Biology subjects

Singhal, M.

Publications and source records attributed to Singhal, M..

6 recordsLinked to original sources

bioRxiv2026

Endothelin-1 signaling regulates chamber-specific mouse atrial cardiomyocyte cytokinesis and polyploidy

Cardiomyocyte (CM) maturation is a central process in postnatal heart development accompanied by profound structural, metabolic, and cell cycle changes. One hallmark of this maturation program is CM polyploidy that is closely associated with the loss of cardiac regenerative capacity. Most insights into ploidy regulation have come from studies of ventricular CMs, whereas the spatiotemporal dynamics and molecular regulation of atrial CM polyploidy remain poorly understood. We show that CM polyploidy in the postnatal mouse heart is highly chamber-specific, with >90% of ventricular CMs are polyploid, compared with [~]30% of left atrial (LA) CMs and [~]15% of right atrial (RA) CMs. These chamber-specific differences correlate with their differential susceptibility to cytokinesis failure and are regulated, at least in part, by endocardial cells. Mechanistically, we identify the endocardial/endothelial-derived factor EDN1 as a postnatally enriched signal in the LA compared with the RA. EDN1 can act directly on primary aCMs to inhibit cytokinesis, in part by suppressing Wnt signaling. Consistently, inhibition of Edn1 signaling in vivo using Bosentan reduced CM cytokinesis failure specifically in the LA. Altogether, our findings reveal a previously unrecognized role for endothelial-myocardial crosstalk in regulating chamber-specific CM polyploidy through Edn1 signaling.

developmental biology↗

bioRxiv2026

Enriched-GF: A Reproducible High-Yield Autologous Blood-Derived Growth Factor Method for Regenerative Medicine

BackgroundPlatelet-derived Growth factors play key roles in tissue repair and regeneration, yet conventional platelet-rich plasma (PRP) formulations release these mediators inconsistently in vivo due to variability in platelet yield and activation dynamics. To overcome this limitation, direct administration of concentrated platelet-derived growth factor preparations has gained interest, though current manufacturing approaches for human platelet lysate (hPL), growth factor concentrates (GFC), and conditioned serum remain constrained by batch variability, incomplete platelet degranulation, and reliance on anticoagulants. Here, we examine alternative platelet activation workflows to establish a standardized, efficient, and reproducible method for high-yield growth factor recovery suitable for translational and clinical applications. MethodsNine GFC production protocols were compared, employing different combinations of freeze-thaw (FT) cycling, glass bead (GB) agitation, calcium (Ca2) activation, and a novel Enriched Growth Factor (Enriched-GF) method. The objective was to identify a protocol capable of maximizing growth factor yield within a three-hour workflow. Optimal Ca2 concentrations and GB conditions were determined from prior optimization studies and integrated into the Enriched-GF processing scheme. Platelet concentrates (n = 10 per protocol) were processed under each condition, and growth factor levels were quantified using ELISA. ResultsGrowth factor yields differed significantly across protocols. The greatest and most consistent increases in growth factor release were observed with the Enriched-GF method combining GB activation, FT cycling, and Ca2 stimulation. This approach resulted in markedly elevated concentrations of key regenerative mediators, including enhanced EGF release, a 4.5-fold increase in PDGF, maximal TGF-{beta} liberation, and a four-fold increase in FGF2 relative to conventional platelet lysate or conditioned serum preparations. These results were reproducible across independent donor pools, demonstrating robustness and batch-to-batch consistency. ConclusionWe describe a rapid and reproducible method for producing highly concentrated platelet-derived growth factors using a combined GB-FT-Ca2 activation strategy. The Enriched-GF protocol consistently outperformed existing platelet lysate, conditioned serum, and conventional GFC preparation methods, yielding a standardized product with enhanced growth factor content. This Enriched-GF approach offers a clinically practicable solution for applications in regenerative medicine requiring reliable and high-yield growth factor delivery. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/712883v2_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@ac60c3org.highwire.dtl.DTLVardef@104d8eborg.highwire.dtl.DTLVardef@13d5afcorg.highwire.dtl.DTLVardef@120645c_HPS_FORMAT_FIGEXP M_FIG C_FIG Schematic overview of platelet concentrate preparation from whole blood and the generation of different platelet lysates and growth factor-enriched serum using freeze-thaw, calcium gluconate, and glass bead activation methods.

biochemistry↗

bioRxiv2024

A perfusion-independent high-throughput method to isolate liver sinusoidal endothelial cells

Liver sinusoidal endothelial cells (LSECs) critically regulate homeostatic liver function and liver pathogenesis. However, the isolation of LSECs remains a major technological bottleneck in studying molecular mechanisms governing LSEC functions. Current techniques to isolate LSECs, relying on perfusion-dependent liver digestion, are cumbersome with limited throughput. We here describe a perfusion-independent high-throughput procedure to isolate LSECs with high purity. Indifferently from previous perfusion-independent approaches, chopped liver tissue was incubated in the digestion mix for 30 minutes with intermittent mixing with a serological pipette. This led to the safeguarding of LSEC integrity and yielded 10 {+/-} 1.0 million LSECs per adult mouse liver, which is far higher than previous perfusion-independent protocols and comparable yield to established perfusion-dependent protocols for isolating LSECs. Combining magnetic and fluorescence-activated cell sorting (FACS), LSECs from different zones of the hepatic sinusoid can now be isolated in high numbers in less than two hours for downstream applications including proteomics. Our protocol enabled the isolation of LSECs from fibrotic liver tissues from mice and healthy liver tissues from higher vertebrate species (pigs), where traditional perfusion-based digestion protocols have very limited application. In conclusion, these technical advancements reduce post-mortem changes in the LSEC state and aid in reliable investigation of LSEC functions.

physiology↗

bioRxiv2023

Statistical Optimization of Hydrazone-Crosslinked Hyaluronic Acid Hydrogels for Protein Delivery

Hydrazone-crosslinked hydrogels are attractive protein delivery vehicles for regenerative medicine. However, each regenerative medicine application requires unique hydrogel properties to achieve an ideal outcome. The properties of a hydrogel can be impacted by numerous factors involved in its fabrication. We used design of experiments (DoE) statistical modeling to efficiently optimize the physicochemical properties of a hyaluronic acid (HA) hydrazone-crosslinked hydrogel for protein delivery for bone regeneration. We modified HA with either adipic acid dihydrazide (HA-ADH) or aldehyde (HA-Ox) functional groups and used DoE to evaluate the interactions of three input variables, the molecular weight of HA (40 or 100 kDa), the concentration of HA-ADH (1-3% w/v), and the concentration of HA-Ox (1-3% w/v), on three output responses, gelation time, compressive modulus, and hydrogel stability over time. We identified 100 kDa HA-ADH3.0HA-Ox2.33 as an optimal hydrogel that met all of our design criteria, including displaying a gelation time of 3.7 minutes, compressive modulus of 62.1 Pa, and minimal mass change over 28 days. For protein delivery, we conjugated affinity proteins called affibodies that were specific to the osteogenic protein bone morphogenetic protein-2 (BMP-2) to HA hydrogels and demonstrated that our platform could control the release of BMP-2 over 28 days. Ultimately, our approach demonstrates the utility of DoE for optimizing hydrazone-crosslinked HA hydrogels for protein delivery. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/549125v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@bd1a4org.highwire.dtl.DTLVardef@1f0766borg.highwire.dtl.DTLVardef@1eea01corg.highwire.dtl.DTLVardef@151bd6b_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Evolution of chemosensory and detoxification gene families across herbivorous Drosophilidae

Herbivorous insects are exceptionally diverse, accounting for a quarter of all known eukaryotic species, but the genetic basis of adaptations that enabled this dietary transition remains poorly understood. Many studies have suggested that expansions and contractions of chemosensory and detoxification gene families - genes directly mediating interactions with plant chemical defenses - underlie successful plant colonization. However, this hypothesis has been challenging to test because the origins of herbivory in many lineages are ancient (>150 million years ago [mya]), obscuring genomic evolutionary patterns. Here, we characterized chemosensory and detoxification gene family evolution across Scaptomyza, a genus nested within Drosophila that includes a recently derived (<15 mya) herbivore lineage of mustard (Brassicales) specialists and carnation (Caryophyllaceae) specialists, and several non-herbivorous species. Comparative genomic analyses revealed that herbivorous Scaptomyza have among the smallest chemosensory and detoxification gene repertoires across 12 drosophilid species surveyed. Rates of gene turnover averaged across the herbivore clade were significantly higher than background rates in over half of the surveyed gene families. However, gene turnover was more limited along the ancestral herbivore branch, with only gustatory receptors and odorant binding proteins experiencing strong losses. The genes most significantly impacted by gene loss, duplication, or changes in selective constraint were those involved in detecting compounds associated with feeding on plants (bitter or electrophilic phytotoxins) or their ancestral diet (yeast and fruit volatiles). These results provide insight into the molecular and evolutionary mechanisms of plant-feeding adaptations and highlight strong gene candidates that have also been linked to other dietary transitions in Drosophila.

evolutionary biology↗

bioRxiv2022

Insights into the evolution of herbivory from a leaf-mining, drosophilid fly

Herbivorous insects and their host plants comprise most known species on Earth. Illuminating how herbivory repeatedly evolved in insects from non-herbivorous lineages is critical to understanding how this biodiversity is created and maintained. We characterized the trophic niche of Scaptomyza flava, a representative of a lineage nested within the Drosophila that transitioned to herbivory [~]15 million years ago. We used natural history studies to determine if S. flava is a true herbivore or a cryptic microbe-feeder. Specifically, we quantified oviposition substrate choice and larval viability across food-types, trophic-related morphological traits, and nitrogen isotope and sterol profiles across putatively herbivorous and non-herbivorous drosophilids. We confirmed that S. flava is an obligate herbivore of living plants. Paired with its genetic model host, Arabidopsis thaliana, S. flava is a novel and powerful system for exploring mechanisms underlying the evolution of herbivory, a complex trait that enabled the exceptional diversification of insects.

ecology↗