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PubMed1993

Rapid, simple method for typing isolates of Mycobacterium tuberculosis by using the polymerase chain reaction.

To develop a molecular typing method for Mycobacterium tuberculosis based on the polymerase chain reaction, oligonucleotide primers were designed to the ends of the insertion sequence IS6110 in an attempt to amplify DNA between clusters of this element on the genome. Although in many strains the copy number of this element is low and is distributed throughout the genome, most strains examined produced a banding pattern which varied between isolates including strains with one copy of IS6110. With strains isolated from patients in epidemiologic clusters of tuberculosis, the banding patterns were similar within each cluster but distinct from those in strains from different clusters. Similarly, multiple isolates from the same patient yielded a consistent banding pattern. Sequencing of four polymerase chain reaction products revealed that amplification was occurring between copies of IS6110 in two of the products and from a single copy of IS6110 to a nonspecific priming site in the other two. This technique provides a rapid and simple means of typing M. tuberculosis isolates for epidemiologic studies.

Bacterial Typing Techniques↗
PubMed1997

Rapid identification of clinically significant species and taxa of aerobic actinomycetes, including Actinomadura, Gordona, Nocardia, Rhodococcus, Streptomyces, and Tsukamurella isolates, by DNA amplification and restriction endonuclease analysis.

A previously described PCR-restriction fragment length polymorphism (RFLP) identification schema for Nocardia that used an amplified 439-bp segment (amplicon) of the 65-kDa heat shock protein gene was evaluated for potential use with isolates of all clinically significant aerobic actinomycetes. The study included 28 reference (American Type Culture Collection) strains and 198 clinical isolates belonging to 20 taxonomic groups. Of these 198 isolates, 188 could be differentiated by this PCR-RFLP method. Amplicons from all aerobic actinomycete isolates lacked BstEII recognition sites, thereby distinguishing them from those of mycobacteria that contain one or more such sites. Of 29 restriction endonucleases, MspI plus HinfI produced RFLP patterns that differentiated 16 of the 20 taxa. A single RFLP pattern was observed for 15 of 20 taxa that included 65% of phenotypically clustered isolates. Multiple patterns were seen with Gordona bronchialis, Nocardia asteroides complex type VI, Nocardia otitidiscaviarum, Nocardia transvalensis, and Streptomyces spp. Streptomyces RFLP patterns were the most heterogeneous (five patterns among 19 isolates), but exhibited a unique HinfI fragment of > 320 bp. RFLP patterns that matched those from type strains of Streptomyces albus, Streptomyces griseus, or Streptomyces somaliensis were obtained from 14 of 19 Streptomyces isolates. Only 10 of 28 isolates of N. otitidiscaviarum failed to yield satisfactory amplicons, while only 6 of 188 (3.2%) clinical isolates exhibited patterns that failed to match one of the 21 defined RFLP patterns. These studies extended the feasibility of using PCR-RFLP analysis as a rapid method for the identification of all clinically significant species and taxa of aerobic actinomycetes.

Actinomyces↗
PubMed2000

Major change in the predominant type of "Norwalk-like viruses" in outbreaks of acute nonbacterial gastroenteritis in Osaka City, Japan, between April 1996 and March 1999.

In Osaka City, Japan, between April 1996 and March 1999, a total of 350 fecal specimens from 64 outbreaks of acute nonbacterial gastroenteritis were examined to investigate infection by "Norwalk-like viruses" (NLVs). By reverse transcription (RT)-PCR, 182 samples (52.0%) from 47 outbreaks (73.4%) were NLV positive. During those three years, the incidence of NLV-associated outbreaks showed seasonality, being higher during January to March (winter to early spring). The ingestion of contaminated oysters was the most common transmission mode (42.6%). The amplicons of the 47 outbreak strains that were NLV positive by RT-PCR were tested using Southern hybridization with four probe sets (Ando et al., J. Clin. Microbiol. 33:64-71, 1995). Forty of the outbreak strains were classified as 4 probe 1-A (P1-A) strains, 6 P1-B strains, 10 P2-A strains, 17 P2-B strains, and 3 untypeable strains, and the other 7 outbreaks were determined to be mixed-probe-type strains. Probe typing and partial sequence analysis of the outbreak strains indicated that a predominant probe type of NLVs in Osaka City had drastically changed; P2-B strains (77.8%) with multiple genetic clusters were observed during the 1996-97 season, the P2-A common strain (81.3%) related to the Toronto virus cluster was observed during the 1997-98 season, and P1-B strains (75.0%) with a genetic similarity were observed during the 1998-99 season. For the three untypeable outbreak strains (96065, 97024, and 98026), the 98026 outbreak strain had Southampton virus (SOV)-like sequences, and each of the other outbreak strains had a unique 81-nucleotide sequence. Newly designed probes (SOV probe for the 98026 outbreak strain and the 96065 probe for the 96065 and 97024 outbreak strains) were hybridized with relative strains and without other probe type strains. The prevalent NLV probe types in Osaka City during those three years were classified in six phylogenetic groups: P1-A, P1-B, P2-A, P2-B, SOV, and 96065 probe types.

Blotting, Southern↗
PubMed2005

Transmission of Burkholderia cepacia complex: evidence for new epidemic clones infecting cystic fibrosis patients in Italy.

To analyze national prevalence, genomovar distribution, and epidemiology of the Burkholderia cepacia complex in Italy, 225 putative B. cepacia complex isolates were obtained from 225 cystic fibrosis (CF) patients attending 18 CF centers. The genomovar status of these isolates was determined by a polyphasic approach, which included whole-cell protein electrophoresis and recA restriction fragment length polymorphism (RFLP) analysis. Two approaches were used to genotype B. cepacia complex isolates: BOX-PCR fingerprinting and pulsed-field gel electrophoresis (PFGE) of genomic macrorestriction fragments. A total of 208 (92%) of 225 isolates belonged to the B. cepacia complex, with Burkholderia cenocepacia as the most prevalent species (61.1%). Clones delineated by PFGE were predominantly linked to a single center; in contrast, BOX-PCR clones were composed of isolates collected either from the same center or from different CF centers and comprised multiple PFGE clusters. Three BOX-PCR clones appeared of special interest. One clone was composed of 17 B. cenocepacia isolates belonging to recA RFLP type H. These isolates were collected from six centers and represented three PFGE clusters. The presence of insertion sequence IS 1363 in all isolates and the comparison with PHDC reference isolates identified this clone as PHDC, an epidemic clone prominent in North American CF patients. The second clone included 22 isolates from eight centers and belonged to recA RFLP type AT. The genomovar status of strains with the latter RFLP type is not known. Most of these isolates belonged to four different PFGE clusters. Finally, a third clone comprised nine B. pyrrocinia isolates belonging to recA RFLP type Se 13. They represented three PFGE clusters and were collected in three CF centers.

Bacterial Typing Techniques↗
PubMed1999

Picornavirus internal ribosome entry site elements target RNA cleavage events induced by the herpes simplex virus virion host shutoff protein.

The herpes simplex virus (HSV) virion host shutoff (vhs) protein (UL41 gene product) is a component of the HSV virion tegument that triggers shutoff of host protein synthesis and accelerated mRNA degradation during the early stages of HSV infection. vhs displays weak amino acid sequence similarity to the fen-1 family of nucleases and suffices to induce accelerated RNA turnover through endoribonucleolytic cleavage events when it is expressed as the only HSV protein in a rabbit reticulocyte in vitro translation system. Although vhs selectively targets mRNAs in vivo, the basis for this selectivity remains obscure, since in vitro activity is not influenced by the presence of a 5' cap or 3' poly(A) tail. Here we show that vhs activity is greatly altered by placing an internal ribosome entry site (IRES) from encephalomyocarditis virus or poliovirus in the RNA substrate. Transcripts bearing the IRES were preferentially cleaved by the vhs-dependent endoribonuclease at multiple sites clustered in a narrow zone located immediately downstream of the element in a reaction that did not require ribosomes. Targeting was observed when the IRES was located at the 5' end or placed at internal sites in the substrate, indicating that it is independent of position or sequence context. These data indicate that the vhs-dependent nuclease can be selectively targeted by specific cis-acting elements in the RNA substrate, possibly through secondary structure or a component of the translational machinery.

Animals↗
PubMed1986

Sequence specificity of point mutations induced during passage of a UV-irradiated shuttle vector plasmid in monkey cells.

A simian virus 40-based shuttle vector was used to characterize UV-induced mutations generated in mammalian cells. The small size and placement of the mutagenesis marker (the supF suppressor tRNA gene from Escherichia coli) within the vector substantially reduced the frequency of spontaneous mutations normally observed after transfection of mammalian cells with plasmid DNA; hence, UV-induced mutations were easily identified above the spontaneous background. UV-induced mutations characterized by DNA sequencing were found primarily to be base substitutions; about 56% of these were single-base changes, and 17% were tandem double-base changes. About 24% of the UV-induced mutants carried multiple mutations clustered within the 160-base-pair region sequenced. The majority (61%) of base changes were the G . C----A . T transitions; the other transition (A . T----G . C) and all four transversions occurred at about equal frequencies. Hot spots for UV mutagenesis did not correspond to hot spots for UV-induced photoproduct formation (determined by a DNA synthesis arrest assay); in particular, sites of TT dimers were underrepresented among the UV-induced mutations. These observations suggest to us that the DNA polymerase(s) responsible for mutation induction exhibits a localized loss of fidelity in DNA synthesis on UV-damaged templates such that it synthesizes past UV photoproducts, preferentially inserting adenine, and sometimes misincorporates bases at undamaged sites nearby.

Animals↗
PubMed2004

Bone marrow engraftment in a rodent model of chemical carcinogenesis but no role in the histogenesis of hepatocellular carcinoma.

BACKGROUND AND AIM: Recent studies indicated that hepatic stem cells in the bone marrow could differentiate into mature hepatocytes, suggesting that bone marrow cells could be used for replacement of damaged hepatocytes in a variety of liver diseases. Hepatocellular carcinoma (HCC) is thought to arise from hepatic stem cells. In this study, we investigated the malignant potential of hepatic stem cells derived from the bone marrow in a mouse model of chemical hepatocarcinogenesis. METHODS: Bone marrow cells were obtained from the male beta-galactosidase (beta-gal) transgenic mouse and transplanted into female recipient mice. Hepatocarcinogenesis was induced by a year of treatment with diethylnitrosamine and phenobarbital (NDEA/PB). One year later, the liver was removed from each treated mouse and evaluated by x-gal staining, immunohistochemistry, and fluorescence in situ hybridisation (FISH). RESULTS: Forty per cent of recipient mice survived and developed multiple HCC. Clusters of beta-gal positive mature hepatocytes were detected sporadically in the entire liver of NDEA/PB treated mice who underwent bone marrow transplantation (BMT) with while no such hepatocytes were identified in the liver of BMT mice that were not treated with NDEA/PB. The Y chromosome was detected with the same frequency as the donor male liver in clusters of beta-gal positive mature hepatocytes by FISH. However, no HCC was positive for beta-gal or the Y chromosome. Immunohistochemically, beta-gal positive mature hepatocytes did not express CD34 or alpha-fetoprotein. CONCLUSIONS: Our results suggest that hepatic stem cells derived from the bone marrow have low malignant potential, at least in our model.

Animals↗
PubMed1977

Intradiploic neural heterotopia. A rare calvarial defect.

Two proved cases of intradiploic neural heterotopia of the skull, a rare cause of calvarial defects, are presented. The literature is reviewed. Common radiographic characteristics include an intradiploic rarefaction, a well-defined margin with or without sclerosis, a lobulated appearance with normal intervening bone, or a cluster of multiple lytic areas with normal bone between them.

Adult↗
PubMed1987

Testicular cysts: US findings.

Testicular cysts have been considered rare. However, of 307 men who underwent high-resolution ultrasonography (10 MHz) of the scrotum, 30 (9.8%) had testicular cysts. Thirty-three testes were involved, 23 with single cysts, eight with clusters of multiple tiny cysts, and two with more than one focus of cyst. One cyst was aspirated intraoperatively, and one cyst of the tunica albuginea was proved at surgery. Eighty cadaveric testes were scanned also, and cysts were found in one. In two cases, the pathologic report, reviewed retrospectively, failed to mention the cyst, but these cysts could have been overlooked. Further work is needed to determine when or if these testicular lesions need follow-up and what significance they have, if any.

Adult↗
PubMed2000

Radio-frequency ablation of renal cell carcinoma: early clinical experience.

PURPOSE: To report the authors' early experience with radio-frequency (RF) ablation of renal cell carcinoma. MATERIALS AND METHODS: Twenty-four percutaneous RF ablation treatments for nine tumors were performed in eight patients with renal cell carcinoma. Indications included coexistent morbidity, previous surgery, or solitary kidney in patients with a life expectancy shorter than 10 years. Smaller (</=3-cm) peripheral lesions (n = 3) were treated with single electrodes. All but one of the larger (>3 cm) and/or central lesions (n = 6) were treated with cluster or multiple electrodes. Patients returned for a second treatment when follow-up imaging depicted tumor enhancement. Follow-up imaging was performed at 1 and 3 months and then at 6-month intervals, with a mean follow-up of 10.3 months. Seven patients were alive at least 6 months after their initial treatment. RESULTS: All five exophytic tumors were free of enhancement. One of three central tumors was free of enhancement. One tumor had both central and exophytic components and was free of enhancement. Three tumors were 3 cm or smaller and free of enhancement. Of the six tumors larger than 3 cm, four were free of enhancement. CONCLUSION: Percutaneous RF ablation is a promising treatment for select patients with renal cell carcinoma. The ultimate role of this modality will continue to evolve and warrants further study.

Aged↗
PubMed1997

Time course of forward masking tuning curves in cat primary auditory cortex.

Nonsimultaneous two-tone interactions were studied in the primary auditory cortex of anesthetized cats. Poststimulatory effects of pure tone bursts (masker) on the evoked activity of a fixed tone burst (probe) were investigated. The temporal interval from masker onset to probe onset (stimulus onset asynchrony), masker frequency, and intensity were parametrically varied. For all of the 53 single units and 58 multiple-unit clusters, the neural activity of the probe signal was either inhibited, facilitated, and/or delayed by a limited set of masker stimuli. The stimulus range from which forward inhibition of the probe was induced typically was centered at and had approximately the size of the neuron's excitatory receptive field. This "masking tuning curve" was usually V shaped, i.e., the frequency range of inhibiting masker stimuli increased with the masker intensity. Forward inhibition was induced at the shortest stimulus onset asynchrony between masker and probe. With longer stimulus onset asynchronies, the frequency range of inhibiting maskers gradually became smaller. Recovery from forward inhibition occurred first at the lower- and higher-frequency borders of the masking tuning curve and lasted the longest for frequencies close to the neuron's characteristic frequency. The maximal duration of forward inhibition was measured as the longest period over which reduction of probe responses was observed. It was in the range of 53-430 ms, with an average of 143 +/- 71 (SD) ms. Amount, duration and type of forward inhibition were weakly but significantly correlated with "static" neural receptive field properties like characteristic frequency, bandwidth, and latency. For the majority of neurons, the minimal inhibitory masker intensity increased when the stimulus onset asynchrony became longer. In most cases the highest masker intensities induced the longest forward inhibition. A significant number of neurons, however, exhibited longest periods of inhibition after maskers of intermediate intensity. The results show that the ability of cortical cells to respond with an excitatory activity depends on the temporal stimulus context. Neurons can follow higher repetition rates of stimulus sequences when successive stimuli differ in their spectral content. The differential sensitivity to temporal sound sequences within the receptive field of cortical cells as well as across different cells could contribute to the neural processing of temporally structured stimuli like speech and animal vocalizations.

Acoustic Stimulation↗
PubMedDate not supplied

Psychosocial indexes and cardiovascular risk factors in a community sample.

BACKGROUND: Psychosocial characteristics might contribute to a more comprehensive understanding of cardiovascular disease as it is increasingly recognised that biomedical risk factors do not fully explain its dynamics. This study aimed to describe psychometric indexes in a Portuguese community sample relating them to known cardiovascular disease risk factors. METHODS: Anthropometric, blood pressure, serum measurements and information on demographic, social, medical and behavioural characteristics were obtained for 215 women and 156 men. Self-administered questionnaires were used for the psychometric evaluation (Bortner scale, Beck Depression Inventory, Hopkins Symptom Distress Checklist 90-Revised, Psychological General Well-Being and the Nottingham Health Profile, NHP). RESULTS: There were significant differences according to gender regarding almost every psychometric dimension assessed. After adjusting for the presence of different biomedical risk factors, significant decreasing mean behaviour pattern scores were found with increasing age. Mean depression scores were significantly higher in women and in individuals with lower educational level. As to general psychological well-being, the highest scores were obtained for men and individuals with higher educational level. For the assessed dimensions of quality of life, as well as indexes of psychopathology, men scored better than women. No significant differences in mean scores of behaviour pattern, depression, psychological general well-being and dimensions of NHP were found according to the presence of an increasing number of cardiovascular risk factors. CONCLUSIONS: The study shows that the clustering of multiple biomedical risk factors does not result in additive deleterious psychological effects and that psychometric indexes are mainly dependent on gender and education, common confounders in most studies evaluating cardiovascular risk factors.

Adult↗
PubMedDate not supplied

Perceived difficult temperament, hostile maternal child-rearing attitudes and insulin resistance syndrome precursors among children: a 3-year follow-up study.

BACKGROUND: We prospectively examined the relationship between latent constructs of perceived difficult temperament and hostile maternal child-rearing attitudes on the one hand, and the cluster of multiple cardiovascular risk factors comprising the insulin resistance syndrome (IRS) on the other. METHODS: A 3-year follow-up study of 451 randomly selected healthy children was used. The temperament of the children and maternal child-rearing attitudes were self-rated by the mothers. The physiological parameters measured were serum insulin, high-density lipoprotein cholesterol (HDL-C), triglycerides, systolic blood pressure, body-mass index and subscapular skinfold thickness. We analyzed the data using structural equation models that involved specific paths allowing separation, and tests of both unique and common (latent) aspects of measured variables. RESULTS: Among boys we found a cross-lagged effect between the latent constructs of perceived difficult temperament and IRS, as well as 1 specific effect from hostile maternal child-rearing attitudes to HDL-C. Among girls there were 6 specific effects from specific measures of perceived temperamental difficultness or hostile maternal child-rearing attitudes that influenced the IRS or its components. CONCLUSIONS: The findings suggest that temperament and maternal child-rearing attitudes may contribute to the early development of the IRS in a gender-specific way among children, thereby potentially increasing the risk of coronary heart disease later in life.

Adolescent↗
PubMedDate not supplied

Prediction of treatment outcome of patients with functional gastrointestinal disorders by the diagnostic criteria for psychosomatic research.

BACKGROUND: The Diagnostic Criteria for Psychosomatic Research (DCPR) have been demonstrated to be useful in identifying specific psychological conditions of medical patients. The aim of this study was to evaluate the clinical utility of the DCPR in predicting the treatment outcome of patients with functional gastrointestinal disorders (FGID). METHODS: FGID outpatients were allocated to improved (n = 65) and unimproved (n = 40) groups on the basis of preestablished criteria following 6 months of treatment. Patients were administered the structured interview for DCPR at baseline and the Gastrointestinal Symptom Rating Scale both at baseline and follow-up. RESULTS: In the unimproved patients, the prevalence of the DCPR categories of alexithymia (82.2%) and persistent somatization (72.5%) was significantly higher while health anxiety was more prevalent in improved patients (21.5%). No unimproved patient lacked a DCPR diagnosis while multiple DCPR diagnoses were significantly higher in the unimproved group (90%). In the regression analysis, alexithymia, persistent somatization, a higher number of DCPR diagnoses for each patient and, to a lesser extent, greater symptom severity at baseline were significant predictors of unimprovement. Health anxiety, even after controlling for gastrointestinal symptoms, was a significant predictor of improvement. CONCLUSIONS: The ability to predict treatment outcome indicates the clinical utility of the DCPR. Clinicians may improve treatment outcome for FGID patients by identifying particular psychosomatic syndromes (alexithymia, persistent somatization, and health anxiety) and patients with multiple DCPR clusters, and attempting to address specific therapeutic interventions.

Adult↗
PubMed2004

Partial unilateral lentiginosis with ipsilateral Lisch nodules and axillary freckling.

A 9-year-old Taiwanese girl had an unusual combination of multiple lentigines clustered on her left face, 3 cafe-au-lait spots on her left arm and ipsilateral axillary freckling. Two Lisch nodules of the iris were recognized in the left eye but none in the right eye, which is why we established a diagnosis of segmental neurofibromatosis 1. No family member had any sign of neurofibromatosis 1. Moreover, the patient had a large speckled lentiginous nevus involving the left side of her trunk, and her father showed a similar skin disorder on his left buttock. These lesions were taken to be unrelated to the patient's segmental neurofibromatosis 1. This case provides further evidence in favor of the concept that partial unilateral lentiginosis represents a mosaic manifestation of type 1 neurofibromatosis.

Axilla↗
PubMed2001

Mobilization of endothelial progenitor cells in patients with acute myocardial infarction.

BACKGROUND: Endothelial progenitor cells (EPCs) circulate in adult peripheral blood (PB) and contribute to neovascularization. However, little is known regarding whether EPCs and their putative precursor, CD34-positive mononuclear cells (MNC(CD34+)), are mobilized into PB in acute ischemic events in humans. METHODS AND RESULTS: Flow cytometry revealed that circulating MNC(CD34+) counts significantly increased in patients with acute myocardial infarction (n=16), peaking on day 7 after onset, whereas they were unchanged in control subjects (n=8) who had no evidence of cardiac ischemia. During culture, PB-MNCs formed multiple cell clusters, and EPC-like attaching cells with endothelial cell lineage markers (CD31, vascular endothelial cadherin, and kinase insert domain receptor) sprouted from clusters. In patients with acute myocardial infarction, more cell clusters and EPCs developed from cultured PB-MNCs obtained on day 7 than those on day 1. Plasma levels of vascular endothelial growth factor significantly increased, peaking on day 7, and they positively correlated with circulating MNC(CD34+) counts (r=0.35, P=0.01). CONCLUSIONS: This is the first clinical demonstration showing that lineage-committed EPCs and MNC(CD34+), their putative precursors, are mobilized during an acute ischemic event in humans.

Aged↗
PubMed1997

Racial differences in risk factors for atherosclerosis. The ARIC Study. Atherosclerosis Risk in Communities.

This paper describes black/white differences in risk factors for atherosclerosis in the large multicenter Atherosclerosis Risk in Communities (ARIC) Project sponsored by the National Heart, Lung, and Blood Institute. It is based on data collected at baseline in ARIC's four geographically distinct clinical centers. Participants were randomly selected (4264 black and 11,479 white men and women, ages forty-five to sixty-four years at entry). There were striking differences in obesity between black and white women, higher fasting glucose and greater prevalence of diabetes in blacks, and lower high-density lipoprotein values in white men. Not unexpectedly, blood pressure in black participants exceeded that in whites. Clustering of multiple risk factors was more common in the black population. Conversely, prevalence of no risk factors was greatest among whites. In conclusion, while African-Americans and Caucasians share much the same group of risk factors for atherosclerosis, there are clinically important racial differences in emphasis.

Arteriosclerosis↗
PubMed2004

New developments in cancer-related computational statistics.

A brief overview is presented of recently developed and currently emerging statistical and computational techniques that have been proved to be highly helpful in handling the avalanche of the new type of data generated by modern high-throughput technologies in experimental biology. The review, in no way comprehensive, focuses attention on Bayesian Networks, Hidden Markov Chain, and methods of chaotic dynamics for time-course genomic data; innovative methods in optimization and clustering; and multiple testing in the context of identification of differentially expressed genes.

Bayes Theorem↗