bluuebunny/crossref_metadata_2025 huggingface dataset:
In silico evaluation of inhibitors of Plasmodium falciparumAP2‐I transcription factor
Authors: Oladejo David , Oduselu Gbolahan , Dokunmu Titilope , Isewon Itunuoluwa , Okafor Esther , Iweala Emeka E. J. , Adebiyi Ezekiel F.
Published: May 2022

Recent treatment failures observed for Artemisinin‐based combination therapy (ACT) have raised concerns about the efficacy of the front‐line drug to treat malaria and the need to develop a new antimalarial drug regimen. Plasmodium falciparum Apicomplexan Apetala 2 Invasion (PfAP2‐I) transcription factor (TF) is a protein that regulates the expression of a subset of gene families involved in Plasmodium falciparum red blood cell (RBC) invasion. Inhibiting PfAP2‐I TF with small molecules represents a potential new antimalarial therapeutic target to combat drug resistance, which is the aim of this study. The 3D model structure of PfAP2‐I was predicted ab initio using ITASSER and ROBETTA prediction tools and was validated using Errat and Procheck from the Save server 6.0. Computed Atlas of Surface Topography of proteins (CASTp) 3.0 and ConCavity were used to predict the active sites of the PfAP2‐I 3D7 modeled structure. Pharmacophore modeling of the control ligand (3W7 from COACH server) and modeled 3D structure of PfAP2‐I was carried out using the Pharmit server to obtain several compounds for docking analysis. Chimera software was used to remove the complexed ligands, and the modeled protein structure was defined as a receptor. Virtual screening and post‐screening studies were conducted using AutoDock vina and LigPlot. The designed ligands’ toxicity predictions and in silico drug‐likeness were performed using the Swiss ADME predictor and OSIRIS Property Explorer. The modeled protein from the ROBETTA prediction tool was prioritized based on structure validation results of 96.827 for ERRAT and 90.2% of the amino acid residues in the most favored region for the Ramachandran plot. A total of 8656 compounds obtained from six (6) databases on the Pharmit server were used to prepare the ligand library and screened against the prepared 3D model structure of PfAP2‐I, considering the active sites predicted from CASTp and ConCavity. Six (6) best hits were selected based on the binding affinity to the active site PfAP2‐I and were considered for post‐screening analyses. The six compounds exhibited dock score values (between ‐9.9 to ‐10.2 kcal/mol), having lower binding energies than the standard drug ‐ Chloroquine (‐5.10 kcal/mol). The best dock score was compound ZINC97139187 (‐10.2 kcal/mol). For the ADMET properties, compound ZINC97139187 had the highest drug score of 0.63, followed by compound 154861216 with a drug score of 0.58 (both higher than that of the standard drug ‐ chloroquine of 0.25). The good, estimated binding energies and drug score values observed for compound ZINC97139187 and compound 154861216 suggest that they can be considered possible PfAP2‐I inhibitors. Further pre‐clinical experimental validations should be carried out to ascertain the efficacy of these predicted best hits.
Teaching Coinciding Remote and In Person Systems Physiology Courses: Lessons Learned
Authors: Nelson Cassandra R.
Published: May 2022

In the fall 2021 semester, my university offered students the option of taking our 3‐credit Systems Physiology course, designed for juniors and seniors, remotely or in person. In charge of both versions of the course, I taught 330 remote and 63 in‐person students. Retention of students was 94% in the in‐person class (4/67 students withdrew during the semester) and 97% in the remote class (9/339 withdrew). My goal was for the students to learn the same content at the same time, so I could invite all students to office hour sessions and have exams on the same day. Online students listened to asynchronous lectures and met with me twice a week for 1 hour to go over those lectures. In person students did not have any required online listening but instead received course content via synchronous lectures. Three, 40‐question exams made up 45% of the grade. For all exams, I gave the in‐person students 70 minutes and the remote students 60 minutes, but remote students were allowed to use their book and notes, while in‐person students were not. The approach of how to assess the remote versus the in‐person students for each of the 3 exams was tricky, and I tried different strategies for each exam. For example, without telling the students, I gave all students the same exam for exam 2 (Cardiovascular & Respiratory Systems), and the in‐person group averaged 68.2% and the remote group averaged 66.3%. With all assignments factored in, the overall class average for the in‐person students was 80.1% and for the remote students, 78.1%. There was a much larger range of grades in the remote course (17.8‐96.8%) compared to the in‐person course (52.0‐92.8%). 16 students (5%) failed the remote course compared to 1 student (1.5%) in the in‐person course. Ultimately, there are ways to teach Systems Physiology effectively and successfully both remotely and in‐person. However, in my fall 2021 case study, I witnessed slightly better outcomes for the students who took the course in person.
5HTR1E receptor interacts with Neurotrophic factor‐α1 and serotonin to activate two distinct signaling pathways
Authors: Sharma Vinay K. , Yang Xuyu , Kim Soo‐Kyung , Maafi Amirhossain , Xiao Lan , Inoue Aska , Goddard William A. , Loh Y. P.
Published: May 2022

Neurotrophic factor‐α1/Carboxypeptidase E (NFα1/CPE) is a prohormone processing enzyme intracellularly and as a neurotrophic factor, extracellularly. Previously we showed that extracellular treatment of hippocampal neurons with NFα1/CPE protected them from oxidative stress‐induced cell death through ERK/BCL2 signaling. Based on these results, we hypothesized that NFα1/CPE activation of the ERK/BCL2 pathway to mediate neuroprotection should involve interaction with receptors at the cell membrane. We have identified 5‐hydroxytryptamine receptor 1E (5HTR1E), a Gi coupled serotonin receptor, as an extracellular binding partner of NFα1/CPE. This interaction was confirmed by co‐IP and pull‐down experiments followed by radioligand binding studies in HEK293‐5HTR1E overexpressing cells. Molecular modeling studies also predicted that NFα1/CPE interacted with HTR1E, not at the serotonin binding pocket, but via the extracellular loop 2 (ECL2) of 5HTR1E, forming 3 stable salt bridges with specific amino acid residues in NFα1/CPE. To identify the HTR1E downstream signaling pathways, we used HEK293 cells stably expressing 5HTR1E. First, we tested the dose dependent effect of both serotonin and NFα1/CPE on ERK, and cAMP signaling and found that both can increase ERK phosphorylation while cAMP pathway activation is serotonin dependent only. To further dissect the 5HTR1E mediated functions of both serotonin and NFα1/CPE we tested Gi, Gq, PKA, and PI3K inhibitors and observed that these proteins are not involved in NFα1/CPE mediated ERK phosphorylation, while serotonin‐stimulated ERK activation was Gi‐PKA dependent. We then explored the involvement of β‐arrestin, an alternative coupling mechanism with GPCRs for ERK activation. Studies in 5HTR1E expressing‐HEK293‐β‐arrestin KO cells revealed that NFα1/CPE‐5HTR1E complex was unable to induce ERK signaling in the absence of β‐arrestin proteins. Furthermore, molecular docking studies revealed a very strong coupling of b‐arrestin to intracellular loop (ICL) 2 and ICL3 of HTR1E. Using LDH cytotoxicity assay we demonstrated that NF‐α1/CPE was able to protect HEK‐293‐5HTR1E expressing cells against H2O2‐induced cytotoxicity, via increasing pro‐survival protein BCL2 expression; however, this protective effect of NFα1/CPE was diminished in the β‐arrestin KO cells. Our study identified two ligands for HTR1E: NFα1/CPE which interacts with HTR1E to activate β‐arrestin/ERK/BCL2 signaling pathway for protecting cells against oxidative stress; and serotonin which activated a Gi‐cAMP‐dependent pathway, the physiological role is under investigation.
Epitope Profiling Reveals the Antibody Immune Response Difference Between COVID‐19 Infected and Vaccinated
Authors: Pierson Lane M. , Yang Li , Liang Te , Fletcher Jesse , Lyon Christopher , Yu Xiaobo , Hu Tony
Published: May 2022

Antibodies play an important part in combating SARS‐CoV‐2 infection whether generated by the infection or vaccination. However, the many epitopes generated by infection have not been fully investigated with only a few epitopes known and these being mostly limited to the S protein’s receptor binding domain (RBD) and the N‐terminal domain which limits vaccine and drug design 1‐4. The difference between epitopes generated by infection and vaccination has also not been studied. To address this, we employed a SARS‐CoV‐2 proteome microarray to screen for linear epitopes recognized by antibodies present in COVID‐19 patients and individuals vaccinated with the Pfizer‐BioNTech mRNA COVID‐19 Vaccine.

The proteome microarray consisted of S, N, and E proteins, as well as spotting peptides that were 15 amino acids in length with overlaps of 5‐amino acids, covering the entire SARS‐CoV‐2 proteome (MN908947.3) (Figure 1). Blood samples were incubated onto the arrays followed by an incubation of fluorescent secondary anti‐human antibodies. Fluorescent intensity data generated and normalized using the Z‐score method and then further analyzed for significance by parametric one‐way ANOVA with Dunnett's post hoc test (COVID‐19 cohort) and repeated measure ANOVAs with Dunnett's post hoc tests (vaccinated cohort).

The full‐length S protein showed a significant increase in COVID‐19 patients at around 20‐23 days after symptom onset and vaccinated individuals over all time points in both IgM and IgG antibodies (Figure 2A). Linear mapping of the IgM epitopes revealed a degree of overlap between infected and vaccinated individuals (22.2%; 6/27 total) with both having epitopes in the RBD and fusion peptide (FP) (Figure 2B). Structural mapping on 3D models of the S protein showed that all epitopes where on the surface of the protein and that COVID‐19 generated epitopes have a different pattern than those generated by vaccination (Figure 2C).

An Epitope identified in this study with future prospects is epitope S481‐495 from COIVD‐19 patients that partially overlapped the binding site of two neutralizing antibodies previously isolated from COVID‐19 patients, S2H135 and F2B‐2F61, and contacted amino acids that interact with ACE2 receptor6,7. One epitope of note from the vaccinated individuals is epitope S811‐825 which mapped adjacent to the fusion‐peptide proximal region. These epitopes may be helpful in future vaccine and antibody therapy development.

1 Ju, B. et al. Nature 584, 115‐119.

2 Robbiani, D. F. et al. Nature 584, 437‐442.

3 Seydoux, E. et al. bioRxiv.

4 Wu, Y. et al. Science 368, 1274‐1278.

5 Piccoli, L. et al. Cell 183, 1024‐1042 e1021.

6 Casalino, L. et al. ACS Cent Sci 6, 1722‐1734.

7 Wang, Q. et al. Cell 181, 894‐904 e899.
Del Nido Cardioplegia or Potassium Induces Nrf2 and Protects Cardiomyocytes Against Oxidative Stress
Authors: Diao Hongting
Published: May 2022

Open heart surgery is often an unavoidable procedure for treatment of cardiovascular disease. Myocardial Ischemia‐Reperfusion Injury can occur as a result of open heart surgery such as cardiopulmonary bypass. The multifaceted mechanisms of MIRI involve the generation of reactive oxygen species (ROS), resulting in cellular damage, ultimately impacting postoperative cardiac performance and complications. Nf‐E2 related factor‐2 (Nrf2), a basic leucine zipper transcription factor, binds to and activates the Antioxidant Response Element (ARE) in the promoters of antioxidant and detoxification genes. We addressed here whether cardioplegic solutions activate Nrf2, therefore serving for cytoprotection. With High K, Low K, Del Nido (DN), histidine‐tryptophan‐ketoglutarate (HTK) and Celsior cardioplegic solutions, we found that High K and DN caused increases of Nrf2 protein in AC16 human cardiomyocytes. Tracing the components in High K and DN led to the finding of K+ at the concentration above 5 mM playing an essential role in Nrf2 protein induction. K+ induced Nrf2 elevation is Ca2+ dependent. ARE luciferase reporter assay confirmed Nrf2 activation by K+ in 10‐40 mM. Profiling transcriptomes using RNA seq showed that oxidation‐reduction process as the main gene ontology group affected by K+. K+induced elevated expression ofclassical downstream targets of Nrf2, including NQO1, AKR, Aldose Reductase, TRXR1, and G6PD. We found that K+ extended the half‐life of Nrf2 from 17.8 mins to 25.1 mins in AC16 cells, and decreased Nrf2 protein ubiquitination. knocking out Keap1 abolished Nrf2 induction by K+. Nrf2 induction by K+ or DN protects cells against loss of viability, apoptosis and ROS production induced by H2O2. Our data support that high K+ concentration in cardioplegic solutions can be cardiac protective via the activation of Nrf2.
Methods of senescence induction in human AC16 Cardiomyocytes and associated pathway and phenotypic changes
Authors: Kastury Nikhitha , Li Lauren , Pozzoli Marina , Hidalgo Veronica , Lau Edward
Published: May 2022

Cellular senescence, induced by stress factors within a cell, is a state of cell growth arrest that has been found to contribute to age‐related diseases. Senescent cells exhibit a particular phenotype that is generically characterized by increased expression of cell‐cycle regulator proteins, increased expression of senescence associated beta‐galactosidase, and increased oxidative mitochondrial stress. Particularly in the cardiomyocyte, this state of growth arrest contributes to many prevalent cardiomyopathies. However, while it is broadly known that oxidative stress and DNA damage inducing conditions can contribute to a cell becoming senescent, methods to induce senescence in cardiomyocytes effectively have yet to be systematically investigated. In particular, the particular lesions and concentrations that can adequately induce senescence as well as the particular pathway through which senescence is induced in each induction method are unknown. Accordingly, the aim of this study is to optimizethe dosage and regimen of three pro‐senescence insults (hydrogen peroxide, doxorubicin, and UV‐C light) that will best induce senescence as measured by the associated senescent phenotype while gaining deeper understanding of differences in the pathways that are employed within a cell in the process of inducing senescence from different forms of stress. To do so, we used western blots and staining techniques in order to analyze relative expression of classic senescence markers in cells that were exposed to stress conditions as compared to healthy cells. The most relevant proteins that were compared were p16 and p21, cell cycle regulator proteins, and H2A.X, a double‐stranded DNA damage protein. We found that in conditions of 500 μM H2O2, 0.1 μM Dox, and 5 mJ/cm^2 UV‐C light, p21 and H2A.X were significantly upregulated and p16 was interestingly found to be significantly downregulated in all conditions. Treated samples were then filtered and tested via mass spectrometry in order to determine relative protein expressions. Preliminary mass spectrometry data used to compare the different insults showed some evidence of activation of different senescent pathways. Samples insulted with Dox showed an enrichment in the apoptotic pathway while those treated with UV‐C or H2O2 showed enrichment in translation elongation and nonsense mediated decay related pathways. Furthermore, we found that combinatorial treatment of H2O2, UV‐C light and doxorubicin increased expressed mitochondrial oxidative stress as seen by MitoSox staining techniques. In summary, our results showed the different concentrations that can induce senescent phenotypes in cardiomyocytes and the differences in the phenotypes through different senescence induction methods.
ARGs Prevalence Among Hospitalized COVID‐19 Patients with Bacterial Infections and Resistome abundance
Authors: Aly Mahmoud
Published: May 2022

Background

The burden of antimicrobial resistance AMR worldwide is substantial and is growing. Many factors play a role in the emergence of resistance. Our microbiome is a significant reservoir for antimicrobial resistance genes (ARGs). The use and misuse of antimicrobials can select multi‐resistant bacteria and modify the repertoire of ARGs in the gut microbiota. There is clear evidence that hospitalized COVID patients have altered gut microbiota that might affect the prevalence and abundance of AMR. The World Health Organization (WHO) has reported 364 million cases worldwide. Moreover, the CDC announced 72 million positives with more than 4.2 million hospitalized cases in the US alone.

Objective

The purpose of the current study is to review AMGs prevalence among hospitalized COVID‐19 patients with bacterial infections and explore the alteration of resistome abundance.

Methodology

We searched the pathogen detection of the National Center for Biotechnology database for hospitalized positive COVID‐19 cases with microbiome sequencing submission using the following keywords: antimicrobial resistance, antibiotic stewardship, prevalence, epidemiology, mechanism of resistance, and COVID‐19, SARS‐CoV‐2, bacterial infection, hospitalization, healthcare‐associated infection, antibiotic resistance, antimicrobial resistance, multi‐drug resistance, Acinetobacter, Klebsiella, Streptococcus, Staphylococcus, Pseudomonas, Escherichia, Enterococcus, Methicillin‐resistant Staphylococcus aureus, New Delhi Metallo‐β‐lactamase‐producing carbapenem‐resistant enterobacterales, Severe acute respiratory syndrome coronavirus 2, Vancomycin‐resistant enterococcus. Web‐based pipelines ResistoXplorer and Qiime2 and different databases, including RESfinder, Card, and ARGminer, are used for the metagenomics analysis. They are mainly based on programming languages ‐ JavaScript (version: 1.2.0) and R (version: 4.1.0).

Results

Our preliminary results showed that, to date, 11,129 isolates were detected worldwide between the declaration of the covid as a pandemic and January 2022. Moreover, the isolates prevalence was Acinetobacter baumannii at 39.7%, followed by Klebsiella pneumonia at 32.2%, then E.coli and Shigella at 11.7%, Enterobacter at 5.4%, and Pseudomonas aeruginosa 2.5%. Finally, infections such as Klebsiella oxytoca, Citrobacter freundii, and Providencia alcalifaciens were around 10%. We explored the genetic diversity of MRGs, including: aac(6′)‐Ib′, aadA1, and(3′′)‐IIa, aph(3′′)‐Ib, aph(3′)‐Ia, aph(6)‐Id, armA, arr‐2, blaADC‐30, blaOXA‐23, blaOXA‐66, blaOXA. Currently, we are working on WGS and 16s sequencing analysis, and our future work includes data analysis on abundance profiles and exploring the resistome signatures generated from AMR metagenomics of the microbiota of positive COVID‐ hospitalized patients.

Conclusion

We are anticipating that there will be a significant change take place in the gut microbiome, hence the ARGs and resistome abundance in patients with COVID‐19.
Characterizing the Therapeutic Potential of the Adropin‐GPR19 Signaling Pathway in MAFLD
Authors: Mushala Bellina , Xie Bingxian , Sipula Ian , Jurczak Michael , Scott Iain
Published: May 2022

Obesity is the second leading cause of preventable death in the United States, and serves as a fundamental risk factor in various pathologies including metabolic‐associated fatty liver disease (MAFLD). The cellular and molecular mechanisms that drive obesity‐related disease in different tissues are not fully elucidated, and this represents an impediment to the development of effective and efficient treatments. Adropin is a nutritionally regulated liver‐ and brain‐derived peptide hormone that modulates metabolic homeostasis in several tissues. Adropin levels are significantly reduced in obese human subjects, and this decrease is linked to increased adiposity, impaired glucose homeostasis, and liver injury. Recent reports show that MAFLD patients exhibit a marked decrease in serum adropin levels, however the functional significance of this is unknown. Various studies suggest that a class A orphan G‐coupled protein receptor, GPR19, mediates adropin signal transduction, therefore we hypothesized that GPR19 may be the putative cellular receptor that permits adropin function in the liver. To investigate this, we used a novel whole‐body GPR19 knockout mouse to perform a preliminary characterization of the effects of GPR19 depletion on hepatic and whole‐body physiology. Using in vivo metabolic approaches, we report that GPR19 KO mice exhibit glucose intolerance, altered hepatic energy metabolism, and histological liver injury. Together, these data suggest that GPR19 KO mice display pathogenic events that promote a MAFLD phenotype, indicating that GPR19 may be critical for the protective effects of adropin signaling in the liver. These findings further define the fundamental role of the adropin‐GPR19 signaling pathway in the regulation of metabolic homeostasis, and thus highlight a potential target for therapeutic interventions in MAFLD.
Ablation of pigment epithelium‐derived factor receptor, PEDF‐R, causes photoreceptor degeneration
Authors: Bernardo‐Colón Alexandra , Dong Lijin , Abu‐Asab Mones , Becerra Patricia
Published: May 2022

Photoreceptor cells produce a receptor protein for pigment epithelium‐derived factor (PEDF‐R), which is encoded by the PNPLA2 gene of the patatin‐like phospholipase domain‐containing 2 family. PEDF‐R is a membrane‐linked lipase detected in the inner segments of photoreceptors. We aim to reveal the physiological role of PEDF‐R in photoreceptors by characterizing CRISPR Pnpla2 knock‐out mouse lines.

CRISPR‐derived Pnpla2 knock‐out mouse lines were generated in a C57BL/6 and rd8 free background. RT‐PCR was performed from dissected retinas. Mice were evaluated for fundoscopy, electroretinography and angiography. Organs of the thorax were harvested and imaged using a LEICA microscope. Plasma was collected for lipid analyses. Eyes were enucleated and processed for histology, immunofluorescence, confocal microscopy, and transmission electron microscopy. Pnpla2‐/‐ mice had undetectable retinal Pnpla2 expression levels, enlarged whiter hearts, and lower plasma free fatty acids and triglycerides levels than their Pnpla2+/+ littermate controls. Their retinal pigment epithelial cells accumulated lipid droplets and their photoreceptors were deformed. Fundi of Pnpla2+/‐ eyes had white spots. The optic nerve area of Pnpla2‐/‐ and Pnpla2+/‐ mice had an excess of pigment. The a‐wave in Pnpla2‐/‐and Pnpla2+/‐ mice, and b‐wave in Pnpla2+/‐ mice were attenuated relative to controls. Retinal rhodopsin and opsin gene expression of Pnpla2‐/‐and Pnpla2+/‐ were lower than in controls. The immunofluorescent intensities of rhodopsin and opsin antibodies decreased in Pnpla2+/‐and Pnpla2‐/‐ photoreceptors relative to controls. The immunofluorescence of PKCα and synaptophysin antibodies (markers for bipolar cells and ribbon synapse) in the Pnpla2+/‐ retinas were lower than in controls. Ablation of Pnpla2 in mice causes malformation of photoreceptors and affects photoreceptor performance, identifying PEDF‐R as an important component for photoreceptor structure and function. These findings suggest that the absence of PEDF‐R in photoreceptor cells interrupts PEDF neurotrophic action, thereby heightening degeneration in the mice.
Menopause Diminishes Female Protection against Vascular Contributions to Cognitive Impairment and Dementia
Authors: Gannon Olivia J. , Naik Janvie S. , Abi‐Ghanem Charly , Mansour Febronia , Salinero Abigail E. , Riccio David , Kelly Richard D. , Brooks Heddwen L. , Zuloaga Kristen L.
Published: May 2022

Around mid‐life, ovarian production of estrogen stops in a sex‐specific endocrine transition called menopause. While most women who have dementia are post‐menopausal, menopause is seldom included in rodent models of dementia despite its significance in dementia risk factors in women. Before, but not after menopause, women are less likely than men to suffer from stroke, obesity, and diabetes: known risk factors for vascular contributions to cognitive impairment and dementia (VCID). Here, we aim to determine if and how menopause worsens VCID. We hypothesize that menopause will increase metabolic dysfunction, cognitive impairment, and brain pathology in a mouse model of VCID. To model menopause, we utilized 4‐vinylcyclohexene diepoxide to induce accelerated ovarian failure. To model VCID, we performed a unilateral common carotid artery occlusion surgery. We assessed cognitive deficits using behavioral tests: open field, novel object recognition, Barnes maze, and nest building. To track metabolic changes, we recorded weight gain, adiposity, and glucose tolerance. We found that menopause led to weight gain, increased visceral adiposity, impaired glucose tolerance and impaired episodic‐like memory (novel object test). While these mice did not have cognitive impairments due to VCID alone, there was a significant correlation between the degree of blood flow deficit and activities of daily living (nest building test). While we have found that menopause had negative metabolic and cognitive consequences, there were no effects of VCID alone on cognitive function in young female mice, potentially due to the protective effect of ovarian estrogen production. Deficits in episodic‐like memory and activities of daily living appeared when menopause and VCID co‐occurred. Additionally, we are examining changes to estrogen receptor expression in the brain and integrity of the brain’s white matter, as white matter is particularly vulnerable to damage due to hypoperfusion. Further investigation into pathological features (brain inflammation, white matter, and vascular damage) may uncover the mechanisms through which menopause impacts cognitive function.
Sexual Dimorphism in Control of Heart Failure in Insulin Resistance
Authors: Yang Wanbao , Yan Hui , Zhou Fenghua , Pan Quan , Allred Kimberly , Allred Clinton , Sun Yuxiang , Threadgill David , Dostal David , Tong Carl , Guo Shaodong
Published: May 2022

The incidence of cardiovascular diseases (CVD) is highly associated with type 2 diabetes (T2D). Women at an early age show reduced incidence of CVD and improved insulin sensitivity compared to men, but these disparities disappear during postmenopausal. Thus, ovary and ovarian hormone, such as estrogen, are expected to protect from these diseases. This study aimed to investigate the role of ovaries and ovarian hormone estrogen in cardiac function and energy metabolism upon loss‐of‐function of cardiac IRS1 and IRS2 in mice and investigate the underlying mechanisms. We found that all male heart‐specific IRS1 and IRS2 double knock‐out (H‐DKO) mice died of heart failure (HF) at 6‐8 weeks with severely impaired energy metabolism. However, all female H‐DKO mice survived more than 1 year and showed improvement in energy metabolism. Removal of ovaries in H‐DKO female mice led to cardiac dysfunction, disruption of energy metabolism, and ultimately death. However, E2 supplement significantly improved cardiac function, enhanced energy metabolism, and prolonged the lifespan in both male and OVX female H‐DKO mice. Finally, we provided evidence that the protective effect of E2 is partially mediated through activation of Akt‐Foxo1 signaling in H‐DKO heart. These results show that estrogen protects mice from heart insulin resistance induced cardiomyopathy. This study indicates that estrogen signaling could be a potential target to prevent insulin resistance induced heart dysfunction in humans. Estrogen signaling is mediated through estrogen receptors, such as estrogen receptor (ER) α, ERβ, and membrane estrogen receptor (GPER). In the future, we will further decipher the role of estrogen receptors in estrogen mediated protection against cardiac dysfunction during insulin resistance.
The Epithelial‐Mesenchymal Transition and Survival in Chromophobe Renal Cell Carcinoma
Authors: Ali Waleed , Jacobs Daniel , Hoang Henry , Kajdacsy‐Balla Andre
Published: May 2022

Introduction and Objective

Advances in our understanding of the epithelial‐mesenchymal transition (EMT), how cells change polarity and surface‐expressed proteins in acquiring a more malignant state, has aided scientists in understanding cancer progression. Using in silico tools, this study examines how EMT‐related gene expression levels are associated with survival outcomes in the rare chromophobe renal cell carcinoma (ChRCC).

Methods

Using dbEMT, a curated database examining EMT literature, 1184 genes were selected as “EMT‐related” for further analysis. Kaplain‐Meier Survival plots were generated based on TCGA data on all recorded ChRCC patients for each gene (n=32 in “high” and “low” groups each based on a median cut‐off), with the step‐up Bonferroni method for multiple hypothesis testing correction being used (significance being established at p<0.004). Genes identified to be statistically significant in terms of survival outcome were selected for enrichment analysis through Cytospace to examine which biological pathways might predict survival outcome. Network analysis based on physical and predicted interactions using GeneMANIA and R was done to generate a list of non‐EMT related genes which might act with those statistically significant EMT‐related genes to affect survival, with cut‐off for for significance of both network and enrichment analysis being p<0.01. DisGeNET was used to observe if the gene expression profile of ChRCC resembled any other kind of cancer.

Results

From the original 1184 genes, 13 showed significant change following multiple hypothesis testing correction (p<0.0038), with all showing an increased expression relating to worse outcomes. Nine distinct processes, ranging from histone modification to SMAD2/3 nuclear pathways among others, were significantly overrepresented (p<0.01) in those 13 genes. The significant EMT‐related gene signature in ChRCC mimicked the profile of mantle cell lymphoma and urothelial carcinoma. Network analysis highlighted more than 20 additional genes which EMT‐related genes could interact with to affect survival.

Conclusions

This study showcases a small subset of EMT‐related genes significantly overexpressed in ChRCC cases with worse prognosis. Analysis highlighted pathway dysfunction regarding microtubule cytoskeletal organization and histone modification (among others) as a possible cause of increased mortality in cases of chromophobe renal cell carcinoma. Such a preliminary in silico analysis would allow for strategically targeted wet‐lab studies.
Interferon Inducible Pseudouridine Modification in Human Transcriptome by Quantitative Nanopore Sequencing
Authors: Huang Sihao , Zhang Wen , Katanski Christopher D. , Pan Tao , Qing Dai , Lolans Karen , Eren A. M. , Dersh Devin , Yewdell Jonathan
Published: May 2022

Pseudouridine (Ψ) is an abundant mRNA modification in the mammalian transcriptome, but its functions have remained elusive due to the difficulty of transcriptome‐wide mapping. The Ψ derivative, N1‐methyl‐Ψ, substitutes every U residue in the Pfizer/BioNTech and Moderna COVID mRNA vaccines. Previous research revealed that Ψ increases translation of reporter genes and this effect depends on Protein kinase R (PKR), an interferon inducible gene. Thus, we hypothesize that endogenous Ψ modification in mRNA may be associated with interferon induced cellular response. In order to map Ψ in the human transcriptome, we developed a nanopore direct RNA sequencing method for quantitative Ψ mapping (NanoPsu, https://github.com/sihaohuanguc/Nanopore_psU) that utilizes native content training on 1329 Ψ and U sites, machine learning modeling and prediction, and single read linkage analysis. We validated our method using quantitative RT‐PCR of Ψ sites in specific mRNAs and through the observation of global Ψ level decrease upon knockdown of TRUB1, a known Ψ writer for mRNA. We apply the nanopore direct RNA sequencing method to interferon β and γ treated human cells and reveal a widespread differential distribution of Ψ in the human transcriptome. Biologically we find interferon inducible Ψ modifications in interferon stimulated gene transcripts which is consistent with a role of Ψ in enabling efficacy of mRNA vaccines. Single read analysis of the nanopore data reveals that multiple Ψ sites within the same transcript can be linked with each other. In summary, our results reveal a potential role of mRNA Ψ modification in interferon induced response and provide a new aspect in the research of mechanism of immune response.
Drp1‐dependent Regulation of RelA/p65 Is Critical for Endothelial Cell Inflammation
Authors: Shadab Mohammad , Najar Rauf A. , Fazal Fabeha , Rahman Arshad
Published: May 2022

Endothelial cell (EC) inflammation is a major pathogenic feature of many diseases including acute lung injury (ALI). However, the role of the dynamin‐related protein 1 (Drp1), a mitochondrial fission factor, in regulating EC inflammation is poorly understood. We show here that Drp1 is a critical mediator of EC inflammation via its ability to control NF‐κB activation. When Drp1 is disabled, expression of adhesion molecules (ICAM‐1, VCAM‐1) and cytokines/chemokines (IL‐6, MCP‐1) is markedly reduced in cells stimulated with thrombin. Drp1‐depleted cells were also impaired in their ability to activate NF‐κB by thrombin. Mechanistic study revealed that thrombin‐induced phosphorylation and nuclear DNA binding of RelAp65 was inhibited in Drp1‐depleted cells. Surprisingly, the thrombin‐induced degradation of IκBα in the cytosol remained unaffected in Drp1‐depleted cells, suggesting a defect in the translocation of released RelA/p65 to the nucleus in these cells. Indeed, nuclear translocation of RelA/p65 caused by thrombin was inhibited in Drp1‐depleted cells. Together, these data uncover a novel function of Drp1 in mediating EC inflammation via regulation of phosphorylation and nuclear translocation of RelA/p65.
Differential regulation of L84F SOD1 in motor neurons and skeletal muscles: an insight into ALS pathology
Authors: Verma Sagar , Vats Abhishek , Devi Mandaville Gourie , Khurana Shiffali , Ganguly Nirmal K. , Chakraborti Pradip , Taneja Vibha
Published: May 2022

Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disorder characterized by progressive deterioration of motor neurons leading to skeletal muscle denervation. Recent evidences have highlighted early involvement of neuromuscular junction (NMJ) in ALS pathogenesis. NMJ is a specialized tripartite chemical synapse which involves a well‐coordinated communication among the presynaptic motor neuron, postsynaptic skeletal muscle and terminal Schwann cells. Despite several evidences, series of events triggering NMJ disassembly in ALS are still obscure.

The aim of our study is to investigate the cellular and molecular mechanisms of NMJ dysfunction in an in vitro Superoxide Dismutase 1 (SOD1) induced model of familial ALS. We have generated an ALS cell model based on a novel SOD1 mutation (L84F) identified previously in a family with a history of ALS. This mutation caused decrease in neurite outgrowth which led us to hypothesize that it may be involved in neuromuscular junction disassembly in ALS pathology.

We have characterized cellular components of NMJ by differentiating hybrid spinal cord cell line (NSC‐34) into motor neurons and myoblast cell line (C2C12) into myotubes. Expression of mutant SOD1 led to decrease in mitochondrial footprints in axons and synaptic terminals and altered mitochondrial dynamics in motor neurons. However mitochondrial fission/fusion is not altered in muscles suggesting differential consequences of mutant protein. L84F SOD1 mutant readily formed detergent‐resistant aggregates in motor neurons but failed to accumulate in skeletal muscles. Skeletal muscles displayed increased levels of LC3‐II compared to motor neurons suggesting a robust proteasome machinery of muscles is responsible for clearance of mutant protein. Furthermore, motor neurons exhibited increased cell death compared to skeletal muscles when subjected to exogenous stress. Interestingly, we observed that condition media from skeletal muscles was able to rescue motor neurons from exogenous stress. We are currently identifying paracrine neuroprotective factors secreted by the muscles. It is noteworthy that mutant SOD1 led to altered expression of post synaptic acetylcholine receptor complex which might hamper neuromuscular transmission in ALS.

To conclude, molecular cues from both neurons and muscles appear to play a role in compromising NMJ integrity in ALS.
In‐Vitro Antioxidant Assays and Computational Investigation of Phytoconstituents from Theobroma cacao Beans as Inhibitors of Neuro‐Modulatory Enzymes
Authors: Shodehinde Sidiqat A. , Omoboyowa Alex D. , Balogun Toheeb A.
Published: May 2022

Neurodegenerative disease (ND) is a heterogeneous group of disorders characterized by progressive deterioration of neuronal cells in the nervous system. Also, there are an increasing number of neurodegenerative disease cases as the number of elderly people increases. Biochemical and molecular investigations have shown that ND pathogenesis is strongly associated with pathologically misfolded proteins. Therefore, blocking altered proteins/therapeutic targets by small molecules may help in the management of neurological disorders such as Alzheimer’s disease and Parkinson’s disease. As such, this study was carried out to evaluate the pharmacological potential of aqueous extract from the medicinal plant, Theobroma cacao, from which bioactive compounds were identified with the potential to inhibit Arginase II and Monoamine Oxidase B (MAO‐B). Chemical profiling and characterization of T. cacao using High‐Performance‐Liquid‐Chromatography reveal the presence of catechin, p‐coumaric acid, epicatechin, gallic acid, caffeic acid, kaempferol, and ellagic acid as the common compounds in the Amazon and Amelonado type of T. cacao. The total phenolic content in Amelonado and Amazon were 1.420 mg and 0.270 mg GAE/g extract, respectively. Flavonoid content was 0.460 mg in Amelonado and 0.720 mg in Amazon. Antioxidant activity of the plant via DPPH radical scavenging effect shows T. cacao has 49.330% in Amelonado and 42.490% in Amazon. The high degree of radical scavenging effect of T. cacao was relatively the same in Amelonado and Amazon, which may be attributed to their high phenolic contents. To further establish the molecular mechanism of T. cacao antioxidant property and inhibitory mechanism against Argainse II and MAO‐B. Computational techniques incorporating molecular docking, pharmacokinetics models, and molecular dynamics simulation were employed. The molecular docking results showed anti‐neurodegenerative potential with Epicatechin showing the highest inhibition against MAO‐B with a docking score of ‐11.156 kcal/mol and kaempferol inhibiting Arginase II with a docking score of ‐7.002 kcal/mol. The lead complexes (epicatechin‐MAOB and Kaempferol‐Arginase II) were subject to MD simulation for a period of 100 ns for stability analysis. The stability shows the compounds established molecular interactions and were found to be stable. The pharmacokinetics model reveals the compound are promising therapeutic agents. Overall, bioactive compounds from T. cacao may be ideal phyto‐drugs in the management of neurological disorders.
TGFβ Induces Changes in Mitochondrial Homeostasis in Skeletal Muscle of Mice with Bone Metastases
Authors: Wilcox‐Hagerty Jenna R. , Xu Haifang , Hain Brian , Waning David
Published: May 2022

Cancer cachexia is a common neoplastic syndrome of advanced cancers characterized by a loss of skeletal muscle mass and decreased function. Patients with cancer cachexia have higher chances of morbidity and mortality. We have shown that mice inoculated with human MDA‐MB‐231 breast cancer cells, which metastasize to bone, have skeletal muscle weakness, cachexia, and altered skeletal muscle metabolism. Mice with bone metastases have increased serum levels of transforming growth factor beta (TGFβ). Serum TGFβ decreased after treatment with the bone anti‐resorptive agent, zoledronic acid (ZA), suggesting TGFβ is, in part, bone‐derived. Additionally, mice with bone metastases treated with ZA had increased skeletal muscle function. TGFβ has also been shown to decrease the master regulator of mitochondrial biogenesis, peroxisome‐activated receptor γ coactivator 1α (PGC1α), in C2C12 myotubes. Mitochondria provide muscle with energy (ATP) via oxidative phosphorylation, so we measured oxygen consumption and energy production using indirect calorimetry in mice with bone metastases. Oxygen consumption and energy production were both decreased in these mice suggesting a decrease in mitochondrial function. We then examined skeletal muscle mitochondria using transmission electron microscopy and western blotting. We found highly dysmorphic mitochondria and altered protein markers of mitochondrial biogenesis and dynamics. Additionally, LC3B protein was increased in skeletal muscle mitochondria demonstrating increased mitophagy in skeletal muscle of mice with bone metastases. From our previous knowledge and data of TGFβ and its role in mice with bone metastases, we hypothesize that TGFβ causes mitochondrial dysfunction in skeletal muscle of mice with bone metastases. We have shown that a 48‐hour treatment with TGFβ decreases protein expression in markers of mitochondrial biogenesis and dynamics in C2C12 myotubes. This data suggests that TGFβ could be causing the mitochondrial dysfunction and dysmorphia we see in our mouse model, but further experiments are needed to prove this hypothesis. Our data demonstrates the importance of mitochondrial function in skeletal muscle cells and skeletal muscle of mice with bone metastases. Understanding the role TGFβ plays in changes of skeletal muscle mitochondria in mice with bone metastases will help contribute to defining treatments for patients with cancer cachexia.
Positive Association between NIRS‐derived measures of Microvascular Reactivity and Mitochondrial Capacity in the Quadriceps and Hamstrings
Authors: Gosalia Jigar G. , Delgado Jocelyn G. , Studinski Matthew G. , McCully Kevin , Vairo Giampietro G. , Proctor David N.
Published: May 2022

BACKGROUND

Delivery of oxygen to the mitochondria is essential for oxidative phosphorylation and the production of ATP during rest and during muscle contraction. Despite widespread application of near infrared spectroscopy (NIRS), to our knowledge it has not been used to evaluate whether oxygen delivery to the mitochondria, as assessed by post‐ischemic reoxygenation parameters, is related to the oxidative capacity of the muscle.

PURPOSE

To determine the relation between microvascular reactivity and mitochondrial oxidative capacity in the knee joint musculature using NIRS.

METHODS

Bilateral measurements of the vastus lateralis (VL) and medial hamstring (MH) muscles were taken in 14 moderate to highly active healthy adults (7M/7F, age: 22 ± 3 yrs, BMI: 21.3 ± 3.5 kg/m2, physical activity levels: 4190 ± 1324 MET minutes/week). Two NIRS probes (Portamon, Artinis) were secured over the respective muscle bellies, which collected data during a 30 second bout of surface electrical stimulation that caused an increase in metabolism followed by brief arterial occlusions to determine the metabolic rate of the muscle over time. A final resting metabolism measurement was taken 5 minutes after the arterial occlusions to obtain the time to half recovery of metabolic rate (NIRS k), which is a valid estimate of mitochondrial oxidative capacity. Microvascular reactivity was measured as 1) the 10‐second reoxygenation slope of the oxygen saturation signal (StO2) and 2) the half time to peak oxygenation following 5 minutes of arterial occlusion in each muscle.

RESULTS

There was a significant positive association between NIRS kand the reoxygenation slope in both the VL (r = 0.64, p = 0.025) and the MH (r = 0.53, p = 0.049) muscles. NIRS kvalues were also positively associated with the time to half peak oxygenation in the VL (r = 0.65, p = 0.021) and the MH (r = 0.60, p = 0.026) muscles. There were no differences between the VL and MH muscles for NIRS kvalues (Mean ± SD: 1.78 ± 0.24 vs. 1.78 ± 0.33, p = 0.99), 10‐second reoxygenation slope (2.16 ± 0.84 vs. 1.99 ± 0.96, p = 0.65), and half time to peak oxygenation (8.4s ± 1.6 vs. 9.1s ± 2.2, p = 0.38).

CONCLUSION

Our results suggest that microvascular reactivity is positively associated with the mitochondrial capacity of the knee joint musculature in this sample of healthy adults. Whether these associations exist in different clinical populations warrants future investigation.
New non‐canonical function of catalytically dead hexokinase 1 in ovarian cancer
Authors: Heneberg Petr , Simcikova Daniela , Gardas Dominik , Zacek Petr
Published: May 2022

Hexokinases (HKs) have been found amplified in primary cancer cells, with the amplification of HK1 reported commonly in ovarian cancer cells. The function of HKs extends beyond glucose phosphorylation, and HKs serve as regulatory proteins in tumorigenesis. We addressed the non‐canonical roles of HK1 in ovarian cancer cells by examining the effects of the deletions of HK1 and HK2 in TOV‐112D ovarian adenocarcinoma cells. We reverted these effects by re‐introducing wild‐type HK1 and HK2, and we compared the HK1 revertant with the knock‐in of catalytically‐dead HK1 p.D656A. We subjected these cells to a battery of metabolic and proliferation assays and targeted GCxGC‐MS metabolomics. When expressed in vitro, the p.D656A mutant did not manifest a measurable KM and exhibited Vmax lower by almost two orders of magnitude when compared to the wild‐type enzyme. Knock‐in of HK1 p.D656A into HK1 KO cells reverted the HK1 KO‐induced effects in a broad range of assays, including the alamarBlue cell viability assay under low glucose conditions, the NADPH/NADP and NAD/NADH ratios, and glutathione levels in metformin‐treated cells. In the presence of 0.4 mM glucose, the phenotype of HK1 p.D656A knock‐in cells resembled the phenotype of cells with deleted HK2 but not those with deleted HK1 with regards to the levels of TCA intermediates, aspartate and cysteine, and glutamate. Moreover, HK1 deletion increased the levels of branched amino acids, which was completely eliminated by the expression of catalytically‐dead HK1. The HK1 deletion (but not HK2 deletion) suppressed the growth of xenotransplanted ovarian cancer cells, and nearly abolished the tumor growth when the mice were fed the glucose‐free diet. In summary, we provided the evidence that HK1 is involved in the so far unknown glycolysis‐independent moonlighting function and influences metabolism even in the glucose‐free conditions.