Biology subjects

Patil, S. B.

Publications and source records attributed to Patil, S. B..

5 recordsLinked to original sources

bioRxiv2026

A robust, reproducible, accessible and scalable protocol for generating three-dimensional human gastruloids

The rapid rise of stem cell-based human embryo models has reignited interest in studying early human development while offering a promising platform to de-risk drugs. Among these, three-dimensional human gastruloids provide a tractable system to model symmetry breaking, germ layer specification and axial organization. However, existing gastruloid protocols remain expensive, specialized, variable and evaluated in a limited number of human pluripotent stem cell (hPSC) lines, restricting broader adoption. Here, we present a simple, robust, standardized gastruloid protocol achieving greater than 90% elongation efficiency with low inter- and intra-experimental variability, developed primarily in BJNhem20, a well-characterized Indian-origin human embryonic stem cell line. Further, we show that the protocol is applicable in a diverse set of hPSC lines. Using a TBXT (Brachyury)-GFP reporter in BJNhem20, we optimized cell seeding density, induction medium and Wnt activation strength, guided by real-time, quantitative assessment of mesoderm induction and symmetry breaking, allowing precise titration of CHIR99021. Comparative testing identified an in-house "Essential 6" medium formulation as the most consistent condition for robust TBXT induction. Optimization of aggregation density produced reproducible gastruloids with polarized TBXT expression and consistent axial elongation, within 72 hours. Single-cell RNA sequencing of individual gastruloids confirmed high transcriptional reproducibility and conserved lineage clusters, aligned with developmental trajectories. Cell line-specific CHIR99021 titration was sufficient to successfully transfer the optimized protocol to two additional lines, BJNhem19 and RUES2-GLR. This simplified and robust protocol reduces costs and improves accessibility, enabling broader application of stem cell-based human embryo models.

developmental biology↗

bioRxiv2025

Deciphering Membrane Pore Formation Mechanisms of Plasmodium falciparum Perforin-Like Protein 1 (PfPLP1)

Membrane Attack Complex/Perforin (MACPF) domain proteins are {beta}-pore forming toxins ({beta}-PFTs) involved in the pathogenesis of various organisms. Among them, Perforin-like proteins (PLPs), produced by Plasmodium species, play essential roles in parasite invasion and egress. Due to increasing drug resistance in Plasmodium, PLPs represent promising but underexplored therapeutic targets, largely due to the lack of structural and mechanistic data. This study investigates the binding and pore formation mechanism of the Plasmodium falciparum PLP1 (PfPLP1), which is expressed during the human life cycle of the parasite. We modeled PfPLP1 structure and performed both all-atom and coarse-grained molecular dynamics simulations in soluble and membrane-associated states. PfPLP1 comprises two domains, a canonical MACPF domain and a {beta}-pleated sheet domain-apicomplexan perforin {beta}-domain (APC{beta}). Initial membrane binding is mediated by cationic residues at the base of the APC{beta} domain, which interact with the polar headgroups of the lipids from the host cell membrane. We analyzed the membrane-inserted tetrameric form where water molecules were observed to penetrate between the tetramer and the lipid bilayer, initiating pore opening. During this process, lipids reorganize into a toroidal edge to shield their hydrophobic tails, while water mixes with lipid headgroups in a disordered, heterogeneous fashion. Larger oligomeric assemblies show lateral displacement of lipids and a clear tendency to form pore-like structures. This study provides molecular insights into PfPLP1s membrane binding and pore-forming behavior in both monomeric and oligomeric forms. The outcome of this study would be applicable in understanding pore formation mechanism in other PLPs and similar toxins. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/682298v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@181ad3borg.highwire.dtl.DTLVardef@940471org.highwire.dtl.DTLVardef@1af9c23org.highwire.dtl.DTLVardef@33fd45_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIStructure of Plasmodium falciparum PLP 1 (PfPLP1) as soluble form and membrane-inserted form. C_LIO_LIPfPLP1 initiates pore formation by binding to the host cell membrane with the help of the APC-{beta} domain through cationic residues. C_LIO_LISmaller oligomers of membrane-inserted PfPLP1 form an arc-like structure by reorganizing membrane lipids into a toroidal edge. C_LIO_LIDecamer and higher oligomers of membrane-inserted PfPLP1 form a pore-like structure by lateral displacement of the membrane lipids. C_LI

biochemistry↗

bioRxiv2022

Multibody kinematic optimisation vs body fat: A performance analysis

We have analysed the performance of mulitbody kinematic optimisation methods in reducing soft tissue artefacts for subject data of varying body fat percentages. Multibody kinematic optimisation methods are a critical aspect of movement analysis using musculoskeletal modelling software. By minimising soft tissue artefacts, they help in achieving higher fidelity joint kinematics and dynamics analyses. Prior studies have not examined the performance of multibody kinematic optimisation on subjects of varying body fat percentages. Herein, we: 1) have analysed the efficacy of three different multibody kinematic optimisation methods on varying body fat percentages, 2) implemented a novel weighting scheme to reduce error irrespective of body fat percentages. Residual error using gait data of 50 participants of varying body fat percentages was calculated through inverse kinematic analysis using OpenSim(c) musculoskeletal modelling software. The analysis was repeated using a time-based weighting scheme. The residual error of participants with higher body fat percentages was greater by 30% when compared to residual error of participants of lower body fat percentages. Additionally, time-based weighting scheme reduced residual error by 20% on average compared to constant-value weighting scheme. Our results indicate that multibody kinematic optimisation methods are adversely affected by higher body fat percentages and that time-based weighting can provide higher fidelity movement analysis irrespective of body fat percentages. Through our results we aim to develop tools which provide greater precision in obesity-related movement analysis. Such tools could also help address the disparities in rates of obesity associated with different ethnic or socioeconomic background.

bioengineering↗

bioRxiv2020

Sucrose represses the expression of the strigolactone signalling gene D3/RMS4/MAX2 to promote tillering

- Shoot branching, a major component of shoot architecture, is regulated by multiple signals. Previous studies have indicated that sucrose may promote branching through suppressing the inhibitory effect of the hormone strigolactone (SL). However, the molecular mechanisms underlying this effect are unknown. - Here we used molecular and genetic tools to identify the molecular targets underlying the antagonistic interaction between sucrose and SL. - We showed that sucrose antagonises the suppressive action of SL on tillering in rice and on the degradation of D53, a major target of SL signalling. Sucrose inhibits the expression of D3, the orthologue of the arabidopsis F-box protein MAX2 required for SL signalling. Over-expression of D3 prevents sucrose from inhibiting D53 degradation and enabled the SL inhibition of tillering under high sucrose. Sucrose also prevents SL-induced degradation of D14, the SL receptor involved in D53 degradation. Interestingly, D14 over-expression enhances D53 protein levels and sucrose-induced tillering. - Our results show that sucrose inhibits SL perception by targeting key components of SL signalling and, together with previous studies reporting the inhibition of SL synthesis by nitrate and phosphate, demonstrate the central role played by strigolactones in the regulation of plant architecture by nutrients.

plant biology↗

bioRxiv2020

Structural insights into loss of function of a pore forming toxin and its role in pneumococcal adaptation to an intracellular lifestyle

The opportunistic pathogen Streptococcus pneumoniae has dual lifestyles: one of an asymptomatic colonizer in the human nasopharynx and the other of a deadly pathogen invading sterile host compartments. The latter triggers an overwhelming inflammatory response, partly driven via pore forming activity of the cholesterol dependent cytolysin (CDC), pneumolysin. Although pneumolysin-induced inflammation drives person-to-person transmission from nasopharynx, the primary reservoir for pneumococcus, it also contributes to high mortality rates, creating a bottleneck that hampers widespread bacterial dissemination, thus acting as a double-edged sword. Serotype 1 ST306, a widespread pneumococcal clone, harbours a non-hemolytic variant of pneumolysin (Ply-NH). Performing crystal structure analysis of Ply-NH, we identified Y150H and T172I as key substitutions responsible for loss of its pore forming activity. We uncovered a novel inter-molecular cation-{pi} interaction, governing formation of the transmembrane {beta}-hairpins (TMH) in the pore state of Ply, which can be extended to other CDCs. H150 in Ply-NH disrupts this interaction, while I172 provides structural rigidity to domain-3, through hydrophobic interactions, inhibiting TMH formation. Loss of pore forming activity enabled improved cellular invasion and autophagy evasion, promoting an atypical intracellular lifestyle for pneumococcus, a finding that was corroborated in in vivo infection models. Attenuation of inflammatory responses and tissue damage promoted tolerance of Ply-NH-expressing pneumococcus in the lower respiratory tract. Adoption of this altered lifestyle may be necessary for ST306 due to its limited nasopharyngeal carriage, with loss of pore forming ability of Ply facilitating a benign association of SPN in an alternative, intracellular host niche. AUTHOR SUMMARYStreptococcus pneumoniae, the main causative agent of pneumonia, triggers inflammation and tissue damage by expressing a pore-forming toxin, pneumolysin (Ply). Ply-induced inflammation drives pneumococcal transmission from nasopharynx (its primary reservoir), but also contributes to host mortality, limiting its occupiable habitats. Here, we uncovered the structural basis for loss of pore-forming activity of a Ply variant, present in Serotype 1 ST306, and observed that this enabled adoption of an intracellular lifestyle, attenuating inflammatory responses and prolonging host tolerance of pneumococcus in the lower airways. This commensal-like lifestyle, resembling that of members of the mitis group of Streptococci, might have evolved within ST306 by loss of function ply mutations, compensating for limited nasopharyngeal carriage capacity by facilitating adaptation to an alternate niche.

microbiology↗