bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Dynamics Investigation of Oncogenic BRAF Mutations
Authors: Markusic Borna , Wang Zhihong , Liu Zhiwei
Published: May 2022

BRAF is one member of the RAF serine/threonine kinase family and a key component of the RAS‐RAF‐MEK‐ERK (MAPK) signaling pathway, controlling cell growth, proliferation, differentiation, migration and survival. BRAF is the most frequently mutated kinase in human cancers, with over 200 mutations discovered, among which V600E and G469A are the most common BRAF mutations. Current therapies with ATP‐competitive inhibitors are potent against BRAFV600E, yet they display “paradoxical activation” and drug resistance toward BRAFG469Aandother non‐V600 BRAF mutants. Thus, novel BRAF therapies towards non‐V600 variants are urgently needed. However, the biochemical properties of non‐V600 BRAF mutants and the cause of drug response are unclear due to the lack of structures of BRAF mutants. In this study, we applied molecular dynamics (MD) simulations to investigate the structure, dynamics and functions of oncogenic BRAFG469A, in comparison with BRAFWT and BRAFV600E. For each BRAF type (WT or mutant), MD simulations are carried out for its monomer and dimer with or without 1 or 2 bound vemurafenib in explicit aqueous solution. Our work provides atomic information on mechanism of aberrant activation by G469A mutation, “paradoxical activation” and structural dynamics of the nucleotide pocket, activation loop, and dimer and inhibitor/BRAF interfaces. G469A mutation makes hydrophobic contact between methyl of A469 with alkyl chain of K483 in the binding pocket. This stabilizes the K483‐E501 salt‐bridge, which functions to bring αC‐helix towards the active site. Thus, G469A stabilizes the active conformation by positioning αC‐helix close to the P‐loop through increasingly stabilized salt‐bridge relative to WT and V600E. Consequently, our simulations illustrate inability of vemurafenib to disrupt active conformation of BRAFG469A by separating αC‐helix from the nucleotide binding pocket. This decreased structural fluctuation upon inhibitor binding to one protomer explains the ability of “paradoxical activation” of BRAF dimer through allosteric activation.
Expression Status of Proton Sensing Receptor GPR68 in Various Breast Cancer Molecular Subtypes
Authors: Elemam Noha M. , Youness Rana , Talaat Iman M. , Maghazachi Azzam A.
Published: May 2022

Background and aim

Breast cancer has become the most diagnosed cancer and the leading cause of global cancer incidence in 2020. It is quite known that highly malignant and invasive cancers have disrupted metabolism that leads to the creation of an acidic milieu of the tumor microenvironment (TME). Among the acid sensing G protein‐coupled receptors is GPR68/OGR1. In this study, we aimed to investigate the expression of GPR68 in breast cancer patients and cell lines in order to understand its role in the breast cancer TME.

Patients, Materials and Methods

In silico tools such as TNMplot and UALCAN were used to assess the expression of GPR68 in breast cancer patients. The expression pattern was validated in fresh and formalin‐fixed paraffin‐embedded tissues obtained from breast cancer patients using qPCR and immunohistochemistry (IHC), respectively. Also, in silico tool EMBEL‐EBI was used to assess GPR68 expression in different breast cancer cell lines. Validation of GPR68 expression was done on the mRNA and protein levels using qPCR and immunofluorescence techniques in four different breast cancer cell lines (MCF‐7, MDA‐MB‐231, BT‐549 and SkBr3).

Results

GPR68 expression was found to be significantly increased in breast cancer patients using the in silico tools and upon validation using qPCR and IHC (figure 1). Additionally, upon classification according to molecular subtypes, the luminal subtype showed highest GPR68 expression followed by triple negative and HER2 enriched classes. Also, in silicoanalysis revealed that the triple negative breast cancer cell line MDA‐MB‐231 showed the highest expression of GPR68, followed by the luminal A, MCF‐7 and Her2+ SkBr3, and lastly BT‐549. Quantitative PCR and immunofluorescence were used to validate this pattern of expression (figure 2), where all four cell lines showed cytoplasmic and membranous expression indicating possible internalization of GPR68 receptor that might be due to its excessive activation.

Conclusion

GPR68 plays a critical role in the TME of breast cancer and its different molecular subtypes. Such a marker could be a potential diagnostic marker and therapeutic target in breast cancer.
Comparative evaluation of methods for determining sensitivity to colistin in E. coli isolates
Authors: Nikitin Georgy , Sulyan Ophelia , Sukhinin Alexander
Published: May 2022

The study aims to compare two colistin susceptibility measurement approaches: by disk diffusion test and using VITEK® 2 Compact instrument. We collected 103 E.coli isolates from 7 livestock farms in St. Petersburg and the Leningrad region. Minimum inhibitory concentration (MIC) of colistin was determined using disk diffusion test and VITEK® 2 Compact instrument and then compared with the reference broth microdilution method results. The processing of the results was based on the EUCAST recommendations. Eight E. coli isolates proved to be resistant to colistin when determining MIC in broth microdilutions. Two isolates (eco‐103 and eco‐151) carried the MCR‐1 gene. The identical isolates were tested by other methods to determine the MIC. The data obtained by the three methods were identical. This suggests that the disc diffusion test can be used to determine the MIC of colistin on E.coli. This method has several advantages over the reference method and VITEK‐2 Compact use since it is simultaneously cheap, fast, and accurate for determining the MIC of colistin.
Anatomical and topographic features of lymphatic outflow of the heart in an Anglo‐Nubian goat
Authors: Melnikov Sergey , Shchipakin Mikhail , Zelenevskiy Nikolay , Khvatov Victor , Bylinskaya Darya , Vasiljev Dmitriy
Published: May 2022

The lymphatic system of the heart is directly involved in the spread and development of many pathological processes in animals, particularly neoplasias of various etiologies. Precise knowledge of the lymphatic outflow pathways from the heart gives access to close study of pathogenesis. It allows to actively influence the elimination of the process through preventive and therapeutic measures. In this regard, our study aims to explore the anatomical and topographic features of the lymphatic outflow of the heart in an Anglo‐Nubian goat. The material for the study was fifteen corpses of Anglo‐Nubian goats aged from one to two years. The research methods were thin anatomical preparation, ink‐gelatin perfusion of lymphatic heart vessels, perfusion with Gerota's mass and yellow lead paint, and subsequent radiography of the material under study. It was established that subepicardial lymph nodes collect lymph from the endocardium, myocardium, and epicardium. In Anglo‐Nubian goats, the subepicardial lymphatic vessels of the left surface of the left ventricle and the caudal part of the left surface of the right ventricle merge in the sulcus interventricularis paraconalis and sulcus coronarius, forming lymphatic vessels of a higher order. After the fusion of the vessels, the left lymph collector of the heart is formed. It follows the ventral surface of a. pulmonalis, bends around it ventrally or dorsally, and then flows into the left tracheobronchial lymph node. Subepicardial lymphatic vessels of the cranial part of the left surface of the right ventricle and the right surfaces of the right and left ventricles merge in sulcus interventricularis subsinuosus and sulcus coronarius, also forming lymphatic vessels of higher orders named right lymph collector. The vessel passes under auricula dextra, goes to truncus brachiocephalicus, and is guided through it cranially until it flows into the cranial mediastinal lymph node, located in the Anglo‐Nubian goat at the beginning of a. subclavia sinistra. The outflowing lymphatic vessels leave the cranial mediastinal and left tracheobronchial nodes, flow either directly into the thoracic duct or into the common trunk of the mediastinal lymph nodes. The atrial lymphatic vessels of the Anglo‐Nubian goat flow either into the lymphatic vessel of sulcus coronarius or directly into the cranial mediastinal lymph nodes. Thus, according to the results of thу study, it was established that the lymphatic drainage of the heart in a goat of the Anglo‐Nubian breed is carried out in two main ways, dividing into the left and right halves of the heart. Also, according to the research data, in a goat of the Anglo‐Nubian breed, the possibility of varying the outflow of lymph from the atria in two ways was determined.
Examining Rates of Evolution Within the Drosophila Insulin Signaling Pathway
Authors: Silverman Jonah , Hark Amy T. , Rele Chinmay P. , Reed Laura K.
Published: May 2022

We are interested in understanding the evolutionary rates of genes that function in a biological network. We focused on the insulin signaling pathway due to its conservation across the animal kingdom, with literature documenting many interactions within this pathway. We used synteny and sequence similarity to identify orthologs of Drosophila melanogaster genes in a variety of Drosophilaspecies. Our focus included genes seen as central to the pathway such as Pdk1 and Tor as well as other genes such as HDAC4and srlwith at least one known connection to insulin signaling. In collaboration with the Genomics Education Partnership, we performed manual gene annotations to derive accurate protein coding sequences within our species of interest. We took a comparative genomics approach using BLAST and data from the UCSC Genome Browser, including RNA‐seq, additional alignment algorithms, and gene predictors to inform identification of exon‐intron boundaries. Comparison of the determined protein coding sequences from the target Drosophila species to the established sequences from D. melanogasterprovides insight into how a gene has been evolving since these two species diverged. Initial analysis suggests large variability in the rates of evolution taking place in our genes of interest and some correlation between higher expression level and greater sequence conservation. We are also considering our data with respect to a gene’s position within its network and its role in biological pathways to help illuminate which factors dictate gene evolution rate. We hope our methodology and results can add to our understanding of gene evolution across biological systems.
Interferon‐γ Induced PD‐L1 Expression in Ovarian Cancer Cells is Regulated by IRF1 Signaling
Authors: Padmanabhan Sveta , Gaire Bijaya , Vancura Ales , Vancurova Ivana
Published: May 2022

The immune checkpoint programmed death ligand‐1 (PD‐L1) is highly expressed in cancer cells, including ovarian cancer (OC) cells. High PD‐L1 expression in ovarian cancer correlates with poor prognosis, but the mechanisms that regulate the PD‐L1 expression in OC remain poorly understood. We have recently shown that IFNγ induces the PD‐L1 expression in OC cells, resulting in their increased proliferation and invasion. Here, we show that the IFNγ‐induced PD‐L1 expression in OC cells is associated with increased levels of STAT1, Tyr‐701 pSTAT1 and Ser‐727 pSTAT1 in OC cells. JAK1 and STAT1 suppression significantly decreases the IFNγ‐induced PD‐L1 mRNA and protein levels in OC cells. In addition, IRF1 suppression significantly decreases the IFNγ‐induced gene and protein levels of PD‐L1, indicating that the IFNγ‐induced PD‐L1 expression in OC cells is dependent on IRF1 signaling. Chromatin immunoprecipitation data demonstrate that IFNγ induces occupancy of STAT1, Ser‐727 pSTAT1 and IRF1 at STAT1 and IRF1 binding sites in human PD‐L1 promoter. Together, these results show that the IFNγ‐induced PD‐L1 expression in OC cells is regulated by IRF1/JAK1/STAT1 signaling, and suggest that targeting the IRF1/JAK1/STAT1 pathway may provide leverage to regulate the PD‐L1 levels in ovarian cancer.
An Introduction to Anatomic Knowledge in Pre‐Hispanic Mesoamerica
Authors: Kruskie Megan
Published: May 2022

Background and Objective

The study of the history of anatomy has primarily focused on contributions from Asia and the Mediterranean, while other civilizations are only briefly mentioned. The purpose of this study was to explore the anatomic knowledge of three Mesoamerican civilizations ‐ the Olmecs, Maya, and Aztecs ‐ based on available literature, as well as identify barriers to research on this subject.

Results

In order to understand the anatomic knowledge of these civilizations, medical researchers have turned to other fields such as archaeology and linguistics. The Olmec heart effigy portrayed a four chambered heart and the great vessels, making it the earliest accurate depiction of the organ. Other Olmec sculptures record observations about human gestation, recognizable stages of fetal development, and congenital abnormalities. Analysis of Mayan dictionaries and other works show that their understanding of anatomy, physiology, and pathology compared to that of pre‐Vesalian Europe. The Aztecs demonstrated their knowledge of anatomy and physiology that was gained through the practice of sacrificial rituals in large ceramic figures and collections of medical texts. Identified barriers to this research include destruction of primary sources by the Spanish during the conquest, substantial linguistic and archaeologic differences across regions, as well as a general lack of investigation.

Conclusion

With the reinterpretation of sculpted figures and by taking a second look at their written works, researchers have begun to reveal that the Olmec, Maya, and Aztec peoples had a much better understanding of anatomy than they have previously been credited. Their knowledge of anatomy and medicine in general should be recognized and discussed in the same regard as that of the Mediterranean and Asia.
Repair of the Murine Tympanic Membrane Displays Hallmarks of Regeneration
Authors: Scaria Sonia , Frumm Stacey , Sheth Amar H. , Tward Aaron
Published: May 2022

Background

The tympanic membrane (TM) has a remarkable ability to repair itself, with perforations typically closing in days to weeks in all mammalian species studied. The cellular and molecular mechanisms underlying TM repair remain largely unknown, however. This study looked to thoroughly characterize the repair process of the injured TM and uncover mechanisms of repair to inform future treatments for TM disorders. We hypothesized that the TM does not undergo canonical “wound healing” but regenerates in response to injury.

Methods

Cells from TMs injured on adult mice 14, 7, 3, and 1 day prior to sacrifice were submitted for single‐cell RNA sequencing using the 10x Genomics scRNA‐seq platform. Using Seurat clustering and the CellFindR algorithm, unbiased cell clusters were generated with matrices of differentially expressed genes for each cluster. Protein detection via immunofluorescence (IF) and RNA detection via RNAscope were used to biologically validate and define the anatomic locations of distinct cellular populations. The K5Cre‐ERT2 promoter was utilized in mouse models to genetically manipulate and fluorescently label keratinocyte (KC) populations. The mTmG and Confetti reporters were utilized as fluorescent labels.

Results

RNA expression data from all timepoints of perforation were merged and analyzed, revealing 8 distinct major populations of cells and revealing time‐dependent transcriptional shifts in each layer of the TM. From both cross‐sectional and whole‐mount views, the TM shows a rapid, proliferative response to injury by 18 hours post‐injury, predominantly in the KCs. 3 days after perforation, there are large transcriptional shifts in the immune, mesenchymal, and mucosal populations. The multi‐layered tissue shows a large volumetric increase by day 7 but quickly remodels and restores the original volume of the TM by day 14. At slightly longer timepoints, the radial and circular collagen patterning of the TM is also restored, creating a scar‐free structure. We identified a regeneration‐induced “wounded epithelial” population, characterized by a combination of distinct marker genes. A K5Cre‐ERT2;Confetti mouse model shows that the population migrates from known stem cell regions of the organ to the site of injury. Based on expression values and immunostaining, EGFR signaling is upregulated during regeneration, corresponding with increased expression of EGFR ligands and processing co‐factors. When EGFR is deleted in vivo, using a K5‐CreERT2;Egfrfl/fl; R26mTmG/mTmG mouse model, TMs no longer display proliferation post‐injury and cannot repair perforations.

Conclusions

TM perforation healing displays a complex and coordinated series of transcriptional, morphological, and patterning events, ultimately culminating in the restoration of normal tissue architecture to the TM. These characteristics are more typical of epimorphic regeneration, rather than more typical scar induced mammalian wound healing.
Establishing Neurospheres in vitro by a 3D Suspension Culture System and Evaluating Neurite Growth to Facilitate Nerve Repair
Authors: Harley‐Troxell Meaghan E. , Dhar Madhu , Anderson David E. , Millet Larry J.
Published: May 2022

The overall goal of this research is to promote nerve repair through applications of carbon‐based nanoparticles and mesenchymal stem cells in a synthetic extracellular matrix. We hypothesize that this combination of nerve tissue engineering technologies will provide us with the optimal environment for neuronal growth. Towards this end, we assess the suitability of neurospheres to be used as an in vitro model to evaluate axonal outgrowth and for the production of cell‐free extracellular matrices to facilitate neuronal growth. Here we employ a new commercial, high throughput 3D culture system to establish neurospheres. A single 3D bioreactor provides a stress‐free environment to produce a 10‐fold increase in the number of neurospheres compared to the conventional 96‐well U‐bottom plate. This in vitro method alleviates the challenges of a co‐culture process, producing neurospheres with a more efficient and cost‐effective approach. Neurospheres were established using primary neurons isolated from 1 to 3‐day old rat hippocampi. Reactors were coated with no‐stick reagents to prevent cell adhesion to the reactor chamber during neurosphere assembly. After 48 hours in the bioreactor, seeded neurospheres were transferred to substrate‐coated (laminin + poly‐D‐lysine (PDL), PDL only) and tissue culture plates (tissue culture treated polystyrene (TCPS)). Immunochemistry and microscopy were used to identify and quantify the neurosphere size and neurite length. The data shows that laminin provides superior neurite outgrowth for neurospheres compared to non‐biological (TCPS) and conventional (PDL) synthetic substrates. Our next steps are to incorporate mesenchymal stem cell and endothelial cell populations to develop an in vitro organoid for nerve repair studies with nanoparticles and synthetic extracellular matrices. The long‐term goal of this project is to generate a regenerative medicine strategy to treat peripheral nerve injuries.
Role of adropin in reducing arterial stiffness in type 2 diabetes
Authors: Jurrissen Thomas J. , Ramirez‐Perez Francisco I. , Cabral Francisco J. , McMillan Neil J. , Fujie Shumpei J. , Butler Andrew A. , Banerjee Subhashis , Sacks Howard S. , Manrique‐Acevedo Camila , Martinez‐Lemus Luis A. , Padilla Jaume
Published: May 2022

Adropin is a peptide primarily expressed and secreted by the liver and is known to regulate energy homeostasis. Increasing evidence also indicates that adropin can exert vascular effects and its low circulating levels are associated with increased arterial stiffness, obesity, and type 2 diabetes. However, whether reduced adropin contributes to arterial stiffening in type 2 diabetes remains unknown. Herein, we tested the hypothesis that loss of adropin in non‐diabetic mice causes arterial stiffening and that the converse is also true. That is, adropin exposure reduces arterial stiffness in diabetic mice. In alignment with this hypothesis, we found that 1) mesenteric arteries from adropin knockout male mice are stiffer than those from wild‐type littermates; 2) exposure of human endothelial cells and mesenteric arteries from diabetic (i.e., db/db) male mice to adropin reduces actin polymerization and stiffness; 3) adropin‐induced reduction of actin polymerization and softening of endothelial cells and diabetic arteries is abrogated by inhibition of nitric oxide synthase; and 4) treatment of diabetic male mice with adropin for four weeks reduces arterial stiffness in mesenteric arteries. Collectively, these findings support the notion that reduced adropin may be implicated in arterial stiffening and thus represent a novel therapeutic target to lessen arterial stiffness in type 2 diabetes.
Use of Nucleoside Modified mRNA Encoding Regenerative Factors Encapsulated with Lipid Nanoparticles to Alleviate Acute and Chronic Murine Liver Diseases
Authors: Gouon‐Evans Valerie , Rizvi Fatima , Everton Elissa , Smith Anna R. , Ying Tam , Pardi Norbert , Weissman Drew
Published: May 2022

The remarkable ability of the liver to regenerate by proliferation of mature hepatocytes constitutes the first mechanism of repair commonly named the hepatocyte‐driven regeneration. Yet, during chronic liver injury or acute severe hepatocyte death, proliferation of mature cells becomes exhausted. In these cases, alternative precursors of hepatocytes that derive from cholangiocytes have been identified in both human and rodent diseased livers. They are presently growing evidence of conversion of these cholangiocytes into hepatocytes as defined as the cholangiocyte‐driven regeneration. Despite these two mechanisms of liver repair, end stage liver disease remains the 12th most common cause of death in the United States, begging for therapeutic strategies to harness mechanisms of liver regeneration. Here we demonstrate that delivery via nucleoside modified mRNA complexed to lipid nanoparticles (mRNA‐LNP) to the liver of the key hepatocyte mitogen hepatocyte growth factor (HGF) and epidermal growth factor (EGF) enhance hepatocyte‐driven repair, while delivery of vascular endothelial growth factor A (VEGFA) mRNA‐LNP promotes cholangiocyte‐driven repair. We confirm specific hepatotropism of mRNA‐LNP via intravenous injection of firefly luciferase encoding mRNA‐LNP, with protein expression lasting about 3 days. In the liver, virtually all hepatocytes are transfected along with a subpopulation of endothelial and Kupffer cells. In homeostasis, HGF mRNA‐LNP efficiently induce hepatocyte proliferation. In a chronic liver injury mouse model recapitulating non‐alcoholic fatty liver disease, injections of both HGF and EGF mRNA‐LNP sharply reverse steatosis and accelerate restoration of liver function. Likewise, HGF and EGF mRNA‐LNP accelerate liver regeneration after acetaminophen‐induced acute liver injury with rapid return to baseline alanine transaminase (ALT) levels. Transient delivery of VEGFA in acute and chronic injured mouse livers via mRNA‐LNP induces robust cholangiocyte conversion to hepatocytes as well as reversion of steatosis and fibrosis. This study introduces mRNA‐LNP as a potentially translatable safe therapeutic intervention to harness both hepatocyte‐ and cholangiocyte‐driven liver regeneration to ultimately treat human acute and chronic liver diseases.
The Effect of Mutations on Protein‐Protein and Protein‐Ligand Interactions for a 17β‐Hydroxysteroid Dehydrogenase
Authors: Roman David , Boldyrev Mary K. , Yang Song‐Yu , Philipp Manfred
Published: May 2022

17beta‐hydroxysteroid dehydrogenase type 10, also known as 3‐hydroxyacyl CoA dehydrogenase type 2, is variously abbreviated as HSD10, ABAD, ERAB, SDR5C1 or HADH2. The gene for this homotetrameric mitochondrial enzyme, designated as HSD17B10, is found on the X chromosome. Mutations in this protein are associated with neurodegenerative disorders.

In addition to its enzymatic activities, HSD10 is a structural component of human ribonuclease P (7ONU.pdb) and is bound to beta‐amyloid during Alzheimer's disease (1SO8.pdb).

This study examines single nucleotide polymorphisms found in HSD17B10 for their possible effects on protein structure, protein‐protein interactions, and protein‐ligand interactions, concentrating on the NAD+ binding site. This study compares the mutations that result from these polymorphisms to the sequence differences between rat and human HSD10.

References: Kissinger et al, JMB, 342, 943 (2004); Powell et al. JMB 303, 311 (2000); Yang et al. J. Steroid Biochem. Mol. Biol. 143, 460 (2013)
CHST11‐mediated microenvironment events contribute to pulmonary fibrosis and cancer progression
Authors: Li Chien Hsiu , Chan Ming‐Hsien , Hsiao Micheal
Published: May 2022

The Carbohydrate sulfotransferase (CHST) family is essential for Chondroitin Sulfate synthesis and is associated with cancer progression. Comparing all CHST families, we found that CHST11 has prognostic value than other families. CHST11 is related to the overall survival rate in the pan‐cancer profile, among which lung adenocarcinoma is the most significant. From pulmonary fibrosis to lung cancer, the expression of CHST11 showed a linear increase. We analyzed the CHST11 interactome, and the results indicated that CHST11 contributes to diverse canonical pathways, including immune microenvironment, fibrosis, and cancer progression. Based on the prediction results of Ingenuity Pathway Analysis (IPA), we speculate that CHST11 can secrete IL1B and IL6 signals to activate the upstream regulator IRF8. This is also the key to the fact that IRF8 has contributed to cystic fibrosis and lung cancer progression. In addition, IRF8 can also transactivate CD80 and CD86 to reform the tumor microenvironments and immune response. By reversing the CHST11‐IL6/IL1B‐IRF8‐CD80/CD86 mediated axis, we confirmed that VE821 and LY2603618 have the value of drug repurposing. Our study proves novel insight into how CHST11 contributes to cystic fibrosis and lung cancer by reprogramming the immune microenvironment.
Emerging Role of Nrf2 in Altering Cardiac Iron Metabolism during Myocardial Infarction
Authors: Jayakumar Deepthy , S Narasimhan Kishore Kumar , Rajkumar Abinayaa , Raghunathan Malathi , Sundaram Sandhya , Periandavan Kalaiselvi
Published: May 2022

Cardiac function demands a combination of iron availability for its high energy requirement and tight regulation of redox signaling. Nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) is a master transcriptional regulator of antioxidant/cytoprotective genes and recent reports has emphasized its role in regulating iron metabolism and ferroptosis in various pathologies including cardiovascular diseases. We hypothesized that iron metabolism is deregulated in myocardial infarction (MI) through alterations in Nrf2/hepcidin/ferroportin axis. Adult Wistar rats weighing ~200g were administered with isoproterenol (ISO; 85mg/kg body weight) subcutaneously for two days to induce MI. After the experimental period, the heart and blood were collected under ketamine/xylazine anesthesia. Morphological, histopathological, serum markers (cTnT, CK & LDH), inflammatory cytokines and western blotting analyses for Nrf2 and key proteins involved in iron trafficking and metabolism were performed. Morphological examination (left ventricle/tibia length; LV/TL) revealed maladaptive remodeling in ISO‐administered rats (P=0.0002). Histopathological analysis demonstrated normal cardiac architecture with a proper myofibrillar arrangement in vehicle‐administered rats, whereas in ISO‐administered rats disoriented myofibril with intense neutrophil infiltration and necrotic impressions were observed. Further, a marked elevation (P<0.05) in the inflammatory markers IL6, TNFα, IL1α, and CRP were observed in ISO‐administered rats which corroborates with the neutrophil infiltration/necrotic impressions in these animals. We also observed elevated cTnT, CK and LDH (P<0.05) whereas the total iron was decreased (P=0.0286) in the serum of ISO‐administered rats. In contrast, we detected that labile redox‐active iron was increased in the heart of ISO‐administered rats. Western blot analysis in the heart tissues from vehicle and ISO‐administered (remote, peri‐infarct and infarct zones) rats revealed an augmentation in HIF1α, hepcidin, and ferritin (P<0.05) levels whereas no significant changes were seen in ferroportin. This increment in hepcidin/ferroportin ratio substantiates the augmented levels of iron within the cardiac tissue. Subsequently, we found elevated accumulation of Nrf2 in the nucleus of ISO‐administered rats highlighting its transcriptional activity which was demonstrated further by increase in the levels of its downstream targets NQO1 and HMOX‐1. Overall, augmented Nrf2‐transcriptional activity during MI regulates iron metabolism through hepcidin/ferroportin axis which in turn leads to iron sequestration within the cardiomyocytes and exacerbates MI.
High Throughput Multi‐omic Characterization of Clonal Hematopoietic Stem Cells During Progression of Myeloid Malignancies
Authors: Son Albert J. , Kroger Benjamin , Huang Yi , Chung Stephen
Published: May 2022

Hematopoietic stem cells (HSCs) are responsible for giving rise to all other lineages of blood cells in the body. Over time, mutations in HSCs can promote the outgrowth of clonal populations that outcompete other HSCs, resulting in a phenomenon called Clonal Hematopoiesis of Indeterminate Potential (CHIP). Though not cancerous in and of itself, CHIP can progress to more serious hematologic disorders, such as the Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML). The mechanisms of clonal expansion, by which certain mutant HSCs acquire a competitive advantage over other HSCs, currently remain largely undeciphered, as are the mechanisms by which clonal HSCs drive the initiation of MDS and contribute to the development of AML. Moreover, previous studies have shown that such mutant HSCs are resistant to conventional therapies and may act as reservoirs for disease relapse and progression. In the past, researchers were hindered by bulk cell analyses and the relative rarity of HSCs, but advances in single‐cell omics have now enabled us to explore the molecular heterogeneity of clonal HSCs and identify distinct clonal populations based on genotype and cell surface phenotype. We used single cell RNA sequencing to examine the transcriptional dynamics of purified HSCs from MDS and AML patients before and after treatment, as well as from age matched elderly controls. Interestingly, dimensionality reduction methods such as UMAP and tSNE revealed a reservoir of control HSCs that clustered with MDS HSCs. Upon comparison with other normal control HSCs, we found that genes associated with aging, mitochondrial function, and particular ion channels were strongly upregulated in these “MDS‐like” control HSCs, while genes involved in ribosomal and translation activity, along with certain surface markers, were substantially downregulated. Additionally, ribosomal transcripts were depleted in MDS HSCs from patients who did not respond to treatment. These results support the notion that the most immature HSCs, which impose the strictest constraints on translation, might clonally expand and initiate CHIP and/or MDS upon acquiring driver mutations, also serving as treatment resistant populations that underlie disease relapse. We also subjected purified MDS HSCs to simultaneous single cell targeted DNA sequencing and cell surface phenotyping, which allowed us to correlate the cell surface phenotype and genotype for specific clonal populations and identify cell surface markers that can be used to isolate HSCs with enhanced engraftment ability. Taken together, these results indicate that combined single cell genotyping and phenotyping can be used to track clonal populations across different stages of pathogenesis, providing further insight about the development of CHIP and its progression to MDS and AML.
Junction adhesion molecule‐A is involved in asthma through JNK and ERK pathway
Authors: Jang Ansoo , An Min‐Hyeok
Published: May 2022

Background

Junctional adhesion molecule (JAM)‐A is an immunoglobulin‐like molecule that colocalizes with tight junctions (TJs) in endothelium and epithelium and is also found on blood leukocytes and platelets.

Objective

We aimed to elucidate the role of JAM‐A on airway hyperresponsiveness and inflammation in asthma using a murine asthma model, and to check blood level of JAM‐A in asthmatic patients.

Methods;

Using mice sensitized and challenged with OVA, as well as mice sensitized and challenged with saline, we investigated whether Jam‐a be involved in the pathogenesis of bronchial asthma. The level of JAM‐A was checked in the plasma of asthmatic patients and control subjects. The relation of JAM‐A with clinical variables in patients with asthma was searched.

Results

JAM‐A level had higher concentrations in plasma from patients with asthma (n = 19) than that of healthy controls (n = 12). JAM‐A level was correlated with FVC%, FEV1%, FEV1/FVC, total IgE, and blood lymphocyte proportion in patients with asthma. Jam‐a protein expression in lung tissue was significantly increased in OVA/OVA mice compared with control mice. Phospho‐JNK and phosphor‐ERK protein expression in lung tissue was significantly increased in OVA/OVA mice compared with control mice. A western blotting was used NHBE cells treated with HDM extracts for 4, 8 and 24 h. NHBE cells showed that HDM extracts resulted in increased JAM‐A, phospho‐JNK, phospho‐ERK expression.

Conclusion;

These data suggest that JAM‐A be elevated in plasma of patients with asthma, which raise the possibility that JAM‐A be involved in asthma through JNK and ERK pathway.
The Minichromosome Maintainance Complex is Up‐regulated in the Placentas of Low‐Insulin Sensitive Mothers in the First Trimester of Pregnancy
Authors: Bandres‐Meriz Julia , Hurtado Irene , Majali‐Martinez Alejandro , Hoch Denise , Honeder Sophie , Birner‐Grünberger Ruth , Dalgaard Louise T. , Desoye Gernot
Published: May 2022

Introduction

Obesity in women at reproductive age is increasing worldwide and is a risk factor for pregnancy complications. In the first trimester of pregnancy the human placenta is rapidly growing making it sensitive to environmental influences. Therefore, we hypothesized that the altered intrauterine milieu associated with maternal obesity modifies placental proteome and function.

Methods

Maternal BMI was calculated and leptin, glucose, C‐peptide and insulin sensitivity (ISHOMA) quantified in maternal serum. To identify obesity‐associated changes in the placental proteome we run untargeted proteomics (LC‐MS/MS) in placentas from lean (n=10) and obese (n=10) women (gestational age 5+0‐ 6+6 weeks). The results were validated using westernblotting and immunohistochemistry. The first trimester trophoblast cell line ACH‐3P was used to perform functional assays in vitro.The cells were challenged with insulin (10nM) or palmitoleoyl ethanolamide (100nM), a metabolite found in higher concentrations in women with low insulin sensitivity (untargeted metabolomics) for 24h at physiological oxygen tension (6.5% O2) in a hypoxia bench. Changes in the concentration of the top identified proteins under exposure to the different stimuli was quantified by qPCR and westernblotting.

Results

Placental proteome differed between lean and obese women with a stronger separation between those with low vs high insulin sensitivity (Principal Component Analysis). Proteins (n=89) enriched (p<0.05, FDR<0.05) in the low insulin sensitive mothers were selected for pathway analysis (KEEG). The top enriched pathways were related to DNA replication and cell cycle (FDR<0.001). Six members of the Minichromosome Maintenance Complex (MCM), involved in DNA damage response and cell cycle arrest, were associated with low maternal insulin sensitivity. There was a significant correlation (R2=0.45 p<0.001) between MCM6 and γH2AX (DNA damage marker) concentration in total tissue. Furthermore, MCM6 co‐localized with γH2AX in the nuclei of cytotrophoblast cells, suggesting increased DNA‐damage in this specific cell type, which was indeed found by semiquantitative γH2AX immmunohistochemistry. Challenging ACH‐3P cells with palmitoleoyl ethanolamide resulted in increased (p<0.05; n=4) MCM6 gene expression after 24h treatment.

Conclusion

Low insulin sensitivity of pregnant women in in the early first trimester of pregnancy associated with obesity induced changes in the placental proteome related to DNA replication and cell cycle. This may reflect trophoblast response to increased DNA damage in these placentas.
EU‐OPENSCREEN: Discovery and development of novel chemical probes through an international, collaborative model
Authors: Stechmann Bahne
Published: May 2022

The majority of molecular biologists does not have ready access to suitable drug screening platforms and compound collections, which are generally expensive to purchase, operate and maintain. This represents a major limitation in the field to advance innovative drug discovery projects and chemical probe discovery projects in academia.

With the aim to address this need, researchers from >20 institutions have joined forces to launch ‘EU‐OPENSCREEN’ (www.eu‐openscreen.eu), which offers international scientists access to a wide range of state‐of‐the‐art technology platforms, resources and expertise. This publicly funded initiative integrates high‐throughput screening (HTS) platforms and medicinal chemistry groups throughout Europe on a not‐for‐profit basis.

The aim of EU‐OPENSCREEN is to collaboratively develop novel chemical probes together with external biologists and to support researchers seeking a better understanding on cellular phenomena by using chemical probes.

Molecular biologists and biochemists who have a robust and HTS amenable assay, and are interested in developing novel potent chemical probes for their target of interest, can access EU‐OPENSCREEN's screening platforms on a collaborative basis and screen comprehensive, rationally designed screening collections with >100,000 compounds. After the primary screening, EU‐OPENSCREEN's medicinal chemists optimise the identified ‘hit’ compounds during the hit‐to‐probe program.

I will explain how international biologists can access and collaborate with EU‐OPENSCREEN to advance their research projects, and highlight some recent collaborative research projects.
Mitochondria Adapt to Diaphragm Muscle Inactivity Imposed by Cervical Spinal Cord Injury
Authors: Brown Alyssa D. , Fogarty Matthew J. , Dasgupta Debanjali , Davis Leah A. , Mantilla Carlos B. , Sieck Gary C.
Published: May 2022

Type I and IIa diaphragm muscle (DIAm) fibers comprise fatigue resistant slow and fast motor units that are recruited to accomplish breathing. In contrast, type IIx/IIb fibers comprise more fatigable fast motor units that are infrequently recruited only during airway protective and straining behaviors. The activity and energetic demand for type I and IIa DIAm fibers is markedly increased compared to type IIx/IIb fibers. Cervical spinal cord hemisection at C2 (C2SH) removes excitatory drive for inspiratory activity of the DIAm and thereby imposing prolonged inactivity of type I and IIa fibers. We hypothesize that mitochondrial structure and function in type I and IIa DIAm fibers adapt in response to imposed inactivity induced by spinal cord hemisection at C2 (C2SH). After C2SH, inactivity was confirmed by the absence of DIAm EMG, and after 14 days, and the effect of inactivity on mitochondrial structure and function was assessed in type identified fibers. Mitochondria in DIAm fibers were labeled using MitoTracker Green and imaged in 3‐D by fluorescence confocal microscopy. Mitochondrial volume density and morphometry were analyzed using ImageJ. In alternate serial sections of the same DIAm fibers, the maximum velocity of the succinate dehydrogenase reaction (SDHmax) was determined using a quantitative histochemical technique. Overall, mitochondrial volume density was higher in type I and IIa DIAm fibers and mitochondria were more filamentous compared to type IIx/IIb fibers. Inactivity reduced mitochondrial volume density in type I and IIa DIAm fibers and induced mitochondrial fragmentation. The SDHmax values normalized for fiber volume and for mitochondrial volume were both higher in type I and IIa DIAm fibers compared to type IIx/IIb fibers. After 14 days of inactivity induced by C2SH, SDHmax (normalized for fiber volume and for mitochondrial volume) decreased in type I and IIa. These results are consistent with mitochondrial structural and functional adaptions to the marked reduction in the energetic demand of type I and IIa DIAm fibers imposed by C2SH.
The Pronator Quadratus Muscle – its Morphology and Functions
Authors: van Schaardenburgh Michel , Poliacu‐Prose Lucian
Published: May 2022

Knowledge of the architecture and nerve supply of the human pronator quadratus muscle (MPQ) appears to be rather scarce and is insufficient for a surgical approach of the muscle. In older French and more recent clinical studies it is stated that the MPQ consists of two or even three parts, each of them with an individual nerve supply and function. Former studies have described a superficial part that is active during pronation, and a deeper part which acts during supination. Furthermore, other authors described that the MPQ regulates the tension of the capsular complex at the distal radio‐ulnar joint.

We studied the existence of subdivisions of the MPQ and their pattern of nerve supply from the anterior interosseous nerve in 15 cadaver forearms (8 M, 7 F, mean age 80 years). The dissection was carried out using a surgical microscope. In all 15 forearms the MPQ consisted of three parts: a superficial (pars superficialis; PQs), a deep proximal (pars profunda proximalis; PQpp) and a deep distal part (pars profunda distalis; PQpd).

Three primary nerve branches were identified (ramus proximalis, ramus intermedius and ramus distalis) as they originated from the anterior interosseous nerve and at entry of the individual parts of the MPQ.

We have not been able to conclude whether the ramus proximalis or the ramus intermedius supplies the superficial (PQs) or the deep proximal (PQpp) part of the MPQ. However, it could be established that the ramus distalis goes to the deep distal (PQpd) part of the MPQ. We could also confirm that only fibers of the deep distal part insert into the distal radio‐ulnar joint.

On the basis of the existing division of the MPQ in three different parts, functional considerations can be made with regard to the complex functions of MPQ, i.e. pronation by the superficial part of the MPQ, fine‐tuning of motion of the radius during pronation and supination by the deep proximal part and capsular tension (stabilization) of the distal radio‐ulnar joint by the deep distal part of the MPQ.
Electrical Stimulated Glut4 Trafficking Reduces Critical Illness‐Associated Muscle Wasting
Authors: Addinsall Alex B. , Cacciani Nicola , Backéus Anders , Hedstöm Yvette , Larsson Lars
Published: May 2022

Critical illness myopathy (CIM) is a debilitating condition characterized by the preferential loss of the motor protein myosin. CIM is a consequence of critical care, impairs recovery and provides long‐term complications, and mortality. CIM pathophysiology is complex and remains incompletely understood, however loss of mechanical stimuli appears central to critical illness associated muscle atrophy and weakness. Passive mechanical loading (ML) and electrical stimulation (ES) augment muscle mass and function, however the mechanisms underpinning these therapies are less known. Here we aimed to assess the hypothesis that chronic supramaximal ES would ameliorate CIM in a unique experimental rat model of critical care. Rats were subjected to deep sedation, controlled mechanical ventilation, and immobilization with and without direct soleus ES for 8 days. Critical care reduced soleus muscle mass, cross‐sectional area (CSA), Myosin: Actin ratio and single muscle fibre specific force. ES reduced the loss of soleus muscle fibre CSA and Myosin: Actin ratio yet failed to effect specific force. Insulin signalling gene pathway was downregulated following critical care and GLUT4 trafficking was reduced leading to muscle glycogen depletion. ES promoted phosphofructokinase and insulin signalling pathways and maintained GLUT4 translocation and glycogen levels. ES evoked AMPK, but not AKT, signalling pathway, where the downstream target TBC1D4 and AMPK‐specific P‐TBC1D4 levels tended to be increased. Reduced muscle protein degradation promoted soleus CSA, as ES reduced E3 ligases Atrogin‐1 and MuRF1 downstream of AMPK‐FoxO3. These results demonstrate chronic supramaximal ES reduces critical care associated muscle wasting, preserved glucose signalling and reduced muscle protein degradation in CIM.
Trabecular bone morphology in felids reflects diversity in locomotor strategy but not home range size
Authors: Assif Layne , ChirChir Habiba
Published: May 2022

A relationship exists between mechanical loading, and the resulting changes in bone morphology. When bones experience increased loading, they undergo change through bone modeling and remodeling in response to loading frequency and strain. Organisms with differing locomotor behaviors are distinguished by differences in limb bone morphology. In other words, an organisms’ biological structure such as limb morphology is adapted for its behavior including locomotor strategy. While there are studies showing a relationship between bone morphology and locomotor strategy in a myriad of taxa none of them have specifically investigated felid species occupying disparate geographical spaces. This study investigates trabecular bone morphology (bone volume fraction, BVF and degree of anisotropy, DA) in the femoral and humeral heads and distal tibia of four felid species to identify whether there is a relationship between these bone properties and their locomotor behavior. We test the hypotheses that felid species with large home ranges i.e., cougars and cheetahs will have significantly greater BVF than those with small home ranges (leopards and jaguars) in the forelimb than hindlimb and will exhibit greater DA because non‐primate mammals have greater vertical ground reaction forces on the fore‐than in the hindlimb. We found no significant differences across species in both the femoral and humeral BVF (p>0.05). Results also showed that in the distal tibia, leopards, cougars and cheetahs have significantly greater (p<0.05) BVF than jaguars. Additionally, humeral and distal tibia DA in the jaguars was significantly greater than in the femur. Interestingly, despite differences in home range size, the proximal limb elements do not reflect differences in BVF, however, the distal most element‐the tibia‐ suggests greater loading among jaguars. In the same vein, humeral and tibia suggest greater stereotypical loading among jaguars. The jaguar results are potentially due to the diversity in locomotor strategy rather than differences in home range size. This implies a cautious approach is warranted in studying how repeated loading influences trabecular morphology and places emphasis on investigating the diversity of locomotor behavior rather than overall loading.