Biology subjects

Singh, I.

Publications and source records attributed to Singh, I..

At least 19 recordsLinked to original sources

bioRxiv2026

A C-terminal Processing Protease Implicated in Flagellin Turnover and Developmental Progression in a Bacterial Predator

Carboxy-terminal processing proteases (CTPs) are widely conserved bacterial proteases implicated in protein maturation, quality control, and stress responses, yet many family members functions remain unclear. Here, we characterise Bd0967, a previously unstudied CTP from the predatory bacterium Bdellovibrio bacteriovorus, and its role in flagellar function during predatory development. Crystal structures reveal a self-compartmentalised protease in which a PDZ domain forms a lid over a large internal cavity accessed through a proteolytic tunnel. Co-purifying peptides occupied two distinct substrate-binding sites, suggesting a coordinated recognition mechanism. Affinity pulldowns coupled with mass spectrometry identified several Bdellovibrio flagellins as candidate substrates, which were subsequently validated by biochemical assays and shown to be selectively degraded through recognition of a conserved C-terminal motif. A Bd0967-mCherry translational fusion localised to periplasmic foci during the intracellular predatory growth, consistent with flagellar resorption and flagellin turnover following prey invasion. Deletion of bd0967 caused developmental defects, including aberrant Bdellovibrio cell morphology and reduced predation efficiency. Together, these findings establish Bd0967 as a specialised CTP that couples flagellin degradation and developmental progression in a predatory bacterium.

biochemistry↗

bioRxiv2026

A Systematic Approach Toward Implementing Machine Learning Techniques to Analyze Gut Microbiome Data

This study investigates the relationship between the gut microbiota and specific diseases. Data was collected from the Human Gut Microbiome Atlas, which examines regional variations across 20 countries on five continents, categorizing microbial species by taxonomy, from genus to species. The Atlas provides color-coded phylum classifications, numerical species counts within the same genus, and an analysis of dysbiosis-related associations with 23 diseases, as well as region-enriched species. The data stratified samples into distinct categories such as westernized, non-westernized, cancerous, and non-cancerous. The findings demonstrate that tree-based ensemble methods, such as Bagging and Boosting prediction methods, achieved the highest accuracies across all categories due to their robustness in handling the complex, high-dimensional data. The XGBoost model yielded the strongest predictive performance, achieving 91% accuracy for westernized cancer-associated samples, 84% accuracy for non-westernized cancer-associated samples, 92% accuracy for westernized samples, and 78% for non-westernized samples. Additionally, advanced topological data analysis was used to assess the global structure and underlying patterns within the dataset. ImportanceThis research aims to connect gut microbiome composition to diseases using global datasets from the Human Gut Microbiome Atlas. The goal was to evaluate how accurately different machine learning algorithms could classify microbiota species and diseases and predict disease associations by comparing westernized and non-westernized populations, including both cancerous and noncancerous groups. These findings can contribute to the future creation of population-specific and disease-specific microbial models.

bioinformatics↗

bioRxiv2026

Leukemia stem cell expansion cultures reveal clonal drivers of leukemogenesis and therapy response

Leukemia stem cells (LSCs) contain the highest capacity for leukemia-reinitiation and therapy-resistance across all leukemic cells, but our understanding of their molecular and cellular properties remains limited due to their relative rarity and ineffective cell culture systems to maintain their purity at scale. Here, we develop Polymer-based Leukemic STem-cell Cultures (PLSTCs) and demonstrate their capacity to derive and propagate large numbers of Npm1cA/Flt3ITD acute myeloid leukemia (AML) stem cells at high purities. Compared to traditional cultures, PLSTCs show more than 1000-fold enrichment in functional LSCs based on single-cell gene expression signatures and leukemia-initiating assays. Tracing LSC clones with genomic LARRY barcodes during ex vivo expansion, we reveal that PLSTCs can sustain a diversity of self-renewing LSC states with stable, heritable transcriptional programs. Using dynamic state-fate analysis, we characterize clonal programs that are linked with enhanced ex vivo self-renewal, in vivo leukemia initiation, and therapeutic response to induction chemotherapy. LSC clones primed to resist treatment were enriched for a rare cell state that underwent a fate-switch and produced megakaryocytic-erythroid-like leukemic cells that expanded in the spleen. Targeting LSC programs through pooled CRISPR and single-cell sequencing (CROPseq) in PLSTCs, we reveal that chondroitin-sulfate synthesis is required to maintain a primitive LSC state and leukemic recovery from chemotherapy. In sum, our studies showcase the powerful application of scalable leukemic stem-cell expansion cultures and dynamic state-fate analysis of AML LSCs. We anticipate these systems will accelerate our understanding and interception of stem cell plasticity in cancer.

cancer biology↗

bioRxiv2026

Diversified Portfolios of Bacterial Communities in Peri-urban Miyawaki Forests and Grassland Shaping Ecosystem Functions and Services

Soil microflora is fundamental to ecosystem functioning, yet their contribution in Miyawaki afforestation, a globally implemented ecological engineering approach, remains poorly characterized. In the present study, we examined the bacterial taxonomic diversity and their functional potential in a peri-urban Miyawaki mini-forest and compared with a nearby grassland the pre-existing ecosystem across dry and wet seasons. The Miyawaki plantation comprised of highly diverse native trees, sub-trees and shrubs spanning evergreen and deciduous varieties, potentiating nitrogen-fixation, diverse litter generation and rooting strategies resulting in pronounced functional heterogeneity. Notably relative to grassland, the Miyawaki forest was intensively managed and supplemented with organic amendments, and supportive irrigation, buffering the seasonal moisture stress. Using 16S rRNA amplicon sequencing of soil eDNA, we characterized seasonal variation in soil bacterial communities in both the systems. The observed soil bacterial community organization in forest as compared to the unmanaged grassland indicates combined influence of vegetation structure, dense canopy cover, continuous litter generation and root exudates. Microbial assemblages in the forest specialised in heterotrophic complex carbon degradation, biofilm formation, exopolysaccharide production and sporulation pathways which suggests adaptive abilities to anoxic microsites and other stressful conditions. In contrast, grassland soils harboured less diversified bacterial communities dominated by phototrophic and oxidative stress adaptation pathways consistent with sun lit, non-irrigated and moisture-variable conditions. Nonetheless, functional divergence in dry season reflects temporal reorganization of microbial communities marking a gradual trend towards soil ecosystem development. Together, these findings establish microbial baseline for Miyawaki forests revealing how tree-dense mini-forests restructure soil bacterial communities relative to grasslands highlighting the value of identifying soil microbial indicators for critically evaluating urban afforestation outcomes over extended time scales to inform sustainable design and policy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/704982v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@15e1bb1org.highwire.dtl.DTLVardef@16c1dddorg.highwire.dtl.DTLVardef@11c99ecorg.highwire.dtl.DTLVardef@bd6d1d_HPS_FORMAT_FIGEXP M_FIG C_FIG Schematic representation of the study depicting the Miyawaki forest and nearby grassland.

ecology↗

bioRxiv2025

DISCO-seq: 3D single-cell transcriptomics of intact biological systems

Single-cell transcriptomics has transformed tissue analysis, yet current methods struggle to integrate whole-tissue 3D architecture. Conventional techniques restrict molecular profiling to pre-selected 2D sections, losing systemic context and introducing anatomical bias by sampling less than 0.001% of a whole organism. To overcome these challenges, we developed DISCO-seq, a tissue-clearing chemistry that enables superior RNA accessibility compared to fresh or fixed tissues. DISCO-seq integrates whole-organ or organism 3D imaging with both untargeted and targeted transcriptomics, yielding high-quality RNA from cleared tissues comparable to standard samples. We demonstrate its versatility by investigating tumor heterogeneity in a syngeneic glioblastoma mouse model, using 3D imaging to identify spatially distinct microenvironments and characterize their unique transcriptomic signatures. Moreover, DISCO-seq enabled unbiased, whole-body mapping of SARS-CoV-2 S1 protein deposition in mice, followed by transcriptomic profiling of spatially defined niches. By bridgingmesoscale 3D imaging with single-cell transcriptomics, DISCO-seq establishes a paradigm for anatomically contextualized, hypothesis-free tissue interrogation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/693352v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@758ee5org.highwire.dtl.DTLVardef@1f862c6org.highwire.dtl.DTLVardef@1cf3b9org.highwire.dtl.DTLVardef@c5082e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDISCO-seq integrates RNA-preserving tissue-clearing chemistry with whole-organ or organism 3D imaging, enabling anatomically unbiased single-cell transcriptomics. C_LIO_LIDISCO-seq yields RNA quality and transcriptome profiles equivalent to those obtained from matched fresh or fixed tissues. C_LIO_LIDISCO-seq identifies discrete glioblastoma microenvironments and defines their transcriptomic states within the intact brain. C_LIO_LIDISCO-seq enables whole-body mapping of SARS-CoV-2 S1 and uncovers region-specific immune and metabolic responses across anatomical niches. C_LI Supplementary movies can be seen at: http://discotechnologies.org/DISCO-seq/

systems biology↗

bioRxiv2025

Mycoelectronics: Bioprinted Living Fungal Bioelectronics for Artificial Sensation

The intelligence of the human biological system is enabled by the highly distributed sensing receptors on soft skin that can distinguish various stimulations or environmental cues, thus establishing the fundamental logic of sensing and physiological regulation or response. To replicate biological perception, two approaches have emerged: artificial nervous systems that utilize soft electronics as biomimetic receptors to convert external stimuli into frequency-encoded signals, and biohybrid solutions that integrate living cells, plants, or even live animals with electronic components to decode environmental cues for life-like sensations. However, most current biohybrid approaches for artificial sensation are based on eukaryotic cells, which suffer from slow growth, stringent culture conditions, environmental susceptibility, and short lifespans, thus limiting their integration into practical wearables or robotic sensory skins. Here, we introduce fungi-based printable "Mycoelectronics", which are created by additive bioprinting of living fungal mycelium networks onto stretchable electronics, as a practical living thermo-responsive sensory platform. This Mycoelectronics approach leverages fungis capacity for rapid biological responsiveness, cultivability with exponential growth, stability and self-healing in ambient conditions, bioprintability for scalable manufacturing, and mechanical flexibility for seamless integration with soft electronics. Critically, we discovered that the thermal responsiveness of the fungal network arises from intrinsic cellular processes--specifically, heat-induced vacuole remodeling and fusion, which modulate ionic transport and thus the electrical conductivity of the mycelial cells and networks, enabling a rapid temperature response. By bridging the gap between cell biology and soft electronics, the Mycoelectronics device with a living mycelium network functions as a thermal sensation system with rapid response and intrinsic self-healing properties, autonomously restoring sensing capabilities after damage or autonomously establishing sensor pathways in hard-to-reach locations. Furthermore, by integrating fungal thermal sensing with electronic circuits, we established a hybrid bioelectronic reflex arc that can actuate muscles and initiate diverse actions, suggesting promising applications in future neurorobotics and neuroprosthetics.

bioengineering↗

bioRxiv2025

Pre-Omicron Immunity Generates IgG⁺ but Not IgA⁺ Memory B Cells Reactive to Omicron Spike Protein

The emergence of the Omicron variant marked a major antigenic shift from previous SARS-CoV-2 variants. While the emergence of new variants of concern was associated with loss of protection from infection, protection from serious disease was maintained. What was less clear was whether this protection was associated with expansion, affinity maturation of pre-existing cross-reactive B cells, or de novo generation of variant-specific B cells. To define the cross-reactivity of memory B cell populations generated through infection and/or vaccination with pre-Omicron variant spike, we utilised archived samples from healthcare workers enrolled in the extensively monitored Panther study cohort. Using samples collected from Omicron-naive individuals approximately three months (106 days) after a third BNT162b2 vaccine dose, we analyzed the spike-specific B cell repertoire via multiparameter flow cytometry and B cell receptor (BCR) sequencing. Reactivity to both Lineage A and Omicron spike proteins was higher for serum IgG than IgA, and serum neutralisation potency was significantly reduced against pseudoviruses bearing the Omicron spike compared to those with Lineage A spike. The frequency of Omicron-reactive IgA+ B cells was markedly lower than that of Lineage A-reactive cells, whereas Omicron-reactive IgG+ memory B cells were more frequent. These responses were predominantly localized to classical memory and double-negative (DN) B cell subsets. BCR sequencing of donors unexposed to Omicron confirmed that Omicron-binding clones were class-switched and somatically mutated, indicating they originated from pre-existing memory B cells primed by vaccination or infection. Our findings demonstrate that memory B cells generated through infection and/or immunisation with pre-Omicron variants were primed for broad antigenic recognition despite Omicrons extensive genetic divergence. However, the diminished IgA+ B cell frequency suggests a weakened mucosal antibody defence, potentially explaining the continued susceptibility to infection despite preserved protection from severe disease.

immunology↗

bioRxiv2025

Understanding proneural-mesenchymal transition using patient-derived glioma stem-like cell (GSC) organoids and engineered extracellular matrix

Glioblastoma multiforme (GBM) is a highly aggressive, angiogenic WHO grade IV glioma marked by rapid progression, therapeutic resistance, and poor prognosis. A defining feature of GBM is the presence of glioma stem-like cells (GSCs), which reside in specialized perivascular niches and drive tumor progression, recurrence, and therapeutic resistance. The blood-brain barrier, coupled with the complex and dynamic tumor microenvironment, poses significant challenges for both treatment and mechanistic investigation. Current in vitro GBM models inadequately recapitulate the structural and biochemical cues of the native perivascular niche due to the absence of functional vasculature and brain-mimetic extracellular matrix (ECM), limiting their physiological relevance and predictive power. To address the limitations of existing in vitro GBM models, we developed a patient-derived glioma stem cells (GSC) derived Matrigel spheroid system that transitions into organoids and enables integration into engineered microenvironments. Our model incorporates GSC organoids representing proneural and mesenchymal GBM subtypes, a synthetic engineered extracellular matrix (eECM), and endothelial cells (ECs) seeded on the matrix surface. We evaluated the expression of subtype-specific, pro-angiogenic, stemness, and differentiation markers under increasingly complex co-culture conditions. Our results show that Matrigel-derived GSC spheroids progressively differentiate into organoids over two weeks, with significantly enhanced expression of cell-specific markers in the presence of ECs. Encapsulation of these organoids within eECM, combined with EC co-culture, further promoted cellular invasion and induction of GBM associated genes. This in situ encapsulation strategy enables real-time observation of GSC behavior in a tunable microenvironment that mimics key features of the native tumor niche. Together, this platform provides a physiologically relevant and modular in vitro system for investigating GBM pathophysiology. It holds promise for uncovering tumor-specific cellular dependencies, studying GSC-vascular interactions, and conducting high-throughput drug screening under controlled, biomimetic conditions. SignificanceThis study establishes a 3D GSC organoid model with engineered matrix to investigate glioblastoma plasticity and vascular mimicry.

cancer biology↗

bioRxiv2025

An in vivo parallelized reporter assay to uncover tissue-specific splicing regulatory sequences in a multicellular animal

Introns play a critical role in regulating alternative splicing. However, identifying functional intronic motifs is challenging due to their short and degenerate sequence composition. Massively parallel reporter assays have provided insights into cis-regulatory logic governing alternative splicing, but these approaches are generally performed in cell culture, limiting their ability to capture tissue-specific contexts. Here, we implemented in vivo Parallelized Reporter Assays in C. elegans neurons and muscle cells to screen for intronic enhancer and silencer motifs among thousands of randomized sequences. We identified nearly 200 sequences regulating splicing in these tissues. We uncovered core sub-sequences with tissue-biased enhancing and silencing activity, including motifs recognized by well-characterized RNA-binding proteins, and orphan motifs with no obvious cognate binding protein. Mapping our PRA-derived motifs to native introns flanking tissue-biased alternative exons revealed their conservation across nematodes, supporting their functional relevance. Additionally, individual intronic regions frequently contained diverse combinations of these motifs, indicative of complex engagement of these sequences by RNA-binding proteins. Finally, we performed targeted mutagenesis of PRA-derived intronic enhancers flanking a neuronal microexon, identifying key cis-regulatory determinants of microexon splicing. Together, our study provides a framework to explore the role of intronic regions in tissue-specific splicing regulation within a multicellular organism. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/670696v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@84d5daorg.highwire.dtl.DTLVardef@f3f625org.highwire.dtl.DTLVardef@a53739org.highwire.dtl.DTLVardef@ccbb03_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Proximal Pulmonary Artery Stiffening as a Biomarker of Cardiopulmonary Aging

The geroscience hypothesis suggests that understanding underlying ageing mechanisms will enable us to delay aging and lessen age-related disability and diseases. While hallmarks of ageing list multiple contributing factors, role of mechanics has only been recently recognized and increasingly appreciated. Here, we use mouse models of ageing to investigate changes in mechanics of the proximal pulmonary artery, lung and right ventricle function in ageing. We found an age-related decline in the capacity to store energy and increased circumferential stiffness of the proximal pulmonary artery with age that associated with a reorientation of collagen towards the circumferential direction, decreased exercise ability, and decreased function of the lung and right ventricle. The observed compromised mechanics in proximal pulmonary artery is consistent across multiple mouse models of accelerated ageing. Further, transcriptional changes in proximal pulmonary artery indicate that aging is associated with senescence of perivascular macrophages, adventitial fibroblasts, and medial smooth muscle cells. Older pulmonary arteries increase expression of genes associated with ECM turnover (including genes in the TGF{beta} pathway) and increased intercellular signaling amongst perivascular macrophages, fibroblasts and smooth muscle cells. Our results provide promising biomarkers of ageing for diagnosis and potential pathways and molecular targets for targeting anti-ageing therapies.

physiology↗

bioRxiv2025

Hypoxia-Induced Cardiopulmonary Remodeling and Recovery: Critical Roles of the Proximal Pulmonary Artery, Macrophages, and Exercise

Hypoxemia impairs cardiopulmonary function. We investigated pulmonary artery remodeling in mice exposed to chronic hypoxia for up to five weeks and quantified associated changes in cardiac and lung function, without or with subsequent normoxic recovery in the absence or presence of exercise or pharmacological intervention. Hypoxia-induced stiffening of the proximal pulmonary artery stemmed primarily from remodeling of the adventitial collagen, which resulted in part from altered inter-cellular signaling associated with phenotypic changes in the mural smooth muscle cells and macrophages. Such stiffening appeared to precede and associate with both right ventricular and lung dysfunction, with changes emerging to similar degrees regardless of the age of onset of hypoxia during postnatal development. Key homeostatic target values of the wall mechanics were recovered by the pulmonary arteries with normoxic recovery while other values recovered only partially. Overall cardiopulmonary dysfunction due to hypoxia was similarly only partially reversible. Remodeling of the cardiopulmonary system due to hypoxia is a complex, multi-scale process that involves maladaptations of the proximal pulmonary artery.

physiology↗

bioRxiv2025

Igf2 regulates early postnatal DPP4+ preadipocyte pool expansion

Adipose tissue is rapidly expanding early in life. Identifying the queues facilitating this process will advance our understanding of metabolically healthy obesity. Single-cell RNA sequencing revealed compositional differences between pre-wean and adult murine subcutaneous adipose tissue. We identify a dipeptidyl peptidase-4 (Dpp4) positive precursor population in pre-wean mice residing at the reticular interstitium of subcutaneous adipose tissue expressing insulin growth factor 2 (Igf2). We show that IGF2 drives proliferation rather than differentiation in these cells. Moreover, loss of Igf2 in Dpp4+ progenitor cells promotes adipogenesis. We show that expression of Igf2 at a limited timeframe during development promotes preadipocyte expansion.

developmental biology↗

bioRxiv2025

BTK-NLRP3-Caspase1 and Gasdermin D-Caspase 11 pathways jointly drive neuroinflammation and functional impairments in a rat model of psychosocial stress

Despite serious health and economic burden, borne more prominently by lower and lower middle-income countries, pharmacotherapy for common psychiatric disorders rely on drugs altering neurotransmission, with partial efficacies. The persistence of a chronic yet sub-threshold inflammation across the periphery and the brain is well documented in the patients and animal models of these disorders and can be investigated for augmenting pharmacotherapy. IL1{beta}, a pleiotropic cytokine and a key regulator of neuroinflammation has been of particular interest in this regard. Previous studies on rodent models of anxiogenic stress show activation of a large multiprotein complex - the NLRP3 inflammasome, which increases IL1{beta} production facilitated by caspase 1, through what is considered a canonical inflammasome activation pathway. However, there exists a second non canonical inflammasome activation pathway whose impact on IL1{beta} release, inflammation and behavioral consequences remain unclear. Using rat models of physical and psychosocial stress, we observed stress-induced sex-specific upregulation of activated caspase 11 and gasdermin D N-terminal fragments, the mediators of the non-canonical inflammasome pathway that facilitates IL1{beta} release through pore formations in the plasma membrane. This is the first report of non-canonical inflammasome pathway being activated in the brain and peripheral immune cells in response to psychosocial stress. Inhibition of caspase 11 with wedelolactone, or Gasdermin D cleavage with Disulfiram reduced stress-induced elevation of IL1{beta} levels, anxiety and fear acquisition, facilitated fear extinction and recall, and improved working memory. Combination treatments targeting both canonical (ibrutinib for pBTK/NLRP3 or MCC950 for NLRP3 inhibition) and non-canonical (wedelolactone for caspase 11 or disulfiram for gasdermin D) pathways proved more efficacious in reducing stress-mediated neuroinflammation, dendritic spine elimination in the CA3 region of the hippocampus and behavioral dysfunction. Furthermore, psychosocial stress drove peripheral inflammation, in peripheral blood mononuclear cells, which was mitigated by the combination treatment. Taken together, this study reveals a novel mechanism underlying psychosocial stress involving both canonical and non-canonical inflammasome signaling to facilitate IL1{beta} induction and behavioral changes. Our finding further suggests that combined targeting of the NLRP3 inflammasome and gasdermin D could contribute to the development of future transdiagnostic therapeutic targets for stress, anxiety, and depression.

neuroscience↗

bioRxiv2024

CCT-217: A Landmark Gene Silencing Therapeutic for Weight Loss That Enhances Lean Mass Composition, Reduces Visceral, Subcutaneous Fat and Restores Metabolic Health in Obesity through Browning

According to the CDC, between 2017 and March 2020, 41.9% of U.S. adults aged 20 and older had obesity, with 9.2% experiencing severe obesity, affecting over 100 million and 22 million adults, respectively. Obesity prevalence increased from 30.5% in 1999-2000 to 41.9% in 2017-2020, while severe obesity rose from 4.7% to 9.2%. As per CDC projection, by 2030, nearly half of the US adults will be obese, with a quarter of them being severely obese. Current therapeutic approaches predominantly focus on appetite suppression and caloric restriction. This approach results in both fat mass and lean mass loss and thus compromises long term metabolic health. Preserving lean mass during weight loss is crucial. Remodeling of the white fat into beige fat through the process called browning offers a promising alternative to appetite suppression dependent weight loss, and complements with reduction of visceral fat, marrow adipose tissue, improve liver steatosis, enhancing insulin sensitivity, reduced cardiovascular risk and suppression of inflammation. Here, we present CCT-217, a groundbreaking siRNA therapy targeting CB1R and ZFP423 genes, with strong adipose tissue-specific tropism with nil brain and liver penetration in diet-induced obese (DIO) C57BL/6J male mice model (n=7 control, n=5 treated). Mice received 7 subcutaneous doses over 20 days (initial half-dose, followed by once after three days CB1R siRNA 2.0 mg/kg and ZFP423 siRNA 1.8 mg/kg dose). CCT-217 induced browning of the WAT and resulted in a significant 26% reduction in body weight with a modest 12% decrease in feed intake after the 4th dose, preserved normal clinical behavior and nutrition. Lean mass composition significantly improved to 79% versus 62% in controls, while total fat mass decreased by 43.5%. Notably, visceral fat depots, including retroperitoneal (59.4%), mesenteric (54.3%), and gonadal (42.0%) fat showed substantial reductions, alongside a reduction of 61.7% inguinal white adipose tissue (iWAT). Histological analysis revealed extensive browning of iWAT, reduced adipocyte size, and increased adipocyte count, indicating enhanced thermogenesis and adipose tissue remodeling. Liver pathology showed significant improvement, with reduced hepatocellular adipose vacuolation and the absence of fibrosis, inflammation, or macrophage infiltration, highlighting the hepatoprotective effects of CCT-217. Metabolic markers demonstrated a 20% reduction in plasma cholesterol and decreased systemic inflammation (21% reduction in CRP), accompanied by significantly improved leptin sensitivity and favorable trends in insulin sensitivity. Mechanistically, CCT-217 achieved 42-fold silencing of CB1R and 14.5-fold silencing of ZFP423 specifically in iWAT, with no off-target effects observed in liver or brain. In conclusion, CCT-217 emerges as a landmark therapy that effectively reduces visceral fat, enhances lean mass composition, and restores metabolic health through precise, tissue-specific gene silencing. These findings position CCT-217 as a transformative and highly promising next generation therapeutic for addressing obesity and its associated complications.

pharmacology and toxicology↗

bioRxiv2024

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

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

cell biology↗

bioRxiv2024

Farmyard Mulching in Regenerative Agriculture Enhances Saprotrophs and Concomitantly Reduces Pathogenic Fungal Genera

Regenerative agriculture (RA) using nature-friendly methods nurtures soil microbial communities. Indian RA farmers use diverse practices to manage their soil. This study compared the fungal communities in RA plots with those in conventional agriculture (CA) and barren land (BL) plots (comprising completely barren-BL and with Eucalyptus - BL-Euc). Two crops were considered - Finger millets and Vegetables (Tomato/ beans) for this study. ITS metagenomic analysis of soil DNA samples obtained from RA, CA and BL plots was done to identify fungal composition in each of the study plots. The fungal communities in RA finger millet and RA vegetable were compared with respective CA finger millet and CA vegetable and with BL plots. Vegetable RA plots observed higher abundances of fungal Operational Taxonomic Units (OTUs) than in CA vegetable and BL plots. Whereas the RA finger millet plots had similar fungal OTUs as in CA finger millet and BL plots. The vegetable RA plot carrying out natural farming for 12 years (maximum length in our samples) recorded the highest fungal OTU (13707) levels while the CA plots had average OTU abundance of (7416). RA plots in both crops showed a significant reduction in plant pathogenic fungal genuses - Bipolaris and Pyrenochaetopsis. Furthermore, RA finger millet plots showed an enhanced representation of saprotrophs while CA (finger millets) had pathotroph-saprotrophs suggesting a favorable increase in decomposer populations in RA.

ecology↗

bioRxiv2024

Pre-existing stem cell heterogeneity dictates clonal responses to acquisition of cancer driver mutations

Cancer cells display wide phenotypic variation even across patients with the same mutations. Differences in the cell of origin provide a potential explanation, but these assays have traditionally relied on surface markers, lacking the clonal resolution to distinguish heterogeneous subsets of stem and progenitor cells. To address this challenge, we developed STRACK, an unbiased framework to longitudinally trace clonal gene expression and expansion dynamics before and after acquisition of cancer mutations. We studied two different leukemia driver mutations, Dnmt3a-R882H and Npm1cA, and found that the response to both mutations was highly variable across different stem cell states. Specifically, a subset of differentiation-biased stem cells, which normally become outcompeted with time, can efficiently expand with both mutations. Npm1c mutations surprisingly reversed the intrinsic bias of the clone-of-origin, with stem-biased clones giving rise to more mature malignant states. We propose a clonal "reaction norm", in which pre-existing clonal states dictate different cancer phenotypic potential. Highlights- Single cell tracing of cancer initiation at the clonal level (STRACK). - Ex vivo expansion cultures sustain intrinsic and heritable HSC heterogeneity. - Premalignant mutations enhance the self-renewal of high-output stem cells, increasing their survival probability. - Transforming mutations reprogram low-output stem cell fates to more mature malignant states.

cancer biology↗

bioRxiv2024

Somatic epimutations enable single-cell lineage tracing in native hematopoiesis across the murine and human lifespan

Current approaches to lineage tracing of stem cell clones require genetic engineering or rely on sparse somatic DNA variants, which are difficult to capture at single-cell resolution. Here, we show that targeted single-cell measurements of DNA methylation at single-CpG resolution deliver joint information about cellular differentiation state and clonal identities. We develop EPI-clone, a droplet-based method for transgene-free lineage tracing, and apply it to study hematopoiesis, capturing hundreds of clonal trajectories across almost 100,000 single-cells. Using ground-truth genetic barcodes, we demonstrate that EPI-clone accurately identifies clonal lineages throughout hematopoietic differentiation. Applied to unperturbed hematopoiesis, we describe an overall decline of clonal complexity during murine ageing and the expansion of rare low-output stem cell clones. In aged human donors, we identified expanded hematopoietic clones with and without genetic lesions, and various degrees of clonal complexity. Taken together, EPI-clone enables accurate and transgene-free single-cell lineage tracing at scale.

genomics↗