Pre-Analytical Conditions in Non-Invasive Prenatal Testing of Cell-Free Fetal RHD

Background

Non-invasive prenatal testing of cell-free fetal DNA (cffDNA) in maternal plasma can predict the fetal RhD type in D negative pregnant women. In Denmark, routine antenatal screening for the fetal RhD gene (RHD) directs the administration of antenatal anti-D prophylaxis only to women who carry an RhD positive fetus. Prophylaxis reduces the risk of immunization that may lead to hemolytic disease of the fetus and the newborn. The reliability of predicting the fetal RhD type depends on pre-analytical factors and assay sensitivity. We evaluated the testing setup in the Capital Region of Denmark, based on data from routine antenatal RHD screening.

Methods

Blood samples were drawn at gestational age 25 weeks. DNA extracted from 1 mL of plasma was analyzed for fetal RHD using a duplex method for exon 7/10. We investigated the effect of blood sample transportation time (n = 110) and ambient outdoor temperatures (n = 1539) on the levels of cffDNA and total DNA. We compared two different quantification methods, the delta Ct method and a universal standard curve. PCR pipetting was compared on two systems (n = 104).

Results

The cffDNA level was unaffected by blood sample transportation for up to 9 days and by ambient outdoor temperatures ranging from -10°C to 28°C during transport. The universal standard curve was applicable for cffDNA quantification. Identical levels of cffDNA were observed using the two automated PCR pipetting systems. We detected a mean of 100 fetal DNA copies/mL at a median gestational age of 25 weeks (range 10–39, n = 1317).

Conclusion

The setup for real-time PCR-based, non-invasive prenatal testing of cffDNA in the Capital Region of Denmark is very robust. Our findings regarding the transportation of blood samples demonstrate the high stability of cffDNA. The applicability of a universal standard curve facilitates easy cffDNA quantification.     

Shadoo/PrP (Sprn0/0/Prnp0/0) double knockout mice

Shadoo (Sho) is a brain glycoprotein with similarities to the unstructured region of PrP^C. Frameshift alleles of the Sho gene, Sprn, are reported in variant Creutzfeldt-Jakob disease (vCJD) patients while Sprn mRNA knockdown in PrP-null (Prnp^0/0) embryos produces lethality, advancing Sho as the hypothetical PrP-like “pi” protein. Also, Sho levels are reduced as misfolded PrP accumulates during prion infections. To penetrate these issues we created Sprn null alleles (Daude et al., Proc. Natl. Acad. Sci USA 2012; 109(23): 9035–40). Results from the challenge of Sprn null and TgSprn transgenic mice with rodent-adapted prions coalesce to define downregulation of Sho as a “tracer” for the formation of misfolded PrP. However, classical BSE and rodent-adapted BSE isolates may behave differently, as they do for other facets of the pathogenic process, and this intriguing variation warrants closer scrutiny. With regards to physiological function, double knockout mice (Sprn^0/0/Prnp^0/0) mice survived to over 600 d of age. This suggests that Sho is not pi, or, given the accumulating data for many activities for PrP^C, that the pi hypothesis invoking a discrete signaling pathway to maintain neuronal viability is no longer tenable.     

Micro-Geographical Heterogeneity in Schistosoma mansoni and S. haematobium Infection and Morbidity in a Co-Endemic Community in Northern Senegal

Background

Schistosoma mansoni and S. haematobium are co-endemic in many areas in Africa. Yet, little is known about the micro-geographical distribution of these two infections or associated disease within such foci. Such knowledge could give important insights into the drivers of infection and disease and as such better tailor schistosomiasis control and elimination efforts.

Methodology

In a co-endemic farming community in northern Senegal (346 children (0–19 y) and 253 adults (20–85 y); n = 599 in total), we studied the spatial distribution of S. mansoni and S. haematobium single and mixed infections (by microscopy), S. mansoni-specific hepatic fibrosis, S. haematobium-specific urinary tract morbidity (by ultrasound) and water contact behavior (by questionnaire). The Kulldorff''s scan statistic was used to detect spatial clusters of infection and morbidity, adjusted for the spatial distribution of gender and age.

Principal Findings

Schistosoma mansoni and S. haematobium infection densities clustered in different sections of the community (p = 0.002 and p = 0.023, respectively), possibly related to heterogeneities in the use of different water contact sites. While the distribution of urinary tract morbidity was homogeneous, a strong geospatial cluster was found for severe hepatic fibrosis (p = 0.001). Particularly those people living adjacent to the most frequently used water contact site were more at risk for more advanced morbidity (RR = 6.3; p = 0.043).

Conclusions/Significance

Schistosoma infection and associated disease showed important micro-geographical heterogeneities with divergent patterns for S. mansoni and S. haematobium in this Senegalese community. Further in depth investigations are needed to confirm and explain our observations. The present study indicates that local geospatial patterns should be taken into account in both research and control of schistosomiasis. The observed extreme focality of schistosomiasis even at community level, suggests that current strategies may not suffice to move from morbidity control to elimination of schistosomiasis, and calls for less uniform measures at a finer scale.     

Tropical Secondary Forest Management Influences Frugivorous Bat Composition,Abundance and Fruit Consumption in Chiapas,Mexico

Most studies on frugivorous bat assemblages in secondary forests have concentrated on differences among successional stages, and have disregarded the effect of forest management. Secondary forest management practices alter the vegetation structure and fruit availability, important factors associated with differences in frugivorous bat assemblage structure, and fruit consumption and can therefore modify forest succession. Our objective was to elucidate factors (forest structural variables and fruit availability) determining bat diversity, abundance, composition and species-specific abundance of bats in (i) secondary forests managed by Lacandon farmers dominated by Ochroma pyramidale, in (ii) secondary forests without management, and in (iii) mature rain forests in Chiapas, Southern Mexico. Frugivorous bat species diversity (Shannon H’) was similar between forest types. However, bat abundance was highest in rain forest and O. pyramidale forests. Bat species composition was different among forest types with more Carollia sowelli and Sturnira lilium captures in O. pyramidale forests. Overall, bat fruit consumption was dominated by early-successional shrubs, highest late-successional fruit consumption was found in rain forests and more bats consumed early-successional shrub fruits in O. pyramidale forests. Ochroma pyramidale forests presented a higher canopy openness, tree height, lower tree density and diversity of fruit than secondary forests. Tree density and canopy openness were negatively correlated with bat species diversity and bat abundance, but bat abundance increased with fruit abundance and tree height. Hence, secondary forest management alters forests’ structural characteristics and resource availability, and shapes the frugivorous bat community structure, and thereby the fruit consumption by bats.     

Aerobic Fitness in Children and Young Adults with Primary Ciliary Dyskinesia

Background

Although aerobic fitness is regarded as an overall prognostic measure of morbidity and mortality, its evaluation in the chronic progressive sinopulmonary disease primary ciliary dyskinesia (PCD) has been infrequently and inconsistently reported. Here we assessed peak oxygen uptake (VO_2peak) in a large well-characterized cohort of PCD patients, and explored whether VO_2peak was associated with parameters of pulmonary function, self-reported physical limitations, and physical activity level.

Methods

VO_2peak, spirometry, diffusing capacity, whole-body plethysmography, and nitrogen multiple breath inert gas washout (N₂ MBW) were assessed in a cross-sectional, single-occasion study of clinically stable children and young adults with PCD. We used a questionnaire including self-reported physical limitations in everyday life or in vigorous activities, and estimation of weekly hours of strenuous physical activity. VO_2peak in PCD patients was compared with that in matched, healthy control subjects and a national reference.

Results

Forty-four PCD patients aged 6–29 years exhibited reduced VO_2peak compared to healthy controls (P<0.001) and the national reference. VO_2peak was abnormal (z-score <–1.96) in 34% of PCD patients. Spirometric values, RV/TLC, and indices of N₂ MBW were significantly abnormal, but VO_2peak only correlated with FEV₁ and DL_CO/V_A. VO_2peak correlated with complaints of moderate or significant limitations in vigorous activities (P = 0.0001), exhibited by 39% of PCD patients.

Conclusion

One-third of PCD patients exhibited substantially lower aerobic fitness than healthy subjects. Aerobic fitness correlated with FEV₁, DL_CO/V_A and self-reported complaints of limitations in vigorous physical activity. These findings are most likely explained by PCD pulmonary disease and its impact on pulmonary function and physical ability. Considering fitness as an important outcome and including regular strenuous physical activity in PCD treatment would probably altogether increase pulmonary clearance, lung function, aerobic fitness, and quality of life, and prevent lifestyle-related diseases.     

Species Diversity,Phylogeny and Large Scale Biogeographic Patterns of the Genus Padina (Phaeophyceae,Dictyotales)

The brown algal genus Padina (Dictyotales, Phaeophyceae) is distributed worldwide in tropical and temperate seas. Global species diversity and distribution ranges, however, remain largely unknown. Species‐level diversity was reassessed using DNA‐based, algorithmic species delineation techniques based on cox3 and rbcL sequence data from 221 specimens collected worldwide. This resulted in estimates ranging from 39 to 61 putative species (ESUs), depending on the technique as well as the locus. We discuss the merits, potential pitfalls, and evolutionary and biogeographic significance of algorithmic species delineation. We unveil patterns whereby ESUs are in all but one case restricted to either the Atlantic or Indo‐Pacific Ocean. Within ocean basins we find evidence for the vast majority of ESUs to be confined to a single marine realm. Exceptions, whereby ESUs span up to three realms, are located in the Indo‐Pacific Ocean. Patterns of range‐restricted species likely arise by repeated founder events and subsequent peripatric speciation, hypothesized to dominate speciation mechanisms for coastal marine organisms in the Indo‐Pacific. Using a three‐gene (cox3, psaA and rbcL), relaxed molecular clock phylogenetic analysis we estimated divergence times, providing a historical framework to interpret biogeographic patterns.     

Risk factors for <Emphasis Type="Italic">Coxiella burnetii</Emphasis> antibodies in bulk tank milk from Danish dairy herds

The aim was to identify risk factors associated with Coxiella burnetii antibody positivity in bulk tank milk (BTM) samples from 100 randomly selected Danish dairy cattle herds. Antibody levels were measured by an enzyme-linked immuno-sorbent assay. Before testing the herds, the farm managers were interviewed about hired labour, biosecurity, housing and herd health during the 12 months prior to the study. Variables considered important for C. burnetii antibody positivity in multivariable logistic regression analysis included the sharing of machines between farms (OR?=?3.6), human contacts (OR?=?4.2), artificial insemination by other people than artificial insemination technicians (OR?=?7.7), routine herd health contract with the veterinarian (OR?=?4.3) and hygiene precautions taken by veterinarians (OR?=?5). In addition, herd size, hired labour, trading of cattle between farms, quarantine and use of calving and disease pens also showed significant association in univariable analysis. This study demonstrates that strict biosecurity is important for the prevention of infections with C. burnetii.     

Activating and Relaxing Music Entrains the Speed of Beat Synchronized Walking

Inspired by a theory of embodied music cognition, we investigate whether music can entrain the speed of beat synchronized walking. If human walking is in synchrony with the beat and all musical stimuli have the same duration and the same tempo, then differences in walking speed can only be the result of music-induced differences in stride length, thus reflecting the vigor or physical strength of the movement. Participants walked in an open field in synchrony with the beat of 52 different musical stimuli all having a tempo of 130 beats per minute and a meter of 4 beats. The walking speed was measured as the walked distance during a time interval of 30 seconds. The results reveal that some music is ‘activating’ in the sense that it increases the speed, and some music is ‘relaxing’ in the sense that it decreases the speed, compared to the spontaneous walked speed in response to metronome stimuli. Participants are consistent in their observation of qualitative differences between the relaxing and activating musical stimuli. Using regression analysis, it was possible to set up a predictive model using only four sonic features that explain 60% of the variance. The sonic features capture variation in loudness and pitch patterns at periods of three, four and six beats, suggesting that expressive patterns in music are responsible for the effect. The mechanism may be attributed to an attentional shift, a subliminal audio-motor entrainment mechanism, or an arousal effect, but further study is needed to figure this out. Overall, the study supports the hypothesis that recurrent patterns of fluctuation affecting the binary meter strength of the music may entrain the vigor of the movement. The study opens up new perspectives for understanding the relationship between entrainment and expressiveness, with the possibility to develop applications that can be used in domains such as sports and physical rehabilitation.     

Multi-Allelic Major Effect Genes Interact with Minor Effect QTLs to Control Adaptive Color Pattern Variation in Heliconius erato

Recent studies indicate that relatively few genomic regions are repeatedly involved in the evolution of Heliconius butterfly wing patterns. Although this work demonstrates a number of cases where homologous loci underlie both convergent and divergent wing pattern change among different Heliconius species, it is still unclear exactly how many loci underlie pattern variation across the genus. To address this question for Heliconius erato, we created fifteen independent crosses utilizing the four most distinct color pattern races and analyzed color pattern segregation across a total of 1271 F2 and backcross offspring. Additionally, we used the most variable brood, an F2 cross between H. himera and the east Ecuadorian H. erato notabilis, to perform a quantitative genetic analysis of color pattern variation and produce a detailed map of the loci likely involved in the H. erato color pattern radiation. Using AFLP and gene based markers, we show that fewer major genes than previously envisioned control the color pattern variation in H. erato. We describe for the first time the genetic architecture of H. erato wing color pattern by assessing quantitative variation in addition to traditional linkage mapping. In particular, our data suggest three genomic intervals modulate the bulk of the observed variation in color. Furthermore, we also identify several modifier loci of moderate effect size that contribute to the quantitative wing pattern variation. Our results are consistent with the two-step model for the evolution of mimetic wing patterns in Heliconius and support a growing body of empirical data demonstrating the importance of major effect loci in adaptive change.