Biology subjects

Kaur, S.

Publications and source records attributed to Kaur, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Global Hypomethylation in Cell-free DNA Enables Non-invasive Colorectal Cancer Screening: Results from a Retrospective Validation Study

Global DNA hypomethylation is a defining hallmark of colorectal cancer (CRC) but is poorly captured by existing cell-free DNA (cfDNA) technologies, which typically interrogate only a fraction of CpG sites and are biased toward CpG islands. We developed Asima Rev, an electrical-impedance cfDNA assay that can differentiate healthy individuals from those with cancer by measuring cfDNA aggregation patterns associated with methylation state, enabling functional detection of genome-wide hypomethylation. In a cohort of 46 treatment-naive CRC patients and 33 controls, Asima Rev achieved 96% sensitivity and 94% specificity, with longitudinal monitoring in six patients fully concordant with clinical outcomes. Interrogation of 11 public methylation array datasets showed that whole array analyses underestimate global changes. However, restricting analyses to OpenSea regions, where cfDNA is enriched, revealed patterns consistent with Asima Rev and a significant 5.8% global hypomethylation in CRC tissue compared to adjacent normal tissue. Hypomethylation was not observed in immune cell genomic DNA (gDNA), a major contributor to cfDNA, supporting a predominantly tumor-derived contribution to the observed cfDNA signal. Together, these results demonstrate that Asima Rev captures a cfDNA signal consistent with true global methylation loss and outperforms locus-specific assays by measuring structural consequences of pan-genomic epigenetic alterations.

bioinformatics↗

bioRxiv2025

Redox-controlled dimerisation regulates ethylene biosynthesis

Ethylene is a central plant hormone that orchestrates growth, development, senescence, and stress responses. Because it is gaseous, ethylene must be synthesised on demand, yet the catalytic and regulatory mechanisms of its biosynthetic enzyme, 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), remain poorly understood. Here, using structural, biophysical, and computational analyses, we uncovered two principles: ACO catalysis relies on an induced-fit mechanism, and disulfide-mediated dimerisation via a conserved cysteine acts as a redox switch toggling ACO between active monomer and inactive dimer. This previously unrecognised regulatory layer positions ACO as a redox sensor in plant cells, revealing a fundamental control point in ethylene biosynthesis. Given ethylenes pivotal role in crop productivity and stress resilience, these findings open new opportunities for precise manipulation of hormone signalling in agriculture and biotechnology.

biochemistry↗

bioRxiv2025

Increased versican and fibrosis in mesenteric lymph nodes disrupts immune surveillance and drives systemic bacterial dissemination in cirrhosis

Background and ObjectiveMesenteric lymph nodes (MLN) are immunological barriers against bacterial translocation (BT). Enhanced gut BT through MLN facilitates bacterial spread and higher mortality in cirrhosis. We aimed to elucidate mechanisms underlying MLN failure to effectively contain BT during advanced cirrhosis. DesignBT and immune cells were analyzed in lymphoid organs and circulation of control and CCl4 models, with and without MLN (MLNx). MLN proteomics identified versican (VCAN) as major upregulated protein in cirrhosis, whose immunomodulatory function was examined in vitro and in vivo in CCl and (Bile duct ligation) BDL models. Plasma VCAN were measured in end-stage cirrhosis patients and analyzed as mortality predictor. ResultsIn control rats, bacteria were confined to MLN, whereas cirrhotics showed BT to MLN, lymph, and portal blood. Compared to control, CCl4 rats had increased activated Th-cells in MLN but reduced in circulation. In control-MLNx rats, activated Th-cells were reduced in circulation vs controls. In BDL models, MLN CFU correlated with VCAN level. In vitro, VCAN enhanced T cell suppression and impaired migration which was reversed by CD44 blockade. In vivo VCAN knockdown reduced fibrosis and bacterial burden in MLN, while restoring Th-cell activation locally and systemically. Clinically, plasma VCAN levels were elevated in advanced cirrhosis patients and remained an independent predictor of 28-day sepsis-related mortality. ConclusionIncreased VCAN impairs T cell activation and migration in MLN, fostering immune suppression and bacterial persistence. Plasma VCAN levels serve as promising biomarker for MLN dysfunction and prognostic factor for predicting sepsis-related mortality in end-stage cirrhosis. What is already known on this topic - Enhanced gut bacterial translocation through mes-enteric lymph nodes (MLN) facilitates systemic bacterial spread and increases mortality in cirrhosis. The mechanisms underlying MLN failure to effectively contain bacterial spread during advanced cirrhosis remain largely unknown. What this study adds - The study unveils a critical role of lymph node fibrosis and elevated versican (VCAN) expression in causing deranged immune responses and bacterial clearance in MLN, increasing systemic bacterial load and immunosuppression. Most importantly, high plasma VCAN emerges as a prognostic biomarker for functional failure of MLN and 28-day mortality predictor in critically ill patients with cirrhosis. How this study might affect research, practice or policy - VCAN, representing enhanced MLN fibrosis and dysfunction, emerges as a predictive biomarker of adverse clinical out-comes in patients with advanced cirrhosis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/690350v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@369f4eorg.highwire.dtl.DTLVardef@14c232org.highwire.dtl.DTLVardef@d99b21org.highwire.dtl.DTLVardef@57ac95_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2025

Alternative splicing of synaptotagmin 7 regulates oligomerization and short-term synaptic plasticity

Synaptic plasticity is crucial for learning and memory. The presynaptic calcium sensor synaptotagmin 7 (syt7) regulates aspects of short-term plasticity (STP), but the underlying mechanisms remain unclear. Here, we show that alternative splicing of the syt7 juxtamembrane linker acts as a molecular switch at both biochemical and functional levels. The and {beta} variants undergo liquid-liquid phase separation to form condensates, while the {gamma} variant forms aggregates. Using iGluSnFR imaging, we found that when expressed at equal levels, these three isoforms also diverge regarding their abilities to regulate two key aspects of STP: paired-pulse facilitation and synaptic depression. STED microscopy showed that all three isoforms form active zone-associated clusters that colocalize with syt1, while MINFLUX super-resolution microscopy resolved syt7 clusters within the active zone, well-positioned to directly control synaptic vesicle dynamics. Thus, alternative splicing might fine-tune STP by differentially impacting syt7 oligomerization.

neuroscience↗

bioRxiv2025

Wnt7a is a Novel Lymphangiocrine Factor Driving Cholangiocyte Proliferation during Liver Regeneration

The significance of lymphatic vessels (LVs) and lymphatic endothelial cells (LyECs) during liver regeneration remains unexplored. We aimed to characterize contribution of LVs and LyECs in liver regeneration using two-thirds partial hepatectomy (PHx) models. An increased number and diameter of CD31+PDPN+ LVs was observed at day 2 and 5 post-PHx. Proteomic analysis of LyECs and ELISA studies in conditioned media of LyECs revealed that one of the secreted proteins, Wnt7a was uniquely expressed at day 2 post PHx. In vitro studies confirmed that Wnt7a is a potent stimulator of proliferation for both hepatocytes and cholangiocytes. In vivo inhibition of lymphangiogenesis and reduction in Wnt7a led to a decline in the percentage of CK19+ cholangiocytes and bile ducts. Western blot analysis showed that Wnt7a activated the downstream Frizzled 7 receptor and non-canonical planar-cell-polarity pathway to drive the proliferation of cholangiocytes. In vivo, Wnt7a loss-of-function experiments in zebrafish along with inhibitor studies in rat models of PHx further re-iterated clear links between Wnt7a and proliferation of bile ducts. Pro-regenerative effects of Wnt7a were demonstrated in a model of small-for-size syndrome (80% PHx), where administering recombinant Wnt7a enhanced both cholangiocyte and hepatocyte proliferation. Elevated Wnt7a levels were seen in donors of liver transplant patients at day 1 and 2 post-hepatectomy and Wnt7a also facilitated growth of human cholangiocyte organoids in vitro. Our findings uncover the novel role of Wnt7 as one of the key lymphangiocrine signals that facilitates proliferation of cholangiocytes during liver regeneration. HighlightsO_LINumber of functional lymphatic Vessels (LVs) and lymphangiogenesis is enhanced between day 2 and day 5 after partial hepatectomy (PHx) in rat models and liver transplant donors. C_LIO_LIWnt7a released from lymphatic endothelial cells (LyECs) acts as a potent lymphangiocrine factor, stimulating proliferation of hepatocytes and cholangiocytes in vitro, across both rodent and human models. C_LIO_LIInhibition of lymphagiogenesis during PHx transiently reduces number of cholangiocytes during early liver regeneration. C_LIO_LIWnt7a signals through Frizzled 7 receptor to activate p-AKT, p-JAK1 and p-STAT3 proteins in cholangiocytes. C_LIO_LIAdministration of recombinant Wnt7a in 80% PHx models induces proliferation of hepatocytes and bile ducts. C_LI

physiology↗

bioRxiv2025

Feasibility of a parent-delivered attention and working memory intervention for early school-aged children born preterm

Preterm birth is a common neurodevelopmental condition that can have lasting impacts on cognition, including attention and working memory. Interventions that strengthen these skills in early childhood could support school readiness. Dino Island is a tablet-based intervention that combines process-specific practice of attention and working memory skills with a compensatory component that teaches metacognitive skills to scaffold learning. In this pilot study, we evaluated the feasibility of parent-delivered Dino Island in young children born preterm (N=10), alongside a control group who played educational games (N=12). Both groups were instructed to play 2-3 times per week for approximately 20 minutes over a 12-week period. Attention and working memory on untrained tasks were assessed before and after completing the intervention. Parents provided fidelity data through tracking sheets and participated in exit interviews to offer feedback and identify barriers and facilitators. We found that the Dino Island program was successfully delivered by parents with high fidelity. Attrition was higher in the Dino Island group, likely reflecting the challenges of delivering cognitive remediation in home settings. Comparison of attention and working memory scores on untrained tasks pre- and post-showed practice effects but no specific benefit of Dino Island. However, parent reports suggested behavioral improvements specific to the Dino Island group, noting far-transfer effects where children applied metacognitive strategies in other contexts. Overall, this work shows feasibility and tolerability of Dino Island in young children born preterm. Future research should examine its potential impact on school readiness and longer-term academic outcomes in this population.

neuroscience↗

bioRxiv2025

Variations in carbapenem resistance associated with the VIM-1 metallo-β-lactamase across the Enterobacterales

The VIM-1 metallo-{beta}-lactamase enzyme, encoded as a cassette within class 1 integrons, is found in Gram-negative clinical isolates worldwide and has been linked to outbreaks of bacterial pathogens in nosocomial settings. Six vim-1+ clinical isolates, from the genera Escherichia, Klebsiella, and Enterobacter, were obtained from Kingston, Ontario, Canada. Whole genome sequencing revealed that vim-1 was plasmid-borne in all strains and situated as the first gene in In916 or In110 integrons. Analysis of related plasmids suggested that these vim-1-containing plasmids are globally disseminated and have spread via horizontal gene transfer and autochthonous vertical spread within Ontario. Interestingly, the minimum inhibitory concentrations of ertapenem and meropenem, two clinically relevant carbapenem antibiotics, against these six isolates varied more than tenfold, suggesting the effects of VIM-1 are dependent on the genomic content of the host microbe. To further study the genomic content dependency of VIM-1, we introduced vim-1 into three common Enterobacterales laboratory strains. Although introduction of vim-1 into Escherichia coli DH5 resulted in little resistance to ertapenem or meropenem, multiple rounds of adaptive laboratory evolution allowed us to identify variants with extremely high levels of resistance to both carbapenems. DNA sequencing revealed that the increase in carbapenem resistance was due to a combination of increased vim-1 gene dosage and epistatic interactions with mutations of ompC that likely would have decreased outer membrane permeability to these antibiotics. Together, these results provide additional support for the role of gene epistasis is modulating the antimicrobial resistance phenotypes of acquired resistance genes, as well as previous results suggesting that the presence of a {beta}-lactamase gene is insufficient to confer strong resistance to carbapenems without being paired with reduced outer membrane permeability.

microbiology↗

bioRxiv2025

Sequence-Based Generative AI-Guided Design of Versatile Tryptophan Synthases

Enzymes offer unparalleled selectivity and sustainability for chemical synthesis, yet their widespread industrial application is often hindered by the slow and uncertain process of discovering and optimizing suitable biocatalysts. While directed evolution remains the gold standard for enzyme optimization, its success hinges on the availability of a starting enzyme with measurable activity, a persistent bottleneck for many desired functions. Designing libraries likely to contain such functional starting points remains a major challenge. In this work, we use the GenSLM protein language model (PLM) along with a series of filters to generate novel sequences of the {beta}-subunit of tryptophan synthase (TrpB) that express in Escherichia coli, are stable, and are catalytically active in the absence of a TrpA partner. Many generated TrpBs also demonstrated significant substrate promiscuity, accepting non-canonical substrates typically inaccessible to natural TrpBs. Remarkably, several outperformed both natural and laboratory-optimized TrpBs on native and non-canonical substrates. Comparative analysis of the most active and promiscuous generated TrpB and its closest natural homolog confirmed that this enhanced functional versatility does not stem from the natural enzyme, highlighting the creative potential of generative models. Our results demonstrate that the model can generate enzymes which not only preserve natural structure and function but also acquire non-natural properties, establishing PLMs as powerful tools for biocatalyst discovery and engineering, with the potential in some cases to bypass further optimization.

biochemistry↗

bioRxiv2025

A framework to enhance the Signal-to-Noise Ratio for quantitative fluorescence microscopy

Single-cell fluorescence characterization has gained much attention for studying the dynamics of individual cells in human diseases such as cancer. Despite the abundance of literature on quantitative fluorescence microscopy and its advantages in measuring cell-to-cell and spatial variation over other high-throughput instruments, it lacks a concise model that one can follow to maximize the quality of images. Here, we used the signal-to-noise ratio (SNR) model to verify camera parameters and optimize microscope settings to maximize SNR for quantitative single cell fluorescence microscopy (QSFM). We determined the microscope cameras readout noise, dark current, photon shot noise, the clock-induced charge, and validated the additive noise model for each noise source. The dark current and the clock-induced charge were both higher than reported in literature, compromising camera sensitivity. We also reduced excess background noise and improved SNR by 3-fold, by adding secondary emission and excitation filters as well as by introducing wait time in the dark before fluorescence acquisition. Additionally, our work opens new avenues for enhancing superresolution microscopy techniques such as small molecule localization microscopy (SMLM).

biophysics↗

bioRxiv2025

Plasmodium Repetome: A mysterious space with a wealth of information

Eukaryotic proteomes harbour repetitive stretches of amino acids that may play critical roles in the biology of that organism. While several tandem repeats (TR) are known to contribute to protein structure and function, information about the vast majority of repeat regions remains obscure. In this article, we have analysed the repeat content of different Plasmodium species and found the leading human malaria-causing P. falciparum (Pf) and P. vivax to be exceptionally rich in TR regions (>40% TR containing proteins). Detailed analysis of Pf repetome showed this intracellular parasite to carry longer TRs, several of which were present in exported proteins important for parasite survival and immune evasion. The repeat regions of Pf were enriched in acidic amino acids and asparagine (Asn), where Asn was more abundant in short and intermediate TRs, suggesting an evolutionary bias influenced by replication slippage and positive selection. Gene ontology analysis of TR containing Pf proteins helped us to understand their cellular localization along with the molecular and biological processes they are involved in. The Pf variable surface antigen families with roles in important pathogenic processes like cytoadherence, immune evasion etc. had low repeat content present within seroreactive peptides. Three-dimensional structure predictions of TR regions revealed several repeats to adopt ordered super-secondary conformations that are known to facilitate intermolecular interactions. Overall, this is a comprehensive study attempting to gain insights on the importance of TRs in malaria parasite biology and suggests a novel route to understanding protein function through the characterization of repeat content.

bioinformatics↗

bioRxiv2025

CEPR2 perceives group II CEPs to regulate cell surface immunity in Arabidopsis

Plant endogenous peptides are crucial for diverse aspects of plant physiology. Among them, C-TERMINALLY ENCODED PEPTIDEs (CEPs) have recently emerged as important regulators of plant growth and stress responses. CEPs are divided into two major subgroups: group I CEPs and the less studied group II CEPs. We recently demonstrated that group I CEPs coordinate cell surface immunity with the nitrogen status in Arabidopsis thaliana (hereafter Arabidopsis). To mount full group I CEP responsiveness, the three phylogenetically related CEP RECEPTOR 1 (CEPR1), CEPR2 and RECEPTOR-LIKE KINASE 7 (RLK7) are required. Here, we provide evidence that biotic stress induces expression of the group II CEP peptide CEP14. CEP14 and the related CEP13 and CEP15 trigger hallmark immune signaling outputs in a proline hydroxylation pattern-dependent manner in Arabidopsis. Genetic data indicates that group II CEP members contribute to cell surface immunity against bacterial infection. We further show that group II CEP perception primarily depends on CEPR2. Our work provides new insights into CEP function during biotic stress and sheds new light on the complexity of sequence-divergent CEP signalling mediated by specific endogenous receptors.

plant biology↗

bioRxiv2024

Identifying the Brain Circuits that Regulate Pain-Induced Sleep Disturbances

Pain therapies that alleviate both pain and sleep disturbances may be the most effective for pain relief, as both chronic pain and sleep loss render the opioidergic system, targeted by opioids, less sensitive and effective for analgesia. Therefore, we first studied the link between sleep disturbances and the activation of nociceptors in two acute pain models. Activation of nociceptors in both acute inflammatory (AIP) and opto-pain models led to sleep loss, decreased sleep spindle density, and increased sleep fragmentation that lasted 3 to 6 hours. This relationship is facilitated by the transmission of nociceptive signals through the spino-parabrachial pathways, converging at the wake-active PBelCGRP (parabrachial nucleus expressing Calcitonin Gene-Related Peptide) neurons, known to gate aversive stimuli. However, it has never been tested whether the targeted blocking of this wake pathway can alleviate pain-induced sleep disturbances without increasing sleepiness. Therefore, we next used selective ablations or optogenetic silencing and identified the key role played by the glutamatergic PBelCGRP in pain-induced sleep disturbances. Inactivating the PBelCGRP neurons by genetic deletion or optogenetic silencing prevented these sleep disturbances in both pain models. Furthermore, to understand the wake pathways underlying the pain-induced sleep disturbances, we silenced the PBelCGRP terminals at four key sites in the substantia innominata of the basal forebrain (SI-BF), the central nucleus of Amygdala (CeA), the bed nucleus of stria terminalis (BNST), or the lateral hypothalamus (LH). Silencing of the SI-BF and CeA also significantly reversed pain-induced sleep loss, specifically through the action on the CGRP and NMDA receptors. This was also confirmed by site-specific blockade of these receptors pharmacologically. Our results highlight the significant potential for selectively targeting the wake pathway to effectively treat pain and sleep disturbances, which will minimize risks associated with traditional analgesics. One sentence summaryParabrachial CGRP neurons regulate awakenings to pain.

neuroscience↗

bioRxiv2024

Mass flowering and flowering asynchrony characterise a seasonal herbaceous community in the Western Ghats

Flowering synchrony within a community may be facilitated by climatic factors and by ecological interactions that promote shared pollination services. In contrast, flowering asynchrony is promoted when there is inter-species competition for pollinators. In this multiyear study, we analyse the flowering phenology of a seasonal, herbaceous community (Kaas plateau) in Western Ghats, India to identify environmental constraints that may influence flowering synchrony. We addressed the questions: (a) Is flowering seasonality correlated with climatic factors? (b) Is there evidence for flowering synchrony within the community? (c) Do plant-pollinator interactions shift with flowering phenology? In Kaas, we recorded flowering phenology of 76 herbaceous species and found that climatic factors influenced their flowering phenology. We also identified the community to be composed of a few mass flowering (MF) species ([~]30%) and several non-mass flowering (nMF) species ([~]70%). Using two novel synchrony indices, temporal overlap (SItemp) and synchrony in abundance (SIabd), we also identified higher asynchronous flowering within the community than expected. Notably, species sharing the same floral colour, showed marked absence of synchrony, thus suggesting that competition and not pollinator-mediated facilitation drives flowering asynchrony within Kaas. Finally, pollination networks were observed to shift with flowering abundances within the community. Our findings reveal that even seasonal landscapes like the laterite plateaus, despite their short flowering season that last only 4-5 months, exhibit an overall asynchronous flowering phenology. And, the synchronous flowering for which the Kaas plateau is famous, was noted to be mostly due to a few mass flowering species that alter across years.

ecology↗

bioRxiv2024

Revisiting Aristotle's observation on bees: High floral constancy is common among bees but it is shaped by the locally abundant flowering species

Floral constancy is the tendency of a pollinator to sequentially visit flowers of the same species despite the availability of other rewarding plants. In a phytodiverse community, resource assurance may lead to pollinators displaying floral constancy to the most abundant plant species. We tested this by investigating if pollinators are floral constant on the abundant or the non-abundant plants within a seasonally flowering tropical community. We quantified floral constancy in three social (Apis spp.) and two solitary (non-Apis) Indian native bees using three approaches, that is by manually tracking the bees, analysing their pollen load, and examining pollen sacs of returning bees at their hive. We next examined in Apis cerana indica if constancy in individual bees translated to hive-level constancy. We found that in our community with distinct co-flowering patches, bees were constant to the most abundant species within a localised patch, and not to the most abundant species in the landscape. While the pollen loads from both the social and solitary bees suggested that they show high floral constancy (> 70% uni-dominant pollen), their values differed significantly (p < 0.0001). Finally, approximately 90% of individuals within a hive showed floral constancy (monolectic), but collectively, a hive displayed polylectic foraging. Our findings highlight that the foraging patterns of native pollinators has been understudied and is a critical first step towards connecting reproductive assurances to plant-pollinator dependencies in large landscapes.

animal behavior and cognition↗

bioRxiv2024

One Health assessment of poultry and cattle farms as reservoirs for ESBL-producing Escherichia coli

BACKGROUNDUnregulated use of antibiotics in animal food production leads to the emergence of multidrug resistant bacteria, notably extended-spectrum beta-lactamases producing Escherichia coli (ESBL E. coli). ESBLs severely limit treatment options and pose a significant threat to public health. Investigating the potential role of livestock and poultry farms in harboring ESBL E. coli is crucial, given the prevalence of ESBL E. coli in humans, animals, and the environment, following the One Health framework. OBJECTIVESThe aim of this study was to assess and compare the potential contribution of cattle and poultry farms along with their respective human handlers to the emergence and spread of ESBL E. coli following One Health approach. METHODSTwenty cattle and twenty poultry farms were surveyed in Ludhiana, India to determine antibiotic usage, prevalent diseases in the farms. Animal faeces/poultry droppings, handler hand swabs, and stool samples were examined for the presence of ESBL E. coli. The isolates were examined for their susceptibility to 16 antibiotics and for the presence of selected antimicrobial resistance genes, including ESBL genes. Generalized linear mixed models were applied to investigate variations in the prevalence of ESBL E. coli associated with farm animal species, farm practices, and farm biosecurity measures. RESULTSOur findings revealed a higher prevalence of ESBL E. coli in poultry farms (53.75%) than in cattle farms (40.72%) and overall lower prevalence (12%) in human handlers. Of the total isolates, 86.52% were multidrug resistant, and 93.91% showed a high multiple antibiotic resistance (MAR) index (>0.2). Isolates from poultry farms exhibited a higher level of resistance with 97% of isolates exhibiting multidrug resistance, 100% isolates with MAR index of more than 0.2, and 90.70% carrying the blaCTXM gene. CONCLUSIONOur findings highlight cattle and poultry farms as potential reservoirs for multidrug-resistant and ESBL E. coli, contributing to the rising environmental burden of antibiotic resistance genes. This emphasizes the urgency of limiting antibiotic use in animal farming, especially in poultry production, to address the escalating problem of antibiotic resistance and protect both animal and human health.

microbiology↗

bioRxiv2024

A functional screen for ubiquitin regulation identifies an E3 ligase secreted by Pseudomonas aeruginosa

Ubiquitin signaling controls many aspects of eukaryotic biology, including targeted protein degradation and immune defense. Remarkably, invading bacterial pathogens have adapted secreted effector proteins that hijack host ubiquitination to gain control over host responses. These ubiquitin-targeted effectors can exhibit, for example, E3 ligase or deubiquitinase activities, often without any sequence or structural homology to eukaryotic ubiquitin regulators. Such convergence in function poses a challenge to the discovery of additional bacterial virulence factors that target ubiquitin. To overcome this, we have developed a workflow to harvest natively secreted bacterial effectors and functionally screen them for ubiquitin regulatory activities. After benchmarking this approach on diverse ligase and deubiquitinase activities from Salmonella Typhimurium, Enteropathogenic Escherichia coli, and Shigella flexneri, we applied it to the identification of a cryptic E3 ligase activity secreted by Pseudomonas aeruginosa. We identified an unreported P. aeruginosa E3 ligase, which we have termed Pseudomonas Ub ligase 1 (PUL-1), that resembles none of the other E3 ligases previously established in or outside of the eukaryotic system. Importantly, in an animal model of P. aeruginosa infection, PUL-1 ligase activity plays an important role in regulating virulence. Thus, our workflow for the functional identification of ubiquitin-targeted effector proteins carries promise for expanding our appreciation of how host ubiquitin regulation contributes to bacterial pathogenesis.

microbiology↗

bioRxiv2024

Active Learning-Assisted Directed Evolution

Directed evolution (DE) is a powerful tool to optimize protein fitness for a specific application. However, DE can be inefficient when mutations exhibit non-additive, or epistatic, behavior. Here, we present Active Learning-assisted Directed Evolution (ALDE), an iterative machine learning-assisted DE workflow that leverages uncertainty quantification to explore the search space of proteins more efficiently than current DE methods. We apply ALDE to an engineering landscape that is challenging for DE: optimization of five epistatic residues in the active site of an enzyme. In three rounds of wet-lab experimentation, we improve the yield of a desired product of a non-native cyclopropanation reaction from 12% to 93%. We also perform computational simulations on existing protein sequence-fitness datasets to support our argument that ALDE can be more effective than DE. Overall, ALDE is a practical and broadly applicable strategy to unlock improved protein engineering outcomes.

bioengineering↗

bioRxiv2024

Exploring the functional, protective, and transcriptomic effects of GIP on cytokine-exposed human pancreatic islets and EndoC-βH5 cells

Immune-mediated beta-cell destruction and lack of alpha-cell responsiveness to hypoglycaemia are hallmarks of type 1 diabetes pathology. The incretin hormone glucose-dependent insulinotropic polypeptide (GIP) may hold therapeutic potential for type 1 diabetes due to its insulinotropic and glucagonotropic effects, as well as its beta-cell protective effects shown in rodent islets. Here, we examined the functional, protective, and transcriptomic effects of GIP treatment upon diabetogenic cytokine exposure to interleukin (IL)-1{beta} {+/-} interferon (IFN)-{gamma} in human EndoC-{beta}H5 beta cells and isolated human islets, respectively. GIP dose-dependently augmented glucose-stimulated insulin secretion from EndoC-{beta}H5 cells and increased insulin and glucagon secretion from human islets during high and low glucose concentrations, respectively. The insulinotropic effect of GIP in EndoC-{beta}H5 cells was abrogated by KN-93, an inhibitor of calcium/calmodulin-dependent protein kinase 2 (CaMK2). GIP did not prevent cytokine-induced apoptosis or cytokine-induced functional impairment of human EndoC-{beta}H5 cells. GIP also did not prevent cytokine-induced apoptosis in human islets. GIP treatment of human islets with or without cytokines for 24 hours did not significantly impact the transcriptome. GIP potentiated cytokine-induced secretion of IL-10 and c-c motif chemokine ligand (CCL)-2 from human islets while decreasing the secretion of c-x-c motif chemokine ligand (CXCL)-8. In EndoC-{beta}H5 cells, GIP reduced IFN-{gamma}-induced secretion of tumor necrosis factor (TNF)-, IL-2, IL-6, and IL-10 but increased the secretion of CXCL8, CCL2, CCL4, and CCL11. In conclusion, our results suggest that the insulinotropic effect of GIP is CaMK2-dependent. Furthermore, our results indicate that GIP does not provide substantial cytoprotective effects against diabetogenic cytokine challenge or significantly modulate the transcriptome of human islets when applied at a supraphysiological level. GIP may, however, still exert selective inflammation-modulatory effects upon diabetogenic cytokine exposure.

cell biology↗