Hydrolysis of Oligodeoxyribonucleotides on the Microarray Surface and in Solution by Catalytic Anti-DNA Antibodies in Systemic Lupus Erythematosus.

Anti-DNA antibodies are known to be classical serological hallmarks of systemic lupus erythematosus (SLE). In addition to high-affinity antibodies, the autoantibody pool also contains natural catalytic anti-DNA antibodies that recognize and hydrolyze DNA. However, the specificity of such antibodies is uncertain. In addition, DNA binding to a surface such as the cell membrane, can also affect its recognition by antibodies. Here, we analyzed the hydrolysis of short oligodeoxyribonucleotides (ODNs) immobilized on the microarray surface and in solution by catalytic anti-DNA antibodies from SLE patients. It has been shown that IgG antibodies from SLE patients hydrolyze ODNs more effectively both in solution and on the surface, compared to IgG from healthy individuals. The data obtained indicate a more efficient hydrolysis of ODNs in solution than immobilized ODNs on the surface. In addition, differences in the specificity of recognition and hydrolysis of certain ODNs by anti-DNA antibodies were revealed, indicating the formation of autoantibodies to specific DNA motifs in SLE. The data obtained expand our understanding of the role of anti-DNA antibodies in SLE. Differences in the recognition and hydrolysis of surface-tethered and dissolved ODNs need to be considered in DNA microarray applications.

Deep-Sea Anemones Are Prospective Source of New Antimicrobial and Cytotoxic Compounds.

The peculiarities of the survival and adaptation of deep-sea organisms raise interest in the study of their metabolites as promising drugs. In this work, the hemolytic, cytotoxic, antimicrobial, and enzyme-inhibitory activities of tentacle extracts from five species of sea anemones (Cnidaria, orders Actiniaria and Corallimorpharia) collected near the Kuril and Commander Islands of the Far East of Russia were evaluated for the first time. The extracts of Liponema brevicorne and Actinostola callosa demonstrated maximal hemolytic activity, while high cytotoxic activity against murine splenocytes and Ehrlich carcinoma cells was found in the extract of Actinostola faeculenta. The extracts of Corallimorphus cf. pilatus demonstrated the greatest activity against Ehrlich carcinoma cells but were not toxic to mouse spleen cells. Sea anemones C. cf. pilatus and Stomphia coccinea are promising sources of antimicrobial and antifungal compounds, being active against Gram-positive bacteria Bacillus subtilis, Staphylococcus aureus, and yeast Candida albicans. Moreover, all sea anemones contain α-galactosidase inhibitors. Peptide mass fingerprinting of L. brevicorne and C. cf. pilatus extracts provided a wide range of peptides, predominantly with molecular masses of 4000-5900 Da, which may belong to a known or new structural class of toxins. The obtained data allow concluding that deep-sea anemones are a promising source of compounds for drug discovery.

A many probes-one spot hybridization oligonucleotide microarray.

A variant of the hybridization oligonucleotide microarray, utilizing the principle of many probes-one spot (MPOS-microarrays), is proposed. A case study based on Orthopoxviruses (Variola, Monkeypox, and Ectromelia viruses) demonstrates a considerable increase in the fluorescence signal (up to 100-fold) when several oligonucleotide probes are printed to one spot. Moreover, the specificity of detection also increases (almost 1000-fold), allowing the use of probes that individually lack such high specificity. The optimal probes have a Tm of 32-37 °C and length of 13-15 bases. We suggest that the high specificity and sensitivity of the MPOS-microarray is a result of cooperativity of DNA binding with all probes immobilized in the spot. This variant of DNA detection can be useful for designing biosensors, tools for point-of-care (POC) diagnostics, microbial ecology, analysis of clustered regularly interspaced short palindromic repeats (CRISPR), and others. Graphical abstract ᅟ.

Effect of Nutrient Sources on the Alginate Accumulation in the Culture Liquid of Azotobacter vinelandii D-05 and Obtaining Biocomposite Materials

ABSTRACT Using the classic biotechnological methods, the dependence of A. vinelandii D-05 culture alginate production from the media carbon and nitrogen content was investigated. The maximal alginate production was observed during cultivation bacterium in the medium with 2 to 4% of sucrose, but the maximal growth was found in the medium with 4% glucose. It was found that for the alginate production the optimal nitrogen contents could take from 0.05% yeast extract (carbon: nitrogen ratio 168:1). For the first time we demonstrated possibility the A. vinelandii growth during the cultivation in a medium with molasses (a by-product of sugar production) and the significant polysaccharide production (16.6 g/l) was obtained. It was established, that A. vinelandii culture broth could be used as a biological binder for obtaining the biocomposite materials.

Unexpected transformation of black hole quenchers in electrophoretic purification of the fluorescein-containing TaqMan probes.

The fluorescence quenchers BHQ1 and BHQ2 can be modified by trace amounts of ammonium persulfate, used for initiating gel polymerization, in electrophoretic purification of TaqMan probes using a denaturing polyacrylamide gel. The case study of BHQ1 quencher has demonstrated that a Boyland-Sims reaction proceeds in the presence of ammonium persulfate to give the corresponding sulfate. The absorption maximum of the resulting quencher shifts to the short-wavelength region relative to the absorption maximum of the initial BHQ1. The TaqMan probe containing such a quencher is less efficient as compared with the probe carrying an unmodified BHQ1. The presence of fluorescein in TaqMan probe plays decisive role in this transformation: the quencher modification proceeds at a considerably lower rate when the fluorescein is absent or replaced with a rhodamine dye (for example, R6G). It is assumed that the observed reaction can take place in two ways-both in darkness and in the reaction of the quencher in an excited state due to energy transfer from the fluorophore irradiated by light.

Universal oligonucleotide microarray for sub-typing of Influenza A virus.

A universal microchip was developed for genotyping Influenza A viruses. It contains two sets of oligonucleotide probes allowing viruses to be classified by the subtypes of hemagglutinin (H1-H13, H15, H16) and neuraminidase (N1-N9). Additional sets of probes are used to detect H1N1 swine influenza viruses. Selection of probes was done in two steps. Initially, amino acid sequences specific to each subtype were identified, and then the most specific and representative oligonucleotide probes were selected. Overall, between 19 and 24 probes were used to identify each subtype of hemagglutinin (HA) and neuraminidase (NA). Genotyping included preparation of fluorescently labeled PCR amplicons of influenza virus cDNA and their hybridization to microarrays of specific oligonucleotide probes. Out of 40 samples tested, 36 unambiguously identified HA and NA subtypes of Influenza A virus.

Actinoporins from the sea anemones, tropical Radianthus macrodactylus and northern Oulactis orientalis: Comparative analysis of structure-function relationships.

Actinoporins Or-A and Or-G from the northern sea anemone Oulactis orientalis and actinoporins RTX-A and RTX-SII from the tropical sea anemone Radianthus macrodactylus (=Heteractis crispa) were compared with each other and with some known actinoporins. In this work the complete amino acid sequence of RTX-SII was determined by molecular biology methods. The following differences were revealed in functionally significant regions of Radianthus, Oulactis, and some other actinoporins: (i) tryptophan is substituted for leucine in the position equivalent to Trp112 in the POC binding site of EqtII; (ii) 13 and 5 residues are truncated in N-terminal regions of Or-A and Or-G, respectively. A possible role of these structural differences in specific regions of the actinoporin sequence was analyzed. Some differences in hydrophobicity parameters, distribution of charged residues, and length of actinoporins' N-terminus apparently cause considerable differences in their hemolytic activities. Homology models of Radianthus and Oulactis actinoporin monomers were generated using crystal structures of equinatoxin II from Actinia equina and sticholysin II from Stichodactyla helianthus as templates. The current data on actinoporin structures and activities, coupled with results of our earlier differential scanning calorimetric and electrophoretic experiments with RTX-A-modified erythrocyte ghosts (Shnyrov et al., 1992), suggests that the exposed RGD motif located near the POC binding site can interact with membrane integrin(s).

Utilization of a two-standard system in real-time PCR for quantification of gene expression in the brain.

In this study, we applied for real-time PCR the two-standard system that we had worked out previously for PCR with gel-detection of products. Genomic DNA of a known concentration was used as external standard and mRNA of the DNA-dependent RNA-polymerase II was used as internal standard. It was shown that PCR with gel-detection of products and real-time PCR provide similar results and demonstrate almost identical accuracy and repeatability when the two-standard system is used. With the help of the both methods and using the two-standard system we have confirmed the link between the genetically determined freezing reaction in mice and reduced 5-HT1A receptor mRNA level in the midbrain. We have also found that the genetically determined freezing reaction in mice is not connected with changes in Tph2 gene expression.

Microarray assay for detection and discrimination of Orthopoxvirus species.

A microarray method was developed for simultaneous detection and identification of six species of Orthopoxvirus (OPV) including Variola, Monkeypox, Cowpox, Camelpox, Vaccinia, and Ectromelia viruses. The method allowed us to discriminate OPV species from varicella-zoster virus (VZV), Herpes Simplex 1 virus (HSV-1), and Herpes Simplex 2 virus (HSV-2) that cause infections with clinical manifestations similar to OPV infections. The nucleotide sequences of the C23L/B29R and the B19R genes identified for 86 and 72 different OPV strains, respectively, were used to design species-specific microarray oligonucleotide probes (oligoprobes). The microarray also contained several oligoprobes selected from the ORF31, US4, and US5 genes of VZV, HSV-1, and HSV-2, respectively. The samples (from HSVs or OPVs) of ssDNAs for analyses were prepared by using asymmetric PCR followed by chemical labeling of ssDNA with Cy3 dye. DNA from 52 samples of various OPV species, two isolates of VZV, two of HSV-1, and three of HSV-2 were tested using the developed microarray assay; all tested viruses were accurately identified. To ensure the robustness of the microarray assay, three additional unrelated variola virus strains with unknown sequences of the C23L/B29R and the B19R genes were tested. In each instance the microarray unambiguously identified them as Variola virus species. The results obtained in this study demonstrated that this new microarray method is a valuable tool for the rapid and accurate detection and differentiation of these important viral pathogens.

Noncapsulated Klebsiella pneumoniae bearing mannose-containing O antigens is rapidly eradicated from mouse lung and triggers cytokine production by macrophages following opsonization with surfactant protein D.

To better understand the relationship between the surface polysaccharides of pulmonary pathogens and components of the lung innate immune system, we employed selected serotypes of Klebsiella pneumoniae expressing distinct capsular polysaccharides and/or O antigen in a murine model of K. pneumoniae infection. In addition, we examined the effect of surfactant protein D (SP-D) on the cytokine response of human monocyte-derived macrophages to these serotypes in vitro. Noncapsulated mannose-containing O3 serotypes (K50/n and K55/n), which react efficiently with SP-D in vitro, triggered high levels of interleukin-1beta (IL-1beta) and IL-6 production. In vivo, they were more efficiently cleared from the lungs of mice but not from macrophage-depleted mice. They also were more efficiently internalized by alveolar macrophages in vivo. In contrast, galactose-containing O1 serotypes (K2/n and K21a/n), which interact poorly with SP-D, exhibited significantly lower cytokine production and less efficient pulmonary clearance and were ineffectively internalized by alveolar macrophages. These findings are consistent with in vitro results showing that production of IL-1beta and IL-6 mRNA and IL-6 protein by human macrophages exposed to mannose-bearing Klebsiella O serotypes is significantly increased by SP-D. Thus, survival of inhaled bacteria in the lung depends partially on the lipopolysaccharide structure of the bacteria and their interactions with innate immunity components. We speculate that an imbalance of host SP-D and therefore cytokine levels may result in high susceptibility of the host to the pathogen.