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bioRxiv2017

Extensive non-canonical phosphorylation in human cells revealed using strong-anion exchange-mediated phosphoproteomics

Protein phosphorylation is a ubiquitous post-translational modification (PTM) that regulates all aspects of life. To date, investigation of human cell signalling has focussed on canonical phosphorylation of serine (Ser), threonine (Thr) and tyrosine (Tyr) residues. However, mounting evidence suggests that phosphorylation of histidine also plays a central role in regulating cell biology. Phosphoproteomics workflows rely on acidic conditions for phosphopeptide enrichment, which are incompatible with the analysis of acid-labile phosphorylation such as histidine. Consequently, the extent of non-canonical phosphorylation is likely to be under-estimated.\n\nWe report an Unbiased Phosphopeptide enrichment strategy based on Strong Anion Exchange (SAX) chromatography (UPAX), which permits enrichment of acid-labile phosphopeptides for characterisation by mass spectrometry. Using this approach, we identify extensive and positional phosphorylation patterns on histidine, arginine, lysine, aspartate and glutamate in human cell extracts, including 310 phosphohistidine and >1000 phospholysine sites of protein modification. Remarkably, the extent of phosphorylation on individual non-canonical residues vastly exceeds that of basal phosphotyrosine. Our study reveals the previously unappreciated diversity of protein phosphorylation in human cells, and opens up avenues for exploring roles of acid-labile phosphorylation in any proteome using mass spectrometry.

biochemistry↗

bioRxiv2022

Identifying the C. elegans vulval transcriptome

Development of the C. elegans vulva is a classic model of organogenesis. This system, which starts with six equipotent cells, encompasses diverse types of developmental event, including developmental competence, multiple signaling events to control precise and faithful patterning of three cell fates, execution and proliferation of specific cell lineages, and a series of sophisticated morphogenetic events. Early events have been subjected to extensive mutational and genetic investigations and later events to cell biological analyses. We infer the existence of dramatically changing profiles of gene expression that accompanies the observed changes in development. Yet except from serendipitous discovery of several transcription factors expressed in dynamic patterns in vulval lineages, our knowledge of the transcriptomic landscape during vulval development is minimal. This study describes the composition of a vulva-specific transcriptome. We used tissue specific harvesting of mRNAs via immunoprecipitation of epitope-tagged poly(A) binding protein, PAB-1, heterologously expressed by a promoter known to express GFP in vulval cells throughout their development. The identified transcriptome was small but tightly interconnected. From this data set we identified several genes with identified functions in development of the vulva and validated more with promoter-GFP reporters of expression. For one target, lag-1, promoter-GFP expression was limited but fluorescent tag of the endogenous protein revealed extensive expression. Thus, we have identified a transcriptome of the C. elegans as a launching pad for exploration of functions of these genes in organogenesis.

genetics↗

bioRxiv2018

Re-expression of SynGAP Protein in Adulthood Improves Translatable Measures of Brain Function and Behavior in a Model of Neurodevelopmental Disorders

BackgroundNeurodevelopmental disorder (NDD) risk genes have pleiotropic biological functions, such as control over both developmental and non-developmental processes that influence disease-related phenotypes. Currently, it remains unclear how developmental versus non-developmental processes influence the duration and/or effectiveness of permissive treatment windows for NDDs. SYNGAP1 haploinsufficiency causes an NDD defined by autistic traits, cognitive impairment, and epilepsy. Syngap1 heterozygosity in mice disrupts a developmental critical period, and, consistent with this, certain behavioral abnormalities are resistant to gene therapy initiated in adulthood. However, the Syngap1 endophenotype is extensive and this protein has diverse cell biological functions. Therefore, SynGAP pleiotropy may influence the permissive treatment window for previously untested disease-relevant phenotypes.\n\nMethodsA whole-body gene restoration technique was used to determine how restoration of SynGAP protein in adult heterozygous mice impacted previously untested phenotypes, such as memory, seizure susceptibility, systems-level cortical hyperexcitability, and hippocampal oscillations linked to mnemonic processes.\n\nResultsAdult restoration of SynGAP protein in haploinsufficient mice reversed long-term contextual memory deficits and behavioral measures of seizure susceptibility. Moreover, SynGAP re-expression in adult mice eliminated brain state-dependent, patient-linked paroxysmal interictal spiking and increased the amplitude of hippocampal theta oscillations.\n\nConclusionsSynGAP protein in the mature brain dynamically regulates neural circuit function and influences disease-relevant phenotypes. The impact of these findings is that treatments targeting certain debilitating aspects of SYNGAP1-related disorders may be effective throughout life. Moreover, the efficacy of experimental treatments for SYNGAP1 patients may be quantifiable through changes in species-conserved, state-dependent pathological electroencephalogram signals.

neuroscience↗

bioRxiv2023

Stable mammalian serum albumins designed for bacterial expression

Albumin is the most abundant protein in the blood serum of mammals and has essential carrier and physiological roles. Albumins are also used in a wide variety of molecular and cellular experiments and in the cultivated meat industry. Despite their importance, however, albumins are challenging for heterologous expression in microbial hosts, likely due to 17 conserved intramolecular disulfide bonds. Therefore, albumins used in research and biotechnological applications either derive from animal serum, despite severe ethical and reproducibility concerns, or from recombinant expression in yeast or rice. We use the PROSS algorithm to stabilize human and bovine serum albumins, finding that all are highly expressed in E. coli. Design accuracy is verified by crystallographic analysis of a human albumin variant with 16 mutations. This albumin variant exhibits ligand binding properties similar to those of the wild type. Remarkably, a design with 73 mutations relative to human albumin exhibits over 40{degrees}C improved stability and is stable beyond the boiling point of water. Our results suggest that proteins with many disulfide bridges have the potential to exhibit extreme stability when subjected to design. The designed albumins may be used to make economical, reproducible, and animal-free reagents for molecular and cell biology. They also open the way to high-throughput screening to study and enhance albumin carrier properties. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/534334v3_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@71ef93org.highwire.dtl.DTLVardef@27a975org.highwire.dtl.DTLVardef@1e657fforg.highwire.dtl.DTLVardef@19b3de3_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- Computational design stabilized human and bovine serum albumins - Designs express solubly in E. coli and exhibit up to 40 {degrees}C increased thermostability - Some designs exhibit identical ligand binding properties - Crystal structure confirms design accuracy - Designs can be used in cell culture and in vitro applications

biochemistry↗

bioRxiv2023

Adenovirus protein VII binds the A-box of HMGB1 to repress interferon responses.

Viruses hijack host proteins to promote infection and dampen host defenses. Adenovirus encodes the multifunctional protein VII that serves both to compact viral genomes inside the virion and disrupt host chromatin. Protein VII binds the abundant nuclear protein high mobility group box 1 (HMGB1) and sequesters HMGB1 in chromatin. HMGB1 is an abundant host nuclear protein that can also be released from infected cells as an alarmin to amplify inflammatory responses. By sequestering HMGB1, protein VII prevents its release, thus inhibiting downstream inflammatory signaling. However, the consequences of this chromatin sequestration on host transcription are unknown. Here, we employ bacterial two-hybrid interaction assays and human cell biological systems to interrogate the mechanism of the protein VII-HMGB1 interaction. HMGB1 contains two DNA binding domains, the A- and B-boxes, that bend DNA to promote transcription factor binding while the C-terminal tail regulates this interaction. We demonstrate that protein VII interacts directly with the A-box of HMGB1, an interaction that is inhibited by the HMGB1 C-terminal tail. By cellular fractionation, we show that protein VII renders A-box containing constructs insoluble, thereby acting to prevent their release from cells. This sequestration is not dependent on HMGB1s ability to bind DNA but does require post-translational modifications on protein VII. Importantly, we demonstrate that protein VII inhibits expression of interferon {beta}, in an HMGB1- dependent manner, but does not affect transcription of downstream interferon- stimulated genes. Together, our results demonstrate that protein VII specifically harnesses HMGB1 through its A-box domain to depress the innate immune response and promote infection.

microbiology↗

bioRxiv2020

Label-free single-instance protein detection in vitrified cells

A general method to map molecular interactions and conformational states in structurally intact cells would find wide application in biochemistry and cell biology. We used a library of images-- calculated on the basis of known structural data--as search templates to detect targets as small as the "head" domain (350 kDa) of the ribosomes small subunit in single-tilt electron cryo-micrographs by cellular high resolution template matching (cHRTM). Atomically precise position and orientation estimates reveal the conformation of individual ribosomes and enable the detection of specifically bound ligands down to 24 kDa. We show that highly head-swivelled states are likely to play a role in mRNA translocation in living cells. cHRTM outperforms cryo-electron tomography three-fold in sensitivity and completely avoids the vicissitudes of exogenous labelling.

biophysics↗

bioRxiv2025

Optical tissue clearing and 3D imaging of intact primate testicular tissue: a novel technology development

Classical histology struggles to preserve three-dimensional spatial context, prompting the emergence of optical tissue clearing techniques that enable imaging of intact specimens at cellular or subcellular resolution. These techniques have revolutionised fields like cell biology, developmental biology, and neuroscience. However, their application in reproductive biology remains unexplored - particularly in studying the complexities of testicular development. We developed a novel, efficient and affordable toolbox for studying intact testicular tissues, PT-CLEAR3D, that stands for primate testis - whole mount staining, tissue clearing and three-dimensional imaging. Intact testicular tissues from humans (transgender model), common marmosets and macaques underwent antibody labelling, clearing with organic solvents, and three-dimensional imaging using light sheet fluorescence microscopy. Marker specificity was confirmed by immunofluorescence staining of 3 and 25 {micro}m testicular sections, followed by imaging with confocal. The testicular structure was evaluated using several markers: spermatogonia (melanoma-associated antigen 4), least differentiated spermatogonia (Piwi-like protein 4), Sertoli cells (vimentin and SRY-Box transcription Factor 9), peritubular myoid cells and vasculature (alpha-smooth muscle actin), and NucSpot as a nuclear dye. PT-CLEAR3D efficiently achieved optical transparency while a commercial kit that was ran in parallel was inefficient. This study presents a pioneering three-dimensional visualization of intact testicular samples of up to 50 mm3 in size and imaging depth of up to 4.5 mm across three primate species. Remarkably, PT-CLEAR3D revealed critical details at both tissue and cellular levels such as the spatial distribution of germ and somatic cells, cellular bridges, and vasculature. Furthermore, PT-CLEAR3D enabled three-dimensional reconstructions that effectively reduce confirmation bias enhancing our observation of spermatogonial clones organized as single cells, pairs, and quartets. Importantly, it adeptly identified testicular pathology and the persistence of germ cell clones in select tubules within the transgender testis following hormonal suppression of spermatogenesis. This technological development offers a versatile toolbox with benefits such as applicability across multiple species, fluorophore multiplexing, compatibility with different fixatives and deep tissue volumetric imaging with cellular resolution. Overall, PT-CLEAR3D establishes a foundation for spatial evaluation of testicular development, presenting substantial potential for advancing our understanding of the intricate kinetics of spermatogenesis in health and disease.

developmental biology↗

bioRxiv2024

FLIPs: Novel Genetically Encoded Probes for Functional Imaging of Cell Signaling by Polarization Microscopy

Genetically encoded fluorescent biosensors convert specific biomolecular events into optically detectable signals. By revealing biochemical processes in situ, they have revolutionized cell biology. However, imaging molecular processes often requires modifying the proteins involved, and many molecular processes are still to be imaged. Here we present a novel, widely applicable design of genetically encoded biosensors that notably expand the observation possibilities, by taking advantage of a hitherto overlooked detection principle: directionality of optical properties of fluorescent proteins. The probes, which we term FLIPs, offer an extremely simple design, high sensitivity, multiplexing capability, ratiometric readout and resilience to bleaching artifacts, without requiring any modifications to the probe targets. We demonstrate their performance on real-time single-cell imaging of activation of G protein-coupled receptors (GPCRs), G proteins, arrestins, small GTPases, as well as receptor tyrosine kinases, even at endogenous expression levels. We also identify a new, pronounced, endocytosis-associated conformational change in a GPCR-{beta}-arrestin complex. By demonstrating a novel detection principle and allowing many more cellular processes to be visualized, FLIPs are likely to inspire numerous future developments and insights.

physiology↗

bioRxiv2021

Automated Spatially Targeted Optical Micro Proteomics (AutoSTOMP) 2.0 identifies proteins enriched within inflammatory lesions in tissue sections and human clinical biopsies.

Tissue microenvironment properties like blood flow, extracellular matrix or proximity to immune infiltrate are important regulators of cell biology. However, methods to study regional protein expression in context of the native tissue environment are limited. To address this need we have developed a novel approach to visualize, purify and measure proteins in situ using Automated Spatially Targeted Optical Micro Proteomics (AutoSTOMP) 2.0. We previously implemented AutoSTOMP to identify proteins localized to the vacuoles of obligate intracellular microbes at the 1-2 m scale within infected host cells1. Here we report custom codes in SikuliX to specify regions of heterogeneity in a tissue section and then biotin tag and identify proteins belonging to specific cell types or structures within those regions. To enrich biotinylated targets from fixed tissue samples we developed a biochemical protocol compatible with LC-MS. These tools were applied to a) identify inflammatory proteins expressed by CD68+ macrophages in rat cardiac infarcts and b) characterize inflammatory proteins enriched in IgG4+ lesions in esophageal tissue. These data indicate that AutoSTOMP is a flexible approach to determine regional protein expression in situ on a range of primary tissues and clinical biopsies where current tools are limited.

immunology↗

bioRxiv2025

Description of a new Telonemia genus and species with novel observations providing insights into its hidden diversity

Telonemia is a fascinating and understudied group of microbial eukaryotes known to have a vast diversity that is still uncharacterized. In fact, although they are thought to be the closest relatives of the eukaryotic supergroup SAR (Stramenopiles, Alveolata and Rhizaria), their diversity and biology are largely unexplored: to date, there are only seven described species in three genera, although there are estimated to be hundreds more unknown lineages. Here, we describe the isolation and characterization of two new strains, including a new genus (Hyaliora molinica gen. et sp. nov.) and a new species (Telonema blandense sp. nov.), and the re-isolation of a previously characterized telonemid, Telonema subtile, accompanied by new behavioral observations. We present morphological measurements highlighting differences among the isolates and a phylogenetic tree incorporating their 18S rRNA gene sequences. Furthermore, key aspects of their cell biology and structure are highlighted to provide insights into the evolution of TSAR. Since they are relevant not only phylogenetically, but also play a crucial role in food webs with some very abundant representatives in aquatic ecosystems, the findings of this study provide a further sampling and culturing of Telonemia to increase the knowledge of the hidden diversity and evolution of this mysterious group.

evolutionary biology↗

bioRxiv2025

Cytotoxic Vδ2+T cell subsets expand in response to malaria in human tonsil and spleen organoids

Vaccine effectiveness against malaria is dramatically reduced in malaria-exposed compared to malaria-naive populations, potentially due to altered immune responses in secondary lymphoid organs following repeated infection. Newly developed human tonsil and spleen organoids, which replicate key features of B and T cell immunity, provide an exciting opportunity to overcome challenges of other models and to improve our understanding of innate-adaptive interactions in lymphoid tissue. The objectives of this study were to use these organoids to investigate the impact of malaria parasites on 1) cells within lymphoid tissues and 2) responses to a heterologous antigen. When we exposed organoids from malaria-naive donors to Plasmodium falciparum-infected red blood cells (iRBC), we observed that iRBC exposure did not disrupt organoid formation and significantly increased V{delta}2+ {gamma}{delta} T cell frequencies in both tonsil and spleen organoids at multiple timepoints. Single-cell RNA/TCR sequencing revealed that iRBC-responsive V{delta}2+ T cells in organoids were clonally expanded and exhibited activated, cytotoxic phenotypes with upregulated expression of granzymes, interferon-stimulated genes, and antigen presentation machinery. TCR repertoire analysis demonstrated that malaria exposure drove clonal expansion of cytotoxic V{delta}2+ T cells, contrasting with the diverse, smaller clones observed in control conditions. To validate these findings, we analyzed tonsils from Ugandan children with asymptomatic malaria infection and found expanded V{delta}2+ T cells with enhanced cytotoxic potential compared to uninfected controls. When we tested whether malaria pre-exposure affected subsequent recall responses to influenza vaccine, malaria pre-exposure or {gamma}{delta} T cell depletion did not significantly alter cellular frequencies or influenza-specific antibody responses in most donors, though modest reductions were observed in some individuals. This work demonstrates the utility of human lymphoid organoids for studying malaria-host interactions and provides novel insights into V{delta}2+ T cell biology, including evidence for antigen-specific clonal expansion and cytotoxic differentiation in response to malaria parasites within secondary lymphoid tissues. Author SummaryMalaria vaccines are significantly less effective in populations with endemic malaria exposure compared to malaria-naive individuals. We used human tonsil and spleen organoids to investigate whether repeated malaria infections alter immune responses in secondary lymphoid organs, potentially contributing to this reduced vaccine efficacy. These organoids create a controlled system that preserves the architecture and cellular interactions of secondary lymphoid tissues. When we exposed organoids to Plasmodium falciparum-infected red blood cells, we observed dramatic expansion of the V{delta}2+ subset of {gamma}{delta} T cells. This finding was particularly noteworthy because V{delta}2+ T cells are not typically considered major participants in immune responses within secondary lymphoid organs. Single-cell analysis revealed that these expanded V{delta}2+ T cells underwent clonal expansion and acquired cytotoxic phenotypes, suggesting antigen-specific responses. Tonsil tissue from Ugandan children with asymptomatic malaria infections showed similar patterns of V{delta}2+ T cell expansion and enhanced cytotoxic potential. Surprisingly, malaria pre-exposure did not affect subsequent recall responses to influenza vaccine in most donors, although this does not discount a possible impact on immune responses to primary vaccination. Our work reveals unexpected roles for {gamma}{delta} T cells in lymphoid tissues during malaria infection and establishes organoids as valuable models for studying host-pathogen interactions.

immunology↗

bioRxiv2021

MultiMAP: Dimensionality Reduction and Integration of Multimodal Data

Multimodal data is rapidly growing in many fields of science and engineering, including single-cell biology. We introduce MultiMAP, an approach for dimensionality reduction and integration of multiple datasets. MultiMAP recovers a single manifold on which all of the data resides and then projects the data into a single low-dimensional space so as to preserve the structure of the manifold. It is based on a framework of Riemannian geometry and algebraic topology, and generalizes the popular UMAP algorithm1 to the multimodal setting. MultiMAP can be used for visualization of multimodal data, and as an integration approach that enables joint analyses. MultiMAP has several advantages over existing integration strategies for single-cell data, including that MultiMAP can integrate any number of datasets, leverages features that are not present in all datasets (i.e. datasets can be of different dimensionalities), is not restricted to a linear mapping, can control the influence of each dataset on the embedding, and is extremely scalable to large datasets. We apply MultiMAP to the integration of a variety of single-cell transcriptomics, chromatin accessibility, methylation, and spatial data, and show that it outperforms current approaches in preservation of high-dimensional structure, alignment of datasets, visual separation of clusters, transfer learning, and runtime. On a newly generated single-cell Assay for Transposase-Accessible Chromatin using sequencing (scATAC-seq) and single-cell RNA-seq (scRNA-seq) dataset of the human thymus, we use MultiMAP to integrate cells along a temporal trajectory. This enables the quantitative comparison of transcription factor expression and binding site accessibility over the course of T cell differentiation, revealing patterns of transcription factor kinetics.

bioinformatics↗

bioRxiv2022

Learning orientation-invariant representations enables accurate and robust morphologic profiling of cells and organelles

Cell and organelle morphology are driven by diverse genetic and environmental factors and thus accurate quantification of cellular phenotypes is essential to experimental cell biology. Representation learning methods for phenotypic profiling map images to feature vectors that form an embedding space of morphological variation useful for clustering, dimensionality reduction, outlier detection, and supervised learning problems. Morphology properties do not change with orientation, and thus we argue that representation learning methods should encode this orientation invariance. We show that prior methods are sensitive to orientation, which can lead to suboptimal clustering. To address this issue, we develop O2-VAE, an unsupervised learning method that learns robust, orientation-invariant representations. We use O2-VAE to discover novel morphology subgroups in segmented cells and mitochondria, detect outlier cells, and rapidly characterise cellular shape and texture in large datasets, including in a newly generated synthetic benchmark.

bioinformatics↗

bioRxiv2026

Hierarchical bounds on RNA chromatin statistical dependence across cellular states in paired single-cell multiome data

Understanding how transcriptional output and chromatin accessibility coordinate across cellular states remains a central challenge in multimodal single-cell biology. Here, we establish explicit hierarchical empirical bounds on RNA-chromatin statistical dependence using a strictly falsification-driven, information-theoretic analysis of paired RNA-seq and ATAC-seq data. Our contribution is not to assert universal coupling, but to quantify the maximum intra-state coupling that survives adversarial nulls, thereby converting qualitative intuition into empirical bounds. Leveraging unimodal latent representations and adversarial null models, we quantify both the existence and the limits of cross-modal dependence across organizational scales. At the population level, global RNA-ATAC mutual information is strong and reproducible across donors, but is shown to be overwhelmingly dominated by cell-type composition rather than fine-grained regulatory coordination. When cellular state is explicitly controlled, intra-state RNA-ATAC coupling collapses to null expectations in the majority of populations, directly falsifying the hypothesis of a universal within-state regulatory channel. Despite this collapse, a weak but statistically robust residual coupling persists in a restricted subset of highly dynamic states, including erythroid differentiation compartments, activated T cells, and NK cells. This residual signal survives stringent local permutation tests and conditional mutual information analysis, demonstrating that it cannot be reduced to compositional mixing alone. Quantitatively, residual within-state dependence is consistently an order of magnitude smaller than global dependence, placing an empirical upper bound on within-state RNA-ATAC coordination in this dataset. Donor-resolved ratios {rho} = I(R;A|S)/I(R;A) indicate that most of the global dependence is removed by conditioning on state; operationally, we refer to the removed fraction (1-{rho}) as composition-dominated dependence. Throughout, "state-contingent statistical dependence" is used strictly as an operational descriptor rather than a causal claim: mutual information and conditional mutual information quantify statistical dependence only, not directionality or mechanism. This framing constrains downstream mechanistic interpretation and future multimodal modeling.

molecular biology↗

bioRxiv2021

The C. albicans virulence factor Candidalysin polymerizes in solution to form membrane pores and damage epithelial cells.

The pathogenic fungus Candida albicans causes severe invasive candidiasis. C. albicans infection requires the action of the virulence factor Candidalysin (CL), which damages the plasma membrane of the target human cells. However, the molecular mechanism that CL uses to permeabilize membranes is poorly understood. We employed complementary biophysical, modeling, microscopy, and cell biology methods to reveal that CL forms membrane pores using a unique molecular mechanism. Unexpectedly, it was observed that CL readily assembles into linear polymers in solution. The basic structural unit in polymer formation is a CL 8-mer, which is sequentially added into a string configuration. Finally, the linear polymers can close into a loop. Our data indicate that CL loops spontaneously insert into the membrane to become membrane pores. We identified a CL mutation (G4W) that inhibited the formation of polymers in solution and prevented formation of pores in different synthetic lipid membranes systems. Studies in epithelial cells showed that G4W CL failed to activate the danger response signaling pathway, a hallmark of the pathogenic effect of CL. These results indicate that CL polymerization in solution is a necessary step for the damage of cellular membranes. Analysis of thousands of CL pores by atomic force microscopy revealed the co-existence of simple depressions and complex pores decorated with protrusions. Imaging and modeling indicate that the two types of pores are formed by CL molecules assembled into alternate orientations. We propose that this structural rearrangement represents a maturation mechanism that might stabilize pore formation to achieve more robust cellular damage. Taken together, the data show that CL uses a previously unknown mechanism to damage membranes, whereby pre-assembly of CL loops in solution directly leads to formation of membrane pores. Our investigation not only unravels a new paradigm for the formation of membrane pores, but additionally identifies CL polymerization as a novel therapeutic target to treat candidiasis.

biophysics↗

bioRxiv2016

P2Y2 purinergic receptor is induced following human cytomegalovirus infection and its activity is required for efficient viral replication

Human cytomegalovirus (HCMV) manipulates many aspects of host cell biology to create an intracellular milieu optimally supportive of its replication and spread. The current study reveals a role for purinergic signaling in HCMV infection. The levels of several components of the purinergic signaling system, including the P2Y2 receptor, were altered in HCMV-infected fibroblasts. P2Y2 receptor RNA and protein are strongly induced following infection. Pharmacological inhibition of receptor activity or knockdown of receptor expression markedly reduced the production of infectious HCMV progeny. When P2Y2 activity was inhibited, the accumulation of most viral RNAs tested and viral DNA was reduced. In addition, the level of cytosolic calcium within infected cells was reduced when P2Y2 signaling was blocked. The HCMV-coded UL37x1 protein was previously shown to induce calcium flux from the smooth endoplasmic reticulum to the cytosol, and the present study demonstrates that P2Y2 function is required for this mobilization. We conclude that P2Y2 supports the production of HCMV progeny, possibly at multiple points within the viral replication cycle that interface with signaling pathways induced by the purinergic receptor.\n\nImportanceHCMV infection is ubiquitous and can cause life-threatening disease in immunocompromised patients, debilitating birth defects in newborns, and has been increasingly associated with a wide range of chronic conditions. Such broad clinical implications result from the modulation of multiple host cell processes. This study documents that cellular purinergic signaling is usurped in HCMV-infected cells and that the function of this signaling axis is critical for efficient HCMV infection. Therefore, we speculate that blocking P2Y2 receptor activity has the potential to become an attractive novel treatment option for HCMV infection.

Microbiology↗

bioRxiv2019

Widespread diversity in the transcriptomes of functionally divergent limb tendons

Tendon is a functionally important connective tissue that transmits force between skeletal muscle and bone. Previous studies have evaluated the architectural designs and mechanical properties of different tendons throughout the body. However, less is known about the underlying transcriptional differences between tendons which may dictate their designs and properties. Therefore, our objective was to develop a comprehensive atlas of the transcriptome of limb tendons in adult mice and rats using systems biology techniques. We selected the Achilles, forepaw digit flexor, patellar, and supraspinatus tendons due to their divergent functions and high rates of injury and tendinopathies in patients. Using RNA sequencing data, we generated the Comparative Tendon Transcriptional Database (CTTDb) that identified substantial diversity in the transcriptomes of tendons both within and across species. Approximately 30% of transcripts were differentially regulated between tendons of a given species, and nearly 60% of the transcripts present in anatomically similar tendons were different between species. Many of the genes that differed between tendons and across species are important in tissue specification and limb morphogenesis, tendon cell biology and tenogenesis, growth factor signaling, and production and maintenance of the extracellular matrix. This study indicates that tendon is a surprisingly heterogenous tissue with substantial genetic variation based on anatomical location and species. Key PointsO_LITendon is a hypocellular, matrix-rich tissue that has been excluded from comparative transcriptional atlases. These atlases have provided important knowledge about biological heterogeneity between tissues, and our manuscript addresses this important gap. C_LIO_LIWe performed measures on four of the most studied tendons, the Achilles, forepaw flexor, patellar, and supraspinatus tendons of both mice and rats. These tendons are functionally distinct and are also among the most commonly injured, and therefore of important translational interest. C_LIO_LIApproximately one-third of the transcriptome was differentially regulated between Achilles, forepaw flexor, patellar, and supraspinatus tendons within either mice or rats. Nearly two thirds of the transcripts that are expressed in anatomically similar tendons were different between mice and rats. C_LIO_LIThe overall findings from this study identified that although tendons across the body share a common anatomical definition based on their physical location between skeletal muscle and bone, tendon is a surprisingly genetically heterogeneous tissue. C_LI

systems biology↗

bioRxiv2018

AmyCo: the Amyloidoses Collection

AmyCo: the Amyloidoses CollectionAmyloid fibrils are formed when soluble proteins misfold into highly ordered insoluble fibrillar aggregates and affect various organs and tissues. The deposition of amyloid fibrils is the main hallmark of a group of disorders, called amyloidoses. Curiously, fibril deposition has been also recorded as a complication in a number of other pathological conditions, including well-known neurodegenerative or endocrine diseases. To date, amyloidoses are roughly classified, owing to their tremendous heterogeneity. In this work, we introduce AmyCo, a freely available collection of amyloidoses and clinical disorders related to amyloid deposition. AmyCo classifies 74 diseases associated with amyloid deposition into two distinct categories, namely 1) amyloidosis and 2) clinical conditions associated with amyloidosis. Each database entry is annotated with the major protein component (causative protein), other components of amyloid deposits and affected tissues or organs. Database entries are also supplemented with appropriate detailed annotation and are referenced to ICD-10, MeSH, OMIM, PubMed, AmyPro and UniProtKB databases. To our knowledge, AmyCo is the largest repository containing information about amyloidoses and diseases related to amyloid deposition. The AmyCo web interface is available at http://bioinformatics.biol.uoa.gr/amyco.\n\nKaterina C. Nastou is a Ph.D. student in Bioinformatics, at the Department of Biology of the National and Kapodistrian University of Athens. She is currently working on computational analysis of membrane and amyloidogenic proteins as part of her Ph.D. thesis. Her research focuses on the study of protein-protein interactions and the visualization and analysis of biological networks for these protein families, on the computational prediction of protein structure and function and the design and development of biological databases.\n\nGeorgia I. Nasi is a Ph.D. student in Biophysics, at the Department of Biology of the National and Kapodistrian University of Athens and a second year student in the Bioinformatics Masters Program, at the same department. She is currently conducting her Masters thesis on the computational analysis and visualization of the interaction network of amyloidoses and proteins associated with these disorders. Her research for her Ph.D. focuses on biophysical and computational analysis of amyloidogenic proteins and peptide-analogues associated with amyloidoses.\n\nDr. Paraskevi L. Tsiolaki is a Biologist with an MSc in Bioinformatics and a PhD in Molecular Biophysics. She is currently working as a postdoctoral fellow in Dr V. Iconomidous group, Assist. Prof. at the National and Kapodistrian University of Athens and her research interests focus on Molecular Biophysics and Structural Biology. Her current research efforts have been directed towards identifying the structural characteristics that underlie the self-assembly mechanisms,governing amyloidogenicity. More specifically she works on the structure and self-assembly of different amyloidogenic proteins or amyloidogenic peptide-analogues, implicated with amyloidoses, utilizing biophysical and biochemical techniques. She is also working on the computational and structural analysis of the anomalous type of protein-protein interactions in protein aggregation, with particular focus on the development of novel therapeutic intervention strategies.\n\nDr.Zoi Litou works as a Special Laboratory Teaching Staff in \"Bioinformatics-Biophysics\" at the Section of Cell Biology and Biophysics, Department of Biology, National &Kapodistrian University of Athens. She has a PhD in Bioinformatics. She is currently working on computational analysis of membrane proteins focusing on the automated recognition and classification of single-spanning membrane proteins, CWPs, GPCRs and Ion channels. Biological Network Analysis, Prediction algorithms, Algorithm Visualization techniques in Bioinformatics, High throughput sequencing analysis and visualization, Clustering Analysis, Knowledge discovery, management and representation, Data integration, Chemoinformatics, Pharmacogenomics, Text Mining in Bioinformatics, Personalized Medicine, Parallel programming.\n\nDr. Vassiliki A. Iconomidou is an Assistant Professor of Structural Biology/Molecular Biophysics and a group leader of Biophysics and Bioinformatics Lab at the Department of Biology of the National and Kapodistrian University of Athens. Her research interests include: 1) Structural and self-assembly studies of fibrous proteins, which form extracellular, proteinaceous structures of physiological importance like lepidopteran, dipteran and fish chorions and arthropod cuticle, 2) Structural and self-assembly studies of silkmoth chorion peptide-analogues as novel self-assembled polymers with amyloid properties, aiming at the construction of novel biomaterials with extraordinary physical properties, 3) Experimental studiesof the role of a great variety of amyloidogenic ( aggregation-prone) peptides, predicted by our AMYLPRED prediction algorithm, in several widespread and also rare pathological amyloidoses. She had been visiting European Molecular Biology Laboratory (EMBL Heidelberg) for more than ten years, conducting research on molecular self-assembly focusing especially on functional, protective and pathological amyloids and amyloidoses, and she was there when she published the first article on natural protective amyloids. She is the author of 41 publications and 6 book chapters which focus mostly on functional and pathological amyloid studies.

bioinformatics↗