Biomedical subjects

V Chizhikov

Publications and source records attributed to V Chizhikov.

13 recordsLinked to original sources

PubMed2004

Phase-matching and femtosecond difference-frequency generation in the quaternary semiconductor AgGaGe5Se12.

We present data on the linear (transmission, index of refraction) and nonlinear (second-order susceptibility) optical properties of the quaternary semiconductor AgGaGe5Se12 with orthorhombic symmetry--a solid solution in the AgxGaxGe1-xSe2 system with x = 0.17. The nonlinear coefficients are estimated from phase-matched second-harmonic generation near 3 microm. After numerical analysis of the phase-matching configurations for three-wave nonlinear interactions, the first experimental results on difference-frequency mixing, producing tunable (4-7.5-microm) femtosecond pulses at a 1-kHz repetition rate, are described. The pulses of only five optical cycles (FWHM = 84 fs) are generated near 5 microm with energy of 0.5 microJ. Because of its higher damage threshold, larger birefringence and bandgap, and greater variety of phase-matching schemes, AgGaGe5Se12 could become an alternative to AgGaS2 and AgGaSe2, more widely used in high-power and specific applications.

Journal Article↗

PubMed2004

Genotyping of Clostridium perfringens toxins using multiple oligonucleotide microarray hybridization.

A microarray-based method for characterization of six Clostridium perfringens toxin genes: iA (iota toxin), cpa (alpha toxin), cpe (enterotoxin E), etxD (epsilon toxin), cpb1 (beta toxin 1),and cpb2 (beta toxin 2) was developed and evaluated using 17 C. perfringens isolates. Three individual oligonucleotide probes (oligoprobes), complementary to the unique sequences of each toxin gene, were designed and immobilized on a surface of aldehyde-coated glass slides. Multiplex PCR was used to simultaneously amplify DNA target regions of all six genes. Single-stranded DNA (ssDNA) samples for microarray analysis were prepared by following a primer extension of amplicons in the presence of one primer. Fluorescent moieties (Cy3) were incorporated into the ssDNA by chemical modification of guanine bases. The presence of toxin genes in C. perfringens was established by hybridization of the fluorescently labeled ssDNA representing different samples to the microarray gene-specific oligoprobes. Results of the study showed sensitivity and specificity of genotyping C. perfringens using multiple microarray-based assays.

ADP Ribose Transferases↗

PubMed2004

Natural atypical Listeria innocua strains with Listeria monocytogenes pathogenicity island 1 genes.

Identification of bona fide Listeria isolates into the six species of the genus normally requires only a few tests. Aberrant isolates do occur, but even then only one or two extra confirmatory tests are generally needed for identification to species level. We have discovered a hemolytic-positive, rhamnose and xylose fermentation-negative Listeria strain with surprising recalcitrance to identification to the species level due to contradictory results in standard confirmatory tests. The issue had to be resolved by using total DNA-DNA hybridization testing and then confirmed by further specific PCR-based tests including a Listeria microarray assay. The results show that this isolate is indeed a novel one. Its discovery provides the first fully documented instance of a hemolytic Listeria innocua strain. This species, by definition, is typically nonhemolytic. The L. innocua isolate contains all the members of the PrfA-regulated virulence gene cluster (Listeria pathogenicity island 1) of L. monocytogenes. It is avirulent in the mouse pathogenicity test. Avirulence is likely at least partly due to the absence of the L. monocytogenes-specific allele of iap, as well as the absence of inlA, inlB, inlC, and daaA. At least two of the virulence cluster genes, hly and plcA, which encode the L. monocytogenes hemolysin (listeriolysin O) and inositol-specific phospholipase C, respectively, are phenotypically expressed in this L. innocua strain. The detection by PCR assays of specific L. innocua genes (lin0198, lin0372, lin0419, lin0558, lin1068, lin1073, lin1074, lin2454, and lin2693) and noncoding intergenic regions (lin0454-lin0455 and nadA-lin2134) in the strain is consistent with its L. innocua DNA-DNA hybridization identity. Additional distinctly different hemolytic L. innocua strains were also studied.

Animals↗

PubMed2003

Genetic and phenotypic analysis of reassortants of high growth and low growth strains of influenza B virus.

The yield of influenza virus in eggs is critical to influenza vaccine production and availability, but the contribution of specific genes to the growth properties of influenza B viruses is not well understood. Influenza B/Beijing/184/93 and B/Shangdong/7/97 were chosen for study because B/Shangdong/7/97 replicated to several fold higher titers in eggs than B/Beijing/184/93 as demonstrated by hemagglutination titers and EID50. A reassortant with the HA, NP and PB2 genes from B/Beijing/184/93 and all other genes from B/Shangdong/7/97 had the high growth phenotype of B/Shangdong/7/97 in eggs, which suggests that NS, M, NA, PB1 or PA, or a combination of these genes derived from B/Shangdong/7/97 were needed for the high growth phenotype of the reassortants. A high degree of homology was found among the genetic sequences of B/Beijing/184/93, B/Shangdong/7/97, and other influenza B viruses. However, differences potentially related to growth characteristics were suggested by analysis of the deduced amino acid (AA) sequences of four genes: NS (NS1, NS2), M (BM2), NA (NA, NB) and PB1. The studies identify multiple genes that may affect growth of influenza B viruses in eggs.

Animals↗

PubMed2003

Microarray analysis of erythromycin resistance determinants.

AIMS: To develop a DNA microarray for analysis of genes encoding resistance determinants to erythromycin and the related macrolide, lincosamide and streptogramin B (MLS) compounds. METHODS AND RESULTS: We developed an oligonucleotide microarray containing seven oligonucleotide probes (oligoprobes) for each of the six genes (ermA, ermB, ermC, ereA, ereB and msrA/B) that account for more than 98% of MLS resistance in Staphylococcus aureus clinical isolates. The microarray was used to test reference and clinical S. aureus and Streptococcus pyrogenes strains. Target genes from clinical strains were amplified and fluorescently labelled using multiplex PCR target amplification. The microarray assay correctly identified the MLS resistance genes in the reference strains and clinical isolates of S. aureus, and the results were confirmed by direct DNA sequence analysis. Of 18 S. aureus clinical strains tested, 11 isolates carry MLS determinants. One gene (ermC) was found in all 11 clinical isolates tested, and two others, ermA and msrA/B, were found in five or more isolates. Indeed, eight (72%) of 11 clinical isolate strains contained two or three MLS resistance genes, in one of the three combinations (ermA with ermC, ermC with msrA/B, ermA with ermC and msrA/B). CONCLUSIONS: Oligonucleotide microarray can detect and identify the six MLS resistance determinants analysed in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that microarray-based detection of microbial antibiotic resistance genes might be a useful tool for identifying antibiotic resistance determinants in a wide range of bacterial strains, given the high homology among microbial MLS resistance genes.

Anti-Bacterial Agents↗

PubMed2002

Detection and genotyping of human group A rotaviruses by oligonucleotide microarray hybridization.

A rapid and reliable method for the identification of five clinically relevant G genotypes (G1 to G4 and G9) of human rotaviruses based on oligonucleotide microarray hybridization has been developed. The genotype-specific oligonucleotides immobilized on the surface of glass slides were selected to bind to the multiple target regions within the VP7 gene that are highly conserved among individual rotavirus genotypes. Rotavirus cDNA was amplified in a PCR with primers common to all group A rotaviruses. A second round of nested PCR amplification was performed in the presence of indodicarbocyanine-dCTP and another pair of degenerate primers also broadly specific for all genotypes. The use of one primer containing 5'-biotin allowed us to prepare fluorescently labeled single-stranded hybridization probe by binding of another strand to magnetic beads. The identification of rotavirus genotype was based on hybridization with several individual genotype-specific oligonucleotides. This approach combines the high sensitivity of PCR with the selectivity of DNA-DNA hybridization. The specificity of oligonucleotide microchip hybridization was evaluated by testing 20 coded rotavirus isolates from different geographic areas for which genotypes were previously determined by conventional methods. Analysis of the coded specimens showed that this microarray-based method is capable of unambiguous identification of all rotavirus strains. Because of the presence of random mutations, each individual virus isolate produced a unique hybridization pattern capable of distinguishing different isolates of the same genotype and, therefore, subgenotype differentiation. This strain information indicates one of several advantages that microarray technology has over conventional PCR techniques.

Antigens, Viral↗

PubMed2001

Two consistently deleted regions within chromosome 1p32-pter in human non-small cell lung cancer.

Allelic losses at 1p32-pter have been reported as frequent events in human non-small cell lung cancer (NSCLC). To further characterize the region of deletions, we studied loss of heterozygosity on a panel of 102 microdissected NSCLC samples with 20 polymorphic markers spanning 1p32-pter. Two shortest regions of the overlap of the deletions (SROs) were found: SRO 2a (D1S417--D1S57) and SRO 2b (D1S450--D1S243). Allelic losses at either region correlated independently with advanced stage of disease and with postoperative metastasis and relapse (P < 0.05), suggesting that crucial genes in these regions are involved in NSCLC progression. Mol. Carcinog. 30:151--158, 2001.

Carcinoma, Non-Small-Cell Lung↗

PubMed2001

Microarray analysis of microbial virulence factors.

Hybridization with oligonucleotide microchips (microarrays) was used for discrimination among strains of Escherichia coli and other pathogenic enteric bacteria harboring various virulence factors. Oligonucleotide microchips are miniature arrays of gene-specific oligonucleotide probes immobilized on a glass surface. The combination of this technique with the amplification of genetic material by PCR is a powerful tool for the detection of and simultaneous discrimination among food-borne human pathogens. The presence of six genes (eaeA, slt-I, slt-II, fliC, rfbE, and ipaH) encoding bacterial antigenic determinants and virulence factors of bacterial strains was monitored by multiplex PCR followed by hybridization of the denatured PCR product to the gene-specific oligonucleotides on the microchip. The assay was able to detect these virulence factors in 15 Salmonella, Shigella, and E. coli strains. The results of the chip analysis were confirmed by hybridization of radiolabeled gene-specific probes to genomic DNA from bacterial colonies. In contrast, gel electrophoretic analysis of the multiplex PCR products used for the microarray analysis produced ambiguous results due to the presence of unexpected and uncharacterized bands. Our results suggest that microarray analysis of microbial virulence factors might be very useful for automated identification and characterization of bacterial pathogens.

Bacterial Proteins↗

PubMed2001

Rotavirus strains bearing genotype G9 or P[9] recovered from Brazilian children with diarrhea from 1997 to 1999.

Human rotavirus strains belonging to genotype G9 or P[9] were detected in a collection of stool specimens from children with diarrhea in two cities of the state of Rio de Janeiro, Brazil, between March 1997 and December 1999. G9 strains were first detected in April 1997 and remained prevalent until the end of the study, at a frequency of 15.9% (n = 157). A high percentage of VP7 nucleotide (99.0 to 99.5%) and deduced amino acid identity (98.6 to 99.1%) was found between three randomly selected Brazilian G9 strains and the American G9 strain US1205. A novel G9:P[4] genotype combination was detected in addition to G9:P[8] and G9:P[6], demonstrating that this G genotype may undergo constant genetic reassortment in nature. The P[9] rotavirus strains constituted 10.2%, the majority of which were detected between April and July 1997. The RNA electrophoretic migration pattern of the G3:P[9] strains resembled that of AU-1 virus (G3:P3[9]), suggesting a genetic similarity between the Brazilian G3:P[9] strains and the Japanese virus, which is similar to a feline rotavirus genetically.

Antigens, Viral↗

PubMed2001

Effect of intragenic rearrangement and changes in the 3' consensus sequence on NSP1 expression and rotavirus replication.

The nonpolyadenylated mRNAs of rotavirus are templates for the synthesis of protein and the segmented double-stranded RNA (dsRNA) genome. During serial passage of simian SA11 rotaviruses in cell culture, two variants emerged with gene 5 dsRNAs containing large (1.1 and 0.5 kb) sequence duplications within the open reading frame (ORF) for NSP1. Due to the sequence rearrangements, both variants encoded only C-truncated forms of NSP1. Comparison of these and other variants encoding defective NSP1 with their corresponding wild-type viruses indicated that the inability to encode authentic NSP1 results in a small-plaque phenotype. Thus, although nonessential, NSP1 probably plays an active role in rotavirus replication in cell culture. In determining the sequences of the gene 5 dsRNAs of the SA11 variants and wild-type viruses, it was unexpectedly found that their 3' termini ended with 5'-UGAACC-3' instead of the 3' consensus sequence 5'-UGACC-3', which is present on the mRNAs of nearly all other group A rotaviruses. Cell-free assays indicated that the A insertion into the 3' consensus sequence interfered with its ability to promote dsRNA synthesis and to function as a translation enhancer. The results provide evidence that the 3' consensus sequence of the gene 5 dsRNAs of SA11 rotaviruses has undergone a mutation causing it to operate suboptimally in RNA replication and in the expression of NSP1 during the virus life cycle. Indeed, just as rotavirus variants which encode defective NSP1 appear to have a selective advantage over those encoding wild-type NSP1 in cell culture, it may be that the atypical 3' end of SA11 gene 5 has been selected for because it promotes the expression of lower levels of NSP1 than the 3' consensus sequence.

Amino Acid Sequence↗

PubMed2000

A four-nucleotide translation enhancer in the 3'-terminal consensus sequence of the nonpolyadenylated mRNAs of rotavirus.

The 5' cap and poly(A) tail of eukaryotic mRNAs work synergistically to enhance translation through a process that requires interaction of the cap-associated eukaryotic initiation factor, eIF-4G, and the poly(A)-binding protein, PABP. Because the mRNAs of rotavirus, and other members of the Reoviridae, contain caps but lack poly(A) tails, their translation may be enhanced through a unique mechanism. To identify translation-enhancement elements in the viral mRNAs that stimulate translation in vivo, chimeric RNAs were prepared that contained an open reading frame for luciferase and the 5' and 3' untranslated regions (UTRs) of a rotavirus mRNA or of a nonviral mRNA. Transfection of the chimeric RNAs into rotavirus-infected cells showed that the viral 3' UTR contained a translation-enhancement element that promoted gene expression. The element did not enhance gene expression in uninfected cells and did not affect the stability of the RNAs. Mutagenesis showed that the conserved sequence GACC located at the 3' end of rotavirus mRNAs operated as an enhancement element. The 3'-GACC element stimulated protein expression independently of the sequence of the 5' UTR, although efficient expression required the RNA to contain a cap. The results indicate that the expression of viral proteins in rotavirus-infected cells is specifically up-regulated by the activity of a novel 4-nt 3' translation enhancer (TE) common to the 11 nonpolyadenylated mRNAs of the virus. The 4-nt sequence of the rotavirus 3' TE represents by far the shortest of any of the sequence enhancers known to stimulate translation.

3' Untranslated Regions↗

PubMed1996

Khabarovsk virus: a phylogenetically and serologically distinct hantavirus isolated from Microtus fortis trapped in far-east Russia.

Two hantavirus strains, MF43 and MF113, isolated from Microtus fortis trapped in the Khabarovsk region of far-eastern Russia, were analysed by direct nucleotide sequencing of PCR generated fragments of the M and S segments, by immunofluorescence and by focus reduction neutralization tests (FRNT). The nucleotide sequences revealed that the two isolates were closely related to each other but distinct from all other hantaviruses. Phylogenetic analysis of the M and S segments showed that the MF strains form a separate branch in the Hantavirus tree, positioned between the branches of Prospect Hill and Puumala viruses. The strains were shown to be serologically distinct from the other hantavirus serotypes by FRNT using immune rabbit sera. Puumala virus was the closest relative, both genetically and serologically. We propose that this new hantavirus serotype should be named Khabarovsk (KBR).

Animals↗

PubMedDate not supplied

[Genetic differentiation of hantaviruses using the polymerase chain reaction and sequencing].

Thirty-two hantavirus strains and 8 samples of lung tissue from rodents collected in different regions of Russia have been examined by molecular biological methods. Two methodological approaches have been employed for the study of genetic relationships between the viruses: nested PCR assay and common RT-PCR with subsequent direct sequencing of 200 and 365 base pair of G2 protein encoding regions of M-segment, respectively, and the resultant sequences were compared with those of the prototype hantavirus. The study revealed a mosaic pattern of distribution of different hantavirus genotypes on the territory of Russia.

Animals↗