Senescent vs. non-senescent cells in the human annulus in vivo: Cell harvest with laser capture microdissection and gene expression studies with microarray analysis

Background  

Senescent cells are well-recognized in the aging/degenerating human disc. Senescent cells are viable, cannot divide, remain metabolically active and accumulate within the disc over time. Molecular analysis of senescent cells in tissue offers a special challenge since there are no cell surface markers for senescence which would let one use fluorescence-activated cell sorting as a method for separating out senescent cells.     

A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and the associated proteins (Cas) comprise a system of adaptive immunity against viruses and plasmids in prokaryotes. Cas1 is a CRISPR-associated protein that is common to all CRISPR-containing prokaryotes but its function remains obscure. Here we show that the purified Cas1 protein of Escherichia coli (YgbT) exhibits nuclease activity against single-stranded and branched DNAs including Holliday junctions, replication forks and 5'-flaps. The crystal structure of YgbT and site-directed mutagenesis have revealed the potential active site. Genome-wide screens show that YgbT physically and genetically interacts with key components of DNA repair systems, including recB, recC and ruvB. Consistent with these findings, the ygbT deletion strain showed increased sensitivity to DNA damage and impaired chromosomal segregation. Similar phenotypes were observed in strains with deletion of CRISPR clusters, suggesting that the function of YgbT in repair involves interaction with the CRISPRs. These results show that YgbT belongs to a novel, structurally distinct family of nucleases acting on branched DNAs and suggest that, in addition to antiviral immunity, at least some components of the CRISPR-Cas system have a function in DNA repair.     

Genetic biodiversity in the Baltic Sea: species-specific patterns challenge management

Information on spatial and temporal patterns of genetic diversity is a prerequisite to understanding the demography of populations, and is fundamental to successful management and conservation of species. In the sea, it has been observed that oceanographic and other physical forces can constitute barriers to gene flow that may result in similar population genetic structures in different species. Such similarities among species would greatly simplify management of genetic biodiversity. Here, we tested for shared genetic patterns in a complex marine area, the Baltic Sea. We assessed spatial patterns of intraspecific genetic diversity and differentiation in seven ecologically important species of the Baltic ecosystem—Atlantic herring (Clupea harengus), northern pike (Esox lucius), European whitefish (Coregonus lavaretus), three-spined stickleback (Gasterosteus aculeatus), nine-spined stickleback (Pungitius pungitius), blue mussel (Mytilus spp.), and bladderwrack (Fucus vesiculosus). We used nuclear genetic data of putatively neutral microsatellite and SNP loci from samples collected from seven regions throughout the Baltic Sea, and reference samples from North Atlantic areas. Overall, patterns of genetic diversity and differentiation among sampling regions were unique for each species, although all six species with Atlantic samples indicated strong resistence to Atlantic-Baltic gene-flow. Major genetic barriers were not shared among species within the Baltic Sea; most species show genetic heterogeneity, but significant isolation by distance was only detected in pike and whitefish. These species-specific patterns of genetic structure preclude generalizations and emphasize the need to undertake genetic surveys for species separately, and to design management plans taking into consideration the specific structures of each species.     

Neuroimmunomodulative properties of dipeptidyl peptidase IV/CD26 in a TNBS-induced model of colitis in mice

Causal connections between dipeptidyl peptidase IV, also known as CD26 molecule (DPP IV/CD26) and inflammatory bowel disease (IBD) have been shown, but mechanisms of these interactions are unclear. Our hypothesis was that DPP IV/CD26 could affect the neuroimmune response during inflammatory events. Therefore, we aimed to evaluate its possible role and the relevance of the gut-brain axis in a model of IBD in mice. Trinitrobenzenesulfonic acid-induced (TNBS) colitis was induced in CD26-deficient (CD26(-/-) ) and wild-type (C57BL/6) mice. Pathohistological and histomorphometrical measurements were done. Concentrations and protein expressions of DPP IV/CD26 substrates neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) were determined. Concentrations of IL-6 and IL-10 were evaluated. Investigations were conducted at systemic and local levels. Acute inflammation induced increased serum NPY concentrations in both mice strains, more enhanced in CD26(-/-) mice. Increased NPY concentrations were found in colon and brain of C57BL/6 mice, while in CD26(-/-) animals only in colon. VIP and IL-6 serum and tissue concentrations were increased in both mice strains in acute inflammation, more pronouncedly in CD26(-/-) mice. IL-10 concentrations, after a decrease in serum of both mice strains, increased promptly in CD26(-/-) mice. Decreased IL-10 concentration was found in brain of C57BL/6 mice, while it was increased in colon of CD26(-/-) mice in acute inflammation. DPP IV/CD26 deficiency affects the neuroimmune response at systemic and local levels during colitis development and resolution in mice. Inflammatory changes in the colon reflected on investigated parameters in the brain, suggesting an important role of the gut-brain axis in IBD pathogenesis.     

Factors Affecting the Tailing of Blunt End DNA with Fluorescent Pyrimidine dNTPs

The transferase activity of non-proofreading DNA polymerases is a well-known phenomenon that has been utilized in cloning and sequencing applications. The non-templated addition of modified nucleotides at DNA blunt ends is a potentially useful feature of DNA polymerases that can be used for selective transformation of DNA 3′ ends. In this paper, we characterized the tailing reaction at perfectly matched and mismatched duplex ends with Cy3- and Cy5-modified pyrimidine nucleotides. It was shown that the best DNA tailing substrate does not have a perfect Watson–Crick base pair at the end. Mismatched duplexes with a 3′ dC were the most efficient in the Taq DNA polymerase-catalysed tailing reaction with a Cy5-modified dUTP. We further demonstrated that the arrangement of the dye residue relative to the nucleobase notably affects the outcome of the tailing reaction. A comparative study of labelled deoxycytidine and deoxyuridine nucleotides showed higher efficiency for dUTP derivatives. The non-templated addition of modified nucleotides by Taq polymerase at a duplex blunt end was generally complicated by the pyrophosphorolysis and 5′ exonuclease activity of the enzyme.     

Stepwise prediction of conformational discontinuous B-cell epitopes using the Mapitope algorithm

Mapping the epitope of an antibody is of great interest, since it contributes much to our understanding of the mechanisms of molecular recognition and provides the basis for rational vaccine design. Here we present Mapitope, a computer algorithm for epitope mapping. The algorithm input is a set of affinity isolated peptides obtained by screening phage display peptide-libraries with the antibody of interest. The output is usually 1-3 epitope candidates on the surface of the atomic structure of the antigen. We have systematically tested the performance of Mapitope by assessing the effect of the algorithm parameters on the final prediction. Thus, we have examined the effect of the statistical threshold (ST) parameter, relating to the frequency distribution and enrichment of amino acid pairs from the isolated peptides and the D (distance) and E (exposure) parameters which relate to the physical parameters of the antigen. Two model systems were analyzed in which the antibody of interest had previously been co-crystallized with the antigen and thus the epitope is a given. The Mapitope algorithm successfully predicted the epitopes in both models. Accordingly, we formulated a stepwise paradigm for the prediction of discontinuous conformational epitopes using peptides obtained from screening phage display libraries. We applied this paradigm to successfully predict the epitope of the Trastuzumab antibody on the surface of the Her-2/neu receptor in a third model system.     

Specific and Selective Bacteriophages in the Fight against Multidrug-resistant Acinetobacter baumannii

Acinetobacter baumannii causes serious infections especially in immunocompromised and/or hospitalized patients. Several A. baumannii strains are multidrug resistant and infect wounds, bones, and the respiratory tract. Current studies are focused on finding new effective agents against A. baumannii. Phage therapy is a promising means to fight this bacterium and many studies on procuring and applying new phages against A. baumannii are currently being conducted. As shown in animal models, phages against multidrug-resistant A. baumannii may control bacterial infections caused by this pathogen and may be a real hope to solve this dangerous health problem.