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PubMed2004

Auto-validation of fluorescent primer extension genotyping assay using signal clustering and neural networks.

BACKGROUND: SNP genotyping typically incorporates a review step to ensure that the genotype calls for a particular SNP are correct. For high-throughput genotyping, such as that provided by the GenomeLab SNPstream instrument from Beckman Coulter, Inc., the manual review used for low-volume genotyping becomes a major bottleneck. The work reported here describes the application of a neural network to automate the review of results. RESULTS: We describe an approach to reviewing the quality of primer extension 2-color fluorescent reactions by clustering optical signals obtained from multiple samples and a single reaction set-up. The method evaluates the quality of the signal clusters from the genotyping results. We developed 64 scores to measure the geometry and position of the signal clusters. The expected signal distribution was represented by a distribution of a 64-component parametric vector obtained by training the two-layer neural network onto a set of 10,968 manually reviewed 2D plots containing the signal clusters. CONCLUSION: The neural network approach described in this paper may be used with results from the GenomeLab SNPstream instrument for high-throughput SNP genotyping. The overall correlation with manual revision was 0.844. The approach can be applied to a quality review of results from other high-throughput fluorescent-based biochemical assays in a high-throughput mode.

Artificial Intelligence↗
PubMed1984

Multiple gene deletions within the human immunoglobulin heavy-chain cluster.

Two subjects, of 11,000 healthy individuals screened, were found to be missing three and four immunoglobulin isotypes, respectively (IgA1, IgG2, and IgG4; IgA1, IgG2, IgG4, and IgE), and have been analyzed at the DNA level by means of Southern blotting and Ig heavy-chain-specific probes. A broad deletion within the heavy-chain constant region (C) gene cluster was found on chromosome 14 of both probands. Two different haplotypes are described: the first has lost the C alpha 1, C psi gamma, C gamma 2, C gamma 4, and C epsilon genes; the second lacks the C psi epsilon, C alpha 1, C psi gamma, C gamma 2, and C gamma 4 genes. These findings confirm the reciprocal order of the Ig heavy-chain genes as derived by molecular cloning. The inclusion of the C psi gamma gene within the deleted regions confirms its location between C alpha 1 and C gamma 2. From the observed frequency of the homozygous genotype, 1%-3% of healthy subjects from our population are expected to be heterozygous for multiple heavy-chain gene deletions. Cross-over between mispaired homologous regions seems to be the favored mechanism of multiple Ig gene deletions and duplications, and, generally, in the evolution of the human Ig heavy-chain gene family.

Alleles↗
PubMed2001

Eradication of multi-drug resistant Acinetobacter from an intensive care unit.

BACKGROUND: Acinetobacter baumannii is a gram-negative coccobacillus that causes outbreaks of nosocomial infections in ICUs. Due to resistance to multiple antibiotics, management of clusters of A. baumannii is useful as a model in eradication of multi-drug resistant infections. We outline the evolution of an A. baumannii outbreak, focusing on methods of transmission and multidisciplinary measures aimed at eliminating it from the ICU. METHODS: Patients in an urban, tertiary care medical center from November 1996 to December 1997 having positive cultures for multi-drug resistant A. baumannii are included in this study. A. baumannii was isolated on blood agar and MacConkey cultures and identified by Vitek panel. Disk diffusion including amikacin, imipenem, polymyxin B, and sulbactam were used to determine resistance. RESULTS: An outbreak of 52 patients (27 infected, 25 colonized) with 68 positive sites began with the transfer of a colonized >50% total body surface area burn patient from an outside hospital. Within 3 days, the index patient was in the burn ICU, coronary care unit, and medical ICU. Soon, clusters of patients with A. baumannii infections sensitive only to polymyxin B were seen in those units and, ultimately, the surgical ICU. On typing, 2 strains were found, PFGE B and C. Given the level of antibiotic resistance, patients with colonization or infection were cohorted and placed on contact isolation. Strict antiseptic measures, such as hand-washing, barrier isolation, equipment and room cleaning, sterilization of ventilator equipment, and dedication of medical equipment to each patient were instituted. Still, positive environmental cultures were found in ventilator water traps, sinks, and bedrails. Sporadic cases continued for a total of 13 months, with 10 deaths resulting from the infections. CONCLUSION: A. baumannii is a mildly virulent organism that becomes resistant to antimicrobials. Because of multiple antibiotic resistance, strict contact isolation cohorting and antiseptic technique are the primary modes of containment. This outbreak serves as a model of eradication of multi-drug resistant organisms from ICUs. These measures will become of greater importance as nosocomial organisms develop increasing resistance to antimicrobials.

Acinetobacter Infections↗
PubMed1994

Clustered charged-to-alanine mutagenesis of poliovirus RNA-dependent RNA polymerase yields multiple temperature-sensitive mutants defective in RNA synthesis.

To generate a collection of conditionally defective poliovirus mutants, clustered charged-to-alanine mutagenesis of the RNA-dependent RNA polymerase 3D was performed. Clusters of charged residues in the polymerase coding region were replaced with alanines by deoxyoligonucleotide-directed mutagenesis of a full-length poliovirus cDNA clone. Following transfection of 27 mutagenized cDNA clones, 10 (37%) gave rise to viruses with temperature-sensitive (ts) phenotypes. Three of the ts mutants displayed severe ts plaque reduction phenotypes, producing at least 10(3)-fold fewer plaques at 39.5 degrees C than at 32.5 degrees C; the other seven mutants displayed ts small-plaque phenotypes. Constant-temperature, single-cycle infections showed defects in virus yield or RNA accumulation at the nonpermissive temperature for eight stable ts mutants. In temperature shift experiments, seven of the ts mutants showed reduced accumulation of viral RNA at the nonpermissive temperature and showed no other ts defects. The mutations responsible for the phenotypes of most of these ts mutants lie in the N-terminal third of the 3D coding region, where no well-characterized mutations responsible for viable mutants had been previously identified. Clustered charged-to-alanine mutagenesis (S. H. Bass, M. G. Mulkerrin, and J. A. Wells, Proc. Natl. Acad. Sci. USA 88:4498-4502, 1991; W. F. Bennett, N. F. Paoni, B. A. Keyt, D. Botstein, J. J. S. Jones, L. Presta, F. M. Wurm, and M. J. Zoller, J. Biol. Chem. 266:5191-5201, 1991; and K. F. Wertman, D. G. Drubin, and D. Botstein, Genetics 132:337-350, 1992) is designed to target residues on the surfaces of folded proteins; thus, extragenic suppression analysis of such mutant viruses may be very useful in identifying components of the viral replication complex.

Alanine↗
PubMed2000

Proliferating ductules are a diagnostic discriminator for intrahepatic cholangiocarcinoma in FNA biopsies.

The histologic distinction of cholangiocarcinoma (CC) from metastatic carcinoma and hepatocellular carcinoma (HCC) is difficult. In particular, the distinction of CC from metastatic carcinoma on morphologic features alone is not possible and is dependent on the identification of an extrahepatic primary carcinoma. The proliferative response to many types of liver injury is characterized by a proliferation of either hepatocyte ductular clusters (HDC) or biliary ductular clusters (BDC). This study examined the presence of such ductular reactions in fine needle aspiration biopsies of 20 consecutive cases each of CC and HCC, and compared the findings to those of 20 cases of hepatic metastases from a wide variety of sites. All 18 cases of CC with adequate smears showed ductular proliferation of either HDC or BDC type, the latter being more common; in 13 cases, there were more than 10 ductular clusters per smear. In contrast, only one case of metastatic carcinoma displayed so many ductular clusters, this being a case with multiple hepatic deposits. Five cases of HCC showed more than 10 clusters. The presence of more than 10 ductular clusters associated with malignant cells is a useful discriminator to separate CC from metastatic carcinoma.

Adenocarcinoma↗
PubMedDate not supplied

Recurrent wavefront morphologies: a method for quantifying the complexity of epicardial activation patterns.

We have developed a method for quantifying the complexity of activation patterns observed during ventricular fibrillation (VF) that is based on our previously reported methodology for decomposing epicardial mapping data into a set of isolated wavefronts. One-half second datasets are acquired from a 21 x 24 array of unipolar electrodes (1 mm spacing), and the wavefronts are isolated. A correlation technique is used to compute the similarity between all possible pairs of the isolated wavefronts. From these data, the wavefronts are sorted into clusters, each of which represents a recurring wavefront morphology. We define multiplicity (M) as the number of clusters needed to account for 90% of the total activations in the VF episode. M measures the complexity of the rhythm. In repetitive patterns (e.g., sinus rhythm), M = 1, indicating that the same morphology repeatedly activates the mapped region. Typically, in VF, M > 1, with larger numbers representing more complex, disorganized patterns. As an example, we computed M at 5, 10, 15, and 20 sec after electrical induction of VF in six pigs. M decreased significantly (p < 0.001), suggesting increasing organization during this period.

Algorithms↗
PubMed2002

Genotypic analysis at multiple loci across Kaposi's sarcoma herpesvirus (KSHV) DNA molecules: clustering patterns, novel variants and chimerism.

BACKGROUND: The genomes of human Kaposi's sarcoma-associated herpesvirus (KSHV) display several levels of DNA sequence heterogeneity and subgrouping that show distinctive clustering patterns in related human populations. The four major subtype patterns for the hypervariable ORF-K1 protein correlate closely with the principal diasporas resulting from the migration of modern humans out of East Africa and suggest that KSHV is an ancient human virus that is transmitted primarily in a familial fashion with consequent very low recombination rates. However, chimeric genomes have also been detected, especially with regard to the presence of P versus M alleles of the ORF-K15 gene. OBJECTIVES: To understand further the genetic organization and evolutionary history of KSHV, especially with regard to possible new subtypes, recombinant genomes, constant region loci and clustering in particular ethnic groups or among classic versus epidemic cases in the same geographic area. STUDY DESIGN: Direct PCR DNA sequencing was carried out on the ORF-K1 and ORF-K15 genes at the extreme left and right hand sides, as well as on six other internal loci of diagnostic samples collected from 70 new KSHV-positive patients in Israel, South Korea, Sicily, Scandinavia, Brazil, Uganda, South Africa and the US. RESULTS AND CONCLUSIONS: Our overall results from more than 135 KSHV genomes from many different human population groups now provides evidence for seven distinct subtypes of KSHV genomes (referred to as A/P, B/P, C/P, D/P, M, N and Q). However, the two most closely related subtypes (A/P and C/P) are only differentiated at the LHS side of the genome, and the three most distantly related forms (M, N and Q) appear to exist only as small chimeric segments that are remnants from the RHS of more ancient forms of the virus. By analyzing multiple conserved loci across the B subtype genomes that predominate in sub-Saharan Africa, we can also now recognize three to four distinct B genome subgroups with varying patterns of inter and intratypic mosaicism. Analysis of classic KS genomes from Israel has revealed that the ORF-K1 clade referred to as A1' predominates in Ashkenazi Jewish immigrants from Russia, whereas C2 and C6 variants predominate in North African Sephardi Jews. A variety of chimeric genomes containing C2 or C3 ORF-K1 genes are disseminated among classic KS cases throughout Europe and Asia including Israel, Sicily, Scandinavia, South Korea, and Taiwan. Comparison of the genomes from classic versus AIDS-associated KSHV in the US indicates that it was derived originally by reactivation and spread of a subset of the endogenous viruses carried by descendants of immigrants from endemic areas of Northern and Eastern Europe, the Mediterranean and sub-Saharan Africa.

Acquired Immunodeficiency Syndrome↗
PubMed2006

Clustering of cardiovascular risk factors associated with the insulin resistance syndrome: assessment by principal component analysis in young hyperandrogenic women.

OBJECTIVE: Hyperinsulinemia is often associated with several metabolic abnormalities and increased blood pressure, which are risk factors for cardiovascular disease. It has been hypothesized that insulin resistance may underlie all these features. However, recent data suggest that some links between insulin resistance and these alterations may be indirect. The aim of our study was to further investigate this issue in a sample of young hyperandrogenic women, who often show insulin resistance and other metabolic abnormalities typical of the insulin resistance syndrome. RESEARCH DESIGN AND METHODS: We tested the hypothesis of a single factor underlying these features by principal component analysis, which should recognize one component if a single mechanism explains this association. The analysis was carried out in a sample of 255 young nondiabetic hyperandrogenic women. Variables selected for this analysis included the basic features of the insulin resistance syndrome and some endocrine parameters related to hyperandrogenism. RESULTS: Principal component analysis identified four separate factors, explaining 64.5% of the total variance in the data: the first included fasting and postchallenge insulin levels, BMI, triglycerides, HDL cholesterol, and uric acid; the second, BMI, blood pressure, and serum free testosterone; the third, fasting plasma glucose, postchallenge glucose and insulin levels, serum triglycerides, and free testosterone; and the fourth, postchallenge plasma insulin, serum free testosterone, and gonadotropin-releasing hormone agonist-stimulated 17-hydroxyprogesterone. CONCLUSIONS: These results support the hypothesis of multiple determinants in the clustering of abnormalities in the so-called insulin resistance syndrome.

Adult↗
PubMed2002

Epidemic decompression sickness: case report, literature review, and clinical commentary.

BACKGROUND: Decompression sickness (DCS) is a syndrome of symptoms caused by bubbles of inert gas. These bubbles are produced by a significant ambient pressure drop. Although cases are usually solitary there have been several episodes of DCS clusters. This paper reports an episode of epidemic decompression sickness and reviews the literature. METHODS: The case reported describes six aircrewmen with DCS following an unpressurized AC-130 flight (maximum altitude 17,000 ft). Two obvious concerns-the low altitude at which DCS was encountered and the potential for epidemic hysteria-are discussed and discounted. In addition, factors contributing to this case are recounted in depth. Moreover, the literature was examined for similar cases of epidemic decompression sickness. Four other instances were discovered. Detailed qualitative analysis of these five reports was performed. RESULTS: With this information epidemic decompression sickness is defined and classified. Two types are described-individual-based (Epi-I) and population-based (Epi-P). Epi-I is a cluster of DCS following a solitary exposure; whereas, Epi-P is a cluster of DCS following multiple exposures over time. Investigation of Epi-P follows the classical rules of outbreak investigation (time, place, person, and environment); whereas, Epi-I does not. In fact, the focus in Epi-I is almost entirely the environment. Following this outline should produce an etiology that control measures can be directed against. However, it is prudent to look beyond the etiology. Enter the Haddon Matrix, a classic public health tool that considers counter-measures before, during, and after the event. CONCLUSION: These many concepts are illustrated with the presented case. Following this template, both the expert and the novice flight surgeon have a systematic and reproducible approach to these difficult puzzles.

Adult↗
PubMed1982

Regional obstetric anesthesia and newborn behavior: a reanalysis toward synergistic effects.

The Brazelton Neonatal Behavioral Assessment Scale was administered to 54 term, healthy infants on days 1, 2, 3, 4, 5, 7, and 10. Infants were divided into 8 groups of maternal obstetrical medication. A separate drug factor score was computed from 4 variables: time from (1) first and (2) last drug administration to delivery, (3) number of different drugs, and (4) drug administrations. In order to determine the additive effects of drug and other obstetric factors on neonatal behavior, the drug group and drug factor score plus length of labor, parity, and the ponderal index were used in a series of multiple regression analyses to predict Brazelton scale cluster scores on each day. In a second set of multiple regressions, these variables were used to predict the infant's rate of behavioral change as estimated by the slopes of the Brazelton scale cluster scores across the 7 days. The results indicated that the combined effect of the predictor variables significantly explained 10%-28% of the variance in neonatal behavior. It was hypothesized that the effects of low levels of medication are subtle but may be unmasked when medication effects are studied in combination with other factors that may potentiate drug effects.

Alphaprodine↗
PubMedDate not supplied

Detection of injury-prone behaviors among internal medicine patients.

OBJECTIVES: 1) To describe the prevalence of eight injury-prone behaviors (IPBs) and the associations of these behaviors with ten standard chronic disease and sociodemographic risk factors (CDSRFs) among internal medicine patients and 2) to identify a subset of patients with multiple IPBs who might be at particularly high risk of injury. DESIGN: Cross-sectional survey. SETTING: The University of Colorado Health Sciences Center general internal medicine clinic. PATIENTS: Four hundred ninety-two consecutive continuity care patients were eligible. The response rate was 94.3% (464/492). INSTRUMENT: A validated, self-administered questionnaire. MAIN RESULTS: Thirty-four percent of patients did not wear safety belts regularly and 32% had no smoke detector in their homes. Nearly 26% of patients had firearms at home and 6% had a loaded and unlocked gun at home. Fourteen percent of patients had seriously thought about suicide and 6% had attempted suicide. In the prior month, 10% had ridden with a drunk driver, and 4% had driven after drinking too much. Of patients aged 65 years or older, 50% had had recent falls. After adjustment for other CDSRFs, problem drinking was the CDSRF most frequently associated with IPBs. Among men, problem drinking was significantly associated with drinking and driving (OR = 35.3), safety belt non-use (OR = 4.3), and previous thoughts of or attempts at suicide (OR = 6.2). Among women, problem drinking was significantly associated with drinking and driving (OR = 8.7). Among men, being unmarried was the demographic risk factor associated with the most IPBs. Of all IPBs, safety belt non-use was most frequently associated with CDSRFs (ORs ranged from 2.8 to 4.4). Men with three or more IPBs were more likely to be problem drinkers (OR = 9.6), smokers (OR = 15.8), obese (OR = 6.3), and unmarried (OR = 67.1). CONCLUSIONS: 1) Injury-prone behaviors are common among patients attending a university-based internal medicine clinic; 2) men and women have substantially different patterns of risk factor associations; 3) CDSRFs, such as problem drinking smoking, and being unmarried, are associated with many IPBs; 4) safety belt non-use is the IPB associated with the most CDSRFs; and 5) CDSRFs appear to cluster in men with multiple IPBs, suggesting that screening for high-risk individuals may be feasible in clinical practice.

Accident Proneness↗
PubMed1998

Evaluating cluster alarms: a space-time scan statistic and brain cancer in Los Alamos, New Mexico.

OBJECTIVES: This article presents a space-time scan statistic, useful for evaluating space-time cluster alarms, and illustrates the method on a recent brain cancer cluster alarms in Los Alamos, NM. METHODS: The space-time scan statistic accounts for the preselection bias and multiple testing inherent in a cluster alarm. Confounders and time trends can be adjusted for. RESULTS: The observed excess of brain cancer in Los Alamos was not statistically significant. CONCLUSIONS: The space-time scan statistic is useful as a screening tool for evaluating which cluster alarms merit further investigation and which clusters are probably chance occurrences.

Brain Neoplasms↗
PubMed1996

Epidemiological typing of isolates from an outbreak of infection with multidrug-resistant Enterobacter cloacae by repetitive extragenic palindromic unit b1-primed PCR and pulsed-field gel electrophoresis.

An outbreak of multidrug-resistant Enterobacter cloacae infection lasted for 4 months in a neonatal intensive care unit (NICU). Forty-six isolates from the NICU and 20 epidemiologically unrelated strains were characterized by pulsed-field gel electrophoresis (PFGE) and repetitive extragenic palindromic unit b1-primed PCR (REPUb1-PCR) typing. The PFGE patterns after XbaI restriction of the bacterial DNA were analyzed by computer software (Gelcompar) using the UPGMA (unweighted pair group method with arithmetic averages) clustering method and the Dice coefficient. The 46 isolates from the NICU were classified by PFGE typing into five clusters: A (further classified into 7 subtypes, A1 to A7), B, C, D, and E. This outbreak was attributed to multiple genetically related strains of cluster A which had a similarity of 85.8% +/- 4.6%. The minor band differences among strains of cluster A were probably due to minor genetic mutations. The type A1 and A3 strains were isolated from the clinical specimens of patients and hands of nurses. It was probable that these outbreak strains were transmitted among patients via the hands of personnel. REPUb1-PCR typing of the 46 isolates also demonstrated five types, in agreement with results obtained by the PFGE technique, but could not detect the minor mutations among the cluster A strains. Twenty epidemiologically unrelated strains were well distinguished by both PFGE and REPUb1-PCR typing. We conclude that PFGE is a highly discriminatory but time-consuming method for epidemiological typing of E. cloacae and that REPUb1-PCR is a more rapid method with good reproducibility and discriminatory power comparable to that of PFGE.

Bacterial Typing Techniques↗
PubMed2000

OEIS complex--a population study.

Using a novel method for the analysis of infants with multiple malformations, we investigated the cluster of associated malformations called the OEIS (omphalocele, bladder exstrophy, imperforate anus, spine defect) complex among 5,260 infants with multiple malformations identified in four large registers of congenital malformations, corresponding to 5.84 million births. The existence of the OEIS complex was clearly demonstrated and malformations entering it could be defined. Other than the four classical malformations, omphalocele, bladder exstrophy, imperforate anus, and spine malformation, a strong association with spina bifida and intersex was stressed. Spine malformations occurred not only in the lumbosacral level but also more cranially, and an association also with upper spina bifida could be demonstrated. No specific association with any other malformation, including cardiac defects, was apparent. The OEIS complex is an unusually clearly defined entity among the various nonrandom associations which have been described.

Abnormalities, Multiple↗
PubMed2005

Small-molecule screening and profiling by using automated microscopy.

Automated fluorescence microscopy provides a powerful tool for analyzing the physiological state of single cells with high throughput and high information content. Here I discuss two types of experiments in which this technology was used to discover and characterize bioactive small molecules. In phenotypic-screening experiments, the goal is to find "hits" with specific effects on cells by screening large libraries of small molecules. An example is screening for small molecules that perturb mitosis by novel mechanisms. In cytological-profiling experiments, the goal is to characterize the bioactivity of a limited number of small molecules in considerable depth, and thus understand their mechanism and toxicities at the cellular level. I discuss an example in which 100 small molecules with known bioactivity were profiled by using multiple fluorescent probes, and clustered into mechanistic classes by automated statistical analysis.

Algorithms↗
PubMed2004

Qualitative and quantitative analysis of amino acids by capillary electrophoresis-electrospray ionization-tandem mass spectrometry.

We describe a method to identify and quantify amino acids using capillary electrophoresis-electrospray ionization-triple-quadrupole tandem mass spectrometry (CE-ESI-MS/MS). Amino acids, including physiological amino acids, were first separated by CE under acidic pH conditions and then detected by MS/MS. To efficiently introduce the whole sample into the capillary, no electrical potential was applied to the electrospray probe until running electrophoresis. The position of the electrosprayer with respect to the MS capillary entrance drastically affected sensitivity and generation of cluster ions. MS/MS with multiple reaction monitoring (MRM) detection was performed to obtain sufficient selectivity and sensitivity. Under optimized CE-MS/MS conditions, the minimum detectable levels for 32 free amino acids normally found in proteins and other physiological amino acids were between 0.1 and 14 micromol/L with pressure injection of 50 mbar for 3 s (3 nL) at a signal-to-noise ratio of 3. For most amino acids, this constitutes a severalfold increase in sensitivity compared to CE-MS. The relative standard deviations (% RSD) for all amino acids were better than 0.4% for migration times and between 1.4% and 8.6% for peak areas (n = 10). Since amino acids exhibited characteristic MS/MS spectra, this approach is useful for the simultaneous, selective, quantitative, and reproducible analysis of amino acids in physiological and biological samples that contain various kinds of matrices. The power of the method was demonstrated by analyzing amino acids in human urine.

Amino Acids↗
PubMed2006

Identification of a new cancer/testis gene family, CT47, among expressed multicopy genes on the human X chromosome.

Cancer/testis (CT) genes are normally expressed in germ cells only, yet are reactivated and expressed in some tumors. Of the approximately 40 CT genes or gene families identified to date, 20 are on the X chromosome and are present as multigene families, many with highly conserved members. This indicates that novel CT gene families may be identified by detecting duplicated expressed genes on chromosome X. By searching for transcript clusters that map to multiple locations on the chromosome, followed by in silico analysis of their gene expression profiles, we identified five novel gene families with testis-specific expression and >98% sequence identity among family members. The expression of these genes in normal tissues and various tumor cell lines and specimens was evaluated by qualitative and quantitative RT-PCR, and a novel CT gene family with at least 13 copies was identified on Xq24, designated as CT47. mRNA expression of CT47 was found mainly in the testes, with weak expression in the placenta. Brain tissue was the only positive somatic tissue tested, with an estimated CT47 transcript level 0.09% of that found in testis. Among the tumor specimens tested, CT47 expression was found in approximately 15% of lung cancer and esophageal cancer specimens, but not in colorectal cancer or breast cancer. The putative CT47 protein consists of 288 amino acid residues, with a C-terminus rich in alanine and glutamic acid. The only species other than human in which a gene homologous to CT47 has been detected is the chimpanzee, with the predicted protein showing approximately 80% identity in its carboxy terminal region.

Antigens, Neoplasm↗