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Application of PCR to distinguish common species of dermatophytes.

This report describes the application of PCR fingerprinting for the identification of species and varieties of common dermatophytes and related fungi utilizing as a single primer the simple repetitive oligonucleotide (GACA)(4). The primer was able to amplify all the strains, producing species-specific profiles for Microsporum canis, Microsporum gypseum, Trichophyton rubrum, Trichophyton ajelloi, and Epidermophyton floccosum. Intraspecific variability was not observed for these species. Instead, three different profiles were observed in the Trichophyton mentagrophytes group.

Animals↗

Evaluation of maize microsatellite markers for genetic diversity analysis and fingerprinting in sugarcane.

The use of maize microsatellite markers as a potential cost-effective method for molecular analysis of sugarcane was evaluated. Of the 34 primer pairs obtained from maize genomic libraries, 14 showed repeatable amplifications in Saccharum species clones, commercial hybrids, and the related genera Erianthus, accounting for 41.17% cross transferability. Complex banding patterns were encountered in sugarcane with the number of amplified fragments ranging from 7 to 14 with an average of 10 per primer, indicating the high polyploidy and heterozygosity existing in sugarcane. Phenetic analysis of the SSR polymorphisms produced by nine primers could clearly differentiate the different species of Saccharum and Erianthus and revealed the relationships that existed between them. Genetic similarity co-efficient indicated low diversity existing among the S. officinarum clones (82%) and a relatively higher level of diversity in the S. spontaneum clones (69.7%). Higher level of divergence of Erianthus from Saccharum was also clearly estabilished. Five primers produced genus- and species-specific fragments for Erianthus, S. spontaneum, S. officinarum, and S. barberi. The polymorphic primers, when tested on a panel of 30 commercial sugarcane cultivars, revealed a broad range (32.4-83.3%) of pair-wise similarity values, indicating their ability to detect high levels of polymorphism. A combination of two primers could differentiate all the varieties, further emphasizing their potential in fingerprinting and varietal identification.

Cluster Analysis↗

Proteomics study of neuropathic and nonneuropathic dorsal root ganglia: altered protein regulation following segmental spinal nerve ligation injury.

Peripheral nerve injury is often followed by the development of severe neuropathic pain. Nerve degeneration accompanied by inflammatory mediators is thought to play a role in generation of neuropathic pain. Neuronal cell death follows axonal degeneration, devastating a vast number of molecules in injured neurons and the neighboring cells. Because we have little understanding of the cellular and molecular mechanisms underlying neuronal cell death triggered by nerve injury, we conducted a proteomics study of rat 4th and 5th lumbar (L4 and L5) dorsal root ganglion (DRG) after L5 spinal nerve ligation. DRG proteins were displayed on two-dimensional gels and analyzed through quantitative densitometry, statistical validation of the quantitative data, and peptide mass fingerprinting for protein identification. Among approximately 1,300 protein spots detected on each gel, we discovered 67 proteins that were tightly regulated by nerve ligation. We find that the injury to primary sensory neurons turned on multiple cellular mechanisms critical for the structural and functional integrity of neurons and for the defense against oxidative damage. Our data indicate that the regulation of metabolic enzymes was carefully orchestrated to meet the altered energy requirement of the DRG cells. Our data also demonstrate that ligation of the L5 spinal nerve led to the upregulation in the L4 DRG of the proteins that are highly expressed in embryonic sensory neurons. To understand the molecular mechanisms underlying neuropathic pain, we need to comprehend such dynamic aspect of protein modulations that follow nerve injury.

Analysis of Variance↗

Molecular techniques in the diagnosis and management of infectious diseases: do they have a role in bacteriology?

The diagnosis and management of bacterial diseases has been done by traditional methods for a century or more. With the advent of molecular methods, however, these traditional approaches are being challenged. This review examines the pros and cons of traditional versus modern methods and tries to answer the question: when are molecular methods useful or essential? The following topics are addressed with appropriate examples: diagnosis; identification, typing and fingerprinting; pathogenesis; patient management; susceptibility to disease, and resistance to antimicrobial agents. It was concluded that there is still a place for both traditional and modern molecular methods, and training of staff must include both methodologies. Innovation is encouraged--but new technologies must be thoroughly tested before introduction into the routine lab. Liaison between laboratory scientist and physician is important, but above all experience is paramount.

Bacteriology↗

Remote Raman spectroscopic detection of minerals and organics under illuminated conditions from a distance of 10 m using a single 532 nm laser pulse.

Raman spectra of several minerals and organics were obtained from a small portable instrument at a distance of 10 m in a well-illuminated laboratory with a single 532 nm laser pulse with energy of 35 mJ/pulse. Remote Raman spectra of common minerals (dolomite, calcite, marble, barite, gypsum, quartz, anatase, fluorapatite, etc.) obtained in a short period of time (1.1 mus) clearly show Raman features that can be used as fingerprints for mineral identification. Raman features of organics (benzene, cyclohexane, 2-propanol, naphthalene, etc.) and other chemicals such as oxides, silicates, sulfates, nitrates, phosphates, and carbonates were also easily detected. The ability to identify minerals from their Raman spectra obtained from a single laser pulse has promise for future space missions where power consumption is critical. Such a system could be reduced in size by minimizing the cooling requirements for the laser unit. The remote Raman system is also capable of performing time-resolved measurements. Data indicate that further improvement in the performance of the system is possible by reducing the gate width of the detector (ICCD) from 1.1 mus to approximately 20 ns, which would significantly reduce the background signal from daylight or a well-illuminated laboratory. The 1.1 mus signal gating was effective in removing nearly all background fluorescence with 532 nm excitation, indicating that the fluorescence in most minerals is probably from long-lifetime inorganic phosphorescence.

Equipment Design↗

Two-dimensional liquid chromatography protein expression mapping for differential proteomic analysis of normal and O157:H7 Escherichia coli.

A multidimensional chromatographic method has been applied for the differential analysis of proteins from different strains of Escherichia coli bacteria. Proteins are separated in the first dimension using chromatofocusing (CF) and further separated by nonporous reversed-phase high-performance liquid chromatography (NPS-RP-HPLC) in the second dimension. A 2-dimensional (2-D) expression map of bacterial protein content is created for virulent O157:H7 and nonvirulent E. coli strains depicting protein isoelectric point (pI) versus protein hydrophobicity. Differentially expressed proteins are further characterized using electrospray/ionization time-of-flight mass spectrometry (ESI-TOF-MS) for intact protein molecular weight (MW) determination and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mass fingerprinting for protein identification. Using this method, no significant differential protein expression is exhibited between the two O157:H7 strains examined over a pH range of 4.0-7.0, and O157:H7 strains could be distinguished from nonvirulent E. coli. Several proteins differentially expressed between O157:H7 and nonvirulent E. coli are identified as potential markers for detection and treatment of O157:H7 infection.

Bacterial Proteins↗

Proteome analysis of resting human neutrophils.

Neutrophils constitute the first line of host defense against pathogens. In the present study 2-D gel electrophoresis-mass spectrometry technology was employed to analyze the human resting neutrophils proteome. One hundred and two conserved spots were subjected to peptide mass fingerprinting, yielding 22 identifications. Among the identified proteins, nine are related to the inflammatory process, two polypeptides are assigned to metabolic functions and five are classified as structural.

Adult↗

Comparison of fatty acid composition and kinetics of phosphorus-accumulating organisms and glycogen-accumulating organisms.

It was demonstrated that glycogen-accumulating organisms (GAOs) were able to compete with phosphorus-accumulating organisms (PAOs) for acetate in a biological phosphorus removal (BPR) process, leading to a loss of BPR capability. Cellular fatty acid composition, which serves as a fingerprint for microbial identification, was used to determine microbial population change and to investigate the competition mechanisms of PAOs and GAOs. Analysis of cellular fatty acid composition indicated that PAOs grown with acetate and glucose were different species and that GAOs and PAOs grown with the same substrate were also different species. Glycogen-accumulating organisms seemed to coexist with PAOs even in a well-developed BPR process. The GAOs were able to accumulate more poly-beta-hydroxybutyrate (PHB) and glycogen than PAOs during the anaerobic stage of the BPR process. The GAOs synthesized more in-cell glycogen than PAOs. The growth rate for PAOs was always greater than that for GAOs at various acetate or glucose concentrations, while GAOs had higher acetate uptake and PHB synthesis rates than PAOs. Therefore, GAOs are thought to compete with PAOs only at long solids retention times (> or = 20 days).

Bacteria↗

[A micro-epidemic of tuberculosis in the South Moravia Region].

In the South Moravian region which is the area with the lowest prevalence of bacillary tuberculosis in the Czech Republic (6.6 per 100,000 population in 1996) in 1993-1996 a total of six local and family microepidemics of tuberculosis were detected. For their identification the RFLP fingerprinting method was used based on evidence of repeated sequence of IS6110 in the chromosomal DNA of the examined strains of Mycobacterium tuberculosis predigested with restrictive enzyme PvuII. The profiles of fingerprints of each microepidemic were included by means of a computer programme into the hierarchy of the fingerprint dendrogram of 184 strains of M. tuberculosis which made it possible to identify possible identical profiles of strains from patients from remote places in the Czech Republic. In three family microepidemics involving always two members no identical fingerprint profiles of other Czech strains of M. tuberculosis were revealed. To the fourth cluster formed by six members of one family an identical RFLP profile of a female patient living in a nearby locality was added. In another microepidemic recorded in three brothers identical fingerprints were found another four patients from the South Moravian region and in one from the Central Bohemian region. The last cluster of two brothers was surprisingly enlarged by six identical RFLP profiles of patients from the West Bohemian region and one from Prague. These findings suggest that in areas with a low prevalence tuberculosis persists more frequently than in areas with a high prevalence as familial or local microepidemics.

Adolescent↗

Identify metastasis-associated genes in hepatocellular carcinoma through clonality delineation for multinodular tumor.

Disease recurrence and metastasis are frequently observed in many successfullytreated localized cancers, including hepatocellular carcinoma in which intrahepatic and extrahepatic recurrence (metastasis) are frequently observed after curative resection. The present study aimed at identifying metastasis-associated genes through delineation of the clonality for multinodular liver cancer. The clonal relationship of 22 tumor foci from six patients was investigated by the genome-wide expression profile via cDNA microarray consisting of 23,000 genes. Tumor molecular properties including p53 protein overexpression and gene mutation, hepatitis B virus integration pattern, and genetic alteration examined by comparative genomic hybridization were compared. Results indicated that gene expression patterns could serve as the molecular fingerprint for clonality identification. Together with the molecular data from p53, hepatitis B virus integration and comparative genomic hybridization profiles, tumor nodules from five patients were confirmed with clonal relationship, and the expression profiles of the primary nodules were compared with their corresponding intrahepatic metastatic nodules. A total of 90 clones were found to be correlated with intrahepatic metastasis by Student's t test (P < 0.05). With reference to the primary tumor, 63 clones (39 known genes and 24 express sequence tags) were down-regulated whereas 27 clones (14 known genes and 13 express sequence tags) were up-regulated in the metastatic nodules. These metastasis-associated genes may provide clues to reveal patients with increased risk of developing metastasis, and to identify novel therapeutic targets for the treatment of metastasis.

Adolescent↗

Identification of genomic signatures for the design of assays for the detection and monitoring of anthrax threats.

Sequences that are present in a given species or strain while absent from or different in any other organisms can be used to distinguish the target organism from other related or un-related species. Such DNA signatures are particularly important for the identification of genetic source of drug resistance of a strain or for the detection of organisms that can be used as biological agents in warfare or terrorism. Most approaches used to find DNA signatures are laboratory based, require a great deal of effort and can only distinguish between two organisms at a time. We propose a more efficient and cost-effective bioinformatics approach that allows identification of genomic fingerprints for a target organism. We validated our approach using a custom microarray, using sequences identified as DNA fingerprints of Bacillus anthracis. Hybridization results showed that the sequences found using our algorithm were truly unique to B. anthracis and were able to distinguish B. anthracis from its close relatives B. cereus and B. thuringiensis.

Animals↗

Analysis of genetic markers by random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR).

RAPD-PCR is a new technique that, starting from genomic DNA allows, with the use of a single primer of "random" base composition to amplify a variable number of sequences that can give important informations if analyzed for linkage studies, gene mapping or phylogenetic purposes. In order to detect the possible application of this simple way of DNA-fingerprinting in individual identification and in cell lineages characterization we analyzed human and non-human Primates DNA. Six different single primers of variable length were used and resulted in individual or specific electrophoretic patterns. As already reported we found a better resolution using "short" primers. The individual electrophoretic patterns obtained by RAPD-PCR can be a simple and reliable approach to DNA analysis.

Animals↗

A technique for developing and photographing ridge impressions on decomposed water-soaked fingers.

One of the most challenging tasks confronting a crime laboratory technician is the fingerprinting and subsequent identification of an unknown homicide or drowning victim whose fingers have been subjected to a long period of exposure to water and the effects of decomposition. If the fingers of the individual have not been exposed to the erosive effects of water and decomposition for a long period of time, they may be allowed to dry, and suitable impressions are often obtainable. In other cases the fingers may have to be removed, with the permission of the Medical Examiners Office, and processed by the Crime Laboratory in an attempt to develop suitable ridge structure for inked impressions or an exact photographic copy of the individual's fingers. In extreme cases the effects of water and decomposition make the fragile ridge structure appear to be nonexistent to the naked eye. The procedure used in this case report, combines the use of cyanoacrylate vapor, commonly called "super glue fuming," and the ninhydrin process in conjunction to develop fragile ridge structure into discernable ridges that are easily seen and photographed for the purpose of making an identification of the individual.

Anthropology, Physical↗

Identification of Candida species by randomly amplified polymorphic DNA fingerprinting of colony lysates.

We have characterized a method that produces simple yet diagnostic fingerprints that are unique to isolates of Candida species. DNA from individual colonies can be amplified from crude single-colony lysates. Randomly amplified polymorphic DNA (RAPD) fingerprints generated from a single primer correctly identified the species of most (>98%) of the isolates identified with CHROMagar Candida plates as non-Candida albicans Candida species. RAPD fingerprints were much more informative than the plates, since they distinguished between all tested species and required less time. Most (91%) of these identifications agreed with those assigned by API 20C tests. In almost every incident of species identity mismatch, electrophoretic karyotyping showed that the RAPD fingerprint was correct. This underscores the improved objectivity and reliability of this method over those of conventional diagnostic tools. The identities of approximately 30% of C. albicans isolates identified in clinical laboratories by positive germ tube tests are not verified by either testing on CHROMagar Candida plates or RAPD fingerprinting. Data suggest that clinical isolates conventionally identified as C. albicans in clinical settings are heterogeneous, consisting of both misidentified and atypical yeasts. RAPD fingerprints obtained from primary culture plate colonies allows for rapid, highly accurate determinations of Candida species, hence permitting earlier selection of appropriate antifungal agents in the clinical setting.

Candida↗

Identification of insect cell lines by DNA amplification fingerprinting (DAF).

Fingerprint profiles were generated from twenty insect cell lines spanning the Orders, Lepidoptera, Diptera, Coleoptera and Homoptera employing DNA amplification fingerprinting (DAF) with arbitrarily selected primers. The fingerprint pattern is a stable characteristic of the cell line because high and low passages generated the same profile. In addition, insect hosts and homologous cell lines generated similar profiles. All cell lines could be distinguished from each other with the following exceptions: Plutella xylostella (BCIRL-PX2-HNU3) and Mamestra brassicae (IZD-MB-0503) produced identical patterns to Trichoplusia ni (TN-CL1). Also Spodoptera exigua (UCR-SE-1C) produced the same profile as Spodoptera frugiperda (SF9) and the parent cell line IPLB-SF21. All these cases of identical patterns are believed to be due to cross contamination or mislabelling of cultures. DAF will serve as an additional, valuable and reliable technique for the identification of insect cell lines.

Aedes↗

Effect of high concentration of Co (II) on Enterobacter liquefaciens strain C-1: a bacterium highly resistant to heavy metals with an unknown genome.

Heavy metals are required as nutrients for essential functions in microorganisms. However, higher concentrations of these cations are generally toxic and may produce contrasting effects on living organisms. Enterobacter liquefaciens strain C-1, a bacterium isolated from the Moa mine in Cuba, is able to survive in the presence of high concentrations of heavy metals. The proteomes of Enterobacter liquefaciens strain C-1, grown under aerobic conditions in the presence and absence of Co (II) were compared using two-dimensional gel electrophoresis analysis in the isoelectric point range of 4-7 and the mass range of 15-120 kDa. Significant changes in the expression level (> two-fold) were detected for 13 spots: seven and six were up- and down-regulated, respectively. Because the genome of this bacterium is unknown, identification by peptide mass fingerprinting only succeeded in four cases and most of the cross-species identifications were supported by de novo sequencing of tryptic peptides followed by sequence alignment using the MS BLAST program. Twelve different proteins were identified, ten are involved in cellular antioxidant defence probably induced by the presence of Co (II). This is the first step towards understanding the role of proteins participating in the mechanism of resistance to heavy metals in this bacterium.

Amino Acid Sequence↗

Determination and characterization of organic explosives using porous graphitic carbon and liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

A new LC-MS method for the determination and characterization of three groups of commonly used organic explosives (nitroaromatic compounds, cyclic nitroamines and nitrate esters) was developed using a porous graphitic carbon (PGC) (Hypercarb) column. Twenty-one different explosive-related compounds--including 2,4,6-trinitrotoluene, its by-products and its degradation products--were chromatographically separated in a single analysis. This efficient separation facilitates the identification of the manufacturer of the explosive using the identified analytes as a fingerprint. A final, conclusive identification of the analytes can be obtained using LC-MS equipped with an atmospheric pressure chemical ionization (APCI) interface. Solvent effects on chromatographic behaviour were investigated, as were the effects of solvent mixtures and mobile phase additives. The number and the relative positions of the nitro groups within analyte molecules influence their order of elution; these effects were investigated. The data thus generated can be interpreted to support a hypothesis concerning the retention mechanism of nitro-containing compounds when using PGC. Limits of detection ranged from 0.5 to 41.2 ng. The new methodology described herein improves the sensitivity and selectivity of explosive detection. The effectiveness of the method is demonstrated by the analysis of soil samples containing explosives residue from test fields in Sweden and Afghanistan.

Atmospheric Pressure↗

Anatomy of fingerprint search calculations on structurally diverse sets of active compounds.

Similarity searching using molecular fingerprints is a widely used approach for the identification of novel hits. A fingerprint search involves many pairwise comparisons of bit string representations of known active molecules with those precomputed for database compounds. Bit string overlap, as evaluated by various similarity metrics, is used as a measure of molecular similarity. Results of a number of studies focusing on fingerprints suggest that it is difficult, if not impossible, to develop generally applicable search parameters and strategies, irrespective of the compound classes under investigation. Rather, more or less, each individual search problem requires an adjustment of calculation conditions. Thus, there is a need for diagnostic tools to analyze fingerprint-based similarity searching. We report an analysis of fingerprint search calculations on different sets of structurally diverse active compounds. Calculations on five biological activity classes were carried out with two fingerprints in two compound source databases, and the results were analyzed in histograms. Tanimoto coefficient (Tc) value ranges where active compounds were detected were compared to the distribution of Tc values in the database. The analysis revealed that compound class-specific effects strongly influenced the outcome of these fingerprint calculations. Among the five diverse compound sets studied, very different search results were obtained. The analysis described here can be applied to determine Tc intervals where scaffold hopping occurs. It can also be used to benchmark fingerprint calculations or estimate their probability of success.

Chemical Phenomena↗