Biology subjects

Prakash, A.

Publications and source records attributed to Prakash, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Integrated View of Baseline Protein Expression in Human Tissues using public Data Independent Acquisition datasets

The PRIDE database is the largest public data repository of mass spectrometry-based proteomics data and currently stores more than 40,000 datasets covering a wide range of organisms, experimental techniques and biological conditions. During the past few years, PRIDE has seen a significant increase in the amount of submitted Data-Independent Acquisition (DIA) proteomics datasets. This provides an excellent opportunity for large scale data reanalysis and reuse. We have reanalysed 15 public label-free DIA datasets across various healthy human tissues, to provide a state-of-the-art view of the human proteome in baseline conditions (without any perturbations). We computed baseline protein abundances and compared them across various tissues, samples and datasets. Our second aim was to compare protein abundances obtained here from the results of previous analyses using human baseline Data-Dependent Acquisition (DDA) datasets. We observed a good correlation across some tissues, especially in liver and colon but weak correlations were found in others, such as lung and pancreas. The reanalysed results including protein abundance values and curated metadata are made available to view and download from the resource Expression Atlas. For TOC Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/613191v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1414dforg.highwire.dtl.DTLVardef@66606aorg.highwire.dtl.DTLVardef@143f862org.highwire.dtl.DTLVardef@1682406_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2024

Mechanical coupling of compartments drives polarity and patterning of mouse auditory epithelium

Morphogenetic information arises from a combination of genetically encoded cellular properties and emergent cellular behaviours. The spatio-temporal implementation of this information is critical to ensure robust, reproducible tissue shapes, yet the principles underlying its organisation remain unknown. We investigated this principle using the mouse auditory epithelium, the organ of Corti (OC). OC consists of a sensory domain, which transduces sound through polar mechanosensory hair cells (HC), part of a mosaic with supporting cells (SC). On either side of the sensory domain are non-sensory domains. These domains undergo cellular rearrangements, which, together, lead to a spiral cochlea that contains planar polarised HCs. This makes the mammalian cochlea a compelling system to understand coordination across spatial scales. Using genetic and ex-vivo approaches, we found patterning of OC into sensory and non-sensory domains is associated with a combinatorial expression of adhesion molecules, which underpins OC into spatially defined compartments, enabling planar cell polarity (PCP) cues to regulate compartment-specific organisation. Through compartment-specific knockouts of the PCP protein, Vangl2, we find evidence of compartment coupling, a non-linear influence on the organisation within one compartment when cellular organisation is disrupted in another. In the OC, compartment coupling originates from vinculin-dependent junctional mechanics, coordinating cellular dynamics across spatial scales. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/613243v2_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@11d31c1org.highwire.dtl.DTLVardef@1ad85dcorg.highwire.dtl.DTLVardef@12f4a0corg.highwire.dtl.DTLVardef@1a663ab_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2024

Mitochondrial activity-driven hematopoietic stem cell fate and lympho-myeloid lineage choice is first established in the aorta-gonad-mesonephros

Mitochondrial metabolism determines bone marrow hematopoietic stem cell (HSC) heterogeneity and influences long-term repopulation potential. However, the origin of this heterogeneity and how it regulates HSC phenotype is unclear. We show that during the endothelial-to-hematopoietic transition (EHT) in the mouse embryo, dynamic changes in mitochondrial activity drive the production of mature HSCs with differing potencies. Lowering mitochondrial activity in the AGM by pharmacological or genetic means activates Wnt signaling to promote HSC expansion. Further, mitochondrial membrane potential (MMP) gives rise to functional heterogeneity in the definitive HSC pool. In-vitro and in-vivo functional assays, and single-cell transcriptomics on AGM HSCs showed that MMPlow HSCs are myeloid-biased, with enhanced differentiation potential, while MMPhigh HSCs are lymphoid-biased with diminished differentiation potential. Mechanistically, low mitochondrial activity upregulates Phosphoinositide 3-kinase (PI3K) signaling to promote HSC differentiation. These insights into the initiation of metabolic heterogeneity could be leveraged to isolate HSCs to efficiently generate desired lineages. HighlightsO_LIMitochondrial changes from HSC emergence to maturation govern embryonic hematopoiesis. C_LIO_LILowering mitochondrial activity promotes Wnt-dependent HSC expansion and PI3K-dependent differentiation. C_LIO_LIMitochondrial membrane potential determines lympho-myeloid fate choice in AGM HSCs. C_LIO_LIAberrant mitochondrial activity in the AGM HSCs perturbs adult hematopoiesis. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/609550v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@10ba1e2org.highwire.dtl.DTLVardef@b10b53org.highwire.dtl.DTLVardef@15eaad0org.highwire.dtl.DTLVardef@2077d5_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2024

Resources modulate developmental shifts but not infection tolerance upon coinfection in an insect system

Energetic resources fuel immune responses and parasite growth within organisms, but it is unclear whether energy allocation is sufficient to explain changes in infection outcomes under the threat of multiple parasites. We manipulated diet in flour beetles (Tribolium confusum) infected with two natural parasites to investigate the role of resources in shifting metabolic and immune responses after single and co-infection. Our results suggest that gregarine parasites alter the within-host energetic environment, and by extension juvenile development time, in a diet- dependent manner. Gregarines do not affect host resistance to acute bacterial infection but do stimulate the expression of an alternative set of immune genes and promote damage to the gut, ultimately contributing to reduced survival regardless of diet. Thus, energy allocation is not sufficient to explain the immunological contribution to coinfection outcomes, emphasizing the importance of mechanistic insight for predicting the impact of coinfection across levels of biological organization.

ecology↗

bioRxiv2024

The DJ-1-derived peptide, ND-13, confers cardioprotection by inhibiting mitochondrial fission and preserving mitochondrial bioenergetics via the RhoA-ROCK1-Drp1 pathway

BackgroundAcute myocardial infarction (AMI) and post-infarct heart failure (HF) are among the leading causes of death and disability worldwide. As such, new treatments are urgently needed to protect the heart against the detrimental effects of acute ischemia/reperfusion injury (IRI), in order to prevent the onset of HF and improve clinical outcomes following AMI. Given that mitochondrial dysfunction is a key determinant of IRI-induced cardiomyocyte death in AMI, we investigated ND-13, a 13-amino acid peptide derived from the pro-survival protein DJ-1, as a novel mitoprotective strategy for limiting myocardial infarct size (IS) following AMI. Methods and ResultsIn isolated adult Dendra-2 mice cardiomyocytes subjected to simulated IRI, treatment with ND-13 peptide reduced cell death by 42%, decreased phosphorylation of Drp1 at Ser616 and inhibited mitochondrial fission, improved mitochondrial respiratory function, decreased oxidative stress, and preserved ATP levels. Treatment of cardiomyocytes with ND-13 decreased levels of RhoA and reduced activity of downstream ROCK1, the latter of which is known to phosphorylate Drp1 at Ser616. In ex vivo Langendorff-perfused hearts subjected to IRI, treatment with ND-13 at reperfusion reduced IS by 43%, attenuated oxidative stress and preserved post-infarct cardiac contractile function. Finally, in vivo administration of ND-13 peptide at reperfusion in mice subjected to acute myocardial IRI reduced IS by 35% at 72-hours of reperfusion, and restored mitochondrial bioenergetics in cardiomyocytes isolated from the area-at-risk following 2-hours reperfusion as evidenced by preservation of key metabolites involved in glucose oxidation and fatty acid oxidation. The cardioprotective phenotype translated to improved cardiac function and less adverse left ventricular remodelling at 28 days post-infarction. ConclusionsWe show for the first time the mitoprotective effects of the DJ-1-derived peptide, ND-13, administered at the onset of reperfusion following AMI. We found that ND-13 protects mitochondria by inhibiting IRI-induced mitochondrial fission and preserved mitochondrial function following IRI via the RhoA-ROCK-Drp1 pathway. These findings highlight ND-13 as a novel mitoprotective agent which has the therapeutic potential for limiting IS and preventing HF in patients with AMI. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIAdministration of the DJ-1-derived peptide, ND-13, at the onset of reperfusion reduced myocardial infarct size and preserved cardiac contractile function following acute myocardial ischemia/reperfusion injury (IRI). C_LIO_LIThe cardioprotective effect of ND-13 treatment was mediated by inhibition of IRI-induced mitochondrial fission and improvement in mitochondrial bioenergetics via the RhoA-ROCK-Drp1 pathway. C_LI What are the Clinical Implications?O_LITreatment with ND-13 peptide at the onset of reperfusion has the therapeutic potential to reduce myocardial infarct size and prevent the onset of heart failure in ST-segment elevation myocardial infarction patients undergoing primary percutaneous coronary intervention. C_LI

physiology↗

bioRxiv2024

Mitochondrial genome variation affects humoral and cell-mediated innate immune responses and infection outcomes

The role of mitochondria in both adaptive and innate immune responses is increasingly recognized, but the role of mitochondrial DNA (mtDNA) variation as an immunomodulatory factor has received less attention. One reason for this is the difficulty of separating the effect of mtDNA from that of the nuclear genome. By utilizing the fruit fly Drosophila melanogaster, a powerful model system, we created cytoplasmic hybrids, aka. cybrid lines, where unique mtDNAs (mitotypes) were introgressed into a controlled isogenic nuclear background. We harnessed a panel of cybrid lines to study the effect of mtDNA variation on humoral and cell-mediated innate immune responses. Mitotypes exhibited heterogeneity in infection outcomes upon bacterial, viral and parasitoid infections. One mitotype of note (mtKSA2) was more immunocompetent when compared to other mitotypes. We performed transcriptomic profiling of uninfected and infected flies to find the mechanistic basis of the immunocompetence of the mtKSA2 line. We found that in uninfected flies mtKSA2 caused an upregulation of oxidative phosphorylation (OXPHOS) and tricarboxylic acid cycle (TCA) related genes and a downregulation of a set of antimicrobial peptides (AMPs). Upon infection, mtKSA2 flies produced transcriptomic changes that were infection type and duration specific. When we examined immune cells (hemocytes) in mtKSA2 larvae, we noted an increase in hemocyte numbers. These hemocytes were activated in the absence of infection, increased their production of ROS, and showed evidence of increased encapsulation efficiency upon parasitoid wasp infection. Overall, our results show that mtDNA variation acts as an immunomodulatory factor in both humoral and cell-mediated innate immunity and that specific mitotypes can provide enhanced protection against various infections.

immunology↗

bioRxiv2023

Rho and a riboswitch regulate mntP expression evading manganese stress and membrane toxicity

The trace metal ion manganese in excess is toxic. Therefore, a small subset of factors tightly maintains its cellular level, among which an efflux protein MntP is the champion. Multiple transcriptional regulators and a manganese-dependent translational riboswitch regulate the MntP expression. As riboswitches are untranslated RNAs, they are often associated with the Rho-dependent transcription termination in bacteria. Here we demonstrate that Rho efficiently terminates transcription at the mntP riboswitch region. The addition of manganese activates the riboswitch, thereby restoring the coupling between transcription and translation to evade Rho-dependent transcription termination partially. Deletion of the riboswitch abolishes Rho-dependent termination and renders bacteria sensitive to manganese due to overexpression of mntP. The high mntP expression is associated with reactive oxygen species (ROS) production, slow growth, and cell filamentation phenotypes. We posit that manganese-dependent transcriptional activation in the absence of Rho-dependent termination leads to the observed toxicity arising from excessive MntP expression, a membrane protein. Thus, we identified a novel regulatory role of Rho in preventing membrane protein toxicity by terminating at the riboswitch element.

biochemistry↗

bioRxiv2023

Phosphorylation in the Plasmodium falciparum proteome: A meta-analysis of publicly available data sets

Malaria is a deadly disease caused by Apicomplexan parasites of the Plasmodium genus. Several species of the Plasmodium genus are known to be infectious to human, of which P. falciparum is the most virulent. Post-translational modifications (PTMs) of proteins coordinate cell signalling and hence, regulate many biological processes in P. falciparum homeostasis and host infection, of which the most highly studied is phosphorylation. Phosphosites on proteins can be identified by tandem mass spectrometry (MS) performed on enriched samples (phosphoproteomics), followed by downstream computational analyses. We have performed a large-scale meta-analysis of 11 publicly available phosphoproteomics datasets, to build a comprehensive atlas of phosphosites in the P. falciparum proteome, using robust pipelines aimed at strict control of false identifications. We identified a total of 28,495 phosphorylated sites on P. falciparum proteins at 5% false localisation rate (FLR) and, of those, 18,100 at 1% FLR. We identified significant sequence motifs, likely indicative of different groups of kinases, responsible for different groups of phosphosites. Conservation analysis identified clusters of phosphoproteins that are highly conserved, and others that are evolving faster within the Plasmodium genus, and implicated in different pathways. We were also able to identify over 180,000 phosphosites within Plasmodium species beyond falciparum, based on orthologue mapping. We also explored the structural context of phosphosites, identifying a strong enrichment for phosphosites on fast evolving (low conservation) intrinsically disordered regions (IDRs) of proteins. In other species, IDRs have been shown to have an important role in modulating protein-protein interactions, particularly in signalling, and thus warranting further study for their roles in host- pathogen interactions. All data has made available via UniProtKB, PRIDE and PeptideAtlas, with visualisation interfaces for exploring phosphosites in the context of other data on Plasmodium proteins. Author SummaryPlasmodium parasites continue to pose a significant global health threat, with a high proportion of the world at risk of malaria. It is imperative to gain new insights into cell signalling and regulation of biological processes in these parasites to develop effective treatments. This study focused on post- translational modifications (PTMs) of proteins, specifically phosphorylation. We conducted a meta- analysis of 11 publicly available phosphoproteomics datasets, identifying over 28,000 phosphorylated sites on P. falciparum proteins, using very rigorous statistics to avoid reporting false positives, and mapping to over 180,000 phosphorylation sites on other species of Plasmodium. The analysis revealed distinct sequence motifs associated with different groups of phosphosites (and likely indicative of different upstream kinases), and differences in the downstream pathways regulated. Conservation analysis highlighted clusters of phosphoproteins evolving at different rates within the Plasmodium genus. Notably, phosphorylation was enriched in regions of proteins lacking distinct structural elements, known as intrinsically disordered regions (IDRs), which are poorly conserved across the genus - we speculate that they are important for modulating protein interactions. The findings provide valuable insights into the molecular mechanisms of P. falciparum, with potential implications for understanding host-pathogen interactions. The comprehensive dataset generated is now publicly accessible, serving as a valuable resource for the scientific community through UniProtKB, PRIDE, and PeptideAtlas.

bioinformatics↗

bioRxiv2023

Integrated analysis of baseline protein expression in pig tissues

The availability of an increasingly large amount of public proteomics datasets presents an opportunity for performing combined analyses to generate comprehensive organism-wide protein expression maps across different organisms and biological conditions. Sus scrofa, the domestic pig, is a model organism relevant for food production and for human biomedical research. Here we reanalyzed 14 public proteomics datasets from the PRIDE database coming from pig tissues to assess baseline (without any biological perturbation) protein abundance in 14 organs, encompassing a total of 20 healthy tissues from 128 samples. The analysis involved the quantification of protein abundance in 599 mass spectrometry runs. We compared protein expression patterns among different pig organs and examined the distribution of proteins across these organs. Then, we studied how protein abundances compared across different datasets and studied the tissue specificity of the detected proteins. Of particular interest, we conducted a comparative analysis of protein expression between pig and human tissues, revealing a high degree of correlation in protein expression among orthologs, particularly in brain, kidney, heart, and liver samples. We have integrated the protein expression results into the Expression Atlas resource for easy access and visualisation of the protein expression data individually or alongside gene expression data.

bioinformatics↗

bioRxiv2023

Protein language models enable prediction of polyreactivity of monospecific, bispecific, and heavy-chain-only antibodies

Early assessment of antibody off-target binding is essential for mitigating developability risks such as fast clearance, reduced efficacy, toxicity, and immunogenicity. The baculovirus particle (BVP) binding assay has been widely utilized to evaluate polyreactivity of antibodies. As a complementary approach, computational prediction of polyreactivity is desirable for counter-screening antibodies from in silico discovery campaigns. However, there is a lack of such models. Herein, we present the development of an ensemble of three deep learning models based on two pan-protein foundational protein language models (ESM2 and ProtT5) and an antibody-specific protein language model (Antiberty). These models were trained in a transfer learning network to predict the outcomes in the BVP assay and the bovine serum albumin (BSA) binding assay which was developed as a complement to the BVP assay. The training was conducted on a large dataset of antibody sequences augmented with experimental conditions, which were collected through a highly efficient application system. The resulting models demonstrated robust performance on normal mAbs (monospecific with heavy and light chain), bispecific Abs, and single-domain Fc (VHH-Fc). Protein language models outperformed a model built using molecular descriptors calculated from AlphaFold 2 predicted structures. Embeddings from the antibody-specific and foundational protein language models resulted in similar performance. To our knowledge, this represents the first application of protein language models to predict assay data on bispecifics and VHH-Fcs. Our study yields valuable insights on building infrastructures to support machine learning activities and training models for critical assays in antibody discovery.

bioinformatics↗

bioRxiv2023

Bicyclomycin generates ROS and blocks cell division in Escherichia coli

The role of reactive oxygen species (ROS) in the killing exerted by antibiotics on bacteria is debated. Evidences attribute the part of toxicity of many antibiotics to their ability to generate ROS by interfering with cellular metabolism, but some studies dismiss the role of ROS. Bicyclomycin (BCM) is a broad-spectrum antibiotic that is the only known compound to inhibit E. coli transcription terminator factor Rho with no known other cellular targets. In the present study, we addressed this question by checking whether the induction of oxidative stress could explain the increased sensitivity to Bicyclomycin in the hns deleted strain even in {Delta}kil background in E. coli. BCM evoked the generation of ROS in E. coli cells. BCM is known to cause the cell filamentation phenotype in E. coli. Performing fluorescence microscopic analysis, we show that bicyclomycin-dependent cell filamentation is associated with SOS response. RecA- GFP filaments were found to colocalize with the damaged DNA sites in the cell. Further analyzes revealed that the genomic DNA was partitioned but the cell septum formation was severely affected under BCM treatment. Furthermore, we observed biofilm formation by E. coli after BCM treatment. We hypothesize that ROS production after BCM treatment could lead to cell filamentation in bacteria. A better understanding of the mode of toxicity of BCM will help us in designing better antibiotic treatment regimes for clinical practices, including combinatorial drug therapies. The cell filamentation phenotype observed after BCM treatment makes this antibiotic a promising drug for PAS therapy.

microbiology↗

bioRxiv2023

Regulation of Lung Immune Tone by the Gut-Lung Axis via Dietary Fiber, Gut Microbiota, and Short-Chain Fatty Acids

Lung immune tone, i.e. the immune state of the lung, can vary between individuals and over a single individuals lifetime, and its basis and regulation in the context of inflammatory responses to injury is poorly understood. The gut microbiome, through the gut-lung axis, can influence lung injury outcomes but how the diet and microbiota affect lung immune tone is also unclear. We hypothesized that lung immune tone would be influenced by the presence of fiber-fermenting short-chain fatty acid (SCFA)-producing gut bacteria. To test this hypothesis, we conducted a fiber diet intervention study followed by lung injury in mice and profiled gut microbiota using 16S sequencing, metabolomics, and lung immune tone. We also studied germ-free mice to evaluate lung immune tone in the absence of microbiota and performed in vitro mechanistic studies on immune tone and metabolic programming of alveolar macrophages exposed to the SCFA propionate (C3). Mice on high-fiber diet were protected from sterile lung injury compared to mice on a fiber-free diet. This protection strongly correlated with lower lung immune tone, elevated propionate levels and enrichment of specific fecal microbiota taxa; conversely, lower levels of SCFAs and an increase in other fatty acid metabolites and bacterial taxa correlated with increased lung immune tone and increased lung injury in the fiber-free group. In vitro, C3 reduced lung alveolar macrophage immune tone (through suppression of IL-1{beta} and IL-18) and metabolically reprogrammed them (switching from glycolysis to oxidative phosphorylation after LPS challenge). Overall, our findings reveal that the gut-lung axis, through dietary fiber intake and enrichment of SCFA-producing gut bacteria, can regulate innate lung immune tone via IL-1{beta} and IL-18 pathways. These results provide a rationale for the therapeutic development of dietary interventions to preserve or enhance specific aspects of host lung immunity.

immunology↗

bioRxiv2023

The molecular basis of macrochaete diversification highlighted by a single-cell atlas of Bicyclus anynana butterfly pupal forewings

Butterfly wings display a large diversity of cell types, including scale cells of different colors, shapes, and sizes, yet the molecular basis of such diversity is poorly understood. Scales are single-cellular projections homologous to sensory bristles in other arthropods, and different scale types are often found intermingled on the wing, alongside other sensory and epidermal cell types, making the analysis of the complete transcriptomes of each scale type difficult to achieve. To explore scale cell diversity at a transcriptomic level we employed single-cell RNA sequencing of [~]5200 large cells (>6 {micro}m) from 22.5-25-hour male pupal forewings of the butterfly Bicyclus anynana. Using single-cell unsupervised, transcriptome clustering, based on shared and differentially expressed genes, followed by in situ hybridization, immunofluorescence, and CRISPR/Cas9 editing of candidate cell type specific genes, we annotate various cell types on the wing. We identify the molecular identities of non-innervated scale cells at various stages of differentiation, innervated sensory cell types, and pheromone producing glandular cells. We further show that senseless, a zinc finger transcription factor, and HR38, a hormone receptor, determine the identity, size, and color of different scale cell types and are important regulators of scale cell differentiation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/554425v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@10624org.highwire.dtl.DTLVardef@13a4963org.highwire.dtl.DTLVardef@1a83a9borg.highwire.dtl.DTLVardef@19f9d1e_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2023

Mapping the functional form of the trade-off between infection resistance and reproductive fitness under dysregulated immune signaling

Immune responses benefit organismal fitness by clearing parasites but also exact costs associated with immunopathology and energetic investment. Hosts manage these costs by tightly regulating the induction of immune signaling to curtail excessive responses and restore homeostasis. Despite the theoretical importance of turning off the immune response to mitigate these costs, experimentally connecting variation in the negative regulation of immune responses to organismal fitness remains a frontier in evolutionary immunology. In this study, we used a doseresponse approach to manipulate the RNAi-mediated knockdown efficiency of cactus (I{kappa}B), a central regulator of Toll pathway signal transduction in flour beetles (Tribolium castaneum). By titrating cactus activity along a continuous gradient, we derived the shape of the relationship between immune response investment and traits associated with host fitness, including infection susceptibility, lifespan, fecundity, body mass, and gut homeostasis. Cactus knock-down increased the overall magintude of inducible immune responses and delayed their resolution in a dsRNA dose-dependent manner, promoting survival and resistance following bacterial infection. However, these benefits were counterbalanced by dsRNA dose-dependent costs to lifespan, fecundity, body mass, and gut integrity. Our results allowed us to move beyond the qualitative identification of a trade-off between immune investment and fitness to actually derive its functional form. This approach paves the way to quantitatively compare the evolution and impact of distinct regulatory elements on life-history trade-offs and fitness, filling a crucial gap in our conceptual and theoretical models of immune signaling network evolution and the maintenance of natural variation in immune systems.

evolutionary biology↗

bioRxiv2023

Deciphering the orthorhombic crystal structure of a novel NEIL1 nanobody with pseudo-merohedral twinning

Nanobodies or VHHs (Variable Heavy domains of Heavy chain) are single domain antibodies that comprise three antigenic complementary determining regions (CDR). Nanobodies are used in numerous scientific applications including, bio-imaging, diagnosis, therapeutics, and macromolecular crystallography. We obtained crystals of a [~]14 kDa nanobody specific for the NEIL1 DNA glycosylase (hereafter called A5) in 0.5 M ammonium sulfate, 0.1 M sodium citrate tribasic dihydrate pH 5.6, and 1.0 M lithium sulfate monohydrate from the Crystal HT Hampton Research screen that were further optimized. Here, we describe the structure determination and refinement of the A5 crystals to a resolution of 2.1 [A]. The data collected were complicated by the presence of anisotropy and twinning, and while initial space group determination pointed to a higher apparent tetragonal crystal system, the data statistics suggested twinning, placing the crystal in an orthorhombic system. Twinning was confirmed by the Padilla and Yeates test, H-test, and Britton test based on local intensity differences with a twin fraction of 0.4. Molecular replacement produced the best solution in the orthorhombic space group P21212 with four molecules in the asymmetric unit and we were able to model over 96% of the residues in the electron density with a final Rwork and Rfree of 0.1988 and 0.2289 upon refinement. SynopsisThe crystal structure of a specific nanobody against NEIL1 was determined to 2.1 [A]. The structure was ultimately solved in an orthorhombic space group after diffraction data analysis revealed mild anisotropy as well as pseudo-merohedral twinning

biochemistry↗

bioRxiv2023

Novel genomic offset metrics account for local adaptation in climate suitability forecasts and inform assisted migration

Local adaptation is increasingly being integrated into macroecological models, offering an evolutionary perspective that has been largely missing from climate change biogeography. Genomic offsets, which quantify the disruption of existing genotype-environment associations under environmental change, are an informative landscape genomic tool that allows for the incorporation of intra-specific adaptive differentiation into forecasts of climate suitability and thus management planning. Gradient forest (GF), a method originally developed to model community turnover along environmental gradients, is now the most commonly used approach in genomic offset estimation. However, major hurdles in the application of GF-derived offsets are (1) an inability to interpret the absolute magnitude of genomic offsets in a biologically meaningful way and (2) uncertainty in how genomic offsets compare to established species-level approaches like Ecological Niche Models (ENMs). We used both ENMs and novel, genomic offset metrics derived from GF modeling of genomic variation along climatic gradients to assess the climate change vulnerability of red spruce (Picea rubens), a cool-temperate tree species endemic to eastern North America. We show how genomic offsets can be standardized relative to contemporary genomic variation across the landscape to better represent their biological significance and facilitate comparisons among studies. In three common gardens, we found a significant negative relationship between standardized genomic offsets and red spruce growth and higher explanatory power for standardized offsets than (raw) climate transfer distances. We also derived new threshold-based metrics that we term Donor and Recipient Importance and which quantify transferability of propagules between donor and recipient localities while minimizing disruption of genotype-environment associations. This approach leverages landscape genomic information to account for local adaptation when predicting climate suitability. ENMs and our novel genomic offset metrics largely agreed in forecasting drastic northward range shifts. Combining several offset-based metrics, we show that the projected northward shift of suitable climate mainly applies to populations located in the center and northern parts of the current range, whereas southern populations might be able to persist in situ owing to specific local climate adaptations. The novel metrics thus yield refined, region-specific prognoses for local persistence and show how management could be improved by considering assisted migration.

ecology↗

bioRxiv2023

Cell-type-specific nuclear morphology predicts genomic instability and prognosis in multiple cancer types

While alterations in nucleus size, shape, and color are ubiquitous in cancer, comprehensive quantification of nuclear morphology across a whole-slide histologic image remains a challenge. Here, we describe the development of a pan-tissue, deep learning-based digital pathology pipeline for exhaustive nucleus detection, segmentation, and classification and the utility of this pipeline for nuclear morphologic biomarker discovery. Manually-collected nucleus annotations were used to train an object detection and segmentation model for identifying nuclei, which was deployed to segment nuclei in H&E-stained slides from the BRCA, LUAD, and PRAD TCGA cohorts. Interpretable features describing the shape, size, color, and texture of each nucleus were extracted from segmented nuclei and compared to measurements of genomic instability, gene expression, and prognosis. The nuclear segmentation and classification model trained herein performed comparably to previously reported models. Features extracted from the model revealed differences sufficient to distinguish between BRCA, LUAD, and PRAD. Furthermore, cancer cell nuclear area was associated with increased aneuploidy score and homologous recombination deficiency. In BRCA, increased fibroblast nuclear area was indicative of poor progression-free and overall survival and was associated with gene expression signatures related to extracellular matrix remodeling and anti-tumor immunity. Thus, we developed a powerful pan-tissue approach for nucleus segmentation and featurization, enabling the construction of predictive models and the identification of features linking nuclear morphology with clinically-relevant prognostic biomarkers across multiple cancer types.

cancer biology↗

bioRxiv2023

The immune regulation and epidemiological consequences of immune priming in Drosophila

Invertebrates lack the immune machinery underlying vertebrate-like acquired immunity. However, in many insects past infection by the same pathogen can prime the immune response, resulting in improved survival upon reinfection. Here, we investigated the generality, specificity and mechanistic basis of innate immune priming in the fruit fly Drosophila melanogaster when infected with the gram-negative bacterial pathogen Providencia rettgeri. We find that priming in response to P. rettgeri infection is a long-lasting and pathogen-specific response. We further explore the epidemiological consequences of immune priming and find it has the potential to curtail pathogen transmission by reducing pathogen shedding and spread. The enhanced survival of individuals previously exposed to a non-lethal bacterial inoculum coincided with a transient decrease in bacterial loads, and we provide strong evidence that the effect of priming requires the IMD-responsive antimicrobial-peptide Diptericin-B in the fat body. Further, we show that while Diptericin B is the main effector of bacterial clearance, it is not sufficient for immune priming, which requires regulation of IMD by peptidoglycan recognition proteins. This work underscores the plasticity and complexity of invertebrate responses to infection, providing novel experimental evidence for the effects of innate immune priming on population-level epidemiological outcomes.

immunology↗