bluuebunny/crossref_metadata_2025 huggingface dataset:
A Study of Unmyelinated Afferent Axons in the Suburothelium in Women with Urinary Stress Incontinence.
Authors: Abtahi Bahareh , Venner Kerrie , Groves Mike , Elneil Sohier
Published: June 2021

Study objective

To study the nerve profile of unmyelinated axons beside the suburothelial myofibroblasts, as neuroeffectors, in comparison to the axons in other sites of the suburothelium.

Methods

This is a prospective study. Bladder biopsies were obtained using rigid cystoscopy from four patients with urinary stress incontinence. Suburothelial nerve bundles were studied at two sites: those adjacent to myofibroblasts (Nmyo) and those located within other sites of the suburothelium (Ns). Axonal contents of unmyelinated nerve bundles and Schwann cells within the suburothelium were studied using transmission electron microscopy.

Results

The mean age of patients was 56 ±15 years. All patients had normal bladder function (maximum cytometric capacity: 554±57 mL). The total of 93 axons beside myofibroblasts and 118 axons in other sites of suburothelium were counted. The mean number of axons per nerve bundle was similar in both groups of nerve bundles (Nmyo: 3.03 vs Ns: 2.95). However, the mean number of large dense cored vesicles (LDCV) and small vesicles (SV) was higher in Nmyo group in comparison to the Ns group

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The values were (Nmyo:16 vs Ns:12.61) for LDCV and (Nmyo: 1.05 vs Ns: 0.79) for SV. The percentage of activated Schwann cells was higher in Nmyo in comparison to the Ns group (52% vs 32.5%).

Conclusion

This study showed that the axonal contents of the nerve bundles adjacent to myofibroblasts had a higher amount of neurotransmitters within them, as compared to nerve bundles elsewhere. This supports the hypothesis that suburothelial myofibroblasts play a role in neuronal signalling.
A Cerebral Clearance Pathway for Amyloid β
Authors: Morris Alan , Hawkes Cheryl , Weller Roy , Carare Roxana
Published: June 2021

Objective

The objective of this study was to define the clearance pathway of Amyloid β (Aβ) from the brain at the ultrastructural level.

Background

Cerebral Amyloid Angiopathy (CAA), the deposition of insoluble Aβ in the walls of cerebral blood vessels, is present in the elderly and Alzheimer's disease (AD) brain. Failure of the clearance of Aβ from the brain along 100‐150nm‐wide basement membranes (BMs) in the walls of capillaries and arteries is thought to contribute to the accumulation of Aβ as CAA. However, the exact localization of Aβ within the BM as it drains from the brain has not yet been determined.

Methods

10 week old male C57/BL6 mice were injected into the hippocampus with 0.5 uL of 10μM biotinylated soluble human Aβ40 before being transcardially perfused with 0.01M PBS, followed by 4% paraformaldehyde. Brains were sectioned and processed by immunocytochemistry for detection of biotin using DAB as chromagen. Sections were then processed for transmission electron microscopy and imaged using a Hitachi H7000 TEM.

Results

Exogenously injected Aβ40 was detected in the BM of hippocampal capillaries 5 minutes after injection.

Conclusions

These results suggest that Aβ40 enters the cerebrovascular BM as it is cleared from the brain and could prove significant in further understanding the pathogenesis of CAA and AD.
Mapping Cortical Plasticity Following Noise Exposure Using c‐Fos Immunoreactivity
Authors: Sirek Paul , Fitzpatrick Sarah , Schormans Ashley , Rajakumar Raj , Allman Brian
Published: June 2021

Using in vivo electrophysiological recordings in rats, our lab has recently observed that high‐intensity noise exposure causes an increase in the number of neurons in the auditory and multisensory cortices that are responsive to visual stimuli (i.e., cortical crossmodal plasticity). To extend this work, the present study is evaluating our hypothesis that this noise‐induced crossmodal plasticity can also be assessed by mapping the activation of the immediate early gene, c‐Fos, across multiple cortical areas in response to visual stimuli. Adult male rats are exposed to a 120dB noise (0.8‐20kHz) for two hours, and the level of hearing loss is assessed with an auditory brainstem response (average hearing loss ~20dB). Fourteen days later, noise‐exposed rats (and age‐matched controls) were subjected to a visual stimulation protocol known to induce c‐Fos activation (200 light flashes; 1‐3 s ITI), followed by transcardial perfusion two hours post‐stimulation. Visually‐responsive neurons in the noise‐exposed rats and controls were confirmed with immunohistochemistry and fluorescent microscopy. If, as hypothesized, we observe an increase in the number of c‐Fos‐immunoreactive neurons in noise‐exposed rats, this would establish that molecular mapping represents a useful tool for studying cortical crossmodal plasticity.
CTCF protein recruits centromeric protein CENP‐E to the centromeric regions through unusual CTCF binding sites
Authors: Xiao Tiaojiang , Wongtrakoongate Patompon , Trainor Cecelia , Felsenfeld Gary
Published: June 2021

Centromere‐associated protein E (CENP‐E) is an integral component of the kinetochore, and has been shown to be essential for chromosome congression during mitosis. However, the molecular mechanism by which CENP‐E is recruited to the kinetochore is poorly understood. In the present study, we report that CENP‐E interacts directly with CTCF, the insulator protein that is involved in large scale organization of chromatin and in insulation. We demonstrate that α‐satellite repeats containing both CENP‐B boxes­­­ and CTCF binding sites in HeLa cells that are arrested in G2/M or Mitosis are co‐occupied by CTCF and CENP‐E. CENP‐E localization appears to depend on CTCF, because deletion of CTCF by siRNA disrupts binding of CENP‐E to the centromeric regions. The CTCF binding sites in centromeric regions are unusual in that most of them carry only the ‘M2′ sequence motif, a 9‐bp long DNA fragment that was originally thought to appear together with the core CTCF binding motif (also called ‘M1′ motif). Furthermore, we provide evidence that the ‘M2′ motif engages only the C‐terminal zinc fingers of CTCF, and binds CTCF quite strongly. Based on these findings, we suggest that CTCF helps recruit CENP‐E to the kinetochore and that the CTCF‐CENP‐E complex may represent a novel structural feature of centromeres.
Variability in HSP90AB1 and HSP90AA1 Are Not Correlated with Phenotypic Differences in Glucocorticoid Receptor‐Mediated Response or Subcellular Hsp90 Localization
Authors: Forst Thomas , Lassalle‐Klein Rose , Knox Rachel , Murphy Patrick
Published: June 2021

Hsp90 is a highly conserved eukaryotic chaperone protein responsible for mediating a multitude of intracellular signaling molecules, including those associated with the glucocorticoid receptor (GR) and stress response. The goal of this study was to identify the extent to which single nucleotide polymorphisms (SNPs) in genes encoding hsp90 correlated with previously observed phenotypic variations in circulating cortisol and hsp90 subcellular localization. Salivary cortisol was measured in healthy volunteers before and after HPA stimulation via mild cardiovascular stress. Whole exome sequencing of a matched participant pair identified HSP90AB1, encoding the stress‐inducible isoform of hsp90, as a potential causal candidate gene warranting further examination. Exons of HSP90AB1 and HSP90AA1 were amplified and sequenced in 20 participants with known differences in cortisol response and hsp90 localization using PCR and the Sanger method. Out of the twenty participants, three HSP90AB1 and eight HSP90AA1 SNPs were found, all of which were registered in the NCBI SNP database. Interestingly, a discrepancy in results was observed between SNPs called by whole exome analysis and those detected by Sanger sequencing. Although no correlation was found between analyzed SNPs, cortisol response, and hsp90 localization, further work utilizing this methodology and consideration of phenotypic polygenicity will likely provide a more comprehensive understanding of hsp90‐mediated GR signaling.
INCREASED RENAL EXPRESSION OF TOLL‐LIKE RECEPTOR‐4 IN GUANYLYL CYCLASE/NATRIURETIC PEPTIDE RECEPTOR‐A GENE‐DISRUPTED FEMALE MICE
Authors: Gogulamudi Venkateswara , Subramanian Umadevi , Mani Indra , Pandey Kailash
Published: June 2021

Toll‐like Receptor‐4 (TLR4) plays a potential role in the innate immune responses by sensing endogenous danger signals associated with the stimulation of pro‐inflammatory molecules. The objective of the present study was to elucidate the impact of guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) gene (Npr1) disruption on TLR4 expression in the kidney tissues of Npr1 null mutant mice. In the present study, 0‐copy (Npr1‐/‐), 1‐copy (Npr1+/‐), and 2‐copy (Npr1+/+) female mice were pre‐treated with rapamycin, the kidneys were collected, for real time quantitative PCR, Western blot, and immunofluorescence analyses. Disruption of Npr1 gene significantly increased TLR4 mRNA by 10‐fold in 0‐copy and 4.5‐fold in 1‐copy mice. The TLR4 protein expression was increased by 5.7‐fold in 0‐copy and 3.2‐fold in 1‐copy mice as analyzed by Western blot; however, immunofluorescence expression of TLR4 was increased by 10‐fold in 0‐copy and 3‐fold in 1‐copy compared with 2‐copy mice. After treatment with rapamycin, TLR4 mRNA expression was reduced by 73% in 0‐copy and 60% in 1‐copy mice and protein expression was decreased by 60 ‐ 64% in 0‐copy and 35 ‐ 38% in 1‐copy mice compared with vehicle‐treated control mice. The present results demonstrate the up‐regulation of TLR4 expression in Npr1 gene‐disrupted mice, which was attenuated after treatment with rapamycin. These findings suggest that Npr1 gene‐disruption promotes the up‐regulation of TLR4 associated pro‐inflammatory immune state.
The Role of Multifunctional RNA Binding Protein OsTudor‐SN in Affecting Storage Protein RNA Transport and Seed Morphology in Rice
Authors: Chou Hong‐Li , Kumamaru Toshihiro , Okita Thomas
Published: June 2021

The major storage proteins of rice, prolamines and glutelins, are synthesized and packaged in the ER lumen and vacuole, respectively. The sorting of these storage proteins into different endomembrane compartments is facilitated by the localization of these RNAs to distinct ER subdomains. A prominent player in RNA localization in rice is Tudor‐SN, a highly conserved RNA binding protein that is well documented to play multiple nuclear and cytoplasmic roles in eukaryotic cells. We hypothesize that Tudor‐SN's multi‐functional properties are due to its modular domain structure consisting of four SN domains, Tudor domain and partial SN domain, where individual domains are responsible for specific processes in gene regulation and RNAs transport. To provide evidence for this view, two TILLING (Targeted Induced Local Lesions in Genomes) mutant lines, EM1084 and PMT64, which have mutations in the third SN and Tudor domains, respectively were identified. Compared to wild type, these mutant lines grow slower and exhibit considerable differences in grain weight and appearance. In situ RT‐PCR showed that RNA localization profile are altered in these mutant lines. PMT64 showed mis‐localization of prolamine RNAs to the cisternal‐ER whereas the targeting of glutelin RNAs was unaffected. EM1084 also showed a similar disruption in prolamine RNAs localization but also exhibited partial mis‐localization of glutelin RNAs to the PB‐ER. These significant differences in RNA localization mediated by mutations in the SN3 domain and Tudor domain, respectively, indicate that different domains of OsTudor‐SN are involved in distinct molecular processes.
Characterization of the Readout Mechanism and the L1 Loop's Role in the p53 Tumor Suppressor Protein Binding Event via Molecular Dynamics Simulations
Authors: Slaw Benjamin , Thayer Kelly
Published: June 2021

A primary target in cancer research, the p53 tumor suppressor protein, is a transcription activator playing an integral role in the regulation of the cell cycle. Through binding to DNA, the protein has the ability to “turn on” other repair proteins to mend damaged DNA, inhibit cellular division, and initiate apoptosis in cells that are damaged beyond repair. Most inactive p53 mutants contain changes primarily localized to the DNA binding domain (DBD). While intensive research has been conducted on different p53 mutants, previous studies have not yet focused on the capability for the system to exhibit direct and indirect readout among various genomic sites. Consequently, the L1 loop, a structural feature of the DBD, participates in binding to sequences exhibiting differences in the readout mechanism. This study aims to characterize the dynamic properties of the p53 binding event. Hydrogen bonding as direct readout will be ascertained from additive energy models and DNA deformation mechanics will be explored as indirect readout from molecular dynamics (MD) simulations. Additionally, this experiment illuminates the contribution of the L1 loop to the p53/DNA binding interface. Molecular models of p53 with DNA expected to have differing hydrogen bonding patterns are constructed and simulated using the Amber12 MD suite. Results from this study inform rational design of novel therapeutics to combat the changes that are hypothesized to cause over 50% of tumorous cancers.
Differential Ankyrin G Binding of Voltage‐gated Sodium and KCNQ2/3 Potassium Channels Is Mediated by An Overlapping N‐terminal Binding Site
Authors: Xu Mingxuan , Cooper Edward
Published: June 2021

Fidelity and robustness of neuronal action potential generation and conduction depends on the co‐localization of voltage‐gated sodium (NaV) and KCNQ2/3 potassium channel conductances at the axon initial segment (AIS) and nodes of Ranvier (NR) in a ratio of approximately 40 to 1. Through molecular convergent evolution, analogous “anchor” sequences within intracellular domains of KCNQ2, KCNQ3 and NaV channels bind Ankyrin G (AnkG), mediating concentration at AISs and NRs. Here, we show that these potassium and sodium channels bind overlapping but distinct sites near the AnkG N‐terminus in a casein kinase 2 (CK2) dependent way. We have identified a minimal AnkG N‐terminal fragment which is sufficient to bind phosphorylated NaV or KCNQ anchors robustly. AnkG's alternatively spliced N‐terminus selectively gates access to those sites. These findings suggest that the NaV: KCNQ channel conductance ratio at the AIS arises from the relative strength of binding to AnkG. Biophysical and electrophysiological data indicate that the balance of these two channel types strongly influences action potential generation at the axon initial segment. Control of this ratio may be a useful therapeutic mechanism for epilepsy and associated mood disturbance. Identification of the binding regions makes possible screening for agents capable of modulating binding and thus neuronal excitability.
Adipocyte‐Breast Cancer Cell Interactions: Preventive Effects of Omega‐3 Fatty Acids
Authors: Aljawadi Arwa , Ouertani Meriam , Siriwardhana Nalin , Scoggin Shane , Gollahon Lauren , Dharmawardhane Surangani , Moustaid‐Moussa Naima
Published: June 2021

According to the American Cancer Society, breast cancer is the most common type of cancer and the second leading cause of death among women in the United States. Menopausal women with obesity face a higher risk of developing breast cancer. Our hypothesis is that omega‐3 fatty acid, eicosapentaenoic acid (EPA) regulates adipocyte‐secreted factors thereby modulating breast cancer cell metabolism. To test this hypothesis, we investigated the interaction between adipocytes and breast cancer cells with emphasis on the role of omega 3 fatty acids in modulating this interaction. To accomplish this, the effects of conditioned media from 3T3L1 preadipocytes/differentiated adipocytes or human mesenchymal stem cells on MCF7 breast cancer cells and vice versa were analyzed. Cells were treated with 100μM of EPA, prior to conditioned media transfer. Conditioned media from EPA‐treated adipocytes reduced interleukin‐6 (IL‐6) and monocyte chemoattractant protein‐1 (MCP‐1) secretion from MCF7 cells. To further determine whether conditioned media affects cell energy metabolism, glycolytic activity was measured using seahorse extracellular flux analyzer. Glycolysis as estimated by extracellular acidification rate was reduced in MCF7 cells exposed to conditioned media from 3T3L1 preadipocytes treated with EPA. These results suggest that adipocyte‐secreted factors modulate breast cancer cell inflammation and glucose metabolism. Importantly, we identified that the anti‐inflammatory nutrient, EPA could be used for a preventive approach in adipocyte‐breast cancer cell interactions. Preventive effects of EPA in post‐menopausal obesity‐associated breast cancer merits further investigation.
Protective mechanism on gastric lesion of Sennoside A and Sennoside B
Authors: Hwang In Young , Lee Sun Yi , Sohn Yoon Ah , Kim Sun Whoe , Kim So Yeon , Moon Aree , Jeong Choonsik
Published: June 2021

Sennoside A and sennoside B is evacuant to increase the sensitivity of the colon. In vivo test, we performed HCl·ethanol‐induced gastritis test, indomethacin‐induced gastric ulcer test, gastric secretion in pylorus‐ligated test, H+/K+ATPase activity test, gastric emptying and intestinal motility test. In vitro test, we examined acid‐neutralizing capacity, quantity of PGE2 and inhibition of H. pylori colonization and we confirmed apoptosis using DAPI nuclear staining, FACS analysis. Sennoside A and B inhibit lesion index in gastritis and gastric ulcer in rats. These results showed that these components reduced gastric juice, an aggressive factor and total acidity and increased pH moderately. In addition, it was made sure that proton pump inhibiton influenced on gastric acid secretion control and that colonization inhibiting activity on H. pylori. As protection enhancing factors to gastric damage, sennoside A and B increased PGE2 in a concentration‐dependent manner. Especially, sennoside B was superior to sennoside A in proton pump inhibitory activity and PGE2 synthesis increase. From gastric emptying rate experiment, both sennoside A and B showed the recovery of stomach adaptability and gastric movement acceleration and that of motility of duodeno‐jejunum disorder from intestinal transporting rate experiment compared to control group. To make use of DAPI nuclear stain method, nuclear change has been identified and the rate of apoptosis increase confirmed concentration dependently with FACS. These results are expected to contribute the standardization and scientific movement of herbal medicines and furtherly to make sure of the capability of development of herbal drugs.
Antioxidant Enzymes Activities and Lipid Peroxidation of Aqueous Extracts of selected Vegetables in Isoproterenol‐induced Myocardial Infarction in Male Wistar Albino Rats
Authors: Okpuzor Joy , Salisu Titilayo
Published: June 2021

Vegetables constitute a substantial portion of most daily diet and also a part of an overall healthy diet worldwide. This present study examined five leafy vegetables generally eaten in some parts of South Western, Nigerian and include L. taraxacifolia (Wild lettuce), C. crepidioides (Fireweed), S. nigrum (Black nightshade), B. alba (Indian spinach) and S. biafrae (English spinach). The vegetables were air dried in the laboratory for two weeks and extracted with water using maceration techniques. The effect of the extract on isoproterenol‐induced (ISO) myocardial infarction model was evaluated in male Wistar rats (150‐200 g).

The rats were randomized into eight groups of six rats each. Groups A and B served as normal and ISO controls and were fed with rat pellet and water ad libitium. Groups C, D, E, F, G and H were the experimental groups pre‐treated orally with 200 mg kg‐1bw‐1 of each extract respectively for 28 days. Group H however, received a combination of all the extracts in ratio 1:1:1:1:1. On day 29 and 30, groups B to H excluding group A received ISO subcutaneously at a dose of 20 mg kg‐1 bw‐1 to induce myocardial infarction based on the preliminary results from our laboratory.

The activities of superoxide dismutase, catalase and reduced glutathione were significantly elevated (P<0.05) in all the pre‐treated groups compared to the ISO control while the malondialdehyde levels decreased significantly (P<0.05) in all the groups including normal control compared to ISO group.

We therefore, suggest that these vegetables extract may be effective in the management and prevention of myocardial infarction and so could be formulated into food supplements
Development of a Nonradioactive Steroid Receptor Ligand Binding Assay Using Fluorescence Polarization for Studying Hsp90‐Mediated Chaperone Protein Activity
Authors: Manlove Nicholas , Walden Jane , Murphy Patrick
Published: June 2021

While glucocorticoid receptor (GR) ligand binding has historically been measured using radiolabeled agonists, such as [3H]dexamethasone, new methods have sought to use fluorescently conjugated ligands, such as dexamethasone‐fluorescein (Dex‐Fl), as more sustainable alternatives. The molecular chaperone protein hsp90 has been shown to be essential for facilitating GR ligand binding, and structurally unrelated hsp90 inhibitors are currently in various phases of clinical investigation. The purpose of our study was to develop a fluorescence polarization (FP)‐based assay for measuring GR binding activity suitable for use in undergraduate research environments, and then apply this method to evaluating the extent to which specific agonists and antagonists of the GR and hsp90 chaperone machinery affect ligand binding. We constructed a versatile FP assay with comparable results to the previously used radiation‐based method. Interestingly, GR agonists dexamethasone and cortisol produced equivalent Dex‐Fl competition to the GR antagonist RU486, and did so in a concentration‐dependent manner. Data from hsp90 and hsp70 inhibitors showed mixed results, with hsp90 antagonists radicicol, NVP‐AUY922, and BIIB012 having limited effects, while the hsp70 antagonist novobiocin showed a potentiation of FP‐detectable steroid binding. Using the described FP method to evaluate steroid binding can be applied to broader studies examining clinically relevant GR and hsp90‐based drug therapies.
Identification of Counterfeit TB Treatment Drugs
Authors: May Elizabeth , Williams Scott
Published: June 2021

Tuberculosis is a disease causes by a bacterial infection, usually of the lungs. It is among the top ten killer infectious diseases, and is the leading cause of death in those infected with HIV. TB can be treated with a cocktail of drugs that destroy the disease‐causing bacteria, but strains of drug‐resistant TB are quickly becoming a problem in countries without advanced health care infrastructure and regulation. There are many factors that contribute to the development of drug‐resistance, including the distribution and widespread availability of substandard and falsified medicines for the treatment of TB. Evidence suggests that antiparasitic and antimicrobial drugs, such as those used in the treatment of TB, are the most counterfeited pharmaceutical products in developing countries, and that these substandard products contribute to anti‐tuberculosis drug resistance. There is a need for a field‐ready assay for the detection of counterfeit medication for the treatment of tuberculosis, and the development of such an assay is the objective of this research project. Targeted reactions will be presented that distinguish the active pharmaceutical ingredients from a list of known counterfeits. Additionally, the assay is intended to be able to quantify the amount of the active chemical present so that the proper dosage can be verified.
Benzoylecgonine, a Major Cocaine Metabolite, Enhances HIV‐1 Integration and Replication in Immune Cells
Authors: Addai Amma , Pandhare Jui , Dash Chandravanu
Published: June 2021

Several studies have indicated an association between drug abuse and an increased risk for HIV/AIDS. The presence of benzoylecgonine (BE), a major cocaine by‐product, is almost a certain indication that a person has consumed the drug. Therefore, BE testing in urine is commoner than testing of cocaine itself in Drug Screening Tests. It has been demonstrated that cocaine influences HIV‐1 replication in peripheral blood mononuclear cells (PBMCs), CD4+T cells, and humanized mice. However, the effects of cocaine and its metabolite on HIV‐1 replication have not been elucidated. The goal of this study is to examine the effects of BE and cocaine on HIV‐1 integration in immune cells that serve as the primary targets HIV in vivo. PBMCs and CD4+ T cells were isolated from human blood by Ficoll‐based reagents. HIV‐1 integration and replication were measured by quantitative nested PCR and flow cytometry. We demonstrate that BE and cocaine increase HIV‐1 integration in immune cells in a dose dependent manner. A direct role of cocaine on HIV‐1 integration was further confirmed by in vitro integration assay using HIV‐1 pre‐integration complexes (PICs) isolated from infected cells treated with cocaine. We observed a maximum increase in integration at 50uM. We also demonstrate that cocaine can localize to the nucleus of CD4+ T cells by utilizing ESI based mass spectrometry. Given that cocaine abuse serves as a powerful cofactor for HIV‐1 infection, replication, and pathogenesis in vivo, our data may have implications in the worsening of disease among cocaine abusing HIV patients.

*Amma Addai is supported by T32 grant #5T32HL007735‐19 from NIH/NHLBI to Dr. S. E. Adunyah. The research is in part supported by grants DA024558, DA30896, DA033892 and DA021471 from NIDA/NIH to CD.
Free Actin Impairs Macrophage Bacterial Defense: Reversal by Plasma Gelsolin
Authors: Ordija Christine , Chiou Terry , de Oliveira Valdo Melina , Yang Zhiping , DeLoid Glen , Kobzik Lester
Published: June 2021

Alveolar macrophage (AM) bacterial clearance is critical to host defense against lung infections. We sought to assess: 1) the effects of free‐actin released during lung injury on AM function and 2) the therapeutic potential of the actin‐binding protein plasma gelsolin (pGSN). AM binding and uptake of bacteria in the presence or absence of free actin and pGSN was measured in vitro using assays based on CFU quantitation or scanning cytometry and fluorescence microscopy of bacteria. We demonstrate impaired binding of Streptococcus pneumoniae by murine B6 AMs in the presence of free actin and reversal of impaired binding by pGSN. In contrast, albumin, heat‐denatured actin or vehicle control do not affect AM bacterial binding, thus establishing specificity. Similarly, primary human AM‐like macrophages also show impaired binding and uptake of Staphylococcus aureus in the presence of free actin or cytochalasin D and improved binding and uptake by pGSN. In conclusion, free actin was found to significantly impair AM binding and phagocytosis of microbial pathogens in vitro, an effect that was reversed in the presence of pGSN. The scanning cytometry method used in these studies will be useful to investigate the mechanism(s) by which free actin interferes with AM bacterial binding. Free actin release after lung injury may contribute to the risk of secondary bacterial pneumonia, and pGSN may offer a therapeutic strategy to reduce this risk.

Funding provided by NIH HL007118
Structural and physical properties of the Hepatitis C Virus (HCV) 1406 TCR and NS31406‐1415/HLA‐A2 pMHC complexes
Authors: Wang Yuan , Baker Brian
Published: June 2021

In the human body, the adaptive immune system provides a highly specialized pathway to eliminate and prevent pathogen growth. The glycoproteins encoded by the major histocompatibility complex (MHC) are located on the outer membrane of antigen presenting cells, or APCs. MHC proteins are used to display peptide antigens to T cells. In the case of MHC class I molecules, peptides are derived from cytosolic proteins, including virally produced proteins that are present in an infected cell. Previous work described the presence of HLA‐A2‐restricted; HCV‐NS31406 specific T cells in an HLA‐A2 negative transplant recipient who received an HLA‐A2 positive HCV infected liver. This is a unique immunological opportunity, as the patient's T cells could not be positively selected on an HLA‐A2 ligand. Such “allospecific” T cells are exceedingly rare, and by some theories of T cell development, should not even exist. We aim to characterize this unusual TCR‐pMHC interaction, which may possess features distinct from more common interactions that have been used to develop the “rules” of TCR‐pMHC engagement. Here, circular dichroism and differential scanning fluorimetry were utilized to study the stability of NS3/HLA‐A2 ligands. Surface plasmon resonance was used to investigate TCR‐pMHC binding, and preliminary work towards a structure of the complex is shown.
Decidual macrophages as an ex‐vivo model to study active HIV‐1 viral reservoir
Authors: Albino Elinette , Godoy Lenin , Ortiz Carmen , Ramirez Fernando , Hill Martin
Published: June 2021

An important step towards the eradication of HIV‐1 infection is deal with latent viral reservoirs. These reservoirs are long‐lived infected cells that replicate and produce virus and/or are infected cells located in sanctuary sites. Current models to investigate ex‐vivo tissue macrophages involve risky and invasive procedures. Decidual macrophages (DM) obtained from placental tissue represent a non‐invasive approach to study HIV‐1 pathogenesis. Macrophages are considering long‐lived cells that can harbor proviral DNA and establish an active viral reservoir. We hypothesize that DM represents an ex‐vivo model to study HIV‐1 active viral reservoir on patients with viral load under limit of detection. Six term placentas were collected from healthy women. After enzymatic digestion, DM were selected using CD14+ microbeads. Purified DM were infected with both CCR5 and CXCR4 tropic HIV‐1 virus for 72 hours after activation. Supernatant were collected every 24 hours and viral RNA was detected by sensitive RT‐PCR and active infection by p24 ELISA. Infected DM were harvested and DNA, RNA and protein were obtained. Copies of proviral DNA was determined by RT‐PCR using HIV‐1 gag gene primers. Host restriction factor SAMHD1 was quantified by western blot. CD4+ T‐cell line was used as a positive control. Results demonstrated that decidual macrophages can be infected and harbor the proviral DNA. These findings will be employed in HIV‐1 infected pregnant women under suppressive cART to demonstrate whether tissue macrophages are a true viral reservoir in HIV‐1 infection and transmission.
A non‐human primate model for Yersinia pestis infection: metabolomic analysis pilot study
Authors: Gautam Aarti , Miller Stacy , Hoke Allison , Hammamieh Rasha , Jett Marti
Published: June 2021

Yersinia pestis, the etiological agent of the plague, is a potential biothreat agent. Upon inhalation, the Gram‐negative bacterium causes pneumonic plague, a rapidly progressing, severe lung infection that is usually fatal. Only limited work using metabolite profiles to study the progression of the disease has been done, and exact mechanisms that facilitate disease are not well‐characterized. In this study, African Green monkeys were exposed to aerosolized highly virulent Y. pestis CO92 and metabolomic profiling of plasma samples was conducted. Comparison of control counterparts to the disease states at earlier time points (6 hours to 18 hours) versus at later time points (24 hours onwards) showed mainly two‐fold higher increases in the levels of certain polyunsaturated fatty acids, long chain fatty acids and metabolites from fatty acid metabolism pathway. Significant alterations in metabolites related to early inflammation and oxidative stress, increased energy demands to the host, and potential renal dysfunction were revealed. The metabolomics of plasma have the potential to provide a more thorough understanding of the Y. pestis pathogenesis, which may assist future treatment of this disease.

The grant # TMTI0029_09_WR_T from Department of Defense Chemical and Biological Defense program through the Defense Threat Reduction Agency is acknowledged.

DISCLAIMER

“Research was conducted in compliance with the Animal Welfare Act and all other Federal requirements. The views expressed are those of the authors and do not constitute endorsement by the U.S. Army.”
Trimeric G Protein Activation by the Non‐Receptor Protein GIV/ Girdin is Required for Integrin Signaling
Authors: Leyme Anthony , Perez‐Gutierrez Lorena , Nguyen Lien , Garcia‐Marcos Mikel
Published: June 2021

Signal transduction via both integrins and G protein‐coupled receptors (GPCRs) is critical to control cell behavior in health and disease. These two receptor classes have been traditionally believed to trigger distinct and independent signaling cascades. Here we report a novel mechanism of integrin signal transduction that requires activation of the trimeric G protein Gαi by the non‐receptor Guanine nucleotide Exchange Factor (GEF) GIV (aka Girdin), a pro‐metastatic protein. We demonstrate that GIV is necessary and sufficient to enhance responses to extracellular matrix (ECM) stimulation and make tumor cells more invasive. These responses include remodeling of the actin cytoskeleton and PI3K‐dependent signaling, resulting in enhanced haptotaxis and haptoinvasion. Tumor cells engineered to express GEF‐deficient GIV fail to transduce integrin signals into pro‐invasive cell responses. Mechanistically, we show that GIV and Gαi3 are recruited to integrin complexes upon ECM stimulation and activate a Gβγ‐PI3K‐Akt signaling cascade. In summary, our discoveries delineate a novel mechanism by which integrin signaling is rewired during metastasis to result in increased tumor invasiveness.

This work is supported by the American Cancer society (RGS‐13‐362‐01‐TBE), the Elsa U. Pardee Foundation and NIH (R01GM108733).
Mechanobiology of the Extracellular Matrix: Learning from Articular Cartilage
Authors: Kearns Sarah , Das Moumita
Published: June 2021

This project aims to study the structure‐function relations in the extracellular matrix (ECM) surrounding cells. The ECM is a complex biopolymer scaffolding that provides structural and mechanical support to cells, and regulates how cells sense and respond to external stimuli. For example, when we walk or run, the cartilage tissue in our knees experiences very large stresses. It is able to bear these stresses mainly due to the supportive nature of the ECM, in which the cells are embedded, and not due to cells because they are very sparsely distributed. As a model system, our study focuses on the ECM in cartilage tissue which has two major mechanobiological components: a network of the biopolymer collagen that acts as a stiff, reinforcing matrix, and a flexible network of Proteoglycans that facilitates swelling and deformability. We model this system as a double network hydrogel made of interpenetrating networks of stiff and flexible biopolymers respectively using Fortran 90. We will study the biomechanical response of the model ECM to shear and compression forces and compare our results with experiments done by our collaborators. This study will provide useful insights into the design principles for the ECM as well as biomimetic hydrogels that are mechanically robust and can, at the same time, easily adapt to cues in their surroundings.
Identification and Characterization of a Group of Polymorphic, Single Domain Peptidoglycan Hydrolases of the NlpC/P60 Superfamily in Dust Mites
Authors: Tang Vivian , Stewart Geoffrey , Chang Barbara
Published: June 2021

A 14 kDa bacteriolytic enzyme, LytFM, previously purified from the growth medium of dust mite Dermatophagoides pteronyssinus, shows high sequence homology with cysteine peptidases belonging to the NlpC/P60 Superfamily. This study aimed to identify genes encoding similar proteins in dust mite species and to characterize the putative encoded proteins. PCR and bioinformatic analysis revealed homologous genes encoding this single domain protein in Psoroptes ovis, Blomia tropicalis, Euroglyphus maynei and D. farinae. A variant, LytFM2, was detected in D. pteronyssinus indicating that the D. pteronyssinus proteins are polymorphic. The lytFM sequence was amplified from a D. pteronyssinus gDNA library as an intronless gene and its 3′ end flanking sequence contained a prokaryotic promoter sequence suggesting a prokaryotic origin. Homology studies showed that the putative mite proteins possessed a cysteine peptidase catalytic triad characteristic of members of the NlpC/P60 Superfamily involved in peptidoglycan processing. However, they all lacked a conserved Asp residue characteristic of NlpC/P60 proteins recognizing a free L‐Ala residue of the peptidoglycan stem peptide suggesting that the mite proteins cleave GlcNAc‐MurNAc‐L‐Ala‐γ‐D‐Glu‐mDAP/Lys peptidoglycan substrates. These enzymes thus represent a conserved group of proteins in several mite species that may have an important role in protection of the gut from pathogens or utilization of bacteria as a food source, or both. As at least two of these mite proteins are secreted, they may represent a novel group of allergenic proteins.

Work supported by Asthma Foundation WA and NH&MRC Australia.
Conservation of Lipid Storage Droplet Proteins in Evolution of Animalia
Authors: Bowman Jacob , Tansey John
Published: June 2021

Lipolysis is a conserved pathway used by organisms for the mobilization of fatty acid from triacylglycerols found in lipid storage droplets (LSDs). Numerous proteins, including the perilipins, CGI‐58, G0S2, and lipases interact and regulate fatty acid release in lipolysis. Because lipolysis is conserved throughout Animalia, and because several of these proteins interact with one another, it stands to reason that genes coding for these proteins are coevolving as species adapt to environmental changes. The present study traces the evolutionary history of lipolysis by examining the evolution of LSD proteins in different species of Animalia. Comparisons were made of fragments of protein sequences coding for LSD proteins previously demonstrated in the literature to interact with one another. Amino acid residues that are conserved between interacting partners and are indicative of potential protein‐protein interactions and will be used in subsequent analysis.
Deciphering Differential Distortion of Purine Substrates by Human and Plasmodium falciparum HGPRT
Authors: Karnawat Vishakha , Gogia Spriha , Balaram Hemalatha , Puranik Mrinalini
Published: June 2021

Enzymes of the salvage pathway are essential to understanding the biochemistry of parasitic protozoa. These parasites lack de novo pathway for nucleotide synthesis and depend solely on purines salvage from their host. Hypoxanthine guanine phosphoribosyltransferase (HGPRT) from Plasmodium falciparum has long been regarded as a potential therapeutic target. Although structurally homologous to human enzyme, PfHGPRT differs from human (h) HGPRT in its expanded substrate specificity and activity towards common substrates. We used substrate analogues, 9‐deazapurines, in combination with UV resonance Raman spectroscopy to capture non‐covalent interactions in enzyme‐substrate complexes. Raman spectral band positions are sensitive to sub‐angstrom changes in bond length. We observed perturbations in Raman spectra that provide quantitative information about hydrogen bonding and stacking‐interactions with the enzyme active‐site. The bound ligands are extensively distorted towards the transition‐state of phosphoribosyltransferase reaction. Differential loss in Raman crosssection of the ligands bound to two enzymes suggests that active‐site is more compact in hHGPRT than in PfHGPRT. The rigidity of active‐site in hHGPRT is attributed to tighter stacking interaction between phenylalanine residue of the active‐site and the nucleobase.

Research was supported by IISER, India and Council for Scientific and Industrial Research, India.
Vimentin Phosphorylation Underlies Corneal Myofibroblast Sensitivity to Withaferin A
Authors: Bargagna‐Mohan Paola , Lei Ling , Thompson Alexis , Shaw Camille , Mohan Royce
Published: June 2021

Objective

To compare vimentin phosphorylation in corneal fibroblasts with myofibroblasts and determine their sensitivity to withaferin A.

Methods: Primary rabbit corneal fibroblasts and myofibroblasts were subjected to cell spreading assays. Wild type and vimentin‐deficient mice were subjected to corneal alkali injury and treated with withaferin A (WFA). Corneas were subjected to immunohistochemistry and western blot analyses.

Results: We show that myofibroblasts produce increased serine‐38 phosphorylated vimentin (pSer38Vim) and display impaired cell spreading and cell polarization compared to fibroblasts. The pSer38Vim isoform is inefficiently incorporated into growing vimentin IFs of myofibroblasts during cell spreading as myofibroblasts maintain higher levels of soluble pSer38Vim compared to fibroblasts. WFA treatment causes a potent blockade of cell spreading selectively in myofibroblasts, targeting pSer38Vim for hyperphosphorylation, which is not induced in fibroblasts. Hyperphosphorylated pSer38Vim in myofibroblasts complexes with adaptor protein filamin A (FlnA), and appears as short squiggles displaced from focal adhesions upon WFA treatment. The extracellular‐signal regulated kinase (ERK) is also phosphorylated (pERK) in response to WFA, but pERK does not enter the nucleus because it remains bound to pSer38Vim in cytoplasmic complexes. Fibrotic corneas of injured wild type mice possess high levels of pERK compared to injured vimentin‐deficient mice. WFA treatment causes a decrease in pERK and downregulates pSer38Vim expressing myofibroblasts in corneas of wild type mice.

Conclusions

We have identified pSer38Vim as a major determinant of myofibroblast sensitivity to WFA.

Support: R01 EY016782
Identification of The Binding Site for Ammonia in GMP Reductase
Authors: YAO Tianjiong , Rosenberg Masha , Hedstrom Lizbeth
Published: June 2021

The reaction of guanosine monophosphate reductase (GMPR) includes two steps: (1) a deamination step that releases ammonia from GMP and forms the intermediate E‐XMP*; (2) a hydride transfer step that converts E‐XMP* to IMP utilizing NADPH as a cofactor. The hydride transfer step is the rate limiting step, yet we failed to observe a burst of ammonia release. This observation suggests that ammonia remains bound to the enzyme during the hydride transfer step. We identified a possible ammonia binding site by inspection of crystal structure of human GMPR type 2. Three candidate amino acids were selected and probed by site directed mutagenesis. The substitution of all 3 residues decreased intermediate production about 4 fold but decreased the reaction with ammonia by at least 100 fold. Therefore, these substitutions behave as expected for mutations at the ammonia binding site.
Kinetic characterization of the α‐2,9 polysialyltransferase of Neisseria meningitidis
Authors: Ming Shonoi , Peterson Dwight , Vann Willie
Published: June 2021

Neisseria meningitidis Serogroup C is responsible for the majority of the meningococcal meningitis outbreaks in the United States in the past 20 years. The serogroup polysaccharide, an α‐2, 9 polysialic acid, of the pathogen is essential for virulence. This highlights the importance understanding of the enzymes involved in its biosynthesis as it may lead to the development of new drugs targeting this disease.

We are interested in obtaining a detailed understanding of the kinetics of the α‐2, 9 polysialyltransferase involved in the formation of the polysaccharide capsule of N. meningitidis. Studies of the α‐2, 8 polysialyltransferase conducted by the Wakarchuk group obtained an apparent Km in the low mM range for CMP‐sialic acid. Using the α‐2, 9 polysialyltransferase we obtained an apparent Kmvalue in the low µM range for CMP‐sialic acid. We believe that this large discrepancy in Km is due primarily to the differences in assay methods and conditions.

Using CMP‐sialic acid as the donor substrate, we compared acceptor substrate specificity of GD3, a ganglioside, with a hexamer of sialic acid. We observed a lower apparent Km value for GD3 compared with the hexamer suggesting that the attachment of a lipid to the oligosaccharide improves substrate binding. However, the enzyme does not appear to follow Michaelis‐Menten kinetics. The complexity of the reaction kinetics may be due to fact that each product formed via the transfer of sialic acid from CMP‐sialic acid in turn produces a new substrate. To address this issue we used CMP‐sialic acid analogs modified at the N‐9 position to observe a single turnover of product, which allowed us to perform detailed kinetic characterization of the enzyme.
Bubristatins inhibit BubR1‐dependent signaling by interrogating BubR1‐CENP‐E interaction
Authors: liu xing , Huang Yuejia , Wang Wenwen , Zhang Yin , Yan Maomao , Du Na , Wang Zhiyong , Ding Xia , Yao Xuebiao
Published: June 2021

The mitotic checkpoint prevents premature anaphase onset through generation of an inhibition to the E3 ubiquitin ligase APC/C, whose ubiquitination of cyclin B and securin targets them for degradation. BUBR1 is a core component of the mitotic checkpoint and is required for formation of stable kinetochore‐microtubule attachments. BubR1 interacts with mitotic kinesin CENP‐E but its mechanism of action has remained elusive. Here we show our identification and characterization of BubR1 chemical inhibitors Bubristatins. Interestingly, the compounds bubristatin‐1 and bubristatin‐3 inhibit BubR1 kinase activity and the binding of BubR1 to its effector CENP‐E. The binding of the bubristatin‐1 to BubR1 was confirmed by enzymatic assay and site‐specific mutagenesis. Bubristatin compounds show selectivity for BubR1 relative to other mitotic kinases such as Bub1. A combination of in vitro reconstitution and cell‐based assays has enabled us to identify Ser2639 of CENP‐E as a cognate substrate of BubR1. Our real‐time imaging analyses show that chromosomes in Bubristatin‐treated cells frequently exhibit segregation errors and deficiency in metaphase alignment, a phenotype seen in CENP‐E inhibited/suppressed cells. Bubristatin is thus a useful tool to study mitotic checkpoint regulation and correction of chromosome mal‐attachment, and contributions of BubR1 signaling to chromosome dynamics and plasticity.
Reciprocal Regulation of Abl kinase by CrkII Y251 and Abi1 Controls Cell Motility and Invasion in Glioblastoma
Authors: Kumar Sushil , Kotula Leszek , Birge Raymond
Published: June 2021

The Src homology 2 (SH2) and Src homology 3 (SH3) domain‐containing adaptor protein, Crk II, positively regulates cell motility and in certain tumor types such as Glioblastoma multiformae (GBM) can promote cell invasion and tumor metastasis. Recently, we reported on a novel paradigm by which Crk II regulates motility in breast cancer cells by virtue of its ability to become phosphorylated at tyrosine 251 in the carboxyl‐terminal SH3 domain and activate Abl kinase. Here we show that Tyr251 phosphorylation and Abl activation is a prominent feature in GBM, which in turn promotes both enhanced motility and invasion of tumor cells, and all these events are potentiated by the loss of the SH3 domain containing adaptor protein Abi‐1. At molecular level, both Crk and Abi‐1 compete with their respective SH3 domains for binding the same proline‐rich domain on the Abl kinase, but do so with different intracellular itineraries, where Crk II is a positive regulator and Abi1 is a negative regulator of Abl kinase activity. Moreover, using Abi‐1 (‐/‐) fibroblasts, genetically‐modified GBM cell lines, and in vitro kinase assays, we show that Crk II mediated transactivation of Abl kinase is enhanced by loss of Abi1 in GBM cell lines, and conversely, that re‐expression of Abi‐1 suppresses Crk‐mediated Abl‐transactivation as well as the motility and invasion‐promoting activities of Crk in GBM. Taken together with expression obtained from GBM tumors, these results show that Abi1 and Crk have opposing biological effects on Abl and that CrkII Y251‐Abl kinase regulatory circuit can be unleashed by loss of Abi1 isoform2 in GBM.