Examination and measurement of coping among adolescents with spinal cord injury.

STUDY DESIGN: Cross-sectional survey. OBJECTIVES: To describe coping strategy use in adolescents with spinal cord injury (SCI), to explore the underlying factor structure of a measure of coping among adolescents with SCI and to assess relationships between coping and psychosocial outcomes. SETTING: Multiple pediatric SCI centers in the United States. METHODS: One hundred and eighty-two participants aged 13-17 years who experienced an SCI completed measures including the Kidcope, Children's Depression Inventory, Revised Children's Manifest Anxiety Scale and the Pediatric Quality of Life Inventory. RESULTS: Participants reported that cognitive restructuring and resignation are the most used coping strategies, whereas social support, emotional regulation (calming) and cognitive restructuring are the most effective coping strategies. An exploratory factor analysis revealed that a three-factor solution provided the most parsimonious model for the relationships between the different coping strategies. However, only one of the three factors had acceptable internal consistency. This factor comprised escape-oriented coping strategies or an avoidant approach to coping with the sequelae of SCI. After controlling for demographic/injury-related factors, higher scores on the escape-oriented factor were associated with the lower quality of life and higher levels of depression and anxiety symptomatology. CONCLUSION: Escape-oriented coping is associated with maladaptive psychosocial outcomes in adolescents with SCI. These adolescents report that active coping strategies are most effective in reducing SCI-related distress. Coping strategy use may mediate psychosocial outcomes in adolescents with SCI and represent an intervention target in adolescents who overly rely on escape-oriented coping.

Real-time PCR in clinical microbiology: applications for routine laboratory testing.

Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods. Real-time PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-time PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory.

Effect of rotation frequency and weaning date on forage measurements and growth performance by cows and calves grazing endophyte-infected tall fescue pastures overseeded with crabgrass and legumes.

A grazing study was initiated in April 2000 and continued through three calving and weaning cycles (ending July 2003) to investigate the effects of rotational grazing management (twice monthly [2M] vs. twice weekly [2W]) and weaning date (mid-April [EARLY] vs. early June [LATE]) on production of fall-calving cow-calf pairs (495 +/- 9.6 kg initial BW) grazing Neotyphodium coenophialum-infected tall fescue (Festuca arundinacea Schreb.) overseeded with legumes and crabgrass. Secondary objectives of the experiment were to monitor differences in quantity and quality of available forage and to evaluate changes in forage species composition. Pastures were dominated by tall fescue throughout the study, and the proportion of basal cover was greater (P < 0.05) in 2M than in 2W pastures. The percentage of legumes was very low across all treatment combinations, but the percentage of crabgrass continued to increase (P < 0.05) linearly and quadratically across years for both summer and fall sampling periods, regardless of rotation or weaning program. In vitro DM disappearance and mineral concentrations varied minimally because of rotation frequency or weaning date. Rotation frequency did not substantially affect (P = 0.11 to 0.97) cow BW, hay offered, milk production, calving interval, calf birth weight, or actual or adjusted weaning weights; however, 2M cows had 0.3 units higher (P < 0.05) BCS at the time of breeding than 2W cows. Calves weaned late had greater (P < 0.05) actual weaning weight and weighed more (P < 0.05) on the LATE weaning date than on the EARLY weaning date, but 205-d adjusted weaning weights did not differ (P = 0.74) across weaning dates. Therefore, rotation frequency and/or weaning date had little effect on forage species composition or forage quality. In addition, the rapid rotation program offered little advantage with respect to animal performance, and weaning fall-born calves grazing endophyte-infected tall fescue pastures at approximately 189 d of age seemed to be detrimental to calf performance compared with delaying weaning until 243 d of age.

Comparison of specimen processing and nucleic acid extraction by the swab extraction tube system versus the MagNA Pure LC system for laboratory diagnosis of herpes simplex virus infections by LightCycler PCR.

A total of 563 specimens (234 dermal and 329 genital swabs) from patients suspected of having herpes simplex virus (HSV) infections were processed using two different extraction methods (the MagNA Pure LC system and the swab extraction tube system [SETS]); HSV DNA was amplified by LightCycler PCR. HSV DNA was detected in 157 of 563 specimens (27.9%) processed by the MagNA Pure LC system and in 179 of 563 specimens (31.8%) processed by SETS (P < 0.0001). There was no specimen processed by the MagNA Pure LC extraction method that was positive only for HSV DNA. Of 157 specimens positive by both methods, HSV DNA copy levels were higher (using cycle crossover points [cycle threshold {C(T)}]) with SETS (mean C(T), 25.9 cycles) than with the MagNA Pure LC system (mean C(T), 32.0 cycles) (P < 0.0001). The time to process 32 samples was longer with the MagNA Pure LC extraction system (90 min) than with SETS (35 min). HSV DNA extraction using SETS is faster, less expensive, and more sensitive than the MagNA Pure LC system and could replace the latter for the laboratory diagnosis of HSV infections using LightCycler PCR.

Comparative quantitation of cytomegalovirus (CMV) DNA in solid organ transplant recipients with CMV infection by using two high-throughput automated systems.

Cytomegalovirus (CMV) DNA quantitation in clinical specimens is progressively becoming a cornerstone in the diagnosis and management of CMV infection in the immunocompromised host. We evaluated two automated and reproducible PCR tests, the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.) and the COBAS AMPLICOR CMV Monitor (Roche Diagnostics, Pleasanton, Calif.), for the detection of CMV DNA in blood samples from transplant recipients with CMV infection as determined by shell vial culture. Following a log transformation analysis, the mean CMV DNA in plasma (PL), whole blood (WB), peripheral blood leukocytes (PBL), and peripheral blood mononuclear cells (PBMC) using the LightCycler was 6.79 copies per ml, 7.23 copies per ml, 6.38 copies per 2 x 10(6) cells, and 6.27 copies per 2 x 10(6) cells, respectively. This compares to 7.86 copies per ml, 8.37 copies per ml, 7.59 copies per 2 x 10(6) cells, and 7.44 copies per 2 x 10(6) cells, respectively, using COBAS AMPLICOR CMV Monitor. While higher CMV DNA levels were observed for the various blood compartments analyzed using COBAS AMPLICOR CMV Monitor, a high degree of correlation was evident between the two automated systems (jackknife correlation r = PL 0.77 [95% confidence interval (CI); 0.64, 0.90], WB 0.77 [95% CI; 0.62, 0.92], PBL 0.77 [95% CI; 0.67, 0.88], and PBMC 0.81 [95% CI; 0.72, 0.89], all P < 0.001). Therefore, we conclude that either automated diagnostic system is accurate for CMV DNA quantitation.

Survey of human mitochondrial diseases using new genomic/proteomic tools.

BACKGROUND: We have constructed Bayesian prior-based, amino-acid sequence profiles for the complete yeast mitochondrial proteome and used them to develop methods for identifying and characterizing the context of protein mutations that give rise to human mitochondrial diseases. (Bayesian priors are conditional probabilities that allow the estimation of the likelihood of an event - such as an amino-acid substitution - on the basis of prior occurrences of similar events.) Because these profiles can assemble sets of taxonomically very diverse homologs, they enable identification of the structurally and/or functionally most critical sites in the proteins on the basis of the degree of sequence conservation. These profiles can also find distant homologs with determined three-dimensional structures that aid in the interpretation of effects of missense mutations. RESULTS: This survey reports such an analysis for 15 missense mutations, one insertion and three deletions involved in Leber's hereditary optic neuropathy, Leigh syndrome, mitochondrial neurogastrointestinal encephalomyopathy, Mohr-Tranebjaerg syndrome, iron-storage disorders related to Friedreich's ataxia, and hereditary spastic paraplegia. We present structural correlations for seven of the mutations. CONCLUSIONS: Of the 19 mutations analyzed, 14 involved changes in very highly conserved parts of the affected proteins. Five out of seven structural correlations provided reasonable explanations for the malfunctions. As additional genetic and structural data become available, this methodology can be extended. It has the potential for assisting in identifying new disease-related genes. Furthermore, profiles with structural homologs can generate mechanistic hypotheses concerning the underlying biochemical processes - and why they break down as a result of the mutations.

Detection of herpes simplex virus DNA in genital and dermal specimens by LightCycler PCR after extraction using the IsoQuick, MagNA Pure, and BioRobot 9604 methods.

We evaluated two automated systems, MagNA Pure (Roche Molecular Biochemicals, Indianapolis, Ind.) and BioRobot 9604 (Qiagen, Inc., Chatsworth, Calif.) as effective replacements for the manual IsoQuick method (Orca Research, Inc., Bothell, Wash.) for extraction of herpes simplex virus (HSV) DNA from dermal and genital tract specimens prior to analysis by LightCycler PCR. Of 198 specimens (152 genital, 46 dermal), 92 (46.2%) were positive for HSV DNA by LightCycler PCR after automated extraction of specimens with either the MagNA Pure or BioRobot 9604 instrument. The manual IsoQuick method yielded HSV DNA (total n = 95) from three additional specimens that were negative by the automated method (P = 0.25, sign test). Although the mean numbers of LightCycler PCR cycles required to reach positivity differed statistically significantly among all three of the methods of extraction, the estimated means differed by no more than 1.5 cycles (P < 0.05). Seventy (76%) of the 92 specimens that were LightCycler PCR positive by all three extraction methods were also positive by shell vial cell culture assay. HSV DNA was detected by a lower LightCycler PCR cycle number (26.1 cycles) in specimens culture positive for the virus than in culture-negative samples (33.3 cycles) (P < 0.0001). The manual IsoQuick and automated MagNA Pure and BioRobot 9604 methods provide standardized, reproducible extraction of HSV DNA for LightCycler PCR. The decision to implement a manual versus an automated procedure depends on factors such as costs related to the number of specimens processed rather than on the minimal differences in the technical efficiency of extraction of nucleic acids among these methods.

A noncanonical WD-repeat protein from the cyanobacterium Synechocystis PCC6803: structural and functional study.

SYNECHOCYSTIS: PCC6803 possesses several open reading frames encoding putative WD-repeat proteins. One, the Hat protein, is involved in the control of a high-affinity transport system for inorganic carbon that is active when the cells are grown under a limiting concentration of this carbon substrate. The protein is composed of two major domains separated by a hydrophobic linker region of 20 amino acid residues. The N-terminal domain of Hat has no homolog in standard databases and does not display any particular structural features. Eleven WD repeats have been identified in the C-terminal moiety. The region encompassing the four terminal WD repeats is essential for growth under a limiting inorganic carbon regime. The region encompassing the two most terminal WD repeats is required for the activity of the high-affinity transport system. However, because the Hat protein is located in the thylakoids, it should not be itself an element of the transport system. The structural organization of the WD-containing domain of Hat was modeled from the crystal structure of the G protein beta subunit (with seven WD repeats) and of hemopexin (a structural analog with four blades). Functional and structural data argue in favor of an organization of the Hat WD moiety in two subdomains of seven and four WD repeats. The C-terminal 4-mer subdomain might interact with another, yet unknown, protein/peptide. This interaction could be essential in modulating the stability of the 4-mer structure and, thus, the accessibility of this subdomain, or at least of the region encompassing the last two WD repeats.

Transcription unit conservation in the three domains of life: a perspective from Escherichia coli.

Here we address the question of the degree to which genes within experimentally characterized operons in one organism (Escherichia coli) are conserved in other genomes. We found that two genes adjacent within an operon are more likely both to have an ortholog in other organisms, regardless of relative position, than genes adjacent on the same strand but in two different transcription units. They are also more likely to occur next to, or fused to, one another in other genomes. Genes frequently conserved adjacent to each other, especially among evolutionarily distant species, must be part of the same transcription unit in most of them.

Clinical evaluation of a new recombinant antigen-based cytomegalovirus immunoglobulin M immunoassay in liver transplant recipients.

BACKGROUND: Human cytomegalovirus (CMV) is a significant cause of morbidity and mortality among transplant recipients. Monitoring transplant recipients by CMV IgM serology has been questioned by several studies due to the reported insensitivity of serologic tests relative to antigen detection methods. METHODS: In this retrospective study, we have evaluated the performance of the new recombinant antigen-based Abbott AxSYM CMV IgM assay and compared it with CMV culture technique in a cohort of 40 liver transplant recipients who did not receive antiviral prophylaxis. RESULTS: The sensitivity, specificity, and positive and negative predictive values for detection of CMV disease by the AxSYM CMV IgM assay were 90.0%, 60.0%, 69.2%, and 85.7%, respectively, and by culture the values were 100%, 55.0%, 69.0%, and 100%, respectively. Detection of CMV IgM occurred before or at the time of CMV disease in only R+ recipients. CONCLUSION: Although this assay is a sensitive test for CMV-specific IgM, detection of CMV IgM preceded detection of virus by culture in patients only when the liver transplant recipient was CMV immune before transplantation (R+).

Susceptibility testing. Viral pathogens.

The control of the global expansion and proliferation of the AIDS pandemic has been complicated by the emergence of resistant strains of HIV-1 to the many new antiviral drugs directed to the genes coding for reverse transcriptase and protease enzymes of the virus. Similarly, new drug regimens for the management of chronic hepatitis B and C infections have been complicated by the lack of sustained clinical responses recently associated with either nucleotide mutation (HBV) or specific genotype of the virus (HCV). Commercial systems for performing and interpreting genotypic analysis will facilitate the recognition of informative mutations, standardize results between laboratories, and produce informative and interpretative result formats for optimal treatment of patients. Drug-resistant strains of herpesviruses (HSV, VZV, CMV) are generally associated with prolonged treatment of these infections in immunocompromised patients. Ultimate relevance of genotypic assays for routine clinical practice will require correlation with phenotypic results and the outcomes of long-term studies associating clinical improvement with antiviral drugs with specific mutation patterns of these viruses.

Indoor environmental quality in six commercial office buildings in the midwest United States.

The aims of this study were to characterize physical, mechanical, and environmental factors influencing indoor environmental quality (IEQ) in commercial office buildings; document occupant perceptions and psychosocial attributes; and evaluate relationships among these parameters. Six large office buildings in metropolitan areas were selected in Iowa, Minnesota, and Nebraska. Comprehensive sampling was conducted over one week in each building, during all four seasons. This paper presents the study methods and selected results from the first round of sampling (November 1996 to April 1997). Air flow and recirculation rates were quite variable, with the proportion of outdoor air provided to occupants ranging from 10 to 79 CFM/person. Carbon dioxide, carbon monoxide, and temperature were within ranges anticipated for nonproblem buildings. Relative humidity was low, ranging from 11.7 to 24.0 percent. Indoor geometric mean concentrations of total volatile organic compounds (TVOCs) ranged from 73 to 235 microg/m3. The most prevalent compounds included xylene, toluene, 2-propanol, limonene, and heptane. Geometric mean formaldehyde concentrations ranged from 1.7 to 13.3 microg/m3, and mean acetaldehyde levels ranged from <3.0 to 7.5 microg/m3. Airborne concentrations of culturable bacteria and fungi were low, with no samples exceeding 150 CFU/m3. Total (direct count) bioaerosols were more variable, ranging from 5010 to 10,700 organisms/m3. Geometric mean endotoxin concentrations ranged from 0.5 to 3.0 EU/m3. Respirable particulates (PM10) were low (14 to 36 microg/m3). Noise levels ranged from 48 to 56 dBA, with mean light values ranging from 200 to 420 lux. Environmental parameters were significantly correlated with each other. The prevalence of upper respiratory symptoms (dry eyes, runny nose), central nervous system symptoms (headache, irritability), and musculoskeletal symptoms (pain/stiffness in shoulders/neck) were elevated compared to other studies using similar questionnaires. Importantly, psychosocial factors were significantly related to increased symptoms in females, while environmental factors were more closely correlated with symptoms in males. Endotoxin concentrations were associated with symptoms in both males and females. These data will help to identify and quantify the relative role of factors that contribute to sick building syndrome. The data collected in this study may also be used to evaluate the effectiveness of current building operation practices, and can be used to prioritize allocations of resources for reduction of risk associated with IEQ complaints.

Human beta-herpesvirus interactions in solid organ transplant recipients.

The replication of beta-herpesviruses-cytomegalovirus (CMV), human herpesvirus (HHV)-6, and HHV-7-and their association with CMV disease and response to antiviral therapy were prospectively investigated in 33 liver transplant recipients not given antiviral prophylaxis. CMV, HHV-6, and HHV-7 DNA were detected within 8 weeks after transplantation in 70%, 33%, and 42% of the patients, respectively. The univariate association between CMV disease and the 3 beta-herpesviruses was more significant by virus load quantitation than by qualitative detection of DNA. This association with high levels of CMV, HHV-6, and HHV-7 (P<.001,.022, and.001, respectively) occurred mainly in CMV-seronegative recipients of transplants from CMV-seropositive donors. Antiviral therapy with ganciclovir (Gcv) reduced the load of CMV and HHV-6 and HHV-7. These results suggest that CMV disease in transplant recipients is related to the unique interaction of the 3 beta-herpesviruses and is ultimately reduced after intravenous Gcv treatment.

Analytical performance and clinical utility of a nucleic acid sequence-based amplification assay for detection of cytomegalovirus infection.

A nucleic acid sequence-based amplification (NASBA) assay for qualitative detection of human cytomegalovirus (CMV) pp67 mRNA was evaluated in a multicenter study. Negative results were obtained for all specimens from 50 CMV-seronegative and 50 CMV-seropositive low-risk whole-blood donors. No interference with CMV mRNA amplification was observed in the testing of 288 specimens containing various potential interfering substances, nonspecifically reacting substances (including mRNA from other herpesviruses), and three anticoagulants. A total of 95% (50 of 51) of CMV-positive (cell culture- and antigenemia immunofluorescence [AG-IFA]-positive) clinical specimens were positive by the NASBA assay. Results from different operators over multiple testing days were consistent for each of four panel members containing different concentrations of CMV mRNA, indicating the reproducibility of the assay. The estimated 95% reliable upper detection limit of the assay was 600 mRNA copies; the lower limit of detection was less than 25 mRNA copies. The clinical utility of the assay was evaluated with longitudinally collected specimens from solid-organ transplant patients (n = 21). A total of 98% (81 of 83) of the specimens from CMV-negative patients were negative by the NASBA assay, while 90% (10 of 11) of patient specimens that were positive by cell culture or AG-IFA were positive by the NASBA assay. Positive NASBA assay results were obtained earlier than AG-IFA or cell culture results for 55% of the patients and at the same time for the remainder of the patients (45%). The overall agreement between the NASBA assay and current reference tests was 86% when active CMV infection was present. These studies indicate that the CMV pp67 mRNA NASBA assay has reproducible and sensitive performance characteristics that should enable more rapid diagnosis of CMV infection.

Diagnosis of varicella-zoster virus infections in the clinical laboratory by LightCycler PCR.

Varicella-zoster virus (VZV) causes vesicular dermal lesions which are clinically evident as varicella (primary infection) or zoster (reactivated) diseases. The LightCycler system (Roche Molecular Biochemicals) is a newly developed commercially available system designed to rapidly perform PCR with real-time detection of PCR products using a fluorescence resonance energy transfer. We compared the detection of VZV from dermal specimens by shell vial cell culture (MRC-5) and by LightCycler PCR. Of 253 specimens, VZV was detected in 23 (9.1%) by shell vial cell cultures and 44 (17.4%) by LightCycler PCR directed to a nucleic acid target sequence in gene 28. Twenty-one of 44 (47.7%) specimens were exclusively positive by LightCycler PCR; the shell vial cell culture assay was never positive when DNA amplification was negative (specificity, 100%). VZV DNA was detected in 39 of 44 (88.6%) specimens positive during cycles 10 through 30 of the LightCycler PCR. These VZV DNA-positive specimens (cycles 10 to 30) and 5 of 11 other PCR positive specimens (cycles 31 to 36) were confirmed by another LightCycler PCR directed to another (gene 29) target of the viral genome. For routine laboratory practice, all specimens yielding amplified DNA to the VZV gene 28 target can be considered positive results. The increased sensitivity (91%) of the LightCycler PCR for detection of VZV, rapid turnaround time for reporting results, virtual elimination of amplicon carryover contamination, and equivalent costs compared to shell vial cell culture for detection of VZV indicate the need for implementation of this technology for routine laboratory diagnosis of this viral infection.

Evaluation of LightCycler PCR for implementation of laboratory diagnosis of herpes simplex virus infections.

Five hundred specimens (288 genital, 192 dermal, and 20 ocular) were extracted by technologists, and the DNA was assayed by LightCycler PCR (DNA polymerase and thymidine kinase [TK] gene targets) and by conventional tube and shell vial cell culture. One hundred fifty-eight confirmed (by cell culture and TK target PCR) positive and LightCycler-positive specimens were detected during the first 30 PCR cycles. LightCycler PCR-positive results for cycles 31 to 45 (39 of 67 [58.2%]) required confirmation by another PCR target (TK). LightCycler PCR is more sensitive (n = 197; 23.1%) than cell cultures (n = 150) for the routine laboratory detection of herpes simplex virus infections.

Eliminating superfluous neighbor pairs while threading fold models.

In this paper we address the problem of identifying which of various possible spatial residue-residue neighbor pairs are plausible physical contacts without reference to the native structure side chain geometry. We propose an algorithm that eliminates most of the implausible physical contacts from the fold models. This algorithm exploits the correlations between the amino acid side chain rotamers and the direction of the physical contacts between the amino acid side chains. We use this algorithm to "filter" the score of the sequence-to-structure alignment. Filtering is dynamic, in the sense that the set of neighbor pairs contributing to the alignment score varies during threading. Whether or not a neighbor pair contributes to the score depends on the threaded amino acids. This score filtering improves the accuracy of the predicted sequence-to-structure alignment.

Prediction of interaction partners for orphan nuclear receptors by prior-based protein sequence profiles.

We present a prior-based profile method for the prediction of protein-protein interaction partners that is here applied to the nuclear receptor superfamily. In this method, the diagnostic features are locally encoded in the physicochemical properties of residues in the interaction surface that are conserved in all proteins belonging to the defining set. The procedure models the positional variation based on that observed in the defining set and a prior-based substitution matrix derived from over 20,000 highly conserved positions in a set of 147 functional protein families. The method clusters sets of nuclear receptors known to interact with retinoid X receptor or corepressor proteins with predictive sets of receptors in C. elegans and higher metazoans. The method effectively reduces the search space of all possible interactions and yields experimentally testable predictions. Applications of this novel approach extend to interaction prediction problems in general, particularly to those that are not amenable to analysis by the rigid-body approximation.

Protein fold recognition by total alignment probability.

We present a protein fold-recognition method that uses a comprehensive statistical interpretation of structural Hidden Markov Models (HMMs). The structure/fold recognition is done by summing the probabilities of all sequence-to-structure alignments. The optimal alignment can be defined as the most probable, but suboptimal alignments may have comparable probabilities. These suboptimal alignments can be interpreted as optimal alignments to the "other" structures from the ensemble or optimal alignments under minor fluctuations in the scoring function. Summing probabilities for all alignments gives a complete estimate of sequence-model compatibility. In the case of HMMs that produce a sequence, this reflects the fact that due to our indifference to exactly how the HMM produced the sequence, we should sum over all possibilities. We have built a set of structural HMMs for 188 protein structures and have compared two methods for identifying the structure compatible with a sequence: by the optimal alignment probability and by the total probability. Fold recognition by total probability was 40% more accurate than fold recognition by the optimal alignment probability. Proteins 2000;40:451-462.