocyanins Improved Mitochondrial Dysfunction in Mice of Non‐alcoholic Fatty Liver Disease Induced by High Fat Diet
Authors: Ling Wenhua , Shen Tianran , Tang Xilan , Jiang Xinwei
Published: November 2023

Recent studies indicate that mitochondrial dysfunction plays a critical role in the pathogenesis of non‐alcoholic fatty liver disease (NAFLD. The goal of this study is to investigate whether anthocyanins (ACNs) from bilberries and blackcurrants possess a protective effect on mitochondrial dysfunction and eventually contribute to attenuation of NAFLD in mice induced by feeding a high fat (HF) diet. Twenty‐four male C57BL6/J mice were randomly divided into three groups with different diets respectively for sixteen weeks: the control group was fed a low‐fat diet and the other two groups were fed a HF diet with ACNs or not. Our results illustrated that ACNs administration at a dosage of 100 mg in 1 kg diet significantly inhibited HF diet‐induced hepatic steatosis and inflammatory cell infiltration. In addition, increase of mitochondrial swelling, decrease of mitochondrial content and damage of mitochondrial biogenesis, oxidative phosphorylation and β‐oxidation were observed in mice fed with a HF diet, which were significantly normalized through ACNs supplementation. This appears to be attributed to activation of phosphorylated AMP‐activated protein kinase (p‐AMPK) and its downstream peroxisome proliferator‐activated receptor‐gamma coactivator‐1α (PGC‐1α). Our work presents the beneficial effects of ACNs on mitochondrial dysfunction in HF‐induced NAFLD mice by promoting activation of AMPK/PGC1α signaling axis, signifying that ACNs may be a useful nutrient for the treatment of NAFLD.

All animal procedures were approved by the Animal Care and Protection Committee of Sun Yat‐Sen University (2013–10).

Support or Funding Information

This research was funded by grants from the National Basic Research Program (973 Program, 2012CB517506) and the National Nature Science Foundation (81172655, 81372994).
Whole Ethanolic Moringa Oleifera Leaf Extract Generates Kinetic Growth Curves of Escherichia coli Comparable to Pharmaceuticals
Authors: Smith Brandon , Orders Tanner , Tallapaneni Jetha , Nair Srijith , Ravi Vivek , Emrani Jahangir , Idassi Joshua , King James , Zwetsloot Kevin A. , Mowa Chishimba
Published: November 2023

Premature births are largely caused by preterm infections. Here, we report the anti‐bacterial activities of the medicinal plant Moringa oleifera (M. oleifera) against Escherichia coli (E. coli), one of the main causes implicated in infections associated with preterm labor. We screened different whole leaf extracts of M. oleifera for anti‐bacterial activities and determined its effects on the overall growth using in vitro kinetic curve liquid broth assays. In our assays, we compared the anti‐bacterial potency of the M. oleifera leaf extracts to commonly utilized antibiotics, such as tetracycline, ampicillin and streptomycin. The pure ethanol leaf extract showed the most anti‐bacterial activity at 20 μg against E. coli, with ethanol and ethanol/water extracts acting in a concentration‐dependent manner, and showed the greatest kinetic change at 6 μg against E. coli and completely inhibited growth, followed by tetracycline at 6 μg compared to control. This study confirms the anti‐bacterial properties of M. oleifera and its potential use as a natural anti‐bacterial agent for the management and prevention of infection‐induced preterm labor.

Support or Funding Information

Office of Student Research, Appalachian State University.