Biology subjects

Alam, R.

Publications and source records attributed to Alam, R..

16 recordsLinked to original sources

bioRxiv2026

RAP2.6 negatively regulates Arabidopsis submergence tolerance through GSNOR1-dependent redox and hormonal signaling

Group VII ETHYLENE RESPONSE FACTORs (ERFVIIs) are central regulators of plant responses to oxygen deprivation. However, the contributions of many other APETALA2 (AP2)/ERF transcription factors to hypoxia and flooding responses remain poorly understood. Here, we identify the group X ERF transcription factor RAP2.6 as a negative regulator of submergence tolerance in Arabidopsis thaliana. RAP2.6 expression declined under hypoxia, and plants overexpressing RAP2.6 exhibited reduced survival following dark submergence, whereas the rap2.6-1 loss-of-function mutant showed enhanced tolerance to submergence, hypoxia, and ethanol treatment. Enhanced tolerance of rap2.6-1 was associated with reduced membrane injury, maintenance of photosynthetic pigments and soluble sugars, and extensive metabolic remodeling. RAP2.6 overexpression altered nitric oxide (NO), S-nitrosothiol (SNO), and ethylene accumulation during submergence and was associated with greater oxidative damage and enhanced abscisic acid (ABA)-responsive gene expression. Genetic analyses using S-NITROSOGLUTATHIONE (GSNO) REDUCTASE 1 (GSNOR1) gain- and loss-of-function backgrounds demonstrated that GSNOR1-dependent GSNO homeostasis strongly modified RAP2.6-dependent submergence responses. Increased GSNOR1 activity suppressed the submergence-sensitive phenotype of RAP2.6-overexpressing plants, reduced nitro-oxidative stress, and restored carbohydrate reserves, whereas loss of GSNOR1 activity abolished the enhanced tolerance of rap2.6-1. GSNOR1 also modified RAP2.6-dependent expression of genes associated with oxygen sensing and ABA signaling, while its protective effect was not explained solely by changes in ethylene accumulation. Together, these findings identify RAP2.6 as a regulatory component of the low-oxygen response and reveal a functional interaction between RAP2.6 and GSNOR1-dependent redox signaling that coordinates plant hormonal, metabolic, and oxidative responses during submergence.

plant biology↗

bioRxiv2026

Safe Redosable Low-Immunogenic In Vivo CAR-T Therapy for B Cell Malignancies and Solid Tumors

In vivo CAR-T cell therapy eliminates manufacturing complexities associated with ex vivo autologous approaches, but safety concerns have limited adoption. We developed viroVbot, a next-generation in vivo CAR-T platform, by combining computational immunogenicity prediction (CIMMEXTM) with envelope engineering. Screening 22,562 glycoprotein sequences, we identified 641 vesiculovirus homologs, from which we selected Piry virus glycoprotein (PIRYV) as the optimal candidate. PIRYV exhibited lower MHC-epitope density, reduced human seroprevalence, with decreased T cell activation compared to VSV-G. To enhance targeting specificity, we engineered receptor-binding-deficient PIRYV (ePIRYVRBD) displaying CD3/CD7 nanobodies for T cell-selective transduction. To maximize safety, we engineered CAR-TRAP producer cells to eliminate unwanted B cell transduction and incorporated machine learning-optimized T cell-specific promoters that restrict CAR activation exclusively to lymphocytes. Additional modifications suppressed hepatocyte expression and prevented phagocytic uptake. In humanized xenograft models, viroVbot3 generated potent BCMA/CD19 specific CAR-T responses against multiple myeloma and Claudin18.2-targeting gastric cancer, demonstrating sequential redosing with alternative envelopes. Critically, viroVbot3 exhibited minimal off-target organ biodistribution with CAR expression restricted to T lymphocytes. These findings establish viroVbot as a low-immunogenic platform for scalable in vivo CAR-T manufacturing with capability for sequential redosing across hematologic and solid tumors.

bioengineering↗

bioRxiv2026

A Deep-Learning Atlas of XPO1-Mediated Nuclear Export at Proteome Scale

Exportin 1 (XPO1/CRM1) is the principal nuclear export receptor for cargos bearing hydrophobic nuclear export sequences (NESs). Dysregulation of XPO1-dependent export is implicated in cancer, neurodegeneration, and other diseases, yet a comprehensive view of XPO1 function remains limited by the poor reliability of sequence-based NES prediction. Existing predictors are largely derived from a small set of XPO1-cargo structures and are therefore biased toward canonical docking geometries, limiting their ability to detect NESs that engage XPO1 through noncanonical pocket-occupancy patterns. We hypothesized that deep learning-based structural modeling could overcome this limitation by directly sampling binding geometries. Using AlphaFold 3, we modeled full-length cargo-XPO1-RanGTP complexes for more than 4,000 human proteins and identified over 3,000 previously uncharacterized, high-confidence NESs. Integration of AlphaFold predictions with unsupervised structural geometry analysis and experimental validation identified both canonical NESs and noncanonical sequence patterns exhibiting atypical anchor-residue usage, expanding the structural language of XPO1-recognized NESs. Groove-resolved contact maps further revealed helix rotation within the export groove as a regulatory feature that can rewire pocket usage without altering the core NES sequence, enabling PTM- and cofactor-sensitive tuning of export strength. This exportome atlas resolves many previously ambiguous or unidentified NESs in disease-associated proteins and across major cellular systems, including centrosome organization, mRNA processing, ubiquitin signaling, kinase networks, ribosome quality control, and macroautophagy. We further identified recurrent NES-NLS tandem motifs encoded in primary sequence, suggesting coordinated regulation of nucleocytoplasmic transport. Together, our deep learning-based exportome atlas, integrated with NLS maps and accessible through a web-searchable resource, defines an expanded and regulatable code of nuclear transport at proteome scale and offers a framework for dissecting nuclear trafficking and its dysregulation in human disease.

cell biology↗

bioRxiv2026

Reprogramming BCMA-Targeted CAR-T Cells through γ-Secretase Modulation Blocks Antigen Shedding and Extends CAR-T Longevity

B-cell maturation antigen (BCMA) shedding by {gamma}-secretase generates soluble BCMA (sBCMA), which diminishes membrane antigen density, and limits the durability of BCMA-directed immunotherapies in multiple myeloma (MM). Here, using AI-driven diffusion modeling, we identify peptide inhibitors that selectively block {gamma}-secretase-mediated BCMA cleavage, stabilizing membrane-bound BCMA (mBCMA) without compromising cellular viability. The lead peptide, P5, suppresses sBCMA and restores mBCMA in vitro and in xenograft models. To create sustained, cell-intrinsic inhibition, we engineered Shedding Deterrent Locked-in (SHEDLOCK) CAR-T cells that locally secrete {gamma}-secretase-modulatory peptides. SHEDLOCK-1 CAR-T cells secreting P5 exhibits enhanced cytotoxicity and persistence relative to conventional CAR-T cells. Building on this, SHEDLOCK-2 CAR-T cells were generated, which secrete a modified, naturally derived peptide (nxP) that directly engages the {gamma}-secretase catalytic site, concurrently preventing BCMA shedding, and surprisingly enhancing CAR-T cell longevity by preserving telomere integrity and metabolic fitness. In MM xenograft and patient-derived models, SHEDLOCK-2 CAR-T cells demonstrate durable antitumor activity with a favorable safety profile. Together, these findings establish SHEDLOCK as a next-generation CAR-T platform and provide a strong preclinical foundation for Phase I clinical evaluation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/700575v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@3cd38aorg.highwire.dtl.DTLVardef@164167borg.highwire.dtl.DTLVardef@1ffed21org.highwire.dtl.DTLVardef@1294d06_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Conserved early steps of stemmadenine biosynthesis

Stemmadenine acetate is a pivotal intermediate in the production of pharmacologically active monoterpene indole alkaloids. Here, we identify orthologs of stemmadenine acetate pathway genes (SGD, GS, GO, Redox1, Redox2, SAT) from Tabernanthe iboga. We characterize these enzymes in vitro, and additionally, we reconstitute stemmadenine acetate biosynthesis in Nicotiana benthamiana, comparing the formation of intermediates and shunt products that are produced when previously characterized orthologs from the related plant Catharanthus roseus are used. Ortholog pairs are catalytically indistinguishable, except in the case of GS. Surprisingly, the T. iboga ortholog catalyzes formation of an alternate stereoisomer 19Z-geissoschizine, seeding a low-flux Z-series in heterologous reconstitution systems in vitro and in planta. We additionally characterize the major shunt products that arose during reconstitution of stemmadenine acetate biosynthesis. We show that the substrate promiscuity of Redox2 results in formation of the shunt products 16(R/S)-isositsirikines, hampering pathway flux and yields. Additionally, we show that stemmadenine can be oxidized by endogenous N. benthamiana enzymes, leading to the shunt product condylocarpine. Nevertheless, we could produce stemmadenine at a 6 mg yield from 19E-geissoschizine by heterologous expression in N. benthamiana. Overall, we highlight the prospects for milligram production of important biosynthetic intermediates in N. benthamiana.

biochemistry↗

bioRxiv2025

B cells specific for polyomavirus-derived oncoprotein are predictive of Merkel cell carcinoma progression

Merkel cell carcinomas typically arise from clonal integration of the Merkel cell polyomavirus. Immunogenic viral oncoproteins then lead to tumorigenesis. Oncoprotein-specific T cells are essential for anti-MCC immunity, but it is unclear whether B cells promote tumor control. Here, we analyzed the frequency and phenotype of viral oncoprotein-specific and total B cells in 47 blood samples and 19 unmatched tumors from MCC patients-- of which 8 out 19 progressed. The phenotype of blood B cells did not correlate with MCC patient outcomes. In contrast, all 11 patients with robust oncoprotein-specific antibody-secreting and/or germinal center B cells in tumors experienced long-term MCC control. In vitro, B cells engineered to be specific for viral oncoproteins increased the sensitivity of oncoprotein-specific CD4+ T cells by over 50-fold. Together, our findings suggest that cancer-specific B cells promote anti-tumor immunity via increased T cell responses and that cancer-specific B cell augmentation could be therapeutically relevant. Statement of SignificanceThe link between cancer-specific B cells in anti-tumor immunity and clinical outcomes remains poorly defined. Here, we show that tumor-associated B cells specific for a viral oncoprotein expressed in MCC patient tumors predict disease control with remarkable accuracy, establishing their potential as active participants in tumor immunity.

immunology↗

bioRxiv2025

AI-Guided Design and AKT3 Degradation Synergize to Enhance Bispecific and Trispecific CAR-T Cell Persistence and Overcome Antigen Escape

The structural design of chimeric antigen receptors (CARs) is critical for achieving robust and durable anti-tumor responses, particularly when targeting multiple antigens to prevent tumor antigen escape. However, increasing CAR complexity can introduce structural vulnerabilities, leading to antigen-independent T cell activation, activation-induced cell death, and reduced CAR-T cell persistence. To overcome these challenges, we designed 10,824 CAR molecules across diverse formats and screened 1,452 constructs in-vitro to develop an artificial intelligence model, termed CAR-Mediated Self-Destruction (CARMSeD), which predicts CAR designs susceptible to self-activation and dysfunction. Guided by CARMSeD and structural CAR-CAR interaction modeling, we identified optimized CAR architectures incorporating ICOS and 4-1BB co-stimulatory domains. Humanized bispecific CARs targeting CD20/CD19 and CD22/CD19 demonstrated superior anti-tumor efficacy and persistence both in-vitro and in various xenograft mice models. To further extend CAR-T cell persistence, we engineered bispecific CARs integrated with an AKT3-targeted PROTAC strategy. Targeted degradation of AKT3 enhanced anti-tumor potency, promoted memory T cell formation, and enabled sustained responses even under tumor rechallenge and CD19 antigen-loss conditions. Mechanistically, these effects were mediated by metabolic reprogramming involving FOXO4; notably, FOXO4-deficient CAR-T cells exhibited impaired long-term persistence. Leveraging these mechanistic insights, we developed a trispecific CAR-T cell platform incorporating a bispecific T cell engager (BiTE) targeting CD22/CD3, combined with AKT3 PROTACs. These trispecific CAR-T cells achieved potent tumor eradication, even against malignancies lacking both CD19 and CD20 expression. Collectively, this study presents a comprehensive strategy combining structure-based design, AI-guided screening, and targeted protein degradation to engineer next-generation bi and trispecific CAR-T cells with enhanced persistence, broad antigen coverage, and superior therapeutic durability.

cancer biology↗

bioRxiv2025

Bacterial extracellular vesicles as a tunable platform for vaginal drug delivery

There is a critical gap in the development of new therapeutic platforms designed to treat gynecologic and obstetric diseases. Compared to systemic drug delivery, vaginal drug administration of nanoparticle formulations limits off-target side effects while increasing therapeutic concentration in target tissues, showing promise for clinical translation. However, these formulations suffer from limited scalability, high-cost reagents, and long optimization timelines. Recent work highlights the potential of bacterial extracellular vesicles (bEVs) as a low-cost, tunable platform for therapeutic applications. Here, we evaluate bEVs as a therapeutic carrier for vaginal drug delivery. We demonstrate the loading of the model protein moxNeonGreen into Escherichia coli Nissle 1917 bEVs. By optimizing growth parameters, we increase protein loading into bEVs. We evaluate the effect of bEVs on the vaginal microenvironment, and observe no negative impact on vaginal epithelial cells, endocervical cells, or vaginal bacteria in vitro. Additionally, we observe the retention of bEVs in the murine female reproductive tract for more than six hours. This study provides a framework for using genetically engineered bEVs to rapidly generate customizable therapies for a range of gynecologic and obstetric conditions, addressing longstanding challenges in womens health therapeutics. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=50 SRC="FIGDIR/small/658669v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@e483d2org.highwire.dtl.DTLVardef@8a6d2corg.highwire.dtl.DTLVardef@5756borg.highwire.dtl.DTLVardef@5fdb67_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

BAHD news from Euphorbia peplus: identification of acyltransferase enzymes involved in ingenane diterpenoid biosynthesis

O_LIThe plant family Euphorbiaceae are an abundant source of structurally complex diterpenoids, many of which have reported anti-cancer, anti-HIV, and anti-inflammatory activities. Among these, ingenol-3-angelate (1a; tradename: Picato(R)), isolated from Euphorbia peplus, has potent anti-tumour activity. C_LIO_LIHere we report the discovery and characterization of the first genes linked to the committed steps of ingenol-3-angelate (1a) biosynthesis in Euphorbia peplus. We identified two genes, the products of which catalyse the addition of angelyl-CoA (9a) to the ingenol (5) scaffold to produce ingenol-3-angelate (1a). C_LIO_LIWe demonstrate using VIGS that just one of these genes, EpBAHD-08, is essential for this angeloylation in E. peplus. VIGS of the second gene, EpBAHD-06, has a significant effect on jatrophanes rather than ingenanes in E. peplus. C_LIO_LIWe also identified three genes whose products can catalyse acetylation of ingenol-3-angelate (1a) to ingenol-3-angelate-20-acetate (2). In this case VIGS indicates considerable functional redundancy in the E. peplus genome of genes encoding this enzymatic step. C_LIO_LIThis work paves the way for increasing ingenol-3-angelate (1a) levels in planta and provides a foundation for the discovery of the remaining genes in the biosynthetic pathway of these important molecules. C_LI

biochemistry↗

bioRxiv2024

Characterizing postoperative T and B cell dysfunction in cancer surgery patients, using COVID-19 as a model antigen

For most cancers, surgery is an effective intervention method for cure but, despite the benefits, many patients recur with metastatic disease. Surgery has been shown to impair immune function by causing suppression of immune cells. In this study, we used a COVID-19 immunological toolkit to answer questions regarding the effects of surgery on antigen specific CD8+ T cells and B cells by exploiting the responses to the spike protein in vaccinated cancer patients. We demonstrate that surgical stress results in a reduction in the number of CD8+ T cell that produce cytokines and B cells that secrete antibodies in response to antigen. This study will improve our understanding of surgery-induced T cell and B cell dysfunction

immunology↗

bioRxiv2024

Enzymatic epimerization of monoterpene indole alkaloids in Kratom

Monoterpene indole alkaloids (MIAs) are a large, structurally diverse class of bioactive natural products. These compounds are biosynthetically derived from a stereoselective Pictet-Spengler condensation that generates a tetrahydro-{beta}-carboline scaffold characterized by a 3S stereocenter. However, a subset of MIAs contain a non-canonical 3R stereocenter. Herein, we report the basis for 3R-MIA biosynthesis in Mitragyna speciosa (Kratom). We discover the presence of the iminium species, 20S-3-dehydrocorynantheidine, which led us to hypothesize that isomerization of 3S to 3R occurs by oxidation and stereoselective reduction downstream of the initial Pictet-Spengler condensation. Isotopologue feeding experiments implicated young leaves and stems as the sites for pathway biosynthesis, facilitating the identification of an oxidase/reductase pair that catalyzes this epimerization. This enzyme pair has broad substrate specificity, suggesting that the oxidase and reductase may be responsible for the formation of many 3R-MIAs and downstream spirooxindole alkaloids in Kratom. These enzymes allow biocatalytic access to a range of previously inaccessible pharmacologically active compounds.

biochemistry↗

bioRxiv2024

Independent evolution of ipecac alkaloid biosynthesis

Ipecac alkaloids are medicinal monoterpenoid-derived tetrahydroisoquinoline alkaloids found in two distantly related plants: Carapichea ipecacuanha (Gentianales) and Alangium salviifolium (Cornales). We have elucidated ipecac alkaloid biosynthesis in both species, conclusively demonstrating that biosynthesis of the structurally complex ipecac alkaloid protoemetine has evolved independently. We show that although protoemetine biosynthesis proceeds via the same chemical logic in both species, each plant uses a distinct monoterpene precursor. Moreover, we provide evidence that both plants initiate ipecac biosynthesis by a non-enzymatic Pictet-Spengler reaction, and we elucidate the biosynthetic fate of both the 1R and 1S stereoisomers that are produced in this non-stereoselective reaction. Phylogenetic analyses clearly show independent pathway evolution through parallel and convergently evolved enzymes. This work provides insight into how nature can capitalize on highly reactive starting substrates, the manner in which multi-step pathways can arise, and also lays the foundation for metabolic engineering of these important medicinal compounds.

plant biology↗

bioRxiv2024

Merkel cell carcinoma-derived macrophage migration inhibitory factor (MIF) may promote persistence of Chronic Lymphocytic Leukemia

While concurrent diagnoses of Merkel cell carcinoma (MCC) and other cancers, like Chronic lymphocytic leukemia (CLL), are rare, patients with MCC have a 30-fold higher incidence of CLL. While these increases have been attributed to the ability of CLL to suppress immune responses allowing for the emergence of MCC, here we found evidence that MCC could support the persistence of CLL. Using single cell sequencing approaches and computational analyses of MCC and CLL from a patient where both cancers were present in the same lymph node, we found that production of macrophage migration inhibitory factor (MIF) by MCC could promote the persistence of CLL through stimulation of CD74 and CXCR4. These results may explain why blood cell counts rapidly normalized after treatment for MCC and were maintained at normal levels despite the absence of treatment for CLL. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/611517v2_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@188968forg.highwire.dtl.DTLVardef@54c084org.highwire.dtl.DTLVardef@2b3a14org.highwire.dtl.DTLVardef@1f20eba_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Pair-EGRET: enhancing the prediction of protein-proteininteraction sites through graph attention networks and protein language models

Proteins are responsible for most biological functions, many of which require the interaction of more than one protein molecule. However, accurately predicting protein-protein interaction (PPI) sites (the interfacial residues of a protein that interact with other protein molecules) remains a challenge. The growing demand and cost associated with the reliable identification of PPI sites using conventional experimental methods call for computational tools for automated prediction and understanding of PPIs. Here, we present Pair-EGRET, an edge-aggregated graph attention network that leverages the features extracted from pre-trained transformer-like models to accurately predict PPI sites. Pair-EGRET works on a k-nearest neighbor graph, representing the three-dimensional structure of a protein, and utilizes the cross-attention mechanism for accurate identification of interfacial residues of a pair of proteins. Through an extensive evaluation study using a diverse array of experimental data, evaluation metrics, and case studies on representative protein sequences, we find that our method outperforms other state-of-the-art methods for predicting PPI sites. Moreover, Pair-EGRET can provide interpretable insights from the learned cross-attention matrix. Pair-EGRET is freely available in open source form at (https://github.com/1705004/Pair-EGRET).

bioinformatics↗

bioRxiv2023

Targeting LINC00152 activates cAMP/Ca2+/ferroptosis axis and overcomes tamoxifen resistance in ER+ breast cancer

Tamoxifen has been the mainstay therapy to treat early, locally advanced, and metastatic estrogen receptor-positive (ER+) breast cancer, constituting around 75% of all cases. However, emergence of resistance is common, necessitating the identification of novel therapeutic targets. Here, we demonstrated that long-noncoding RNA LINC00152 confers tamoxifen resistance via blocking tamoxifen-induced ferroptosis, an iron-mediated cell death. Mechanistically, inhibiting LINC00152 reduces the mRNA stability of phosphodiesterase 4D (PDE4D), leading to activation of cAMP/PKA/CREB axis and increased expression of TRPC1 Ca2+ channel. This causes cytosolic Ca2+ overload and generation of reactive oxygen species (ROS) that is, on one hand, accompanied by downregulation of FTH1, a member of the iron sequestration unit, thus increasing intracellular Fe2+ levels; and on the other hand, inhibition of the peroxidase activity upon reduced GPX4 and xCT levels. These ultimately induce lipid peroxidation and ferroptotic cell death in combination with tamoxifen. Overexpressing PDE4D rescues LINC00152 inhibition-mediated tamoxifen sensitization by de-activating the cAMP/Ca2+/ferroptosis axis. Importantly, high LINC00152 expression is significantly correlated with high PDE4D/low ferroptosis and worse survival in multiple cohorts of tamoxifen- or tamoxifen-containing endocrine therapy-treated ER+ breast cancer patients. Overall, we identified LINC00152 inhibition as a novel mechanism of ferroptosis induction and tamoxifen sensitization, thereby revealing LINC00152 and its effectors as actionable therapeutic targets to improve clinical outcome in refractory ER+ breast cancer.

cancer biology↗

bioRxiv2018

PI3Kδ hyper-activation promotes the development of B cells that exacerbate Streptococcus pneumoniae infection in an antibody-independent manner

Streptococcus pneumoniae is a major cause of pneumonia and a leading cause of death world-wide. Antibody-mediated immune responses can offer protection against repeated exposure to S. pneumoniae, yet vaccines only offer partial protection. Patients with Activated PI3K{delta} Syndrome (APDS) are highly susceptible to S. pneumoniae. We generated a conditional knockin mouse model of this disease and identified a CD19+B220- B cell subset that is induced by PI3K{delta} signaling, is resident in the lungs, and which promotes increased susceptibility to S. pneumoniae during the early phase of infection via an antibody-independent mechanism. We show that an inhaled PI3K{delta} inhibitor improves survival rates following S. pneumoniae infection in wild-type mice and in mice with activated PI3K{delta}. These results suggest that a subset of B cells in the lung can promote the severity of S. pneumoniae infection, representing a novel therapeutic target.

immunology↗