Biology subjects

Singh, S.

Publications and source records attributed to Singh, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Neuroprotective activity of sub-anesthetic ketamine in a preclinical model of Parkinson's disease is not blocked by antagonizing brain-derived neurotrophic factor signaling

Treatment with low-dose ketamine is neuroprotective in rodent models of acute damage, while high doses of ketamine can be neurotoxic. Our group has previously shown that sub-anesthetic ketamine treatment leads to long-term reduction of L-DOPA-induced dyskinesia (LID) via brain-derived neurotrophic factor (BDNF)-signaling in a rat 6-hydroxydopamine (6-OHDA) model of Parkinson's disease (PD). In this report, we investigated if sub-anesthetic ketamine is also neuroprotective in a progressive 6-OHDA model of PD via neurotrophic action through the tropomyosin receptor kinase B (TrkB) receptor. We demonstrated that ketamine-treatment (20 mg/kg, i.p.) prior to striatal administration of the neurotoxin 6-OHDA is neuroprotective, providing near complete protection against development of parkinsonian motor deficits. This robust behavioral efficacy occurred in the presence of only modest preservation of tyrosine hydroxylase (TH) striatal levels (~50-60% loss). Interestingly, based on data from an additional cohort treated with ketamine and ANA-12 (TrkB antagonist) we found that the neuroprotective effects of ketamine are not mediated by TrkB receptors. Semi-quantitative western analysis showed that the relative tyrosine hydroxylase content in the striatum was not significantly increased in ketamine-treated rats compared to vehicle. Unbiased stereology of dopamine neurons in the substantia nigra pars compacta (SNc) revealed no significant differences between any of the groups. The role of BDNF was evaluated with in situ hybridization for RNA of BDNF and TrkB. While BDNF expression was not changed, co-administration of ketamine and ANA-12 led to a significant decrease in TrkB receptor expression compared to ketamine-only on the lesioned but not the intact hemisphere in the SNc. In conclusion, we show that ketamine at the low dose currently pursued for therapeutic benefit for LID led to robust behavioral motor improvement in this rodent PD degeneration model, yet these protective effects were not driven by neurotrophic BDNF-signaling, indicating complex mechanisms are underlying neuroprotective activities of low-dose ketamine.

neuroscience↗

bioRxiv2026

AlphaGenome informed machine learning for pathogenicity prediction of germline mutations

Accurate interpretation of germline variants remains a central challenge in clinical genomics. Existing in silico pathogenicity predictors often provide incomplete coverage or restricted to missense variants from the coding region, leaving the variants from splice sites, -UTR, and intronic regions under-scored. We present AGVarPred, a gradient-boosted classifier designed to predict the pathogenicity of germline mutants, with a broad consequence-class coverage across SNVs and indels. Here, the population allele frequency from gnomAD, functional consequence annotations from Ensembl VEP, and cell-type-specific functional genomic scores from the AlphaGenome platform were integrated to develop the classifier. Using a gene-level split of 134,002 high-confidence ClinVar variants across 9,222 genes, AGVarPred achieved an AUC-ROC of 0.9753 (95% CI 0.9731-0.9775) and a AUC-PR of 0.9606 on 12,385 scored internal test variants. A paired DeLong analysis showed that the full model significantly outperformed an allele-frequency-plus-VEP model ({Delta}AUC = 0.1539, 95% CI 0.1489-0.1589; p < 1 x 10-300), supporting a substantial incremental contribution from AlphaGenome-derived features. Performance remained strong when restricted to SNVs (AUC-ROC 0.9558) and was also high for indels (AUC-ROC 0.9532). On five external benchmarks, AUC-ROCs ranged from 0.528 on a functional-assay-derived benchmark to 0.977 on a clinically curated deafness database, reflecting its dependence on benchmark composition and label source. A separate analysis of the VIP Database, whose pathogenic labels are ClinVar-derived, showed consistent performance (AUC-ROC of 0.892) in held-out genes. AGVarPred scored 100% of evaluated variants in the external benchmarks, whereas AlphaMissense and REVEL covered only small fractions of non-missense variants. Model interpretation indicated that gnomAD allele frequency was the dominant individual predictor; the 111 AlphaGenome features provided complementary functional signal, with splicing and chromatin assays contributing to the largest share of the AlphaGenome SHAP mass. A web-based interface for AGVarPred is available at https://pankaj357.github.io/AGVarPred/ to facilitate the submission of normalized GRCh38 germline variants and retrieval of pathogenicity predictions using the frozen AGVarPred model.

bioinformatics↗

bioRxiv2026

PlasmoRhythm: A comprehensive database for malaria-associated rhythmic transcripts, proteins, and metabolites

Abstract: Motivation: The malaria transmission cycle is temporally organized across three interacting biological systems: the parasite, vertebrate host, and mosquito vector. Rhythms in parasite development, host physiology, and vector behavior govern infection dynamics, transmission, and the efficacy of stage-specific antimalarial interventions. However, the molecular architecture connecting these rhythms remains difficult to interrogate because time-resolved omics datasets are distributed across species, experimental paradigms, and analytical frameworks, with no unified resource for comparative analysis. Results: We developed PlasmoRhythm, a curated database integrating 67 transcriptomic, proteomic, and metabolomic datasets from 15 studies, encompassing 1,126 sampling events across Plasmodium species, mammalian hosts, and Anopheles vectors. Harmonized metadata and rhythmicity outputs from JTK_CYCLE, ARSER, Lomb-Scargle, and the meta2d function support interactive cross-study interrogation of molecular profiles. Dedicated modules enable the exploration of rhythmic virulence factors and antimalarial drug interactors, providing a unified framework for hypothesis generation across the malaria transmission cycle. Availability: PlasmoRhythm is freely accessible at https://project.iith.ac.in/cgntlab/PlasmoRhythm/.

bioinformatics↗

bioRxiv2026

Evolutionarily Conserved Plasmodium falciparum HSP70-3 Enhances the Immunogenicity of the Established Malaria Vaccine Antigen Circumsporozoite Protein through Cross-Presentation

Background: Clinically approved P. falciparum circumsporozoite protein (PfCSP)-based pre-erythrocytic stage malaria vaccines suffer from sub-optimal efficacy and rapidly waning memory. By decoding these limitations, we fast-tracked vaccine optimization by correcting CSPs fragile immunogenicity using a parasite-derived chaperone. Unlike other orthologues of P. falciparum heat shock protein 70 (PfHSP70), substrate-binding domain (SBD) of PfHSP70-3 is an evolutionarily conserved internal mitochondrial matrix chaperone. We demonstrate that PfHSP70-3-SBD hydrophobic region complexes with PfCSP and targets host CD91 on antigen-presenting cells to trigger cross-presentation, resulting in enhanced immune responses to PfCSP. Methods: In silico analyses predicted interactions among PfHSP70-3-SBD, PfCSP, and CD91 receptor. Recombinant PfHSP70-3-SBD and PfCSP proteins were purified and assessed for in vitro interaction using ELISA and far-western dot blot assays. BALB/c mice were immunized with alum-formulated PfCSP, with or without PfHSP70-3-SBD. PfCSP-specific IgG and IFN-{gamma} expression were evaluated by ELISA and RT-qPCR, respectively. Functional activity was assessed using transgenic Plasmodium berghei expressing PfCSP (Pb/Pf CSP), by sporozoite neutralization assay. Findings: PfHSP70-3-SBD demonstrated a specific interaction with PfCSP. The complex primarily engages CD91 receptor cluster IV, providing a potential mechanistic basis for antigen cross-presentation. Incorporation of PfHSP70-3-SBD enhanced PfCSP-specific total IgG responses and increased IFN-gamma expression in BALB/c mice, indicating improved immunogenicity upon co-formulation. Notably, immune sera neutralized Pb/Pf CSP sporozoites, delaying blood-stage patency until day 12 in 40% of C57BL/6 mice, with no detectable parasitemia in surviving mice for up to 2 months, indicating sustained efficacy against the sporozoite stage. Interpretation: Molecular chaperones, including HSPs, are currently under intense investigation for their immune-potentiating activity, leading to next-generation vaccine development. By utilizing chaperonic domains derived directly from the pathogenic agent itself to serve an essential adjuvant function, this approach reduces reliance on heterologous adjuvants while enhancing antigen-specific immunity. Funding: This work was supported by funding from Indian Council of Medical Research (IIRPSG-2024-01-03940). Keywords: Substrate-binding domain, PfHSP70-3, Adjuvant, Cross-presentation, Pre-erythrocytic stage, Plasmodium falciparum

microbiology↗

bioRxiv2026

Sugarcane bZIP16 regulates internode elongation by targeting gibberellic acid biosynthesis pathway

Modern sugarcane cultivars are vegetatively propagated interspecific hybrids developed by extensive breeding and selection method to enhance sugar content, culm dimensions, desired plant architecture and disease resistance. Internode length greatly varies among existing sugarcane cultivars which determines culm length, biomass and sugar storage. Molecular factors responsible for this trait remains largely unknown in sugarcane. In this study, we characterized short and long internode sugarcane varieties and elucidated gene regulatory module interplaying with hormonal-network underneath the internodal growth. The genome size, pigment content and juice characteristics of short and long internode varieties differed with each other and correlated with gene expressions pattern. We established tissue and spatial transcriptome dataset, and comparative analyses demonstrated number of genes involved in cell cycle, cell expansion, transcription factors and hormone pathway including bZIP16 are differentially expressed between short and long internode varieties. Biochemical, DNA-protein interactions and molecular evidences revealed that ScbZIP16 inversely related to GA content by repressing transcripts of ent-isokaurene C2/C3-hydroxylase and GA20 Oxidase in sugarcane. The further genetic evidences confirmed role of ScbZIP16 in reducing GA biosynthesis pathway, flowering time and growth in Arabidopsis. This report of ScbZIP16 regulatory module offers a potential candidate for sugarcane improvement programs.

plant biology↗

bioRxiv2026

Differential utilization of PACAP and glutamate in parabrachial neurons shapes pain and stress

The external parabrachial nucleus (elPBN) contributes to diverse behaviors, including pain and stress. Neurons in this region use glutamatergic and neuropeptidergic signaling, with pituitary adenylate cyclase-activating polypeptide (PACAP) expressed in a subset of glutamatergic neurons. Deletion of PACAP in elPBN neurons attenuates behavioral responses to stress, but the contribution of PACAP to pain sensitization and of glutamatergic neurotransmission to pain- and stress-related responses remain poorly understood. Here, we show that constitutive loss of PACAP markedly attenuated nerve injury-induced hypersensitivity across heat, pressure, cold and tactile modalities without altering baseline nociception, while also reducing inflammation-induced spontaneous pain-related behavior. Notably, selective deletion of PACAP within elPBN neurons phenocopied the suppression of injury-induced hypersensitivity, but left inflammation- induced pain-related behavior unaffected. Ex vivo optogenetic-assisted circuit mapping showed that activation of PACAP-expressing elPBN terminals elicited glutamatergic transmission onto central amygdala (CeA) PKC{delta}-expressing neurons. Selective deletion of VGluT2 in elPBN neurons reduced nerve injury-induced hypersensitivity but, unlike PACAP deletion, also reduced inflammation-induced pain-related behavior, while leaving stress-induced hypophagia intact. Together, these findings reveal differential requirements for PACAP and glutamatergic neurotransmission within the elPBN across pain- and stress-related behaviors.

neuroscience↗

bioRxiv2026

PDF2.2 links constitutive defensin expression to disease resistance in Arabidopsis

Background: Plant defensins are small cysteine-rich antimicrobial peptides. In Arabidopsis thaliana, the inducible Class I gene AtPDF1.2a is widely used as a transcriptional marker of jasmonate/ethylene immunity, whereas the constitutively expressed Class II members have received less functional attention. AtPDF2.2 was compared with AtPDF1.2a during infection of Arabidopsis by the vascular fungal pathogen Verticillium longisporum, the necrotrophic fungus Sclerotinia sclerotiorum and the bacterium Pseudomonas syringae pv. tomato DC3000. Results: Profiling of the AtPDF family showed that AtPDF2.2, together with the Class II genes AtPDF2.3 and AtPDF2.5, was significantly reduced during V. longisporum infection, whereas AtPDF1.2a was induced. A promoter-reporter line showed a corresponding decrease in AtPDF2.2 promoter activity during V. longisporum infection, strongest in infected roots. KD-pdf2.2 plants contained more V. longisporum DNA, whereas OE-PDF2.2 plants contained less. The same reciprocal disease pattern was observed during S. sclerotiorum and Pst DC3000 infection. Histochemical Prom::AtPDF2.2::GUS reporter signal decreased during S. sclerotiorum infection but increased following Pst DC3000 infection. Basal AtPDF2.2 expression was lower in JA and ET-signaling mutants and higher in the SA-deficient sid2 background, while the analyzed promoter lacked the GCC-box that mediates inducible AtPDF1.2a regulation. AtPDF1.2a overexpression reduced symptoms across the three pathosystems, but loss of AtPDF1.2a increased disease only during V. longisporum infection under the conditions tested and did not alter Pst DC3000 DNA abundance. AtPDF2.2 overexpression was not associated with reduced vegetative growth under the conditions tested, whereas AtPDF1.2a overexpression reduced rosette size and leaf number. Conclusions: AtPDF2.2 is a constitutively expressed Class II defensin whose expression level is associated with disease outcome across two fungal pathogens and a bacterium. Its promoter shows tissue-dependent regulation during V. longisporum infection, while histochemical reporter signal decreases during S. sclerotiorum infection and increases during Pst DC3000 infection. This regulatory behavior differs from that of the inducible reference gene AtPDF1.2a. The reciprocal disease phenotypes of the AtPDF2.2 transgenic backgrounds support a protective association that may involve direct antimicrobial activity, modulation of host responses or both. The absence of reduced vegetative growth in OE-PDF2.2 under the conditions tested supports further evaluation of PDF2-type genes in Brassica.

plant biology↗

bioRxiv2026

Structural Insights into the Expanded Substrate Scope of a Cysteine-Dependent Hydrolase from Rhodococcus opacus for Polyethylene, Polypropylene, and Polystyrene Biodegradation

Microplastic pollution from homopolymers such as polypropylene (PP), polystyrene (PS), and polyethylene (PE) poses growing health and environmental concerns, highlighting the need for robust polymer-degrading biocatalysts. We previously identified a naturally occurring cysteine-dependent hydrolase (CsdH) from Rhodococcus opacus, featuring a unique Cys160-Lys127-Asp39 catalytic triad and providing a novel mechanism for polyethylene terephthalate (PET) degradation. In this study, we have determined crystal structures of CsdH in complex with PP, PS, PE, and styrene and demonstrated the potential of the enzyme to directly degrade these homopolymers into their respective low molecular weight degradation products using LC-MS. Structural analysis reveals that the enzyme retains a conserved principal access tunnel while undergoing subtle substrate-dependent changes to accommodate different substrates. The enzyme exhibited broad plastic degradation activity with the highest weight loss observed for PE, followed by PP and PS. SEM analysis reveals surface roughness, cracks and grooves, confirming polymer degradation. Further, these results corroborate through FTIR and TGA analysis. These findings establish a strong foundation for enzyme engineering and aim at developing efficient microplastic degradation.

biophysics↗

bioRxiv2026

MICROSTRUCTURAL ORGANIZATION OF CORTICO-SUBCORTICAL FUNCTIONAL CONVERGENCE

The human subcortex integrates information from distributed cortical regions through functionally organized cortico-subcortical systems. Using the publicly available MICA-MICs multimodal MRI dataset of 50 healthy adults, we characterized the microstructural organization of functionally converging cortical regions. First, using resting-state fMRI., we identified cortical regions converging on subcortical targets, including thalamic, basal ganglia, and subcortical limbic structures. Then, using depth-resolved quantitative T1 profiles, we constructed a convergence-restricted microstructural profile covariance matrix, followed by diffusion-map embedding. The first microstructural gradient predominantly differentiated primary and association cortex, whereas the second gradient showed target-related differentiation beyond differences in primary versus association cortex. Cytoarchitectural analysis further revealed target-related differences in cortical composition. Together, these findings reveal complementary dimensions of cortico-subcortical functional organization, spanning broad cortical hierarchy, target-related microstructural differentiation, and cytoarchitectural composition.

neuroscience↗

bioRxiv2026

Activation of α7 Nicotinic Acetylcholine Receptor Inhibits Neutrophil ex vivo Swarming and Pulmonary Recruitment in Inflamed Mice through an Integrin αMβ2-dependent Mechanism

Background. The cholinergic anti-inflammatory pathway regulates inflammation through vagus signaling that activates the 7 nicotinic acetylcholine receptor (7nAChR) on leukocytes. Suppression of macrophage inflammatory cytokine production through 7nAChR-mediated inhibition of NF-{kappa}B activation is considered a central mechanism of this pathway. However, the effects of cholinergic activation of 7nAChR on neutrophils remain poorly understood. Here, we investigated whether and how activation of 7nAChR in neutrophils regulates neutrophil migration, defined the underlying molecular mechanism, and evaluated its contribution to anti-inflammatory protection during endotoxemia. Results. The ex vivo swarming assays revealed that activation of 7nAChR with the selective agonists PNU-282987 and AR-R17779 significantly inhibited human and mouse neutrophil migration. The inhibitory effect of PNU-282987 was abolished in 7nAChR-deficient neutrophils, confirming receptor specificity. Mechanistically, 7nAChR activation reduced integrin M{beta}2 activation, decreased F-actin polymerization, and impaired neutrophil polarization. These effects required M{beta}2, as PNU-282987 failed to inhibit actin remodeling in M-deficient neutrophils. Modulation of M{beta}2 activation with an activating antibody (MEM48) demonstrated that 7nAChR regulates neutrophil migration by limiting excessive M{beta}2 activation and maintaining an optimal adhesive state. In vivo, adoptively transferred fluorescently labeled PNU-282987-treated neutrophils exhibited reduced recruitment to the lungs compared with vehicle-treated neutrophils during LPS-induced endotoxemia. Furthermore, neutrophil depletion markedly diminished the protective effect of PNU-282987 on survival, demonstrating that neutrophils contribute to 7nAChR-mediated protection during systemic inflammation. Conclusions. Our findings identify neutrophils as an important cellular target of the cholinergic anti-inflammatory pathway and demonstrate that 7nAChR activation limits neutrophil migration by suppressing integrin M{beta}2 activation and actin cytoskeletal remodeling. Together, these findings establish M{beta}2 as a critical downstream effector of 7nAChR signaling in neutrophils and identify regulation of integrin activation as a previously unrecognized mechanism of the cholinergic anti-inflammatory pathway.

immunology↗

bioRxiv2026

Designing enzymes for new-to-nature chemistry and non-natural substrates with AlphaProtein Novo

Creating highly active enzymes for arbitrary reactions is a transformative goal for the molecular sciences (1). Traditional protein engineering (2,3) is constrained by a reliance on existing natural starting points, which are often challenging to discover. De novo design offers the potential to overcome this by creating enzymes from first principles (4), but has yet to achieve practically relevant catalytic properties. Here we present AlphaProtein Novo (AP Novo), a machine-learning pipeline that demonstrates, for the first time, that de novo enzyme design can outperform natural sequence mining in addressing challenging chemistry. We used AP Novo to design new-to-nature nitrene transferases to synthesise the pharmacophore piperidine with unprecedented product selectivity, and to create enzymes that degrade the environmental toxin DEHP under conditions that denature natural enzymes. We also obtained state-of-the-art catalytic efficiencies on two well-studied model reactions, and through iterative design and analysis found that key enablers of success were mechanism-inspired metrics based on AlphaFold 3 predictions and sequence-ensemble-based scoring of designed backbones. Our results show that de novo design has become a powerful complement to natural enzyme diversity for discovering catalysts for a range of applications.

synthetic biology↗

bioRxiv2026

Cytokinin Mediates Photosynthesis and Senescence Action Through CHLOROPLAST IMPORT APPARATUS 2 (CIA2) and CIA2-like (CIL)

Progression of leaf senescence is regulated by internal factors like the plant hormone cytokinin (CK) that acts to delay senescence and retain photosynthesis by maintaining chloroplast-functionality. While initial CK-signaling components controlling senescence have been described, downstream targets regulating physiological performance have remained less characterized. Here we present the CHLOROPLAST IMPORT APARATUS 2 (CIA2) and CIA2-like (CIL) transcription factors (TFs) as mediators of CK senescence activity, enabling chloroplast-function. Utilizing loss-of-function mutants, we show CIA2 and CIL negatively regulate senescence progression and alter physiological performance in Arabidopsis thaliana. Integrated transcriptomic-metabolomic analysis shows that CIA2 and CIL action is distinct from other known chloroplast-related TFs (like GLKs/GATAs) and can boost antioxidant response over senescence. We find that CIA2 and CIL are essential in relaying CK activity during chloroplast biogenesis, photosynthesis, rubisco function, and metabolite accumulation. In turn, ARR10/12 CK signaling components were needed for CK induction of CIA2 and CIL themselves - a cascade that induced chloroplast- and photosynthesis-associated nuclear genes, to boost chloroplast-function and metabolism. We also show that CK acts to repress senescence through accumulation of the polyamine, spermidine, in a CIA2/CIL dependent manner. This work presents a CIA2/CIL-cascade as a new anterograde signal for CK antisenescence effects, indicating a mechanism through which CK prolongs leaf function. This fundamental understanding of CK action has implications towards engineering plants with elevated photosynthesis performance and delayed senescence.

plant biology↗

bioRxiv2026

Design-Evolution Synergy Enables New-to-Nature Enzyme Chemistry

Directed evolution and machine-learning-assisted evolution have transformed biocatalysis by enabling rapid optimisation of catalytic performance within compatible protein scaffolds. In parallel, advances in deep-learning-driven de novo protein design have expanded accessible protein architectural space, enabling the generation of functional catalysts for increasingly challenging transformations. We herein report a synergistic design-evolution strategy to encode new-to-nature regio- and enantioselective C-H amination reactions using engineered de novo haemproteins to synthesise chiral piperidines, a privileged pharmacophore in drug discovery. By integrating motif scaffolding, computational re-design, and directed evolution, we demonstrate de novo enzyme design can enable challenging, high-energy-barrier enzyme chemistry with programmable regio- and enantioselectivity, high catalytic efficiency, excellent enzyme stability, and scaffold diversity.

synthetic biology↗

bioRxiv2026

Association between breeding-habitat bacterial communities and oviposition behavior of Aedes aegypti: Insights from arboviral outbreak-affected areas of Pune, Western India

Aedes aegypti, an important vector of several arboviral diseases, is highly adapted to human-associated environments and commonly exploits anthropogenic water-holding containers for breeding. Understanding the ecological factors governing oviposition-site selection is essential for developing sustainable vector control strategies. Previous studies have established that microbial communities in breeding waters influence mosquito oviposition-site selection through the production of volatile organic compounds. Importantly, rapid urbanization in India has increased the availability of artificial breeding habitats for Ae. aegypti, potentially facilitating the transmission and persistence of arboviral diseases. However, the composition of bacterial communities in these larval habitats and their potential role in shaping the oviposition behavior of Ae. aegypti remain poorly characterized in the Indian context. Here, we investigated the bacterial diversity and community composition of Aedes larval habitats during the 2024 Zika and chikungunya outbreaks in Pune, India. Water samples, collected from artificial urban containers and natural freshwater habitats, were subjected to 16S rRNA amplicon sequencing. Artificial breeding habitats exhibited significantly higher bacterial diversity and were enriched with Enterobacter spp. compared with natural habitats. Oviposition bioassays further demonstrated a strong preference of gravid Ae. aegypti females for Enterobacter-conditioned water. GCMS analysis of bacterial suspension identified indole as a prominent volatile metabolite associated with Enterobacter, suggesting its possible role in mediating oviposition site selection. Together, these findings provide insights into the microbial and chemical ecology of Ae. aegypti breeding habitats and establish a basis for investigating microbial volatile cues as potential targets for vector surveillance and environmentally sustainable attract-and-kill strategies.

genomics↗

bioRxiv2026

Argonaute 15 regulates immunity of potato to filamentous pathogens through transposon-derived small RNAs

Canonical ribonucleases such as Argonaute (AGO) participate in RNA-based gene regulatory processes. They are important for small RNA (sRNA) association and the formation of RNA-induced RNA silencing complexes. Potato (Solanum tuberosum) has 14 AGO-encoding genes, including the Solanum-specific StAGO15, which is activated upon infection by filamentous pathogens. The resistant phenotype conferred by ectopic expression of StAGO15 was abolished in plants in which a 21-nt sequence in the PAZ-encoding region of StAGO15 was targeted using an artificial miRNA. We then analyzed the sRNAs associated with StAGO15 in Phytophthora infestans leaf infection. Small RNAs were coimmunoprecipitated, subjected to deep sequencing, and analyzed against the P. infestans and potato genomes, in which cleavage sites were predicted by applying the R package, smartPARE. The most notable difference between pathogen- and water-inoculated StAGO15 samples was observed in the transposon (TE) category. Here, two categories of nonautonomous TE dominated: the miniature inverted-repeat transposable elements (MITEs) and short interspersed nuclear elements. The StAGO15-associated sRNAs bind and target complementary endogenous potato RNAs. One example is siRNA122, which acts on a cleavage site in the resistance gene PGSC0003DMG400015681 (StRES1). siRNA122 is derived from a MITE; upon deletion of this source sequence, resistance is restored. Our findings provide new insights into the plant immune surveillance network and demonstrate that deleting a 21 nt RNA sequence from a MITE transposon released a defense response, against two important filamentous pathogens.

plant biology↗

bioRxiv2026

Thermodynamic, Electrochemical and Practical Constraints on Electromicrobial Formate Assimilation

Electromicrobial production (EMP) technologies aim to combine renewable electricity, CO2, and engineered microbes to make energy-dense molecules at efficiencies exceeding photosynthesis. CO2 can be electrochemically reduced to formate, which is far easier to handle at the bench than H2 or an electrode, but formate carries only two electrons per carbon against the six in a biofuel. The remaining electrons must come from oxidizing additional formate, from H2 oxidation, or from extracellular electron uptake (EEU), and no rigorous comparison of these options coupled to the choice of carbon assimilation pathway currently exists. We calculate upper-limit efficiencies for butanol production by six carbon assimilation pathways, each paired with all three electron delivery mechanisms, using electrochemical parameters drawn from a survey of the recent literature. Electrical to butanol energy conversion efficiencies range from 35.5 to 51.7%, corresponding to solar-to-fuel efficiencies of 11.7 to 17%, so even the least efficient route exceeds the 8% theoretical ceiling of algal photosynthesis. The serine variant of the reductive glycine pathway reaches an electrical energy conversion efficiency of when using H2 oxidation, within 1.9 points of the most efficient pathway, and is the only high-efficiency option that tolerates O2. This makes an EMP system that combines electron delivery by formate coupled with the serine variant of reductive glycine pathway highly attractive, as it presents few barriers to rapid, iterative engineering in the lab, and a high theoretical ceiling. Drawing both carbon and electrons from formate costs 6.2 points against H2 at a state-of-the-art whole-cell voltage (2.2 V). However, this small penalty is amplified three-fold by any rise in the CO2-to-formate cell voltage, and reaches 13.5 points at the highest whole-cell voltages reported for scaled-up CO2-to-formate electrolyzers, where formate-only operation falls to 11.2 electrical-to-fuel and 3.7% solar-to-fuel efficiency, below the ceiling of photosynthesis, against 24.7 and 8.1% for H2 (only just above algal photosynthesis). Our choice between a formate-only system and one coupled to H2 oxidation or EEU therefore depends on our belief about the trajectory of CO2 reduction technology. If whole-cell voltages continue to fall at the rate of the past decade, formate alone is the right target, and the simplicity of its workflow is bought at low cost. However, if that improvement plateaus, the electron delivery mechanism must be swappable, and a system should be designed from the outset so that it can be. At the US Department of Energy SunShot target of 2 cents per kilowatt hour, the electricity to make a US gallon of butanol costs $1.40 for a formate-only system at the state of the art, rising to $5.45 at the highest scaled-up electrolyzer voltage reported, against $1.23 and $2.47 for formate and H2 system.

synthetic biology↗

bioRxiv2026

Physical priors improve performance of structure-based binding affinity models

Structure-based drug discovery is a widely used paradigm for the rational design of novel small molecule therapeutics. However, the benefits conferred by the use of structural information has seen limited adoption in machine learning, where ligand-only ("2D") models are still the industry standard for molecular property or binding affinity prediction. Structure-based ("3D") ML models for binding-affinity prediction promise to present a clear advantage, but have not yet overtaken existing 2D models. Here, we show that physics-based priors can improve predictive performance of structure-based models by comparing different model architectures with varying physical priors on several prediction tasks. We present the Modular Training and Evaluation of Neural Networks (mtenn) package, where we decompose affinity prediction into separate steps of embedding structure into learned representations and combining those embeddings into a predicted binding affinity. We consider both E(3)-invariant and E(3)-equivariant architectures to determine the importance of encoding roto-translational inductive biases, as well as different methods for combining learned embeddings. By first optimizing several aspects of model construction using the general purpose PDBBind dataset, we are able to improve the performance and data efficiency of structure-based models. When subsequently trained and evaluated on the COVID Moonshot small molecule drug discovery dataset, our tuned models perform on par with industry standard ligand-only models. Our decomposed model framework highlights that encoding some physical priors improves model performance, while more complex biases such as equivariance offer limited benefit. Additionally, structure-based models generalize better to an unseen target and display higher training efficiency. Overall, these results emphasize that structure-based models benefit from their ability to incorporate physics-informed constraints, giving promising directions for model architecture development. These results also suggest that the strength of these models may be in tasks specifically aimed at generalizability, providing guidelines for their use in early-stage drug discovery campaigns.

bioinformatics↗

bioRxiv2026

Neuronal lipid composition regulates distinct states of full-length tau assembly, mechanics and membrane interactions

Aggregation of tau into fibrillar assemblies and neurofibrillary tangles is a defining feature of tauopathies. However, the role of the membrane-rich neuronal environment on the assembly and mechanics of full-length tau remains unclear. Here, we examined how neuronal lipid membranes affect the assembly and membrane interaction of full-length human tau (hTau40). Lipid composition redirected hTau40 assembly, with total brain extract and brain phosphatidylcholine membrane producing shorter, more flexible fibrils, whereas brain phosphatidylserine membrane favoured a more heterogeneous and relatively rigid fibrillar population. Tau-membrane interaction depended strongly on the assembly state of tau. At the same protein concentration, preformed oligomers accumulated on membranes much more rapidly than monomeric tau, indicating that oligomer formation increases membrane recruitment. However, strong membrane binding did not directly predict the extent of membrane perturbation. Pronounced changes in lipid organisation were observed when tau was undergoing continued higher-order assembly at the membrane. Thus, the ability of tau to bind a membrane and its ability to reorganise that membrane appear to be related, but distinct, features of tau-membrane interaction. In cells, internalised hTau40 formed stable, low-mobility assemblies associated with the endolysosomal compartment and changes in its physical properties. These assemblies could also be transferred between cells in a membrane-derived vesicle model. Together, our results show that local membrane composition shapes tau assembly and mechanics, while the state of tau assembly influences how it binds to and remodels membranes. Key words: Amyloid, Tauopathy, Tau protein (Tau), Lipid Vesicle, Membrane Biophysics

biophysics↗