Molecular Recognition of Canonical and Deaminated Bases by P. abyssi Family B DNA Polymerase

Euryarchaeal polymerase B can recognize deaminated bases on the template strand, effectively stalling the replication fork 4 nt downstream the modified base. Using Pyrococcus abyssi DNA B family polymerase (PabPolB), we investigated the discrimination between deaminated and natural nucleotide(s) by primer extension assays, electrophoretic mobility shift assays, and X-ray crystallography. Structures of complexes between the protein and DNA duplexes with either a dU or a dH in position + 4 were solved at 2.3 Å and 2.9 Å resolution, respectively. The PabPolB is found in the editing mode. A new metal binding site has been uncovered below the base-checking cavity where the + 4 base is flipped out; it is fully hydrated in an octahedral fashion and helps guide the strongly kinked template strand. Four other crystal structures with each of the canonical bases were also solved in the editing mode, and the presence of three nucleotides in the exonuclease site caused a shift in the coordination state of its metal A from octahedral to tetrahedral. Surprisingly, we find that all canonical bases also enter the base-checking pocket with very small differences in the binding geometry and in the calculated binding free energy compared to deaminated ones. To explain how this can lead to stalling of the replication fork, the full catalytic pathway and its branches must be taken into account, during which the base is checked several times. Our results strongly suggest a switch from elongation to editing modes right after nucleotide insertion when the modified base is at position + 5.     

A tetravalent dengue nanoparticle stimulates antibody production in mice

Background

Dengue is a major public health problem worldwide, especially in the tropical and subtropical regions of the world. Infection with a single Dengue virus (DENV) serotype causes a mild, self-limiting febrile illness called dengue fever. However, a subset of patients experiencing secondary infection with a different serotype progresses to the severe form of the disease, dengue hemorrhagic fever/dengue shock syndrome. Currently, there are no licensed vaccines or antiviral drugs to prevent or treat dengue infections. Biodegradable nanoparticles coated with proteins represent a promising method for in vivo delivery of vaccines.

Findings

Here, we used a murine model to evaluate the IgG production after administration of inactivated DENV corresponding to all four serotypes adsorbed to bovine serum albumin nanoparticles. This formulation induced a production of anti-DENV IgG antibodies (p < 0.001). However, plaque reduction neutralization assays with the four DENV serotypes revealed that these antibodies have no neutralizing activity in the dilutions tested.

Conclusions

Our results show that while the nanoparticle system induces humoral responses against DENV, further investigation with different DENV antigens will be required to improve immunogenicity, epitope specicity, and functional activity to make this platform a viable option for DENV vaccines.     

Chloroplast Activity and 3′phosphadenosine 5′phosphate Signaling Regulate Programmed Cell Death in Arabidopsis

Programmed cell death (PCD) is a crucial process both for plant development and responses to biotic and abiotic stress. There is accumulating evidence that chloroplasts may play a central role during plant PCD as for mitochondria in animal cells, but it is still unclear whether they participate in PCD onset, execution, or both. To tackle this question, we have analyzed the contribution of chloroplast function to the cell death phenotype of the myoinositol phosphate synthase1 (mips1) mutant that forms spontaneous lesions in a light-dependent manner. We show that photosynthetically active chloroplasts are required for PCD to occur in mips1, but this process is independent of the redox state of the chloroplast. Systematic genetic analyses with retrograde signaling mutants reveal that 3′-phosphoadenosine 5′-phosphate, a chloroplast retrograde signal that modulates nuclear gene expression in response to stress, can inhibit cell death and compromises plant innate immunity via inhibition of the RNA-processing 5′-3′ exoribonucleases. Our results provide evidence for the role of chloroplast-derived signal and RNA metabolism in the control of cell death and biotic stress response.Programmed cell death (PCD) is a universal process in multicellular organisms, contributing to the controlled and active degradation of the cell. In plants, PCD is required for processes as diverse as development, self-incompatibility, and stress response. One well-documented example is the induction of PCD upon pathogen attack, allowing the confinement of the infection, and resistance of the plant. The signaling events leading to the onset of PCD have been extensively studied: pathogen recognition triggers activation of mitogen-activated protein kinase cascades, as well as production of reactive oxygen species (ROS) and salicylic acid (SA), which lead to a hypersensitive response (Coll et al., 2011).From a cellular point of view, several classes of plant PCD have been described and compared with the ones found in animal cells (van Doorn, 2011). PCD is thought to have evolved independently in plants and animals, and genes underlying these mechanisms are therefore poorly conserved between the two kingdoms. However, most cellular features are conserved between plant and animal PCD that are both characterized by cell shrinkage, chromatin condensation, DNA laddering, mitochondria permeabilization, and depolarization (Dickman and Fluhr, 2013). In animal cells, mitochondria play a central role in the regulation of apoptosis (Czabotar et al., 2014; Mariño et al., 2014), and this role is likely shared between the two kingdoms (Lord and Gunawardena, 2012). That said, additional mitochondria-independent PCD pathways have clearly evolved in plants.Genetic approaches have greatly contributed to our understanding of cellular pathways governing PCD in plants. For example, the isolation of lesion mimic mutants (LMMs), in which cell death occurs spontaneously, has allowed the identification of several negative regulators of cell death (for review, see Bruggeman et al., 2015b). Interestingly, lesion formation is light dependent in several of these mutants, which include one of the best characterized LMMs—lesions simulating disease1 (lsd1; Dietrich et al., 1994). The LSD1 protein is required for plant acclimation to excess excitation energy (Mateo et al., 2004): when plants are exposed to excessive amounts of light, the redox status of the plastoquinone pool in the chloroplastic electron transfer chain is thought to influence LSD1-dependent signaling to modulate cell death (Mühlenbock et al., 2008). Additionally, we have previously identified the myoinositol phosphate synthase1 (mips1) mutant as a LMM, in which lesion formation is also light dependent (Meng et al., 2009). This mutant is deficient in the myoinositol (MI) phosphate synthase that catalyzes the first committed step of MI biosynthesis and displays pleiotropic defects such as reduced root growth, abnormal vein development, and spontaneous cell death on leaves, together with severe growth reduction after lesions begin to develop (Meng et al., 2009; Donahue et al., 2010). The light-dependent PCD in the mips1 mutant, as observed for lsd1, suggests that chloroplasts may play a role in the MI-dependent cell death regulation. Accumulating evidence suggests that chloroplasts may play a central role in PCD regulation like mitochondria in animal cells (Wang and Bayles, 2013). First, as described in the case of lsd1, excess light energy received by the chloroplast can function as a trigger for PCD. Furthermore, singlet oxygen (¹O₂), a ROS, can activate the EXECUTER1 (EX1) and EX2 proteins in the chloroplasts to initiate PCD (Lee et al., 2007). Likewise, ROS generated by chloroplasts play a major role for PCD onset during nonhost interaction between tobacco (Nicotiana tabacum) and Xanthomonas campestris (Zurbriggen et al., 2009). Finally, functional chloroplasts have also been shown to be required for PCD in cell suspensions (Gutierrez et al., 2014) and in a number of LMMs (Mateo et al., 2004; Meng et al., 2009; Bruggeman et al., 2015b). Thus, chloroplasts are now recognized as important components of plant defense response against pathogens (Stael et al., 2015) and are proposed to function with mitochondria in the execution of PCD (Van Aken and Van Breusegem, 2015). However, the exact signaling and metabolic contribution of chloroplasts to PCD remain to be elucidated. Furthermore, cross talk between chloroplasts and mitochondria does occur, such as during photorespiration (Sunil et al., 2013), but whether such communication functions sequentially or in parallel in the control of PCD remains to be determined (Van Aken and Van Breusegem, 2015).To further investigate how chloroplasts contribute to the regulation of cell death, we performed both forward and reverse genetics on the mips1 mutant. An extragenic secondary mutation in divinyl protochlorophyllide 8-vinyl reductase involved in chlorophyll biosynthesis leads to chlorophyll deficiency that abolishes the mips1 cell death phenotype, as do changes in CO₂ availability. These findings provide evidence for a link between photosynthetic activity and PCD induction in mips1. Additionally, we investigated the contribution of several retrograde signaling pathways (Chan et al., 2015) to the control of PCD in mips1. This process was independent of GENOMES UNCOUPLED (GUN) and EX signaling pathways, but we found that the SAL1-PAP_XRN retrograde signaling pathway inhibits cell death as well as basal defense reactions in Arabidopsis (Arabidopsis thaliana).     

Antithrombin III: structural and functional aspects

Antithrombin III is a plasma glycoprotein responsible for thrombin inhibition in the blood coagulation cascade. The X-ray structure of its cleaved form has been determined and refined to 3.2 A resolution. The overall topology is similar to that of alpha 1-antitrypsin, another member of the serpin (serine protease inhibitor) superfamily. The biological activity of antithrombin III is mediated by a polysaccharide, heparin. The binding site of this effector is described. A possible structural transition from the native to the cleaved structure is discussed.     

Time‐shifted reproductive behaviours among fall armyworm (Noctuidae: Spodoptera frugiperda) host strains: evidence for differing modes of inheritance

The noctuid moth Spodoptera frugiperda consists of two strains associated with different larval host plants (most notably corn and rice). These strains exhibit differential temporal patterns of female calling and copulation during scotophase, with the corn strain more active earlier in the night. We investigated strain‐specific constraints in reproductive timing, mating interactions between the two strains, and the mode of inheritance of timing of female calling, male calling, copulation and oviposition. We observed an allochronic shift of all reproductive behaviours by approximately 3 h and a parallel shift of nonreproductive locomotor activity, suggesting involvement of the circadian clock. The corn strain was more variable in the timing of calling and copulation than the rice strain. Rice strain females were more restricted in the timing of copulation than rice strain males, while such differences between the sexes were not apparent in the corn strain. There were significant interactions between the strains affecting onset times of copulation and male calling. The four investigated reproductive traits differed in their modes of inheritance: timing of female and male calling exhibited strong maternal effects, timing of copulation was controlled by a combination of maternal effects and corn strain dominant autosomal factors, and timing of oviposition was inherited in a corn strain dominant fashion. We conclude that the allochronic separation of reproduction between fall armyworm strains is asymmetric, less pronounced than previously thought, and under complex genetic control.     

Prognostic Significance of New Immunohistochemical Markers in Refractory Classical Hodgkin Lymphoma: A Study of 59 Cases

Although most classical Hodgkin lymphoma patients are cured, a significant minority fail after primary therapy and may die as result of their disease. To date, there is no consensus on biological markers that add value to usual parameters (which comprise the International Prognostic Score) used at diagnosis to predict outcome. We evaluated 59 patients (18 with primary refractory or early relapse disease and 41 responders) for bcl2, Ki67, CD20, TiA1 and c-kit expression by semi-quantitative immunohistochemical study and correlated the results with the response to treatment.The results showed that expression of bcl2 and CD20 in Hodgkin and Reed Sternberg cells, and expression of TiA1 in micro-environmental lymphocytes, and c-kit positive mast cells in microenvironment, were independent prognostic markers. These novel cHL markers could be used in association with clinical parameters to identify newly diagnosed patients with favorable or unfavorable prognosis and to better tailor treatment for different risk groups.     

Mental patients in prisons

Mental conditions usually affect cognitive, emotional and volitional aspects and functions of the personality, which are also functions of interest in law, as they are essential at the time of adjudicating guilt, labeling the accused a criminal, and proffering a sentence. A relationship between mental illness and criminality has, thus, been described and given as one of the reasons for the large number of mental patients in prisons. Whether this relationship is one of causality or one that flows through many other variables is a matter of debate, but there is no debating that prisons have become a de facto part, and an important one, of mental health systems in many countries. This paper deals with the issue of the relationship and provides estimates of prevalence of mental patients in prisons culled from many studies in different countries. It also provides some direction for the management of mental patients as they crowd correctional systems.     

Oligodeoxynucleotides covalently linked to intercalating dyes as base sequence-specific ligands. Influence of dye attachment site.

New molecules with high and specific affinity for nucleic acid base sequences have been synthesized. They involve an oligodeoxynucleotide covalently attached to an intercalating dye. Visible absorption spectroscopy and fluorescence have been used to investigate the binding of poly(rA) to octadeoxythymidylates substituted by a 9-aminoacridine derivative in different positions along the oligonucleotide chain. The 9-amino group of the acridine dye was linked through a polymethylene bridge to the 3''-phosphate, the 5''-phosphate, the fourth internucleotidic phosphate or to both the 3''- and 5''-phosphates. Different interactions of the acridine dye were exhibited by these different substituted oligodeoxynucleotides when they bind to poly(rA). The interaction was shown to be specific for adenine-containing polynucleotides. The stability of these complexes was compared with that of oligodeoxynucleotides substituted by an alkyl group on the 3''-phosphate. The increase in stability due to the presence of the intercalating dye has been determined from the comparison of melting temperatures. These results are discussed with respect to the strategy of synthesis of a new class of molecules with high affinity and high specificity for nucleic acid base sequences.