Biology subjects

Venkataraman, S.

Publications and source records attributed to Venkataraman, S..

18 recordsLinked to original sources

bioRxiv2026

Simultaneous Proportional Control of Two Degrees-of-Freedom Human Machine Interface Using Highly Sparse Sonomyography

Non-invasive human-machine interfaces (HMIs) are critical in developing prosthetic systems that offer intuitive, simultaneous, and proportional control over multiple degrees of freedom (DOFs). This study introduces a novel system for intuitive concurrent control of hand and wrist movements using sonomyography based imaging of muscle activity. Our method uses a sparse set of ultrasound scanlines to reduce computational complexity while enhancing usability. We evaluated four regression techniques for wrist and hand angle prediction, focusing on performance with a reduced sonomyographic feature set. We also explored the feasibility of a sonomyography-based system by simulating various factors that could affect prediction, including feature selection and scanline count. Our findings demonstrate that Gaussian process regression excels in predicting wrist and hand angles with just eight equispaced transducers in offline settings. Real-time evaluations with 10 non-disabled participants showed a 93 % success rate for two-DOF tasks using linear regression. The system was tested with an individual with amputation, achieving a 46 % success rate for two-DOF control in a 2D space, even though the ground truth data for model training was collected from the contralateral limb. This study validates our sonomyography-based approach for accurate wrist and hand angle estimation, reducing complexity and demonstrating potential in real-world scenarios.

bioengineering↗

bioRxiv2025

Targeting processive transcription for Myc-driven circuitry in medulloblastoma

AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSBackgroundC_ST_ABSMedulloblastoma is the most common malignant brain tumor of childhood. The highest-risk tumors are driven by recurrent Myc amplifications (Myc-MB) and experience poorer outcomes despite intensive multimodal therapy. The Myc transcription factor defines core regulatory circuitry for these tumors and acts to broadly amplify downstream pro-survival transcriptional programs. Therapeutic targeting of Myc directly has proven elusive, but inhibiting transcriptional cofactors may present an indirect means of drugging the oncogenic transcriptional circuitry sustaining Myc-MB. MethodsIndependent CRISPR-Cas9 screens were pooled to identify conserved dependencies in Myc-MB. We performed chromatin conformation capture (Hi-C) from primary patient Myc-MB samples to map enhancer-promoter interactions. We then treated in vitro and xenograft models with CDK9/7 inhibitors to evaluate effect on Myc-driven programs and tumor growth. ResultsEight CRISPR-Cas9 screens performed across three independent labs identify CDK9 as a conserved dependency in Myc-MB. Myc-MB cells are susceptible to CDK9 inhibition, which is synergistic with concurrent inhibition of CDK7. Inhibition of transcriptional CDKs disrupts enhancer-promoter activity in Myc-MB and downregulates Myc-driven transcriptional programs, exerting potent anti-tumor effect. ConclusionsOur findings identify CDK9 inhibition as a translationally promising strategy for the treatment of Myc-MB. KO_SCPLOWEYC_SCPLOW PO_SCPLOWOINTSC_SCPLOWO_LICDK9 is an intrinsic dependency in Myc-driven medulloblastoma C_LIO_LIDual CDK9/7 inhibition disrupts Myc-driven transcriptional circuitry C_LIO_LICDK9 inhibitors should be developed as pharmaceutical agents for Myc-MB C_LI IO_SCPLOWMPORTANCEC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWOFC_SCPLOWO_SCPCAP C_SCPCAPO_SCPLOWTHEC_SCPLOW SO_SCPLOWTUDYC_SCPLOWMedulloblastoma is the most common malignant brain tumor of childhood, and outcomes for high-risk subgroups remain unsatisfactory despite intensive multimodal therapy. In this study, we pool multiple independent CRISPR-Cas9 screens to identify transcriptional cofactors such as CDK9 as conserved dependencies in Myc-MB. Using Hi-C from primary patient samples, we map Myc enhancer-promoter interactions and show that they can be disrupted using inhibition of transcriptional CDKs. CDK9 inhibitor treatment depletes Myc-driven transcriptional programs, leading to potent anti-tumor effect in vitro and prolongation of xenograft survival in vivo. With a large number of CDK9 inhibitory compounds now in clinical development, this study highlights the opportunity for clinical translation of these for children diagnosed with Myc-MB.

cancer biology↗

bioRxiv2025

Differential Phagocytosis induces Diverse Macrophage Activation States in Malignant Gliomas

Diffuse midline glioma (DMG) and Glioblastoma are malignant brain tumors in pediatric and adult patients. The current standard-of-care treatment for DMG is radiotherapy (RT), whereas GBM treatment includes surgery, followed by RT and chemotherapy. Although RT is known to modulate immune responses in cancer and enhance the effectiveness of myeloid checkpoint blockade, the downstream macrophage responses to differential phagocytosis induction remain poorly understood. This study examined macrophage-mediated phagocytosis caused by either RT, anti-CD47 checkpoint blockade, or their combination. We found that RT increased the expression of several damage-associated molecular patterns on the surface of glioma cell lines. Furthermore, RT enhanced anti-CD47-mediated macrophage phagocytosis of glioma cell lines in vitro. Single-cell RNA-sequencing revealed the diverse transcriptional and functional signatures of human macrophage subsets that either promoted or inhibited phagocytosis of glioma cells pretreated with RT, anti-CD47 therapy, or both. Consistent with these results, the combination therapy significantly reduced tumor growth, prolonged survival in glioma-bearing mice, and induced distinct macrophage activation states in vivo compared to either treatment alone. These findings highlight the plasticity and heterogeneity of macrophage responses during phagocytosis and provide compelling evidence for combining RT with anti-CD47 therapy as a promising therapeutic strategy for glioma treatment.

cancer biology↗

bioRxiv2025

Characterisation of CD4 Th subsets as well as dietary, exercise and lifestyle factors in an established Rheumatoid Arthritis cohort; pilot study.

BackgroundSex disparities in the incidence and severity of Rheumatoid Arthritis (RA) are well-documented, with females experiencing more aggressive disease and different responses to treatment. While underlying mechanisms remain elusive, immune factors and lifestyle components, such as diet and physical activity, could play a role in shaping disease outcomes. Since Th cells are key drivers of RA, this study characterised CD4 Th subset frequencies, including Th1, Th2, Th17 and Th1/17 cells, in a cohort of males and females with established RA. Considering the role of diet and lifestyle in RA development, we also evaluated participant diet and lifestyle patterns. MethodsAn observational cross-sectional cohort study profiling 39 individuals (19 RA and 20 age and sex matched controls) with no major co-morbidities was conducted. Percentages of Th1, Th2, Th17 and Th1/17 cells from peripheral blood were determined by flow cytometry as well as serum levels of RF and cytokines IL17, IFN-{gamma}, TNF-, GM-CSF, TGF-{beta}, IL-6, and CRP by ELISA. Dietary intake was assessed by Food Frequency Questionnaire (FFQ), the Dietary inflammatory Index (DII) calculated from nutrient intake. Physical activity levels were evaluated by Global Physical Activity Questionnaire (GPAQ). ResultsSex-specific differences of Th17, Th1 an Th2 cells were observed in RA groups. Males had lower levels of Th1 cells and higher levels of Th17 cells than control subjects, while females had lower levels of Th2 cells than controls. Female RA subjects had higher ratio of Th1:Th2 cells than controls, while male RA subjects had higher Th17:Th1 ratio than controls. Interestingly, circulating CD4 Th subset levels were predictive of RA in this cohort. No changes in cytokine levels were observed. The male RA group consumed 30% less carbohydrate (RA = 156g +/-57g, Control = 253g +/-59g; p=0.003), 37% less sugar (RA = 74.1g +/-29g, Control = 118g +/-33g; p=0.004) and 30% less dietary fibre (R = 33g +/-8g, Control = 48g +/-13 g; p=0.008) than the male control group. The female RA group had the lowest consumption of alcohol in comparison to female control group (RA = 0.31g, Control = 8.97g; p=0.047). Male subjects had a 4x higher DII than females (p=0.012). ConclusionsOur findings highlight potential sex-specific differences in CD4+ Th cell subsets in individuals with established RA, indicating that sex may influence immune cell composition in this disease. Furthermore, dietary components showed sex-specific consumption patterns, underscoring the potential role of tailored lifestyle interventions in modulating immune responses in RA. Evaluating immunological sex differences and dietary and lifestyle factors may be important for enhancing management strategies in RA.

immunology↗

bioRxiv2024

H3K27M diffuse midline glioma is homologous recombination defective and sensitized to radiotherapy and NK cell-mediated antitumor immunity by PARP inhibition

BackgroundRadiotherapy (RT) is the primary treatment for diffuse midline glioma (DMG), a lethal pediatric malignancy defined by histone H3 lysine 27-to-methionine (H3K27M) mutation. Based on the loss of H3K27 trimethylation producing broad epigenomic alterations, we hypothesized that H3K27M causes a functional double-strand break (DSB) repair defect that could be leveraged therapeutically with PARP inhibitor and RT for selective radiosensitization and antitumor immune responses. MethodsH3K27M isogenic DMG cells and orthotopic brainstem DMG tumors in immune deficient and syngeneic, immune competent mice were used to evaluate the efficacy and mechanisms of PARP1/2 inhibition by olaparib or PARP1 inhibition by AZD9574 with concurrent RT. ResultsH3K27M mutation caused an HRR defect characterized by impaired RT-induced K63-linked polyubiquitination of histone H1 and inhibition of HRR protein recruitment. H3K27M DMG cells were selectively radiosensitized by olaparib in comparison to isogenic controls, and this effect translated to efficacy in H3K27M orthotopic brainstem tumors. Olaparib and RT induced an innate immune response and induction of NK cell (NKG2D) activating ligands leading to increased NK cell-mediated lysis of DMG tumor cells. In immunocompetent syngeneic orthotopic DMG tumors, either olaparib or AZD9574 in combination with RT enhanced intratumoral NK cell infiltration and activity in association with NK cell-mediated therapeutic responses and favorable activity of AZD9574. ConclusionsThe HRR deficiency in H3K27M DMG can be therapeutically leveraged with PARP inhibitors to radiosensitize and induce an NK cell-mediated antitumor immune response selectively in H3K27M DMG, supporting the clinical investigation of best-in-class PARP inhibitors with RT in DMG patients. Key pointsO_LIH3K27M DMG are HRR defective and selectively radiosensitized by PARP inhibitor. C_LIO_LIPARP inhibitor with RT enhances NKG2D ligand expression and NK cell-mediated lysis. C_LIO_LINK cells are required for the therapeutic efficacy of PARP inhibitor and RT. C_LI Importance of the StudyRadiotherapy is the cornerstone of H3K27M-mutant diffuse midline glioma treatment, but almost all patients succumb to tumor recurrence with poor overall survival, underscoring the need for RT-based precision combination therapy. Here, we reveal HRR deficiency as an H3K27M-mediated vulnerability and identify a novel mechanism linking impaired RT-induced histone H1 polyubiquitination and the subsequent RNF168/BRCA1/RAD51 recruitment in H3K27M DMG. This model is supported by selective radiosensitization of H3K27M DMG by PARP inhibitor. Notably, the combination treatment results in NKG2D ligand expression that confers susceptibility to NK cell killing in H3K27M DMG. We also show that the novel brain penetrant, PARP1-selective inhibitor AZD9574 compares favorably to olaparib when combined with RT, prolonging survival in a syngeneic orthotopic model of H3K27M DMG. This study highlights the ability of PARP1 inhibition to radiosensitize and induce an NK cell-mediated antitumor immunity in H3K27M DMG and supports future clinical investigation.

cancer biology↗

bioRxiv2024

Targeting Diffuse Midline Glioma with a novel anti-CD99 Antibody

Diffuse midline gliomas (DMGs) are devastating brain tumors that occur primarily in children. The salient feature of these tumors is the presence of a H3K27M mutation (K27M), associated with the worst prognosis. We identified the cell surface antigen CD99 as notably expressed in DMGs, particularly in K27M+DMGs. We found that the increased expression of CD99 in K27M+DMGs was a result of the onco-histone K27M mutation. In K27M+DMG cells, CD99 inactivation impaired tumor growth by inducing cell differentiation, indicating an oncogenic role of CD99 enabled by blocking differentiation. We then developed a novel therapeutic anti-CD99 chimeric antibody, 10D1, with a membrane-proximal binding epitope, and evaluated its antitumor efficacy in preclinical models of K27M+DMG. 10D1 suppressed DMG growth in vitro and in vivo by inducing apoptosis. When combined with radiation treatment, 10D1 exhibited improved antitumor efficiency and xenograft survival, providing a strong justification for its clinical development as a therapy for DMGs. Statement of SignificanceThis study emphasizes that CD99 overexpression occurs due to the H3K27M mutation in Diffuse Midline Gliomas (DMGs). This heightened expression suppresses apoptosis, inhibits differentiation, and induces radio-resistance in DMGs. This research justifies using a novel CD99 antibody alone or combined with radiation therapy in human pediatric clinical trials.

cancer biology↗

bioRxiv2024

Transcriptional Regulation of Protein Synthesis by Mediator Kinase in MYC-driven Medulloblastoma

MYC-driven medulloblastoma (MB) is a highly aggressive cancer type with poor prognosis and limited treatment options. Through CRISPR-Cas9 screening across MB cell lines, we identified the Mediator-associated kinase CDK8 as the top dependence for MYC-driven MB. Loss of CDK8 markedly reduces MYC expression and impedes MB growth. Mechanistically, we demonstrate that CDK8 depletion suppresses ribosome biogenesis and mRNA translation. CDK8 regulates occupancy of phospho-Polymerase II at specific chromatin loci facilitating an epigenetic alteration that promotes transcriptional regulation of ribosome biogenesis. Additionally, CDK8-mediated phosphorylation of 4EBP1 plays a crucial role in initiating eIF4E-dependent translation. Targeting CDK8 effectively suppresses cancer stem and progenitor cells, characterized by increased ribosome biogenesis activity. We also report the synergistic inhibition of CDK8 and mTOR in vivo and in vitro. Overall, our findings establish a connection between transcription and translation regulation, suggesting a promising therapeutic approach targets multiple points in the protein synthesis network for MYC-driven MB.

cancer biology↗

bioRxiv2024

PRMT5 maintains tumor stem cells to promote pediatric high-grade glioma tumorigenesis

BackgroundPediatric high-grade gliomas (PHGG) are aggressive, undifferentiated CNS tumors with poor outcomes, for which no standard-of-care drug therapy currently exists. Through a screen for epigenetic regulators, we identified PRMT5 as essential for PHGG growth. We hypothesized that, similar to its effect in normal cells, PRMT5 promotes self-renewal of stem-like PHGG tumor initiating cells (TICs) essential for tumor growth. Methods. We conducted in vitro assays, including limiting dilution studies of self-renewal, to determine the phenotypic effects of PRMT5 KD. We performed ChIP-Seq to identify PRMT5-mediated epigenetic changes and gene set enrichment analysis to identify pathways that PRMT5 regulates. Using an orthotopic xenograft model of PHGG, we tracked survival and histological characteristics resulting from PRMT5 KD or administration of a PRMT5 inhibitor {+/-} radiation therapy (RT). ResultsIn vitro, PRMT5 KD slowed cell cycle progression, tumor growth and self-renewal. PRMT5 KD reduced H3K4me3 occupancy at genes associated with self-renewal, tumor formation and growth. In vivo, PRMT5 KD increased survival and reduced tumor aggressiveness; however, pharmacological inhibition of PRMT5 with or without RT did not improve survival. ConclusionPRMT5 KD epigenetically reduced TIC self-renewal, leading to increased survival in preclinical models. Pharmacological inhibition of PRMT5 enzymatic activity may have failed in vivo due to insufficient reduction of PRMT5 activity by chemical inhibition, or this failure may suggest that non-enzymatic activities of PRMT5 are more relevant. ImplicationsOur findings show the importance of PRMT5 to maintain and promote the growth of stemlike cells that initiate and drive tumorigenesis in pediatric high grade glioma.

cancer biology↗

bioRxiv2023

Mobilisation and analyses of publicly available SARS-CoV-2 data for pandemic responses

The COVID-19 pandemic has seen large-scale pathogen genomic sequencing efforts, becoming part of the toolbox for surveillance and epidemic research. This resulted in an unprecedented level of data sharing to open repositories, which has actively supported the identification of SARS-CoV-2 structure, molecular interactions, mutations and variants, and facilitated vaccine development and drug reuse studies and design. The European COVID-19 Data Platform was launched to support this data sharing, and has resulted in the deposition of several million SARS-CoV-2 raw reads. In this paper we describe (1) open data sharing, (2) tools for submission, analysis, visualisation and data claiming (e.g. ORCiD), (3) the systematic analysis of these datasets, at scale via the SARS-CoV-2 Data Hubs as well as (4) lessons learned. As a component of the Platform, the SARS-CoV-2 Data Hubs enabled the extension and set up of infrastructure that we intend to use more widely in the future for pathogen surveillance and pandemic preparedness.

bioinformatics↗

bioRxiv2023

Rapid PTEFb-dependent transcriptional reorganization underpins the glioma adaptive response to radiotherapy

AO_SCPLOWBSTRACTC_SCPLOWDynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. PTEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma, we show that rapid genome-wide reorganization of active chromatin facilitates PTEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of PTEFb disrupts this chromatin reorganization and blunts transcriptional induction, abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for PTEFb underpinning the early adaptive response to radiotherapy, opening new avenues for combinatorial treatment in these lethal malignancies.

cancer biology↗

bioRxiv2022

Integrated analysis of Wnt signalling system component gene expression

Wnt signalling controls patterning and differentiation across many tissues and organs of the developing embryo via temporally and spatially restricted expression of multi-gene families encoding ligands, receptors, pathway modulators and intracellular components. Here we report an integrated analysis of key encoding genes in the 3D space of the mouse embryo across multiple stages of development. We applied a method for 3D/3D image transformation to map all gene expression patterns to a single reference embryo for each stage providing both visual analysis and volumetric mapping allowing computational methods to interrogate the combined expression patterns. We identify novel territories where multiple Wnt and Fzd genes are co-expressed and cross-compare all patterns, including all seven Wnt paralogous gene pairs. The comprehensive analysis allows regions in the embryo where no Wnt or Fzd gene expression is detected, and where single Wnt genes are uniquely expressed, to be revealed. This work provides insight into a level of organisation of the patterns not previously possible, as well as presenting a resource that can be utilised further by the research community for whole system analysis.

developmental biology↗

bioRxiv2022

Next-generation intranasal Covid-19 vaccine: a polymersome-based protein subunit formulation that provides robust protection against multiple variants of concern and early reduction in viral load of the upper airway in the golden Syrian hamster model

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of coronavirus disease 2019 (Covid-19), an ongoing global public health emergency. Despite the availability of safe and efficacious vaccines, achieving herd immunity remains a challenge due in part to rapid viral evolution. Multiple variants of concern (VOCs) have emerged, the latest being the heavily mutated Omicron, which exhibits the highest resistance to neutralizing antibodies from past vaccination or infection. Currently approved vaccines generate robust systemic immunity, yet poor immunity at the respiratory tract. We have demonstrated that a polymersome-based protein subunit vaccine with wild type (WT) spike protein and CpG adjuvant induces robust systemic immunity (humoral and T cell responses) in mice. Both antigen and adjuvant are encapsulated in artificial cell membrane (ACM) polymersomes - synthetic, nanoscale vesicles that substantially enhance the immune response through efficient delivery to dendritic cells. In the present study, we have formulated a vaccine candidate with the spike protein from Beta variant and assessed its immunogenicity in golden Syrian hamsters. Two doses of ACM-Beta spike vaccine administered via intramuscular (IM) injection evoke modest serum neutralizing titers that are equally efficacious towards WT and Beta viruses. In contrast, the ACM-WT spike vaccine induces a predominantly WT-specific serum neutralizing response with pronounced reduction in potency towards the Beta variant. Remarkably, immunogenicity of the ACM-Beta spike vaccine is greatly enhanced through intranasal (IN) administration. Following IN challenge with the Beta variant, IM-immunized hamsters are fully protected from disease but not infection, displaying similar peak viral RNA loads in oral swabs as non-vaccinated controls. In contrast, hamsters IN vaccinated with ACM-Beta spike vaccine are protected from disease and infection, exhibiting a [~]100-fold drop in total and subgenomic RNA load as early as day 2 post challenge. We further demonstrate that nasal washes from IN-but not IM-immunized animals possess virus neutralizing activity that is broadly efficacious towards Delta and Omicron variants. Altogether, our results show IN administration of ACM-Beta spike vaccine to evoke systemic and mucosal antibodies that cross-neutralize multiple SARS-CoV-2 VOCs. Our work supports IN administration of ACM-Beta spike vaccine for a next-generation vaccination strategy that not only protects against disease but also an infection of the respiratory tract, thus potentially preventing asymptomatic transmission.

immunology↗

bioRxiv2020

Neoplastic and Immune single cell transcriptomics define subgroup-specific intra-tumoral heterogeneity of childhood medulloblastoma.

Medulloblastoma (MB) is a heterogeneous disease in which neoplastic cells and associated immune cells contribute to disease progression. To better understand cellular heterogeneity in MB we use single-cell RNA sequencing, immunohistochemistry and deconvolution of transcriptomic data to profile neoplastic and immune populations in childhood MB samples. Neoplastic cells cluster primarily according to individual sample of origin which is in part due to the effect of chromosomal copy number gains and losses. Harmony alignment reveals novel MB subgroup/subtype-associated subpopulations that recapitulate neurodevelopmental processes and are associated with clinical outcomes, including discrete photoreceptor-like cells in MB subgroups GP3 and GP4 and nodule-associated neuronally-differentiated cells in subgroup SHH. We definitively chart the spectrum of MB immune cell infiltrates, which include subgroup/subtype-associated developmentally-related neuron-pruning and antigen presenting myeloid cells. MB cellular diversity is recapitulated in genetically engineered mouse subgroup-specific models of MB. These findings provide a clearer understanding of both the neoplastic and immune cell heterogeneity in MB.

cancer biology↗

bioRxiv2020

LAMA: Automated image analysis for developmentalphenotyping of mouse embryos

Advanced 3D imaging modalities such as micro computed tomography (micro-CT), high resolution episcopic microscopy (HREM), and optical projection tomography (OPT) have been readily incorporated into high-throughput phenotyping pipelines, such as the International Mouse Phenotyping Consortium (IMPC). Such modalities generate large volumes of raw data that cannot be immediately harnessed without significant resources of manpower and expertise. Thus, rapid automated analysis and annotation is critical to ensure that 3D imaging data is able to be integrated with other multi-dimensional phenotyping data. To this end, we present an automated computational mouse phenotyping pipeline called LAMA, based on image registration, which requires minimal technical expertise and human input to use. Designed predominantly for developmental biologists, our software performs image pre-processing, registration, statistical and gene function annotation, and segmentation of 3D micro-CT data. We address several limitations of current methods and create an easy to use, fast solution application for mouse embryo phenotyping. We also present a highly granular, novel anatomical E14.5 (14.5 days post coitus) atlas of a population average that integrates with our pipeline to allow a range of dysmorphologies to be automatically annotated as well as results from the validation of the pipeline.

bioinformatics↗

bioRxiv2020

Transcriptional control of DNA repair networks by CDK7 regulates sensitivity to radiation in Myc-driven Medulloblastoma

Myc-driven Medulloblastoma remains a major therapeutic challenge due to frequent metastasis and a poor 5-year survival rate. Myc gene amplification results in transcriptional dysregulation, proliferation, and survival of malignant cells. To identify therapeutic targets in Myc-amplified medulloblastoma we performed a CRISPR-Cas9 essentiality screen targeting 1140 genes annotated as the druggable genome. CDK7 was identified as a mediator of medulloblastoma tumorigenesis. Using covalent inhibitors and genetic depletion of CDK7 we observe the cessation of tumor growth in xenograft mouse models and increase in apoptotic mechanisms. The results are attributed to repression of a core set of Myc-driven transcriptional programs mediating DNA repair. We further establish that blocking CDK7 activity sensitizes cells to ionizing radiation leading to accrual of DNA damage and extended survival and tumor latency in medulloblastoma xenograft mouse models. Our studies establish a mechanism for selective inhibition of Myc-driven MB by CDK7 inhibition combined with radiation as a viable therapeutic strategy for Myc-amplified medulloblastoma.

cancer biology↗

bioRxiv2020

Towards understanding the structure of the capsid of Banana Bunchy Top Virus

Banana is the major staple food crop for approximately 400 million people. Bunchy Top disease of Banana is one of the most devastating diseases caused by Banana Bunchy Top Virus (BBTV) that results in a significant loss of yield, stunting and bunchy appearance of leaves. While many isolates of BBTV from various regions of India have been characterized by different groups, no structural study exists for this important virus. To pursue structural studies, the pET28a clone of coat protein (CP) gene from BBTV isolate of Hill Banana grown in lower Pulney Hills (Virupakshi) of Tamilnadu was expressed in BL21 (DE3) pLysS. Purification of the CP was done using Ni-NTA affinity chromatography. In vitro capsid assembly studied using sucrose density gradient centrifugation suggested that the CP did not assemble as virus like particle (VLPs) but remained as smaller oligomers. Studies using dynamic light scattering (DLS) indicates that the purified protein is poly-dispersed represented majorly as pentamers. Studies using both homology modelling and ab initio structure determination gave useful insights into the probable fold of the CP suggesting it is a {beta}-sandwich fold similar to that seen in majority of plant viruses. In silico capsid reconstruction aided understanding of the quaternary organization of subunits in the capsid and molecular interactions present between the subunits. The location of aphid binding EAG motif was identified on the surface loops close to the pentameric axis indicating their role in vector mediated transmission.

bioinformatics↗

bioRxiv2020

Super elongation complex as a targetable dependency in diffuse midline glioma

Mutations in the histone 3 gene (H3K27M) are the eponymous drivers in diffuse intrinsic pontine gliomas (DIPGs) and other diffuse midline gliomas (DMGs), aggressive pediatric brain cancers for which no curative therapy currently exists. The salient molecular consequence of these recurrent oncohistones is a global loss of repressive H3K27me3 residues and broad epigenetic dysregulation. In order to identify specific, therapeutically targetable epigenetic dependencies within this disease context, we performed an shRNA screen targeting 408 genes classified as epigenetic/chromatin-associated molecules in patient-derived DMG cultures. This approach identified AFF4, the scaffold protein of the super elongation complex (SEC), as a previously-undescribed dependency in DMG. Interrogation of SEC function demonstrated a key role for maintaining DMG cell viability and clonogenic potential while promoting self-renewal of DMG tumor stem cells. Small-molecule inhibition of the SEC with the highly-specific, clinically relevant CDK9 inhibitors atuveciclib and AZD4573 restores regulatory RNA polymerase II pausing, promotes cellular differentiation, and leads to potent anti-tumor effect both in vitro and in patient-derived xenograft models. These studies present a biologic rationale for translational exploration of CDK9 inhibition as a promising therapeutic approach in a disease which currently has no effective medical therapies.

cancer biology↗

bioRxiv2018

Direct determination and differentiation of carbapenemases of A. baumannii from uncultured tracheal samples

The emergence of multiple carbapenemases and the consequent multi drug resistance in bacteria constitute a grave concern in the management of critical care patients and also in community acquired infections. Detection of carbapenamase activity helps to understand the possible mechanism(s) of carbapenem resistance in the microorganism. Identification of carbapenemases is currently being done by various phenotypic methods and molecular methods. However, innovative biochemical and spectrophotometric methods are desirable as they will be easy to perform, affordable and rapid. Recently a novel chromogenic method called CarbaNP test was introduced to screen for carbapenemases in clinical isolates of gram negative pathogens. We adopted this assay: (i) to detect the total carbapenemase activity (ii) to measure the relative rates of hydrolysis of imipenem by class A, B and D carbapenemases with inhibitors (iii) to confirm the genotype by PCR and (iv)for direct differential detection of various carbapenemases in uncultured clinical sample like tracheal aspirate. The study included 75 culture isolates and 153 purulent tracheal aspirates. All isolates were screened by our optimized protocol and also genotyped by PCR. This adopted assay showed good sensitivity and correlation with conventional phenotyping and genotyping. Our protocol offers the fastest way to identify the pathogen by PCR but also its carbapenemase profile directly from uncultured clinical samples in less than 4h. Our protocol is currently being validated on other types of clinical specimens in our laboratory.

microbiology↗