Role of EXO1 nuclease activity in genome maintenance, the immune response and tumor suppression in Exo1D173A mice

ABSTRACTDNA damage response pathways rely extensively on nuclease activity to process DNA intermediates. Exonuclease 1 (EXO1) is a pleiotropic evolutionary conserved DNA exonuclease involved in various DNA repair pathways, replication, antibody diversification, and meiosis. But, whether EXO1 facilitates these DNA metabolic processes through its enzymatic or scaffolding functions remains unclear. Here we dissect the contribution of EXO1 enzymatic versus scaffolding activity by comparing Exo1DA/DA mice expressing a proven nuclease-dead mutant form of EXO1 to entirely EXO1-deficient Exo1−/− and EXO1 wild type Exo1+/+ mice. We show that Exo1DA/DA and Exo1−/− mice are compromised in canonical DNA repair processing, suggesting that the EXO1 enzymatic role is important for error-free DNA mismatch and double-strand break repair pathways. However, in non-canonical repair pathways, EXO1 appears to have a more nuanced function. Next-generation sequencing of heavy chain V region in B cells showed the mutation spectra of Exo1DA/DA mice to be intermediate between Exo1+/+ and Exo1−/− mice, suggesting that both catalytic and scaffolding roles of EXO1 are important for somatic hypermutation. Similarly, while overall class switch recombination in Exo1DA/DA and Exo1−/− mice was comparably defective, switch-switch junction analysis suggests that EXO1 might fulfill an additional scaffolding function downstream of class switching. In contrast to Exo1−/− mice that are infertile, meiosis progressed normally in Exo1DA/DA and Exo1+/+ cohorts, indicating that a structural but not the nuclease function of EXO1 is critical for meiosis. However, both Exo1DA/DA and Exo1−/− mice displayed similar mortality and cancer predisposition profiles. Taken together, these data demonstrate that EXO1 has both scaffolding and enzymatic functions in distinct DNA repair processes and suggest a more composite and intricate role for EXO1 in DNA metabolic processes and disease.

Engaging the ‘A’ Class Antibody: Variable-Heavy (VH) region influencing IgA1&2 engagement of FcαRI and superantigen proteins G, A, and L

Interest in IgA as an alternative therapeutic and diagnostic antibody has increased over the years, yet much remains to be investigated especially given their importance in activating immune cells in blood and in mucosal immunity. Recent whole antibody-based investigations have shown significant distal effects between the variable (V) and constant (C)-regions that can be mitigated by the different hinge regions of the human IgA subtypes A1 and A2. Diving deeper into the mechanisms underlying this, systematic VH manipulations retaining the CDRs were performed on a panel of 28 IgA1s and A2s across the Trastuzumab and Pertuzumab models, revealed distal effects on FcαRI binding. Further insights from structural modelling showed these effects to also be mitigated by the differing glycosylation patterns in IgA1 and 2 to explain reversal of trends of IgA1s and 2s effected by slight changes in the CDRs. IgAs bound at the Fc showed similar trends but magnitudes better binding to Her2 with that bound by ppL, showing that ppL can sterically hinder Her2 antigen binding. Contrary to canonical knowledge, we found strong evidence of IgAs binding SpG that was narrowed to be at the CH2-3 region, and that the likely binding with SpA was beyond VH3 FWR and most likely at the CH1. VH1 was found to be the most suitable framework (FWRs) for CDR-grafting for both IgA1 and 2. With relevance to interactions with the microbiome at mucosal surfaces, mechanistic insight of how these IgAs can interact bacterial superantigens proteins G, A, and L are also discovered for potential future interventions.One Sentence SummaryAn insight into the mechanism of distal V-region effects on FCAR and superantigens proteins G, A, and L by both IgA1 and A2.

STAT3-Specific Single Domain Nanobody Inhibits Expansion of Pathogenic Th17 Responses and Suppresses Uveitis in Mice

STAT3 activates transcription of genes that regulate cell growth, differentiation, and survival of mammalian cells. Genetic deletion of Stat3 in T cells has been shown to abrogate Th17 differentiation, suggesting that STAT3 is a potential therapeutic target for Th17-mediated diseases. However, a major impediment to therapeutic targeting of intracellular proteins such as STAT3 is the lack of efficient methods for delivering STAT3 inhibitors into cells. In this study, we developed a novel antibody (SBT-100) comprised of the variable (V) region of a STAT3-specific heavy chain molecule and demonstrate that this 15 kDa STAT3-specific nanobody enters human and mouse cells, and induced suppression of STAT3 activation and lymphocyte proliferation in a concentration-dependent manner. To investigate whether SBT-100 would be effective in suppressing inflammation in vivo, we induced experimental autoimmune uveitis (EAU) in C57BL/6J mice by active immunization with peptide from the ocular autoantigen, interphotoreceptor retinoid binding protein (IRBP651-670). Analysis of the retina by fundoscopy, histological examination, or optical coherence tomography showed that treatment of the mice with SBT-100 suppressed uveitis by inhibiting expansion of pathogenic Th17 cells that mediate EAU. Electroretinographic (ERG) recordings of dark and light adapted a- and b-waves showed that SBT-100 treatment rescued mice from developing significant visual impairment observed in untreated EAU mice. Adoptive transfer of activated IRBP-specific T cells from untreated EAU mice induced EAU, while EAU was significantly attenuated in mice that received IRBP-specific T cells from SBT-100 treated mice. Taken together, these results demonstrate efficacy of SBT-100 in mice and suggests its therapeutic potential for human autoimmune diseases.

Role of Dot1L and H3K79 methylation in regulating somatic hypermutation of immunoglobulin genes

Somatic hypermutation (SHM) and class-switch recombination (CSR) of the immunoglobulin (Ig) genes allow B cells to make antibodies that protect us against a wide variety of pathogens. SHM is mediated by activation-induced deaminase (AID), occurs at a million times higher frequency than other mutations in the mammalian genome, and is largely restricted to the variable (V) and switch (S) regions of Ig genes. Using the Ramos human Burkitt’s lymphoma cell line, we find that H3K79me2/3 and its methyltransferase Dot1L are more abundant on the V region than on the constant (C) region, which does not undergo mutation. In primary naïve mouse B cells examined ex vivo, the H3K79me2/3 modification appears constitutively in the donor Sμ and is inducible in the recipient Sγ1 upon CSR stimulation. Knockout and inhibition of Dot1L in Ramos cells significantly reduces V region mutation and the abundance of H3K79me2/3 on the V region and is associated with a decrease of polymerase II (Pol II) and its S2 phosphorylated form at the IgH locus. Knockout of Dot1L also decreases the abundance of BRD4 and CDK9 (a subunit of the P-TEFb complex) on the V region, and this is accompanied by decreased nascent transcripts throughout the IgH gene. Treatment with JQ1 (inhibitor of BRD4) or DRB (inhibitor of CDK9) decreases SHM and the abundance of Pol II S2P at the IgH locus. Since all these factors play a role in transcription elongation, our studies reinforce the idea that the chromatin context and dynamics of transcription are critical for SHM.

Evaluación de raleos en monte bajo de Eucalyptus globulus (Labill) V Región

Se evalúa el comportamiento de un rodal de monte bajo de Eucalyptus globulus sometido a dos prácticas de raleo, y que está ubicado en la comuna de Santo Domingo, Región de Valparaíso. Para cumplir con este objetivo se instaló en 1982 un dispositivo experimental en bloques al azar, con los siguientes tratamientos: testigo, raleo por lo bajo y raleo por lo alto. En ambas intervenciones se extrajo el 30% del número de árboles. Se concluye, bajo las condiciones de esta experiencia, que el área basal ideal residual está próxima a 9,9 m²/ha y el área basal crítica cercana a 7,2 m²/ha o menor. La mayor producción en términos de volumen, biomasa total, astillas para leña y aceites esenciales por unidad de superficie, se presenta en los tratamientos testigo y raleo por lo bajo.

Characterization of DNA G-Quadruplex Structures in Human Immunoglobulin Heavy Variable (IGHV) Genes

Activation-induced deaminase (AID) is a key enzyme involved in antibody diversification by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) of the Immunoglobulin (Ig) loci. AID preferentially targets WRC (W=A/T, R=A/G) hotspot motifs and avoids SYC (S=C/G, Y=C/T) coldspots. G-quadruplex (G4) structures are four-stranded DNA secondary structures with key functions in transcription, translation and replication. In vitro studies have shown G4s to form and bind AID in Ig switch (S) regions. Alterations in the gene encoding AID can further disrupt AID-G4 binding and reduce CSR in vivo. However, it is still unclear whether G4s form in the variable (V) region, or how they may affect SHM. To assess the possibility of G4 formation in human V regions, we analyzed germline human Ig heavy chain V (IGHV) sequences, using a pre-trained deep learning model that predicts G4 potential. This revealed that many genes from the IGHV3 and IGHV4 families are predicted to have high G4 potential in the top and bottom strand, respectively. Different IGHV alleles also showed variability in G4 potential. Using a high-resolution (G4-seq) dataset of biochemically confirmed potential G4s in IGHV genes, we validated our computational predictions. G4-seq also revealed variation between S and V regions in the distribution of potential G4s, with the V region having overall reduced G4 abundance compared to the S region. The density of AGCT motifs, where two AGC hotspots overlap on both strands, was roughly 2.6-fold greater in the V region than the Constant (C) region, which does not mutate despite having predicted G4s at similar levels. However, AGCT motifs in both V and C regions were less abundant than in S regions. In silico mutagenesis experiments showed that G4 potentials were generally robust to mutation, although large deviations from germline states were found, mostly in framework regions. G4 potential is also associated with higher mutability of certain WRC hotspots on the same strand. In addition, CCC coldspots opposite a predicted G4 were shown to be targeted significantly more for mutation. Our overall assessment reveals plausible evidence of functional G4s forming in the Ig V region.

Expansion of an Unusual Virtual Memory CD8+ Subpopulation Bearing Vα3.2 TCR in Themis-Deficient Mice

Deletion of the gene for Themis affects T cell selection in the thymus, which would be expected to affect the TCR repertoire. We found an increased proportion of cells expressing Vα3.2 (TRAV9N-3) in the peripheral CD8+ T cell population in mice with germline Themis deficiency. Analysis of the TCRα repertoire indicated it was generally reduced in diversity in the absence of Themis, whereas the diversity of sequences using the TRAV9N-3 V-region element was increased. In wild type mice, Vα3.2+ cells showed higher CD5, CD6 and CD44 expression than non-Vα3-expressing cells, and this was more marked in cells from Themis-deficient mice. This suggested a virtual memory phenotype, as well as a stronger response to self-pMHC. The Vα3.2+ cells responded more strongly to IL-15, as well as showing bystander effector capability in a Listeria infection. Thus, the unusually large population of Vα3.2+ CD8+ T cells found in the periphery of Themis-deficient mice reflects not only altered thymic selection, but also allowed identification of a subset of bystander-competent cells that are also present in wild-type mice.

Caracterización de personas con enfermedad de chagas en el Centro de Salud Familiar Valle Los Libertadores y establecimientos dependientes en Putaendo

Introducción: La enfermedad de Chagas es una patología parasitaria crónica y sistémica; Chile tiene una incidencia de 11,6 por 100.000 habitantes, presentándose mayoritariamente en zonas rurales y precordilleranas. Putaendo es una comuna de la V región ubicada en una zona endémica, donde se desconoce la realidad epidemiológica actual de la enfermedad. El objetivo del estudio es caracterizar a los pacientes con Chagas inscritos en el Centro de Salud Familiar (CESFAM) Valle Los Libertadores en Putaendo. Material y Métodos: El estudio tuvo un diseño transversal. Se incluyeron todos los registros de pacientes con diagnóstico de enfermedad de Chagas hasta el 30 de junio de año 2019, excluyéndose a pacientes fallecidos. Los datos fueron obtenidos de una base de datos anonimizada de dicho centro. Resultados: Se encontró una prevalencia de Chagas de 108 enfermos por 100.000 personas registradas vivas; 98 de sexo femenino y 56 masculino, con mayor frecuencia entre los 50-59 años. Respecto al Fondo Nacional de Salud (FONASA), los tramos A y B (las personas con menores ingresos) concentran la mayor cantidad de pacientes con Chagas (87%). Discusión: Si bien se pudo caracterizar a la enfermedad, siendo más prevalente en personas adultas, mujeres y de bajos recursos, se requieren más estudios para objetivar el real estado de salud de estos pacientes.

Structural and Electrochemical Properties of the High Ni Content Spinel LiNiMnO4

This work presents a contribution to the study of a new Ni-rich spinel cathode material, LiNiMnO4, for Li-ion batteries operating in the 5-V region. The LiNiMnO4 compound was synthesized by a sol-gel method assisted by ethylene diamine tetra-acetic acid (EDTA) as a chelator. Structural analyses carried out by Rietveld refinements and Raman spectroscopy, selected area electron diffraction (SAED) and X-ray photoelectron (XPS) spectroscopy reveal that the product is a composite (LNM@NMO), including non-stoichiometric LiNiMnO4-δ spinel and a secondary Ni6MnO8 cubic phase. Cyclic voltammetry and galvanostatic charge-discharge profiles show similar features to those of LiNi0.5Mn1.5O4 bare. A comparison of the electrochemical performances of 4-V spinel LiMn2O4 and 5-V spinel LiNi0.5Mn1.5O4 with those of LNM@NMO composite demonstrates the long-term cycling stability of this new Ni-rich spinel cathode. Due to the presence of the secondary phase, the LNM@NMO electrode exhibits an initial specific capacity as low as 57 mAh g−1 but shows an excellent electrochemical stability at 1C rate for 1000 cycles with a capacity decay of 2.7 × 10−3 mAh g−1 per cycle.

Mink SLAM V-Region V74I Substitutions Contribute to the Formation of Syncytia Induced by Canine Distemper Virus

The Signal lymphatic activation molecule (SLAM, also known as CD150) as the cellular receptor of canine distemper virus (CDV) plays an important role in the virus-host interaction. However, it is still unknown whether amino acid differences in the SLAM variable (V) region affect the formation of syncytia. Here, using raccoon dog SLAM (rSLAM) and mink SLAM (mSLAM), we performed SLAM-V homologous modeling, site-directed mutagenesis, and surface expression analysis, as well as a cell fusion assay, to study the interaction between SLAM and CDV. More specifically, our investigation focused on two amino acid residues (74 and 129) of SLAM, previously predicted to play a relevant role in receptor-ligand interaction. Our results indicated that only residues at position 60, 74, and 129 were different between rSLAM and mSLAM among the 29 amino acids that might interact with CDV H, and residues 74 and 129 were located in the interface region interacting with CDV H. The amino acid substitution at the positions of 74 have a significant effect on the expression of mSLAM. The SLAM-V74I mutation in mink significantly improved the cell fusion efficiency of CDV. In contrast, the SLAM-I74V mutation in the raccoon dog significantly decreased cell fusion efficiency. We conclude that residue 74 of SLAM plays an important role during the the formation of syncytia. Only when implementing CDV infection analysis, the rSLAM-Q129R can significantly decreased the mean number of syncytia, but the mSLAM-R129Q can't. Additionally, residue 60 show variability between rSLAM and mSLAM. We believe that our study makes a significant contribution to the literature because we provide molecular data, partially accounting for the differences in host membrane and virus interaction laying the foundation for further molecular work.