Biology subjects

Sharma, S.

Publications and source records attributed to Sharma, S..

At least 163 records · Page 9Linked to original sources

bioRxiv2025

Smoke exposure triggers rapid transcriptional changes and reveals glycosyltransferases linked to smoke taint in ripening grape berries

With the increasing frequency of wildfires, vineyards are more often exposed to smoke, resulting in a higher risk of smoke taint in wine. This taint imparts undesirable "smoky" and "ashy" aromas and causes significant economic losses. Smoke-derived volatile phenols (VPs) in grape berries, metabolized into stable, non-volatile glycoconjugates via glycosyltransferases (GTs), underlie smoke taint formation. Here, we present the dwarf grapevine cultivar Pixie as a model system for smoke taint research. We generated a phased, telomere-to-telomere diploid genome using HiFi long-read sequencing, along with an expression atlas spanning 30 replicated samples across various tissues and developmental stages. We also constructed a second telomere-to-telomere assembly for Pinot Noir to assess GT expression during berry ripening in the field. Controlled smoke-exposure experiments profiled GT expression and enabled transcriptome-wide analyses at multiple time points. We measured VPs in both smoke-exposed and control samples, revealing 12 GT1 genes significantly upregulated by smoke, named Vitis vinifera smoke-inducible UGTs (VviSIUGTs). Their expression peaked one day post-exposure and declined by day three, mirroring glycoside accumulation dynamics. Gene co-expression network analyses highlighted stress-responsive modules enriched in these VviSIUGTs, while transcription factor binding site analyses pinpointed stress-related regulatory elements in their promoters. These findings provide new insights into the molecular basis of smoke taint and identify potential targets for breeding and biotechnological interventions aimed at developing grapevine cultivars with reduced susceptibility to smoke damage.

plant biology↗

bioRxiv2025

Metagenomic Profiling of Drinking Water Microbiomes: Insights into Microbial Diversity and Antimicrobial Resistance

Monitoring microbial components in drinking water is as essential as tracking its chemical composition. Although traditional culture-based methods provide valuable insight into microbial morphology and behaviour, their scope is restricted to culturable species. With the advent of high-throughput sequencing, we can now detect a wider range of microbes in any ecosystem, along with efficient insights into their functional potential and metabolic capabilities. In this study, metagenomic analyses were performed to fully understand the microbiome of drinking water supplied through public distribution systems in an Indian city. Our findings identified bacteria from the phyla Pseudomonadota, Planctomycetota, Bacteroidota, and Actinomycetota, consistent with previous studies of drinking water microbiomes of other countries. At the species level, Afipia carboxidovorans, Klebsiella pneumoniae, Pseudomonas aeruginosa, Sphingopyxis macrogoltabida, and Variovorax paradoxus were identified as members of the core microbiome. It was observed that the temperature of the water samples, even as little as a 5{o}C increase, influenced the composition and diversity of the microbial communities. No significant correlation was detected between the abundance of microbial species and the metal concentration in the sample. In addition, we traced the distribution of antibiotic resistance genes (ARGs), finding widespread resistance to aminoglycosides, tetracyclines, and macrolides in samples. In particular, ARGs such as adeF and ermR, which are known to be associated with multidrug resistance, were detected. Although this study did not directly assess the pathogenicity or mobility of these genes, their presence in potable water raises potential public health concerns due to the possibility of horizontal gene transfer (HGT) in environmental settings. Therefore, continuous monitoring of antibiotic resistance genes (ARGs) is imperative to accurately evaluate long-term risks and to guide evidence-based water quality management strategies. In summary, this study provides a comprehensive metagenomic overview of drinking water microbiota, ARGs, and water quality, offering a foundation for future surveillance and risk mitigation strategies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/636216v3_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@819c0org.highwire.dtl.DTLVardef@1d29372org.highwire.dtl.DTLVardef@1ce3cf9org.highwire.dtl.DTLVardef@1050997_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

B7-H3 as a Dual Clinically Relevant Checkpoint and Antibody Drug Conjugate Target Expressed Across Adenocarcinoma and Neuroendocrine Prostate Cancers

Background and ObjectivesCD276 (B7-H3) has recently emerged as a promising presumptive immune checkpoint inhibitor (ICI) and a potential antibody-drug conjugate (ADC) target for prostate cancer (PCa). We evaluated B7-H3 and 15 other clinically relevant ADC and ICI targets for expression at the RNA and protein level across the PCa continuum--hormone-sensitive, castration-resistant, and neuroendocrine. MethodsCCLE data analysis and western blot experiments were performed for quantifying RNA and protein expression variability across PCa cell lines. Inter- and intratumoral heterogeneity was evaluated by integrating single-cell RNA sequencing data across 595k cells and 102 patients, spanning the disease continuum. AR and B7-H3 knockouts of PCa cell lines were developed and investigated using R1881 and Enzalutamide to elucidate the AR-CD276 signaling pathway through qRT-PCR and flow cytometry. Key FindingsB7-H3 showed high expression in tumor and myeloid cells (tumor microenvironment - TME), lowest heterogeneity across all ADC and ICI targets, and was negatively regulated by androgen signaling. LimitationsFurther validation of ADC and ICI target protein expression in human samples and more exhaustive exploration of the AR-CD276 signaling cascade is required. ConclusionB7-H3 demonstrates the least susceptibility to selective pressure due to its stable expression across PCa disease states. Clinical ImplicationsB7-H3 represents an important ADC target for PCa due to its potential to minimize drug resistance and likely ability to be a valid target across the prostate cancer continuum. Additionally, its expression in myeloid cells supports a dual role as an ICI, further enhancing its therapeutic relevance. Graphical AbstractDiscovery of optimal checkpoints and antibody drug conjugates (ADCs) in Prostate Cancer (PCa). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/632253v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@1af1115org.highwire.dtl.DTLVardef@8a9d70org.highwire.dtl.DTLVardef@6659aeorg.highwire.dtl.DTLVardef@189e200_HPS_FORMAT_FIGEXP M_FIG C_FIG Patient SummaryIn this study, we demonstrate that prostate cancer expresses high amounts of a cell surface protein called B7-H3 both when first diagnosed and throughout various stages of metastatic disease. Furthermore, we demonstrate a clear and yet to be fully resolved cross-talk between B7-H3 and the androgen signaling pathway central to prostate cancer development. We conclude that B7-H3 is therefore a highly promising therapeutic cell surface protein expressed by prostate cancer due to its stable expression potentially leading to low selective pressure and reduced drug resistance--thereby having significant implications for clinical trial design and patient inclusion considerations. Advancing PracticePCa is a highly heterogeneous disease with an increasingly wide treatment landscape, governed by various factors including clinical covariates, histopathological features, and molecular factors. B7-H3 has recently emerged as a promising clinical target for immunotherapy in the localized setting and antibody drug targeting in the metastatic setting. Despite the clinical significance, B7-H3 expression has not yet been thoroughly evaluated against other clinically relevant immune checkpoint and antibody drug targets, especially along the continuum of PCa treatment stages--hormone sensitive, castration resistant, and neuroendocrine. To our knowledge, this is the first study to characterize B7-H3 in this continuum using representative cell lines and the largest collection of PCa patient single cell sequencing data yet assembled in the literature, thus accounting for the substantial heterogeneity across PCa disease states as well as between datasets. Take Home MessageThis article explores the therapeutic relevance of clinical stage ADC targets and immune checkpoints for Prostate Cancer (PCa). We show that B7-H3 is a highly promising therapeutic candidate for PCa due to its stable expression across various disease states.

cancer biology↗

bioRxiv2025

Investigation of All Disease-Relevant Lysine Acetylation Sites in α-Synuclein Enabled by Non-canonical Amino Acid Mutagenesis

Aggregates of -synuclein (S) are hallmarks of synucleinopathies, including Parkinsons Disease (PD) and Multiple System Atrophy (MSA). We have recently shown that S lysine acetylation in the soluble monomer pool varies between healthy controls, PD, and MSA patients. We used non-canonical amino acid (ncAA) mutagenesis to express and purify all 12 currently known disease-relevant variants of S and studied their binding to membranes as well as their aggregation propensities. We found that acetylation of lysine 12, 43, and 80 had particularly strong effects. To understand the implications for acetylation of monomeric S found in healthy cells, we performed NMR experiments to study protein conformation and fluorescence correlation spectroscopy experiments to quantify lipid binding. We also investigated the effects of acetylation at lysine 12, 43, and 80 on fibril seeding in neurons. Collectively, our biochemical and cell biological investigations indicated that acetylation of K80 could inhibit aggregation without conferring negative effects on monomer function in healthy cells. Therefore, we studied the structures of fibrils with K80 acetylation through cryo-electron microscopy to uncover the structural basis for these effects. Finally, we identified inhibition of HDAC8 as a way of potentially increasing acetylation at K80 and other inhibitory sites for therapeutic benefit.

biochemistry↗

bioRxiv2025

Damage signals preferentially activate killer CD8+/- regulatory T cells to protect injured tissue

Self-antigens that are obscure to immune cells under homeostasis, are exposed following tissue damage and can trigger autoimmunity. Our previous work showed that conventional type 1 dendritic cells (cDC1s) mediate immunoregulation after traumatic skeletal muscle injury. Here, we found that, immune responses to injury in cDC1-depleted mice (Batf3-/-) mirror those of autoimmune mice (Aire-/-). Mechanistically, we determined that cDC1s prime killer regulatory T cells (CD8+/-Ly49+Tregs) which express Ly49 inhibitory receptors and HELIOS. These killer-like Tregs are clonally diverse and carry T cell receptors associated with self-reactivity and response to hydrophobicity. cDC1 or CD8 deletion promoted CD62L+CCR7+ naive T cell retention and B cell recruitment to injured muscle. These naive T cells, which are implicated in autoimmunity, strongly correlate with B cell abundance in the muscle and are selectively pruned by CD8+/-Ly49+ Tregs. Furthermore, clinically used materials that promote wound healing enrich CD8+/-Ly49+ Treg function whereas those that are associated with pathology promote naive T cell and B cell accumulation. We hypothesize that CD8+/-Ly49+ Tregs maintain self-tolerance after tissue damage and avert autoimmunity by eliminating naive T cells and preventing pathogenic B cell activation.

immunology↗

bioRxiv2025

Evolutionary sparse learning with paired species contrast reveals the shared genetic basis of convergent traits

Cases abound in which nearly identical traits have appeared in distant species facing similar environments. These unmistakable examples of adaptive evolution offer opportunities to gain insight into their genetic origins and mechanisms through comparative analyses. Here, we present a novel comparative genomics approach to build genetic models that underlie the independent origins of convergent traits using evolutionary sparse learning. We test the hypothesis that common genes and sites are involved in the convergent evolution of two key traits: C4 photosynthesis in grasses and echolocation in mammals. Genetic models were highly predictive of independent cases of convergent evolution of C4 photosynthesis. These results support the involvement of sequence substitutions in many common genetic loci in the evolution of convergent traits studied. Genes contributing to genetic models for echolocation were highly enriched for functional categories related to hearing, sound perception, and deafness (P < 10-6); a pattern that has eluded previous efforts applying standard molecular evolutionary approaches. We conclude that phylogeny-informed machine learning naturally excludes apparent molecular convergences due to shared species history, enhances the signal-to-noise ratio for detecting molecular convergence, and empowers the discovery of common genetic bases of trait convergences.

evolutionary biology↗

bioRxiv2025

MLC1 alteration in iPSCs give rise to disease-like cellular vacuolation phenotype in the astrocyte lineage

BackgroundMegalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare and progressive neurodegenerative disorder involving the white matter, is not adequately recapitulated by current disease models. Somatic cell reprogramming, along with advancements in genome engineering, may allow the establishment of in-vitro human models of MLC for disease modeling and drug screening. In this study, we utilized cellular reprogramming and gene-editing techniques to develop induced pluripotent stem cell (iPSC) models of MLC to recapitulate the cellular context of the classical MLC-impacted nervous system. MethodsSomatic cell reprogramming of peripheral patient-derived blood mononuclear cells (PBMCs) was used to develop iPSC models of MLC. CRISPR-Cas9 system-based genome engineering was also utilized to create the MLC1 knockout model of the disease. Directed differentiation of iPSCs to neural stem cells (NSCs) and astrocytes was performed in a 2D cell culture format, followed by various cellular and molecular biology approaches, to characterize the disease model. ResultsMLC iPSCs established by somatic cell reprogramming and genome engineering were well characterized for pluripotency. iPSCs were subsequently differentiated to disease-relevant cell types: neural stem cells (NSCs) and astrocytes. RNA sequencing profiling of MLC NSCs revealed a set of differentially expressed genes related to neurological disorders and epilepsy, a common clinical finding within MLC disease. This gene set can serve as a target for drug screening for the development of a potential therapeutic for this disease. Upon differentiation to the more disease relevant cell type-astrocytes, MLC-characteristic vacuoles were clearly observed, which were distinctly absent from controls. This emergence recapitulated a distinguishing phenotypic marker of the disease. ConclusionThrough the creation and analyses of iPSC models of MLC, our work addresses a critical need for relevant cellular models of MLC for use in both disease modeling and drug screening assays. Further investigation can utilize MLC iPSC models, as well as generated transcriptomic data sets and analyses, to identify potential therapeutic interventions for this debilitating disease.

cell biology↗

bioRxiv2024

Phototropin localization and interactions regulate photophysiological processes in Chlamydomonas reinhardtii

Phototropin, a blue-light sensing serine/threonine kinase, plays a pivotal role in regulating diverse photophysiological processes in both plants and algae. In Chlamydomonas reinhardtii, phototropin (CrPhot) localizes to the eyespot and flagella, coordinating key cellular functions such as phototaxis, photosynthesis, gametogenesis, and chlorophyll biosynthesis. While previous research has identified phototropin interactions with signaling proteins such as channelrhodopsins and light-harvesting complex proteins, many aspects of its interaction network and regulatory mechanisms remain unresolved. In this study, we explored novel interacting protein partners of phototropin and their roles in modulating its regulatory functions in Chlamydomonas reinhardtii. Employing a suite of intraflagellar transport (IFT) mutants of C. reinhardtii such as IFT172, IFT52, IFT88, IFT139, kinesin/dynein, CEP290 etc., we elucidate that phototropin localization within the flagella and eyespot is IFT-mediated. Our study highlights interaction of phototropin with other photoreceptors-channelrhodopsins (ChR1 and ChR2), chlamyopsin 6, LOV-histidine kinases (LOV-HK1, LOV-HK2) and signaling protein-14-3-3. CRISPR-Cas9 knockouts of phototropin showed reduced ChR1, 14- 3-3 levels and exhibited impaired photomotility. Moreover, two LOV-domain containing histidine kinases, LOV-HK1 and LOV-HK2, were identified in C. reinhardtii. Gene expression of LOV-HK1 and LOV-HK2 were found to be elevated in UV-light in C. reinhardtii and their genes expression was found to be altered in phototropin CRISPR-Cas9 knockouts. This study provides new insights into phototropin signalosome and highlights molecular mechanisms governing its function. The research outcomes advances our understanding of phototropin trafficking and signal modulation in Chlamydomonas reinhardtii, and sets the stage for further exploration into the broader physiological roles of phototropin in cellular responses. Graphical abstractPhototropin, a blue-light receptor in Chlamydomonas reinhardtii, localizes to the flagella and eyespot, mediates phototaxis and photosynthesis. Its trafficking is mediated by intraflagellar transport (IFT) machinery, with mutations in IFT components (kinesin, dynein, IFT172, IFT52, IFT88, IFT139, CEP290) disrupting phototropin localization. Phototropin interacts with other photoreceptors like channelrhodospins (ChR1/2), chlamyopsin 6, LOV-histidine kinases (LOV-HK1, LOV-HK2) and signaling proteins (14-3-3), coordinating light-driven responses. These findings underscore the details of phototropin trafficking and phototropin signaling impacting light-induced physiological processes in C. reinhardtii. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/630506v1_ufig1.gif" ALT="Figure 1000"> View larger version (51K): org.highwire.dtl.DTLVardef@29f6b3org.highwire.dtl.DTLVardef@10382aeorg.highwire.dtl.DTLVardef@1786f0dorg.highwire.dtl.DTLVardef@1c2ed65_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights* Phototropin localizes in eyepot and flagella in Chlamydomonas reinhardtii. * Intraflagellar transport (IFT) mutants of C. reinhardtii suggest role of different IFT proteins in phototropin trafficking and localization. * Phototropin interacts with other photoreceptors (ChR1 & ChR2, COP6, LOV-HK1 & LOV-HK2) and signaling proteins (14-3-3), contributing to various physiological processes. * CRISPR-Cas9 knockouts of phototropin showed reduced 14-3-3 protein content and photomotility response in C. reinhardtii.

plant biology↗

bioRxiv2024

IL-1β-driven NF-κB transcription of ACE2 as a Mechanism of Macrophage Infection by SARS-CoV-2

Coronavirus disease 2019 (COVID-19), caused by infection with the enveloped RNA betacoronavirus, SARS-CoV-2, led to a global pandemic involving over 7 million deaths. Macrophage inflammatory responses impact COVID-19 severity; however, it is unclear whether macrophages are infected by SARS-CoV-2. We sought to identify mechanisms regulating macrophage expression of ACE2, the primary receptor for SARS-CoV-2, and to determine if macrophages are susceptible to productive infection. We developed a humanized ACE2 (hACE2) mouse whereby hACE2 cDNA was cloned into the mouse ACE2 locus under control of the native promoter. We validated the susceptibility of hACE2 mice to SARS-CoV-2 infection relative to wild-type mice and an established K18-hACE2 model of acute fulminating disease. Intranasal exposure to SARS-CoV-2 led to pulmonary consolidations with cellular infiltrate, edema, and hemorrhage, consistent with pneumonia, yet unlike the K18-hACE2 model, hACE2 mice survived and maintained stable weight. Infected hACE2 mice also exhibited a unique plasma chemokine, cytokine, and growth factor inflammatory signature relative to K18-hACE2 mice. Infected hACE2 mice demonstrated evidence of viral replication in infiltrating lung macrophages, and infection of macrophages in vitro revealed a transcriptional profile indicative of altered RNA and ribosomal processing machinery as well as activated cellular antiviral defense. Macrophage IL-1{beta}-driven NF-{kappa}B transcription of ACE2 was an important mechanism of dynamic ACE2 upregulation, promoting macrophage susceptibility to infection. Experimental models of COVID-19 that make use of native hACE2 expression will allow for mechanistic insight into factors that can either promote host resilience or increase susceptibility to worsening severity of infection.

immunology↗

bioRxiv2024

Genetic ancestry and population structure in the All of Us Research Program cohort

The NIH All of Us Research Program (All of Us) aims to build one of the worlds most diverse population biomedical datasets in support of equitable precision medicine. For this study, we analyzed participant genomic variant data to assess the extent of population structure and to characterize patterns of genetic ancestry for the All of Us cohort (n=297,549). Unsupervised clustering of genomic principal component analysis (PCA) data revealed a non-uniform distribution of genetic diversity and substantial population structure in the All of Us cohort, with dense clusters of closely related participants interspersed among less dense regions of genomic PC space. Supervised genetic ancestry inference was performed using genetic similarity between All of Us participants and global reference population samples. Participants show diverse genetic ancestry, with major contributions from European (66.4%), African (19.5%), Asian (7.6%), and American (6.3%) continental ancestry components. Participant genetic similarity clusters show group-specific genetic ancestry patterns, with distinct patterns of continental and subcontinental ancestry among groups. We also explored how genetic ancestry changes over space and time in the United States (US). African and American ancestry are enriched in the southeast and southwest regions of the country, respectively, whereas European ancestry is more evenly distributed across the US. The diversity of All of Us participants genetic ancestry is negatively correlated with age; younger participants show higher levels of genetic admixture compared to older participants. Our results underscore the ancestral genetic diversity of the All of Us cohort, a crucial prerequisite for genomic health equity.

bioinformatics↗

bioRxiv2024

Embryonic exposures to flame retardant tetrabromobisphenol A (TBBPA) disrupts dorsoventral patterning in zebrafish

Tetrabromobisphenol A (TBBPA), a widely used flame retardant in commercial products such as synthetic textiles, plastics, and electronics poses potential toxicity risks through indoor exposure. This study aims to leverage zebrafish as a model to study TBBPA impacts on zebrafish dorsoventral patterning--a process that lays the foundation of an embryos axial determination and localization of specific tissues and organs. Zebrafish embryos were exposed to varying concentrations of TBBPA (0-10 {micro}M) at either 0.75- or 6-hours post-fertilization (hpf) and phenotyped at 8 or 24 hpf. Following this, whole-mount immunohistochemistry (IHC) was conducted out to quantify various proteins important in the BMP signaling pathway, epithelial-to-mesenchymal transition (EMT) and ectoderm and mesoderm germ layers. Importantly, these assessments were done at environmentally relevant concentrations ranging down to nM and pM levels. Our data showed a significant concentration-dependent increase in ventralization phenotypes, marked by enlarged blood island area, coupled with a disruption of the ventral-to-dorsal gradient of pSMAD protein levels, indicating BMP signaling disruptions. Perturbations in epithelial-to-mesenchymal transition (EMT), as evidenced by changes in E- and N-cadherin expression and Snail2 transcription factor levels, indicated impaired cell migration on TBBPA exposures. We then interrogated if TBBPA impacts germ layers and structures derived from specific germ layers. We observed significant concentration-dependent changes in levels of Sox2 and Sox10- both indicators of neural crest cell formation and differentiation from the ectoderm. We also observed a significant reduction of Tbx16- a marker of paraxial mesodermal cells. Collectively, both these data show TBBPA-induced impact on germ layers. Finally, we examined specific cell types derived from ectoderm and mesoderm and showed TBBPA-induced inhibition of cartilage development (derived from ectodermal neural crest cells) and blood cell development (derived from mesodermal cells). Our findings collectively demonstrate that TBBPA-induced disruptions in early developmental signaling and dorsoventral patterning may contribute to systemic toxicity in zebrafish embryos. Importantly, we see these disruptions at environmentally relevant concentrations, reinforcing the importance of continued interrogation of TBBPA in targeting early embryogenesis.

pharmacology and toxicology↗

bioRxiv2024

Extracellular Exosomal RNAs are Glyco-Modified

Epitranscriptomic modifications play pivotal roles in regulating RNA function, encompassing base alterations and the addition of both canonical m7G and noncanonical nucleotide metabolite caps. Recently, the spectrum of modifications has extended to include glyco modification at the 5 cap or within the RNA. Despite this expansion, the functional implications of glyco modification on RNA remain elusive. Our study reveals that mammalian cells labeled with N-azidoacetylgalactosamine-tetraacylated (Ac4GalNAz) produce small noncoding and nonpolyadenylated glyco-modified RNA (glycoRNA), primarily localized within exosome vesicles. The resistance of glycoRNA-containing exosomes to RNase treatment suggests that Ac4GalNAz-derived glycoRNA constitutes intraluminal cargo, distinct from recently reported cell surface glycoRNAs. Furthermore, we demonstrate that exosome cargo can be transferred to naive cells, underscoring exosome-mediated intercellular communication of glycoRNAs. Inhibition of exosome biogenesis leads to the accumulation of intracellular glycoRNA while blocking glycan transfer to proteins concomitantly reduced the targeting of glycoRNA within exosomes. These findings highlight a correlation between protein and RNA glycosylation, suggesting that the accumulation of glycoRNAs within exosomes is a regulated process. Our results support a functional role for glyco-modification in mediating RNA targeting into exosomes, offering new insights for enhancing recent advances in exosome-directed diagnostics and therapeutic applications.

cell biology↗

bioRxiv2024

Preclinical Proof of Concept for SNAPTM-TIL: a personalized, next-generation tumor-infiltrating lymphocyte therapy platform

Tumor-infiltrating lymphocyte (TIL) therapy, which involves extracting, expanding, and reinfusing immune cells to target cancer cells, has shown promise in melanoma treatment, but requires optimization for broader efficacy. The success of TIL therapy depends on the recognition of tumor-associated antigens, but neoantigen-reactive T-cells are often rare and exhausted in less immunogenic malignancies. Isolating T cells enriched in neoantigen reactivity prior to in vitro expansion and reinfusion may improve the response rates. To this end, our proprietary Specific Neo-Antigen Peptides (SNAP) technology platform improves the accuracy of neoantigen prediction and validation by combining advanced computational modeling and PepSeq, a high-throughput screen for the physical credentialing of putative neoantigens based on their affinity to bind patient-specific HLA class II proteins. This approach allows for the education and enrichment of TILs (SNAP-TILs) with personalized, predefined, highly immunogenic neoantigens prior to expansion. Using the SNAP platform, we consistently achieved, on average, an SNAP-TIL product comprising 96% CD3+ cells, with a mixture of 75% effector and 23% central memory cells. SNAP-TILs exhibited greater efficacy and selectivity in immune infiltration than TIL, which was expanded by the rapid expansion protocol alone using ex vivo models. SNAP-TIL was also reactive in highly and poorly immunogenic tumors, with 70% and 50% tumor growth inhibition in melanoma and pancreatic patient-derived xenograft models, respectively. This study demonstrates the novel benefit of our Personalized Neoantigen Pipeline approach, potentially providing a durable antitumor immune response for a larger proportion of cancer patients.

immunology↗

bioRxiv2024

MEGA12: Molecular Evolutionary Genetic Analysis version 12 for adaptive and green computing

We introduce the 12th version of the Molecular Evolutionary Genetics Analysis (MEGA) software. This latest version brings many significant improvements by reducing the computational time needed for selecting optimal substitution models and conducting bootstrap tests on phylogenies using maximum likelihood (ML) methods. These improvements are achieved by implementing heuristics that minimize likely unnecessary computations. Analyses of empirical and simulated datasets show substantial time savings by using these heuristics without compromising the accuracy of results. MEGA12 also implements an evolutionary sparse learning approach to identify fragile clades and associated sequences in evolutionary trees inferred through phylogenomic analyses. In addition, this version includes fine-grained parallelization for ML analyses, support for high-resolution monitors, and an enhanced Tree Explorer. The MEGA12 beta version can be downloaded from https://www.megasoftware.net/beta_download.

evolutionary biology↗

bioRxiv2024

Catalytic inhibition of KAT6/KAT7 enhances the efficacy and overcomes primary and acquired resistance to Menin inhibitors in MLL leukaemia

Understanding the molecular pathogenesis of MLL fusion oncoprotein (MLL-FP) leukaemia has spawned epigenetic therapies that have improved clinical outcomes in this often-incurable disease. Using genetic and pharmacological approaches, we define the individual and combined contribution of KAT6A, KAT6B and KAT7, in MLL-FP leukaemia. Whilst inhibition of KAT6A/B is efficacious in some pre-clinical models, simultaneous targeting of KAT7, with the novel inhibitor PF-9363, increases the therapeutic efficacy. KAT7 interacts with Menin and the MLL complex and is co-localised at chromatin to co-regulate the MLL-FP transcriptional program. Inhibition of KAT6/KAT7 provides an orthogonal route to targeting Menin to disable the transcriptional activity of MLL-FP. Consequently, combined inhibition rapidly evicts the MLL-FP from chromatin, potently represses oncogenic transcription and overcomes primary resistance to Menin inhibitors. Moreover, PF-9363 or genetic depletion of KAT7 can also overcome acquired genetic/non-genetic resistance to Menin inhibition. These data provide the molecular rationale for rapid clinical translation of combination therapy in MLL-FP leukaemia.

cancer biology↗

bioRxiv2024

Scaling ecological niches from individuals to populations and beyond

The niche is a key concept that unifies ecology and evolutionary biology. However, empirical and theoretical treatments of the niche are mostly performed at the species level, neglecting individuals as important units of ecological and evolutionary processes. So far, a formal mathematical link between individual-level niches and higher organismal-level niches has been lacking, hampering the unification of ecological theories and more accurate forecasts of biodiversity change. To fill in this gap, we propose a bottom-up approach to derive population and higher organismal-level niches from individual niches. We demonstrate the power of our framework by showing that 1) the statistical properties of higher organismal-level niches (e.g. niche breadth, skewness etc.) can be partitioned into individual contributions; 2) the species-level niche shifts can be estimated by tracing the responses of individuals. Our method paves the way for a unifying niche theory and enables mechanistic assessments of organism-environment relationships across organismal scales.

ecology↗

bioRxiv2024

ARID1A-induced transcriptional reprogramming rewires signalling responses to drug treatment in melanoma

Resistance to BRAF and MAPK inhibitors is a significant challenge in melanoma treatment, driven by adaptive and acquired mechanisms that allow tumour cells to evade therapy. Here, we examined early signalling responses to single and combined BRAF and MAPK inhibition in a BRAFV600E, drug-sensitive melanoma cell line and a drug-resistant ARID1A-knockout (KO) derivative. ARID1A, frequently mutated in melanoma, is associated with resistance and immune evasion. Using an innovative systems biology approach that integrates transcriptomics, proteomics, phosphoproteomics, and functional kinomics through matrix factorization and network analysis, we identified key signalling alterations and resistance mechanisms. We found that ARID1A-KO cells exhibited transcriptional rewiring, sustaining MAPK1/3 and JNK activity post-treatment, bypassing feedback sensitivity observed in parental cells. This rewiring suppressed PRKD1 activation, increased JUN activity--a central resistance network node--and disrupted PKC dynamics through elevated basal RTKs (e.g., EGFR, ROS1) and Ephrin receptor activity post-treatment. ARID1A mutations also reduced HLA-related protein expression and enriched extracellular matrix components, potentially limiting immune infiltration and reducing immunotherapy efficacy. Our graph-theoretical multi-omics approach uncovered novel resistance-associated signalling pathways, identifying PRKD1, JUN, and NCK1 as critical nodes. While receptor activation redundancies complicate single-target therapies, they also present opportunities for combination strategies. This study highlights ARID1As role in reshaping signalling and immune interactions, offering new insights into melanoma resistance mechanisms. By identifying actionable targets, including JUN and immune pathways, we provide a foundation for developing integrated therapeutic strategies to overcome resistance in BRAF/MAPK inhibitor-treated melanoma. One sentence summaryThis study reveals how ARID1A-mediated transcriptional rewiring drives resistance to MAPK inhibitors in melanoma by altering signalling pathways, immune interactions, and receptor dynamics, highlighting potential targets for combinatorial therapies.

systems biology↗

bioRxiv2024

Editing cis-elements of OsPHO1;2 improved phosphate transport and yield in rice

Increasing grain yield is the main aim of crop improvement, which is globally impacted by the low availability of soil phosphate (Pi). Overexpressing Pi transporters for increasing Pi uptake often result in Pi toxicity and growth retardation. Despite advancements in genetic engineering, targeting cis-regulatory motifs of Pi transporters remains underexplored for understanding plant mechanisms and improving Pi status. Here, we demonstrated that the excision of the transcription inhibitor motif from the promoter of Pi transporter, OsPHO1;2, enhanced its expression and increased root-to-shoot Pi transport, leading to improved grain yield. Using, in-silico and DNA-protein interaction studies, we show the role of OsWRKY6 transcription factor in the negative regulation of OsPHO1;2 expression by binding to the cis-regulatory element (W-box) present in its promoter. oswrky6 knockout lines displayed a higher OsPHO1;2 expressions, and improved shoot Pi levels. Further, we engineered the OsPHO1;2 promoter for precisely removing the W-box and enhancing OsPHO1;2 expression. The phenotypic and physiological evaluation at the vegetative stage shows that OsPHO1;2 promoter edited (OsPHO1;2:PE) lines have increased shoot length, plant biomass and more root-shoot Pi export under low and normal P conditions. Notably, the 33Pi uptake assay reveals that OsPHO1;2:PE lines exhibited enhanced root Pi uptake, supported by higher root-associated Pi transporters (OsPHTs) expression. An extensive agronomical assessment revealed that OsPHO1;2:PE lines acquire increased seed and panicle numbers, thereby increasing yield without affecting seed quality. Our findings offer valuable insight into the potential of promoter editing to raise plants with better Pi use and improved crop yield.

plant biology↗