Biology subjects

Kumar, S.

Publications and source records attributed to Kumar, S..

At least 217 records · Page 12Linked to original sources

bioRxiv2024

4-Phenylbutyric acid modulates Connexin 43 expression restricting murine-β-coronavirus infectivity and virus-induced demyelination

Gap junction intercellular communication, particularly involving Connexin 43 and Connexin 47, plays a critical role in maintaining CNS homeostasis and has been implicated in Multiple Sclerosis (MS) pathology. Thus, warranting further studies in experimental animal models to understand how modulation of Cx43 expression can influence MS pathology. Intracranial infection with murine-{beta}-coronavirus Mouse Hepatitis Virus (MHV-A59) in mice results in acute pathology characterized by high viral titers, glial activation in the brain and chronic neuroinflammatory demyelination, effectively mimicking key pathological hallmarks of MS and serving as a robust model to investigate its viral etiology. MHV-A59 infection leads to a pronounced downregulation of Cx43 during the acute phase, emphasizing its critical role in virus-induced CNS pathology. In this study, we investigated the potential of in vivo 4-phenylbutyric acid (4-PBA) administration in modulating Cx43 expression in this MHV-induced model and its consequence on chronic virus-induced demyelination. Our results reveal that 4-PBA treatment reduced acute MHV-A59 infectivity and viral spread in the brain while modulating the glial cell response, mounting host immunity. Treatment with 4-PBA effectively preserved the expression of both Cx43 and Cx47 in infected CNS cells, counteracting their infection-induced downregulation. Furthermore, MHV-A59 infection downregulated the expression of ER-resident thioredoxin family protein (ERp29), a well-known molecular chaperone of Cx43, which was rescued by 4-PBA treatment. We further validated if such downregulation of ERp29 is also evident in MS demyelinating plaques. In human MS patient-derived brain tissue, reduced Cx43 and ERp29 staining was observed in demyelinating plaques. Our studies revealed that 4-PBA treatment not only limits viral replication and spread throughout the brain but also protects the mice against severe chronic neuroinflammatory demyelination. These findings suggest that targeting Cx43 with 4-PBA holds significant therapeutic potential for addressing virus-induced neuroinflammatory demyelination and MS by preserving gap junction intercellular communication.

neuroscience↗

bioRxiv2024

Inferring gene-pathway associations from consolidated transcriptome datasets: an interactive gene network explorer for Tetrahymena thermophila

Although an established model organism, Tetrahymena thermophila remains comparatively inaccessible to high throughput screens, and alternative bioinformatic approaches still rely on unconnected datasets and outdated algorithms. Here, we report a new approach to consolidating RNA-seq and microarray data based on a systematic exploration of parameters and computational controls, enabling us to infer functional gene associations from their co-expression patterns. To illustrate the power of this approach, we took advantage of new data regarding a previously studied pathway, the biogenesis of a secretory organelle called the mucocyst. Our untargeted clustering approach recovered over 80% of the genes that were previously verified to play a role in mucocyst biogenesis. Furthermore, we tested four new genes that we predicted to be mucocyst-associated based on their co-expression and found that knocking out each of them results in mucocyst secretion defects. We also found that our approach succeeds in clustering genes associated with several other cellular pathways that we evaluated based on prior literature. We present the Tetrahymena Gene Network Explorer (TGNE) as an interactive tool for genetic hypothesis generation and functional annotation in this organism and as a framework for building similar tools for other systems. Key PointsO_LIur approach integrates nearly 20-year-old microarray and contemporary RNA-seq datasets. C_LIO_LIrigorously compare co-expression clustering parametrization by way of computational controls. C_LIO_LICo-expression clustering identifies known and novel functionally associated genes in Tetrahymena. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/627356v2_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1dbe655org.highwire.dtl.DTLVardef@1ef09aaorg.highwire.dtl.DTLVardef@63b670org.highwire.dtl.DTLVardef@5e9209_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2024

Eukaryotic Translation Initiation Factor Loaded Extracellular Vesicles Promotes Macrophage Cholesterol Metabolism in ovarian cancer

Tumor-driven immune suppression poses a significant impediment to the success of immunotherapy in ovarian cancer. Among the various mechanisms contributing to immune suppression, intracellular communication facilitated by tumor-derived extracellular vesicles (EVs) within the tumor microenvironment (TME) emerges as a pivotal factor influencing tumor growth. We discovered that EVs from both ovarian tumor cell lines and the plasma of ovarian cancer patients are encapsulated with eukaryotic translation initiation factor 4E (eIF4E). Our study revealed a new mechanism showing how these EVs are loaded with eIF4E and its impact on ovarian cancer progression. We also demonstrated that eIF4E-containing EVs (eIF4E-EVs) alter protein translation in macrophages, contributing to anti-tumor immune response. Treatment of macrophages with eIF4E-EVs induces an immunosuppressive phenotype marked by the release of cytokines such as IL-6 and an elevated expression of Programmed death-ligand 1 (PD-L1). Notably, eIF4E-packaged EVs enhance the expression of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) a pivotal enzyme in cholesterol biosynthesis, resulting in increased cholesterol levels within macrophages. Inhibition of HMGCR or reduction of cholesterol in macrophages effectively restores their antitumor activity by decreasing PD-L1 on macrophages. Analysis of tumor tissue from ovarian cancer patients revealed a positive correlation between HMGCR and TAM in ovarian cancer. In summary, we have characterized the mechanism of how eIF4E loaded EVs induced cholesterol synthesis, creating an immunosuppressive environment by upregulating PD-L1 expression in macrophages.

cancer biology↗

bioRxiv2024

Investigating the Functional Role of the DI-DII Linker in Nav1.5 Channel Function

The cardiac voltage-gated sodium channel, Nav1.5 initiates the cardiac action potential. Its dysfunction can lead to dangerous arrhythmias, sudden cardiac arrest, and death. The functional Nav1.5 core consists of four homologous repeats (I, II, III, and IV), each formed from a voltage sensing and a pore domain. The channel also contains three cytoplasmic linkers (I-II, II-III, and III-IV). While Nav1.5 structures have been published, the I-II and II-III linkers have remained absent, are predicted to be disordered, and their functional role is not well understood. We divided the I-II linker into eight regions ranging in size from 32 to 52 residues, chosen based on their distinct properties. Since these regions had unique sequence properties, we hypothesized that they may have distinct effects on channel function. We tested this hypothesis with experiments with individual Nav1.5 constructs with each region deleted. These deletions had small effects on channel gating, though two (430 - 457del and 556 - 607del) reduced peak current. Phylogenetic analysis of the I-II linker revealed five prolines (P627, P628, P637, P640, P648) that were conserved in mammals but absent from the Xenopus sequence. We created mutant channels, where these were replaced with their Xenopus counterparts. The only mutation that had a significant effect on channel gating was P627S, which depolarized channel activation (10.13 +/- 2.28 mV). Neither a phosphosilent (P627A) nor a phosphomimetic (P627E) mutation had a significant effect, suggesting that either phosphorylation or another specific serine property is required. Since deletion of large regions had little effect on channel gating while a point mutation had a conspicuous impact, the I-II linker role may be to facilitate interactions with other proteins. Variants may have a larger impact if they create or disrupt these interactions, which may be key in evaluating pathogenicity of variants.

physiology↗

bioRxiv2024

Gut MicrobiotAware: how much do we know about gut microbiota? An international questionnaire.

Over the past two decades, our understanding of human gut microbiota composition and function has grown significantly. The surge in studies and data has unveiled the profound influence of gut microbiota on host nutrition, health, and behavior, bridging biology, medicine, and ecology. The dynamic interaction between daily choices, life events, and gut microbiota composition makes it a fascinating subject for scientists to communicate to diverse audiences. Effective scientific communication involves adapting the delivery of knowledge to different audiences, using precise language in academic settings and accessible concepts in public forums like science festivals or social media. With the growing interest in gut microbiota beyond academic circles, there is an increased risk of disseminating information lacking scientific rigor. This study aims to assess the general publics knowledge about gut microbiota and evaluate healthcare professionals understanding of its links to various health conditions, ultimately informing better communication strategies for both groups.

scientific communication and education↗

bioRxiv2024

Targeting Endoplasmic Reticulum Stress and Nitroso-Redox Imbalance in Neuroendocrine Prostate Cancer: The Therapeutic Role of Nitric Oxide

Neuroendocrine prostate cancer (NEPC) is an aggressive and therapy-resistant subtype of prostate cancer. Current standard-of-care treatment for NEPC involves chemotherapies, which largely exert their cytotoxic effects by forming DNA crosslinks, disrupting DNA replication and transcription in NEPC cells. However, these therapies are often met with resistance, partly due to increased endoplasmic reticulum (ER) stress, which facilitates cancer cell survival and adaptive mechanisms. Despite its critical role, the molecular landscape underlying ER stress in NEPC remains inadequately understood. Here we showed that ER stress is intimately linked to the metabolic reprogramming of NEPC cells, a process that supports their transition from adenocarcinoma to a neuroendocrine phenotype. We identified MYCN as a key driver of this process, promoting unfolded protein response (UPR) elements that enhance ER stress by increasing the efflux of calcium ions through the ER which later is absorbed by the mitochondria and assist in increasing the overall glycolytic stress, thereby adding to the extended survival and metastatic potential of an NEPC cell. Our previous studies highlighted the importance of S-nitrosylation as a protein modification that is dysregulated in high-grade PCa. In this context, structural analysis of MYCN revealed potential S-nitrosylation sites at the positions Cys4, 186, and 464, respectively. However, similar to the castration-resistant stage, this modification is hindered in NEPC due to impaired nitric oxide (NO) production from dysregulated endothelial nitric oxide synthases (eNOS). We found that exogenous NO supplementation S-nitrosylates MYCN, reducing its binding to protein molecules which are essential to assist with increasing ER stress in NEPC cells. Exogenous supplementation of NO reduced the overall tumor burden in the mice harboring orthotopic NEPC cells and reduced the metastasis to the brain and liver. In conclusion, the findings from this study enrich our understanding of the mechanisms driving the ER stress responses in NEPC phenotype and how NO supplementation could pave the way as potential therapeutics for this challenging cancer. HIGHLIGHTSO_LIEndoplasmic reticulum (ER) stress is intricately linked to metabolic reprogramming, which supports the transition from prostate adenocarcinoma to neuroendocrine prostate cancer (NEPC). C_LIO_LIMYCN increases the ER stress in NEPC cells and is correlated with increased nitroso-redox imbalance. C_LIO_LIStructural analysis reveals potential S-nitrosylation sites on MYCN. Exogenous nitric oxide (NO) supplementation induces S-nitrosylation, disrupting MYCNs role in enhancing ER stress. C_LIO_LINO supplementation reduced tumor burden and metastasis in NEPC-bearing mice, highlighting its potential as a therapeutic avenue for NEPC. C_LIO_LIExogenous NO supplementation inhibits ER stress by targeting unfolded protein response (UPR) elements and decreasing calcium ion efflux, inhibiting the glycolytic stress in NEPC. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=191 SRC="FIGDIR/small/624202v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@1fb292forg.highwire.dtl.DTLVardef@4d00f7org.highwire.dtl.DTLVardef@17a7f43org.highwire.dtl.DTLVardef@1393286_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Leveraging AlphaFold 3 for Structural Modeling of Neurological Disorder-Associated Proteins: A Pathway to Precision Medicine

Accurate structural modeling of neurological disorder-causing proteins provides an important layer in unraveling the mechanism of disease and identifying therapeutic targets. This study utilizes AlphaFold 3, a state-of-the-art protein structure prediction platform, to model and interpret cis- and trans-pQTL-derived proteins associated with Alzheimers disease, Parkinsons disease, and stroke. Using the NG00102 dataset, we created a high-resolution structure for more than 1,200 proteins expressed in Brain, CSF, and Plasma, providing tissue-specific protein structure analysis with associated functional implications. AlphaFold 3 predictions have illuminated key structure parameters including sequence length, average pLDDT confidence scores, and overall distribution of residues with confidence of >75% pLDDT. We used these features to determine the set of druggable proteins having optimal sequence lengths of 100-3000 residues, high structural reliability as evidenced by an average pLDDT > 80, and contain large regions of high-confidence residues. Tissue-specific mapping revealed unique mechanisms characterized by both cis and trans-pQTL effects, that have critical functional implications for how these genetic variants act in neurological disease pathways. Protein clusters by structural properties then led to more defined subgroups with potential implications for drug intervention. This integrated effort captures the strength of AlphaFold 3 in linking genetic variation to protein structure and function, providing a scalable pipeline for prioritizing therapeutic targets. Coupling our results with advanced predictive modeling and tissue-specific data sets provides a robust framework for uncovering new mechanisms and druggable targets in the research of Alzheimers, Parkinsons, and stroke. This advances the field toward precision medicine.

bioinformatics↗

bioRxiv2024

Combined analysis of somatic mutations and gene expression reveals nuclear speckles-associated enhanced stemness in gingivobuccal carcinoma under DNA damage response

Smokeless tobacco chewing habits in India lead to a high prevalence of Gingivobuccal oral squamous cell carcinoma (OSCC-GB). Cancer stem cells (CSCs) are a sub-population of cancer cells within a tumor with stem-like properties and are believed to contribute to tumor initiation, progression, increased resistance to drug therapy, and promote post-therapeutic cancer relapse. An RNA-seq data-based integrative analysis of somatic mutation and gene expression was performed to explore the role of CSCs in disease progression using the novel Indian-origin OSCC-GB cell line IIOC019 from a patient with tobacco-chewing habit. The identified DNA damage-related known mutational signature (ID1, SBS5) indicates the impact of smokeless tobacco-related carcinogens in the IIOC019 cell line. The differentially expressed somatic variants, functional impact predictions, and survival analysis reveal the role of DNA damage response (DDR)-related genes in OSCC-GB, with the SON gene as a significant player. The study suggests that the loss of function in a somatic variant of the SON gene is associated with enhanced stemness and increased risk of disease progression in OSCC-GB under DDR conditions. The newly identified CSC-associated somatic variants appear to promote cancer spread, local recurrence, and resistance to chemotherapy or radiotherapy, contributing to the high mortality rates among Indian OSCC-GB patients.

genomics↗

bioRxiv2024

DreaM: A Computational Pipeline for Enhanced Short-Read Sequence Analysis in Repetitive Genomic Regions

Mapping short sequencing reads to repetitive genomic regions, such as centromeres, presents significant challenges, primarily due to PCR duplicates, which can be erroneously mapped multiple times within these regions. Conventional bioinformatics pipelines often overlook this issue, potentially leading to misinterpretation as signal enrichment. To address this, we developed DreaM (Deduplication of Reads for Enhanced and Accurate Mapping), a computational pipeline that prioritises the preprocessing of raw sequencing data. DreaM firstly identifies and removes PCR duplicates, which is followed by read trimming to reduce noise from multiply mapped reads. When applied to ChIP-Seq and CUT&RUN datasets targeting CENP-A, a key marker of centromeres, DreaM demonstrated improved peak detection within centromeres. Overall, DreaM provides a robust solution for enhancing the analysis of DNA-protein binding sites in repetitive genomic regions using short-read sequencing.

bioinformatics↗

bioRxiv2024

Ultrafast elastocapillary fans control agile maneuvering in ripple bugs and robots

Millimeter-sized ripple bugs in the genus Rhagovelia exhibit exceptional agility and rapid maneuvers in fast, unsteady streams, comparable to animal fliers. Their remarkable interfacial transit and turning skills stem from a specialized fan structure on their middle legs. While researchers have suggested active fan actuation, the role of capillary forces and unique microstructure in self-spreading remains unclear. We reveal that Rhagovelias fans possess a flat-ribbon architecture with directional stiffness, enabling ultrafast elastocapillary morphing for passive actuation in under 10 ms, independent of muscle control, while producing high-thrust momentum through unsteady vortical wakes. These self-morphing fans allow Rhagovelia to execute [~]90 {degrees} turns at a rate of [~]4200 {degrees}/s, in [~]50 ms, with speeds reaching [~]120 BL/s - on par with the fastest recorded turns in animal fliers like fruit flies. Inspired by these, we develop an ultralight, ultrafast elastocapillary robotic fan ([~]1 mg, [~]100 ms opening/closing time) and integrate it into an insect-scaled robot (Rhagobot, [~]0.2 g). The engineered fans passively balance surface tension and water drag through stiffness anisotropy, enabling the robot to achieve high agility with speeds up to [~]2 BL/s and turning rates of 206 {degrees}/sec. Experiments with both insects and robots, with and without fans, show that a self-spreading passive fan significantly improves thrust, braking, and turning - key factors for controlled, high-speed maneuvers. This elastocapillary innovation enables ripple bugs to survive and thrive in turbulent streams and offers new insights for agile aquatic robotics.

biophysics↗

bioRxiv2024

Measuring Growth, Resistance and Recovery after Artemisinin Treatment of Plasmodium falciparum in a semi-high-throughput Assay

BackgroundArtemisinin partial resistance (ART-R) has spread throughout Southeast Asia and mutations in pfKelch13, the molecular marker of resistance, are widely reported in East Africa. Effective in vitro assays and robust phenotypes are crucial for monitoring populations for the emergence and spread of resistance. The recently developed extended Recovery Ring-stage Survival Assay used a qPCR-based readout to reduce the labor intensiveness for in vitro phenotyping of ART-R and improved correlation with the clinical phenotype of ART-R. Here, we extend and refine this assay to include measurements of parasite growth and recovery after drug exposure. Clinical isolates and progeny from two genetic crosses were used to optimize and validate the reliability of a straight-from-blood, SYBR Green-based qPCR protocol in a 96-well plate format to accurately measure phenotypes for Growth, Resistance, and Recovery. ResultsThe assay determined growth between 6 h and 96 h, resistance at 120 h, and recovery from 120 h and 192 h. Growth can be accurately captured by qPCR and is shown by reproduction of previous growth phenotypes from HB3 x Dd2. Resistance measured at 120 h continually shows the most consistent phenotype for ring stage susceptibility. Recovery identifies an additional response to drug than parasites that are determined sensitive by Fold Change at 120 h. Comparison of progeny phenotypes for Growth vs Resistance showed a minor but significant correlation, whereas Growth vs Recovery and Resistance vs Recovery showed no significant correlation. Additionally, dried blood spot (DBS) samples matched Fold Change measured from liquid samples demonstrating Resistance can be easily quantified using either storage method. ConclusionsThe qPCR-based methodology provides the throughput needed to quickly measure large numbers of parasites for multiple relevant phenotypes. Growth can reveal fitness defects and illuminate relationships between proliferation rates and drug response. Recovery serves as a complementary phenotype to resistance that quantifies the ability of sensitive parasites to tolerate drug exposure. All three phenotypes offer a comprehensive assessment of parasite-drug interaction each with independent genetic determinants of main effect and overlapping secondary effects that should be further. By adapting our method to include DBS, readouts can be easily extended to ex vivo surveillance applications.

microbiology↗

bioRxiv2024

PolSpec: polarisation-based detection for spectral classification of optical signals

Spectrally resolved imaging is typically realised using bandpass filters, which are inefficient when they reject "out-of-band" photons or using angularly dispersive devices with at least one image dimension requiring scanning to acquire a full hyperspectral dataset and therefore sequential data acquisition, unless cascades of dichroic beamsplitters are employed, for which cost and experimental complexity scale with the number of spectral channels. Here we present a new approach, "PolSpec", to realise rapid and flexible widefield hyperspectral imaging with lower cost and complexity using polarisation optics instead of dielectric coatings or dispersive devices. PolSpec utilises Lyot filters that provide continuously varying transmission across the desired spectral range to generate orthogonal "spectral modulation vectors" that can represent specific spectral signatures with significantly lower data volumes than full spectral profiles. We demonstrate single-shot widefield hyperspectral imaging using a polarisation-resolving camera and rapid, electronically reconfigurable, more photon efficient, hyperspectral imaging using a liquid crystal variable retarder.

biophysics↗

bioRxiv2024

Engineered Bxb1 variants improve integrase activity and fidelity

Many current genome editing technologies rely on the action of large serine integrases (LSIs) to insert gene-sized DNA sequences into the genome. Bxb1 is the most commonly used LSI for therapeutic efforts, including PASTE, PASSIGE and I-PGI. While Bxb1 demonstrated good activity in vitro in cycling cells, the activity in non-dividing hepatocytes was significantly less efficient. Further, wild-type Bxb1 is known to have detectable off-target activity at cryptic genomic sites, which presents a potential safety risk for therapeutic development. To address these issues, we developed Bxb1 variants that demonstrate increased specificity and potency in vitro and have engineered stabilized Bxb1 variants that increase in vivo activity over 25-fold enabling targeted integration at therapeutically relevant levels.

biochemistry↗

bioRxiv2024

CXCL10/CXCR3 Signaling Induces Neural Senescence and Cognitive Impairments

Chemokine receptors belong to the G-protein-coupled receptors family and multiple lines of emerging evidence suggest that several chemokines are elevated in aging associated with central nervous system disorders. Increased level of CXCL10 in central nervous system is reported in several neurodegenerative diseases, including Multiple sclerosis, Alzheimers disease, and Virus-associated dementia. We also observed significantly increased expression of CXCL10 and CXCR3 in the prefrontal cortex and hippocampus of aged C57BL/6J mice (12- and 18-month-old mice). This leads us to hypothesize that CXCL10, being a component of SASPs, may aggravate/perpetuate the brain aging process and, finally, neurodegenerative diseases. To test this hypothesis, we administered CXCL10 (intracerebroventricular: ICV, 0.5 pg/ hrs, 28 days) in 8-month-old C57BL/6J mice. We observed increased expression of senescent marker proteins p16INK4a, p21Cip1, p53 and decreased expression of pRB in the prefrontal cortex, which was blocked by CXCR3-specific antagonist AMG487. Furthermore, chronic infusion of CXCL10 induced learning and memory deficits in Y-maze, social recognition, contextual freeze tests and c-FOS expression in the prefrontal cortex. To further determine the specificity of CXCL10/CXCR3 signaling, we treated the primary cortical neuron (Days in vitro: DIV 7-8) with CXCL10 and found increased senescence in CXCR3 dependent fashion. Using RFP-EGFP-LC3-tagged transgenic mice, we also showed CXCL10/CXCR3 signaling attenuates autophagic flux in primary cortical neurons. Lastly, using a c-FOS-iRFP reporter, we observed that increased CXCL10/CXCR3 signaling impairs glutamatergic signaling in primary cortical neurons. These results support the hypothesis that increased CXCL10/CXCR3 facilitates brain aging and could be targeted for the management of aging-associated CNS disorders.

neuroscience↗

bioRxiv2024

Emergence of Dip2-mediated Specific DAG-based PKC Signalling Axis in Eukaryotes

Diacylglycerols (DAGs) are used for metabolic purposes and are tightly regulated secondary lipid messengers in eukaryotes. DAG subspecies with different fatty-acyl chains are proposed to be involved in the activation of distinct PKC isoforms, resulting in diverse physiological outcomes. However, the molecular players and the regulatory origin for fine-tuning the PKC pathway are unknown. Here, we show that Dip2, a conserved DAG regulator across Fungi and Animalia, has emerged as a modulator of PKC signalling in yeast. Dip2 maintains the level of a specific DAG subpopulation, required for the activation of PKC-mediated cell wall integrity pathway. Interestingly, the canonical DAG-metabolism pathways, being promiscuous, are decoupled from PKC signalling. We demonstrate that these DAG subspecies are sourced from a phosphatidylinositol pool generated by the acyl-chain remodelling pathway. Furthermore, we provide insights into the intimate coevolutionary relationship between the regulator (Dip2) and the effector (PKC) of DAG-based signalling. Hence, our study underscores the establishment of Dip2-PKC axis about 1.2 billion years ago in Opisthokonta, which marks the rooting of the first specific DAG-based signalling module of eukaryotes.

cell biology↗

bioRxiv2024

Model-free machine learning-based 3D single molecule localisation microscopy

Single molecule localisation microscopy (SMLM) can provide two-dimensional super-resolved image data from conventional fluorescence microscopes, while three dimensional (3D) SMLM usually involves a modification of the microscope, e.g. to engineer a predictable axial variation in the point spread function. Here we demonstrate a 3D SMLM approach (we call "easyZloc") utilising a lightweight Convolutional Neural Network that is generally applicable, including with "standard" (unmodified) fluorescence microscopes, and which we consider may be practically useful in a high throughput SMLM workflow. We demonstrate the reconstruction of nuclear pore complexes with comparable performance to previously reported methods but with a significant reduction in computational power and execution time. 3D reconstructions of the nuclear envelope and a tubulin sample over a larger axial range are also shown.

biophysics↗

bioRxiv2024

Curative levels of endogenous gene replacement achieved in non-human primate liver using programmable genomic integration

The ability to efficiently place a large piece of DNA in a specific genomic location has been a goal for the gene therapy field since its inception; however, despite significant advances in gene editing technology, this had yet to be achieved. Here we describe two methods of programmable genomic integration (PGI) that overcome some of the limitations of current approaches. Using a combination of clinically validated delivery technologies (LNP, AAV), we demonstrate the ability to specifically integrate large (>2 kb) DNA sequences into endogenous introns in the liver of non-human primates (NHP). PGI was effective across multiple genomic locations and transgenes, and insertion led to expression from the endogenous promoter. PGI was highly efficient, achieving expression in >50% of liver cells after a single course of treatment, which would be curative for most monogenic recessive liver diseases. This is the first report of clinically curative level of gene insertion at endogenous loci in NHP.

bioengineering↗

bioRxiv2024

Optimal Prescriptive Treatments for Ovarian Cancer with Genetic Data

Among the cancers affecting the female population, Ovarian Cancer (OC), while being relatively rare, is the leading cause of gynecological cancer-related deaths, with overall 5-year survival rates of approximately 50% for all stages combined. This is because of the challenges associated with the diagnosis, resulting in detection at advanced stages of OC, coupled with the slow progress in effective treatment options since the approval of platinum-based chemotherapy in the late 1970s. There has been a relative lack of sophisticated methods based on Machine Learning (ML) models that use genetic data for better prediction of Ovarian Cancer outcomes and result in more effective treatment recommendations. Therefore, there is an unmet clinical need to create models that allow physicians to make informed decisions based on all available data, including patient demographic, social, health, and genomic data. Hence, we develop new techniques for leveraging genetic information in prescribing optimal treatments for patients with OC, using a publicly available dataset from the Prostate, Lung, Colorectal and Ovarian Cancer (PLCO) trial. Our approach is able to transform genotype sequencing information into a simple tabular form that can then be used as the input to any ML model. Coupled with the recorded treatment regimen and clinical parameters of matched patients from the genetic dataset, we estimate the treatment effect in terms of mortality prediction and use it to prescribe the optimal treatment for any given patient. By including the genetic features engineered through our proposed method, our models have a higher accuracy than the models without genetic information embedded. The increase in predictive accuracy demonstrates the improved efficacy of our method in the predictive setting. Furthermore, in the prescriptive setting, the models including genetic features output different treatment choices for patients, showing the impact of their inclusion. This is further highlighted by the feature importance of the genetic features such as mutations in the FAT3, BRCA1, BRCA2, and NF1 genes, where they rank highly with a tighter aggregation of the top features, relative to the sharp drop-off in feature importance after the top feature in the models without genetic data. Taken together, in summary, our models will allow oncologists to make more informed and accurate decisions, incorporating a patients genetic data with all other available clinical information, which has the potential for improved prognosis and better long-term survival outcomes for Ovarian Cancer patients.

cancer biology↗