ABSTRACT
The identification of Mycobacterium tuberculosis genes, specifically expressed during infection is a key step in understanding molecular mechanism of mycobacterial pathogenesis. Such genes likely encode proteins required for mycobacterium's survival and progressive infection within the host. In this study, we applied in-vivo-induced antigen technology (IVIAT) to M. tuberculosis and identified 11 putative in-vivo induced genes encoding for immunogenic proteins of diverse functions; these included transcriptional regulators (Rv1460 and Rv2565), biosynthesis and macromolecule metabolism (leuD, guaB1, plcC, hupB and glyS), polyketide synthases (pks6 and pks9), cell processes (ctpA) and one with unknown function (Rv3701c). Quantitative real time-PCR analysis of these genes in the specimens obtained from TB patients demonstrated induced expression of eight genes as compared with bacteria grown in-vitro. In addition, distribution of these genes in different strains of M. tuberculosis was analyzed using PCR and their nucleotide sequence alignments and they were found to be widely distributed among M. tuberculosis isolates including multiple-drug resistant (MDR) and extensively-drug resistant (XDR). This study identified several antigenic determinants of M. tuberculosis expressed during infection, which might help pathogens adapt to or counter hostile environments and suggesting their role during disease process.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Tuberculosis/microbiology , Adolescent , Adult , Aged , Antibodies, Bacterial/blood , Antigens, Bacterial/genetics , Antigens, Bacterial/isolation & purification , Antigens, Bacterial/metabolism , Child , Child, Preschool , Humans , Male , Middle Aged , Molecular Sequence Data , Reverse Transcriptase Polymerase Chain Reaction , Species Specificity , Young AdultABSTRACT
BACKGROUND: Hydroxychavicol, isolated from the chloroform extraction of the aqueous leaf extract of Piper betle L., (Piperaceae) was investigated for its antifungal activity against 124 strains of selected fungi. The leaves of this plant have been long in use tropical countries for the preparation of traditional herbal remedies. METHODS: The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of hydroxychavicol were determined by using broth microdilution method following CLSI guidelines. Time kill curve studies, post-antifungal effects and mutation prevention concentrations were determined against Candida species and Aspergillus species "respectively". Hydroxychavicol was also tested for its potential to inhibit and reduce the formation of Candida albicans biofilms. The membrane permeability was measured by the uptake of propidium iodide. RESULTS: Hydroxychavicol exhibited inhibitory effect on fungal species of clinical significance, with the MICs ranging from 15.62 to 500 microg/ml for yeasts, 125 to 500 microg/ml for Aspergillus species, and 7.81 to 62.5 microg/ml for dermatophytes where as the MFCs were found to be similar or two fold greater than the MICs. There was concentration-dependent killing of Candida albicans and Candida glabrata up to 8 x MIC. Hydroxychavicol also exhibited an extended post antifungal effect of 6.25 to 8.70 h at 4 x MIC for Candida species and suppressed the emergence of mutants of the fungal species tested at 2 x to 8 x MIC concentration. Furthermore, it also inhibited the growth of biofilm generated by C. albicans and reduced the preformed biofilms. There was increased uptake of propidium iodide by C. albicans cells when exposed to hydroxychavicol thus indicating that the membrane disruption could be the probable mode of action of hydroxychavicol. CONCLUSIONS: The antifungal activity exhibited by this compound warrants its use as an antifungal agent particularly for treating topical infections, as well as gargle mouthwash against oral Candida infections.
Subject(s)
Antifungal Agents/pharmacology , Eugenol/analogs & derivatives , Fungi/drug effects , Piper betle/chemistry , Antifungal Agents/chemistry , Antifungal Agents/isolation & purification , Aspergillus/drug effects , Biofilms/drug effects , Biofilms/growth & development , Candida/drug effects , Candida/physiology , Eugenol/chemistry , Eugenol/isolation & purification , Eugenol/pharmacology , Microbial Sensitivity Tests , Mutation/drug effects , Plant Leaves/chemistryABSTRACT
The archaeal diversity in salt pan sediment from Mumbai, India, was investigated by 16S rDNA-dependent molecular phylogeny. Small-subunit rRNA (16S rDNA) from salt pan sediment metagenome were amplified by polymerase chain reaction (PCR) using primers specific to the domain archaea. Thirty-two unique phylotypes were obtained by PCR-based RFLP of 16S rRNA genes using endonucleases Hae111 and Msp1, which were most suitable to score the genetic diversity. These phylotypes spanned a wide range within the domain Archaea including both Crenarchaeota and Euryarcheaota. However, none of the retrieved Crenarchaeota sequences could be grouped with previously cultured Crenarchaeota. Of all the Euryarcheaota sequences, three sequences were related to Haloarchaea, two were related to cultured Methanosarcina sp., and the remaining sequences were affiliated with uncultured Methanosarcina sp., Methanogenium sp., and Methanolobus sp. Most of the sequences determined were closely related to the sequences that had been previously obtained from metagenome of a variety of marine environments. The phylogenetic study of a site investigated for the first time revealed the presence of a highly diverse archaeal population and may represent novel sequences and organisms specially adapted to the salt pan sediment of Mumbai. These findings are of fundamental value for understanding the complexity of salt pan ecosystems.
Subject(s)
Archaea/classification , Archaea/isolation & purification , Biodiversity , Geologic Sediments/microbiology , Phylogeny , Archaea/genetics , DNA Fingerprinting , DNA Primers/genetics , DNA, Archaeal/chemistry , DNA, Archaeal/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Genes, rRNA , Genotype , India , Molecular Sequence Data , Polymerase Chain Reaction/methods , Polymorphism, Restriction Fragment Length , RNA, Archaeal/genetics , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Sequence Homology, Nucleic AcidABSTRACT
Cryoprecipitate and the more purified factor VIII concentrates are all heterogeneous preparations that contain not only a high concentration of factor VIII but also various other materials, some of which might be injurious, causing liver damage after long-term exposure. The efficiency of three standard cryoprecipitate filters, two microaggregate filters and the appropriate factor VIII concentrate filters in reducing the amount of particulate matter delivered to the patient was assessed. Filtration of cryoprecipitate through the standard filters removed less than 20% of the contaminating microaggregates and very few of the large number of intact platelets, although the total dose of factor VIII was delivered. Microaggregate filters were no better in reducing the platelet contamination, although the total number of particles delivered was halved. However, 25% of the factor VIII was retained in the bed volume of the filter. The concentrate preparations also contained significant amounts of particulate matter that was unrelated to factor VIII and was not removed following filtration through the designated filter. These findings indicate that a new filter should be developed for administration of factor VIII concentrate that would remove the particulate matter while delivering all of the factor VIII to the patient.
