Biology subjects

Kumar, S.

Publications and source records attributed to Kumar, S..

At least 289 records · Page 16Linked to original sources

bioRxiv2024

Nuclear compression-mediated DNA damage drives ATR-dependent Lamin expression and mouse ESC differentiation

Embryonic stem cells (ESCs) which are susceptible to DNA damage depend on a robust and highly efficient DNA damage response (DDR) mechanism for their survival. However, the implications of physical force-mediated DNA damage on ESC fate remains unclear. We show that stiffness-dependent spreading of mouse ESCs (mESCs) induces DNA damage through nuclear compression, with DNA damage causing differentiation through early induction of Lamin A/C expression. Interestingly, differentiation is associated with rescue of DNA damage and activation of the DDR factor ATR. While ATR is typically known to play roles in DDR pathway, its role during stiffness-mediated nuclear compression and mESC differentiation is unknown. Nuclear enrichment of activated ATR on stiff substrates and reduction of Lamin A/C expression upon ATR inhibition suggests that mESC differentiation is driven by nuclear compression-mediated DNA damage and involves ATR-dependent modulation of Lamin A/C.

cell biology↗

bioRxiv2024

Fruit-In-Sight: a deep learning-based framework for secondary metabolite class prediction using fruit and leaf images

Fruits produce a wide variety of secondary metabolites of great economic value. Analytical measurement of secondary metabolites is tedious, time-consuming and expensive. Additionally, metabolite concentration varies greatly from tree to tree, making it difficult to choose trees for fruit collection. The current study tested whether deep learning-based models can be developed using fruit and leaf images alone to predict a metabolites concentration class (high or low). We collected fruits and leaves (n = 1045) from neem trees grown in the wild across 0.6 million sq km, imaged those, measured concentration of five metabolites (azadirachtin, deacetyl-salannin, salannin, nimbin and nimbolide) using high-performance liquid chromatography and used those to train deep learning models for metabolite class prediction. The best model out of the seven tested (YOLOv5, GoogLeNet, InceptionNet, EfficientNet_B0, Resnext_50, Resnet18, and SqueezeNet) provided a validation F1 score of 0.93 and a test F1 score of 0.88. The sensitivity and specificity of the fruit model alone in the test set were 83.52 {+/-} 6.19 and 82.35 {+/-} 5.96 and 79.40 {+/-} 8.50 and 85.64 {+/-} 6.21, for the low and the high class, respectively. The sensitivity was further boosted to 92.67{+/-} 5.25 for the low class and 88.11 {+/-} 9.17 for the high class and the specificity to 100% for both classes, using a multi-analyte framework. We incorporated the model in an Android mobile App Fruit-In-Sight that uses fruit and leaf images to decide whether to pick or not pick the fruits from a specific tree based on the metabolite concentration class. Our study provides evidence that images of fruits and leaves alone can predict the concentration class of a secondary metabolite without using extensive analytical laboratory procedures and equipment and makes the process of choosing the right tree for fruit collection easy and free of equipment and additional cost.

bioinformatics↗

bioRxiv2024

Pyruvate Kinase M2 regulates Japanese encephalitis virus replication by interacting with NS1 protein

Pyruvate kinase isoform M2 (PKM2) is a key modulator of glucose metabolism. While the major role of PKM2 is to facilitate the breakdown of glucose, it is potentially associated with other additional non-glycolytic functions as well. The role of PKM2 in the autoimmune response and inflammatory process is increasingly being acknowledged as a crucial modulator of cellular pathophysiological activity. However, its role in modulating viral replication has not been explored in detail. In the present study, we have shown a significant increase in endogenous PKM2 expression in JEV-infected mouse neuroblastoma cells. Furthermore, overexpression of PKM2 significantly reduced JEV replication, suggesting a negative effect of PKM2 on JEV replication. This was further confirmed by siRNA-mediated downregulation of endogenous PKM2 expression, which resulted in enhanced JEV replication. In silico studies revealed the potential interaction between PKM2 and NS1 protein of JEV. The microscopic studies also showed cellular colocalization of PKM2 and NS1 in the ER of infected cells. The interaction was further validated in vitro by co-immunoprecipitation assay. The present study suggests that PKM2 negatively regulates the JEV replication by its possible interaction with NS1. ImportanceJapanese encephalitis (JE) is a neuroinflammatory disease caused by the Japanese encephalitis virus (JEV). JE is a major threat to public health not only because it causes many deaths but also for its permanent neuropsychiatric sequelae in children. Out of all non-structural proteins of JEV, NS1 is highly immunogenic. A wide range of possible interactive partners has been identified for the NS1, many of those have been linked to immune evasion and regulating viral replication. In the current study, we have described a novel host cell factor, PKM2 modulating JEV replication by interacting with NS1 protein. Considering PKM2s central role in regulating host cell metabolism, our findings suggest a previously unrecognized role for PKM2 in JEV neuropathogenesis. The identification and characterization of previously unknown host factors, as well as the elucidation of their regulatory mechanisms, are of utmost importance in the development of innovative treatments and antivirals against JEV.

microbiology↗

bioRxiv2024

Using RNA-targeting CRISPR-Cas13 and engineered U1 systems to reduce ABCA4 splice variants in Stargardt disease

Dysregulation of the alternative splicing process results in aberrant mRNA transcripts, leading to dysfunctional proteins or nonsense-mediated decay that cause a wide range of mis-splicing diseases. Development of therapeutic strategies to target the alternative splicing process could potentially shift the mRNA splicing from disease isoforms to a normal isoform and restore functional protein. As a proof of concept, we focus on Stargardt disease (STGD1), an autosomal recessive inherited retinal disease caused by biallelic genetic variants in the ABCA4 gene. The splicing variants c.5461-10T>C and c.4773+3A>G in ABCA4 cause the skipping of exon 39-40 and exon 33-34 respectively. In this study, we compared the efficacy of different RNA-targeting systems to modulate these ABCA4 splicing defects, including four CRISPR-Cas13 systems (CASFx-1, CASFx-3, RBFOX1N-dCas13e-C and RBFOX1N-dPspCas13b-C) as well as an engineered U1 system (ExSpeU1). Using a minigene system containing ABCA4 variants in the human retinal pigment epithelium ARPE19, our results show that RBFOX1N-dPspCas13b-C is the best performing CRISPR-Cas system, which enabled up to 80% reduction of the mis-spliced ABCA4 c.5461-10T>C variants and up to 78% reduction of the ABCA4 c.4773+3A>G variants. In comparison, delivery of a single ExSpeU1 was able to effectively reduce the mis-spliced ABCA4 c.4773+3A>G variants by up to 84%. We observed that the effectiveness of CRISPR-based and U1 splicing regulation is strongly dependent on the sgRNA/snRNA targeting sequences, highlighting that optimal sgRNA/snRNA designing is crucial for efficient targeting of mis-spliced transcripts. Overall, our study demonstrated the potential of using RNA-targeting CRISPR-Cas technology and engineered U1 to reduce mis-spliced transcripts for ABCA4, providing an important step to advance the development of gene therapy to treat STGD1.

molecular biology↗

bioRxiv2024

Transcriptomic characterization of recombinant Clostridium beijerinckii NCIMB 8052 expressing methylglyoxal synthase and glyoxal reductase from Clostridium pasteurianum ATCC 6013

Bioconversion of abundant lactose-replete whey permeate to value added chemicals holds promise for valorization of this increasing food processing waste. Efficient conversion of whey-permeate-borne lactose requires adroit microbial engineering to funnel carbon to the desired chemical. Having engineered a strain of Clostridium beijerinckii NCIMB 8052 (C. beijerinckii_mgsA+mgR) that produces 87% more butanol on lactose than the control strain, in this study, we deployed RNA sequencing to profile the global transcriptome of C. beijerinckii_mgsA+mgR. The results revealed broadly contrasting gene expression patterns in C. beijerinckii_mgsA+mgR relative to the control strain. These were characterized by widespread downregulation of Fe-S proteins in C. beijerinckii_mgsA+mgR, coupled with increased expression of lactose uptake and catabolic genes, iron and phosphate uptake genes, two component signal transduction and motility genes, and genes involved in the biosynthesis of vitamin B5 and B12, aromatic amino acids, particularly tryptophan; arginine, and pyrimidines. Conversely, L-aspartate-dependent de novo biosynthesis of NAD as well as biosynthesis/metabolism of glycine, threonine, lysine, isoleucine and asparagine were downregulated. Furthermore, genes involved in cysteine and methionine biosynthesis and metabolism, including cysteine desulfurase--a central player in Fe-S cluster biosynthesis--were equally downregulated. Genes involved in biosynthesis of capsular polysaccharides and stress response were also downregulated in C. beijerinckii_mgsA+mgR. The results suggest that remodeling of cellular and metabolic networks in C. beijerinckii_mgsA+mgR to counter likely effect of methylglyoxal production following heterologous expression of methyl glyoxal synthase led to enhanced growth and butanol production in C. beijerinckii_mgsA+mgR. IMPORTANCEBiological production of commodity chemicals from abundant waste streams such as whey permeate represents a rational approach for decarbonizing chemical production. Whey permeate remains a vastly underutilized feedstock for bioproduction purposes. Thus, enhanced understanding of the cellular and metabolic repertoires of lactose-mediated production of chemicals such as butanol, promises to arm researchers with new engineering targets that can be fine-tuned in recombinant and native microbial strains to engender stronger coupling of whey permeate-borne lactose to value-added chemicals. Our results highlight new genetic targets for future engineering of C. beijerinckii_mgsA+mgR and indeed, C. beijerinckii for improved butanol production on lactose, and ultimately in whey permeate.

microbiology↗

bioRxiv2024

Rapid generation and screening of transgenic black soldier fly (Hermetia illucens)

BackgroundThe black soldier fly (BSF), Hermetia illucens is a widely used, and mass-produced insect that fulfils an important role in both the management of organic waste and as a component of animal feed formulations. They also have significant potential as a platform for converting organic waste into high-value proteins, and lipids for the production of biofuels. Applying synthetic biology to BSF provides even more potential for improvement through the generation of transgenic BSF to enhance animal feed, produce and fine tune high-value industrial biomolecules, and to expand their waste conversion capabilities. ResultsTo enable the rapid generation and screening of transgenic BSF, we utilised microinjections of piggyBac mRNA with donor plasmids. We have found preliminary screening of G0 BSF to identify mosaics for outcrossing can be completed less than 2 weeks after microinjection. Stable transgenic lines were reliably generated with effective transformation rates of 30-33%, and transmission of the transgene could be confirmed 3 days after outcrossing the G0 adults. We also present a protocol for identifying the location of integrated transgenes. ConclusionsThe methods presented here expedite the screening process for BSF transgenesis and further expand the toolkit for BSF synthetic biology.

synthetic biology↗

bioRxiv2024

Unravelling the roadblocks to 1,2-propanediol biosynthesis in select solventogenic Clostridium species

BackgroundThe compound 1,2-propanediol is an important industrial bulk chemical that has proven particularly recalcitrant to bio-production. Solvent-producing Clostridium species represent promising candidates for engineering 1,2-propaediol production. Co-production of 1,2-popanediol and butanol has the potential to improve the economics of the acetone-butanol-ethanol (ABE) fermentation. ResultsIn this study, the methylglyoxal synthase gene (mgsA) from Clostridium beijerinckii NCIMB 8052 was homologously expressed in this organism. Additionally, a separate strain of Clostridium beijerinckii NCIMB 8052 was engineered by cloning and expressing mgsA and methylglyoxal/glyoxal reductase (mgR) from Clostridium pasteurianum ATCC 6013 as a fused protein linked by polyglycine linker in the former. Both strains of C. beijerinckii NCIMB 8052 failed to produce 1,2-propaneol. Instead, traces of acetol--the precursor of 1,2-propanediol--were detected in cultures of both strains. When the recombinant strains were exposed to acetol, both strains exhibited [~]100% acetol-to-1,2-propanediol conversion efficiency. Conversely, methylglyoxal supplementation led to the production of traces of acetol but not lactaldehyde or 1,2-propanediol. When wildtype C. beijerinckii NCIMB 8052, C. pasteurianum ATCC 6013 and Clostridium tyrobutyricum ATCC 25755 were challenged with methylglyoxal, C. beijerinckii produced [~]0.1 g/L (S)-(+)-1,2-Propanediol, while C. tyrobutyricum produced traces of lactate. C. pasteurianum produced neither 1,2-propanediol nor lactate. The wild types of all three species above exhibited [~]100% acetol-to-1,2-propanediol conversion efficiency. The recombinant strain of C. beijerinckii expressing fused MgsA and MgR from C. pasteurianum ATCC 6013 showed enhanced growth and solvent production, producing as high as 88% more butanol on both glucose and lactose than the control strain and the recombinant strain of the same organism expressing the native MgsA. ConclusionsRecombinant and native strains of C. beijerinckii, C. pasteurianum and C. tyrobutyricum studied in this work exhibit extremely poor capacity to catalyze the conversion of the intermediates of the methylglyoxal bypass to 1,2-propanediol. This is indicative of lack of appropriate enzymes to catalyze the reactions from methylglyoxal to acetol or lactaldehyde. Inability to detect methylglyoxal in the recombinant strains harboring mgsA (both homologous and heterologous)-- whereas the strain expressing both mgsA and mgR from C. pasteurianum, under the same promoter (Padc) produced higher concentrations of butanol--suggests that C. beijerinckii might possess a regulatory mechanism that limits the activity of methylglyoxal-producing MgsA. The protein product of mgR from C. pasteurianum represents a promising metabolic engineering candidate towards increasing butanol production.

microbiology↗

bioRxiv2024

UBA1-CDK16: A Sex-Specific Chimeric RNA and Its Role in Immune Sexual Dimorphism

RNA processing mechanisms, such as alternative splicing and RNA editing, have been recognized as critical means to expand the transcriptome. Chimeric RNAs formed by intergenic splicing provide another potential layer of RNA diversification. By analyzing a large set of RNA-Seq data and validating results in over 1,200 blood samples, we identified UBA1-CDK16, a female-specific chimeric transcript. Intriguingly, both parental genes, are expressed in males and females. Mechanistically, UBA1-CDK16 is produced by cis-splicing between the two adjacent X-linked genes, originating from the inactive X chromosome. A female-specific chromatin loop, formed between the junction sites, facilitates the alternative splicing of its readthrough precursor. This unique chimeric transcript exhibits evolutionary conservation, evolving to be female-specific from non-human primates to humans. Furthermore, our investigation reveals that UBA1-CDK16 is enriched in the myeloid lineage and plays a regulatory role in myeloid differentiation. Notably, female COVID-19 patients who tested negative for this chimeric transcript displayed higher counts of neutrophils, highlighting its potential role in disease pathogenesis. These findings support the notion that chimeric RNAs represent a new repertoire of transcripts that can be regulated independently from the parental genes, and a new class of RNA variance with potential implications in sexual dimorphism and immune responses.

genomics↗

bioRxiv2024

Microenvironment Shapes Cell State, Plasticity, and Heterogeneity of Small Cell Lung Cancer

Small-cell lung cancer (SCLC) is the most fatal form of lung cancer. Intra-tumoral heterogeneity, marked by neuroendocrine (NE) and non-neuroendocrine (non-NE) cell states, defines SCLC, but the drivers of SCLC plasticity are poorly understood. To map the landscape of SCLC tumor microenvironment (TME), we apply spatially resolved transcriptomics and quantitative mass spectrometry-based proteomics to metastatic SCLC tumors obtained via rapid autopsy. The phenotype and overall composition of non-malignant cells in the tumor microenvironment (TME) exhibits substantial variability, closely mirroring the tumor phenotype, suggesting TME-driven reprogramming of NE cell states. We identify cancer-associated fibroblasts (CAF) as a crucial element of SCLC TME heterogeneity, contributing to immune exclusion, and predicting exceptionally poor prognosis. Together, our work provides a comprehensive map of SCLC tumor and TME ecosystems, emphasizing their pivotal role in SCLCs adaptable nature, opening possibilities for re-programming the intercellular communications that shape SCLC tumor states.

cancer biology↗

bioRxiv2024

A proteome-wide quantitative guide for nanoscale spatially resolved extraction of membrane proteins into native nanodiscs

The intricate molecular environment of the native membrane profoundly influences every aspect of membrane protein (MP) biology. Despite this, the most prevalent method of studying MPs uses detergent-like molecules that disrupt and remove this vital local membrane context. This severely impedes our ability to quantitatively decipher the local molecular context and comprehend its regulatory role in the structure, function, and biogenesis of MPs. Using a library of membrane-active polymers we have developed a platform for the high-throughput analysis of the membrane proteome. The platform enables near-complete spatially resolved extraction of target MPs directly from their endogenous membranes into native nanodiscs that maintain the local membrane context. We accompany this advancement with an open-access database that quantifies the polymer-specific extraction variability for 2065 unique mammalian MPs and provides the most optimized condition for each of them. Our method enables rapid and near-complete extraction and purification of target MPs directly from their endogenous organellar membranes at physiological expression levels while maintaining the nanoscale local membrane environment. Going beyond the plasma membrane proteome, our platform enables extraction from any target organellar membrane including the endoplasmic reticulum, mitochondria, lysosome, Golgi, and even transient organelles such as the autophagosome. To further validate this platform, we took several independent MPs and demonstrated how our resource can enable rapid extraction and purification of target MPs from different organellar membranes with high efficiency and purity. Further, taking two synaptic vesicle MPs, we show how the database can be extended to capture multiprotein complexes between overexpressed MPs. We expect these publicly available resources to empower researchers across disciplines to efficiently capture membrane nano-scoops containing a target MP and interface with structural, functional, and other bioanalytical approaches. We demonstrate an example of this by combining our extraction platform with single-molecule TIRF imaging to demonstrate how it can enable rapid determination of homo-oligomeric states of target MPs in native cell membranes.

biophysics↗

bioRxiv2024

Characterisation of RNA editing and gene therapy with a compact CRISPR-Cas13 in the retina

CRISPR-Cas13 nucleases are programmable RNA-targeting effectors that can silence gene expression in a reversible manner. Recent iterations of Cas13 nucleases are compact for adeno-associated virus (AAV) delivery to achieve strong and persistent expression in various organs in a safe manner. Here, we report significant transcriptomic signatures of Cas13bt3 expression in retinal cells and show all-in-one AAV gene therapy with Cas13bt3 can effectively silence VEGFA mRNA in human retinal organoids and humanised VEGF transgenic mouse (trVEGF029, Kimba) models. Specifically, human embryonic stem cells (hESC)-derived retinal pigment epithelium cells show high expression of Cas13bt3 from virus delivery corresponding to a significant reduction of VEGFA mRNA. We further show that intravitreal delivery of Cas13bt3 can transduce mouse retinal cells efficiently, reaching the photoreceptors for specific knockdown of human VEGFA in the Kimba mouse. Our results reveal important considerations for assessing Cas13 activity and establish Cas13bt3 as a potential anti-VEGF agent that can achieve long-term control of VEGFA for the treatment of retinal neovascularization.

molecular biology↗

bioRxiv2024

The genetic landscape, gene flow and adaptation in Ladakh highlanders of trans-Himalayas

Trans-Himalayan Ladakh has witnessed complex cultural movements and demographic changes since the Neolithic period, which is still continue despite the harsh, inhospitable and cold climate. Although geographically isolated from mainland South Asia, Ladakh has historic trade routes and is well connected and accessible to travelers from Tibet and Central Asia. Despite its rich cultural heritage, a detailed description of the genetic landscape of the Ladakh region is completely lacking, particularly with regard to genome-wide analysis and larger sample sizes. Therefore, in the current study, we genotyped 80 individuals from Kargil and Leh districts of the Union Territory of Ladakh, India. Here, we performed a comprehensive genetic analysis based on allele frequency and haplotype sharing. Our analysis revealed the presence of two distinct genetic lineages in the region with quite distinct genetic composition. The population of Leh Region is more similar to East Asian and Southeast Asian populations. In contrast, the population of the Kargil Region (LDKLA) is more similar to Indo-European populations. Demographic modeling suggests that the Leh group shares a genetic history with Tibetans, while the Kargil group showed great affinities with Kashmiri Muslims, Gujjars and Nepalese Brahmins, and both showed recent admixture. Both groups have experienced a founder event around during 11th to 22nd generations ago, the duration of which coincided with the Mughal invasion. The genome-wide scan for a signal of positive selection revealed genetic signatures of high-altitude adaptation (EPAS1 and ELMO2) in the Leh population, while in the Kargil population the key gene signatures were associated with immunity and female fertility.

genetics↗

bioRxiv2024

MAGIC matrices: freeform bioprinting materials to support complex and reproducible organoid morphogenesis

Complex and robust self-organization requires defined initial conditions and dynamic boundaries - neighboring tissues and extracellular matrix (ECM) that actively evolve to guide morphogenesis. A major challenge in tissue engineering is identifying material properties that mimic dynamic tissue boundaries but that are compatible with the engineering tools necessary for controlling the initial conditions of culture. Here we describe a highly tunable granular biomaterial, MAGIC matrix, that supports long-term bioprinting and gold-standard tissue self-organization. MAGIC matrix is designed for two temperature regimes: at 4 {degrees}C it exhibits reversible yield-stress behavior to support hours-long high-fidelity 3D printing without compromising cell viability; when transferred to cell culture at 37 {degrees}C, the material cross-links and exhibits viscoelasticity and stress relaxation that can be tuned to match numerous conditions, including that of reconstituted basement membrane matrices like Matrigel. We demonstrate that the timescale of stress relaxation and loss tangent are decoupled in MAGIC matrices, allowing us to test the role of stress relaxation rate and strain-dependence across formulations with identical storage and loss moduli. We find that fast absolute stress relaxation rates and large relative deformation magnitudes are required to optimize for morphogenesis. We apply optimized MAGIC matrices toward precise extrusion bioprinting of saturated cell suspensions directly into 3D culture. The ability to carefully control initial conditions for tissue growth yields dramatic increases in organoid reproducibility and complexity across multiple tissue types. We also fabricate perfusable 3D microphysiological systems that experience large strains in response to pressurization due to the compliant and dynamic tissue boundaries. Combined, our results both identify key parameters for optimal organoid morphogenesis in an engineered material and lay the foundation for fabricating more complex and reproducible tissue morphologies by canalizing their self-organization in both space and time.

bioengineering↗

bioRxiv2024

XBB.1.5 monovalent booster improves antibody binding and neutralization against emerging SARS-CoV-2 Omicron variants

The rapid emergence of divergent SARS-CoV-2 variants has led to an update of the COVID-19 booster vaccine to a monovalent version containing the XBB.1.5 spike. To determine the neutralization breadth following booster immunization, we collected blood samples from 24 individuals pre- and post-XBB.1.5 mRNA booster vaccination ([~]1 month). The XBB.1.5 booster improved both neutralizing activity against the ancestral SARS-CoV-2 strain (WA1) and the circulating Omicron variants, including EG.5.1, HK.3, HV.1, XBB.1.5 and JN.1. Relative to the pre-boost titers, the XBB.1.5 monovalent booster induced greater total IgG and IgG subclass binding, particular IgG4, to the XBB.1.5 spike as compared to the WA1 spike. We evaluated antigen-specific memory B cells (MBCs) using either spike or receptor binding domain (RBD) probes and found that the monovalent booster largely increases non-RBD cross-reactive MBCs. These data suggest that the XBB.1.5 monovalent booster induces cross-reactive antibodies that neutralize XBB.1.5 and related Omicron variants.

immunology↗

bioRxiv2024

Novel spirocyclic dimer, SpiD3, targets chronic lymphocytic leukemia survival pathways with potent preclinical effects

Chronic lymphocytic leukemia (CLL) cell survival and growth is fueled by the induction of B-cell receptor (BCR) signaling within the tumor microenvironment (TME) driving activation of NF- {kappa}B signaling and the unfolded protein response (UPR). Malignant cells have higher basal levels of UPR posing a unique therapeutic window to combat CLL cell growth using pharmacological agents that induce accumulation of misfolded proteins. Frontline CLL therapeutics that directly target BCR signaling such as Bruton-tyrosine kinase (BTK) inhibitors (e.g., ibrutinib) have enhanced patient survival. However, resistance mechanisms wherein tumor cells bypass BTK inhibition through acquired BTK mutations, and/or activation of alternative survival mechanisms have rendered ibrutinib ineffective, imposing the need for novel therapeutics. We evaluated SpiD3, a novel spirocyclic dimer, in CLL cell lines, patient-derived CLL samples, ibrutinib-resistant CLL cells, and in the E{micro}-TCL1 mouse model. Our integrated multi-omics and functional analyses revealed BCR signaling, NF-{kappa}B signaling, and endoplasmic reticulum stress among the top pathways modulated by SpiD3. This was accompanied by marked upregulation of the UPR and inhibition of global protein synthesis in CLL cell lines and patient-derived CLL cells. In ibrutinib-resistant CLL cells, SpiD3 retained its anti-leukemic effects, mirrored in reduced activation of key proliferative pathways (e.g., PRAS, ERK, MYC). Translationally, we observed reduced tumor burden in SpiD3-treated E{micro}-TCL1 mice. Our findings reveal that SpiD3 exploits critical vulnerabilities in CLL cells including NF-{kappa}B signaling and the UPR, culminating in profound anti-tumor properties independent of TME stimuli. STATEMENT OF SIGNIFICANCESpiD3 demonstrates cytotoxicity in CLL partially through inhibition of NF-{kappa}B signaling independent of tumor-supportive stimuli. By inducing the accumulation of unfolded proteins, SpiD3 activates the UPR and hinders protein synthesis in CLL cells. Overall, SpiD3 exploits critical CLL vulnerabilities (i.e., the NF-{kappa}B pathway and UPR) highlighting its use in drug-resistant CLL. VISUAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=137 HEIGHT=200 SRC="FIGDIR/small/578283v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1dd7a38org.highwire.dtl.DTLVardef@1831c85org.highwire.dtl.DTLVardef@c571f9org.highwire.dtl.DTLVardef@bdce53_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Etiology of Craniofacial and Cardiac Malformations in a Mouse Model of SF3B4-Related Syndromes

Pathogenic variants in SF3B4 are responsible for the acrofacial disorders Nager and Rodriguez Syndrome, also known as SF3B4-related Syndromes, associated with malformations in the head, face, limbs, vertebrae as well as the heart. To uncover the etiology of craniofacial malformations found in SF3B4-related syndromes, mutant mouse lines with homozygous deletion of Sf3b4 in neural crest cells (NCC) were generated. Like in human patients, these embryos had craniofacial and cardiac malformations with variable expressivity and penetrance. The severity and survival of Sf3b4 NCC mutants was modified by the level of Sf3b4 in neighbouring non NCC. RNA sequencing analysis of heads of embryos prior to morphological abnormalities showed significant changes in expression of genes forming the NCC regulatory network, as well as an increase in exon skipping. We identified several key transcription factors and histone modifiers involved in craniofacial and cardiac development with increased exon skipping. Increased exon skipping was also associated with use of a more proximal branch point, as well as an enrichment in thymidine bases in the 50bp around the branch points. We propose that decrease in Sf3b4 causes changes in the expression and splicing of transcripts required for proper craniofacial and cardiac development, leading to abnormalities.

developmental biology↗

bioRxiv2024

P66Shc Mediates SUMO2-induced Endothelial Dysfunction

BackgroundSumoylation is a post-translational modification that can regulate different physiological functions. Increased sumoylation, specifically conjugation of SUMO2/3 (small ubiquitin-like modifier 2/3), is detrimental to vascular health. However, the molecular mechanism mediating this effect is poorly understood. MethodsWe used cell-based assays and mass spectrometry to show that p66Shc is a direct target of SUMO2 and SUMO2 regulates p66Shc function via lysine-81 modification. To determine the effects of SUMO2-p66ShcK81 on vascular function, we generated p66ShcK81R knockin mice and crossbred to LDLr-/- mice to induce hyperlipidemia. Next, to determine p66ShcK81-SUMO2ylation-induced changes in endothelial cell signaling, we performed mass spectrometry followed by Ingenuity Pathway Analysis. ResultsOur data reveal that p66Shc mediates the effects of SUMO2 on endothelial cells. Mass spectrometry identified that SUMO2 modified lysine-81 in the unique collagen homology-2 domain of p66Shc. SUMO2ylation of p66Shc increased phosphorylation at serine-36, causing it to translocate to the mitochondria, a step critical for oxidative function of p66Shc. Notably, sumoylation-deficient p66Shc (p66ShcK81R) was resistant to SUMO2-induced p66ShcS36 phosphorylation and mitochondrial translocation. P66ShcK81R knockin mice were resistant to endothelial dysfunction induced by SUMO2ylation and hyperlipidemia. Ingenuity Pathway Analysis revealed multiple signaling pathways regulated by p66ShcK81-SUMO2ylation in endothelial cells, highlighting JAK-STAT as a major pathway affected by SUMO2-p66ShcK81. ConclusionsCollectively, our work reveals SUMO2-p66Shc signaling as a fundamental regulator of vascular endothelial function. We discovered that p66ShcK81 is an upstream modification regulating p66Shc signaling and mediates hyperlipidemia-induced endothelial dysfunction and oxidative stress.

cell biology↗

bioRxiv2024

Shear flow as a tool to distinguish microscopic activities of molecular machines in a chromatin loop

Several types of molecular machines move along biopolymers like chromatin. However, the details about the microscopic activity of these machines and how to distinguish their modes of action are not well understood. We propose that the activity of such machines can be classified by studying looped chromatin under shear flow. Our simulations show that a chromatin-like polymer with two types of activities (constant or local curvature-dependent tangential forces) exhibits very different behavior under shear flow. We show that one can distinguish both activities by measuring the nature of a globule-to-extended coil transition, tank treading, and tumbling dynamics.

biophysics↗