Caught bending the A-rule: crystal structures of translesion DNA synthesis with a non-natural nucleotide

Damage to DNA involving excision of the nucleobase at the N-glycosidic bond forms abasic sites. If a nucleotide becomes incorporated opposite an unrepaired abasic site during DNA synthesis, most B family polymerases obey the A-rule and preferentially incorporate dAMP without instruction from the template. In addition to being potentially mutagenic, abasic sites provide strong blocks to DNA synthesis. A previous crystal structure of an exonuclease deficient variant of the replicative B family DNA polymerase from bacteriophage RB69 (RB69 gp43 exo-) illustrated these properties, showing that the polymerase failed to translocate the DNA following insertion of dAMP opposite an abasic site. We examine four new structures depicting several steps of translesion DNA synthesis by RB69 gp43 exo-, employing a non-natural purine triphosphate analogue, 5-nitro-1-indolyl-2'-deoxyriboside-5'-triphosphate (5-NITP), that is incorporated more efficiently than dAMP opposite abasic sites. Our structures indicate that a dipole-induced dipole stacking interaction between the 5-nitro group and base 3' to the templating lesion explains the enhanced kinetics of 5-NITP. As with dAMP, the DNA fails to translocate following insertion of 5-NIMP, although distortions at the nascent primer terminus contribute less than previously thought in inducing the stall, given that 5-NIMP preserves relatively undistorted geometry at the insertion site following phosphoryl transfer. An open ternary configuration, novel in B family polymerases, reveals an initial template independent binding of 5-NITP adjacent to the active site of the open polymerase, suggesting that closure of the fingers domain shuttles the nucleotide to the active site while testing the substrate against the template.     

Effect of salt stress on physiological and antioxidative responses in two species of <Emphasis Type="Italic">Salicornia</Emphasis> (<Emphasis Type="Italic">S. persica</Emphasis> and <Emphasis Type="Italic">S. europaea</Emphasis>)

The effects of salt stress on growth parameters, free proline content, ion accumulation, lipid peroxidation, and several antioxidative enzymes activities were investigated in S. persica and S. europaea. The seedlings were grown for 2 months in half-strength Hoagland solution and treated with different concentrations of NaCl (0, 85, 170, 340, and 510 mM) for 21 days. The fresh and dry weights of both species increased significantly at 85 and 170 mM NaCl and decreased at higher concentrations. Salinity increased proline content in both the species as compared to that of control. Sodium (Na⁺) content in roots and shoots increased, whereas K⁺ and P_i content in both organs decreased. At all NaCl concentrations, the total amounts of Na⁺ and K⁺ were higher in shoots than in roots. Malondialdehyde (MDA) content declined at moderate NaCl concentrations (85 and 170 mM) and increased at higher levels. With increased salinity, superoxide dismutase (SOD), catalase (CAT), and guaiacol peroxidase (GPX) activities also increased gradually in both species. In addition, it seems that GPX, CAT, and SOD activities play an essential protective role in the scavenging reactive oxygen species (ROS) in both species. Native polyacrylamide gel electrophoresis (PAGE) indicated different isoform profiles between S. persica and S. europaea concerning antioxidant enzymes. These results showed that S. persica exhibits a better protection mechanism against oxidative damage and it is more salt-tolerant than S. europaea possibly by maintaining and/or increasing growth parameters, ion accumulation, and antioxidant enzyme activities.     

Adjuvant activity of GP96 C-terminal domain towards Her2/neu DNA vaccine is fusion direction-dependent

The Her2 is one of tumor-associated antigens (TAA), regarded as an ideal target of immunotherapy. DNA encoding full-length or truncated rat Her2/neu have shown protective and therapeutics potentials against Her2/neu-expressing mammary tumors. However, the efficacy of active vaccination is limited since Her2 is a self-tolerated antigen. Hence, new strategies are required to enhance both the quality and quantity of the immune response against Her2-expressing tumors. Many studies have used Her2/neu gene with cytokine or other molecules involved in regulation of immune response to enhance the potency of Her2/neu DNA vaccines. Some studies fused adjuvant gene to C-terminal domain of Her2/neu gene, while others fused the adjuvant gene N-terminally to Her2/neu gene, but no comparison on how direction of fusion could affect efficiency of DNA vaccine has ever been made. Based on previous reports demonstrating potent adjuvant activity of gp96 C-terminal domain, we chose it as adjuvant. The aim of this study was to investigate if direction of fusion could affect adjuvant activity of gp96 C-terminal domain or potency of Her2/neu DNA vaccination. To do so, we fused C-terminal domain of gp96 to downstream or C-terminal end of transmembrane and extracellular domain (TM+ECD) of rat Her2/neu and resultant immune response to DNA vaccination was evaluated. The results were compared with that of N-terminally fusion of gp96 C-terminal domain to TM+ECD of rat Her2/neu. Our results revealed that adjuvant activity of gp96 C-terminal domain is enhanced when fused N-terminally to TM+ECD of rat Her2/neu. It suggests that adjuvant activity of gp96 C-terminal domain towards Her2/neu is fusion direction-dependent.     

The Human Leukocyte Antigen–presented Ligandome of B Lymphocytes

Peptides presented by human leukocyte antigen (HLA) molecules on the cell surface play a crucial role in adaptive immunology, mediating the communication between T cells and antigen presenting cells. Knowledge of these peptides is of pivotal importance in fundamental studies of T cell action and in cellular immunotherapy and transplantation. In this paper we present the in-depth identification and relative quantification of 14,500 peptide ligands constituting the HLA ligandome of B cells. This large number of identified ligands provides general insight into the presented peptide repertoire and antigen presentation. Our uniquely large set of HLA ligands allowed us to characterize in detail the peptides constituting the ligandome in terms of relative abundance, peptide length distribution, physicochemical properties, binding affinity to the HLA molecule, and presence of post-translational modifications. The presented B-lymphocyte ligandome is shown to be a rich source of information by the presence of minor histocompatibility antigens, virus-derived epitopes, and post-translationally modified HLA ligands, and it can be a good starting point for solving a wealth of specific immunological questions. These HLA ligands can form the basis for reversed immunology approaches to identify T cell epitopes based not on in silico predictions but on the bona fide eluted HLA ligandome.Peptides presented by human leukocyte antigen (HLA)¹ molecules on the cell surface play a crucial role in immunology and mediate the communication between T cells and antigen presenting cells. Knowledge of these peptides is of pivotal importance in fundamental studies of T cell action, the design of T-cell-mediated therapies such as tumor immunotherapy (1), and the treatment of hematological malignancies through a combination of hematopoietic stem cell transplantation and donor lymphocyte infusion (2). In addition, T cells can play an important role in organ rejection following transplantation.The presented HLA class I ligands are the products of the intracellular processing machinery, with its continuous cycle of protein synthesis and degradation (3). Much is known about the proteins involved in antigen processing, but high fidelity ligand/epitope predictions are at present not possible. The discovery of additional involved enzymes (3, 4) and the exciting discovery of peptide splicing (5) have shown that antigen processing is even more complex than was previously thought. Moreover, gene expression studies have shown many nonstandard, unexpected protein products, including the production of antigens derived from aberrant protein fragments as a result of expression in alternative reading frames (6). Several studies report the identification of HLA ligands (7–10). Many results have been collected and discussed in a recent review on the large-scale analysis of HLA class I ligands (11). Collectively, these reports illustrate the need for in-depth elucidation of the HLA ligandome.Elucidation of T cell epitopes has traditionally been achieved with the use of a forward immunological approach, as pioneered by Hunt and coworkers (12, 13). In this approach, the cognate peptide of T cells with the appropriate activity profile is elucidated via repeated rounds of chromatographic separation in combination with T cell recognition assays. Because T cells are not always available from the start, reverse immunological approaches (14–17) have been developed to predict T cell epitopes through a combination of bioinformatics and in vitro proteasome digests. Predicted epitopes are synthesized and tested for their capability to activate T cells. The main disadvantage of this approach is that less than 0.1% of the peptides that survive intracellular processing are presented on HLA class I molecules (3).Therefore, we developed a large-scale peptidomics approach that is a reverse immunology approach based not on algorithms but on the bona fide eluted ligandome, which means that the identified peptides are known to have survived processing and are bona fide HLA ligands. Once the ligandome has been identified as comprehensively as possible, T cells can subsequently be selected on the basis of the immunological question at hand, as will be illustrated in a separate paper.² The development of MHC exchange tetramers for finding relevant T cell epitopes is instrumental to this approach (18, 19).To improve ligandome coverage, we applied and compared three off-line first dimension separation techniques, followed by on-line nano-HPLC-tandem MS.The tandem mass spectra were interrogated by being matched against the International Protein Index (IPI) human database (20). In a second step, post-translation modifications (phosphorylation, cysteinylation) were allowed in the database search. In a third step, the tandem mass spectra were matched against a newly in-house developed database for the optimal identification of polymorphic ligands to find potential minor histocompatibility antigens (21). This led to the identification of ∼14,000 HLA class I ligands, the majority of which also were relatively quantitated. Next, we analyzed the peptides constituting our ligandome in as much detail as possible to confirm the correct identification of the vast majority of the ligands. We achieved this through a combination of several physicochemical and biological checks and comparison with existing ligand and epitope databases.Finally, as an additional quality check, we illustrated the functional relevance of the ligandome through the identification of both previously known and new minor histocompatibility antigens, virus-derived epitopes, and post-translationally modified HLA ligands (phosphorylated ligands and cysteinylated ligands) (22–24). This is the largest ligandome reported to date, and it allows general insight into the presented peptide repertoire. This study supports the building of the “immunopeptidome” as has recently been suggested (25). A proteomics approach can be used as a starting point for contributions to immunology by providing a peptidome landscape in many immunological studies, both fundamental and applied.     

Trypanosome Letm1 Protein Is Essential for Mitochondrial Potassium Homeostasis

Letm1 is a conserved protein in eukaryotes bearing energized mitochondria. Hemizygous deletion of its gene has been implicated in symptoms of the human disease Wolf-Hirschhorn syndrome. Studies almost exclusively performed in opisthokonts have attributed several roles to Letm1, including maintaining mitochondrial morphology, mediating either calcium or potassium/proton antiport, and facilitating mitochondrial translation. We address the ancestral function of Letm1 in the highly diverged protist and significant pathogen, Trypanosoma brucei. We demonstrate that Letm1 is involved in maintaining mitochondrial volume via potassium/proton exchange across the inner membrane. This role is essential in the vector-dwelling procyclic and mammal-infecting bloodstream stages as well as in Trypanosoma brucei evansi, a form of the latter stage lacking an organellar genome. In the pathogenic bloodstream stage, the mitochondrion consumes ATP to maintain an energized state, whereas that of T. brucei evansi also lacks a conventional proton-driven membrane potential. Thus, Letm1 performs its function in different physiological states, suggesting that ion homeostasis is among the few characterized essential pathways of the mitochondrion at this T. brucei life stage. Interestingly, Letm1 depletion in the procyclic stage can be complemented by exogenous expression of its human counterpart, highlighting the conservation of protein function between highly divergent species. Furthermore, although mitochondrial translation is affected upon Letm1 ablation, it is an indirect consequence of K⁺ accumulation in the matrix.     

Length‐weight relationships of three fish species from Jask mangrove protected area in northern coastline of Gulf of Oman (Hormozgan,Iran): Liza klunzingeri (Day, 1888), Cociella crocodilus (Cuvier, 1829) and Platycephalus indicus (Linnaeus, 1758)

This study presents length‐weight relationships (LWRs) of Liza klunzingeri (Day, 1888), Cociella crocodilus (Cuvier, 1829), Platycephalus indicus (Linnaeus, 1758) belonging to two Families (Mugilidae and Platycephalidae) from northern coastline of Gulf of Oman (Hormozgan province). Samples were collected by artisanal trawl and beach seine (both with 11 mm effective mesh size) monthly during 2010 to 2015. The presented models were highly significant (p < 0.01) with a reliable coefficient of determination (R² > 0.90) that provides a reliable basic information for ichthyologists and fisheries scientists.     

Insecticidal Potential of α-Pinene and β-Caryophyllene against <i>Myzus persicae</i> and Their Impacts on Gene Expression

Myzus persicae (M. persicae) is now considered a threat to agricultural crops due to economic losses. Numerous synthetic insecticides applied every year against M. persicae, are reported to be unsafe for environment, humans, and beneficial insects. Furthermore, several species of Myzus have been found to develop resistance due to over application of these insecticides. Therefore, it is required to find some novel insecticide that would be safe for the environment as well as for humans. In the current study, two major pure constituents α-pinene and β-caryophyllene were evaluated for their insecticidal potential against M. persicae using a fumigant toxicity assay. Furthermore, impact of α-pinene and β-caryophyllene on expression of five different genes, e.g., HSP 60, FPPS I, OSD, TOL and ANT responsible for reproduction, dispersion, and growth of M. persicae has also been investigated. To perform fumigant toxicity assay, five different concentrations (3.5, 4, 4.5, 5 and 6 μL L⁻¹) of α-pinene and β-caryophyllene were prepared. Lethal concentration (LC) was calculated, and gene expression studies were executed through qRT PCR at LC₃₀ of α-pinene and β-caryophyllene. Both constituents demonstrated excellent fumigant toxicity effects against M. persicae at all five concentrations. However, α-pinene shows significantly better results (98%) as compared to β-caryophyllene (80%) after 72 h at 6 μL L⁻¹ of dose. The highest upregulation in expression was demonstrated at LC₃₀ dose of α-pinene in five in three out of five genes understudy (TOL, ANT, and FPPS I). Conversely, two genes HSP 60 and OSD demonstrated downregulation at LC₃₀ dose of β-caryophyllene. Conclusively, our results highlighted the promising insecticidal potential of both compounds α-pinene and β-caryophylleneby interfering with the reproduction and development related processes in M. persicae, allowing us to recommend the phytoconstituents under investigation as an ecofriendly alternative to synthetic insecticides.     

Recent trends using natural polymeric nanofibers as supports for enzyme immobilization and catalysis

All the disciplines of science, especially biotechnology, have given continuous attention to the area of enzyme immobilization. However, the structural support made by material science intervention determines the performance of immobilized enzymes. Studies have proven that nanostructured supports can maintain better catalytic performance and improve immobilization efficiency. The recent trends in the application of nanofibers using natural polymers for enzyme immobilization have been addressed in this review article. A comprehensive survey about the immobilization strategies and their characteristics are highlighted. The natural polymers, e.g., chitin, chitosan, silk fibroin, gelatin, cellulose, and their blends with other synthetic polymers capable of immobilizing enzymes in their 1D nanofibrous form, are discussed. The multiple applications of enzymes immobilized on nanofibers in biocatalysis, biosensors, biofuels, antifouling, regenerative medicine, biomolecule degradation, etc.; some of these are discussed in this review article.     

Synthesis of sulfadiazinyl acyl/aryl thiourea derivatives as calf intestinal alkaline phosphatase inhibitors,pharmacokinetic properties,lead optimization,Lineweaver-Burk plot evaluation and binding analysis

To seek the new medicinal potential of sulfadiazine drug, the free amino group of sulfadiazine was exploited to obtain acyl/aryl thioureas using simple and straightforward protocol. Acyl/aryl thioureas are well recognized bioactive pharmacophore containing moieties. A new series (4a–4j) of sulfadiazine derived acyl/aryl thioureas was synthesized and characterized through spectroscopic and elemental analysis. The synthesized derivatives 4a–4j were subjected to calf intestinal alkaline phosphatase (CIAP) activity. The derivative 4a–4j showed better inhibition potential compared to standard monopotassium phosphate (MKP). The compound 4c exhibited higher potential in the series with IC₅₀ 0.251?±?0.012?µM (standard KH₂PO₄ 4.317?±?0.201?µM). Lineweaver-Burk plots revealed that most potent derivative 4c inhibition CIAP via mixed type pathway. Pharmacological investigations showed that synthesized compounds 4a–4j obey Lipinsk’s rule. ADMET parameters evaluation predicted that these molecule show significant lead like properties with minimum possible toxicity and can serve as templates in drug designing. The synthetic compounds show none mutagenic and irritant behavior. Molecular docking analysis showed that compound 4c interacts with Asp273, His317 and Arg166 amino acid residues.     

Oligomeric forms of insulin amyloid aggregation disrupt outgrowth and complexity of neuron-like PC12 cells

Formation of protein amyloid fibrils consists of a series of intermediates including oligomeric aggregates, proto-fibrillar structures, and finally mature fibrils. Recent studies show higher toxicity for oligomeric and proto-fibrillar intermediates of protein relative to their mature fibrils. Here the kinetic of the insulin amyloid fibrillation was evaluated using a variety of techniques including ThT fluorescence, Congo red absorbance, circular dichroism, and atomic force microscopy (AFM). The solution surface tension changes were attributed to hydrophobic changes in insulin structure and were detected by Du Noüy Ring method. Determination of the surface tension of insulin oligomeric, proto-fibrillar and fibrillar forms indicated that the hydrophobicity of solution is enhanced by the formation of the oligomeric forms of insulin compared to other forms. In order to investigate the toxicity of the different forms of insulin we monitored morphological alterations of the differentiated neuron-like PC12 cells following incubation with native, oligomeric aggregates, proto-fibrillar, and fibrillar forms of insulin. The cell body area, average neurite length, neurite width, number of primary neurites, and percent of bipolar cells and node/primary neurite ratios were used to assess the growth and complexity of PC12 cells exposed to different forms of insulin. We observed that the oligomeric form of insulin impaired the growth and complexity of PC12 cells compared to other forms. Together our data suggest that the lower surface tension of oligomers and their perturbation affects the morphology of PC12 cells, mainly due to their enhanced hydrophobicity and detergent-like structures.