Differential DNA extraction of challenging simulated sexual-assault samples: a Swiss collaborative study

In sexual-assault cases, autosomal DNA analysis of gynecological swabs is a challenge, as the presence of a large quantity of female material may prevent detection of the male DNA. A solution to this problem is differential DNA extraction, but there is no established best practice for this. We decided to test the efficacy of a number of different protocols on simulated casework samples. Four difficult samples were sent to the nine Swiss laboratories active in forensic genetics. In each laboratory, staff used their routine protocols to separate the epithelial-cell fraction, enriched with the non-sperm DNA, from the sperm fraction. DNA extracts were then sent to the organizing laboratory for analysis. Estimates of male:female DNA ratio without differential DNA extraction ranged from 1:38 to 1:339, depending on the semen used to prepare the samples. After differential DNA extraction, most of the ratios ranged from 1:12 to 9:1, allowing detection of the male DNA. Compared with direct DNA extraction, cell separation resulted in losses of 94-98% of the male DNA. As expected, more male DNA was generally present in the sperm than in the epithelial-cell fraction. However, for about 30% of the samples, the reverse trend was seen. The recovery of male and female DNA was highly variable, depending on the laboratory involved. An experimental design similar to the one used in this study may be of assistance for local protocol testing and improvement

Pollen exposure is associated with risk of respiratory symptoms during the first year of life

BACKGROUND: Pollen exposure is associated with respiratory symptoms in children and adults. However, the association of pollen exposure with respiratory symptoms during infancy, a particularly vulnerable period, remains unclear. We examined whether pollen exposure is associated with respiratory symptoms in infants and whether maternal atopy, infant's sex or air pollution modifies this association. METHODS: We investigated 14,874 observations from 401 healthy infants of a prospective birth cohort. The association between pollen exposure and respiratory symptoms, assessed in weekly telephone interviews, was evaluated using generalized additive mixed models (GAMMs). Effect modification by maternal atopy, infant's sex, and air pollution (NO$_{2}$ , PM$_{2.5}$ ) was assessed with interaction terms. RESULTS: Per infant, 37 ± 2 (mean ± SD) respiratory symptom scores were assessed during the analysis period (January through September). Pollen exposure was associated with increased respiratory symptoms during the daytime (RR [95% CI] per 10% pollen/m$^{3}$ : combined 1.006 [1.002, 1.009]; tree 1.005 [1.002, 1.008]; grass 1.009 [1.000, 1.23]) and nighttime (combined 1.003 [0.999, 1.007]; tree 1.003 [0.999, 1.007]; grass 1.014 [1.004, 1.024]). While there was no effect modification by maternal atopy and infant's sex, a complex crossover interaction between combined pollen and PM$_{2.5}$ was found (p-value 0.003). CONCLUSION: Even as early as during the first year of life, pollen exposure was associated with an increased risk of respiratory symptoms, independent of maternal atopy and infant's sex. Because infancy is a particularly vulnerable period for lung development, the identified adverse effect of pollen exposure may be relevant for the evolvement of chronic childhood asthma

A global analysis of Y-chromosomal haplotype diversity for 23 STR loci

In a worldwide collaborative effort, 19,630 Y-chromosomes were sampled from 129 different populations in 51 countries. These chromosomes were typed for 23 short-tandem repeat (STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643) and using the PowerPlex Y23 System (PPY23, Promega Corporation, Madison, WI). Locus-specific allelic spectra of these markers were determined and a consistently high level of allelic diversity was observed. A considerable number of null, duplicate and off-ladder alleles were revealed. Standard single-locus and haplotype-based parameters were calculated and compared between subsets of Y-STR markers established for forensic casework. The PPY23 marker set provides substantially stronger discriminatory power than other available kits but at the same time reveals the same general patterns of population structure as other marker sets. A strong correlation was observed between the number of Y-STRs included in a marker set and some of the forensic parameters under study. Interestingly a weak but consistent trend toward smaller genetic distances resulting from larger numbers of markers became apparent.Peer reviewe

Role of the Cytoplasmic C Terminus of the FliF Motor Protein in Flagellar Assembly and Rotation

Twenty-six FliF monomers assemble into the MS ring, a central motor component of the bacterial flagellum that anchors the structure in the inner membrane. Approximately 100 amino acids at the C terminus of FliF are exposed to the cytoplasm and, through the interaction with the FliG switch protein, a component of the flagellar C ring, are essential for the assembly of the motor. In this study, we have dissected the entire cytoplasmic C terminus of the Caulobacter crescentus FliF protein by high-resolution mutational analysis and studied the mutant forms with regard to the assembly, checkpoint control, and function of the flagellum. Only nine amino acids at the very C terminus of FliF are essential for flagellar assembly. Deletion or substitution of about 10 amino acids preceding the very C terminus of FliF resulted in assembly-competent but nonfunctional flagella, making these the first fliF mutations described so far with a Fla+ but Mot− phenotype. Removal of about 20 amino acids further upstream resulted in functional flagella, but cells carrying these mutations were not able to spread efficiently on semisolid agar plates. At least 61 amino acids located between the functionally relevant C terminus and the second membrane-spanning domain of FliF were not required for flagellar assembly and performance. A strict correlation was found between the ability of FliF mutant versions to assemble into a flagellum, flagellar class III gene expression, and a block in cell division. Motile suppressors could be isolated for nonmotile mutants but not for mutants lacking a flagellum. Several of these suppressor mutations were localized to the 5′ region of the fliG gene. These results provide genetic support for a model in which only a short stretch of amino acids at the immediate C terminus of FliF is required for flagellar assembly through stable interaction with the FliG switch protein

Identification of the Protease and the Turnover Signal Responsible for Cell Cycle-Dependent Degradation of the Caulobacter FliF Motor Protein

Flagellar ejection is tightly coupled to the cell cycle in Caulobacter crescentus. The MS ring protein FliF, which anchors the flagellar structure in the inner membrane, is degraded coincident with flagellar release. Previous work showed that removal of 26 amino acids from the C terminus of FliF prevents degradation of the protein and interferes with flagellar assembly. To understand FliF degradation in more detail, we identified the protease responsible for FliF degradation and performed a high-resolution mutational analysis of the C-terminal degradation signal of FliF. Cell cycle-dependent turnover of FliF requires an intact clpA gene, suggesting that the ClpAP protease is required for removal of the MS ring protein. Deletion analysis of the entire C-terminal cytoplasmic portion of FliF C confirmed that the degradation signal was contained in the last 26 amino acids that were identified previously. However, only deletions longer than 20 amino acids led to a stable FliF protein, while shorter deletions dispersed over the entire 26 amino acids critical for turnover had little effect on stability. This indicated that the nature of the degradation signal is not based on a distinct primary amino acid sequence. The addition of charged amino acids to the C-terminal end abolished cell cycle-dependent FliF degradation, implying that a hydrophobic tail feature is important for the degradation of FliF. Consistent with this, ClpA-dependent degradation was restored when a short stretch of hydrophobic amino acids was added to the C terminus of stable FliF mutant forms

Multiday acute sodium bicarbonate intake improves endurance capacity and reduces acidosis in men

BACKGROUND: The purpose was to investigate the effects of one dose of NaHCO3 per day for five consecutive days on cycling time-to-exhaustion (Tlim) at 'Critical Power' (CP) and acid-base parameters in endurance athletes. METHODS: Eight trained male cyclists and triathletes completed two exercise periods in a randomized, placebo-controlled, double-blind interventional crossover investigation. Before each period, CP was determined. Afterwards, participants completed five constant-load cycling trials at CP until volitional exhaustion on five consecutive days, either after a dose of NaHCO3 (0.3 g·kg-1 body mass) or placebo (0.045 g·kg-1 body mass NaCl). RESULTS: Average Tlim increased by 23.5% with NaHCO3 supplementation as compared to placebo (826.5 ± 180.1 vs. 669.0 ± 167.2 s; P = 0.001). However, there was no time effect for Tlim (P = 0.375). [HCO3-] showed a main effect for condition (NaHCO3: 32.5 ± 2.2 mmol·l-1; placebo: 26.2 ± 1.4 mmol·l-1; P < 0.001) but not for time (P = 0.835). NaHCO3 supplementation resulted in an expansion of plasma volume relative to placebo (P = 0.003). CONCLUSIONS: The increase in Tlim was accompanied by an increase in [HCO3-], suggesting that acidosis might be a limiting factor for exercise at CP. Prolonged NaHCO3 supplementation did not lead to a further increase in [HCO3-] due to the concurrent elevation in plasma volume. This may explain why Tlim remained unaltered despite the prolonged NaHCO3 supplementation period. Ingestion of one single NaHCO3 dose per day before the competition during multiday competitions or tournaments might be a valuable strategy for performance enhancement

iTRAQ-based and label-free proteomics approaches for studies of human adenovirus infections

Both isobaric tags for relative and absolute quantitation (iTRAQ) and label-free methods are widely used for quantitative proteomics. Here, we provide a detailed evaluation of these proteomics approaches based on large datasets from biological samples. iTRAQ-label-based and label-free quantitations were compared using protein lysate samples from noninfected human lung epithelial A549 cells and from cells infected for 24 h with human adenovirus type 3 or type 5. Either iTRAQ-label-based or label-free methods were used, and the resulting samples were analyzed by liquid chromatography (LC) and tandem mass spectrometry (MS/MS). To reduce a possible bias from quantitation software, we applied several software packages for each procedure. ProteinPilot and Scaffold Q+ software were used for iTRAQ-labeled samples, while Progenesis LC-MS and ProgenesisF-T2PQ/T3PQ were employed for label-free analyses. R (2) correlation coefficients correlated well between two software packages applied to the same datasets with values between 0.48 and 0.78 for iTRAQ-label-based quantitations and 0.5 and 0.86 for label-free quantitations. Analyses of label-free samples showed higher levels of protein up- or downregulation in comparison to iTRAQ-labeled samples. The concentration differences were further evaluated by Western blotting for four downregulated proteins. These data suggested that the label-free method was more accurate than the iTRAQ method