Connexin and Pannexin Large-Pore Channels in Microcirculation and Neurovascular Coupling Function

Microcirculation homeostasis depends on several channels permeable to ions and/or small molecules that facilitate the regulation of the vasomotor tone, hyperpermeability, the blood–brain barrier, and the neurovascular coupling function. Connexin (Cxs) and Pannexin (Panxs) large-pore channel proteins are implicated in several aspects of vascular physiology. The permeation of ions (i.e., Ca²⁺) and key metabolites (ATP, prostaglandins, D-serine, etc.) through Cxs (i.e., gap junction channels or hemichannels) and Panxs proteins plays a vital role in intercellular communication and maintaining vascular homeostasis. Therefore, dysregulation or genetic pathologies associated with these channels promote deleterious tissue consequences. This review provides an overview of current knowledge concerning the physiological role of these large-pore molecule channels in microcirculation (arterioles, capillaries, venules) and in the neurovascular coupling function.     

miRNA and mRNA Expression Profiles Associated with Lymph Node Metastasis and Prognosis in Penile Carcinoma

Penile cancer (PeC) is a rare disease, and no prognostic biomarkers have been adopted in clinical practice yet. The objective of the present study was to identify differentially expressed miRNAs (DEmiRs) and genes (DEGs) as potential biomarkers for lymph node metastasis and other prognostic factors in PeC. Tumor samples were prospectively obtained from 24 patients with squamous cell carcinoma of the penis. miRNA microarray analysis was performed comparing tumors from patients with inguinal lymph node metastatic and localized disease, and the results were validated by qRT-PCR. Eighty-three gene expression levels were also compared between groups through qRT-PCR. Moreover, DEmiRs and DEGs expression levels were correlated with clinicopathological variables, cancer-specific (CSS), and overall survival (OS). TAC software, TM4 MeV 4.9 software, SPSS v.25.0, and R software v.4.0.2 were used for statistical analyses. We identified 21 DEmiRs in microarray analysis, and seven were selected for validation. miR-744-5p and miR-421 were overexpressed in tissue samples of metastatic patients, and high expression of miR-421 was also associated with lower OS. We found seven DEGs (CCND1, EGFR, ENTPD5, HOXA10, IGF1R, MYC, and SNAI2) related to metastatic disease. A significant association was found between increased MMP1 expression and tumor size, grade, pathological T stage, and perineural invasion. Other genes were also associated with clinicopathological variables, CSS and OS. Finally, we found changes in mRNA–miRNA regulation that contribute to understanding the mechanisms involved in tumor progression. Therefore, we identified miRNA and mRNA expression profiles as potential biomarkers associated with lymph node metastasis and prognosis in PeC, in addition to disruption in mRNA–miRNA regulation during disease progression.     

Extracellular Cysteines Are Critical to Form Functional Cx46 Hemichannels

Connexin (Cxs) hemichannels participate in several physiological and pathological processes, but the molecular mechanisms that control their gating remain elusive. We aimed at determining the role of extracellular cysteines (Cys) in the gating and function of Cx46 hemichannels. We studied Cx46 and mutated all of its extracellular Cys to alanine (Ala) (one at a time) and studied the effects of the Cys mutations on Cx46 expression, localization, and hemichannel activity. Wild-type Cx46 and Cys mutants were expressed at comparable levels, with similar cellular localization. However, functional experiments showed that hemichannels formed by the Cys mutants did not open either in response to membrane depolarization or removal of extracellular divalent cations. Molecular-dynamics simulations showed that Cys mutants may show a possible alteration in the electrostatic potential of the hemichannel pore and an altered disposition of important residues that could contribute to the selectivity and voltage dependency in the hemichannels. Replacement of extracellular Cys resulted in “permanently closed hemichannels”, which is congruent with the inhibition of the Cx46 hemichannel by lipid peroxides, through the oxidation of extracellular Cys. These results point to the modification of extracellular Cys as potential targets for the treatment of Cx46-hemichannel associated pathologies, such as cataracts and cancer, and may shed light into the gating mechanisms of other Cx hemichannels.     

Probing the Conformational States of Thimet Oligopeptidase in Solution

Thimet oligopeptidase (TOP) is a metallopeptidase involved in the metabolism of oligopeptides inside and outside cells of various tissues. It has been proposed that substrate or inhibitor binding in the TOP active site induces a large hinge-bending movement leading to a closed structure, in which the bound ligand is enclosed. The main goal of the present work was to study this conformational change, and fluorescence techniques were used. Four active TOP mutants were created, each equipped with a single-Trp residue (fluorescence donor) and a p-nitro-phenylalanine (pNF) residue as fluorescence acceptor at opposite sides of the active site. pNF was biosynthetically incorporated with high efficiency using the amber codon suppression technology. Inhibitor binding induced shorter Donor-Acceptor (D-A) distances in all mutants, supporting the view that a hinge-like movement is operative in TOP. The activity of TOP is known to be dependent on the ionic strength of the assay buffer and D-A distances were measured at different ionic strengths. Interestingly, a correlation between the D-A distance and the catalytic activity of TOP was observed: the highest activities corresponded to the shortest D-A distances. In this study for the first time the hinge-bending motion of a metallopeptidase in solution could be studied, yielding insight about the position of the equilibrium between the open and closed conformation. This information will contribute to a more detailed understanding of the mode of action of these enzymes, including therapeutic targets like neurolysin and angiotensin-converting enzyme 2 (ACE2).     

Distributed text search using suffix arrays

Text search is a classical problem in Computer Science, with many data-intensive applications. For this problem, suffix arrays are among the most widely known and used data structures, enabling fast searches for phrases, terms, substrings and regular expressions in large texts. Potential application domains for these operations include large-scale search services, such as Web search engines, where it is necessary to efficiently process intensive-traffic streams of on-line queries. This paper proposes strategies to enable such services by means of suffix arrays. We introduce techniques for deploying suffix arrays on clusters of distributed-memory processors and then study the processing of multiple queries on the distributed data structure. Even though the cost of individual search operations in sequential (non-distributed) suffix arrays is low in practice, the problem of processing multiple queries on distributed-memory systems, so that hardware resources are used efficiently, is relevant to services aimed at achieving high query throughput at low operational costs. Our theoretical and experimental performance studies show that our proposals are suitable solutions for building efficient and scalable on-line search services based on suffix arrays.     

P2P OLAP: Data model,implementation and case study

It is a common situation nowadays that business groups own different companies that operate in an autonomous way. Nevertheless, these companies must be requested to provide the headquarters with summarized information for decision-making. An architecture for cooperative interchange of decision-making information seems to be a natural solution for this problem. We propose the use of a peer-to-peer (P2P) architecture for addressing the problem of processing OLAP data in a distributed environment, in a way that all companies involved can maintain full autonomy over the use of its own data resources. In a scenario like this, data exchange between peers occurs when one of them, in the role of a local peer, receives a query and, for answering it, requests data available in other nodes, denoted acquaintances. No global schema is assumed to exist for any data under this computing paradigm. Henceforth, data provided by an acquaintance of a local peer must be adapted, in a manner that answers to queries posed by local peer users conform the view those users have of their data. Because multidimensional data normally consist of a collection of views of aggregated data, a careful translation process is needed in this case, in order to transform any summary concept that appears in a peer acquaintance into a summary concept meaningful to the requesting peer. We first present a model for multidimensional data distributed in a P2P network, and a query rewriting technique, that allows a local peer to propagate OLAP queries among its acquaintances, obtaining a meaningful and correct answer. Mappings are performed using a novel technique called revise and map, based on belief revision concepts. Revising a dimension instance allows to produce consistent aggregations when an OLAP query is answered at more than one node. We then describe an implementation of a P2P system for answering OLAP queries over a network of data warehouses. We apply our proposal to a real-world case study of an insurance group. Finally, we report the results of an experimental evaluation of our implementation, and discuss the issues that must be accounted for in this setting.     

GABAergic Regulation of Astroglial Gliotransmission through Cx43 Hemichannels

Gamma-Aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the brain. It is produced by interneurons and recycled by astrocytes. In neurons, GABA activates the influx of Cl^- via the GABA_A receptor or efflux or K⁺ via the GABA_B receptor, inducing hyperpolarization and synaptic inhibition. In astrocytes, the activation of both GABA_A and GABA_B receptors induces an increase in intracellular Ca²⁺ and the release of glutamate and ATP. Connexin 43 (Cx43) hemichannels are among the main Ca²⁺-dependent cellular mechanisms for the astroglial release of glutamate and ATP. However, no study has evaluated the effect of GABA on astroglial Cx43 hemichannel activity and Cx43 hemichannel-mediated gliotransmission. Here we assessed the effects of GABA on Cx43 hemichannel activity in DI NCT1 rat astrocytes and hippocampal brain slices. We found that GABA induces a Ca²⁺-dependent increase in Cx43 hemichannel activity in astrocytes mediated by the GABA_A receptor, as it was blunted by the GABA_A receptor antagonist bicuculline but unaffected by GABA_B receptor antagonist {"type":"entrez-protein","attrs":{"text":"CGP55845","term_id":"875097176","term_text":"CGP55845"}}CGP55845. Moreover, GABA induced the Cx43 hemichannel-dependent release of glutamate and ATP, which was also prevented by bicuculline, but unaffected by CGP. Gliotransmission in response to GABA was also unaffected by pannexin 1 channel blockade. These results are discussed in terms of the possible role of astroglial Cx43 hemichannel-mediated glutamate and ATP release in regulating the excitatory/inhibitory balance in the brain and their possible contribution to psychiatric disorders.     

Methylation in the CHH Context Allows to Predict Recombination in Rice

DNA methylation is the most studied epigenetic trait. It is considered a key factor in regulating plant development and physiology, and has been associated with the regulation of several genomic features, including transposon silencing, regulation of gene expression, and recombination rates. Nonetheless, understanding the relation between DNA methylation and recombination rates remains a challenge. This work explores the association between recombination rates and DNA methylation for two commercial rice varieties. The results show negative correlations between recombination rates and methylated cytosine counts for all contexts tested at the same time, and for CG and CHG contexts independently. In contrast, a positive correlation between recombination rates and methylated cytosine count is reported in CHH contexts. Similar behavior is observed when considering only methylated cytosines within genes, transposons, and retrotransposons. Moreover, it is shown that the centromere region strongly affects the relationship between recombination rates and methylation. Finally, machine learning regression models are applied to predict recombination using the count of methylated cytosines in the CHH context as the entrance feature. These findings shed light on the understanding of the recombination landscape of rice and represent a reference framework for future studies in rice breeding, genetics, and epigenetics.     

Quantum Biochemistry and MM-PBSA Description of the ZIKV NS2B-NS3 Protease: Insights into the Binding Interactions beyond the Catalytic Triad Pocket

The Zika virus protease NS2B-NS3 has a binding site formed with the participation of a H51-D75-S135 triad presenting two forms, active and inactive. Studies suggest that the inactive conformation is a good target for the design of inhibitors. In this paper, we evaluated the co-crystallized structures of the protease with the inhibitors benzoic acid (5YOD) and benzimidazole-1-ylmethanol (5H4I). We applied a protocol consisting of two steps: first, classical molecular mechanics energy minimization followed by classical molecular dynamics were performed, obtaining stabilized molecular geometries; second, the optimized/relaxed geometries were used in quantum biochemistry and molecular mechanics/Poisson–Boltzmann surface area (MM-PBSA) calculations to estimate the ligand interactions with each amino acid residue of the binding pocket. We show that the quantum-level results identified essential residues for the stabilization of the 5YOD and 5H4I complexes after classical energy minimization, matching previously published experimental data. The same success, however, was not observed for the MM-PBSA simulations. The application of quantum biochemistry methods seems to be more promising for the design of novel inhibitors acting on NS2B-NS3.