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Patra, A.

Publications and source records attributed to Patra, A..

10 recordsLinked to original sources

Model Validation Protocols for Machine Learning in Small Molecule Drug Discovery

Machine learning (ML) models for molecular property prediction are increasingly deployed in drug discovery, yet their adoption in real-world scenarios requires an understanding of the conditions in which a model succeeds or fails. While standardized benchmarks are powerful instruments to measure and unlock progress in ML research, they should not be blindly treated as the end goal. Especially static and retrospective benchmarks, in which no true unknown test set is employed, limit our ability to robustly validate a model's performance. Building on the collective expertise of a cross-industry consortium, we present a model validation framework consisting of five recommendations that would enable the community to move beyond aggregate metrics toward understanding where and why molecular property prediction models fail. We connect evaluation choices to real-world applications and case studies encountered in pharmaceutical research. The framework proposes splitting strategies that mimic realistic distribution shifts and expose common failure modes. We apply the recommended framework to a recently released dataset of absorption, distribution, metabolism, and excretion (ADME) properties. Across two complementary model algorithms, our case studies reveal four distinct failure modes (extrapolation, interpolation, representation, and evaluation), showing that model errors arise not only from distribution shift but also from limitations in molecular representations. Our results show that commonly used evaluation protocols can significantly overestimate performance and may not detect important model failure modes. All software and data are released via https://github.com/srijitseal/polaris.

bioinformatics↗

QuickStainer: a rapid negative staining device for improved preservation of molecular structure

Negative staining is a widely used technique for observing macromolecules and their assemblies by transmission electron microscopy. It is commonly employed to optimize specimens for cryo-EM. The stain, typically a uranyl salt, surrounds the structure, providing an outline view at about 20 [A] resolution. Many macromolecules are relatively stable and rigid, and negative stain images provide a good representation of their structure. However, some are labile or flexible and their structure or assembly state is altered by binding to the carbon substrate on the grid before specimen staining. In these cases, the negatively stained appearance does not faithfully represent the structure in solution. This problem is reduced when samples are incubated on the carbon surface for short times (5 s) rather than typical times (30-60 s) before staining. To reduce disruption to a minimum, we have developed a rapid negative staining device (QuickStainer) using 3D-printed components, a stepper motor for precisely timed movements, and an Arduino-controlled interface to execute commands. QuickStainer produces consistent, reproducible results, achieving sample incubation times as low as 10 ms before staining. Tests show rapid adherence of molecules to the grid and greatly improved structural preservation of labile specimens compared with standard preparation protocols. The design of QuickStainer can accommodate inclusion of additional steps, such as timed incubation with enzyme substrate, before staining.

biophysics↗

Dengue virus suppression by 25-hydroxycholesterol is mediated by a combination of membrane fusion inhibition, innate immune activation and cholesterol biosynthesis dysregulation

Physicochemical properties and composition of cellular membranes are crucial for regulating broad cellular responses including signaling and defense against pathogens. Dengue virus (DENV) exploits cholesterol-rich membranes and host lipid pathways, such as cholesterol biosynthesis, lipid raft organization, and lipid droplet formation, for entry, replication, and assembly. Additionally, lipid-based plasma membrane signaling can trigger innate immune responses that attenuate viral growth, underscoring the dual role of lipids in facilitating and restricting DENV infection. Here, we demonstrate that 25-hydroxycholesterol (25-HC), an oxidized cholesterol metabolite, inhibits DENV infection through a multifaceted mechanism. 25-HC disrupts viral membrane fusion by altering cholesterol distribution and lipid raft organization, impairing the binding and fusion of the DENV envelope (E) protein with host membranes. Additionally, 25-HC modulates host cholesterol metabolism by suppressing biosynthesis pathways essential for viral replication while enhancing lipid droplet formation and stress-response pathways. Transcriptomic analyses reveal that 25-HC primes innate immune responses, activating proinflammatory pathways such as the NLRP3 inflammasome and MAPK signaling, while selectively modulating interferon-stimulated gene expression. Notably, 25-HC exhibits synergistic antiviral effects when combined with direct-acting antivirals like Remdesivir, underscoring its potential in combination therapies. These findings establish 25-HC as a promising candidate for host-directed antiviral strategies against DENV and other enveloped viruses.

cell biology↗

Snake venom-inspired novel peptides protect Caenorhabditis elegans against paraquat induced Parkinson's pathology

The in vivo protective mechanisms of two low molecular mass ([~]1.4 kDa) novel custom peptides (CPs) against paraquat (PT)-induced neurodegenerative dysfunction in the Caenorhabditis elegans model were deciphered. CPs prevent the PT binding to the nerve ring adjacent to the pharynx in C. elegans (N2 strain) by stable and high-affinity binding to the tyrosine-protein kinase receptor CAM-1, resulting in significant inhibition of PT-induced toxicity by reducing enhanced reactive oxygen species production, mitochondrial membrane depolarization, and chemosensory dysfunction. The CPs inhibited PT-induced dopaminergic (DAergic) neuron degeneration and alpha-synuclein aggregation, the hallmarks of Parkinsons Disease, in transgenic BZ555 and NL5901 strains of C. elegans. The transcriptomic, functional proteomics, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses show that CPs prevented the increased expression of the genes involved in the skn-1 downstream pathway, thereby restoring PT-mediated oxidative stress, apoptosis, and neuronal damage in C. elegans. The CPs ability to repair PT-induced damage was demonstrated by a network of gene expression profiles illustrating the molecular relationships between the regulatory proteins. Further, CPs (10 mg/kg, parental route) did not show toxicity or induce inflammatory mediators in the mouse model.

neuroscience↗

Rapid, high-resolution, non-destructive assessments of metabolic and morphological homogeneity uniquely identify high-grade cervical precancerous lesions

PurposeTwo-photon microscopy (2PM) is an emerging clinical imaging modality with the potential to non-invasively assess tissue metabolism and morphology in high-resolution. This study aimed to assess the translational potential of 2PM for improved detection of high-grade cervical precancerous lesions. Experimental Design2P images attributed to reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and oxidized flavoproteins (FP) were acquired from the full epithelial thickness of freshly excised human cervical tissue biopsies (N = 62). Fifteen biopsies harbored high-grade squamous intraepithelial lesions (HSILs), 14 biopsies harbored low-grade SILs (LSILs), and 33 biopsies were benign. Quadratic discriminant analysis (QDA) leveraged morphological and metabolic functional metrics extracted from these images to predict the presence of HSILs. We performed gene set enrichment analysis (GSEA) using datasets available on the Gene Expression Omnibus (GEO) to validate the presence of metabolic reprogramming in HSILs. ResultsIntegrating metabolic and morphological 2P-derived metrics from finely sampled, full-thickness epithelia achieved a high 90.8 {+/-} 6.1% sensitivity and 72.3 {+/-} 11.3% specificity of HSIL detection. Notably, sensitivity (91.4 {+/-} 12.0%) and specificity (77.5 {+/-} 12.6%) were maintained when utilizing metrics from only two images at 12- and 72-{micro}m from the tissue surface. Upregulation of glycolysis, fatty acid metabolism, and oxidative phosphorylation in HSIL tissues validated the metabolic reprogramming captured by 2P biomarkers. ConclusionLabel-free 2P images from as few as two epithelial depths enable rapid and robust HSIL detection through the quantitative characterization of metabolic and morphological reprogramming, underscoring the potential of this tool for clinical evaluation of cervical precancers. Translational Relevance StatementThe colposcopy and biopsy paradigm for cervical pre-cancer detection leads to an excessive number of unnecessary biopsies, with significant economic and psychological costs. This study highlights the potential of label-free, high-resolution two photon imaging to improve this paradigm by introducing real-time morphofunctional tissue assessments. In an extensive dataset comprising freshly excised high-grade and low-grade cervical intraepithelial lesions, along with benign metaplastic and inflamed human cervical tissue biopsies, we successfully characterize a loss of morphofunctional heterogeneity indicative of high-grade precancerous changes. Leveraging a combination of two-photon imaging-derived quantitative morphofunctional metrics, our findings showcase a substantial improvement in both sensitivity and specificity of high-grade lesion detection compared to the current gold standard of colposcopy followed by a single biopsy. The demonstrated enhancement in sensitivity and specificity highlights the prospect of integrating non-invasive, label-free metabolic imaging into clinical practice, offering a more effective and efficient approach to identify and manage cervical precancerous lesions.

bioengineering↗

Deep Learning-Enabled, Detection of Rare Circulating Tumor Cell Clusters in Whole Blood Using Label-free, Flow Cytometry

Metastatic tumors have poor prognoses for progression-free and overall survival for all cancer patients. Rare circulating tumor cells (CTCs) and rarer circulating tumor cell clusters (CTCCs) are potential biomarkers of metastatic growth, with CTCCs representing an increased risk factor for metastasis. Current detection platforms are optimized for ex vivo detection of CTCs only. Microfluidic chips and size exclusion methods have been proposed for CTCC detection; however, they lack in vivo utility and real-time monitoring capability. Confocal backscatter and fluorescence flow cytometry (BSFC) has been used for label-free detection of CTCCs in whole blood based on machine learning (ML) enabled peak classification. Here, we expand to a deep-learning (DL) -based, peak detection and classification model to detect CTCCs in whole blood data. We demonstrate that DL-based BSFC has a low false alarm rate of 0.78 events/min with a high Pearson correlation coefficient of 0.943 between detected events and expected events. DL-based BSFC of whole blood maintains a detection purity of 72% and a sensitivity of 35.3% for both homotypic and heterotypic CTCCs starting at a minimum size of two cells. We also demonstrate through artificial spiking studies that DL-based BSFC is sensitive to changes in the number of CTCCs present in the samples and does not add variability in detection beyond the expected variability from Poisson statistics. The performance established by DL-based BSFC motivates its use for in vivo detection of CTCCs. Further developments of label-free BSFC to enhance throughput could lead to critical applications in the clinical detection of CTCCs and ex vivo isolation of CTCC from whole blood with minimal disruption and processing steps.

bioengineering↗

Restoration of metabolic functional metrics from label-free, two-photon cervical tissue images using multiscale deep-learning-based denoising algorithms

Label-free, two-photon imaging captures morphological and functional metabolic tissue changes and enables enhanced understanding of numerous diseases. However, this modality suffers from low signal arising from limitations imposed by the maximum permissible dose of illumination and the need for rapid image acquisition to avoid motion artifacts. Recently, deep learning methods have been developed to facilitate the extraction of quantitative information from such images. Here, we employ deep neural architectures in the synthesis of a multiscale denoising algorithm optimized for restoring metrics of metabolic activity from low-SNR, two-photon images. Two-photon excited fluorescence (TPEF) images of reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavoproteins (FAD) from freshly excised human cervical tissues are used. We assess the impact of the specific denoising model, loss function, data transformation, and training dataset on established metrics of image restoration when comparing denoised single frame images with corresponding six frame averages, considered as the ground truth. We further assess the restoration accuracy of six metrics of metabolic function from the denoised images relative to ground truth images. Using a novel algorithm based on deep denoising in the wavelet transform domain, we demonstrate optimal recovery of metabolic function metrics. Our results highlight the promise of denoising algorithms to recover diagnostically useful information from low SNR label-free two-photon images and their potential importance in the clinical translation of such imaging.

bioengineering↗

Deciphering therapeutic efficacy of mycosynthesized silver nanoparticles using entomopathogenic fungi Metarhizium anisopliae against MCF-7 breast cancer cells in vitro

Green synthesis of silver nanoparticles has gained much interest over few decades in the field of biomedical research due to their ease of synthesis, cost effectiveness, enhanced bioactivity and biocompatibility compared to the chemical synthesis. Recent studies on silver nanoparticles have shown their potential in various fields like antimicrobial, anticancer, larvicidal, catalytic, and wound healing properties. In the present study, entomopathogenic fungi Metarhizium anisopliae was used to synthesize silver nanoparticles. These silver nanoparticles were synthesized and characterized using UV-Vis spectroscopy, FESEM, FETEM and FTIR. Compared to the chemically synthesized silver nanoparticles, the mycosynthesized silver nanoparticles (MaAgNP) showed high yield. The size of mycosynthesized silver nanoparticles was found to be 5-20 nm and was spherical in shape. FTIR results confirmed the possible functional groups that are responsible for the reduction of silver ions. The mycosynthesized silver nanoparticles showed cytotoxicity on human breast cancer cell line (MCF-7) and the calculated IC50 value for MaAgNP was found to be 16.50 g ml-1 whereas the chemically synthesized silver nanoparticles showed cytotoxicity but with increasing concentration, no further significant reduction in cell viability was observed. The possible reason behind improved cytotoxicity of MaAgNP can be the presence of extracellular secondary metabolites present in the fungal filtrate used to synthesize the nanoparticles. The MaAgNP was also observed to induce cell death through reactive oxygen species (ROS) generation.

cancer biology↗

Antioxidant enriched fraction from Pueraria tuberosa alleviates ovariectomized-induced osteoporosis in rats, and inhibits growth of breast and ovarian cancer cell lines in vitro

Pueraria tuberosa (P. tuberosa), known as Indian Kudzu belongs to family Fabaceae and it is solicited as "Rasayana" drugs in Ayurveda. In the present study, we analyzed the efficacy an antioxidant enriched fraction (AEF) from the tuber extract of P. tuberosa against menopausal osteoporosis and breast and ovarian cancer cell lines. The AEF from P. tuberosa was identified by determining phenolic composition (total phenolic and flavonoid amount). Antioxidant property (in vitro assays) was also carried out followed by analysis of the AEF for its antiosteoporotic and anticancer potentials. Antiosteoporotic activity of AEF was investigated in ovariectomy-induced osteoporosis in rats and in vitro anticancer activity by MTT assay. Also, the GC/MS analysis of AEF was performed to determine various phytoconstituents. A docking analysis was performed to verify the interaction of bioactive molecules with estrogen receptors (ERs). Ethyl acetate fraction of the mother extract was proved as the AEF. AEF significantly improved various biomechanical and biochemical parameters in a dose dependent manner in the ovariectomized animals. AEF also controlled the increased body weight and decreased uterus weight following ovariectomy. Histopathology of femur revealed the restoration of typical bone structure and trabecular width in ovariectomized animals after AEF and raloxifene treatment. AEF also exhibited in vitro cytotoxicity in breast (MCF-7 and MDA-MB-231) and ovarian (SKOV-3) cancer cells. Further, genistein and daidzein exhibited a high affinity towards both estrogen receptors ( and {beta}) in docking study revealing the probable mechanism of the antiosteoporotic activity. GC/MS analysis confirmed the presence of bioactive molecules such as stigmasterol, {beta}-sitosterol, and stigmasta-3,5-dien-7-one. The observations of this study vindicate the potency of AEF from P. tuberosa in the treatment of menopausal osteoporosis and cancer.

pharmacology and toxicology↗

Whole Cell Phenotypic Screening Of MMV Pathogen Box identifies Specific Inhibitors of Plasmodium falciparum merozoite maturation and egress.

We report a systematic, cellular phenotype-based antimalarial screening of the MMV Pathogen Box collection, which facilitated the identification of specific blockers of late stage intraerythrocytic Plasmodium falciparum maturation. First, from standard growth inhibition asays, we discovered 62 additional antimalarials (EC50 [≤] 10M) over previously known antimalarial candidates from Pathogen Box. A total of 90 potent molecules (EC50 [≤] 1M) were selected for evaluating their stage-specific effects during the intra-erythrocytic development of P. falciparum. None of these molecules had significant effect on ring-trophozoite transition, 10 molecules inhibited trophozoite-schizont transition, and 21 molecules inhibited schizont-ring transition at 1M. These compounds were further validated in secondary assays by flow cytometry and microscopic imaging of treated cells to prioritize 12 molecules as potent and selective blockers of schizont-ring transition. Seven of these were found to strongly inhibit calcium ionophore induced egress of Toxoplasma gondii, a related apicomplexan parasite, suggesting that the inhibitors may be acting via similar mechanism in the two parasites, which can be further exploited for target identification studies. Two of these molecules, with previously unknown mechanism of action, MMV020670 and MMV026356, were found to induce fragmentation of DNA in developing merozoites. Further mechanistic studies would facilitate therapeutic exploitation of these molecules as broadly active inhibitors targeting development and egress of apicomplexan parasites relevant to human health.

microbiology↗