Method for Determining the Level of Centralization in BTC Lightning Nodes: A Centrality Analysis of the Lightning Network

This study explores the Bitcoin Lightning Network (BLN), a Layer 2 solution for faster and cheaper transactions. Concerns about centralization have emerged due to the increasing concentration of power among specific nodes, named “hubs.” Statistical measures like the Gini coefficient reveal a trend towards centralization, challenging the LN’s decentralized nature. Consequently, further research is necessary to address this issue and ensure the integrity of the LN architecture. This paper aims to establish a method for determining the level of centralization within the BLN by applying centrality analysis techniques. Study revealed that over the six-year period Gini coefficient increased from 0.87 to 0.955, indicating significant inequality and apparent centralization of BLN nodes

Mechanisms of adaptation and evolution in Toxoplasma gondii

Toxoplasma has high host flexibility, infecting all nucleated cells of mammals and birds. This implies that duringits infective process the parasite must constantly adapt to different environmental situations, which in turn leadsto modifications in its metabolism, regulation of gene transcription, translation of mRNAs and stage specificfactors. There are conserved pathways that support these adaptations, which we aim to elucidate in this review.We begin by exploring the widespread epigenetic mechanisms and transcription regulators, continue with thesupportive role of Heat Shock Proteins (Hsp), the translation regulation, stress granules, and finish with theemergence of contingency genes in highly variable genomic domains, such as subtelomeres. Within epigenetics,the discovery of a new histone variant of the H2B family (H2B.Z), contributing to T. gondii virulence and differentiation, but also gene expression regulation and its association with the metabolic state of the parasite, ishighlighted. Associated with the regulation of gene expression are transcription factors (TFs). An overview of themain findings on TF and development is presented. We also emphasize the role of Hsp90 and Tgj1 in T. gondiimetabolic fitness and the regulation of protein translation. Translation regulation is also highlighted as amechanism for adaptation to conditions encountered by the parasite as well as stress granules containing mRNAand proteins generated in the extracellular tachyzoite. Another important aspect in evolution and adaptabilityare the subtelomeres because of their high variability and gene duplication rate. Toxoplasma possess multigenefamilies of membrane proteins and contingency genes that are associated with different metabolic stresses.Among them parasite differentiation and environmental stresses stand out, including those that lead tachyzoiteto bradyzoite conversion. Finally, we are interested in positioning protozoa as valuable evolution models,focusing on research related to the Extended Evolutionary Synthesis, based on models recently generated, such asextracellular adaptation and ex vivo cyst recrudescence.Fil: Ángel, Sergio Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Vanagas, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Alonso, Andrés Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin

Apicomplexa and Histone Variants: What’s New?

Plasmodium spp. and Toxoplasma gondii present a conserved nucleosome composition based on canonical H3 and variants, H4, canonical H2A and variants, and H2B. One-off, the phylum has also a variant H2B, named H2B.Z, which was shown to form a double variant nucleosome H2A.Z/H2B.Z. These histones also present conserved and unique post-translational modifications (PTMs). Histone variants have shown particular genomic localization and PTMs along euchromatin and heterochromatin, including telomere-associated sequences (TAS), suggesting fine-grained chromatin structure modulation. Several other nonhistone proteins present remarkable participation in controlling chromatin state, especially at TAS. Based on that, we discuss the role of epigenetics (PTMs and histone variants) in Plasmodium and Toxoplasma gene expression, replication, and DNA repair. We also discuss TAS structures and chromatin composition and its impact on antigenic variant expression in Plasmodium

Evaluation of topotecan and 10-hydroxycamptothecin on Toxoplasma gondii: Implications on baseline DNA damage and repair efficiency

Toxoplasma gondii is an obligate intracellular parasite in the phylum Apicomplexa that causes toxoplasmosis in humans and animals worldwide. Despite its prevalence, there is currently no effective vaccine or treatment for chronic infection. Although there are therapies against the acute stage, prolonged use is toxic and poorly tolerated. This study aims to explore the potential of repurposing topotecan and 10-hydroxycamptothecin (HCPT) as drugs producing double strand breaks (DSBs) in T. gondii. DSBs are mainly repaired by Homologous Recombination Repair (HRR) and Non-Homologous End Joining (NHEJ). Two T. gondii strains, RHΔHXGPRT and RHΔKU80, were used to compare the drug's effects on parasites. RHΔHXGPRT parasites may use both HRR and NHEJ pathways but RHΔKU80 lacks the KU80 protein needed for NHEJ, leaving only the HRR pathway. Here we demonstrate that topotecan and HCPT, both topoisomerase I venoms, affected parasite replication in a concentration-dependent manner. Moreover, variations in fluorescence intensity measurements for the H2A.X phosphorylation mark (γH2A.X), an indicator of DNA damage, were observed in intracellular parasites under drug treatment conditions. Interestingly, intracellular replicative parasites without drug treatment show a strong positive staining for γH2A.X, suggesting inherent DNA damage. Extracellular (non-replicating) parasites did not exhibit γH2A.X staining, indicating that the basal level of DNA damage is likely to be associated with replicative stress. A high rate of DNA replication stress possibly prompted the evolution of an efficient repair machinery in the parasite, making it an attractive target. Our findings show that topoisomerase 1 venoms are effective antiparasitics blocking T. gondii replication.Fil: Cristaldi, Constanza. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Saldarriaga Cartagena, Ana María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Ganuza, Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Sullivan, William J.. Indiana University School Of Medicine; Estados UnidosFil: Angel, Sergio Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Vanagas, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin

Emerging Therapeutic Targets Against Toxoplasma gondii: Update on DNA Repair Response Inhibitors and Genotoxic Drugs

Toxoplasma gondii is the causative agent of toxoplasmosis in animals and humans. This infection is transmitted to humans through oocysts released in the feces of the felines into the environment or by ingestion of undercooked meat. This implies that toxoplasmosis is a zoonotic disease and T. gondii is a foodborne pathogen. In addition, chronic toxoplasmosis in goats and sheep is the cause of recurrent abortions with economic losses in the sector. It is also a health problem in pets such as cats and dogs. Although there are therapies against this infection in its acute stage, they are not able to permanently eliminate the parasite and sometimes they are not well tolerated. To develop better, safer drugs, we need to elucidate key aspects of the biology of T. gondii. In this review, we will discuss the importance of the homologous recombination repair (HRR) pathway in the parasite's lytic cycle and how components of these processes can be potential molecular targets for new drug development programs. In that sense, the effect of different DNA damage agents or HHR inhibitors on the growth and replication of T. gondii will be described. Multitarget drugs that were either associated with other targets or were part of general screenings are included in the list, providing a thorough revision of the drugs that can be tested in other scenarios.Fil: Ángel, Sergio Oscar. Universidad Nacional de San Martin. Instituto Tecnológico de Chascomús - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Tecnológico de Chascomús; ArgentinaFil: Vanagas, Laura. Universidad Nacional de San Martin. Instituto Tecnológico de Chascomús - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Tecnológico de Chascomús; ArgentinaFil: Ruiz, Diego Mario. Universidad Nacional de San Martin. Instituto Tecnológico de Chascomús - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Tecnológico de Chascomús; ArgentinaFil: Cristaldi, Constanza. Universidad Nacional de San Martin. Instituto Tecnológico de Chascomús - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Tecnológico de Chascomús; ArgentinaFil: Saldarriaga Cartagena, Ana María. Universidad Nacional de San Martin. Instituto Tecnológico de Chascomús - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Tecnológico de Chascomús; ArgentinaFil: Sullivan, William J.. Indiana University. School of Medicine; Estados Unido

Canonical histone H2Ba and H2A.X dimerize in an opposite genomic localization to H2A.Z/H2B.Z dimers in Toxoplasma gondii

Histone H2Ba of Toxoplasma gondii was expressed as recombinant protein (rH2Ba) and used to generate antibody in mouse that is highly specific. Antibody recognizing rH2Ba detects a single band in tachyzoite lysate of the expected molecular weight (12kDa). By indirect immunofluorescence (IFA) in in vitro grown tachyzoites and bradyzoites, the signal was detected only in the parasite nucleus. The nucleosome composition of H2Ba was determined through co-immunoprecipitation assays. H2Ba was detected in the same immunocomplex as H2A.X, but not with H2A.Z. Through chromatin immunoprecipitation (ChIP) assays and qPCR, it was observed that H2Ba is preferentially located at promoters of inactive genes and silent regions, accompanying H2A.X and opposed to H2A.Z/H2B.Z dimers

Genome-wide localization of histone variants in Toxoplasma gondii implicates variant exchange in stage-specific gene expression.

BACKGROUND: Toxoplasma gondii is a protozoan parasite that differentiates from acute tachyzoite stages to latent bradyzoite forms in response to environmental cues that modify the epigenome. We studied the distribution of the histone variants CenH3, H3.3, H2A.X, H2A.Z and H2B.Z, by genome-wide chromatin immunoprecipitation to understand the role of variant histones in developmental transitions of T. gondii parasites. RESULTS: H3.3 and H2A.X were detected in telomere and telomere associated sequences, whereas H3.3, H2A.X and CenH3 were enriched in centromeres. Histones H2A.Z and H2B.Z colocalize with the transcriptional activation mark H3K4me3 in promoter regions surrounding the nucleosome-free region upstream of the transcription start site. The H2B.Z/H2A.Z histone pair also localizes to the gene bodies of genes that are silent but poised for activation, including bradyzoite stage-specific genes. The majority of H2A.X and H2A.Z/H2B.Z loci do not overlap, consistent with variant histones demarcating specific functional regions of chromatin. The extent of enrichment of H2A.Z/H2B.Z (and H3.3 and H2A.X) within the entire gene (5'UTR and gene body) reflects the timing of gene expression during the cell cycle, suggesting that dynamic turnover of H2B.Z/H2A.Z occurs during the tachyzoite cell cycle. Thus, the distribution of the variant histone H2A.Z/H2B.Z dimer defines active and developmentally silenced regions of the T. gondii epigenome including genes that are poised for expression. CONCLUSIONS: Histone variants mark functional regions of parasite genomes with the dynamic placement of the H2A.Z/H2B.Z dimer implicated as an evolutionarily conserved regulator of parasite and eukaryotic differentiation

Evaluation of ATM Kinase Inhibitor KU-55933 as Potential Anti-Toxoplasma gondii Agent

Toxoplasma gondii is an apicomplexan protozoan parasite with a complex life cycle composed of multiple stages that infect mammals and birds. Tachyzoites rapidly replicate within host cells to produce acute infection during which the parasite disseminates to tissues and organs. Highly replicative cells are subject to Double Strand Breaks (DSBs) by replication fork collapse and ATM, a member of the phosphatidylinositol 3-kinase (PI3K) family, is a key factor that initiates DNA repair and activates cell cycle checkpoints. Here we demonstrate that the treatment of intracellular tachyzoites with the PI3K inhibitor caffeine or ATM kinase-inhibitor KU-55933 affects parasite replication rate in a dose-dependent manner. KU-55933 affects intracellular tachyzoite growth and induces G1-phase arrest. Addition of KU-55933 to extracellular tachyzoites also leads to a significant reduction of tachyzoite replication upon infection of host cells. ATM kinase phosphorylates H2A.X (γH2AX) to promote DSB damage repair. The level of γH2AX increases in tachyzoites treated with camptothecin (CPT), a drug that generates fork collapse, but this increase was not observed when co-administered with KU-55933. These findings support that KU-55933 is affecting the Toxoplasma ATM-like kinase (TgATM). The combination of KU-55933 and other DNA damaging agents such as methyl methane sulfonate (MMS) and CPT produce a synergic effect, suggesting that TgATM kinase inhibition sensitizes the parasite to damaged DNA. By contrast, hydroxyurea (HU) did not further inhibit tachyzoite replication when combined with KU-55933

Epichromatin is conserved in Toxoplasma gondii and labels the exterior parasite chromatin throughout the cell cycle

Toxoplasma gondii is an apicomplexan intracellular protozoan parasite responsible for toxoplasmosis, a disease with considerable medical and economic impact worldwide. Toxoplasma gondii cells never lose the nuclear envelope and their chromosomes do not condense. Here, we tested the murine monoclonal antibody PL2-6, which labels epichromatin (a conformational chromatin epitope based on histones H2A and H2B complexed with DNA), in T. gondii cultured in human fibroblasts. This epitope is present at the exterior chromatin surface of interphase nuclei and on the periphery of mitotic chromosomes in higher eukaryotes. PL2-6 reacted with T. gondii H2A and H2B histones in Western blot (WB) assays. In addition, the antibody reacted with the nuclear fraction of tachyzoites, as a single band coincident with H2B histone. In the T. gondii tachyzoite stage, PL2-6 also had peripheral nuclear localization, as observed by epifluorescence/confocal microscopy and immunoelectron microscopy. Confocal analysis showed that epichromatin is slightly polarized to one face of the parasite exterior chromatin surface. In replicating tachyzoites, PL2-6 also labels the exterior chromatin surface, covering the face of both segregating nuclei, facing the plasma membrane of the mother cell. The possible role of epichromatin in T. gondii is discussed.Facultad de Ciencias Veterinaria

Analysis of the interactome of the Toxoplasma gondii Tgj1 HSP40 chaperone

Toxoplasma gondii is an obligate intracellular apicomplexan that causes toxoplasmosis in humans and animals. Central to its dissemination and pathogenicity is the ability to rapidly divide in the tachyzoite stage and infect any type of nucleated cell. Adaptation to different cell contexts requires high plasticity in which heat shock proteins (Hsps) could play a fundamental role. Tgj1 is a type I Hsp40 of T. gondii, an ortholog of the DNAJA1 group, which is essential during the tachyzoite lytic cycle. Tgj1 consists of a J-domain, ZFD, and DNAJ_C domains with a CRQQ C-terminal motif, which is usually prone to lipidation. Tgj1 presented a mostly cytosolic subcellular localization overlapping partially with endoplasmic reticulum. Protein–protein Interaction (PPI) analysis showed that Tgj1 could be implicated in various biological pathways, mainly translation, protein folding, energy metabolism, membrane transport and protein translocation, invasion/pathogenesis, cell signaling, chromatin and transcription regulation, and cell redox homeostasis among others. The combination of Tgj1 and Hsp90 PPIs retrieved only 70 interactors linked to the Tgj1-Hsp90 axis, suggesting that Tgj1 would present specific functions in addition to those of the Hsp70/Hsp90 cycle, standing out invasion/pathogenesis, cell shape motility, and energy pathway. Within the Hsp70/Hsp90 cycle, translation-associated pathways, cell redox homeostasis, and protein folding were highly enriched in the Tgj1-Hsp90 axis. In conclusion, Tgj1 would interact with a wide range of proteins from different biological pathways, which could suggest a relevant role in them.Fil: Múnera López, Jonathan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Alonso, Andrés Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Figueras López, María Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Saldarriaga Cartagena, Ana María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Hortua Triana, Miryam A.. University of Georgia; Estados UnidosFil: Diambra, Luis Anibal. Centro Regional de Estudios Genomicos (creg) ; Facultad de Cs.exactas ; Universidad Nacional de la Plata; . Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Vanagas, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Deng, Bin. University of Vermont; Estados UnidosFil: Moreno, Silvia N. J.. University of Georgia; Estados UnidosFil: Ángel, Sergio Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentin