Showing papers by "Fundación Instituto Leloir published in 2016"
TL;DR: In this paper, the authors present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macro-autophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes.
Abstract: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.
For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.
Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.
5,187 citations
TL;DR: It is demonstrated that the phytochrome B (phyB) photoreceptor participates in temperature perception through its temperature-dependent reversion from the active Pfr state to the inactive Pr state, and proposed that in addition to its photorecept functions, phyB is a temperature sensor in plants.
Abstract: Ambient temperature regulates many aspects of plant growth and development, but its sensors are unknown. Here, we demonstrate that the phytochrome B (phyB) photoreceptor participates in temperature perception through its temperature-dependent reversion from the active Pfr state to the inactive Pr state. Increased rates of thermal reversion upon exposing Arabidopsis seedlings to warm environments reduce both the abundance of the biologically active Pfr-Pfr dimer pool of phyB and the size of the associated nuclear bodies, even in daylight. Mathematical analysis of stem growth for seedlings expressing wild-type phyB or thermally stable variants under various combinations of light and temperature revealed that phyB is physiologically responsive to both signals. We therefore propose that in addition to its photoreceptor functions, phyB is a temperature sensor in plants.
569 citations
Nencki Institute of Experimental Biology1, Federal University of Rio de Janeiro2, Leibniz Institute for Neurobiology3, Fundación Instituto Leloir4, Pompeu Fabra University5, Virginia Commonwealth University6, Instituto Politécnico Nacional7, Florey Institute of Neuroscience and Mental Health8, Cross River University of Technology9, Hebrew University of Jerusalem10, Baylor College of Medicine11, University of Copenhagen12, University College London13, University of the Witwatersrand14, West Bengal University of Health Sciences15, University of Würzburg16, University of Connecticut Health Center17, University of Texas MD Anderson Cancer Center18
TL;DR: Basic concepts of synapse structure and function are discussed, and a critical view of how aberrant synapse physiology may contribute to neurodevelopmental disorders as well as neurodegenerative disorders are provided.
Abstract: Synapses are essential components of neurons and allow information to travel coordinately throughout the nervous system to adjust behavior to environmental stimuli and to control body functions, memories, and emotions. Thus, optimal synaptic communication is required for proper brain physiology, and slight perturbations of synapse function can lead to brain disorders. In fact, increasing evidence has demonstrated the relevance of synapse dysfunction as a major determinant of many neurological diseases. This notion has led to the concept of synaptopathies as brain diseases with synapse defects as shared pathogenic features. In this review, which was initiated at the 13th International Society for Neurochemistry Advanced School, we discuss basic concepts of synapse structure and function, and provide a critical view of how aberrant synapse physiology may contribute to neurodevelopmental disorders (autism, Down syndrome, startle disease, and epilepsy) as well as neurodegenerative disorders (Alzheimer and Parkinson disease). We finally discuss the appropriateness and potential implications of gathering synapse diseases under a single term. Understanding common causes and intrinsic differences in disease-associated synaptic dysfunction could offer novel clues toward synapse-based therapeutic intervention for neurological and neuropsychiatric disorders. In this Review, which was initiated at the 13th International Society for Neurochemistry (ISN) Advanced School, we discuss basic concepts of synapse structure and function, and provide a critical view of how aberrant synapse physiology may contribute to neurodevelopmental (autism, Down syndrome, startle disease, and epilepsy) as well as neurodegenerative disorders (Alzheimer's and Parkinson's diseases), gathered together under the term of synaptopathies. Read the Editorial Highlight for this article on page 783.
230 citations
TL;DR: This review focuses on how developing neurons remodel the adult dentate gyrus and discusses key aspects that illustrate the potential of neurogenesis as a mechanism for circuit plasticity and function.
Abstract: The adult hippocampus generates functional dentate granule cells (GCs) that release glutamate onto target cells in the hilus and cornus ammonis (CA)3 region, and receive glutamatergic and γ-aminobutyric acid (GABA)ergic inputs that tightly control their spiking activity. The slow and sequential development of their excitatory and inhibitory inputs makes them particularly relevant for information processing. Although they are still immature, new neurons are recruited by afferent activity and display increased excitability, enhanced activity-dependent plasticity of their input and output connections, and a high rate of synaptogenesis. Once fully mature, new GCs show all the hallmarks of neurons generated during development. In this review, we focus on how developing neurons remodel the adult dentate gyrus and discuss key aspects that illustrate the potential of neurogenesis as a mechanism for circuit plasticity and function.
146 citations
TL;DR: A novel function of NS5 in the nucleus where it interferes with cellular splicing is demonstrated and it is proposed that NS5 binding to U5 snRNP proteins hijacks the splicing machinery resulting in a less restrictive environment for viral replication.
Abstract: Dengue virus NS5 protein plays multiple functions in the cytoplasm of infected cells, enabling viral RNA replication and counteracting host antiviral responses. Here, we demonstrate a novel function of NS5 in the nucleus where it interferes with cellular splicing. Using global proteomic analysis of infected cells together with functional studies, we found that NS5 binds spliceosome complexes and modulates endogenous splicing as well as minigene-derived alternative splicing patterns. In particular, we show that NS5 alone, or in the context of viral infection, interacts with core components of the U5 snRNP particle, CD2BP2 and DDX23, alters the inclusion/exclusion ratio of alternative splicing events, and changes mRNA isoform abundance of known antiviral factors. Interestingly, a genome wide transcriptome analysis, using recently developed bioinformatics tools, revealed an increase of intron retention upon dengue virus infection, and viral replication was improved by silencing specific U5 components. Different mechanistic studies indicate that binding of NS5 to the spliceosome reduces the efficiency of pre-mRNA processing, independently of NS5 enzymatic activities. We propose that NS5 binding to U5 snRNP proteins hijacks the splicing machinery resulting in a less restrictive environment for viral replication.
142 citations
TL;DR: New hypotheses of how these RNA elements specialize for replication in vertebrate and invertebrate hosts are discussed, and new ideas associating the significance of RNA structure duplication, small subgenomic flavivirus RNA formation, and host adaptation are presented.
135 citations
TL;DR: The results reveal a mechanism for dynamic remodeling in which experience activates dentate networks that “prime” young GCs through a disynaptic feedback loop mediated by PV-INs.
Abstract: Experience shapes the development and connectivity of adult-born granule cells (GCs) through mechanisms that are poorly understood We examined the remodeling of dentate gyrus microcircuits in mice in an enriched environment (EE) Short exposure to EE during early development of new GCs accelerated their functional integration This effect was mimicked by in vivo chemogenetic activation of a limited population of mature GCs Slice recordings showed that mature GCs recruit parvalbumin γ-aminobutyric acid–releasing interneurons (PV-INs) that feed back onto developing GCs Accordingly, chemogenetic stimulation of PV-INs or direct depolarization of developing GCs accelerated GC integration, whereas inactivation of PV-INs prevented the effects of EE Our results reveal a mechanism for dynamic remodeling in which experience activates dentate networks that “prime” young GCs through a disynaptic feedback loop mediated by PV-INs
112 citations
TL;DR: Modulation of Sec61-mediated cotranslational translocation selectively impaired glycoprotein proteostasis of influenza as well as HIV and dengue viruses and led to inhibition of viral growth and infectivity, and targetable host factors for broad-spectrum antiviral therapies are identified.
106 citations
TL;DR: The gaps in understanding of how ROS impact on the oscillatory Ca2+ and pH signatures that, coordinately, allow root hair cells and pollen tubes to expand in a controlled manner to several hundred times their original size toward specific signals are discussed.
Abstract: Root hair cells and pollen tubes, like fungal hyphae, possess a typical tip or polar cell expansion with growth limited to the apical dome. Cell expansion needs to be carefully regulated to produce a correct shape and size. Polar cell growth is sustained by oscillatory feedback loops comprising three main components that together play an important role regulating this process. One of the main components are reactive oxygen species (ROS) that, together with calcium ions (Ca(2+)) and pH, sustain polar growth over time. Apoplastic ROS homeostasis controlled by NADPH oxidases as well as by secreted type III peroxidases has a great impact on cell wall properties during cell expansion. Polar growth needs to balance a focused secretion of new materials in an extending but still rigid cell wall in order to contain turgor pressure. In this review, we discuss the gaps in our understanding of how ROS impact on the oscillatory Ca(2+) and pH signatures that, coordinately, allow root hair cells and pollen tubes to expand in a controlled manner to several hundred times their original size toward specific signals.
102 citations
TL;DR: It is believed that a deeper understanding of how the capsid protein works during infection will create opportunities for novel antiviral strategies, which are urgently needed to control dengue virus infections.
Abstract: Dengue virus affects hundreds of millions of people each year around the world, causing a tremendous social and economic impact on affected countries. The aim of this review is to summarize our current knowledge of the functions, structure, and interactions of the viral capsid protein. The primary role of capsid is to package the viral genome. There are two processes linked to this function: the recruitment of the viral RNA during assembly and the release of the genome during infection. Although particle assembly takes place on endoplasmic reticulum membranes, capsid localizes in nucleoli and lipid droplets. Why capsid accumulates in these locations during infection remains unknown. In this review, we describe available data and discuss new ideas on dengue virus capsid functions and interactions. We believe that a deeper understanding of how the capsid protein works during infection will create opportunities for novel antiviral strategies, which are urgently needed to control dengue virus infections.
102 citations
TL;DR: Genetic analysis using biofilm formation-related Pseudomonas mutants confirmed the relevance of bacterial root adhesion in the increase in nitrogen content, biomass accumulation and nitrogen fixation rates in wheat roots, the first report of robust BNF in major cereal crops.
Abstract: A main goal of biological nitrogen fixation research has been to expand the nitrogen-fixing ability to major cereal crops. In this work, we demonstrate the use of the efficient nitrogen-fixing rhizobacterium Pseudomonas protegens Pf-5 X940 as a chassis to engineer the transfer of nitrogen fixed by BNF to maize and wheat under non-gnotobiotic conditions. Inoculation of maize and wheat with Pf-5 X940 largely improved nitrogen content and biomass accumulation in both vegetative and reproductive tissues, and this beneficial effect was positively associated with high nitrogen fixation rates in roots. 15 N isotope dilution analysis showed that maize and wheat plants obtained substantial amounts of fixed nitrogen from the atmosphere. Pf-5 X940-GFP-tagged cells were always reisolated from the maize and wheat root surface but never from the inner root tissues. Confocal laser scanning microscopy confirmed root surface colonization of Pf-5 X940-GFP in wheat plants, and microcolonies were mostly visualized at the junctions between epidermal root cells. Genetic analysis using biofilm formation-related Pseudomonas mutants confirmed the relevance of bacterial root adhesion in the increase in nitrogen content, biomass accumulation and nitrogen fixation rates in wheat roots. To our knowledge, this is the first report of robust BNF in major cereal crops.
TL;DR: The fate of nucleocapsid components (capsid protein and viral genome) during the infection process is investigated and found that capsid is degraded by the ubiquitin-proteasome system, in contrast to that observed for other RNA and DNA viruses.
Abstract: Fil: Byk, Laura Andrea. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires. Fundacion Instituto Leloir. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina
TL;DR: A conserved role for the TIP60 complex as a HIF1 transcriptional cofactor in Drosophila and human cells is reported, which is required for Hif1-dependent gene expression in fly cells and embryos and colorectal cancer cells.
TL;DR: This work focuses on auxin-induced cellular elongation in root hairs, exposing a mechanistic view of plant growth regulation and highlighting a complex interplay between auxin metabolism and transport, steering root hair development in response to internal and external triggers.
Abstract: Auxin is a crucial growth regulator in plants. However, a comprehensive understanding of how auxin induces cell expansion is perplexing, because auxin acts in a concentration- and cell type-dependent manner. Consequently, it is desirable to focus on certain cell types to exemplify the underlying growth mechanisms. On the other hand, plant tissues display supracellular growth (beyond the level of single cells); hence, other cell types might compromise the growth of a certain tissue. Tip-growing cells do not display neighbor-induced growth constraints and, therefore, are a valuable source of information for growth-controlling mechanisms. Here, we focus on auxin-induced cellular elongation in root hairs, exposing a mechanistic view of plant growth regulation. We highlight a complex interplay between auxin metabolism and transport, steering root hair development in response to internal and external triggers. Auxin signaling modules and downstream cascades of transcription factors define a developmental program that appears rate limiting for cellular growth. With this knowledge in mind, the root hair cell is a very suitable model system in which to dissect cellular effectors required for cellular expansion.
TL;DR: COP1 promotes the degradation of HFR1 under shade, thus increasing the ability of PIFs to control gene expression, increase auxin levels and promote stem growth.
Abstract: Shade-avoidance responses require CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1) but the mechanisms of action of COP1 under shade have not been elucidated. Using simulated shade and control conditions, we analysed: the transcriptome and the auxin levels of cop1 and phytochrome interacting factor 1 (pif1) pif3 pif4 pif5 (pifq) mutants; the dynamics of ELONGATED HYPOCOTYL 5 (HY5) and LONG HYPOCOTYL IN FAR-RED (HFR1) proteins; and the epistatic relationships between cop1 and pif3, pif4, pif5, hy5 and hfr1 mutations in Arabidopsis thaliana. Despite severely impaired shade-avoidance responses, only a few genes that responded to shade in the wild-type failed to do so in cop1. Shade enhanced the convergence between cop1 and pifq transcriptomes, mainly on shade-avoidance marker genes. Shade failed to increase auxin levels in cop1. Residual shade avoidance in cop1 was not further reduced by the pif3, pif4 or pif5 mutations, suggesting convergent pathways. HFR1 stability decreased under shade in a COP1-dependent manner but shade increased HY5 stability. The cop1 mutant retains responses to shade and is more specifically impaired in shade avoidance. COP1 promotes the degradation of HFR1 under shade, thus increasing the ability of PIFs to control gene expression, increase auxin levels and promote stem growth.
TL;DR: It is shown that cerebrospinal fluid-contacting neurons (CSF-cNs), an anatomically discrete cell type of the ependymal area, originate from surprisingly late neurogenic events in the ventral spinal cord, highlighting that spatiotemporal mechanisms are instrumental in creating neural cell diversity in the vents to produce distinct classes of interneurons, motoneurons and CSF- cNs.
Abstract: Considerable progress has been made in understanding the mechanisms that control the production of specialized neuronal types. However, how the timing of differentiation contributes to neuronal diversity in the developing spinal cord is still a pending question. In this study, we show that cerebrospinal fluid-contacting neurons (CSF-cNs), an anatomically discrete cell type of the ependymal area, originate from surprisingly late neurogenic events in the ventral spinal cord. CSF-cNs are identified by the expression of the transcription factors Gata2 and Gata3, and the ionic channels Pkd2l1 and Pkd1l2. Contrasting with Gata2/3(+) V2b interneurons, differentiation of CSF-cNs is independent of Foxn4 and takes place during advanced developmental stages previously assumed to be exclusively gliogenic. CSF-cNs are produced from two distinct dorsoventral regions of the mouse spinal cord. Most CSF-cNs derive from progenitors circumscribed to the late-p2 and the oligodendrogenic (pOL) domains, whereas a second subset of CSF-cNs arises from cells bordering the floor plate. The development of these two subgroups of CSF-cNs is differentially controlled by Pax6, they adopt separate locations around the postnatal central canal and they display electrophysiological differences. Our results highlight that spatiotemporal mechanisms are instrumental in creating neural cell diversity in the ventral spinal cord to produce distinct classes of interneurons, motoneurons, CSF-cNs, glial cells and ependymal cells.
TL;DR: Structural analyses suggest that the photoconversion between the Pr and Pfr states in the full-length XccBphP may involve changes in the relative positioning of the output module, contributing to understand the light-induced structural changes propagated from the photosensor to the output modules in phytochrome signaling.
TL;DR: The importance of the analysis of macroheterogeneity for a complete understanding of glycoprotein biosynthesis and function is highlighted, and how advances in mass spectrometry glycoproteomics will enable analysis of this critical facet of glyCoprotein structural diversity is emphasized.
Abstract: Glycosylation is a co- and post-translational modification that is critical for the regulation of the biophysical properties and biological activities of diverse proteins. Biosynthetic pathways for protein glycosylation are inherently inefficient, resulting in high structural diversity in mature glycoproteins. Macroheterogeneity is the structural diversity due to the presence or absence of glycans at specific glycosylation sites, and is caused by inefficiency in the initial transfer of glycans to proteins. Here, we review the enzymatic and evolutionary mechanisms controlling macroheterogeneity, its biological consequences in physiological and disease states, its relevance to heterologous production and glycoengineering of glycoproteins, and mass spectrometry based methods for its analysis. We highlight the importance of the analysis of macroheterogeneity for a complete understanding of glycoprotein biosynthesis and function, and emphasize how advances in mass spectrometry glycoproteomics will enable analysis of this critical facet of glycoprotein structural diversity.
TL;DR: Evaluating peripheral blood NK cells from untreated patients and their possible role in metastasis progression determined that NKp30 and NKp46 are the key receptors involved in detriment of CRC-NK cells’ antitumor activity, and potential strategies to enhance CRC- NK cell activity are shown.
Abstract: The clinical outcome of colorectal cancer (CRC) is associated with the immune response, thus these tumors could be responsive to different immune therapy approaches. NK cells are key anti-tumor primary effectors that can eliminate CRC cells without prior immunization. We previously determined that NK cells from the local tumor environment of CRC tumors display a profoundly altered phenotype compared to circulating NK cells from healthy donors. In this study, we evaluated peripheral blood NK cells from untreated patients and their possible role in metastasis progression. We observed profound deregulation in receptor expression even in early stages of disease compared to healthy donors. CRC-NK cells displayed under-expression of CD16, NKG2D, DNAM-1, CD161, NKp46 and NKp30 activating receptors while inhibitory receptors CD85j and NKG2A were over-expressed. This inhibited phenotype affected cytotoxic functionality against CRC cells and IFN-γ production. We also determined that NKp30 and NKp46 are the key receptors involved in detriment of CRC-NK cells anti-tumor activity. Moreover, NKp46 expression correlated with relapse-free survival of CRC patients with a maximum follow-up of 71 months. CRC-NK cells also exhibited altered antibody-dependent-cellular cytotoxicity function responding poorly to cetuximab. IL-2 and IL-15 in combination with cetuximab stimulated NK cell improving cytotoxicity. These results show potential strategies to enhance CRC-NK cell activity.
TL;DR: It is proposed that in natural conditions where the light environment is not homogeneous, the uncovered phytochrome-phototropin co-action is important for plants to adapt their growth strategy to optimize photosynthetic light capture.
TL;DR: An updated view of cell surface proteins that include modular domains with an extensin (EXT)-motif followed by a cytoplasmic kinase-like domain, known as PERKs, and the EXT-motifs with the short sequence Ser-Pro(3-5), which is found in several different protein contexts within the same extracellular space, highlighting a putative conserved structural and functional role.
Abstract: Fil: Borassi, Cecilia. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires. Fundacion Instituto Leloir. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina
TL;DR: It is found that the G9a methyltransferase is required for differentiation of the mouse neuronal cell line N2a and that E10 inclusion increases during neuronal differentiation of cultured cells, as well as in the developing mouse brain.
TL;DR: Author(s): Klionsky, DJ; Abdelmohsen, K; Abe, A; Abedin, MJ; Abeliovich, H; A Frozena, AA; Adachi, H, Adeli, K, Adhihetty, PJ; Adler, SG; Agam, G; Agarwal, R; Aghi, MK; Agnello, M; Agostinis, P; Aguilar, PV; Aguirre-Ghis
Abstract: Author(s): Klionsky, DJ; Abdelmohsen, K; Abe, A; Abedin, MJ; Abeliovich, H; Arozena, AA; Adachi, H; Adams, CM; Adams, PD; Adeli, K; Adhihetty, PJ; Adler, SG; Agam, G; Agarwal, R; Aghi, MK; Agnello, M; Agostinis, P; Aguilar, PV; Aguirre-Ghiso, J; Airoldi, EM; Ait-Si-Ali, S; Akematsu, T; Akporiaye, ET; Al-Rubeai, M; Albaiceta, GM; Albanese, C; Albani, D; Albert, ML; Aldudo, J; Algul, H; Alirezaei, M; Alloza, I; Almasan, A; Almonte-Beceril, M; Alnemri, ES; Alonso, C; Altan-Bonnet, N; Altieri, DC; Alvarez, S; Alvarez-Erviti, L; Alves, S; Amadoro, G; Amano, A; Amantini, C; Ambrosio, S; Amelio, I; Amer, AO; Amessou, M; Amon, A; An, Z; Anania, FA; Andersen, SU; Andley, UP; Andreadi, CK; Andrieu-Abadie, N; Anel, A; Ann, DK; Anoopkumar-Dukie, S; Antonioli, M; Aoki, H; Apostolova, N; Aquila, S; Aquilano, K; Araki, K; Arama, E; Aranda, A; Araya, J; Arcaro, A; Arias, E; Arimoto, H; Ariosa, AR; Armstrong, JL; Arnould, T; Arsov, I; Asanuma, K; Askanas, V; Asselin, E; Atarashi, R; Atherton, SS; Atkin, JD; Attardi, LD; Auberger, P; Auburger, G; Aurelian, L; Autelli, R
TL;DR: A novel integrative network model is developed to prioritize and identify candidate drug targets in neglected pathogen proteomes, and bioactive drug-like molecules, and showcase the application of this network to suggest targets for orphan compounds that are active against Plasmodium falciparum in high-throughput screens.
Abstract: Drug development for neglected diseases has been historically hampered due to lack of market incentives. The advent of public domain resources containing chemical information from high throughput screenings is changing the landscape of drug discovery for these diseases. In this work we took advantage of data from extensively studied organisms like human, mouse, E. coli and yeast, among others, to develop a novel integrative network model to prioritize and identify candidate drug targets in neglected pathogen proteomes, and bioactive drug-like molecules. We modeled genomic (proteins) and chemical (bioactive compounds) data as a multilayer weighted network graph that takes advantage of bioactivity data across 221 species, chemical similarities between 1.7 105 compounds and several functional relations among 1.67 105 proteins. These relations comprised orthology, sharing of protein domains, and shared participation in defined biochemical pathways. We showcase the application of this network graph to the problem of prioritization of new candidate targets, based on the information available in the graph for known compound-target associations. We validated this strategy by performing a cross validation procedure for known mouse and Trypanosoma cruzi targets and showed that our approach outperforms classic alignment-based approaches. Moreover, our model provides additional flexibility as two different network definitions could be considered, finding in both cases qualitatively different but sensible candidate targets. We also showcase the application of the network to suggest targets for orphan compounds that are active against Plasmodium falciparum in high-throughput screens. In this case our approach provided a reduced prioritization list of target proteins for the query molecules and showed the ability to propose new testable hypotheses for each compound. Moreover, we found that some predictions highlighted by our network model were supported by independent experimental validations as found post-facto in the literature.
TL;DR: The effect of a red‐light pulse on AS of a gene encoding a splicing factor was not impaired in a quintuple phytochrome mutant, providing unequivocal evidence that nonphotosensory photoreceptors control AS in light‐grown plants.
Abstract: Light modulates plant growth and development to a great extent by regulating gene expression programs. Here, we evaluated the effect of light on alternative splicing (AS) in light-grown Arabidopsis thaliana plants using high-throughput RNA sequencing (RNA-seq). We found that an acute light pulse given in the middle of the night, a treatment that simulates photoperiod lengthening, affected AS events corresponding to 382 genes. Some of these AS events were associated with genes involved in primary metabolism and stress responses, which may help to adjust metabolic and physiological responses to seasonal changes. We also found that several core clock genes showed changes in AS in response to the light treatment, suggesting that light regulation of AS may play a role in clock entrainment. Finally, we found that many light-regulated AS events were associated with genes encoding RNA processing proteins and splicing factors, supporting the idea that light regulates this posttranscriptional regulatory layer through AS regulation of splicing factors. Interestingly, the effect of a red-light pulse on AS of a gene encoding a splicing factor was not impaired in a quintuple phytochrome mutant, providing unequivocal evidence that nonphotosensory photoreceptors control AS in light-grown plants.
TL;DR: A family of VHHs against the B subunit of Stx 2 (Stx2B) that neutralize Stx2 in vitro at subnanomolar concentrations is identified that should offer new therapeutic options for treating STEC infections to prevent or ameliorate HUS outcome.
Abstract: Shiga toxin (Stx)-producing Escherichia coli (STEC) infections are implicated in the development of the life-threatening Hemolytic Uremic Syndrome (HUS). Despite the magnitude of the social and economic problems caused by STEC infections, no licensed vaccine or effective therapy is presently available for human use. Single chain antibodies (VHH) produced by camelids exhibit several advantages in comparison with conventional antibodies, making them promising tools for diagnosis and therapy. In the present work, the properties of a recently developed immunogen, which induces high affinity and protective antibodies against Stx type 2 (Stx2), were exploited to develop VHHs with therapeutic potential against HUS. We identified a family of VHHs against the B subunit of Stx2 (Stx2B) that neutralize Stx2 in vitro at subnanomolar concentrations. One VHH was selected and was engineered into a trivalent molecule (two copies of anti-Stx2B VHH and one anti-seroalbumin VHH). The resulting molecule presented extended in vivo half-life and high therapeutic activity, as demonstrated in three different mouse models of Stx2-toxicity: a single i.v. lethal dose of Stx2, several i.v. incremental doses of Stx2 and intragastrical STEC infection. This simple antitoxin agent should offer new therapeutic options for treating STEC infections to prevent or ameliorate HUS outcome.
TL;DR: It is shown that illumination of Xcc, prior to plant infection, attenuates its virulence in an XccBphP‐dependent manner, eliciting bacterial virulence attenuation via downregulation ofacterial virulence factors.
Abstract: Fil: Bonomi, Hernan Ruy. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires. Fundacion Instituto Leloir. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina
TL;DR: In vivo and in vitro observations reveal that Crisp2-knockout mice exhibit significant defects in fertility-associated parameters under demanding conditions, as well as deficiencies in sperm fertilizing ability, hyperactivation development and intracellular Ca(2+) regulation.
Abstract: STUDY HYPOTHESIS We hypothesize that fertility disorders in patients with aberrant expression of Cysteine-RIch Secretory Protein 2 (CRISP2) could be linked to the proposed functional role of this protein in fertilization. STUDY FINDING Our in vivo and in vitro observations reveal that Crisp2-knockout mice exhibit significant defects in fertility-associated parameters under demanding conditions, as well as deficiencies in sperm fertilizing ability, hyperactivation development and intracellular Ca(2+) regulation. WHAT IS KNOWN ALREADY Testicular CRISP2 is present in mature sperm and has been proposed to participate in gamete fusion in both humans and rodents. Interestingly, evidence in humans shows that aberrant expression of CRISP2 is associated with male infertility. STUDY DESIGN, SAMPLES/MATERIALS, METHODS A mouse line carrying a deletion in the sixth exon of the Crisp2 gene was generated. The analyses of the reproductive phenotype of Crisp2(-/-) adult males included the evaluation of their fertility before and after being subjected to unilateral vasectomy, in vivo fertilization rates obtained after mating with either estrus or superovulated females, in vitro sperm fertilizing ability and different sperm functional parameters associated with capacitation such as tyrosine phosphorylation (by western blot), acrosome reaction (by Coomassie Blue staining), hyperactivation (by computer-assisted sperm analysis) and intracellular Ca(2+) levels (by flow cytometry). MAIN RESULTS AND THE ROLE OF CHANCE Crisp2(-/-) males presented normal fertility and in vivo fertilization rates when mated with estrus females. However, the mutant mice showed clear defects in those reproductive parameters compared with controls under more demanding conditions, i.e. when subjected to unilateral vasectomy to reduce the number of ejaculated sperm (n = 5; P< 0.05), or when mated with hormone-treated females containing a high number of eggs in the ampulla (n ≥ 5; P< 0.01). In vitro fertilization studies revealed that Crisp2(-/-) sperm exhibited deficiencies to penetrate the egg vestments (i.e. cumulus oophorus and zona pellucida) and to fuse with the egg (n ≥ 6; P< 0.01). Consistent with this, Crisp2-null sperm showed lower levels of hyperactivation (n = 7; P< 0.05), a vigorous motility required for penetration of the egg coats, as well as a dysregulation in intracellular Ca(2+) levels associated with capacitation (n = 5; P< 0.001). LIMITATIONS, REASONS FOR CAUTION The analysis of the possible mechanisms involved in fertility disorders in men with abnormal expression of CRISP2 was carried out in Crisp2 knockout mice due to the ethical and technical problems inherent to the use of human gametes for fertilization studies. WIDER IMPLICATIONS OF THE FINDINGS Our findings in mice showing that Crisp2(-/-) males exhibit fertility and fertilization defects under demanding conditions support fertilization defects in sperm as a mechanism underlying infertility in men with aberrant expression of CRISP2. Moreover, our observations in mice resemble the situation in humans where fertility disorders can or cannot be detected depending on the accumulation of own individual defects or the fertility status of the partner. Finally, the fact that reproductive defects in mice are masked by conventional mating highlights the need of using different experimental approaches to analyze male fertility. STUDY FUNDING AND COMPETING INTERESTS This study was supported by the World Health Organization (H9/TSA/037), the National Research Council of Argentina (PIP 2009-290), the National Agency for Scientific and Technological Promotion of Argentina (PICT 2011, 2023) and the Rene Baron Foundation to P.S.C. and by the MEXT of Japan to M.I. The authors declare that there are no conflicts of interest.
TL;DR: It is shown here that endogenous p21, instead of being residual, it is functional and necessary to preserve the genomic stability of unstressed cells and prevents a type of genomic instability which is not triggered by endogenous DNA lesions but by a dysregulation in the DNA polymerase choice during genomic DNA synthesis.
Abstract: The levels of the cyclin-dependent kinase (CDK) inhibitor p21 are low in S phase and insufficient to inhibit CDKs. We show here that endogenous p21, instead of being residual, it is functional and necessary to preserve the genomic stability of unstressed cells. p21depletion slows down nascent DNA elongation, triggers permanent replication defects and promotes the instability of hard-to-replicate genomic regions, namely common fragile sites (CFS). The p21's PCNA interacting region (PIR), and not its CDK binding domain, is needed to prevent the replication defects and the genomic instability caused by p21 depletion. The alternative polymerase kappa is accountable for such defects as they were not observed after simultaneous depletion of both p21 and polymerase kappa. Hence, in CDK-independent manner, endogenous p21 prevents a type of genomic instability which is not triggered by endogenous DNA lesions but by a dysregulation in the DNA polymerase choice during genomic DNA synthesis.
TL;DR: This study demonstrates entrainable, endogenous, and temperature-dependent circadian rhythms in gene expression as well as part of the pathway for synchronization in C. elegans, opening the way for novel research in neuroscience and molecular pathways in general.
Abstract: Circadian rhythms are based on endogenous clocks that allow organisms to adjust their physiology and behavior by entrainment to the solar day and, in turn, to select the optimal times for most biological variables. Diverse model systems—including mice, flies, fungi, plants, and bacteria—have provided important insights into the mechanisms of circadian rhythmicity. However, the general principles that govern the circadian clock of Caenorhabditis elegans have remained largely elusive. Here we report robust molecular circadian rhythms in C. elegans recorded with a bioluminescence assay in vivo and demonstrate the main features of the circadian system of the nematode. By constructing a luciferase-based reporter coupled to the promoter of the suppressor of activated let-60 Ras (sur-5) gene, we show in both population and single-nematode assays that C. elegans expresses ∼24-h rhythms that can be entrained by light/dark and temperature cycles. We provide evidence that these rhythms are temperature-compensated and can be re-entrained after phase changes of the synchronizing agents. In addition, we demonstrate that light and temperature sensing requires the photoreceptors LITE and GUR-3, and the cyclic nucleotide-gated channel subunit TAX-2. Our results shed light on C. elegans circadian biology and demonstrate evolutionarily conserved features in the circadian system of the nematode.