Biology subjects

Roy, A.

Publications and source records attributed to Roy, A..

At least 73 records · Page 4Linked to original sources

bioRxiv2025

SKiD: A Structure-Oriented Kinetics Database of Enzyme-Substrate Interactions

Enzymes are essential biological catalysts that drive nearly all biochemical reactions. Understanding their efficiency and specificity involves studying enzyme kinetics, particularly the parameters kcat and Km. However, there is limited data linking these kinetic parameters with the three-dimensional (3D) structures of enzyme-substrate complexes. Since enzyme function is determined by its structure, such mapping enhances insight into structural basis of enzymatic function and supports applications in enzyme design, synthetic biology and metabolic engineering. To address this critical gap, this work presents SKiD (Structure-oriented Kinetics Database), a comprehensive, structured database integrating kcat and Km values with the corresponding 3D structural data. This is accomplished by integrating data from existing bioinformatics resources using automated programs to process the data and enhancing it with computational predictions. The erroneous data encountered during data integration is manually resolved. Metadata such as literature and assay conditions (e.g., pH and temperature) are preserved. The 3D coordinates of the modelled enzyme-substrate complexes are provided along with their UniProtKB identifier. The database is freely accessible from: https://doi.org/10.5281/zenodo.15355031.

bioinformatics↗

bioRxiv2025

Mutations differentially affecting the coronavirus Mac1 ADP-ribose binding and hydrolysis activities indicate that it promotes multiple stages of the viral replication cycle

All coronaviruses (CoVs) encode a conserved macrodomain, termed Mac1, in non-structural protein 3 (nsp3) that binds and hydrolyzes ADP-ribose covalently attached to proteins. Mac1 is a key virulence factor that counters antiviral ADP-ribosyltransferase (PARP) activity. Previously, we found that MHV strain JHM (JHMV) with a mutation in the adenine binding site, JHMV-D1329A, was extremely attenuated in all tested cell types as opposed to JHMV-N1347A, which only has a replication defect in bone-marrow derived macrophages (BMDMs). Interestingly, an N1347A/D1329A double mutant was unrecoverable, indicating an essential role for Mac1 in JHMV infection. We hypothesized that these mutations may impact different stages of the MHV lifecycle. First, to clarify how these mutations affected the biochemical activities of Mac1, we generated Mac1 proteins encoding the same mutations. As expected, the D-A mutation was extremely defective in ADP-ribose binding but maintained enzyme activity. In contrast, we previously found that the N-A mutation had WT levels of ADP-ribose binding but low enzyme activity, confirming that these mutations differentially effect the biochemical functions of Mac1. Following infection, D1329A displayed a large defect in the accumulation of viral RNA compared to WT or N1347A in all cells tested. Alternatively, N1347A infection produced normal levels of viral RNA but produced reduced levels of viral protein in interferon competent bone-marrow derived macrophages (BMDMs). These results suggest that Mac1 ADP-ribose binding and enzymatic activities promote different stages of the viral lifecycle, demonstrating the critical importance of Mac1 for JHMV replication. IMPORTANCEOver the last three decades, coronaviruses have repeatedly demonstrated their potential to become significant veterinary and public health threats. Zoonotic transmission of the myriad known coronavirus strains will remain a concern, regardless of the advances in vaccines and treatment. One difficulty in anticipating the next coronavirus outbreak is their diverse lineage and high propensity for mutation and recombination. The coronavirus macrodomain, Mac1, is conserved amongst all known coronaviruses and is also conserved in the Togaviridae and Hepeviridae families. Mac1 is a key factor in viral replication and pathogenesis, but its role in the replication cycle remains unclear. A deeper investigation of Mac1 function will identify conserved antiviral mechanisms and aid in the development of Mac1 inhibitors that represent a novel strategy for antiviral therapeutics.

microbiology↗

bioRxiv2025

Hyperactivation of TAK1 causes skeletal muscle pathology reminiscent of inflammatory myopathies

Loss of skeletal muscle mass and strength is a debilitating consequence of various chronic diseases, inflammatory myopathies, and neuromuscular disorders. Inflammation plays a major role in the perpetuation of myopathy in degenerative muscle diseases. TAK1 is a major signaling protein that mediates the activation of multiple signaling pathways in response to inflammatory cytokines and microbial products. Recent studies have demonstrated that TAK1 is essential for the growth and maintenance of skeletal muscle mass in adult mice. However, the effects of overstimulation of TAK1 activity in the regulation of skeletal muscle mass remain unknown. In the present study, using AAV vectors, we investigated the effect of varying levels of TAK1 activation on skeletal muscle in adult mice. Our results demonstrate that while low levels of TAK1 activation improve skeletal muscle mass, sustained hyperactivation of TAK1 causes myopathy in adult mice. Excessive stimulation of TAK1 manifests pathological features, such as myofiber degeneration and regeneration, cellular infiltration, increased expression of proinflammatory molecules, and interstitial fibrosis. Hyperactivation of TAK1 also upregulates proteolytic systems and various catabolic signaling pathways in skeletal muscle of adult mice. Altogether, our study demonstrates that physiological levels of activation of TAK1 lead to myofiber hypertrophy, whereas its hyperactivation results in myofiber damage and other pathological features resembling inflammatory myopathies.

cell biology↗

bioRxiv2025

Discovery of a VHL molecular glue degrader of GEMIN3 by Picowell RNA-seq

Targeted protein degradation (TPD) is an emerging therapeutic modality in which small molecules are used to recruit targets to the natural protein degradation machinery of the cell. Molecular glue degraders (MGD) are monovalent small molecules that accomplish this by redirecting E3 ubiquitin ligases to target proteins, offering the potential to degrade previously unliganded and "undruggable" proteins in cancer, neurodegenerative, and other diseases. While attractive due to their drug-like properties, MGDs are exceptionally hard to discover and have largely been identified serendipitously. The Von Hippel-Lindau (VHL) E3 ligase is the second most widely used effector for TPD, though current VHL-based degraders are primarily large heterobifunctional PROTACs (proteolysis-targeting chimeras) designed using target-based ligands. Here, we have instead pursued target-agnostic discovery of VHL MGDs leveraging proprietary ultra-miniaturized microfluidics devices (Picowells) to facilitate unbiased RNA-seq screening of a biased E3-focused library. This resulted in dGEM3, a novel VHL molecular glue that targets the survival of motor neuron (SMN) complex member GEMIN3 for degradation. Through a combination of cellular, biochemical, and biophysical assays, we have characterized the GEMIN3 degron within its helicase ATP-binding domain, and how the kinetics of ternary complex formation impact degradation. These findings provide insights on the re-programmability of VHL for novel targets using drug-like molecular glues.

biochemistry↗

bioRxiv2025

comBO: A combined human bone and lympho-myeloid bone marrow organoid for pre-clinical modelling of haematopoietic disorders

The bone marrow supports lifelong blood and immune cell production. Current human bone marrow organoid models do not include both lymphoid and myeloid elements and lack the complexity of stromal cell types present in native haematopoietic tissues, precluding the accurate ex vivo modelling of human pathologies. Here we introduce "comBOs" (combined bone and lympho-myeloid bone marrow organoids) that include osteolineage, vascular, lymphoid and myeloid cells. comBOs are generated by the differentiation of induced pluripotent stem cells guided by physiologically-relevant oxygen and cytokine exposures within an innovative granular microgel scaffold to increase scalability and reproducibility. We demonstrate that comBOs can be used to generate "chimeroids" - incorporating healthy or aberrant cells from adult donors - and recapitulate features of diseased microenvironments. ComBOs are one of the most physiologically-relevant human organoid systems to date, and this study showcases the potential of 3D in vitro disease models for discovery science and translational studies.

cancer biology↗

bioRxiv2025

Bi-specific CAR-iNKT cell immunotherapy for high-risk KMT2A-rearranged leukemia outperforms CAR-T in an NKG2D-dependent manner and eradicates leptomeningeal disease

Current therapies, including autologous CAR-T immunotherapy, fail to cure half of infants with KMT2A-rearranged acute lymphoblastic leukemia (KMT2Ar-ALL). Here we deploy allogeneic iNKT cells, innately more powerful effectors than T cells, and equip them with CD19- and/or CD133-targeting CARs. Compared to mono-specific counterparts and bi-specific CAR-T, CD19-CD133 bi-specific CAR-iNKT have more potent anti-leukemia activity, they effectively target CAR antigen-low leukemia, eradicate medullary and leptomeningeal leukemia and induce sustained remissions without discernible hematologic toxicity. Mechanistically, dynamic CAR- and CAR antigen-dependent upregulation of the activating innate receptor NKG2D and its engagement by corresponding ligands on KMT2Ar-ALL cells lead to more potent anti-leukemia effect of CAR-iNKT over CAR-T cells, including against CAR antigen-negative leukemia. Thus, by engaging with two different types of leukemia-associated targets, CAR-iNKT provide a powerful platform for the treatment of KMT2Ar-ALL. This approach can be readily adapted for other high-risk malignancies, including those with otherwise difficult to target leptomeningeal involvement.

cancer biology↗

bioRxiv2025

Two independent translocation modes drive neural stem cell dissemination into the human fetal cortex

The strong size increase of the human neocortex is supported both by the amplification and the basal translocation of a neural stem cell population, the basal radial glial cells (or bRG cells). Using live imaging of second trimester human fetal tissue and cortical organoids, we identify two independent translocation modes for bRG cell colonization of the human neocortex. On top of an actomyosin-dependent movement called mitotic somal translocation (MST), we identify a microtubule-dependent motion occurring during interphase, that we call interphasic somal translocation (IST). We show that IST is driven by the LINC complex, through the nuclear envelope recruitment of the dynein motor and of its activator LIS1. Consequently, IST severely altered in LIS1 patient-derived cortical organoids. We also demonstrate that MST occurs during prometaphase and is a mitotic spindle translocation event. MST is controlled by the mitotic cell rounding molecular pathway, via Moesin and Vimentin, driving translocation. We report that 85% of bRG cell translocation is due to IST, for a total movement of 0,67 mm per month of human fetal gestation. Our work identifies how bRG cells colonize the human fetal cortex, and further shows that IST and MST are conserved in bRG-related migrating glioblastoma cells.

neuroscience↗

bioRxiv2025

Studies of all-trans retinoic acid transport in myopigenesis

PurposeMyopia incidence is increasing globally. All-trans retinoic acid (atRA) is important in myopigenic retinoscleral signaling, motivating research on its ocular transport. However, atRAs weak autofluorescence limits its direct visualization in tissues. Further, atRA is hydrophobic and must bind to protein carriers for transport. We assessed a fluorescent analog of atRA (LightOxTM14, CAS:198696-03-6, referred as floRA), as an experimentally accessible atRA surrogate by: (i) evaluating its binding to carrier proteins and (ii) visualizing its distribution in ocular tissues. MethodsO_ST_ABSBindingC_ST_ABSWe assessed atRA-carrier protein binding using fluorescence quenching assays with bovine serum albumin (BSA), high density lipoprotein (HDL), apolipoprotein A-I (Apo A-I) and retinol binding protein 4 (RBP4). Direct visualizationWild-type C57BL/6J mice were euthanized, eyes enucleated, and wedges containing sclera and choroid incubated for specific durations in 50 M floRA+BSA. The wedge centers were cryo-sectioned and counterstained for nuclei. Fluorescent micrographs were acquired and analyzed using ImageJ. ResultsAssociation constants (Ka) for atRA and floRA binding to carrier proteins were similar and ranged from 2-13 x 105 M-1, indicating non-specific binding. floRA could be visualized in sclera and choroid, yet showed significant spatial heterogeneity (enhanced fluorescence often colocalizing with nuclei). ConclusionsfloRA is a reasonable surrogate for atRA binding to BSA, HDL, Apo A-I and RBP4. Considering these proteins relative serum and extravascular abundances, and their similar binding affinity to atRA, we predict that serum albumin is an important atRA carrier. Use of floRA in whole tissue tracer studies shows promise but requires further refinement.

biochemistry↗

bioRxiv2024

Enhancer heterogeneity in acute lymphoblastic leukemia drives differential gene expression between patients

Genetic alterations alone cannot account for the diverse phenotypes of cancer cells. Even cancers with the same driver mutation show significant transcriptional heterogeneity and varied responses to therapy. However, the mechanisms underpinning this heterogeneity remain under-explored. Here, we find that novel enhancer usage is a common feature in acute lymphoblastic leukemia (ALL). In particular, KMT2A::AFF1 ALL, an aggressive leukemia with a poor prognosis and a low mutational burden, exhibits substantial transcriptional heterogeneity between individuals. Using single cell multiome analysis and extensive chromatin profiling, we reveal that much transcriptional heterogeneity in KMT2A::AFF1 ALL is driven by novel enhancer usage. Using high resolution Micro-Capture-C in primary patient samples, we also identify patient-specific enhancer activity at key oncogenes such as MEIS1 and RUNX2, driving high levels of expression of both oncogenes in a patient-specific manner. Overall, our data show that enhancer heterogeneity is highly prevalent in KMT2A::AFF1 ALL and may also be a mechanism that drives transcriptional heterogeneity in cancer more generally. Key PointsO_LILeukemia patients with the same driver mutations often display gene expression differences C_LIO_LIUsing chromatin profiling and high resolution 3C methods we show that enhancer heterogeneity drives gene expression differences C_LI

cancer biology↗

bioRxiv2024

Cellular moonlighting function of Hsp20 directs morphological and pathogenic development in Ustilago maydis

Ustilago maydis Hsp20 is involved in the pathogenicity of the fungus. In this study we have investigated the molecular basis of contribution of Hsp20 to U. maydis pathogenicity. Through biochemical studies we have demonstrated environment-dependent oligomeric plasticity associated with Hsp20. Hsp20 was also found to form higher order oligomers that undergo phase separation in vitro. Within cells Hsp20 was found to form distinct punctate structures that we believe play a pivotal role in its function. These punctate structures were demonstrated to sequester proteins such as actin and septin within it. Absence of Hsp20 was found to significantly affect key cellular processes like endocytosis, budding, cell polarity determination and mating in U. maydis cells. The deletion mutant failed to sporulate and complete pathogenic life cycle. This study presents a comprehensive understanding of the pathogenic development of U. maydis in reference to the moonlighting function of Hsp20 within the cell.

microbiology↗

bioRxiv2024

Identification of a series of pyrrolo-pyrimidine based SARS-CoV-2 Mac1 inhibitors that repress coronavirus replication

Coronaviruses (CoVs) can emerge from zoonotic sources and cause severe diseases in humans and animals. All CoVs encode for a macrodomain (Mac1) that binds to and removes ADP-ribose from target proteins. SARS-CoV-2 Mac1 promotes virus replication in the presence of interferon (IFN) and blocks the production of IFN, though the mechanisms by which it mediates these functions remain unknown. Mac1 inhibitors could help elucidate these mechanisms and serve as therapeutic agents against CoV-induced diseases. We previously identified compound 4a (a.k.a. MCD-628), a pyrrolo-pyrimidine that inhibited Mac1 activity in vitro at low micromolar levels. Here, we determined the binding mode of 4a by crystallography, further defining its interaction with Mac1. However, 4a did not reduce CoV replication, which we hypothesized was due to its acidic side chain limiting permeability. To test this hypothesis, we developed several hydrophobic derivatives of 4a. We identified four compounds that both inhibited Mac1 in vitro and inhibited murine hepatitis virus (MHV) replication: 5a, 5c, 6d, and 6e. Furthermore, 5c and 6e inhibited SARS-CoV-2 replication only in the presence of IFN{gamma}, similar to a Mac1 deletion virus. To confirm their specificity, we passaged MHV in the presence of 5a to identify drug-resistant mutations and identified an alanine-to-threonine and glycine-to-valine double mutation in Mac1. Recombinant virus with these mutations had enhanced replication compared to WT virus when treated with 5a, demonstrating the specificity of these compounds during infection. However, this virus is highly attenuated in vivo, indicating that drug-resistance emerged at the expense of viral fitness. IMPORTANCECoronaviruses (CoVs) present significant threats to human and animal health, as evidenced by recent outbreaks of MERS-CoV and SARS-CoV-2. All CoVs encode for a highly conserved macrodomain protein (Mac1) that binds to and removes ADP-ribose from proteins, which promotes virus replication and blocks IFN production, though the exact mechanisms remain unclear. Inhibiting Mac1 could provide valuable insights into these mechanisms and offer new therapeutic avenues for CoV-induced diseases. We have identified several unique pyrrolo-pyrimidine-based compounds as Mac1 inhibitors. Notably, at least two of these compounds inhibited both murine hepatitis virus (MHV) and SARS-CoV-2 replication. Furthermore, we identified a drug-resistant mutation in Mac1, confirming target specificity during infection. However, this mutant is highly attenuated in mice, indicating that drug-resistance appears to come at a fitness cost. These results emphasize the potential of Mac1 as a drug target and the promise of structure-based inhibitor design in combating coronavirus infections.

microbiology↗

bioRxiv2024

The TWEAK/Fn14 signaling promotes skeletal muscle wasting during cancer cachexia

Cachexia is an involuntary loss of body weight mostly due to skeletal muscle wasting. The proinflammatory cytokine TWEAK and its receptor Fn14 constitute a major signaling system that regulates skeletal muscle mass in diverse conditions. However, the role of TWEAK/Fn14 system in the regulation of skeletal muscle mass during cancer-induced cachexia remains poorly understood. In this study, we demonstrate that the levels of Fn14, but not TWEAK, are induced in skeletal muscle of multiple mouse models of cancer cachexia. Targeted deletion of Fn14 inhibits muscle wasting and gene expression of multiple components of the ER stress-induced unfolded protein response (UPR) in the KPC mouse model of pancreatic ductal adenocarcinoma (PDAC) cancer cachexia. The TWEAK/Fn14 signaling activates PERK and IRE1 arm of the UPR and inhibits protein synthesis in cultured primary myotubes. Inhibition of PERK using pharmacological or molecular approaches improves protein synthesis and inhibits atrophy in TWEAK-treated cultured myotubes. Silencing of Fn14 in KPC cells prior to their inoculation in pancreas of mice also attenuates tumor growth without having any significant effect on muscle atrophy. The knockdown of Fn14 inhibits proliferation, migration, and invasion of cultured KPC cells. Finally, our results demonstrate that targeted ablation of Fn14 also attenuates muscle atrophy in the Lewis lung carcinoma model of cancer cachexia. Altogether, our study provides initial evidence that the inhibition of TWEAK/Fn14 signaling can prevent tumor growth and skeletal muscle wasting during cancer-induced cachexia.

cell biology↗

bioRxiv2024

Lamins regulate nuclear mechanics and shape to control glioblastoma cell proliferation, migration and invasion

Glioblastoma (GBM) is known as the most aggressive brain tumor and is characterized by a high heterogeneity and a median patient survival time around 15 months. Phenotyping based on cell mechanics is increasingly recognized as a potential prognostic marker for tumor aggressiveness. We have previously shown that cell and nuclear mechanical properties vary between different grades of gliomas and may be used to differentiate between GBM of different aggressiveness. Here, we find that the levels of lamin proteins can serve as an indicator of GBM aggressiveness. In patient-derived GBM cell lines, we found that cells from different GBM express different lamin levels. Nuclear size correlates positively with the ratio between lamin A and lamin B1 while nuclear stiffness increases with the levels of both lamin A and lamin B1. A simple mechanical model suggests that lamin A and lamin B1 act like springs in series. We also show that cells proliferate faster in GBM cell lines expressing higher lamin A levels. Downregulating lamin A expression in these cells reverse the aggressive phenotype. In contrast with breast cancer cells for which reduced lamin A levels favor cell migration in a confined environment, increased levels of lamin A may facilitate the invasion of more aggressive GBM cell lines in the soft environment of the brain. Furthermore, since nuclear deformation is a hallmark of malignancy in cancer cells, our results suggest that nuclear shape and mechanics may serve as prognosis biomarkers for GBM.

biophysics↗

bioRxiv2024

The fetal specific gene LIN28B is essential for human fetal B-lymphopoiesis and initiation of KMT2A::AFF1 infant leukemia

Infant ALL (iALL) is initiated in utero, most often by rearrangement of the KMT2A gene (KMT2Ar). It carries a very poor prognosis despite a lack of additional oncogenic driver mutations common in childhood ALL. Here, we aimed to identify specific properties of human fetal hematopoietic stem/progenitor cells (HSPC) that promote leukemic transformation in KMT2Ar iALL using molecular, functional and in vivo assays. First, by comparing transcriptomes of human fetal HSPC to adult HSPC we derived a fetal-specific gene signature and identified the fetal oncogene LIN28B and its downstream effectors among the top hits. These genes were also expressed in iALL. Functional assays revealed that LIN28B was essential in human fetal liver (FL) CD34+ cells to maintain proliferation and stemness, and support B- and NK-lymphopoiesis. To interrogate the role of LIN28B in iALL, we utilised a human FL-derived CRISPR-Cas9 KMT2A::AFF1 model. In this model, LIN28B-expressing leukemias were more proliferative in vitro and in vivo, with this advantage being lost upon LIN28B knockdown. Mechanistic studies showed that LIN28B acts by stabilizing key early B-lymphoid genes, epigenetic regulators, and cell cycle and anti-apoptotic genes. Finally, In the absence of LIN28B, human FL CD34+ cells fail to transform upon induction of KMT2A::AFF1 translocation. Thus, LIN28B has an essential role in normal human fetal B-lymphopoiesis, and is necessary for the initiation of KMT2A::AFF1 iALL in fetal cells in the absence of co-operating mutations. It has a role in making leukemias more aggressive, suggesting it is a potential target in LIN28B-expressing leukemias.

cancer biology↗

bioRxiv2024

Screening and identification of gene expression in large cohorts of clinical lung cancer samples unveils the major involvement of EZH2 and SOX2

Lung adenocarcinoma (LUAD), the primary subtype of Non-Small Cell Lung Cancer (NSCLC), accounts for 80% to 85% of cases. Due to suboptimal screening method, LUAD is often detected in late stage, leading to aggressive progression and poor outcomes. Therefore, early disease prognosis for the LUAD is high priority. In order to identify early detection biomarkers, we conducted a meta-analysis of mRNA expression TCGA and GTEx datasets from LUAD patients. A total of 795 differentially expressed genes (DEGs) were identified by exploring the Network-Analyst tool and utilizing combined effect size methods. DEGs refer to genes whose expression levels are significantly different (either higher or lower) compared to their normal baseline expression levels. KEGG pathway enrichment analysis highlighted the TNF signaling pathway as being prominently associated with these DEGs. Subsequently, using the MCODE and CytoHubba plugins in Cytoscape software, we filtered out the top 10 genes. Among these, SOX2 was the only gene exhibiting higher expression, while the others were downregulated. Consequently, our subsequent research focused on SOX2. Further transcription factor-gene network analysis revealed that enhancer of zeste homolog 2 (EZH2) is a significant partner of SOX2, potentially playing a crucial role in euchromatin-heterochromatin dynamics. Structure of SOX2 protein suggest that it is a non-druggable transcription factor, literature survey suggests the same; hence, we drove our focus to investigate on potential drug(s) targeting EZH2. Molecular docking analyses predicted most probable inhibitors of EZH2. We employed several predictive analysis tools and identified GSK343, as a promising inhibitor of EZH2.

genetics↗

bioRxiv2024

Reducing oxidative stress improves ex vivo polymer-based human haematopoietic stem and progenitor cell culture and gene editing

Self-renewing multipotent haematopoietic stem cells (HSCs) have the unique capacity to stably regenerate the entire blood and immune systems following transplantation. HSCs are used clinically to reconstitute a healthy blood system in patients suffering from a range of haematological diseases. However, HSCs are very rare and have been challenging to grow ex vivo, which has hampered efforts to collect large numbers of HSCs for both basic research and clinical therapies. Polymer-based culture conditions have recently been developed to support expansion of mouse and human haematopoietic stem and progenitor cells (HSPCs). While mouse HSPCs expanded rapidly in polymer-based cultures, growth speeds for human HSPCs in polymer-based cultures was limited to [~]70-fold over 4-weeks. Here we have found that reducing oxidative stress improves human HSPC growth in these conditions. We describe an optimised culture condition that improves growth to 250-1400-fold over 4-weeks through reducing oxygen tension and the addition of antioxidants. These conditions also enable efficient gene editing in these polymer-based cultures. We envision these improved culture conditions will support a range of research into human HSPC biology and provide a platform for clinical-scale HSPC expansion and gene editing.

cell biology↗

bioRxiv2024

Exploring American Elderberry Compounds for Antioxidant, Antiviral, and Antibacterial Properties Through High-Throughput Screening Assays Combined with Untargeted Metabolomics

American elderberry (Sambucus nigra subsp. canadensis) is a rapidly emerging new perennial crop for Missouri, recognized for its high level of bioactive compounds with significant health benefits, including antibacterial, antiviral, and antioxidant properties. A high-throughput screening assay combined with untargeted metabolomics analysis was utilized on American elderberry juice from 21 genotypes to explore and characterize these bioactive compounds. Our metabolomics study has identified 32 putative bioactive compounds in the American Elderberry juices. An array of high-throughput screening bioassays was conducted to evaluate 1) total antioxidant capacity, 2) activation of antioxidant response elements (ARE), 3) antiviral activity, and 4) antibacterial activity of the putatively identified compounds. Our results revealed that 14 of the 32 American elderberry compounds exhibited strong antioxidant activity. Four compounds (isorhamnetin 3-O-glucoside, kaempferol, quercetin, and naringenin) activated ARE activity and were found to be non-cytotoxic to cells. Notably, six of the 32 compounds demonstrated significant antiviral activity in an in vitro TZM-bl assay against two strains of HIV-1 virus, CXCR4-dependent NL4-3 virus and CCR5-dependent BaL virus. Luteolin showed the most potent anti-HIV activity against the NL4-3 virus (IC50 = 1.49 {micro}M), followed by isorhamnetin (IC50 = 1.67 {micro}M). The most potent anti-HIV compound against the BaL virus was myricetin (IC50 = 1.14 {micro}M), followed by luteolin (IC50 = 4.38 {micro}M). Additionally, six compounds were found to have antibacterial activity against gram-positive bacteria S. aureus, with cyanidin 3-O-rutinoside having the most potent antibacterial activity in vitro (IC50 = 2.9 {micro}M), followed by cyanidin 3-O-glucoside (IC50 = 3.7 {micro}M). These findings support and validate the potential health benefits of compounds found in American elderberry juices and highlight their potential for use in dietary supplements as well as innovative applications in health and medicine.

biochemistry↗

bioRxiv2024

Early atrial remodelling drives arrhythmia in Fabry Disease

BackgroundFabry disease (FD) is an X-linked lysosomal storage disorder caused by -galactosidase A (-Gal A) deficiency, resulting in multi-organ accumulation of sphingolipid, namely globotriaosylceramide (Gb3). This triggers ventricular myocardial hypertrophy, fibrosis, and inflammation, driving arrhythmia and sudden death, a common cause of FD mortality. Atrial fibrillation (AF) is common in FD, yet the cellular mechanisms accounting for this are unknown. To address this, we conducted electrocardiography (ECG) analysis from a large cohort of adults with FD at varying stages of cardiomyopathy. Cellular contractile and electrophysiological function were examined in an atrial FD model, developed using gene-edited atrial cardiomyocytes and imputed into in-silico atrial models to provide insight into arrhythmia mechanisms. MethodsIn 115 adults with FD, ECG P-wave characteristics were compared with non-FD controls. Induced pluripotent stem cells (iPSCs) were genome-edited using CRISPR-Cas9 to introduce the GLA p. N215S variant and differentiated into atrial cardiomyocytes (iPSC-CMs). Contraction, calcium handling and electrophysiology experiments were conducted to explore proarrhythmic mechanisms. A bi-atrial in-silico model was developed with the cellular changes induced by GLA p. N215S iPSC-CMs. ResultsECG analysis demonstrated P-wave duration and PQ interval shortening in FD adults before onset of cardiomyopathy on imaging and biochemical criteria. FD patients exhibited a higher incidence of premature atrial contractions and increased risk of developing AF. In our cellular model, GLA p. N215S iPSC-CMs were deficient in -Gal A and exhibited Gb3 accumulation. Atrial GLA p. N215S iPSC-CMs demonstrated a more positive diastolic membrane potential, faster action potential upstroke velocity, greater burden of delayed afterdepolarizations, greater contraction force, slower beat rate and dysfunction in calcium handling compared to wildtype iPSC-CMs. Inputting these changes into the in-silico model resulted in similar P-wave morphology changes to those seen in early FD cardiomyopathy and increased the action potential duration (APD) restitution slope, causing APD alternans and inducing AF. ConclusionsThese findings enhance our understanding of atrial myopathy in FD by providing novel insights into underpinning mechanisms for atrial arrhythmia and a rationale for early P-wave changes. These may be targeted in future research to develop therapeutic strategies to reduce the arrhythmic burden in FD and other atrial cardiomyopathies.

pathology↗