Forensic botany and forensic chemistry working together: application of plant DNA barcoding as a complement to forensic chemistry-a case study in Brazil.

Recently, Brazilian Federal Police used forensic chemistry and forensic botany techniques on a case. Two packets containing fragmented plant matter were seized and sent for forensic analysis. Forensic chemistry, the gold standard for evaluating plant material suspected to contain illicit substances, did not find illicit materials. Gas chromatography coupled mass spectrometry (GC-MS) identified thujone in the botanical material. Thujone is a chemical compound naturally found in many plant species, notably Artemisia absinthium. Because doubt remained, we next used plant DNA barcoding methods. Total DNA from plant tissue fragments was extracted and five different DNA regions were amplified, sequenced, and analyzed using plant DNA barcoding methods. Genetic analysis yielded 30 good quality sequences representing five taxa. Most specimens were identified as A. absinthium. Few studies focus on practical forensic applications of plant DNA barcoding methods using a case solved in a forensic laboratory with its difficulties and limitations. To the best of our knowledge, this is the first study to report an effective joint effort of forensic chemistry and botany techniques to assess plant material in Brazil. The availability of a new technical approach for the genetic sequencing of plant species will enhance many forensic investigations and inspire similar initiatives.

Refining the continuum of CFTR-associated disorders in the era of newborn screening.

Clinical heterogeneity in cystic fibrosis (CF) often causes diagnostic uncertainty in infants without symptoms and in older patients with milder phenotypes. We performed a cross-sectional evaluation of a comprehensive set of clinical and laboratory descriptors in a physician-defined cohort (N = 376; Children's Hospital of Wisconsin and the American Family Children's Hospital CF centers in Milwaukee and Madison, WI, USA) to determine the robustness of categorizing CF (N = 300), cystic fibrosis transmembrane conductance regulator (CFTR)-related disorder (N = 19), and CFTR-related (CRMS) metabolic syndrome (N = 57) according to current consensus guidelines. Outcome measures included patient demographics, clinical measures, sweat chloride levels, CFTR genotype, age at diagnosis, airway microbiology, pancreatic function, infection, and nutritional status. The CF cohort had a significantly higher median sweat chloride level (105 mmol/l) than CFTR-related disorder patients (43 mmol/l) and CFTR-related metabolic syndrome patients (35 mmol/l; p ≤ 0.001). Patient groups significantly differed in pancreatic sufficiency, immunoreactive trypsinogen levels, sweat chloride values, genotype, and positive Pseudomonas aeruginosa cultures (p ≤ 0.001). An automated classification algorithm using recursive partitioning demonstrated concordance between physician diagnoses and consensus guidelines. Our analysis suggests that integrating clinical information with sweat chloride levels, CFTR genotype, and pancreatic sufficiency provides a context for continued longitudinal monitoring of patients for personalized and effective treatment.

Depression among adults with neurofibromatosis type 1: prevalence and impact on quality of life.

Neurofibromatosis type 1 (NF1) carries a significant psychosocial burden for affected individuals. The objective of this study was to measure the prevalence of depressive symptoms among a large sample of adults with NF1 and to quantify the impact of depressive symptoms on quality of life (QoL). This cross-sectional study used an Internet-based questionnaire to collect data from 498 adults who self-reported as having NF1. Using the Center for Epidemiologic Studies Depression (CESD) scale, 55% of all participants (61% of females and 43% of males) scored above 16, indicating a high likelihood of clinical depression. In a multivariate regression model controlling for demographics and potential confounders, depressive symptoms accounted for 32% of the variance in QoL as measured by the Quality of Life Index. This study is the largest to date and found the highest prevalence of depression compared to prior studies. Our data provide more compelling evidence that individuals with NF1 are at increased risk for psychiatric morbidity and suggest that this population should be routinely screened for depression. Because depression was found to be strongly associated with QoL and accounted for nearly one-third of the variance in QoL, it is likely that effectively treating depression may significantly enhance QoL for individuals with NF1.

Derivation and validation of Transform equations to convert historical Enhanced liver fibrosis (ELF) scores to modern equivalents.

BACKGROUND AND AIMS: The Siemens Healthineers ELF™ Test was designed in 2004 with 2 algorithms to allow choice in histological alignment. Consequently, historical and modern algorithms are not fully harmonized, complicating comparisons involving early datasets. We derived transform equations to equate all ELF score versions, allowing historical data to be used in systematic reviews and meta-analyses. METHODS: Historical ELF equations were graphed pairwise versus their modern equivalent to assess correlation and derive four transforms. Transforms were validated using multiple datasets and evaluated for median absolute bias, number of samples reflecting clinically significant bias, number of discordant samples, bias at established cutoffs, and regression slope and y-intercept. RESULTS: Three transforms were validated equating Scheuer-aligned and/or age-included historical ELF equations (Immuno 1) to later equations aligned to Ishak and omitting age. A fourth transform corrected ADVIA Centaur® / Atellica® IM ELF scores miscalculated using the Scheuer Immuno 1 equation. Transformed data were well within allowable ELF bias limits. CONCLUSIONS: All transforms enabled accurate comparison of ELF scores generated by all historical algorithms to the current ADVIA Centaur / Atellica IM Analyzer ELF score. The transforms presented in this report should be used in systematic reviews and meta-analyses to facilitate comparisons to historical data.

Reliability of the Siemens Enzygnost and Novagnost Epstein-Barr virus assays for routine laboratory diagnosis: agreement with clinical diagnosis and comparison with the Merifluor Epstein-Barr virus immunofluorescence assay.

BACKGROUND: Diagnosis of Epstein-Barr virus (EBV) infection is routinely conducted by clinical laboratories, especially to diagnose infectious mononucleosis. At an estimated general population incidence of 1:200, this represents a potentially significant testing burden. We evaluated the reliability of the Siemens Novagnost® and Enzygnost® EBV microtiter assays measuring VCA IgM and IgG, and EBNA-1 IgG for clinical diagnosis of EBV-related infectious mononucleosis. METHODS: Remnant sera from 537 patients tested for EBV infection were used to compare the Siemens assays to each other and to the Merifluor assay. The Siemens assays are qualitative/semiquantitative, automatable enzyme immunoassays. The Merifluor assays are manual, qualitative indirect immunofluorescent assays. Testing was conducted on the Siemens and Merifluor assays in parallel. All assays were conducted and interpreted according to each manufacturer's specifications. Agreement of serostatus between each of the three assays was assessed. Discrepant results were resolved using a third method (Mikrogen recomLine). RESULTS: Final EBV serostatus indicated 2.9% of the population had an acute infection, 89.6% had a past infection, and 7.5% were EBV naive. All three assays demonstrated 100% agreement with acute infection. Agreement with past-infection serostatus was 99.1% for Enzygnost, between 86% and 98.8% for Novagnost, and 98.1% for Merifluor. Seronegative agreement was 100% for Enzygnost, 89.7% for Novagnost, and 92.3% for Merifluor. CONCLUSIONS: The Siemens Enzygnost and Novagnost EBV microtiter assays are suitable for clinical rule-in of acute EBV infection and for identifying EBV-naive individuals. Both assays also adequately identify remote EBV infections. Because these assays can be automated, they can improve speed and efficiency of EBV testing, especially in high-volume laboratories.

Transcriptional signatures as a disease-specific and predictive inflammatory biomarker for type 1 diabetes.

The complex milieu of inflammatory mediators associated with many diseases is often too dilute to directly measure in the periphery, necessitating development of more sensitive measurements suitable for mechanistic studies, earlier diagnosis, guiding therapeutic decisions and monitoring interventions. We previously demonstrated that plasma samples from recent-onset type 1 diabetes (RO T1D) patients induce a proinflammatory transcriptional signature in freshly drawn peripheral blood mononuclear cells (PBMCs) relative to that of unrelated healthy controls (HC). Here, using cryopreserved PBMC, we analyzed larger RO T1D and HC cohorts, examined T1D progression in pre-onset samples, and compared the RO T1D signature to those associated with three disorders characterized by airway infection and inflammation. The RO T1D signature, consisting of interleukin-1 cytokine family members, chemokines involved in immunocyte chemotaxis, immune receptors and signaling molecules, was detected during early pre-diabetes and found to resolve post-onset. The signatures associated with cystic fibrosis patients chronically infected with Pseudomonas aeruginosa, patients with confirmed bacterial pneumonia, and subjects with H1N1 influenza all reflected immunological activation, yet each were distinct from one another and negatively correlated with that of T1D. This study highlights the remarkable capacity of cells to serve as biosensors capable of sensitively and comprehensively differentiating immunological states.

Cystic fibrosis mutations for p.F508del compound heterozygotes predict sweat chloride levels and pancreatic sufficiency.

Cystic fibrosis (CF) is a monogenetic disease with a complex phenotype. Over 1500 mutations in the CFTR gene have been identified; however, the p.F508del mutation is most common. There has been limited correlation between the CFTR mutation genotype and the disease phenotypes. We evaluated the non-p.F508del mutation of 108 p.F508del compound heterozygotes using the biological classification method, Grantham and Sorting Intolerant from Tolerant (SIFT) scores to assess whether these scoring systems correlated with sweat chloride levels, pancreatic sufficiency, predicted FEV(1) , and risk of infection with Pseudomonas aeruginosa in the last year. Mutations predicted to be 'mild' by the biological classification method are associated with more normal sweat chloride levels (p < 0.001), pancreatic sufficiency (p < 0.001) and decreased risk of infection with Pseudomonas in the last year (p = 0.014). Lower Grantham scores are associated with more normal sweat chloride levels (p < 0.001), and pancreatic sufficiency (p = 0.014). Higher SIFT scores are associated with more normal sweat chloride levels (p < 0.001) and pancreatic sufficiency (p = 0.011). There was no association between pulmonary function measured by predicted FEV(1) and the biological classification (p = 0.98), Grantham (p = 0.28) or SIFT scores (p = 0.62), which suggests the pulmonary disease related to CF may involve other modifier genes and environmental factors.

A limited spectrum of phenylalanine hydroxylase mutations is observed in phenylketonuria patients in western Poland and implications for treatment with 6R tetrahydrobiopterin.

Phenylketonuria (PKU) is an autosomal recessive defect in hepatic metabolism of phenylalanine, which is secondary to mutations in the phenylalanine hydroxylase (PAH) gene. Sixty-seven ethnically Polish PKU patients, followed at the Outpatient Department of Pediatrics and Developmental Medicine in Poznan, Poland, were assessed for mutations in the PAH gene. Two mutations were identified in 61 of 67 patients and a single mutation was identified in the remaining six patients. The four most prevalent mutations (p.R408W, 68%; c.1066-11G>A, 6%; c.1315+1G>A, 5.2%; c.822-832delGCCCATGTATA, 3.7%) accounted for 83% of the mutant alleles. Fifteen additional mutations were identified of which most (13/15) were observed in an individual patient. Before knowledge of PAH genotypes, 19 patients were challenged with a 20 mg kg(-1) dose of 6R tetrahydrobiopterin (BH(4)) and serum phenylalanine concentration was monitored in hospital over 24 h. Two patients responded to the BH(4) challenge with a reduction of serum phenylalanine concentration >30% from baseline. PAH genotypes of the two responsive patients would have been predicted, as they contained mutations recognized as BH(4) responsive, whereas the 17 patients who were unresponsive would have been predicted as their mutations were either recognized as non-responsive or were highly deleterious frame-shift mutations. Overall, only 7.5% (5/ 67) of patients had PAH mutations recognized as responsive to co-factor therapy. Among the PKU patients from western Poland, PAH mutations responsive to BH(4) therapy are poorly represented; therefore, genotyping may be useful for identifying candidate patients likely to respond to BH(4) before physiological challenge.

Normal atmosphere: large radical and formaldehyde concentrations predicted.

A steady-state model of the normal (unpolluted) surface atmosphere predicts a daytime concentration of hydroxyl, hydroperoxyl, and methylperoxyl radicals approaching 5 x 10(8)molecules per cubic centimeter and a formaldehyde concentration of 5 x 10(10) molecules per cubic centimeter or 2 parts per billion. A radical chain reaction is proposed for the rapid removal of carbon monoxide, leading to a carbon monoxide lifetime as low as 0.2 year in the surface atmosphere.

Heat inactivation of the relaxing site of actomyosin: prevention and reversal with dithiothreitol.

Adenosine triphosphate and magnesium (MgATP) inhibit contraction by binding to a specific relaxing site on natural actomyosin gel. This inhibitory control site is distinct from the active sites where MgATP causes contraction. In high concentrations of MgATP, calcium triggers contraction by releasing the protein from substrate inhibition, allowing the contractile reactions to occur. Heating the protein for 5 minutes at 43 degrees C selectively inactivates the relaxing site. After this treatment, actomyosin with MgATP contracts as well without calcium as with it. That this effect of heat is prevented and reversed by dithiothreitol (an agent that reduces disulfide bonds) indicates that the structure of the relaxing site depends on certain labile sulfhydryl groups, which may be those of tropomyosin. When these are oxidized to disulfide bonds, the site loses its activity; when the disulfide bonds are reduced, the site regains its activity.

Ribosomes: effect of interferon on their interaction with rapidly labeled cellular and viral RNA's.

Rapidly labeled RNA of mouse L cells and labeled RNA of Mengo virus, unlike cellular RNA labeled under steady-state conditions, form detectable complexes with L-cell ribosomes. These ribosome-RNA complexes formed in vitro appear analogous to those assembled during polysome formation in vivo. When ribosomes are prepared from L cells exposed to homologous interferon, their capacity to associate with cell messenger is preserved, while their ability to interact with viral RNA is markedly reduced. The ribosomes from cells exposed to interferon are thus altered selectively to permit only certain messages to be bound and translated.

Interferon: lack of detectable uptake by cells.

No interferon, or at most a small fraction of that applied, is taken up by cells during the period of induction of antiviral activity. The lack of detectable adsorption was not influenced either by the concentration of interferon or the volume. The results were similar in both chicken and mouse systems.

Attention in the newborn: Effect on motility and skin potential.

Newborn infants showed lower motility and greater reactivity of the skin potential while attending to a visual target than when equally alert but inattentive.

Effect of interferon on early interferon production.

Chick embryo cells given prior treatment with interferon make new interferon earlier and in larger quantities upon stimulation with Chikungunya virus than cells not so treated. By the criterion of loss of sensitivity to actinomycin, the time needed for formation of messenger RNA for interferon was decreased in the primed cells. Thus interferon affects virus action within 1 hour after infection.

Amnesia produced by spreading depression and ECS: evidence for time-dependent memory trace localization.

Rats were given electroconvulsive shock and bilateral cortical spreading depression, either alone or in combination, at various times after a single passive avoidance training trial. Assessment of retention deficits, 24 hours after training, revealed a U-shaped amnesic function for cortical spreading depression as compared with the short linear function consistently obtained with electroconvulsive shock in this situation. Induction of cortical spreading depression immediately after training resulted in an extension of the amnesic gradient produced by electroconvulsive shock, presumably by disruption of the subcortically confined memory trace. In addition to indicating a stibcortical locus of action for the amnesic effects of electroconvulsive shock, these results are interpreted as favoring a hypothesis of time-dependent memory trace localization in short-term memory processing, which involves an initial subcortical localization of the trace followed by a phase involving either direct or indlirect cortical participation in a mulltistage memory fixation process.

Polyinosinic-polycytidylic acid inhibits chemically induced tumorigenesis in mouse skin.

Polyinosinic-polycytidylic acid administered intraperitoneally inhibits (i) the formation of skin tumors induced by a single topical application of 9,10-dimethylbenzanthracene followed by weekly applications of croton oil and (ii) the formation of skin tumors induced by a single large application of 9,10-dimethylbenzanthracene.

Branched threadlike micelles in an aqueous solution of a trimeric surfactant.

Very long threadlike micelles observed in aqueous solutions of some surfactants have attracted much attention because of the peculiar rheological properties of these systems. Molecular dynamics simulations have suggested that branched threadlike micelles should exist in concentrated solutions of dimeric surfactants. Here experimental evidence, obtained from transmission electron microscopy at cryogenic temperature, is presented of branched threadlike micelles in aqueous solutions of a triquaternary ammonium (trimeric) surfactant made up of three amphiphilic moieties connected at the level of the head-groups by two propanediyl spacers.

Growth of continental-scale metro-agro-plexes, regional ozone pollution, and world food production.

Three regions of the northern mid-latitudes, the continental-scale metro-agro-plexes, presently dominate global industrial and agricultural productivity. Although these regions cover only 23 percent of the Earth's continents, they account for most of the world's commercial energy consumption, fertilizer use, food-crop production, and food exports. They also account for more than half of the world's atmospheric nitrogen oxide (NOx,) emissions and, as a result, are prone to ground-level ozone (O(3)) pollution during the summer months. On the basis of a global simulation of atmospheric reactive nitrogen compounds, it is estimated that about 10 to 35 percent of the world's grain production may occur in parts of these regions where ozone pollution may reduce crop yields. Exposure to yield-reducing ozone pollution may triple by 2025 if rising anthropogenic NOx emissions are not abated.

Swine influenza virus vaccine: potentiation of antibody responses in rhesus monkeys.

Polyriboinosinic-polyribocytidylic acid stabilized with poly-L-lysine and carboxymethylcellulose [poly(ICLC)] enhances the antibody response in rhesus monkeys immunized with swine influenza virus subunit vaccine. Monkeys given the vaccine-adjuvant combination had earlier and significantly (P less than .05) higher titers by 14 days compared to those that received vaccine alone. The potentiation of the antibody response of young monkeys given a split-virus vaccine in combination with poly(ICLC) suggests that this vaccine-adjuvant combination may similarly provide a potentially useful alternative approach to the immunization of pediatric and young adult age groups against swine influenza.

Electron microscope tomography: transcription in three dimensions.

Three-dimensional reconstruction of an asymmetric biological ultrastructure has been achieved by tomographic analysis of electron micrographs of sections tilted on a goniometer specimen stage. Aligned micrographs could be displayed as red-green three-dimensional movies. The techniques have been applied to portions of in situ transcription units of a Balbiani ring in the polytene chromosomes of the midge Chironomus tentans. Current data suggest a DNA compaction of about 8 to 1 in a transcription unit. Nascent ribonucleoprotein granules display an imperfect sixfold helical arrangement around the chromatin axis.