Biology subjects

Singh, M.

Publications and source records attributed to Singh, M..

At least 91 records · Page 5Linked to original sources

bioRxiv2024

Longitudinal Multi-omic Immune Profiling Reveals Age-Related Immune Cell Dynamics in Healthy Adults

The generation and maintenance of protective immunity is a dynamic interplay between host and environment that is impacted by age. Understanding fundamental changes in the healthy immune system that occur over a lifespan is critical in developing interventions for age-related susceptibility to infections and diseases. Here, we use multi-omic profiling (scRNA-seq, proteomics, flow cytometry) to examined human peripheral immunity in over 300 healthy adults, with 96 young and older adults followed over two years with yearly vaccination. The resulting resource includes scRNA-seq datasets of >16 million PBMCs, interrogating 71 immune cell subsets from our new Immune Health Atlas. This study allows unique insights into the composition and transcriptional state of immune cells at homeostasis, with vaccine perturbation, and across age. We find that T cells specifically accumulate age-related transcriptional changes more than other immune cells, independent from inflammation and chronic perturbation. Moreover, impaired memory B cell responses to vaccination are linked to a Th2-like state shift in older adults memory CD4 T cells, revealing possible mechanisms of immune dysregulation during healthy human aging. This extensive resource is provided with a suite of exploration tools at https://apps.allenimmunology.org/aifi/insights/dynamics-imm-health-age/ to enhance data accessibility and further the understanding of immune health across age.

immunology↗

bioRxiv2024

DNMT1-Mediated Regulation of Inhibitory Interneuron Migration Impacts Cortical Architecture and Function

The fine-tuned establishment of neuronal circuits during the formation of the cerebral cortex is pivotal for its functionality. Developmental abnormalities affecting the composition of cortical circuits, which consist of excitatory neurons and inhibitory cortical interneurons (cINs), are linked to a spectrum of neuropsychiatric disorders. Excitatory neurons originate in cortical proliferative zones, while inhibitory interneurons migrate from discrete domains of the basal telencephalon into the cortex. This migration is intricately governed by intrinsic genetic programs and extrinsic cues. Our current study reveals the role of the DNA methyltransferase 1 (DNMT1) in regulating the expression of key genes implicated in mouse cIN development and in guiding the migration of somatostatin (SST)-expressing interneurons at postmitotic level within the developing cortex. Dnmt1 deletion causes SST+ cINs to exit prematurely from the superficial migratory stream. In addition to the perturbed migration pattern and altered gene expression signatures, Dnmt1-deficient SST+ cINs had a discernible non-cell autonomous effect on cortical progenitors, which culminated in nuanced alterations of layer thicknesses in the adult cortex. Our study uncovers that DNMT1 governs the migration of SST+ cINs and through this, their instructive role in sculpting the intricate cortical layer architecture by signaling to cortical progenitors, with pronounced effects on neuronal network function.

neuroscience↗

bioRxiv2024

Commensal Yeast Promotes Salmonella Typhimurium Virulence

Enteric pathogens engage in complex interactions with the host and the resident microbiota to establish gut colonization. Although mechanistic interactions between enteric pathogens and bacterial commensals have been extensively studied, whether and how commensal fungi affect pathogenesis of enteric infections remains largely unknown. Here we show that colonization with the common human gut commensal fungus Candida albicans worsened infections with the enteric pathogen Salmonella enterica serovar Typhimurium. Presence of C. albicans in the mouse gut increased Salmonella cecum colonization and systemic dissemination. We investigated the underlying mechanism and found that Salmonella binds to C. albicans via Type 1 fimbriae and uses its Type 3 Secretion System (T3SS) to deliver effector proteins into C. albicans. A specific effector, SopB, was sufficient to manipulate C. albicans metabolism, triggering increased arginine biosynthesis in C. albicans and the release of millimolar amounts of arginine into the extracellular environment. The released arginine, in turn, induced T3SS expression in Salmonella, increasing its invasion of epithelial cells. C. albicans deficient in arginine production was unable to increase Salmonella virulence in vitro or in vivo. In addition to modulating pathogen invasion, arginine also directly influenced the host response to infection. Arginine-producing C. albicans dampened the inflammatory response during Salmonella infection, whereas C. albicans deficient in arginine production did not. Arginine supplementation in the absence of C. albicans increased the systemic spread of Salmonella and decreased the inflammatory response, phenocopying the presence of C. albicans. In summary, we identified C. albicans colonization as a susceptibility factor for disseminated Salmonella infection, and arginine as a central metabolite in the cross-kingdom interaction between fungi, bacteria, and host.

microbiology↗

bioRxiv2024

Solvatochromic reporter to image plasma membrane order leaflet by leaflet reveals ahighly asymmetric bilayer locally modulated by transbilayer interactions

The plasma membrane (PM) of cells is an asymmetric bilayer composed of a complex mixture of lipids and proteins. The emergent biophysical properties of its leaflets and their consequence on cellular function remain unexplored owing to the limitation in available probes. In this manuscript, we report on the design and characterisation of a PM-localised solvatochromic probe, C3L, based on the established reporter Laurdan. C3L is retained at the inner-leaflet due to flippase and scramblase activity, as determined by Bovine Serum Albumin-based back exchange experiments. By measuring the Generalized Polarization of C3L we are able to determine membrane order, leaflet-by-leaflet at the PM. Our results reveal that the PM is made of a tightly-packed outer/exofacial-leaflet in contrast to a disordered inner/cytoplasmic-leaflet. This differential packing is established by the maintenance of lipid asymmetry, the presence of specific lipids, Sphingomyelin in the outer-leaflet and Phosphatidylserine at the inner-leaflet, and exaggerated during mesenchymal to epithelial transition. We find spatial signatures of local modulation of the inner leaflet by protein scaffolds which promote trans-bilayer coupling of lipids. C3L thus informs on steady state lipid organisation at the asymmetric membrane while allowing further exploration of spatial regulation of lateral heterogeneity at each leaflet.

cell biology↗

bioRxiv2024

Disruption of Immune Responses By Type I Diabetes Exacerbates SARS-CoV-2 Mediated Lung Injury

COVID-19 commonly presents as pneumonia, with those most severely affected progressing to respiratory failure. Patient responses to SARS-CoV-2 infection are varied, with comorbidities acting as major contributors to varied outcomes. Focusing on one such major comorbidity, we assessed whether pharmacological induction of Type I Diabetes Mellitus (T1DM) would increase the severity of lung injury in a murine model of COVID-19 pneumonia utilizing wild type mice infected with mouse-adapted SARS-CoV-2. Hyperglycemic mice exhibited increased weight loss and reduced blood oxygen saturation in comparison to their euglycemic counterparts, suggesting that these animals indeed experienced more severe lung injury. Transcriptomic analysis revealed a significant impairment of the adaptive immune response in the lungs of diabetic mice compared to those of control. In order to expand the limited options available for tissue analysis due to biosafety restrictions, we also employed a novel technique to digest highly fixed tissue into a single cell suspension, which allowed for flow cytometric analysis as well as single cell RNA sequencing. Flow immunophenotyping and scRNA-Seq confirmed impaired recruitment of T cells into the lungs of T1DM animals. Additionally, scRNA-Seq revealed a distinct, highly inflammatory macrophage profile in the diabetic cohort that correlates with the more severe infection these mice experienced clinically, allowing insight into a possible mechanism for this phenomenon. Recognizing the near certainty that respiratory viruses will continue to present significant public health concerns for the foreseeable future, our study provides key insights into how T1DM results in a much more severe infection and identifies possible targets to ameliorate comorbidity-associated severe disease. NEW AND NOTEWORTHYWe define the exacerbating effects of Type I Diabetes Mellitus (T1DM) on COVID-19 pneumonia severity in mice. Hyperglycemic mice experienced increased weight loss and reduced oxygen saturation. Transcriptomic analysis revealed impaired immune responses in diabetic mice, while flow cytometry and single-cell RNA sequencing confirmed reduced T cell recruitment and an inflammatory macrophage profile. Additionally, we introduced a novel technique for tissue analysis, enabling flow cytometric analysis and single-cell RNA sequencing on highly fixed tissue samples.

immunology↗

bioRxiv2024

Synergistic Hypoxia and Apoptosis Conditioning Unleashes Superior Mesenchymal Stem Cells Efficacy in Acute Graft-versus-Host-Disease

Mesenchymal stem cells (MSCs) have emerged as promising candidates for immune modulation in various diseases that are associated with dysregulated immune responses like Graft-versus-Host-Disease (GVHD). MSCs are pleiotropic and the fate of MSCs following administration is a major determinant of their therapeutic efficacy. In this context, we here demonstrate that hypoxia preconditioned apoptotic MSCs [bone marrow (BM), Whartons Jelly (WJ)] bear more immune programming ability in a cellular model of acute Graft-versus-Host-Disease (aGVHD). To this purpose, we programmed MSCs by exposing them to hypoxia and inducing apoptosis both sequentially as well as simultaneously. Our findings demonstrated that WJ MSCs that were conditioned with indicated approaches simultaneously induced the differentiation of CD4+T-cell towards Tregs, enhanced Th2 effector, and concomitantly mitigated Th1 and Th17, with polarization of M1 effector macrophages towards their M2 phenotype, and more interestingly enhanced efferocytosis by macrophages indicated Th2 programming ability of MSCs programmed by conjunctional approaches Overall, our study highlights the potential of WJ-MSCs conditioned with hypoxia and apoptosis concurrently, as a promising therapeutic strategy for aGVHD and underscores the importance of considering MSC apoptosis in optimizing MSCs-based cellular therapy protocols for enhanced therapeutic efficacy in aGvHD. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=123 HEIGHT=200 SRC="FIGDIR/small/588248v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@92c102org.highwire.dtl.DTLVardef@bd895forg.highwire.dtl.DTLVardef@185e041org.highwire.dtl.DTLVardef@45ef0f_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

A benchmarked, high-efficiency prime editing platform for multiplexed dropout screening

Prime editing installs precise edits into the genome with minimal unwanted byproducts, but low and variable editing efficiencies have complicated application of the approach to high-throughput functional genomics. Leveraging several recent advances, we assembled a prime editing platform capable of high-efficiency substitution editing across a set of engineered prime editing guide RNAs (epegRNAs) and corresponding target sequences (80% median intended editing). Then, using a custom library of 240,000 epegRNAs targeting >17,000 codons with 175 different substitution types, we benchmarked our platform for functional interrogation of small substitution variants (1-3 nucleotides) targeted to essential genes. Resulting data identified negative growth phenotypes for nonsense mutations targeted to [~]8,000 codons, and comparing those phenotypes to results from controls demonstrated high specificity. We also observed phenotypes for synonymous mutations that disrupted splice site motifs at 3' exon boundaries. Altogether, we establish and benchmark a high-throughput prime editing approach for functional characterization of genetic variants with simple readouts from multiplexed experiments.

genomics↗

bioRxiv2024

Bridging fungal resistance and plant growth through constitutive overexpression of Thchit42 gene in Pelargonium graveolens

Pelargonium graveolens essential oil possesses significant attributes, known for perfumery and aromatherapy. However, optimal yield and propagation are predominantly hindered by biotic stress. All biotechnological approaches have yet to prove effective in addressing fungal resistance. The current study developed transgenic geranium bridging molecular mechanism of fungal resistance and plant growth by introducing cassette 35S::Thchit42. Furthermore, 120 independently putative transformed explants were regenerated on kanamycin fortified medium. Primarily transgenic lines were demonstrated peak pathogenicity and antifungal activity against formidable Colletotrichum gloeosporioides and Fusarium oxysporum. Additionally, phenotypic analysis revealed [~]2fold increase in leaf size and [~]2.1fold enhanced oil content. To elucidate the molecular mechanisms for genotypic cause, De novo transcriptional profiles were analyzed to indicate that the auxin-regulated longifolia gene is accountable for augmentation in leaf size, and ZF RICESLEEPER attributes growth upregulation. Collectively, data provides valuable insights into unravelling the mechanism of Thchit42-mediated crosstalk between morphological and chemical alteration in transgenic plants. This knowledge might create novel opportunities to cultivate fungal-resistant geranium throughout all seasons to fulfil demand.

plant biology↗

bioRxiv2024

Tunable Macroscopic Alignment of Self-Assembling Peptide Nanofibers

Fibrous proteins that comprise the extracellular matrix (ECM) guide cellular growth and tissue organization. A lack of synthetic strategies able to generate aligned, ECM-mimetic biomaterials has hampered bottom-up tissue engineering of anisotropic tissues and led to a limited understanding of cell-matrix interactions. Here, we present a facile extrusion-based fabrication method to produce anisotropic, nanofibrous hydrogels using self-assembling peptides. The application of shear force coinciding with ion-triggered gelation is used to kinetically trap supramolecular nanofibers into aligned, hierarchical structures. We establish how modest changes in phosphate buffer concentration during peptide self-assembly can be used to tune their alignment and packing. In addition, increases in the nanostructural anisotropy of fabricated hydrogels are found to enhance their strength and stiffness under hydrated conditions. To demonstrate their utility as an ECM-mimetic biomaterial, aligned nanofibrous hydrogels are used to guide directional spreading of multiple cell types, but strikingly, increased matrix alignment is not always correlated with increased cellular alignment. Nanoscale observations reveal differences in cell-matrix interactions between variably aligned scaffolds and implicate the need for mechanical coupling for cells to understand nanofibrous alignment cues. In total, innovations in the supramolecular engineering of self-assembling peptides allow us to generate a gradient of anisotropic nanofibrous hydrogels, which are used to better understand directed cell growth.

bioengineering↗

bioRxiv2024

Towards Equitable MHC Binding Predictions: Computational Strategies to Assess and Reduce Data Bias

Deep learning tools that predict peptide binding by major histocompatibility complex (MHC) proteins play an essential role in developing personalized cancer immunotherapies and vaccines. In order to ensure equitable health outcomes from their application, MHC binding prediction methods must work well across the vast landscape of MHC alleles. Here we show that there are alarming differences across individuals in different racial and ethnic groups in how much binding data are associated with their MHC alleles. We introduce a machine learning framework to assess the impact of this data disparity for predicting binding for any given MHC allele, and apply it to develop a state-of-the-art MHC binding prediction model that additionally provides per-allele performance estimates. We demonstrate that our MHC binding model successfully mitigates much of the data disparities observed across racial groups. To address remaining inequities, we devise an algorithmic strategy for targeted data collection. Our work lays the foundation for further development of equitable MHC binding models for use in personalized immunotherapies.

bioinformatics↗

bioRxiv2024

The Streptococcus phage protein paratox is an intrinsically disordered protein.

The bacteriophage protein paratox blocks quorum sensing in its streptococcal host by directly binding the signal receptor and transcription factor ComR. This reduces ability of Streptococcus to uptake environmental DNA and protects phage DNA from damage by recombination. Past work characterizing the paratox:ComR molecular interaction revealed that paratox adopts a well-ordered globular fold when bound to ComR. However, solution-state biophysical measurements suggested that paratox may be conformationally dynamic. To address this discrepancy, we investigated the stability and dynamic properties of paratox in solution using circular dichroism, nuclear magnetic resonance, and several fluorescence-based protein folding assays. Our work shows that under dilute buffer conditions paratox is intrinsically disordered. We also show that the addition of kosmotropic salts or protein stabilizing osmolytes induces paratox folding. However, only the addition of ComR was able to induce paratox to adopt its previously characterized globular fold. Furthermore, as we can induce different paratox folding-states we characterize Prx folding thermodynamics and folding kinetics using stopped flow measurements. Based upon the kinetic results, paratox is a highly dynamic protein in dilute solution, folding and refolding within the 10 ms timescale. Overall, our results demonstrate that the streptococcal phage protein paratox is an intrinsically disordered protein in a two-state equilibrium with a solute-stabilized folded form. Furthermore, the solute-stabilized paratox fold is likely the predominant form of paratox in a solute-crowded bacterial cell. Finally, our work suggests that Prx binds and inhibits ComR, and thus quorum sensing in Streptococcus, by a combination of conformational selection and induced-fit binding mechanisms.

biochemistry↗

bioRxiv2024

Self-reactive B cells traverse a perfect storm of somatic mutagenesis to cause a virus-induced autoimmune disease

The unexplained association between infection and autoimmune disease is strongest for hepatitis C virus-induced cryoglobulinemic vasculitis (HCV-CV). We traced the evolution of the pathogenic rheumatoid factor (RhF) autoantibodies in four HCV-CV patients by deep single cell multi-omic analysis, revealing three sources of B cell somatic mutation converged to drive accumulation of a large disease causing clone. A sensitive method for quantifying low affinity binding revealed three recurring heavy/light chain combinations created by V(D)J recombination bound self IgG but not viral E2 antigen. Whole genome sequencing revealed accumulation of thousands of somatic mutations, at levels comparable to CLL and normal memory B cells, but with 1-2 corresponding to driver mutations found recurrently in B cell leukemia/lymphoma. V(D)J hypermutation created autoantibodies with compromised solubility. In this virus-induced autoimmune disease, infection promotes a perfect storm of somatic mutagenesis in the descendants of a single B cell.

immunology↗

bioRxiv2023

The molecular basis of sugar detection by an insect taste receptor

Animals crave sugars because of their energy potential and the pleasurable sensation of tasting sweetness. Yet all sugars are not metabolically equivalent, requiring mechanisms to detect and differentiate between chemically similar sweet substances. Insects use a family of ionotropic gustatory receptors to discriminate sugars, each of which is selectively activated by specific sweet molecules. To gain insight into the molecular basis of sugar selectivity, we determined structures of Gr9, a gustatory receptor from the silkworm Bombyx mori (BmGr9), in the absence and presence of its sole activating ligand, D-fructose. These structures, along with structure-guided mutagenesis and functional assays, illustrate how specificity for D-fructose is seemingly achieved by a ligand-binding pocket that precisely matches the overall shape and pattern of chemical groups in D-fructose. However, our computational docking and experimental binding assays revealed that other sugars also bind BmGr9, yet they are unable to activate the receptor. We identified the conformational change required to open the channel gate that provides an additional layer of receptor tuning in BmGr9; only D-fructose can both fit into the pocket and simultaneously engage a bridge of two conserved aromatic residues that connects the pocket to the ion conducting pore. Thus, chemical specificity does not depend solely on the selectivity of the ligand-binding pocket, but it is an emergent property arising from a combination of receptor-ligand interactions and allosteric coupling. Our results support a model whereby coarse receptor tuning is derived from the size and chemical characteristics of the pocket, whereas fine-tuning of receptor activation is achieved through the selective engagement of an allosteric pathway that regulates ion conduction.

neuroscience↗

bioRxiv2023

Transposable elements may enhance antiviral resistance in HIV-1 elite controllers

Less than 0.5% of people living with HIV-1 are elite controllers (ECs) - individuals who have a replication-competent viral reservoir in their CD4+ T cells but maintain undetectable plasma viremia without the help of antiretroviral therapy. While the EC CD4+ T cell transcriptome has been investigated for gene expression signatures associated with disease progression (or, in this case, a lack thereof), the expression and regulatory activity of transposable elements (TEs) in ECs has not been explored. Yet previous studies have established that TEs can directly impact the immune response to pathogens, including HIV-1. Thus, we hypothesize that the regulatory activities of TEs could contribute to the natural resistance of ECs against HIV-1. We perform a TE-centric analysis of previously published multi-omics data derived from EC individuals and other populations. We find that the CD4+ T cell transcriptome and retrotranscriptome of ECs are distinct from healthy controls, treated patients, and viremic progressors. However, there is a substantial level of transcriptomic heterogeneity among ECs. We categorize individuals with distinct chromatin accessibility and expression profiles into four clusters within the EC group, each possessing unique repertoires of TEs and antiviral factors. Notably, several TE families with known immuno-regulatory activity are differentially expressed among ECs. Their transcript levels in ECs positively correlate with their chromatin accessibility and negatively correlate with the expression of their KRAB zinc-finger (KZNF) repressors. This coordinated variation is seen at the level of individual TE loci likely acting or, in some cases, known to act as cis-regulatory elements for nearby genes involved in the immune response and HIV-1 restriction. Based on these results, we propose that the EC phenotype is driven in part by the reduced availability of specific KZNF proteins to repress TE-derived cis-regulatory elements for antiviral genes, thereby heightening their basal level of resistance to HIV-1 infection. Our study reveals considerable heterogeneity in the CD4+ T cell transcriptome of ECs, including variable expression of TEs and their KZNF controllers, that must be taken into consideration to decipher the mechanisms enabling HIV-1 control.

immunology↗

bioRxiv2023

How did UGA codon translation as tryptophan evolve in certain ciliates? A critique of Kachale et al. 2023 Nature

Ciliates are a widespread clade of microbial eukaryotes with the greatest diversity of nuclear genetic codes (at least eight) following a recent addition1. All non-standard ciliate genetic codes involve stop codon reassignments1,2,3. Two of these codes are ambiguous1-3, with "stop" codons either translated or terminating translation depending on their context2,3. Ambiguous genetic codes have arisen not only in ciliates, but also independently in trypanosomatids from the genus Blastocrithidia4 and an alveolate species from the genus Amoebophrya5. Two ambiguous genetic codes in ciliates share translation of UGA "stop" codons as tryptophan with Blastocrithidia and the Amoebophrya species. tRNA genes with complementary anticodons to reassigned UAA and UAG stop codons have invariably been found in ciliate species that translate these codons1,2. Furthermore, though a UGA-cognate tRNACysUCA was reported in Euplotes6, a ciliate genus that translates UGA as cysteine, vexingly, no nuclear genome-encoded tRNATrpUCA has been found in ciliate species with UGA tryptophan codons. Recently, Kachale et al. provided evidence for UGA translation as tryptophan in Blastocrithidia nonstop and the ciliate Condylostoma magnum using 4 base pair anticodon stem (AS) near-cognate tryptophan tRNATrpCCAs, rather than the typical 5 base pair stem tRNAs7. New tRNA data we report from additional ciliates bolsters this hypothesis. Kachale et al. also hypothesised that a particular amino acid substitution in the key stop codon recognition protein, eRF1 (eukaryotic Release Factor 1), favours translation of UGA as tryptophan instead of termination7. Contrary to Kachale et al, we propose such substitutions favouring reduced eRF1 competition enhancing "stop" codon translation do not need to occur concomitantly with tRNA alterations or acquisitions to evolve new genetic codes via stop codon reassignment. We report multiple instances of the substitution investigated in Kachale et al. 2023 that have not led to UGA translation, and multiple ciliate species with UGA tryptophan translation but without the substitution, indicating it is not necessary. Consistent with the ambiguous intermediate hypothesis for genetic code evolution, experimental evidence and our observations suggest continued potential ciliate eRF1-tRNA competition.

evolutionary biology↗

bioRxiv2023

Erythropoietin restrains the inhibitory potential of interneurons in the mouse hippocampus

Erythropoietin (EPO) aids in rectifying hippocampal transcriptional networks and synaptic structures of pyramidal lineages, thereby mitigating mood and cognition-associated disorders. An imminent conundrum is how EPO restores synapses by involving interneurons. By analyzing [~] 12,000 single-nuclei transcriptomic data, we generated a comprehensive molecular atlas of hippocampal interneurons, resolved into 15 interneuron subtypes. Next, we studied molecular alterations upon recombinant human (rh)EPO and saw that gene expression changes relate to synaptic structure, trans-synaptic signaling and intracellular catabolic pathways. Putative ligand-receptor interactions between pyramidal and inhibitory neurons, regulating synaptogenesis, are altered upon rhEPO. An array of in/ex vivo experiments confirms that specific interneuronal populations exhibit reduced dendritic complexity, synaptic connectivity, and changes in plasticity-related molecules. Metabolism and inhibitory potential of interneuron subgroups are compromised, leading to greater excitability of pyramidal neurons. To conclude, improvement by rhEPO of neuropsychiatric phenotypes may partly owe to restrictive control over interneurons, facilitating re-connectivity and synapse development.

neuroscience↗

bioRxiv2023

Conformational dynamics of a nicotinic receptor neurotransmitter binding site

Agonists increase receptor activity by providing net-favorable binding energy to active versus resting conformations of their target sites. We used molecular dynamics (MD) simulations to explore dynamics of the low-to-high affinity conformational change (L[->]H) at the Torpedo -{delta} nicotinic acetylcholine receptor neurotransmitter site, using 4 agonists. Alternative conformations were identified in trajectories generated from a single starting structure by matching approximate binding energies calculated in silico with exact values measured experimentally in vitro. In all simulations, the L[->]H transition started with a rotation of the agonist about its cationic center (flip), followed by a staged downward displacement of loop C (flop) and the formation of a compact, hydrophobic and stable high-affinity pocket (fix). Agonist rotation and a transient intermediate state are only in simulations but can be confirmed or refuted, for example by time-resolved structures.

biophysics↗

bioRxiv2023

Development and Characterization of Co-crystals Assisted with In-silico Screening for Solubility and Permeability Enhancement of Curcumin

Despite being a promising phytochemical, Curcumins potential applications are limited due to its classification in BCS class IV, which is associated with low water solubility and permeability. Enhancing the bioavailability of BCS class IV drugs presents a significant challenge, but crystal chemistry provides a hopeful avenue for overcoming this hurdle. In this research, co-crystals of Curcumin were developed to improve both solubility and permeability. Unlike traditional methods that require extensive trial-based lab work and time-consuming screening of co-formers, the use of molecular docking in In-silico co-former screening offers a scientific and rational approach to selecting suitable partners. In this study, two distinct co-crystals were synthesized using a solvent evaporation technique with methanol as the solvent, employing a 1:1 molar ratio. L-proline and piperine were chosen as co-formers to enhance solubility and permeability, respectively. The co-crystals underwent optimization and characterization through Design of Experiments (DOE). Comparing the dissolution study results for the same curcumin concentration, the cumulative drug release (CDR) after 8 hours was 20% for pure curcumin and an impressive 71% for curcumin-L-proline co-crystals. The permeability study, conducted over four hours using the everted gut sac method in phosphate buffer pH 6.8, revealed curcumins permeability to be less than 0.05 mg/mL, while curcumin-piperine co-crystals exhibited a five-fold increase (0.2545 mg/mL) in permeability. The co-crystals formed through a molecular ratio of 1:1 for curcumin-L-proline to enhance solubility and 1:1 for curcumin-piperine to enhance permeability, both demonstrated positive outcomes with support from optimization analysis, FTIR, DSC, SEM, PXRD analysis, and dissolution studies.

pharmacology and toxicology↗