Significance of Gardnerella vaginalis genotyping in diagnosis of recurrent bacterial vaginosis

Objective. To assess the importance of identifying different genotypes of Gardnerella vaginalis in the diagnosis of recurrent bacterial vaginosis.Materials and methods. The study involved 299 women of reproductive age. All patients were divided into three groups (healthy women, women with the first episode of bacterial vaginosis, and women with recurrent bacterial vaginosis). DNA of Gardnerella vaginalis in vaginal discharge was detected by real-time PCR. The detection of four genotypes of G. vaginalis was performed using real-time multiplex PCR. To quantify the amplified PCR fragments, quantitative standard samples were constructed. Statistical analysis of the results was carried out using the statistical package NCSS 11 (NCSS, LCC).Results. In 38.2 % of healthy women, any one genotype of G. vaginalis was identified in the vaginal biotope, most often it was genotype 4 (35.2 %), while the concentration of G. vaginalis DNA was low (102–103 geqs/ml). When several genotypes of gardnerella were detected simultaneously in healthy women, the DNA concentration did not exceed 104 geqs/ml. A completely different picture was observed among women with bacterial vaginosis (BV). In the first episode of BV, genotype 4 of G. vaginalis prevailed, both as a single genotype and in combination with 1 or 2, or 3 genotypes. In the recurrent course of BV, only 3–4 genotypes of G. vaginalis were detected at once, and in 78 % of cases it had place is a combination of 1, 2 and 4 genotypes, and the DNA concentration was 107–108 geqs/ml.Conclusion. To diagnose recurrent forms of BV, it is necessary to develop and introduce into practice laboratory diagnostics a test system for detecting different genotypes of G. vaginalis by real-time PCR.

Effect of prophylactic dextrose gel on the neonatal gut microbiome

ObjectiveTo determine the effect of prophylactic dextrose gel on the infant gut microbiome.DesignObservational cohort study nested in a randomised trial.SettingThree maternity hospitals in New Zealand.PatientsInfants at risk of neonatal hypoglycaemia whose parents consented to participation in the hypoglycaemia Prevention in newborns with Oral Dextrose trial (hPOD). Infants were randomised to receive prophylactic dextrose gel or placebo gel, or were not randomised and received no gel (controls). Stool samples were collected on days 1, 7 and 28.Main outcome measuresThe primary outcome was microbiome beta-diversity at 4 weeks. Secondary outcomes were beta-diversity, alpha-diversity, bacterial DNA concentration, microbial community stability and relative abundance of individual bacterial taxa at each time point.ResultsWe analysed 434 stool samples from 165 infants using 16S rRNA gene amplicon sequencing. There were no differences between groups in beta-diversity at 4 weeks (p=0.49). There were also no differences between groups in any other microbiome measures including beta-diversity (p=0.53 at day 7), alpha-diversity (p=0.46 for day 7 and week 4), bacterial DNA concentration (p=0.91), microbial community stability (p=0.52) and microbial relative abundance at genus level. There was no evidence that exposure to any dextrose gel (prophylaxis or treatment) had any effect on the microbiome. Mode of birth, type of milk fed, hospital of birth and ethnicity were all associated with differences in the neonatal microbiome.ConclusionsClinicians and consumers can be reassured that dextrose gel used for prophylaxis or treatment of neonatal hypoglycaemia does not alter the neonatal gut microbiome.Trial registration number12614001263684.

DNA damage modeled with Geant4-DNA: effects of plasmid DNA conformation and experimental conditions

The chemical stage of the Monte Carlo track-structure code Geant4-DNA was extended for its use in DNA strand break (SB) simulations and compared against published experimental data. Geant4-DNA simulations were performed using pUC19 plasmids (2686 base pairs) in a buffered solution of DMSO irradiated by 60Co or 137Cs γ-rays. A comprehensive evaluation of SSB yields was performed considering DMSO, DNA concentration, dose and plasmid supercoiling. The latter was measured using the super helix density value used in a Brownian Dynamics (BD) plasmid generation algorithm. The Geant4-DNA implementation of the Independent Reaction Times method (IRT), developed to simulate the reaction kinetics of radiochemical species, allowed to score the fraction of supercoiled, relaxed and linearized plasmid fractions as a function of the absorbed dose. The percentage of the number of strand breaks after •OH + DNA and H• + DNA reactions, referred as SSB efficiency, obtained using MCTS were 13.77% and 0.74% respectively. This is in reasonable agreement with published values of 12% and 0.8%. The SSB yields as a function of DMSO concentration, DNA concentration and super helix density recreated the expected published experimental behaviors within 5%, one standard deviation. The dose response of SSB and DSB yields agreed with published measurements within 5%, one standard deviation. We demonstrated that the developed extension of IRT in Geant4-DNA, facilitated the reproduction of experimental conditions. Furthermore, its calculations were strongly in agreement with experimental data. These two facts will facilitate the use of this extension in future radiobiological applications, aiding the study of DNA damage mechanisms with a high level of detail.

Cell Free DNA as a Potential Biomarker of Blast-Wave Mild Traumatic Brain Injury and Posttraumatic Stress Disorder.

BackgroundIndividuals being within non-lethal distance from explosion may present with blast induced mild traumatic brain injury, post traumatic stress disorder, or combination of the two conditions. Early diagnosis to enable interventions is important. This study tested the possible role of cell free DNA in the diagnosis of blast related head injuries in a rat model.MethodsA rat controlled model of a blast. Cell free DNA concentrations were determined in the serum by a direct fluorescence method. Cognitive and behavioral tests were used to diagnose affected rats. ResultsMean cell free DNA concentration increased significantly at 2 hours following the blast compared to baseline level and remained high throughout the follow-up period (665.43±159.15 ng/ml vs. 344.20±69.62 ng/ml, p<0.0001). The rate of affected rats among the blast exposed animals was 42.5%. A significant increase in mean cell free DNA concentration was found at 2 hours after exposure in the affected group (741.40±47.18 ng/ml) compared to both the baseline concentration (372.42±149.11 ng/ml), p<0.0001 and to the well-adapted group (517.47 ng/ml), p<0.0045. ConclusionThis rat model of blast demonstrated that the incidence of mild brain injury and or PTSD is significant and that affected animals demonstrated increased serum concentrations of cell free DNA. Cell free DNA may potentially serve as a biomarker to diagnose brain psychopathology early in individuals exposed to blast.

Variability in ITS1 and ITS2 sequences of historic herbaria and extant (fresh) Phalaris species (Poaceae)

Background Phalaris species (Poaceae) occupy diverse environments throughout all continents except Antarctica. Phalaris arundinacea is an important forage, ornamental, wetland restoration and biofuel crop grown globally as well as being a wetland invasive. The nuclear ribosomal internal transcribed spacer (ITS) region has been used for Phalaris barcoding as a DNA region with high nucleotide diversity for Phalaris species identification. Recent findings that P. arundinacea populations in Minnesota USA are most likely native and not European prompted this analysis to determine whether Eurasian vs. native North American P. arundinacea differed in ITS regions. Our objectives were to amplify and compare ITS regions (ITS1 and ITS2) of historic herbaria (1882–2001) and extant (fresh) Phalaris specimens; analyze ITS regions for species-specific polymorphisms (diagnostic SNPs) and compare ITS regions of historic Phalaris specimens with known, extant Phalaris species. Results We obtained complete ITS1 and ITS2 sequences from 31 Phalaris historic (herbaria samples, 1908 to 2001) and five extant (fresh) specimens. Herbaria Phalaris specimens did not produce new SNPs (single nucleotide polymorphisms) not present in extant specimens. Diagnostic SNPs were identified in 8/12 (66.6%) Phalaris species. This study demonstrates the use of herbaria tissue for barcoding as a means for improved species identification of Phalaris herbaria specimens. No significant correlation between specimen age and genomic DNA concentration was found. Phalaris arundinacea showed high SNP variation within its clade, with the North American being distinctly different than other USA and most Eurasian types, potentially allowing for future identification of specific SNPs to geographic origin. Conclusions While not as efficient as extant specimens to obtain DNA, Phalaris herbaria specimens can produce high quality ITS sequences to evaluate historic genetic resources and facilitate identification of new species-specific barcodes. No correlation between DNA concentration and age of historic samples (119 year range) occurred. Considerable polymorphism was exhibited in the P. arundinacea clade with several N. American accessions being distinct from Eurasian types. Further development of within species- and genus-specific barcodes could contribute to designing PCR primers for efficient and accurate identification of N. American P. arundinacea. Our finding of misidentified Phalaris species indicates the need to exercise stringent quality control measures on newly generated sequence data and to approach public sequence databases in a critical way.

Evagreen Dye Fluorescence Verification v1

This protocol is to establish the relationship between Evagreen Dye fluorescent intensity and DNA concentration.

Relationship Between the Xylem Anatomy of Grapevine Rootstocks and Their Susceptibility to Phaeoacremonium minimum and Phaeomoniella chlamydospora

Fungal grapevine trunk diseases (GTDs) are some of the most pressing threats to grape production worldwide. While these diseases are associated with several fungal pathogens, Phaeomoniella chlamydospora and Phaeoacremonium minimum are important contributors to esca and Petri diseases. Recent research has linked grapevine xylem diameter with tolerance to Pa. chlamydospora in commercial rootstocks. In this study, we screen over 25 rootstocks for xylem characteristics and tolerance to both Pa. chlamydospora and Pm. minimum. Tolerance was measured by fungal incidence and DNA concentration (quantified via qPCR), while histological analyses were used to measure xylem characteristics, including xylem vessels diameter, density, and the proportion of the stem surface area covered by xylem vessels. Rootstocks were grouped into different classes based on xylem characteristics to assess the potential association between vasculature traits and pathogen tolerance. Our results revealed significant differences in all the analyzed xylem traits, and also in DNA concentration for both pathogens among the tested rootstocks. They corroborate the link between xylem vessels diameter and tolerance to Pa. chlamydospora. In Pm. minimum, the rootstocks with the widest xylem diameter proved the most susceptible. This relationship between vasculature development and pathogen tolerance has the potential to inform both cultivar choice and future rootstock breeding to reduce the detrimental impact of GTDs worldwide.