Biology subjects

Kumari, A.

Publications and source records attributed to Kumari, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Engineered RVG29-Conjugated Chitosan for Enhanced Brain Delivery of Kisspeptin Agonists and Antagonists in Reproductive Health.

Efficient delivery of intact therapeutic peptides across the blood-brain barrier (BBB) remains a central obstacle in translating neuroendocrine modulators to clinical practice. Kisspeptin receptor (KISS1R) agonists and antagonists precisely regulate gonadotropin-releasing hormone (GnRH) pulsatility, thereby controlling luteinizing hormone (LH) release, steroidogenesis, and fertility. While the KISS1R agonist KP10 accelerates GnRH-LH pulses to promote ovulation, the antagonist KP234 suppresses pulse generation, inducing reversible infertility. However, their poor BBB permeability limits clinical application. Here, we engineered a non-invasive brain delivery platform by covalently coupling the rabies virus glycoprotein-derived peptide RVG29 to oxidized chitosan nanoparticles via Schiff base chemistry. Sodium periodate oxidation introduced aldehyde groups, FT-IR confirmed aldehyde formation, and UV-Vis spectroscopy validated RVG29 conjugation through characteristic absorbance shifts. The resulting RVG29 - chitosan carriers demonstrated biocompatibility, enhanced neuronal uptake [~]2.1-fold in vitro, and achieved [~]2.5-fold higher brain accumulation in vivo compared to unconjugated controls. Systemic delivery of RVG29-chitosan-KP10 restored and amplified LH pulsatility in ovariectomized mice, accelerated estrous cycling, increased preovulatory follicle numbers, and enhanced litter size. In contrast, RVG29-chitosan-KP234 abolished LH pulses, maintained animals in persistent diestrus, depleted corpora lutea, and produced complete yet reversible infertility. Longitudinal dosing demonstrated stable neuroendocrine modulation without systemic toxicity, and ovarian histology confirmed ovulatory arrest in antagonist-treated animals. By enabling programmable, bidirectional control of a central hypothalamic circuit through systemic delivery of unmodified peptides, this platform addresses a long-standing barrier in neuroendocrine therapeutics. Beyond fertility regulation, this approach can be adapted to other hypothalamic-driven processes, including metabolic, stress-axis, and circadian control.

bioengineering↗

bioRxiv2025

Role of zinc in growth, stress response and virulence gene expression of pathogenic Mucorale Rhizopus arrhizus

During COVID-19 pandemic, the cases of mucormycosis increased substantially, with rhino-orbito-cerebral form linked to uncontrolled diabetes being the predominant manifestation. Some clinical and epidemiological studies associated the usage of zinc supplements with the occurrence of COVID-19 associated mucormycosis, but experimental evidence remains limited. This study aimed to elucidate the impact of zinc enrichment on Rhizopus arrhizus, the predominant causative agent of mucormycosis. The effect of zinc supplementation (5 to 150 {micro}M) on fungal growth, metabolic activity, antifungal susceptibility and biofilm formation, along with cell-wall (Congo red), oxidative (hydrogen peroxide) and osmotic (sodium chloride) stress was evaluated in RPMI-1640. Morphological changes were visualized by scanning electron microscopy. Gene expression was monitored by RNA sequencing. Zinc supplementation promoted R. arrhizus growth in a concentration-dependent manner, with the effect being particularly pronounced under less-favorable conditions, such as alkaline conditions resembling the nasal pH of diabetics. Exposure to zinc enhanced the metabolic activity, and partly alleviated the cell-wall and oxidative stress. Susceptibility to amphotericin B and posaconazole was unchanged. Zinc-supplemented cultures over-expressed multiple genes, notably those associated with respiratory electron transport chain, ergosterol biosynthesis, chitosan production, oxidative stress response, mucoricin, high-affinity iron permease, iron transport multi-copper oxidase and ferric-reductase-like protein. Zinc also augmented the growth/metabolism of several other Mucorales but not Aspergillus fumigatus and Candida albicans. The findings suggest that zinc supplementation supports mucoralean growth and enhances the expression of key virulence factors, implying that excessive intake of zinc supplements during COVID-19 pandemic likely contributed to the emergence of mucormycosis. IMPORTANCEMucormycosis remains a highly devastating fungal infection that presented additional challenges during the COVID-19 pandemic. Several risk factors were implicated in the emergence of COVID-19 associated mucormycosis (CAM), including inappropriate corticosteroid usage and COVID-19 associated glycemic imbalance. The excessive intake of nutritional supplements, especially that of zinc, owing to self-medication and over-prescription, was also proposed as a plausible factor in the rise of CAM in many clinical/epidemiological studies. This study experimentally evaluated the potential impact of zinc enrichment on pathogenic Mucorales. The work demonstrates that zinc supplementation supports Rhizopus arrhizus growth, metabolism and stress response, and enhances the expression of key virulence factors, including mucoricin and iron transporters. Notably, the augmentation was specifically observed in pathogenic Mucorales but not Aspergillus fumigatus and Candida albicans. The work provides experimental evidence for the potential association of zinc over-availability with the occurrence of CAM, and strengthens evidence-based therapeutic practices in clinical settings.

microbiology↗

bioRxiv2025

Molecular characterization of unique multi-domain harbouring fungal rhodopsin for establishing their novel opto-synthetic biological usages

Organisms employ light as an external stimulus for regulating cellular functions. The light-sensitive photoreceptors detect light at varying wavelengths, activating signaling cascades and triggering a range of physiological responses. Rhodopsin is a transmembrane heptahelical protein that functions as an ion channel, or a pump, and sensory receptor, respectively. It consists of a light-sensing chromophore, a retinal that upon absorbing light, initiates a series of signaling pathways of sensory perception, growth and survival. Modular rhodopsin (Different from Rhodopsin-Cyclase Module) has been reported in lower eukaryotes, its identification, characterisation and functional significance in the Fungal Kingdom largely unknown. Here, we report the identification of novel modular rhodopsins in fungi, which highlights their potential usages towards the unexplored opto-biotechnological applications (e.g., biomanufacturing of terpenoids, cytoskeleton regulation, DNA metabolism, light-controlled acetyltransferase, etc.) simply by illumination. Furthermore, identification of novel modular rhodopsins augments the expansion of the new optogenetic tools for a wide range of relevant applications. The structural and homology analysis of these identified domains sheds light on their evolutionary lineage and relatedness with the well-characterised bacteriorhodopsin, sensory and channelrhodopsin. The interactome analysis effector domain coupled with the microbial rhodopsin (Rh) reveals RPEL-mediated gene expression and metabolite regulation, which further modulates the retinol synthesis pathway. The role of the fungal Rh-RPEL effector domain in modulating the terpenoid and sphingolipid metabolism in response to light was successfully elucidated via protein-protein interaction and Biosynthesis Gene Cluster (BGC) analysis. This highlights the potential of these novel opto-synthetic biological usages that can induce the light-dependent production of commercially relevant fungal bioactive(s).

bioinformatics↗

bioRxiv2025

Adolescent intermittent ethanol exposure induces sex-specific and time-dependent changes in affective behaviors and metabolomic profiles.

Given the adverse effects of adolescent binge alcohol consumption in humans, the present work explores the lasting behavioral and metabolomic impacts following adolescent binge ethanol exposure. Here we determine short- and long-term changes in affective behaviors and metabolomic profiles in male and female adolescent binge ethanol-exposed mice. Male and female C57BL/6J mice were exposed to adolescent intermittent ethanol (AIE) as a model of binge ethanol exposure using intermittent vapor inhalation from postnatal day (PND) 28-42. Mice were tested in a battery of behavioral tests and metabolomic analyses were conducted following short-term and long-term withdrawal from AIE exposure. Mice were tested for affective behaviors using the open field test (OFT), the light/dark test (LDT), and the tail suspension test (TST) one week following AIE exposure from PND 49-53 and again from PND 91-95. Serum samples were collected on PND 43, corresponding to 24 hours after the last exposure, fecal samples were collected during each OFT, and liver samples were collected approximately 80 days after AIE exposure for metabolomic analysis. We show modest incubation of behavioral differences in anxiety-like behavior in males after adolescent binge ethanol exposure; an effect that was absent in female mice. In contrast, metabolomic differences in male mice that were more pronounced shortly after adolescent binge ethanol exposure waned as time progressed since last exposure. Male mice appear to be more susceptible to the persistent changes in adolescent binge ethanol exposure that varies over time. It is possible that short-term metabolomic changes may predict long-term behavioral changes in affective behaviors. Contribution to the field statementAdolescent binge alcohol exposure causes long-lasting changes in behavior, however the underlying biology mediating these changes have not readily been assessed in a sex-specific manner. Metabolomic assays are a powerful tool that can be used as biomarkers to determine changes in biological pathways that are linked to specific behavioral phenotypes. Here we explored the relationship between adolescent binge alcohol exposure, changes in anxiety-like behavior, and metabolomic profiles in male and female C57BL/6J mice. We find robust changes in metabolomic profiles following short-term withdrawal in male mice exposed to binge alcohol during adolescence. We also see modest changes in anxiety-like behavior following long-term withdrawal. Although female mice did not show any robust changes in anxiety-like behavior nor global changes in metabolomic profiles, in both sexes we do observe persistent changes in some amino acids, which may serve as specific biomarkers associated with changes in alcohol-induced behavioral changes. These data add to the field by conducting a novel longitudinal study following male and female mice from adolescence to early adulthood and measuring physiological biomarkers (metabolomics) coupled with behavioral changes following binge ethanol exposure.

neuroscience↗

bioRxiv2025

Aerobic glycolysis and lactate regulate histone H3K18Lactylation occupancy to fine-tune gene expression in developing and mature retina

High aerobic glycolysis in retinal photoreceptors, as in cancer cells, is implicated in mitigating energy and metabolic demands. Lactate, a product of glycolysis, plays a key role in epigenetic regulation through histone lactylation in cancer. Here, we demonstrate that increased ATP production during retinal development is achieved primarily through augmented glycolysis. Histone lactylation, especially H3K18La, parallels enhanced glycolysis and lactate in developing retina and in retinal explants. Multi-omics analyses, combined with confocal imaging, reveal the localization of H3K18La near H3K27Ac in euchromatin at promoters of active retinal genes. H3K18La and gene expression are also concordant with glucose metabolism in retinal explants. Evaluation of accessible chromatin at H3K18La marked promoters uncovers an enrichment of GC-rich motifs for transcription factors of SP, KMT and KLF families, among others, indicating specificity of H3K18La-mediated gene regulation. Our results highlight glycolysis/lactate/H3K18La as a regulatory axis in fine-tuning gene expression in developing and mature retina.

molecular biology↗

bioRxiv2025

Structural basis of multitasking by the apicoplast DNA polymerase from Plasmodium falciparum

Plasmodium falciparum is a unicellular eukaryotic pathogen responsible for the majority of malaria-related fatalities. Plasmodium belongs to the phylum Apicomplexa and like most members of this phylum, contains a non-photosynthetic plastid called the apicoplast. The apicoplast has its own genome, which is replicated by a dedicated apicoplast replisome. Unlike other cellular replisomes, the apicoplast replisome uses a single DNA polymerase (apPol) for copying the apicoplast DNA. Being the only DNA polymerase in the apicoplast, apPol is expected to multitask, catalysing both replicative and lesion bypass synthesis. Replicative synthesis typically relies on a restrictive active site for high accuracy while lesion bypass requires an open active site. This raises the question how does apPol combine the structural features of multiple DNA polymerases in a single protein. Using single particle electron cryomicroscopy (cryoEM), we have solved the structures of apPol bound to its DNA and nucleotide substrates in five pre-chemistry conformational states, allowing us to describe the events leading up to nucleotide incorporation and answer how apPol incorporates features of multiple polymerases. We found that, unlike most replicative polymerases, apPol can accommodate a nascent base pair with the fingers in an open configuration, which might facilitate the lesion bypass activity. In the fingers open state we identified a nascent base pair checkpoint that can preferentially select a Watson-Crick base pair, an essential requirement for replicative synthesis. Taken together these structural features explain how apPol may balance replicative and lesion bypass synthesis.

biochemistry↗

bioRxiv2025

Interaction of the mitochondrial calcium/proton exchanger TMBIM5 with MICU1

Ion transport within mitochondria influences their structure, energy production, and cell death regulation. TMBIM5, a conserved calcium/proton exchanger in the inner mitochondrial membrane, contributes to mitochondrial structure, ATP synthesis, and apoptosis regulation. The relationship of TMBIM5 with the mitochondrial calcium uniporter complex formed by MCU, MICU1-3, and EMRE remains undefined. We generated Tmbim5-deficient Drosophila that exhibit disrupted cristae architecture, premature mitochondrial permeability transition pore opening, reduced calcium uptake, and mitochondrial swelling - resulting in impaired mobility and shortened lifespan. Crossing these with flies lacking mitochondrial calcium uniporter complex proteins was generally detrimental, but partial MICU1 depletion ameliorated the Tmbim5-deficiency phenotype. In human cells, MICU1 rescues morphological defects in TMBIM5-knockout mitochondria, while TMBIM5 overexpression exacerbates size reduction in MICU1-knockout mitochondria. Both proteins demonstrated opposing effects on submitochondrial localization and coexisted in the same macromolecular complex. Our findings establish a functional interplay between TMBIM5 and MICU1 in maintaining mitochondrial integrity, with implications for understanding calcium homeostasis mechanisms.

cell biology↗

bioRxiv2024

14-3-3 epsilon inhibits premature centriole disengagement by inhibiting the activity of Plk1 and Separase.

The 14-3-3 protein family regulates several pathways in mammalian cells, including centrosome duplication. However, the precise mechanisms by which 14-3-3 paralogs regulate the centrosome cycle remain unclear. To identify the mechanisms by which 14-3-3{varepsilon} regulates centrosome duplication, we altered two conserved acidic residues in the 14-3-3{varepsilon} phospho-peptide-binding pocket that regulate complex formation and dissociation with the associated ligands, D127 and E134, to Alanine. Altering these residues to Alanine led to opposing effects on centrosome duplication; the D127A mutant inhibited centrosome duplication, while cells expressing the E134A mutant showed the presence of supernumerary centrosomes. We demonstrate that 14-3-3{varepsilon} does not inhibit centriole duplication, as reported for 14-3-3{gamma}, but inhibits centriole disengagement. Using a combination of pharmacological and genetic approaches, we demonstrate that 14-3-3{varepsilon} inhibits the activity of Plk1 and Separase, leading to disengagement defects that ultimately lead to decreased proliferation and cell death. Our work demonstrates that different 14-3-3 paralogs regulate different steps in the centrosome cycle, and disrupting complex formation between 14-3-3{varepsilon} and Plk1 or Separase could be a novel therapeutic strategy in tumor cells.

cell biology↗

bioRxiv2024

Convergent olfactory circuits for courtship in Drosophila revealed by ds-Tango

Animals exhibit sex-specific behaviors that are governed by sexually dimorphic circuits. One such behavior in male Drosophila melanogaster, courtship, is regulated by various sensory modalities, including olfaction. Here, we reveal how sexually dimorphic olfactory pathways in male flies converge at the third-order, onto lateral horn output neurons, to regulate courtship. To achieve this, we developed ds-Tango, a modified version of the monosynaptic tracing and manipulation tool trans-Tango. In ds-Tango, two distinct configurations of trans-Tango are positioned in series, thus providing selective genetic access not only to the monosynaptic partners of starter neurons but also to their disynaptic connections. Using ds-Tango, we identified a node of convergence for three sexually dimorphic olfactory pathways. Silencing this node results in deficits in sex recognition of potential partners. Our results identify lateral horn output neurons required for proper courtship behavior in male flies and establish ds-Tango as a tool for disynaptic circuit tracing.

neuroscience↗

bioRxiv2024

Single-molecule tracking reveals the dynamics of Ipl1 recruitment to the kinetochores and spindles in S. cerevisiae

Aurora kinase B, Ipl1 in Saccharomyces cerevisiae, is the master regulator of cell division required for checkpoint regulation, spindle assembly and disassembly, chromosome segregation, and cytokinesis. Decades of research employed ensemble averaging methods to understand its dynamics and function; however, the dynamic information was lost due to population-based averaging. Here, we use single-molecule imaging and tracking (SMIT) to quantify the recruitment dynamics of Ipl1 at the kinetochores and spindles in live cells. Our data suggest that Ipl1 is recruited to these locations with different dynamics. We have demonstrated how the recruitment dynamics of Ipl1 at the kinetochores during metaphase changes in the presence and absence of tension across the kinetochore, in the absence of protein phosphatase 1 (Glc7), and the absence of its known recruiters (Ctf19 and Bub1). The SMIT of other chromosome passenger complex members suggests its hierarchical assembly at the kinetochore. Hence, SMIT provides a dynamic view of the Ipl1 trafficking at the kinetochores and spindles.

cell biology↗

bioRxiv2024

Cathepsin B regulates ovarian reserve quality and quantity via mitophagy by modulating IGF1R turnover.

The quality and quantity of the ovarian reserve are meticulously regulated through various cell death pathways to guarantee the availability of high-quality oocytes for fertilization. While apoptosis is recognized for contributing to maintaining ovarian reserve, the involvement of other cell death pathways remains unclear. Employing chemical genetics and proteomics, this study reveals the crucial involvement of Cathepsin B in maintaining the ovarian reserve. Results indicate that apoptosis and autophagy play pivotal roles, and inhibiting these pathways significantly increases follicle numbers. Proteomics reveals a dynamic shift from apoptosis to autophagy during follicular development, with Cathepsin B emerging as a key player in this transition. Inhibiting Cathepsin B not only mimics the augmented oocyte reserve observed with autophagy inhibition but also upregulated IGF1R and AKT-mTOR pathways without compromising fertility. Further, IGF1R inhibition partially compromised the protective effects of Cathepsin B inhibition on oocyte reserves, suggesting their interdependence. This association is further supported by the finding that Cathepsin B can degrade IGF1R in vitro. Moreover, the increased IGF1R levels enhance the oocyte mitochondrial membrane potential via transcriptional regulation of mitochondrial biogenesis and mitophagy genes. Remarkably, this Cathepsin B-dependent ovarian reserve maintenance mechanism is conserved in higher-order vertebrates. Cumulatively, our study sheds valuable light on the intricate interplay of autophagy, Cathepsin B, and growth factors in ovarian reserve maintenance, offering potential implications for fertility research.

cell biology↗

bioRxiv2023

Development of adaptive anoikis resistance promotes metastasis that can be overcome by CDK8/19 Mediator kinase inhibition

Anoikis resistance or evasion of cell death triggered by matrix detachment is a hallmark of cancer cell survival and metastasis. We show that repeated exposure to suspension stress followed by recovery under attached conditions leads to development of anoikis resistance. The acquisition of anoikis resistance is associated with enhanced invasion, chemoresistance, and immune evasion in vitro and distant metastasis in vivo. This acquired anoikis resistance is not genetic, persisting for a finite duration without detachment stress, but is sensitive to CDK8/19 Mediator kinase inhibition that can also reverse anoikis resistance. Transcriptomic analysis reveals that CDK8/19 kinase inhibition induces bidirectional transcriptional changes in both sensitive and resistant cells, disrupting the balanced reprogramming required for anoikis adaptation and resistance by reversing some resistance associated pathways and enhancing others. Both anoikis resistance and in vivo metastatic growth of ovarian cancers are sensitive to CDK8/19 inhibition, thereby providing a therapeutic opportunity to both prevent and suppress ovarian cancer metastasis.

cancer biology↗

bioRxiv2023

TNF-alpha promotes cilia elongation via Mixed Lineage Kinases signaling.

The primary cilium is characteristic of most of non-immune cells and acts as an environmental signal transduction sensor. The defects in primary cilium have profound consequences on the developmental program, including the maturation of retinal epithelium. The ciliary length is tightly regulated during ciliogenesis. Additionally, many features of ciliogenesis are shared with an immune synapse formation. While the interaction between the cells within an immune synapse is well-characterized, the impact of inflammatory stresses on ciliogenesis in non-immune cells remains elusive. The current study investigates the outcome of inflammatory stimuli for the primary cilium in human retinal epithelial cells. Here, we report that the exposure of retinal epithelium cells to pro-inflammatory cytokine TNF-alpha elongates cilia in a Mixed-Lineage Kinase (MLK) - dependent manner. In contrast, febrile condition-mimicking heat stress dramatically reduced the number of ciliated cells regardless of TNF-alpha exposure, suggesting distinct but rapid effects of inflammatory stresses on ciliogenesis.

cell biology↗

bioRxiv2023

Distinct expression patterns of Hedgehog signaling components in mouse gustatory system during postnatal tongue development and adult homeostasis

It is well established that the Hedgehog (HH) pathway regulates embryonic development of anterior tongue taste fungiform papilla (FP) and the posterior circumvallate (CVP) and foliate (FOP) taste papillae, and taste organ maintenance and regeneration in adults. However, there are knowledge gaps in determining HH signaling regulation in postnatal taste organ differentiation and maturation. Importantly, the HH transcription factors GLI1, GLI2 and GLI3 have not been investigated in early postnatal stages; and, the receptors PTCH1, GAS1, CDON and HHIP, required to either drive HH pathway activation or antagonism, remain unexplored. Using lacZ reporter mouse models, we mapped expression of the HH ligand SHH, receptors, and transcription factors in FP, CVP and FOP in early and late postnatal and adult stages. In adults we also studied the soft palate, and the geniculate and trigeminal ganglia which extend afferent fibers to the anterior tongue. Shh and Gas1 are the only components that were consistently expressed within taste buds of all three papillae and the soft palate. In the first postnatal week, we observed a broad expression of HH signaling components in FP and adjacent, non-taste filiform (FILIF) papillae in epithelium or stroma and tongue muscles. Remarkably, we observed elimination of Gli1 in FILIF and Gas1 in muscles, and downregulation of Ptch1 in lingual epithelium and of Cdon, Gas1 and Hhip in stroma from late postnatal stage. Further, HH receptor expression patterns in CVP and FOP epithelium differed from anterior FP. Among all the components, only known positive regulators of HH signaling, SHH, Ptch1, Gli1 and Gli2, were expressed in the ganglia. Our studies emphasize differential regulation of HH signaling in distinct postnatal developmental periods and in anterior versus posterior taste organs, and lay the foundation for functional studies to understand the roles of numerous HH signaling components in postnatal tongue development.

developmental biology↗

bioRxiv2023

Translocation of gut commensal bacteria to the brain

The gut-brain axis, a bidirectional signaling network between the intestine and the central nervous system, is crucial to the regulation of host physiology and inflammation. Recent advances suggest a strong correlation between gut dysbiosis and neurological diseases, however, relatively little is known about how gut bacteria impact the brain. Here, we reveal that gut commensal bacteria can translocate directly to the brain when mice are fed an altered diet that causes dysbiosis and intestinal permeability, and that this also occurs without diet alteration in distinct murine models of neurological disease. The bacteria were not found in other systemic sites or the blood, but were detected in the vagus nerve. Unilateral cervical vagotomy significantly reduced the number of bacteria in the brain, implicating the vagus nerve as a conduit for translocation. The presence of bacteria in the brain correlated with microglial activation, a marker of neuroinflammation, and with neural protein aggregation, a hallmark of several neurodegenerative diseases. In at least one model, the presence of bacteria in the brain was reversible as a switch from high-fat to standard diet resulted in amelioration of intestinal permeability, led to a gradual loss of detectable bacteria in the brain, and reduced the number of neural protein aggregates. Further, in murine models of Alzheimers disease, Parkinsons disease, and autism spectrum disorder, we observed gut dysbiosis, gut leakiness, bacterial translocation to the brain, and microglial activation. These data reveal a commensal bacterial translocation axis to the brain in models of diverse neurological diseases.

neuroscience↗

bioRxiv2023

Heparan sulfate modifications of betaglycan promote TIMP3-dependent ectodomain shedding to fine-tune TGF-β signaling.

In pathologies such as cancer, aberrant Transforming Growth Factor-{beta} (TGF-{beta}) signaling exerts profound tumor intrinsic and extrinsic consequences. Intense clinical endeavors are underway to target this pivotal pathway. Central to the success of these interventions is pinpointing factors that decisively modulate the TGF-{beta} responses. Betaglycan/type III TGF-{beta} receptor (T{beta}RIII), is an established co-receptor for the TGF-{beta} superfamily known to bind directly to TGF-{beta}s 1-3 and inhibin A/B. While betaglycan can be membrane-bound, it can also undergo ectodomain cleavage to produce soluble-betaglycan that can sequester its ligands. The extracellular domain of betaglycan undergoes heparan sulfate and chondroitin sulfate glycosaminoglycan modifications, transforming betaglycan into a proteoglycan. Here we report the unexpected discovery that the heparan sulfate modifications are critical for the ectodomain shedding of betaglycan. In the absence of such modifications, betaglycan is not shed. Such shedding is indispensable for the ability of betaglycan to suppress TGF-{beta} signaling and the cells responses to exogenous TGF-{beta} ligands. Using unbiased transcriptomics, we identified TIMP3 as a key regulator of betaglycan shedding and thereby TGF-{beta} signaling. Our results bear significant clinical relevance as modified betaglycan is present in the ascites of patients with ovarian cancer and can serve as a marker for predicting patient outcomes and TGF-{beta} signaling responses. These studies are the first to demonstrate a unique reliance on the glycosaminoglycan modifications of betaglycan for shedding and influence on TGF-{beta} signaling responses. Dysregulated shedding of TGF-{beta} receptors plays a vital role in determining the response and availability of TGF-{beta}s, which is crucial for prognostic predictions and understanding of TGF-{beta} signaling dynamics.

biochemistry↗

bioRxiv2023

Establishing Ac/Ds Starter Lines for Large-Scale Transposon-Tagged Mutagenesis in Tomato

Tomato (Solanum lycopersicum), a model system for ripening of fleshy fruits, has [~]40,000 genes predicted by in silico homology-based annotation. However, functional validation is lacking for most annotated tomato genes. Among the strategies for functional annotations, transposon-tagged mutagenesis is the most powerful approach. Transposon- tagged genes can be functionally validated by phenotyping and activation tagging. However, the lack of a robust in planta transformation system precludes large-scale transposon- mutagenesis of tomato. To overcome this limitation, we developed two sets of starter lines in tomato, each carrying maize transposon element (Ds) and transposase (Ac), respectively. The Ds and Ac lines were crossed to allow the Ac-mediated transposition of the Ds in the F1 generation. In the F2 generation, the location of excised Ds at new sites in the tomato genome was monitored. The Ds transposition was interspersed on different chromosomes of the tomato, indicating unlinked transposition of the Ds. The analysis of DNA sequences flanking Ds showed random integration of Ds in intergenic, genes, and the promoter region of the genome. Our study paves the way for the generation of large-scale transposon-tagged tomato lines using Ac/Ds starter lines and provides a potential tool for the functional validation of genes in tomato. Short summaryWe developed two sets of starter lines in tomato, carrying maize transposon element (Ds) and transposase (Ac), to enable large-scale transposon-mutagenesis, facilitating functional validation of tomato genes and for generating an insertional mutant resource in tomato.

plant biology↗

bioRxiv2023

Efficient self-organization of blastoids solely from mouse ESCs is facilitated by transient reactivation of 2C gene network

Human pluripotent stem cells (hPSCs) can self-organize into a blastocyst-like structure (blastoid) by virtue of their full developmental potential. The pluripotent mouse embryonic stem cells (mESC) are considered to lack this potential and hence can form blastoids only when combined with trophoblast stem cells. We performed a small molecule and cytokine screen to demonstrate that mESC have full potential to efficiently self-organize themselves into E-blastoids (ESC-blastoids). The morphology, cell lineages and the transcriptome of these blastoids resemble the mouse blastocyst. The E-blastoids undergo implantation and in utero development in mice. The transient reactivation of the 2C-gene network by retinoid signaling is essential for E-blastoid generation. GSK3{beta} activity is critical for retinoid signaling and consequent 2C gene network activation. Collectively, the mESC possess full developmental potential to generate blastoids similar to hPSCs and other mammals. The plasticity of PSCs to self-organize into blastoids is not exclusive to humans or larger mammals; rather, it could be a general feature shared by most mammals, including rodents.

developmental biology↗