Biology subjects

Roy, S.

Publications and source records attributed to Roy, S..

At least 145 records · Page 8Linked to original sources

bioRxiv2024

A Multitrait Genome-Wide Association Study Reveals a Requirement for the Strigolactone Receptor DWARF14 in Optimal GOLVEN10 Signaling

GOLVEN/ROOT MERISTEM GROWTH FACTOR family of signaling peptides have been shown to control root lateral organ number, density and positioning in plants, although the signaling pathways involved remain obscure. A diverse set of 171 Medicago truncatula HapMap accessions with variation in responses to the GOLVEN 10 peptide, GLV10, were used to identify 74 significant loci controlling seven traits related to nodule formation and root architecture. Importantly, a single nucleotide polymorphism (SNP) in the upstream region of the MtGLV10 peptide-inducible strigolactone receptor gene, MtDWARF14 was significantly associated with insensitivity of nodule density to GLV10, suggesting a link between strigolactone signaling and GLV10 responsiveness. Three independent d14 mutants of the DWARF14 gene were found to hypernodulate, while overexpression of the gene led to reduction in nodule number, phenocopying GLV10. A null mutant, mtd14-1, remained sensitive to GLV10s effect on nodule density. However, at the transcriptional level, the mutant failed to effectively induce the expression of the GOLVEN marker genes, MtPLETHORA3 and MtPINLIKES2. Our study uncovers a hitherto unknown link between the strigolactone and GLV peptide signaling pathways using genotype x environment analysis of Medicago HapMap lines and provides a putative molecular mechanism for recovery from frost damage to fine roots.

plant biology↗

bioRxiv2024

NMR structures of small molecules bound to a model of an RNA CUG repeat expansion

Trinucleotide repeat expansions fold into long, stable hairpins and cause a variety of incurable RNA gain-of-function diseases such as Huntingtons disease, the myotonic dystrophies, and spinocerebellar ataxias. One approach for treating these diseases is to bind small molecules to the structured RNAs. Both Huntingtons disease-like 2 (HDL2) and myotonic dystrophy type 1 (DM1) are caused by a r(CUG) repeat expansion, or r(CUG)exp. The RNA folds into a hairpin structure with a periodic array of 1x1 nucleotide UU loops (5CUG/3GUC; where the underlined nucleotides indicate the Us in the internal loop) that sequester various RNA-binding proteins (RBP) and hence the source of its gain-of-function. Here, we report NMR-refined structures of single 5CUG/3GUC motifs in complex with three different small molecules, a diguandinobenzoate (1), a derivative of 1 where the guanidino groups have been exchanged for imidazole (2), and a quinoline with improved drug-like properties (3). These structures were determined using nuclear magnetic resonance (NMR) spectroscopy and simulated annealing with restrained molecular dynamics (MD). Compounds 1, 2, and 3 formed stacking and hydrogen bonding interactions with the 5CUG/3GUC motif. Compound 3 also formed van der Waals interactions with the internal loop. The global structure of each RNA-small molecule complexes retains an A-form conformation, while the internal loops are still dynamic but to a lesser extent compared to the unbound form. These results aid our understanding of ligand-RNA interactions and enable structure-based design of small molecules with improved binding affinity for and biological activity against r(CUG)exp. As the first ever reported structures of RNA r(CUG) repeats bound to ligands, these structures can enable virtual screening campaigns combined with machine learning assisted de novo design.

biochemistry↗

bioRxiv2024

Long term rescue of Alzheimer deficits in vivo by one-time gene-editing of App C-terminus.

Gene-editing technologies promise to create a new class of therapeutics that can achieve permanent correction with a single intervention. Besides eliminating mutant alleles in familial disease, gene-editing can manipulate upstream pathophysiologic events and alter disease-course in wider patient populations. Here we use CRISPR-Cas9 to edit the last exon of amyloid precursor protein (App), relevant for Alzheimers disease (AD). Our strategy effectively eliminates an endocytic (YENPTY) motif at APP C-terminus in mouse and human neurons, while preserving N-terminus and compensatory APP-homologues. This manipulation favorably alters events along the amyloid-pathway; inhibiting toxic APP-beta-cleavage fragments (including Abeta) and upregulating neuroprotective APP-alpha-cleavage. AAV-editing ameliorates neuropathologic, electrophysiologic, and behavioral deficits in an AD knockin mouse model. Effects persist for many months with no detectable abnormalities in germline-edited WT mice, and pathologic alterations in glial-transcriptomes are also normalized. Our strategy takes advantage of innate transcriptional rules that render terminal exons insensitive to nonsense-decay, and this upstream manipulation is expected to be effective for all forms of AD.

neuroscience↗

bioRxiv2024

Spectrin condensates provide a nidus for assembling the periodic axonal structure

Coordinated assembly of individual components into higher-order structures is a defining theme in biology, but underlying principles are not well-understood. In neurons, /{beta} spectrins, adducin, and actinfilaments assemble into a lattice wrapping underneath the axonal plasma membrane, but mechanistic events leading to this periodic axonal structure (PAS) are unclear. Visualizing PAS components in axons as they develop, we found focal patches in distal axons containing spectrins and adducin (but sparse actin filaments) with biophysical properties reminiscent of biomolecular condensation. Overexpressing spectrin-repeats - constituents of /{beta}-spectrins - in heterologous cells triggered condensate formation, and preventing association of {beta}II-spectrin with actin-filaments/membranes also facilitated condensation. Finally, overexpressing condensate-triggering spectrin repeats in neurons before PAS establishment disrupted the lattice, presumably by competing with innate assembly, supporting a functional role for biomolecular condensation. We propose a condensation-assembly model where PAS components form focal phase-separated condensates that eventually unfurl into a stable lattice-structure by associating with subplasmalemmal actin. By providing local depots of assembly parts, biomolecular condensation may play a wider role in the construction of intricate cytoskeletal structures.

cell biology↗

bioRxiv2024

Cyclin O controls entry into the cell-cycle variant required for multiciliated cell differentiation

Multiciliated cells (MCC) ensure proper fluid circulation in various organs in metazoans. Their differentiation is marked by the massive ampliication of cilia-nucleating centrioles and is known to be controlled by various cell cycle components. Tn a companion study, we show that the differentiation of MCC is driven by a genuine cell-cycle variant characterized by sequential and wave-like expression of canonical and non-canonical cyclins such as Cyclin O (CCNO). Patients with CCNO mutations exhibit a subtype of Primary Ciliary Dyskinesia (PCD) designated as Reduced Generation of Multiple Motile Cilia (RGMC), yet the role of CCNO during MCC differentiation remains unclear. Here, using mice and human cellular models, single cell transcriptomics and functional studies, we show that Cena is activated during a strategic temporal window at the crossroads between the onset of MCC differentiation, the entry into the MCC cell cycle variant, and the activation of the centriole biogenesis program. We ind that the absence of Cena leads to a block of MCC progenitor differentiation at the G1/S-like transition, just before the beginning of centriole formation. This leads to a complete lack of centrioles and cilia in mouse brain and human airway MCC. Altogether, our study identifies CCNO as a core regulator of entry into the MCC cell cycle variant and shows that the coupling of centriole biogenesis to an S-like phase, maintained in MCC, is dependent on CCNO. One sentence summaryCyclin O is necessary for multiciliated cells to enter their differentiation cell cycle variant and allows the massive amplification of centrioles, which serve as basal bodies for cilia nucleation.

cell biology↗

bioRxiv2024

ARHGEF17/TEM4 regulates the cell cycle through control of G1 progression

The Ras homolog (Rho) small GTPases, via their role in regulating the actin cytoskeleton, coordinate diverse cellular functions including cell morphology, adhesion and motility, as well as cell cycle progression, survival and apoptosis. The upstream Rho regulators for many of these functions are unknown. ARHGEF17 (also known as TEM4) is a Rho family guanine nucleotide exchange factor (GEF) that been implicated in cell migration, cell-cell junction formation and the mitotic checkpoint. In this study we characterize the regulation of the cell cycle by TEM4. We demonstrate that TEM4 depleted cells exhibit multiple defects in mitotic entry and duration, spindle morphology, and spindle orientation. In addition, we find that TEM4 insufficiency leads to excessive cortical actin polymerization and cell rounding defects. Mechanistically, we demonstrate that TEM4 depleted cells delay in G1 as a consequence of elevated levels of the G1/S inhibitor p21waf1/cip1 and that TEM4 depleted cells that progress through to mitosis, do so with decreased transcription of CCNB1 and thus attenuated levels of cyclin B. Importantly, cyclin B overexpression in TEM4-depleted cells largely rescues mitotic progression and chromosome segregation defects in anaphase. Our study thus illustrates the consequences of Rho signalling imbalance on cell cycle progression and identifies TEM4 as the first GEF governing Rho GTPase-mediated regulation of G1/S.

cell biology↗

bioRxiv2024

Novel roles for pirin proteins and a 2-ketoglutarate:ferredoxin oxidoreductase ortholog in Bacteroides fragilis central metabolism and comparison of metabolic mutants susceptibility to antimicrobials metronidazole and amixicile.

The regulation of the central metabolism and fermentation pathways and its effect on antimicrobial susceptibility in the anaerobic pathogen Bacteroides fragilis is not completely understood. In this study we show that B. fragilis encodes for two iron-dependent redox-sensitive regulatory pirin protein genes, pir1 and pir2, whose mRNA expression are upregulated following oxygen exposure and growth in iron-limiting conditions.{Delta} pir1 and{Delta} pir2 mutants showed altered short-chain fatty acids production compared to the parent strain. Overexpression of Pir1 and Pir2 increased susceptibility to metronidazole (MTZ), and to amixicile (AMIX), a novel inhibitor of pyruvate:ferredoxin oxidoreductase (PFOR) in anaerobes. We hypothesized these observations were a result of a modulatory effect of Pir1 and Pir2 proteins. Consistent with this, we showed that Pir1 forms direct protein-protein interactions with PFOR. In addition, AlphaFold2-based structural analysis predicts that Pir1 and Pir2 form stable interactions with several enzymes of the central metabolism including the 2-ketoglutate:ferredoxin oxidoreductases (KFOR), Kor1AB and Kor2CDAEBG. A series of metabolic mutants and electron transport chain inhibitors were used to show a wide-ranging effect of bacterial metabolism on MTZ and AMIX susceptibility. There was no cross-resistance between MTZ resistant strains with AMIX susceptibility. Furthermore, we show that AMIX is an effective antimicrobial against B. fragilis in an experimental model of intra-abdominal infection. This investigation led to the discovery that the kor2AEBG genes are essential for growth, and we present evidence that kor2AEBG genes have dual-functions including the reductive synthesis of 2-ketoglutarate via reverse TCA cycle. Support for a novel Kor2AEBG activity comes from the findings showing that addition of compounds containing oleic acid stimulated growth of the{Delta} kor2AEBG mutant. However, the metabolic activity that bypasses KorAEBG function remains to be defined. Collectively our investigation reveals new information on B. fragilis central metabolism and its modulatory control by pirin proteins. These data provide new genetic and metabolic knowledge which may be leveraged for the future development of new narrow-spectrum antimicrobials.

microbiology↗

bioRxiv2024

Multimodal assessment of acute stress dynamics using an Aversive Video Paradigm (AVP)

This study explored the efficacy of inducing stress through aversive video clips and investigated its impact on psychological processes, brain, and vegetative physiology. It had a randomized, single-blinded, crossover design, where participants were exposed in separate sessions to aversive or neutral video clips. Subjective feelings of stress were assessed via questionnaires. Electroencephalography (EEG) with 62 electrodes was recorded continuously. EEG power and connectivity changes based on coherence were analyzed. Heart rate (HR) and heart rate variability (HRV) data were obtained during the whole experiment, and saliva was collected for cortisol and cytokine analysis at different time intervals. Subjective data showed increased anxiety and negative affect induced by the aversive video clips, accompanied by elevated salivary cortisol levels after exposure to the stressful clips, and decreased heart rate variability. Cytokine levels however increased over time in both control and stress conditions, which argues against a stress-specific alteration of cytokines in this specific stress protocol. EEG alterations during stress induction suggest a disruption of top-down control and increased bottom-up processing. These results show that aversive video clips are suited to induce psychological stress in an experimental setting reliably, and are associated with stress-specific emotional, and physiological changes.

neuroscience↗

bioRxiv2024

Experimental type 1 diabetes metabolically rejuvenates CD8+ T cells for improved control of tumor growth through an IGF1-IGF1R axis

AbstractEpidemiological studies suggest that patients with pre-existing type 1 diabetes (T1D) have a decreased risk of developing melanoma, prostate cancer, and breast cancer, although the underlying mechanism remains to be elucidated. In translational modelling, we observed that streptozotocin (STZ) induced T1D mice exhibited restricted melanoma and carcinoma (mammary, lung and colon) growth in association with extended overall survival. Tumor-infiltrating CD8+ T cells were found to be responsible for tumor growth restriction. Tumor infiltrating CD8+ T cells but not tumor cells themselves exhibited higher glycolytic and cytotoxic activities in T1D hosts. Such improved anti-tumor T cell function was linked to selective upregulated expression of insulin-like growth factor 1, insulin-like growth factor 1 receptor, and phospho-mTOR in CD8+ T cells in the TME. T1D patient derived CD8+ T cells displayed superior activation in vitro after tumor antigen stimulation vs. non-diabetic CD8+ T cells. Activation of T1D patient derived CD8+ T cells was sensitive to targeted antagonism of IGF1R and mTOR, supporting the operational involvement of the IGF1R-mTOR signaling axis. Our results suggest that selective activation of the intrinsic IGF1R-mTOR signaling axis in CD8+ T cells represents a preferred endpoint to achieving more effective immunotherapy outcomes and improved cancer patient management. SignificanceExperimental type 1 diabetes decelerates tumor growth through metabolic activation of cytotoxic T cells dependent on an IGF1R-mTOR signaling pathway. CD8+IGF1R+IGF1+ T cells play a crucial role in T1D dependent tumor control.

cancer biology↗

bioRxiv2024

An ab initio information-theoretic approach to proteins and protein-ligand interactions

Differing conformational structure of a protein, associated with two distinct signaling states or between ligand-free and ligand-bound states, leads to differing inter-residue interactions and consequently different biological function. We propose a fresh first-principles information-theoretic approach for studying such proteins and their interactions. A de novo measure called protein residue information (PRI), which incorporates details of interactions between all pairs of atoms within and across all residues of the protein, is introduced herein. We formulate a method to calculate the intrastate and inter-state entropy of every residue, needed to determine PRI across any two states of a protein. The intra-state entropy can be determined for every state of a protein possessing one or more states. The inter-state entropy can be calculated pairwise for proteins possessing more than one state. We analyze twenty eight distinct pairs of protein structures from ten different classes. PRI successfully identifies important residues displaying significant conformational changes bearing influence with respect to itself and all other residues. Furthermore, it also successfully identifies important residues displaying rather subtle conformational changes. The identified residues exhibit influential roles in diverse performative features of proteins like stability, allostery, signaling, etc. PRI successfully recovers known experimental results from literature and predicts important roles for many hitherto unstudied residues.

biophysics↗

bioRxiv2024

Non-Electrostatic Basis for an Artificial Metalloenzyme Catalysis

The artificial metalloenzyme (referred to as Ir-Q) reported by Hartwig and coworkers presented an important milestone in merging the extraordinary efficiency of biocatalyst with the versatility of small molecule chemical catalyst in catalyzing a new-to-nature carbene insertion reaction. The artificial enzyme results from formal replacement of the Fe by an Ir(Me) moiety along with four C317G, T213G, L69V, V254L mutations in a natural Cytochrome enzyme CYP119 by directed evolution method. Importantly, this is a show-stopper enzyme as it exhibits a catalytic rate enhancement similar to that of natural enzymes. Despite this remarkable discovery, there is no mechanistic understanding as to why it displays extraordinary efficiency, so far been intractable to experimental methods. In this study, we have deciphered the catalytically active conformation of Ir-Q using large-scale molecular dynamics simulations and rigorous quantum chemical calculations. Our study reveals how directed evolution mutations precisely position the cofactor-substrate in an unusual orientation within a reshaped active site that emerged during evolution and fostered by C-H...{pi} interactions from more ordered mutated L69V and V254L residues. This productive conformation correctly reproduces the experimental barrier height and the catalytic effect of 2.7 kcal/mol, in excellent agreement with observed rate enhancement. Moreover, the active conformation features an unprecedented bonding interaction in a metal-carbene species that preferentially stabilizes the rate determining formation of an Iridium-Porphyrin Carbene intermediate to render the observed high catalytic rate acceleration. While the electrostatic criteria are widely established, this study suggests a new design paradigm towards realization of fully programmable protein catalysis.

biophysics↗

bioRxiv2024

Profound synthetic lethality between SMARCAL1 and FANCM

DNA replication stress is a threat to genome integrity. The large SNF2-family of ATPases participates in preventing and mitigating DNA replication stress by employing their ATP-driven motor to remodel DNA or DNA-bound proteins. To understand the contribution of these ATPases in genome maintenance, we undertook CRISPR-based synthetic lethality screens with three SNF2-type ATPases: SMARCAL1, ZRANB3 and HLTF. Here we show that SMARCAL1 displays a profound synthetic lethal interaction with FANCM, another ATP-dependent translocase involved in DNA replication and genome stability. Their combined loss causes severe genome instability that we link to chromosome breakage at loci enriched in simple repeats, which are known to challenge replication fork progression. Our findings illuminate a critical genetic buffering mechanism that provides an essential function for maintaining genome integrity.

molecular biology↗

bioRxiv2024

Gut feeling: host and habitat as drivers of the microbiome in blackbuck (Antilope cervicapra)

The gut microbiome can be shaped by both intrinsic host factors and extrinsic environmental factors. However, the relative importance of intrinsic and extrinsic factors in gut microbial composition has rarely been investigated, particularly for a single host across its natural range. Here, we characterise the gut microbiome of an endemic, endangered antelope, the blackbuck or Antilope cervicapra. We evaluated the influence of seven predictor variables, which were classified into intrinsic and extrinsic factors, on the gut microbiome. We determined which of these seven variables explains greater variation in the microbiome within (-diversity) and between ({beta}-diversity) the blackbuck host. We analysed the microbiome of 60 blackbuck hosts from ten different populations across India. We recorded 11800 unique OTUs across 30 known phyla and 2.9 million reads. We find an average of 2056 OTUs per individual, with firmicutes and bacteroidetes being the most dominant phyla. We find that nucleotide diversity (intrinsic), blackbuck population density (intrinsic), distance to human settlement (extrinsic), and anthropogenic land-use (extrinsic) explain within-host variation. In contrast, precipitation (extrinsic), nucleotide diversity, distance to human settlement, and anthropogenic land-use explain between-host variation. Overall, we also show that the genetic diversity of the host is more important than their environment for both within- and between-host variation in the microbiome, in blackbuck. Therefore, conservation efforts should be directed to not only preserve natural habitats but also increase the genetic pool of the blackbuck populations, which will positively impact their survival through diverse gut microbiomes.

ecology↗

bioRxiv2024

Human iPSC-derived Committed Cardiac Progenitors Generate Cardiac Tissue Grafts in a Swine Ischemic Cardiomyopathy Model without Triggering Ventricular Arrhythmias

BackgroundIntramyocardial injection of human pluripotent stem cell-derived cardiomyocytes following a myocardial infarction (MI) improves cardiac function in large animal models, but associated ventricular arrhythmias are major safety concern. We hypothesized that transendocardial injection of human induced pluripotent stem cell (hiPSC)-derived committed cardiac progenitor cells (CCPs), combined with cardiac fibroblast-derived extracellular matrix (cECM) to enhance cell retention, will generate cardiac tissue grafts improving contractility without triggering ventricular arrhythmias. MethodshiPSCs were differentiated using bioreactors and small molecules to produce committed cardiac progenitor cells (CCPs). MI was created using a coronary artery balloon occlusion and reperfusion model in Yucatan mini pigs. Four weeks later, epicardial needle injections of CCPs+cECM were performed in a small initial feasibility cohort (n=6), and then transendocardial injections of CCPs+cECM (n=14), CCPs alone (n=14), cECM alone (n=4) or vehicle control (n=13) into the peri-infarct region in a randomized cohort. Arrhythmias were evaluated using implanted event recorders. Magnetic resonance imaging (MRI) and invasive pressure-volume assessment were used to evaluate left ventricular anatomic and functional performance. Detailed histology was performed to detect and characterize human grafts. ResultsA scalable biomanufacturing protocol was developed generating CCPs which can efficiently differentiate into cardiomyocytes or endothelial cells in vitro. Intramyocardial delivery of CCPs to post-MI porcine hearts resulted in engraftment and differentiation of CCPs to form ventricular cardiomyocyte rich grafts. There was no significant difference in cardiac MRI-based measured cardiac volumes or function between control, CCP and CCP+cECM groups; however, pressure-volume analysis showed an improvement in dobutamine-stimulated functional reserve in CCP and CCP+cECM groups. Delivery of CCPs did not result in tumors or ventricular arrhythmias. ConclusionsTransendocardial delivery of CCPs with or without cECM into post-MI porcine hearts resulted in comparable human cardiomyocyte grafts which did not improve resting LV function but did improve stress-induced contractile reserve without triggering ventricular arrhythmias.

pathology↗

bioRxiv2024

Localization and function of key axonemal microtubule inner proteins and dynein docking complex members reveal extensive diversity among vertebrate motile cilia

Vertebrate motile cilia are broadly classified as (9+2) or (9+0), based on the presence or absence of the central pair apparatus, respectively. Cryogenic electron microscopy (cryo-EM) analyses of (9+2) cilia have uncovered an elaborate axonemal protein composition; whether these features are relevant to (9+0) cilia remain unclear. We previously demonstrated that Cfap53, a key microtubule inner protein (MIP) as well as centriolar-satellites component, is essential for motility of (9+0), but not (9+2) cilia. Here, we show that in (9+2) cilia, Cfap53 functions redundantly with a paralogous MIP, Mns1. Mns1 localizes to ciliary axonemes, and combined loss of both proteins in zebrafish and mice, caused severe loss of outer dynein arms (ODAs) of (9+2) cilia, significantly affecting their motility. Moreover, using immunoprecipitation, we demonstrate that while Mns1 can self-associate and interact with Cfap53, Cfap53 is unable to self-associate. Finally, we show that multiple additional dynein interacting proteins, such as the ODA docking complex (ODA-DC) members, show strikingly distinct localization patterns between various motile cilia-types. Our findings clarify how paralogous MIPs, Cfap53 and Mns1, function in regulating motility of (9+2) versus (9+0) cilia, and establish that localization pattern of other key motility proteins also differ between these cilia-types, further emphasizing extensive structural variations among these organelles.

developmental biology↗

bioRxiv2024

E41K Mutation Activates Brutons Tyrosine Kinase by Stabilizing an Inositol Hexakisphosphate Dependent Invisible Dimer

Brutons tyrosine kinase (BTK) regulates diverse cellular signaling of the innate and adaptive immune system in response to microbial pathogens. Downregulation or constitutive activation of BTK is reported in patients with autoimmune diseases or various B-cell leukemias. BTK is a multidomain protein tyrosine kinase that adopts an Src-like autoinhibited conformation maintained by the interaction between the kinase and PH-TH domains. The PH-TH domain plays a central role in regulating BTK function. The BTK is activated by binding to PIP3 at the plasma membrane upon stimulation by the B-cell receptor (BCR). The PIP3 binding allows dimerization of the PH-TH domain and subsequent transphosphorylation of the activation loop. Alternatively, a recent study shows that the multivalent T-cell-independent (TI) antigen induces BCR response by activating BTK independently of PIP3 binding. It was proposed that a transiently stable IP6-dependent PH-TH dimer may activate BTK during BCR activation by the TI antigens. However, no IP6-dependent PH-TH dimer has been identified yet. Here, we investigated a constitutively active PH-TH mutant (E41K) to determine if the elusive IP6-dependent PH-TH dimer exists. We showed that the constitutively active E41K mutation activates BTK by stabilizing the IP6-dependent PH-TH dimer. We observed that a downregulating mutation in the PH-TH domain (R28H) linked to X-linked agammaglobulinemia impairs BTK activation at the membrane and in the cytosol by preventing PH-TH dimerization. We conclude that the IP6 dynamically remodels the BTK active fraction between the membrane and cytoplasm. Stimulating with IP6 increases the cytosolic fraction of the activated BTK.

biochemistry↗

bioRxiv2024

Towards a phylogenomic classification of Magnoliidae

PremiseMagnoliidae are a strongly supported clade of angiosperms. Previous phylogenetic studies based primarily on analyses of a limited number of mostly plastid markers have led to the current classification of magnoliids into four orders and 18 families. However, uncertainty remains regarding the placement of several families. MethodsHere we present the first comprehensive phylogenomic analysis of Magnoliidae as a whole, sampling 235 species from 199 (74%) genera and representing all families and most previously accepted subfamilies and tribes. We analyze newly generated data from the Angiosperms353 probe set using both coalescent and concatenation analyses and testing the impact of multiple filtering and alignment strategies. ResultsWhile our results generally provide further support for previously established phylogenetic relationships in both magnoliids as a whole and large families including Annonaceae and Lauraceae, they also provide new evidence for previously ambiguous relationships. In particular, we find support for the position of Hydnoraceae as sister to the remainder of Piperales and, for the first time, resolve the backbone of relationships among most genera of Myristicaceae. ConclusionsAlthough some of our results are limited by low gene recovery for a number of taxa and significant gene tree conflict for some relationships, this study represents a significant step towards reconstructing the evolutionary history of a major lineage of angiosperms. Based on these results, we present an updated phylogenetic classification for Magnoliidae, recognizing 21 families, summarizing previously established subfamilies and tribes, and describing new tribes for Myristicaceae.

evolutionary biology↗

bioRxiv2024

3-epicaryoptin induces G2/M phase cell cycle arrest and apoptosis in human breast cancer cells by disrupting the microtubule network, an in vitro and in silico investigation

Breast cancer (BC) is a prevalent form of cancer observed in women across the globe, constituting over a quarter of all female BC cases. The treatment of BC continues to require significant efficacy, aiming to achieve high success rates while minimizing adverse effects on the body as a whole. In the current study, 3-epicaryoptin was tested for the molecular mechanism of its anti-cancer activity in the human breast cancer cell line, MCF-7. We investigated cell viability by MTT assay, cell cycle kinetics and apoptosis, immunofluorescence straining, molecular modelling, and ADMET profiling. MTT assay results showed that 3-epicaryoptin was found cytotoxic against MCF-7 cells with an IC50 value of 344.64 {micro}g mL-1 for 48 h. Flow cytometric analysis exhibited that 3-epicaryoptin halted the MCF-7 cells in the G2/M phase and subsequently induced apoptosis in a time-dependent manner. Our immunofluorescence studies indicated that 3-epicaryoptin inhibited microtubule polymerization in MCF-7 cells. Furthermore, molecular docking followed by molecular dynamics (MD) simulation studies demonstrated the ability of 3-epicaryoptin to interact with the tubulin protein at the colchicine binding pockets. Overall, our results suggest that 3-epicaryoptin can inhibit the proliferation of human breast cancer cells by depolymerizing of cellular microtubule networks, which causes cell cycle arrest and promotes apoptotic cell death. Therefore, it has been indicated that the natural product 3-epicaryoptin exhibited considerable promise as a potent therapeutic agent capable of inducing apoptosis in breast cancer cells.

cancer biology↗