Biology subjects

Agarwal, N.

Publications and source records attributed to Agarwal, N..

At least 19 recordsLinked to original sources

bioRxiv2026

SWI/SNF Alterations Define a Chromatin-Dependent Subtype of Urothelial Carcinoma

PurposeSWI/SNF (BAF) chromatin remodeling complex alterations are common in urothelial carcinoma, yet no biomarker-directed therapeutic strategies have been established for this population. We investigated whether BAF alterations delineate a biologically distinct, therapeutically actionable urothelial carcinoma subtype. Experimental DesignWe performed integrative genomic and transcriptomic analyses of 792 urothelial carcinoma tumors from the Oncology Research Information Exchange Network (ORIEN) and validated findings in the TCGA-BLCA cohort. Mechanistic studies incorporated RNA sequencing and ATAC-seq following histone deacetylase (HDAC) inhibition. Functional dependencies were assessed using patient-derived xenograft organoids and cell line models. Clinical relevance was explored in a biomarker-enriched investigator-initiated trial. ResultsApproximately half of urothelial carcinoma tumors exhibited BAF alterations, defining a previously unrecognized chromatin-altered molecular subtype characterized by activation of proliferative programs, loss of lineage identity, and altered metabolic signaling. This subtype was enriched for transcriptomic programs associated with HDAC inhibitor sensitivity and depleted of HDAC inhibitor resistance signatures. Mechanistically, HDAC inhibition induced widespread chromatin remodeling with reduced accessibility at AP-1 and TEAD-associated regions, and downregulation of E2F- and MYC-driven transcriptional networks. Functional studies confirmed enhanced HDAC inhibition sensitivity in ARID1A-mutated cell lines and a patient-derived organoid model. Early clinical observations demonstrated a durable responder treated with HDAC inhibitors and immunotherapy. ConclusionsBAF alterations define a chromatin-dependent tumor state in urothelial carcinoma that is selectively vulnerable to HDAC inhibition. Integrating genomic, epigenomic, functional, and early clinical evidence, these findings provide a rationale for biomarker-enriched clinical trials and HDAC inhibitor-based combination strategies in urothelial carcinoma.

cancer biology↗

bioRxiv2026

Defining biologically directed therapy target signatures in urothelial carcinoma: A transcriptomic framework for precision therapy

IntroductionNectin-4 targeting antibody-drug conjugate (ADC) enfortumab vedotin (EV), in combination with pembrolizumab, is the first-line treatment for patients with locally advanced or metastatic urothelial carcinoma (UC). Optimal treatment strategies for patients who are non-responders or progress on EV with pembrolizumab remain an unmet clinical need. We sought to characterize ADC and immunotherapy (IO)-associated target expression profiles to identify candidate therapeutic vulnerabilities beyond EV. MethodsWe conducted a literature review to identify ADC and IO targets with approved or investigational relevance in UC. Unsupervised hierarchical clustering was used to identify clusters of target gene expression in RNA-seq data. Transcriptomic clustering analyses were performed in 434 patients from The Cancer Genome Atlas Bladder Urothelial Carcinoma cohort (TCGA-BLCA) and validated in an independent cohort of 478 patients from the Oncology Research Information Exchange Network (ORIEN) consortium. Proteomic interrogation of these targets was performed using mass spectrometry data from additional cohort of 116 patients. Differential gene expression analyses evaluated associations between target expression patterns, histologic variants, and consensus molecular subtypes of muscle-invasive bladder cancer (CMIBC). ResultsWe identified 13 ADC and 10 IO-associated targets with translational relevance in UC. Transcriptomic analyses revealed three reproducible clusters of overexpressed target genes across independent cohorts: 1) a luminal/epithelial-associated cluster enriched for VTCN1, SLITRK6, FGFR3, NECTIN4, TACSTD2, ERBB2, and ERBB3; 2) an immune target predominant cluster enriched for BTLA, LAG3, PDCD1, TIGIT, CTLA4, TNFRSF9, TNFRSF18, TNFRSF4; and 3) a basal/neuroendocrine-associated cluster characterized by CD274, F3, NT5E, EGFR, MET and DLL3. Similar clusters were largely conserved at the proteomic level. Adenocarcinomas overexpressed ERBB3 compared to neuroendocrine and squamous cell carcinomas. Pure squamous cell carcinomas overexpressed TACSTD2 compared to adenocarcinomas. In CMIBC subtypes, basal/squamous tumors expressed higher levels of CD274, EGFR, F3, LAG3, NT5E, and TNFRSF18, whereas luminal tumors demonstrated higher ERBB2 and ERBB3 expression. Neuroendocrine-like tumors showed higher DLL3 expression compared to all other subtypes. Tumors with low expression of NECTIN4, TACSTD2, and FGFR3 were enriched for alternative targets including DLL3, CD274, and CD276. Our findings provide a framework for hypothesis-driven therapeutic prioritization in advanced UC. Conclusions: UC is characterized by reproducible, biologically distinct patterns of ADC and IO target expressions. The degree of expression of NECTIN4 was positively associated with TACSTD2, FGFR3 and inversely associated with DLL3, CD276, and CD274, supporting alternative biologically informed treatment strategies besides EV . Histologic variants and molecular subtypes of UC also display distinct patterns of target expression. This study provides the first integrated transcriptomic framework linking ADC and IO target co-expression patterns for hypothesis-driven therapeutic prioritization. These findings provide a basis for rational ADC and immunotherapy development in advanced UC and support prospective proteomic validation in treatment stratified cohorts. Statement of Translational RelevanceEnfortumab vedotin plus pembrolizumab has redefined first-line therapy for advanced urothelial carcinoma, yet treatment selection following resistance or progression remains undefined. In this study, we integrate transcriptomic and proteomic analyses across independent cohorts to define reproducible patterns of antibody-drug conjugate (ADC) and immunotherapy target co-expression in urothelial carcinoma. We identify biologically distinct target-expression patterns that are associated with histologic and molecular subtypes and demonstrate coordinated and, in some cases, mutually exclusive relationships among therapeutically actionable targets. These findings have direct translational implications. First, they provide biologic rationale for rational sequencing and combination strategies based on co-expressed targets in NECTIN4-enriched tumors. Second, they identify alternative therapeutic vulnerabilities, including DLL3- and CD274-associated pathways, in tumors with low NECTIN4 expression, a population potentially enriched for resistance to EV-based therapy. Finally, this framework establishes a foundation for biomarker-driven clinical trials in urothelial carcinoma and supports the development of precision therapeutic approaches beyond current standards.

cancer biology↗

bioRxiv2025

LoAR-Exo and HiAR-Exo: One-step label-free isolation of extracellular vesicles using inertial microfluidic devices

Extracellular vesicles (EVs) are lipid membrane-bound nanoscale (20 nm - 1000 nm) objects shed by all cells. As EVs play an important role in intercellular signaling, these have emerged as promising biomarkers for many diseases including cancer and neurodegenerative disorders. A major bottleneck in research into EVs and their smooth translation to clinic as disease biomarkers is a lack of access to affordable and user-friendly technologies to quickly isolate EVs from complex biological and clinical samples with acceptable purity and yield. Here, we report two different designs of inertial microfluidic devices (e.g. LoAR-Exo and HiAR-Exo) that can isolate EVs from different kinds of biological samples in a single label-free step using a spiral microchannel design with different aspect ratios. LoAR-Exo has a height-to-width aspect ratio [~] 1, while HiAR-Exo has a height-to-width aspect ratio of 2.5, optimized by COMSOL simulations. We tested both designs by separating polystyrene particles of size < 1 m from a heterogeneous mixture. We then benchmarked the performance of the microfluidic chips against ultracentrifugation and a precipitation kit by isolating EVs from the cell culture-conditioned media (CCM) of MDA-MB-231 cells. We also compared the microchip with SEC by using the CCM from H1975/OR cells. The size distribution of EVs isolated by the microfluidic chip was comparable with ultracentrifugation, precipitation and SEC. In summary, both devices isolated EVs from as little as 1 mL of sample volume using a label-free technique in a continuous manner and without any user intervention. Both microfluidic platforms offer a simple, efficient, and scalable alternative to conventional methods for EV isolation.

bioengineering↗

bioRxiv2025

ER oxidoreductin-1α and unfolded protein response as sex-dependent drivers of cardiorenal dysfunction in experimental autoimmune encephalomyelitis

BackgroundMultiple sclerosis (MS) is associated with increased cardiovascular and renal morbidity, but mechanisms linking CNS autoimmunity to peripheral organ injury remain poorly defined. We tested the hypothesis that experimental autoimmune encephalomyelitis (EAE) induces cardiorenal dysfunction via sex-specific dysregulation of endoplasmic reticulum (ER) oxidoreductases and unfolded protein response (UPR) signaling. MethodsAdult female and male C57BL/6J mice [10-12 weeks] and IRE1C148S knock-in mice underwent non-pertussis toxin EAE (nPTX-EAE) induced with 100{micro}g MOG35-55 in CFA and 200{micro}g heat-inactivated MTB with a booster at 7 days. Controls received all components except MOG. Motor scoring was done daily and EN460 (ERO1 inhibitor, 10 mg/kg IP) was given twice weekly beginning at 10DPI. Echocardiography and renal Doppler (Vevo 2100) were performed at 36-38DPI with primary outcomes of LV systolic/diastolic function and renal perfusion with tissue collected at 40DPI. LV and kidney were analyzed via western blot for ERO1, PDIA1, Prdx4, 4HNE, and BiP. Data are expressed as mean {+/-} SEM with two-way ANOVA/Tukeys post hoc or Mann Whitney test performed and outliers identified by ROUT (Q=1%). ResultsSixty-seven percent of immunized mice developed motor deficits. At 36-38DPI, EAE reduced ejection fraction and fractional shortening while increasing IVCT, IVRT, and myocardial performance index without hypertrophy. Renal resistive index increased and end-diastolic velocity decreased in addition to reduced Bowmans space at 40DPI. Females showed upregulated LV ERO1 and 4HNE while males exhibited reduced PDI and Prdx4 paired with elevated BiP. EN460 attenuated cardiac and renal dysfunction and lowered LV ERO1/4HNE in females, but not males. IRE1C148S mitigated cardiac dysfunction in males and restore renal indices in both sexes. ConclusionsnPTX-EAE causes clinically relevant, sex-specific cardiorenal dysfunction linked to distinct ER stress/UPR alterations. ERO1 inhibition protects females, whereas enhanced IRE1 activity protects males and kidneys across sexes, supporting sex-specific ER stress-targeted therapies for MS-associated cardiorenal disease.

molecular biology↗

bioRxiv2025

Sec and Tat mediated secretion safeguards Mycobacterium tuberculosis membrane homeostasis

Protein secretion drives Mycobacterium tuberculosis (Mtb) physiology and pathogenesis, yet a unified picture of the machinery and its role in cell membrane homeostasis is still lacking. By comprehensively curating published evidence, we assembled a systems-level map of Mtb secretion encompassing 92 components and 198 mechanistic reactions across Sec, Tat, and ESX pathways. The secretory components identified were integrated with high-throughput ChIP-Seq and transcriptome datasets to elucidate the regulation of the secretion system. Using CRISPRi, conditional depletion of SecA1 or TatA impaired growth in vitro and survival ex vivo. Quantitative secretome revealed decreased export of SecA1- and TatA-dependent substrates, with enrichment of cytosolic proteins in culture filtrates, indicating increased membrane permeability. Membrane proteomics showed depletion dependent increased metabolic/lipid-degrading proteins and decreased cell-wall/cell-process proteins, consistent with loss of membrane stability. Ultrastructural defects and increased ethidium bromide uptake confirmed impaired membrane integrity. Together, our multi-omics and functional genetics established SecA1 and TatA as essential guardians of Mtb membrane integrity which provided valuable datasets and a framework for secretion-dependent Mtb pathogenesis.

microbiology↗

bioRxiv2025

Identifying a novel mechanism of L-leucine uptake in Mycobacterium tuberculosis using a chemical genomic approach

Amino acid biosynthesis is vital for Mycobacterium tuberculosis (Mtb) proliferation and tuberculosis pathogenesis. However, it is not clear how amino acids are transported in Mtb, particularly the branched chain amino acids (BCAAs) that contribute to the production of the cell-wall lipid component precursors such as acetyl-CoA and propionyl-CoA. While performing the screening of an FDA-approved repurposed library of small molecule inhibitors against the auxotrophic strain Mtb mc2 6206, which lacks leuC-leuD and panC-panD genes, we identified a molecule namely semapimod, which exclusively inhibits growth of the auxotrophic strain, whereas no effect is observed against the wild-type Mtb H37Rv. Interestingly, 24 h of exposure of Mtb mc2 6206 to semapimod causes massive transcriptional reprogramming with differential expression of >450 genes associated with a myriad of metabolic activities. By performing a series of experiments, we affirm that semapimod indeed inhibits the L-leucine uptake in Mtb mc2 6206 by targeting a protein involved in the cell-wall lipid biosynthesis pathway. Remarkably, semapimod treatment of mice infected with Mtb H37Rv causes a significant reduction of bacterial load in lungs and spleen, despite showing no efficacy against the pathogenic strain in vitro. Overall findings of our study reveal that together with an endogenous pathway for L-leucine biosynthesis, a well-orchestrated machinery for its uptake is functional in Mtb which is important for intracellular survival of the TB pathogen.

microbiology↗

bioRxiv2024

Beats of the Brain: Mapping Neural Activation During Candombe Engagement

This research delves into the complex connection between traditional music in Uruguay, specifically candombe, and its cognitive advantages. Relying on real-life studies, we explore the impact of involvement with candombe music on diverse cognitive abilities such as cognitive flexibility, emotional regulation, and social cognition. Our results show that playing music, especially drumming, activates certain brain pathways related to cognitive function and emotional regulation. The sense of community and distinct rhythmic patterns found in candombe not only boost individual cognitive skills but also foster social bonding, ultimately leading to better mental well-being and strength. This study highlights how cultural heritage influences cognitive functions, showing that candombe is crucial for mental well-being in addition to being a significant cultural symbol. In summary, our research supports incorporating traditional Uruguayan music in educational and therapeutic environments, offering a new framework for cognitive and emotional growth. This research focuses on how Uruguays diverse musical traditions can be used to enhance cognitive abilities and mental health, offering possibilities for application in different cultural settings globally.

neuroscience↗

bioRxiv2023

Elucidation of the essentiality of lumazine synthase (RibH) for Mycobacterium tuberculosis survival and discovery of potent inhibitors for enhanced antimycobacterial therapy

Tuberculosis (TB) continues to be a global health crisis, necessitating urgent interventions to address drug resistance and improve treatment efficacy. In this study, we validate the indispensable role of lumazine synthase (RibH), a vital enzyme in the riboflavin biosynthetic pathway, in the survival of Mycobacterium tuberculosis (M. tb) using a CRISPRi-based conditional gene knockdown strategy. We show that genetic and functional ablation of RibH in M. tb cannot be compensated by exogenous supply of Riboflavin, or co-factors Flavin Adenine Dinucleotide (FAD) or Flavin Mononucleotide (FMN). Capitalizing on the essentiality of RibH, we employ a high-throughput molecular docking approach to screen [~]600,000 compounds and identify inhibitors of RibH. Through in vitro screening of 55 shortlisted compounds, we discover 3 inhibitors that exhibit potent antimycobacterial activity. These compounds effectively also eradicate intracellular M. tb during macrophage infection and prevent the resuscitation of the nutrient-starved persister bacteria. Moreover, these 3 compounds synergistically enhance the bactericidal effect of first-line anti-TB drugs, Isoniazid and Rifampicin. Corroborating with the in silico predicted high docking scores along with favorable ADME and toxicity profiles, all 3 compounds demonstrate exceptional binding affinity towards purified lumazine synthase enzyme in vitro, and display an acceptable safety profile in mammalian cells, with a high selective index. By providing mechanistic evidence for the essentiality of RibH in M. tb survival, and discovering potent RibH inhibitors with outstanding antimycobacterial activity, our study contributes to the development of superior TB treatment strategies and advances the global fight against this devastating disease.

microbiology↗

bioRxiv2022

DNA origami 2.0

DNA origami is a technique that allows the creation of precise, modular, and programmable nanostructures using DNA. These nanostructures have found use in several fields like biophysics, molecular biology, nanoelectronics, and nanophotonic due to their programmable nature as well as ability to organize other nanomaterials with high accuracy. However, they are fragile and unstable when removed from their optimal aqueous conditions. In contrast, other commonly used bottom-up methods for creating inorganic nanoparticles do not have these issues, but it is difficult to control the shape or spatial organization of ligands on these nanoparticles. In this study, we present a simple, highly controlled method for templated growth of silica on top of DNA origami while preserving all the salient features of DNA origami. Using the polyplex micellization (PM) strategy, we create DNA nanostructures that can withstand salt-free, buffer-free, alcohol-water mixtures, enabling us to control the material growth conditions while maintaining the monodispersity and organization of nanoelements. We demonstrate the growth of silica shells of different thicknesses on brick and ring-shaped DNA origami structures using the standard Stober process. We also demonstrate the thermostability of the silica-coated nanostructures as well as accessibility of surface sites programmed into the DNA origami after the silica growth in the final inorganic nanostructure.

bioengineering↗

bioRxiv2022

Designing a Cas9/gRNA-assisted quantitative Real-Time PCR (CARP) assay for identification of point mutations leading to rifampicin resistance in the human pathogen Mycobacterium tuberculosis

A simple, rapid and low-cost diagnostic test, which can detect both the drug-sensitive and the drug-resistant tuberculosis (TB) cases is the need of the hour. Here, we developed a Cas9/gRNA-assisted quantitative Real-Time PCR (qRT-PCR) (CARP) assay to detect single nucleotide mutations causing drug resistance in the TB pathogen, Mycobacterium tuberculosis (Mtb). Guide RNAs (gRNAs) were designed against S531 and H526 positions in the rifampicin (RIF)-resistance-determining region (RR-DR) of the Mtb rpoB gene that exhibit frequent mutations in the RR clinical isolates of Mtb. Conditions were optimised for in vitro Cas9 cleavage such that single nucleotide changes at these positions can be recognised by Cas9/gRNA complex with 100% specificity. Further estimation of Cas9/gRNA-based cleavage of target DNA by qRT-PCR led to rapid detection of drug-resistant sequences. The newly designed CARP assay holds a great deal of promise in the diagnosis and prognosis of patients suffering from TB, in a cost-effective manner.

microbiology↗

bioRxiv2022

ISCA2 inhibition decreases HIF and induces ferroptosis in clear cell renal carcinoma

Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, is typically initiated by inactivation of the von Hippel Lindau (VHL) gene, which results in the constitutive activation of the hypoxia inducible factors, HIF-1 and HIF-2. Using a high throughput screen, we identify novel compounds that decrease HIF-1/2 levels and induce ferroptosis by targeting Iron Sulfur Cluster Assembly 2 (ISCA2), a component of the late mitochondrial Iron Sulfur Cluster (L-ISC) assembly complex. ISCA2 inhibition either pharmacologically or using siRNA decreases HIF-2 protein levels by blocking iron-responsive element (IRE)-dependent translation, and at higher concentrations, also decreases HIF-1 translation through unknown mechanisms. ISCA2 inhibition also triggers the iron starvation response, resulting in iron/metals overload and death via ferroptosis. ISCA2 levels are decreased in ccRCC compared to normal kidney, and decreased ISCA2 levels are associated with pVHL loss and with sensitivity to ISCA2 inhibition. Strikingly, pharmacological inhibition of ISCA2 using an orally available ISCA2 inhibitor significantly reduced ccRCC xenograft growth in vivo, decreased HIF- levels and increased lipid peroxidation, suggesting increased ferroptosis in vivo. Thus, the targeting of ISCA2 may be a promising therapeutic strategy to inhibit HIF-1/2 and to induce ferroptosis in pVHL deficient cells.

cancer biology↗

bioRxiv2022

Human anelloviruses produced by recombinant expression of synthetic genomes.

Human anelloviruses are acquired universally in infancy, highly prevalent, abundant in blood, and extremely diverse. Their apparent lack of pathogenicity indicates that they are a major component of the commensal human virome. Despite their being extensively intertwined with human biology, these viruses are poorly understood. A major impediment in studying anelloviruses is the lack of an in vitro system for their production and/ or propagation. Here we show that the T cell-derived human cell line MOLT-4 can be transfected with plasmids comprising tandem anellovirus genomes to produce viral particles visualized by electron microscopy. We found that a previously described human anellovirus of the Betatorquevirus genus (LY2), as well as a second Betatorquevirus detected by sequencing DNA extracted from a human retinal pigmental epithelium (nrVL4619), can be synthesized and produced by these means, enabling further molecular virology studies. Southern blot was used to demonstrate replication, and site-directed mutagenesis of the viral genome was performed to show that the production of anellovirus in this cell line is dependent on the expression of certain viral proteins. Finally, experiments performed in mice using purified nrVL4619 particles produced in MOLT-4 cells demonstrated infectivity in vivo in the tissue of origin. These results indicate that anelloviruses can be produced in vitro and manipulated to improve our understanding of this viral family which is ubiquitous in humans and many other mammals. Applications of this work to gene therapy and other therapeutic modalities are currently under investigation. IMPORTANCEAnelloviruses are a major component of the human virome. However, their biology is not well understood mainly due to the lack of an in vitro system for anellovirus production and/or propagation. In this study, we used multiple orthogonal measures to show that two different anelloviruses belonging to the Betatorquevirus genus can be produced in a T-cell-derived human cell line, MOLT-4, via recombinant expression of synthetic genomes. Additionally, we show that anellovirus particles generated in this in vitro system demonstrate infectivity in vivo. Our findings enable new molecular virology studies of this highly prevalent, non-pathogenic, and weakly immunogenic family of viruses, potentially leading to therapeutic applications.

microbiology↗

bioRxiv2022

Mapping phenotypic heterogeneity in melanoma onto the epithelial-hybrid-mesenchymal axis

Epithelial to mesenchymal transition (EMT) is a well-studied hallmark of epithelial-like cancers that is characterized by loss of epithelial markers and gain of mesenchymal markers. Interestingly, melanoma, which is derived from melanocytes of the skin, also undergo phenotypic plasticity toward mesenchymal-like phenotypes under the influence of various micro-environmental cues. Our study connects EMT to the phenomenon of de-differentiation (i.e., transition from proliferative to more invasive phenotypes) observed in melanoma cells during drug treatment. By analyzing 78 publicly available transcriptomic melanoma datasets, we found that de-differentiation in melanoma is accompanied by upregulation of mesenchymal genes, but not necessarily a concomitant loss of an epithelial program, suggesting a more "one-dimensional" EMT that leads to a hybrid epithelial/ mesenchymal phenotype. Samples lying in the hybrid epithelial/mesenchymal phenotype also correspond to the intermediate phenotypes in melanoma along the proliferative-invasive axis - neural crest and transitory ones. Interestingly, as melanoma cells progress along the invasive axis, the mesenchymal signature does not increase monotonically. Instead, we observe a peak in mesenchymal scores followed by a decline, as cells further de-differentiate. This biphasic response recapitulates the dynamics of melanocyte development, suggesting close interactions among genes controlling differentiation and mesenchymal programs in melanocytes. Similar trends were noted for metabolic changes often associated with EMT in carcinomas in which progression along mesenchymal axis correlates with the downregulation of oxidative phosphorylation, while largely maintaining glycolytic capacity. Overall, these results provide an explanation for how EMT and dedifferentiation axes overlap with respect to their transcriptional and metabolic programs in melanoma.

cancer biology↗

bioRxiv2021

Computational analysis reveals similarities and differences between SCLC subtypes

Small cell lung cancer (SCLC) is a neuroendocrine malignancy with dismal survival rates. Previous studies have revealed inter and intra tumoral heterogeneity of SCLC driven by neuroendocrine differentiation and multiple gene expression signatures have been proposed to classify the distinct SCLC molecular subtypes However, few questions remain unanswered: a) how many SCLC subtypes exist? b) how similar or different are these subtypes?, c) which gene list(s) can be used to identify those specific subtypes? Here, we show that irrespective of the three gene sets (33 genes, 50 genes, 105 genes) proposed in different studies to classify SCLC into different subtypes, the markers of phenotypic heterogeneity in SCLC form a "teams" like pattern of co-expressed modules. Moreover, the 105 geneset could classify SCLC cell lines into five clusters, three of which can be distinctly mapped to the SCLC-A, SCLC-N and SCLC-Y subtypes. Intriguingly, we noticed a high degree of similarity in the transcriptional landscape of two non-neuroendocrine subtypes: SCLC-Y and SCLC-I*, as well as in their enrichment of EMT. Thus, our analysis elucidates the landscape of phenotypic heterogeneity enabling diverse SCLC subtypes.

cancer biology↗

bioRxiv2021

Versatile Encapsulation and Synthesis of Potent Therapeutic Liposomes by Thermal Equilibration

The wide-scale use of liposomal delivery systems is hampered by difficulties in obtaining potent liposomal suspensions. Passive and active loading strategies have been proposed to formulate drug encapsulated liposomes, but are limited by low efficiencies (passive) or high drug specificities (active). Here, we present an efficient and universal loading strategy for synthesizing therapeutic liposomes. Integrating a thermal equilibration technique with our unique liposome synthesis approach, co-loaded targeting liposomes can be engineered in an efficient and scalable manner with potencies 200-fold higher than typical passive encapsulation techniques. We demonstrate this capability through simultaneous co-loading of hydrophilic and hydrophobic small molecules and through targeted delivery of liposomal Doxorubicin to a metastatic breast cancer cell line MDA-MB-231. Molecular dynamic simulations are used to explain interactions between Doxorubicin and liposome membrane during thermal equilibration. By addressing the existing challenges, we have developed an unparalleled approach that will facilitate the formulation of novel theranostic and pharmaceutical strategies.

bioengineering↗

bioRxiv2021

Co-deletion of ATAD1 with PTEN primes cells for BIM-mediated apoptosis

PTEN is a potent tumor suppressor gene that is frequently mutated or deleted in human cancers. Such deletions often include portions of the 10q23 locus beyond the bounds of PTEN itself, in many cases resulting in the disruption of additional genes. Coincidental loss of PTEN-adjacent genes might impose vulnerabilities that could either affect patient outcome basally or be exploited therapeutically. Here we describe how the loss of ATAD1, which is adjacent to and frequently co-deleted with PTEN, predisposes cancer cells to apoptosis and correlates with improved survival in cancer patients. ATAD1 directly and specifically extracts the pro-apoptotic BIM protein from mitochondria to inactivate it. Cells lacking ATAD1 are hypersensitive to clinically used proteasome inhibitors, which increase BIM and trigger apoptosis. Thus, we demonstrate that mitochondrial protein quality control interfaces with cell death in a clinically actionable manner.

cancer biology↗

bioRxiv2021

EEG-Based Focus Estimation Using Neurable's Enten Headphones and Analytics Platform

We introduce Neurables research on focus using our recently developed Enten EEG headphones. First we quantify Entens performance on standard EEG protocols, including eyes-closed alpha rhythms, auditory evoked response and the P300 event-related potential paradigm. We show that Entens performance is on-par with established industry-standard hardware. We then introduce a series of experimental tasks designed to mimic how focus might be maintained or disrupted in a real-world office setting. We show that (A) these tasks induce behavioral changes that reflect underlying changes in focus levels and (B) our proprietary algorithm detects these changes across a large number of sessions without needing to adjust the model per participant or recording session. Through manipulation of our experimental protocol, we show that our algorithm is not dependent on gross EMG artifacts and it is driven by changes in EEG. Finally, we evaluated the models performance on the same subject across several days, and show that performance remained consistent over time. Our model correctly captured 80% {+/-} 4.1% of distractions present in our experiments with statistical significance. This indicates that our model generalizes across subjects, time points, and conditions. Our findings are based on EEG data collected from 132 participants across 337 sessions and 45 different experiments.

neuroscience↗

bioRxiv2020

Disulfiram inhibits M. tuberculosis growth by altering methionine pool, redox status and host-immune response

Methionine biosynthetic pathway, essential for the growth of Mycobacterium tuberculosis (Mtb) in the host, represents an attractive target for the development of novel anti-tuberculars. Here, we have biochemically characterized homoserine acetyl transferase (HSAT viz. MetA) of Mtb, which catalyses the first committed step of methionine and S-adenosylmethionine (SAM) biosynthesis. High-throughput screening of a 2300 compound library resulted in identification of thiram, an anti-fungal organosulfur compound, as the most potent MetA inhibitor. Further analysis of thiram analogs led to the identification of orally bioavailable disulfiram (DIS, an anti-alcoholism FDA approved drug) as a novel inhibitor of MetA. Both thiram and DIS restricted the growth of drug-sensitive and drug-resistant Mtb strains in a bactericidal manner. ThermoFlour assay demonstrated direct binding of DIS with MetA. Metabolomic and transcriptomic studies showed DIS mediated perturbation of methionine and redox homeostasis, respectively, in Mtb. In concordance, the effect of DIS on Mtb growth was partially rescued by supplementation with either L-methionine as well as N-acetyl cysteine, suggesting a multi-target killing mechanism. In Mtb-infected mice, DIS administration restricted bacterial growth, increased efficacy of isoniazid, ameliorated lung pathology, modulated lung immune cell landscape and protective immune response. Taken together, our results demonstrate that DIS can be repurposed for designing an effective anti-tubercular therapy.

microbiology↗