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bioRxiv2017

Active site alanine substitutions can convert deubiquitinating enzymes into avid ubiquitin-binding domains

A common strategy for studying the biological role of deubiquitinating enzymes (DUBs) in different pathways is to study the effects of replacing the wild type DUB with a catalytically inactive mutant in cells. We report here that a commonly studied DUB mutation, in which the catalytic cysteine is replaced with alanine, can dramatically increase the affinity of some DUBs for ubiquitin. Overexpression of these tight-binding mutants thus has the potential to sequester cellular pools of monoubiquitin and ubiquitin chains. As a result, cells expressing these mutants may display unpredictable dominant negative physiological effects that are not related to loss of DUB activity. The structure of the SAGA DUB module bound to free ubiquitin reveals the structural basis for the 30-fold higher affinity of Ubp8C146A for ubiquitin. We show that an alternative option, substituting the active site cysteine with arginine, can inactivate DUBs while also decreasing the affinity for ubiquitin.

biochemistry↗

bioRxiv2017

Variations in δ13C of different plant organs: implications for post-photosynthetic fractionation

Compared to photosynthetic fractionation, the mechanism of post-photosynthetic carbon isotope fractionation is not well understood. The aim of this study was to investigate post-photosynthetic fractionation in both above and below ground tissues and to evaluate potential hypotheses explaining differences in carbon isotope composition ({delta}13C) among different plant organs, which can provide valuable insights into plant physiology. The results revealed that there is no significant day-night difference in{delta} 13C of twig phloem water soluble organic materials (WSOM), which could be explained by the unrestricted exchange of triose-phosphates between the chloroplast and cytoplasm and a time lag for carbohydrate exportation. Further, we found that{delta} 13C of twig phloem WSOM is more sensitive to plant water status than leaf WSOM. Analysis of{delta} 13C in different plant organs showed that the greatest 13C enrichment was recorded in stem phloem. Divergences in{delta} 13C of phloem WSOM among different plant organs were not likely to be explained by respiratory fractionation or time lag and were ascribed to transport of carbohydrates across organ boundaries and metabolic processes. Our study demonstrated that post-photosynthesis fractionation could not be ascribed to a single, unifying hypotheses; instead, it is the result of multiple processes.\n\nHighlight{delta}13C of twig phloem water soluble organic materials varied no clear diel pattern. In the leaf-twig-stem-root sequence, the greatest 13C enrichment was recorded in stem phloem.

ecology↗

bioRxiv2017

Modulatory upregulation of an insulin peptide gene by different pathogens in C. elegans

When an animal is infected, its innate immune response needs to be tightly regulated across tissues and coordinated with other aspects of organismal physiology. Previous studies with Caenorhabditis elegans have demonstrated that insulin-like peptide genes are differentially expressed in response to different pathogens. They represent prime candidates for conveying signals between tissues upon infection. Here, we focused on one such gene, ins-11 and its potential role in mediating cross-tissue regulation of innate immune genes. While diverse bacterial intestinal infections can trigger the up-regulation of ins-11 in the intestine, we show that epidermal infection with the fungus Drechmeria coniospora triggers an upregulation of ins-11 in the epidermis. Using the Shigella virulence factor OpsF, a MAP kinase inhibitor, we found that in both cases, ins-11 expression is controlled cell autonomously by p38 MAPK, but via distinct transcription factors, STA-2/STAT in the epidermis and HLH-30/TFEB in the intestine. We established that ins-11, and the insulin signaling pathway more generally, are not involved in the regulation of antimicrobial peptide gene expression in the epidermis. The up-regulation of ins-11 in the epidermis does, however, affect intestinal gene expression in a complex manner, and has a deleterious effect on longevity. These results support a model in which insulin signaling, via ins-11, contributes to the coordination of the organismal response to infection, influencing the allocation of resources in an infected animal.

genetics↗

bioRxiv2017

Analysis of RDR1/RDR2/RDR6-independent small RNAs in Arabidopsis thaliana improves MIRNA annotations and reveals novel siRNA loci

Plant small RNAs regulate key physiological mechanisms through post-transcriptional and transcriptional silencing of gene expression. sRNAs fall into two major categories: those that are reliant on RNA Dependent RNA Polymerases (RDRs) for biogenesis and those that arent. Known RDR-dependent sRNAs include phased and repeat-associated short interfering RNAs, while known RDR-independent sRNAs are primarily microRNAs and other hairpin-derived sRNAs. In this study, we produced and analyzed small RNA-seq libraries from rdr1/rdr2/rdr6 triple mutant plants. Only a small fraction of all sRNA loci were RDR1/RDR2/RDR6-independent; most of these were microRNA loci or associated with predicted hairpin precursors. We found 58 previously annotated microRNA loci that were reliant on RDR1, -2, or -6 function, casting doubt on their classification. We also found 38 RDR1/2/6-independent small RNA loci that are not MIRNAs or otherwise hairpin-derived, and did not fit into other known paradigms for small RNA biogenesis. These 38 small RNA-producing loci have novel biogenesis mechanisms, and are frequently located in the vicinity of protein-coding genes. Altogether, our analysis suggest that these 38 loci represent one or more new types of small RNAs in Arabidopsis thaliana.\n\nSignificance StatementSmall RNAs regulate gene expression in plants and are produced through a variety of previously-described mechanisms. Here, we examine a set of previously undiscovered small RNA-producing loci that are produced by novel mechanisms.

plant biology↗

bioRxiv2017

The PTPRT pseudo-phosphatase domain is a denitrase

Protein tyrosine nitration occurs under both physiological and pathological conditions1. However, enzymes that remove this protein modification have not yet been identified. Here we report that the pseudo-phosphatase domain of protein tyrosine receptor T (PTPRT) is a denitrase that removes nitro-groups from tyrosine residues in paxillin. PTPRT normally functions as a tumor suppressor and is frequently mutated in a variety of human cancers including colorectal cancer2,3. We demonstrate that some of the tumor-derived mutations located in the pseudophosphatase domain impair the denitrase activity. Moreover, PTPRT mutant mice that inactivate the denitrase activity are susceptible to carcinogen-induced colon tumor formation. This study uncovers a novel enzymatic activity that is involved in tumor suppression.

cancer biology↗

bioRxiv2017

Caste-biased movements by termites in isolation

The caste system of termites is an example of phenotypic plasticity. The castes differ not only in morphology and physiology, but also in behavior. As most of their behaviors within colonies involve nestmates, it is difficult to extract innate differences among castes. In this study, we focused on movement patterns of isolated individuals of Hodotermopsis sjostedti. We observed distinct clusters in movement patterns over 30 min, which indicates that termites have multiple innate modes of movement. The use of these modes is biased among castes, among which neotenics had a caste-specific mode and soldiers moved more actively than workers or neotenics. These caste biases may reflect different adaptive responses to social isolation. Our study provides a basis for a deeper understanding of the roles of individual movements in social behaviors.\n\nSummary StatementMovement patterns of termites in isolation were described for different castes. We proposed movements as a novel caste-specific characteristics in social insects.

animal behavior and cognition↗

bioRxiv2017

Self-assembling all-enzyme hydrogels for biocatalytic flow processes

We describe the construction of binary self-assembling all-enzyme hydrogels that are comprised entirely of two tetrameric globular enzymes, the stereoselective dehydrogenase LbADH and the cofactor-regenerating glucose 1-dehydrogenase GDH. The enzymes were genetically fused with a SpyTag or SpyCatcher domain, respectively, to generate two complementary homo-tetrameric building blocks that polymerise under physiological conditions into porous hydrogels. The biocatalytic gels were used for the highly stereoselective reduction of a prochiral diketone substrate where they showed the typical behaviour of the coupled kinetics of coenzyme regenerating reactions in the substrate channelling regime. They effectively sequestrate the NADPH cofactor even under continuous flow conditions. Owing to their sticky nature, the gels can be readily mounted in simple microfluidic reactors without the need for supportive membranes. The reactors revealed extraordinary high space-time yields with nearly quantitative conversion (>95%), excellent stereoselectivity (d.r. > 99:1), and total turnover numbers of the expensive cofactor NADP(H) that are amongst the highest values ever reported.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC=\"FIGDIR/small/240325_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (30K):\norg.highwire.dtl.DTLVardef@19a66b9org.highwire.dtl.DTLVardef@1ce1a41org.highwire.dtl.DTLVardef@1dac96org.highwire.dtl.DTLVardef@2db317_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2017

Isoxanthopterin: An Optically Functional Biogenic Crystal in the Eyes of Decapod Crustaceans

The eyes of some aquatic animals form images through reflective optics. Shrimp, lobsters, crayfish and prawns possess reflecting superposition compound eyes, composed of thousands of square-faceted eye-units (ommatidia). Mirrors in the upper part of the eye (the distal mirror) reflect light collected from many ommatidia onto the underlying photosensitive elements of the retina, the rhabdoms. A second reflector, the tapetum, underlying the retina, back-scatters dispersed light onto the rhabdoms. Using microCT and cryo-SEM imaging accompanied by in situ micro-X-ray diffraction and micro-Raman spectroscopy, we investigated the hierarchical organization and materials properties of the reflective systems at high resolution and under close to physiological conditions. We show that the distal mirror consists of three or four layers of sparse plate-like nano-crystals. The tapetum is a diffuse reflector composed of hollow nanoparticles constructed from concentric lamellae of crystals. Isoxanthopterin, a pteridine analog of guanine, forms both the reflectors in the distal mirror and in the tapetum. The crystal structure of isoxanthopterin was determined from crystal structure prediction calculations and verified by comparison with experimental X-ray diffraction. The extended hydrogen bonded layers of the molecules results in an extremely high calculated refractive index in the H-bonded plane, n = 1.96, which makes isoxanthopterin crystals an ideal reflecting material. The crystal structure of isoxanthopterin, together with a detailed knowledge of the reflector superstructures, provide a rationalization of the reflective optics of the crustacean eye.\n\nSignificanceAquatic animals use reflectors in their eyes either to form images or to increase photon capture. Guanine is the most widespread molecular component of these reflectors. Here we show that crystals of isoxanthopterin, a pteridine analogue of guanine, form both the image-forming distal mirror and the intensity-enhancing tapetum reflector in the compound eyes of some decapod crustaceans. The crystal structure of isoxanthopterin was determined, providing an explanation for why these crystals are so well suited for efficient reflection. Pteridines were previously known only as pigments and our discovery raises the question of which other organic molecules may be used to form crystals with superior reflective properties either in organisms or in artificial optical devices.

bioengineering↗

bioRxiv2017

An ordered pattern of Ana2 phosphorylation by Plk4 is required for centriole assembly

Polo-like kinase 4 (Plk4) initiates an early step in centriole assembly by phosphorylating Ana2/STIL, a structural component of the procentriole. Here, we show that Plk4 binding to the central coiled-coil (CC) of Ana2 is a conserved event, involving Polo-box 3 and a previously unidentified putative CC located adjacent to the kinase domain. Ana2 binding stimulates Plk4 kinase activity in vitro, and, in turn, is phosphorylated along its length. Previous studies showed that Plk4 phosphorylates the C-terminal STAN domain of Ana2/STIL, triggering binding and recruitment of the cartwheel protein Sas6 to the procentriole assembly site. However, the physiological relevance of N-terminal phosphorylation was unknown. We found that Plk4 first phosphorylates the extreme N-terminus of Ana2 which is critical for subsequent STAN domain modification. Phosphorylation of the central region then breaks the Plk4-Ana2 interaction. This phosphorylation pattern is important for centriole assembly and integrity because replacement of endogenous Ana2 with phospho-Ana2 mutants disrupts distinct steps in Ana2 function and inhibits centriole duplication.

cell biology↗

bioRxiv2017

Decoding the gene co-expression network underlying the ability of Gevuina avellana Mol. to live in diverse light conditions

O_LIGevuina avellana Mol. (Proteaceae) is a typical tree from the South American temperate rainforest. Although this species mostly regenerates in shaded understories, it exhibits an exceptional ecological breadth, being able to live under a wide range of light conditions. Here we studied the genetic basis regulating physiological acclimation of the photosynthetic responses of G. avellana under contrasting light conditions.\nC_LIO_LIWe analyzed carbon assimilation and light energy used for photochemical process in plants acclimated to contrasting light conditions. Also, we used a transcriptional profile of leaf primordia from G. avellana saplings growing under different light environments to identify the gene co-expression network underpinning photosynthetic performance and light-related processes.\nC_LIO_LIThe photosynthetic parameters revealed optimal performance regardless of light conditions. Strikingly, the mechanism involved in dissipation of excess light energy showed no significant differences between high and low-light acclimated plants. The gene co-expression network defined a community structure consistent with the photochemical responses, including genes involved mainly in assembly and functioning of photosystems, photoprotection, and retrograde signaling.\nC_LIO_LIOur ecophysiological genomics approach provides an understanding of the molecular regulatory mechanisms that allows this tree to have an optimal balance between photochemical, photoprotective and antioxidant performance in the diverse light habitats it encounters in nature.\nC_LI

plant biology↗

bioRxiv2017

Binocular System with Asymmetric Eyes

I elaborate binocular geometry with a novel eye model that incorporates the foveas temporalward displacement and the cornea and the lens misalignment. The formulated binocular correspondence results in longitudinal horopters that are conic sections resembling empirical horopters. When the eye models asymmetry parameters range is that which is observed in healthy eyes, abathic distance also falls within its experimentally observed range. This range in abathic distance is similar to that of the vergence resting position distance. Further, the conics orientation is specified by the eyes version angle, integrating binocular geometry with eye movement. This integration presents the possibility for modeling 3D perceptual stability during physiological eye movements.\n\nOCIS codes: (330.1400) Vision - binocular and stereopsis; (330.4060) Vision modeling

neuroscience↗

bioRxiv2017

Converting a broad matrix metalloproteinase family inhibitor into a specific inhibitor of MMP-9 and MMP-14

MMP-14 and MMP-9 are two well established cancer targets for which no specific clinically relevant inhibitor is available. Using a powerful combination of computational design and yeast surface display technology, we engineered such an inhibitor starting from a non-specific MMP inhibitor, N-TIMP2. The engineered purified N-TIMP2 variants showed enhanced specificity towards MMP-14 and MMP-9 relative to a panel of off-target MMPs. MMP-specific N-TIMP2 sequence signatures were obtained that could be understood from the structural perspective of MMP/N-TIMP2 interactions. Our MMP-9 inhibitor exhibited 1000-fold preference for MMP-9 vs. MMP-14, which is likely to translate into significant differences under physiological conditions. Our results provide new insights regarding evolution of promiscuous proteins and optimization strategies for design of inhibitors with single-target specificities.

biophysics↗

bioRxiv2017

Multivariate analysis reveals environmental and genetic determinants of element covariation in the maize grain ionome

Plants obtain elements from the soil through genetic and biochemical pathways responsive to physiological state and environment. Most perturbations affect multiple elements which leads the ionome, the full complement of mineral nutrients in an organism, to vary as an integrated network rather than a set of distinct single elements. To examine the genetic basis of covariation in the accumulation of multiple elements, we analyzed maize kernel ionomes from Intermated B73 x Mo17 (IBM) recombinant inbred populations grown in 10 environments. We compared quantitative trait loci (QTL) determining single-element variation to QTL that predict variation in principal components (PCs) of multiple-element covariance. Single-element and multivariate approaches detected partially overlapping sets of loci. In addition to loci co-localizing with single-element QTL, multivariate traits within environments were controlled by loci with significant multi-element effects not detectable using single-element traits. Gene-by-environment interactions underlying multiple-element covariance were identified through QTL analyses of principal component models of ionome variation. In addition to interactive effects, growth environment had a profound effect on the elemental profiles and multi-element phenotypes were significantly correlated with specific environmental variables.\n\nAuthor SummaryA multivariate approach to the analysis of element accumulation in the maize kernel shows that elements are not regulated independently. By describing relationships between element accumulation we identified new genetic loci invisible to single-element approaches. The mathematical combinations of elements distinguish groups of plants based on environment, demonstrating that observed variation derives from interactions between genetically controlled factors and environmental variables. These results suggest that successful application of ionomics to improve human nutrition and plant productivity requires simultaneous consideration of multiple-element effects and variation of such effects in response to environment.

plant biology↗

bioRxiv2017

A deep convolutional neural network approach for astrocyte detection

Astrocytes are involved in brain pathologies such as trauma or stroke, neurodegenerative disorders like Alzheimers and Parkinsons disease, chronic pain, and many others. Determining cell density and timing of morphological and biochemical changes is important for a proper understanding of the role of astrocytes in physiological and pathological conditions. One of the most important of such analyses is astrocytes count within a complex tissue environment in microscopy images. The most widely used approaches for the quantification of microscopy images data are either manual stereological cell counting or semi-automatic segmentation techniques. Detecting astrocytes automatically is a highly challenging computational task, for which we currently lack efficient image analysis tools. In this study, we developed a fast and fully automated software that assesses the number of astrocytes using Deep Convolutional Neural Networks (DCNN). The method highly outperforms state-of-the-art image analysis and machine learning methods and provides detection accuracy and precision comparable to that of human experts. Additionally, the runtime of cell detection is significantly less than other three analyzed computational methods, and it is faster than human observers by orders of magnitude. We applied DCNN-based method to examine the number of astrocytes in different brain regions of rats with opioid-induced hyperalgesia/tolerance (OIH/OIT) as morphine tolerance is believed to activate glial cells in the brain. We observed strong positive correlation between manual cell detection and DCNN-based analysis method for counting astrocytes in the brains of experimental animals.

bioinformatics↗

bioRxiv2018

meaRtools: an R package for the Comprehensive Analysis of Neuronal Networks Recorded on Multi-Electrode Arrays

Here we present an open-source R package meaRtools that provides a platform for analyzing neuronal networks recorded on Microelectrode Arrays (MEAs). Cultured neuronal networks monitored with MEAs are now being widely used to characterize in vitro models of neurological disorders and to evaluate pharmaceutical compounds. meaRtools provides core algorithms for MEA spike train analysis, feature extraction, statistical analysis and plotting of multiple MEA recordings with multiple genotypes and treatments. meaRtools functionality covers novel solutions for spike train analysis, including algorithms to assess electrode cross-correlation using the spike train tiling coefficient (STTC), mutual information, synchronized bursts and entropy within cultured wells. Also integrated is a solution to account for bursts variability originating from mixed-cell neuronal cultures. The package provides a statistical platform built specifically for MEA data that can combine multiple MEA recordings and compare extracted features between different genetic models or treatments. We demonstrate the utilization of meaRtools to successfully identify epilepsy-like phenotypes in neuronal networks from Celf4 knockout mice. The package is freely available under the GPL license (GPL>=3) and is updated frequently on the CRAN web-server repository. The package, along with full documentation can be downloaded from: https://cran.r-project.org/web/packages/meaRtools/.\n\nAuthor summaryCultured neuronal networks are widely used to study and characterize neuronal network activity. Among the many uses of neuronal cultures are the capabilities to evaluate neurotoxicity and the effects of pharmacological compounds on cellular physiology. Multi-well microelectrode arrays (MEAs) can collect high-throughput data from multiple neuronal cultures simultaneously, and thereby make possible hypotheses-driven inquiries into neurobiology and neuropharmacology. The analysis of MEA-derived information presents many computational challenges. High frequency data recorded simultaneously from hundreds of electrodes can be difficult to handle. The need to compare network activity across various drug treatments or genotypes recorded on the same plate from experiments lasting several weeks presents another challenge. These challenges inspired us to develop meaRtools; an MEA data analysis package that contains new methods to characterize network activity patterns, which are illustrated here using examples from a genetic mouse model of epilepsy. Among the highlights of meaRtools are novel algorithms designed to characterize neuronal activity dynamics and network properties such as bursting and synchronization, options to combine multiple recordings and use a robust statistical framework to draw appropriate statistical inferences, and finally data visualizations and plots. In summary, meaRtools provides a platform for the analyses of singular and longitudinal MEA experiments.

neuroscience↗

bioRxiv2018

A Multi-layer, Self-aligning Hydrogel Micro-molding Process Offering a Fabrication Route to Perfusable 3D In-Vitro Microvasculature

The in-vitro fabrication of hierarchical biological systems such as human vasculature, which are made up of two or more cell types with intricate co-culture architectures, is by far one of the most complicated challenges that tissue engineers have faced. Here, we introduce a versatile method to create multi-layered, cell-laden hydrogel microstructures with coaxial geometries and heterogeneous mechanical and biological properties. The technique can be used to build in-vitro vascular networks that are fully embedded in hydrogels of physiologically realistic mechanical stiffness. Our technique produces free-standing 3D structures, eliminating rigid polymeric surfaces from the vicinity of cells and allowing layers of multiple cell types to be defined with tailored extracellular matrix (ECM) composition and stiffness, and in direct contact with each other. We demonstrate co-axial geometries with diameters ranging from 200-2000 m and layer thicknesses as small as 50-200 {micro}m in agarose- collagen (AC) composite hydrogels. Coaxial geometries with such fine feature sizes are beyond the capabilities of most bioprinting techniques. A potential application of such a structure is to simulate vascular networks in the brain with endothelial cells surrounded by multiple layers of pericytes and other glial cells. For this purpose, the composition and mechanical properties of the composite AC hydrogels have been optimized for cell viability and biological performance of endothelial and glial cell types in both 2D and 3D culture modes. Multi-layered vascular constructs with an endothelial layer surrounded by layers of glial cells have been fabricated. This prototype in-vitro model resembles vascular geometries and opens the way for complex multi-luminal blood vessels to be fabricated.

bioengineering↗

bioRxiv2018

The genome of the water strider Gerris buenoi reveals expansions of gene repertoires associated with adaptations to life on the water

The semi-aquatic bugs conquered water surfaces worldwide and occupy ponds, streams, lakes, mangroves, and even open oceans. As such, they inspired a range of scientific studies from ecology and evolution to developmental genetics and hydrodynamics of fluid locomotion. However, the lack of a representative water strider genome hinders thorough investigations of the mechanisms underlying the processes of adaptation and diversification in this group. Here we report the sequencing and manual annotation of the Gerris buenoi (G. buenoi) genome, the first water strider genome to be sequenced so far. G. buenoi genome is about 1 000Mb and the sequencing effort recovered 20 949 predicted protein-coding genes. Manual annotation uncovered a number of local (tandem and proximal) gene duplications and expansions of gene families known for their importance in a variety of processes associated with morphological and physiological adaptations to water surface lifestyle. These expansions affect key processes such as growth, vision, desiccation resistance, detoxification, olfaction and epigenetic components. Strikingly, the G. buenoi genome contains three Insulin Receptors, a unique case among metazoans, suggesting key changes in the rewiring and function of the insulin pathway. Other genomic changes include wavelength sensitivity shifts in opsin proteins likely in association with the requirements of vision in water habitats. Our findings suggest that local gene duplications might have had an important role during the evolution of water striders. These findings along with the G. buenoi genome open exciting research opportunities to understand adaptation and genome evolution of this unique hemimetabolous insect.

genomics↗

bioRxiv2018

Root Responses to Heterogeneous Nitrate Availability are Mediated by trans-Zeatin in Arabidopsis Shoots

Plants are subjected to variable nitrogen (N) availability including frequent spatial nitrate (NO3-) heterogeneity in soil. Thus, plants constantly adapt their genome expression and root physiology in order to optimize N acquisition from this heterogeneous source. These adaptations rely on a complex and long-distance root-shoot-root signaling network that is still largely unknown. Here, we used a combination of reverse genetics, transcriptomic analysis, NO3- uptake experiments and hormone profiling under conditions of homogeneous or heterogeneous NO3- availability to characterize the systemic signaling involved. We demonstrate the important role of the trans-zeatin form of cytokinin (CK) in shoots, in particular using a mutant altered for ABCG14-mediated trans-zeatin-translocation from the root to the shoot, in mediating: (i) rapid long distance N-demand signaling and (ii) long term functional adaptations to heterogeneous NO3- supply, including changes in NO3- transport capacity and root growth modifications. We also provide insights into the potential CK-dependent and independent shoot-to-root signals involved in root adaptation to heterogeneous N availability.

plant biology↗