Characterization of a Novel Flavivirus from Mosquitoes in Northern Europe That Is Related to Mosquito-Borne Flaviviruses of the Tropics

A novel flavivirus was isolated from mosquitoes in Finland, representing the first mosquito-borne flavivirus from Northern Europe. The isolate, designated Lammi virus (LAMV), was antigenically cross-reactive with other flaviviruses and exhibited typical flavivirus morphology as determined by electron microscopy. The genomic sequence of LAMV was highly divergent from the recognized flaviviruses, and yet the polyprotein properties resembled those of mosquito-borne flaviviruses. Phylogenetic analysis of the complete coding sequence showed that LAMV represented a distinct lineage related to the Aedes sp.-transmitted human pathogenic flaviviruses, similarly to the newly described Nounané virus (NOUV), a flavivirus from Africa (S. Junglen et al., J. Virol. 83:4462-4468, 2009). Despite the low sequence homology, LAMV and NOUV were phylogenetically grouped closely, likely representing separate species of a novel group of flaviviruses. Despite the biological properties preferring replication in mosquito cells, the genetic relatedness of LAMV to viruses associated with vertebrate hosts warrants a search for disease associations.The genus Flavivirus in the family Flaviviridae consists of 53 recognized virus species that are enveloped, positive-sense single-stranded RNA viruses. The virion consists of three structural proteins: capsid (C), membrane (M), and envelope (E). In addition, seven nonstructural proteins are present in infected cells (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Based on their antigenic properties and vector associations, flaviviruses have been grouped into mosquito-borne, tick-borne, and no-known-vector viruses and have been isolated from vertebrates, bats, and rodents (15, 25). The grouping of flaviviruses according to their transmission mode is strongly supported by phylogenetic analyses of their genomic sequences (9, 18, 31).Mosquito-borne flaviviruses are a large and divergent group of viruses that can be differentiated phylogenetically into those associated either with encephalitic disease and transmission by Culex spp. mosquitoes or with diseases with hemorrhagic complications and transmission by Aedes spp. (18). Seven groups of mosquito-borne flaviviruses, namely, the Aroa, dengue, Japanese encephalitis, Kokobera, Ntaya, Spondweni, and yellow fever virus groups are recognized (15, 25). These groups include important animal and human pathogens such as dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and yellow fever virus (YFV).Unclassified insect flaviviruses that have no recognized association with vertebrates have been isolated from a variety of mosquito species and also from mosquito cell lines. These insect flaviviruses do not appear to infect vertebrate cells and are not associated with human or animal disease. The cell fusing agent virus (CFAV), a tentative species in the genus Flavivirus, was the first of these insect viruses to be characterized (5, 40), Although CFAV was originally identified in cultured mosquito cells, it was later isolated from mosquitoes collected in Puerto Rico (7). This as-yet-unclassified insect flavivirus group now also includes Kamiti river virus (KRV) isolated in Kenya (10, 38) and a virus isolated from Culex spp. in Japan, designated culex flavivirus (22). In addition, related viral sequences or isolates have been recently reported from mosquitoes in Spain (1), the United States and Trinidad (26), and Mexico (14). Moreover, the identification of flaviviruslike sequences integrated within the genomes of Aedes mosquitoes further complicates the evolutionary history of the flaviviruses. These sequences, currently referred to as cell silent agent are genetically most closely related to CFAV and possibly share common evolutionary origin (11). Phylogenetically, the insect viruses form a divergent outgroup that may represent a primordial flavivirus lineage. Apart from the insect flaviviruses, the other recently discovered novel flaviviruses represent highly divergent lineages, such as Tamana bat virus (13), and Ngoye virus (20). Recently, a novel flavivirus, Nounané virus (NOUV) was isolated from a novel mosquito vector species, Uranotaenia mashonaensis in Côte d''Ivoire (23), and was shown to be phylogenetically related to the human pathogenic mosquito-borne flaviviruses.Several arboviruses have been reported from Northern Europe including the flavivirus tick-borne encephalitis virus (24, 36) but, to date, no mosquito-borne flaviviruses have been isolated. Our aim was to screen for arboviruses in Finland by studying mosquitoes using virus isolation and subsequent arbovirus antigen detection, which resulted in the identification of a novel flavivirus. We present here the isolation and characterization of this isolate, designated Lammi virus (LAMV), and discuss the implications of our findings.     

A Novel Basal Body Protein That Is a Polo-like Kinase Substrate Is Required for Basal Body Segregation and Flagellum Adhesion in Trypanosoma brucei

The Polo-like kinase (PLK) in Trypanosoma brucei plays multiple roles in basal body segregation, flagellum attachment, and cytokinesis. However, the mechanistic role of TbPLK remains elusive, mainly because most of its substrates are not known. Here, we report a new substrate of TbPLK, SPBB1, and its essential roles in T. brucei. SPBB1 was identified through yeast two-hybrid screening with the kinase-dead TbPLK as the bait. It interacts with TbPLK in vitro and in vivo, and is phosphorylated by TbPLK in vitro. SPBB1 localizes to both the mature basal body and the probasal body throughout the cell cycle, and co-localizes with TbPLK at the basal body during early cell cycle stages. RNAi against SPBB1 in procyclic trypanosomes inhibited basal body segregation, disrupted the new flagellum attachment zone filament, detached the new flagellum, and caused defective cytokinesis. Moreover, RNAi of SPBB1 confined TbPLK at the basal body and the bilobe structure, resulting in constitutive phosphorylation of TbCentrin2 at the bilobe. Altogether, these results identified a basal body protein as a TbPLK substrate and its essential role in promoting basal body segregation and flagellum attachment zone filament assembly for flagellum adhesion and cytokinesis initiation.     

ROC Generated Thresholds for Field-Assessed Aerobic Fitness Related to Body Size and Cardiometabolic Risk in Schoolchildren

Objectives

1. to investigate whether 20 m multi-stage shuttle run performance (20mSRT), an indirect measure of aerobic fitness, could discriminate between healthy and overweight status in 9–10.9 yr old schoolchildren using Receiver Operating Characteristic (ROC) analysis; 2. Investigate if cardiometabolic risk differed by aerobic fitness group by applying the ROC cut point to a second, cross-sectional cohort.

Design

Analysis of cross-sectional data.

Participants

16,619 9–10.9 year old participants from SportsLinx project and 300 11–13.9 year old participants from the Welsh Schools Health and Fitness Study.

Outcome Measures

SportsLinx; 20mSRT, body mass index (BMI), waist circumference, subscapular and superilliac skinfold thicknesses. Welsh Schools Health and Fitness Study; 20mSRT performance, waist circumference, and clustered cardiometabolic risk.

Analyses

Three ROC curve analyses were completed, each using 20mSRT performance with ROC curve 1 related to BMI, curve 2 was related to waist circumference and 3 was related to skinfolds (estimated % body fat). These were repeated for both girls and boys. The mean of the three aerobic fitness thresholds was retained for analysis. The thresholds were subsequently applied to clustered cardiometabolic risk data from the Welsh Schools study to assess whether risk differed by aerobic fitness group.

Results

The diagnostic accuracy of the ROC generated thresholds was higher than would be expected by chance (all models AUC >0.7). The mean thresholds were 33 and 25 shuttles for boys and girls respectively. Participants classified as ‘fit’ had significantly lower cardiometabolic risk scores in comparison to those classed as unfit (p<0.001).

Conclusion

The use of the ROC generated cut points by health professionals, teachers and coaches may provide the opportunity to apply population level ‘risk identification and stratification’ processes and plan for “at-risk” children to be referred onto intervention services.     

Changes in Eating Attitudes,Body Esteem and Weight Control Behaviours during Adolescence in a South African Cohort

Failure to consume an adequate diet or over consumption during adolescence can disrupt normal growth and development, resulting in undesirable weight change. This leads to an increase in unhealthy weight control practices related to eating and exercise among both adolescent girls and boys to meet the societal ‘ideal’ body shape. This study therefore aims to examine the longitudinal changes in eating attitudes, body-esteem and weight control behaviours among adolescents between 13 and 17 years; and, to describe perceptions around body shape at age 17 years. A total of 1435 urban South African black and mixed ancestry boys and girls, who had data at both age 13 and 17 years from the Birth to Twenty cohort were included. Data were collected through self-administered questionnaires on eating attitudes (EAT-26), body esteem and weight control behaviours for either weight loss or muscle gain attempts. Height and weight were measured at both time points and BMI was calculated. Black females had a higher BMI (p<0.001) and an increased risk of developing eating disorders as well as significant increase in the prevalence of weight loss practices between the ages 13 and 17 years. At age 17 years both Mixed ancestry adolescents had lower body-esteem compared to black adolescents. The prevalence of possible eating disorders was 11% and 13.1% in early and late adolescents respectively. Males and females shared similar opinions on normal silhouettes being the ‘best’, ‘getting respect’ and being the ‘happiest’, while the obese silhouette was associated with the ‘worst’ and the ‘unhappiest’, and the underweight silhouette with the “weakest”. Black females had a higher BMI and an increased risk of developing eating disorders. Adolescent females engaged more in weight loss practices whereas, males in muscle gain practices indicating that Western norms of thinness as the ideal are becoming more common in South Africa.     

C1q/TNF-related Protein 4 (CTRP4) Is a Unique Secreted Protein with Two Tandem C1q Domains That Functions in the Hypothalamus to Modulate Food Intake and Body Weight

CTRP4 is a unique member of the C1q family, possessing two tandem globular C1q domains. Its physiological function is poorly defined. Here, we show that CTRP4 is an evolutionarily conserved, ∼34-kDa secretory protein expressed in the brain. In human, mouse, and zebrafish brain, CTRP4 expression begins early in development and is widespread in the central nervous system. Neurons, but not astrocytes, express and secrete CTRP4, and secreted proteins form higher-order oligomeric complexes. CTRP4 is also produced by peripheral tissues and circulates in blood. Its serum levels are increased in leptin-deficient obese (ob/ob) mice. Functional studies suggest that CTRP4 acts centrally to modulate energy metabolism. Refeeding following an overnight fast induced the expression of CTRP4 in the hypothalamus. Central administration of recombinant protein suppressed food intake and altered the whole-body energy balance in both chow-fed and high-fat diet-fed mice. Suppression of food intake by CTRP4 is correlated with a decreased expression of orexigenic neuropeptide (Npy and Agrp) genes in the hypothalamus. These results establish CTRP4 as a novel nutrient-responsive central regulator of food intake and energy balance.     

Polarity in Migrating Neurons Is Related to a Mechanism Analogous to Cytokinesis

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Aspergillus Fabm Encodes an Essential Product That Is Related to Poly(a)-Binding Proteins and Activates Development When Overexpressed

Overexpression of several different Aspergillus nidulans developmental regulatory genes has been shown to cause inappropriate developmental activation and growth inhibition. We previously exploited this observation that induced development caused growth inhibition in designing a screen to identify other genes that could activate development when overexpressed. We identified 16 mutants in which induced expression of different random genomic DNA sequences caused growth inhibition, accumulation of mRNA corresponding to the brlA developmental regulatory locus, and in several cases sporulation. This phenotype was designated FAB for Forced expression Activation of brlA and the genes were called fabA through fabP. Here we describe one of these genes, fabM, which is predicted to encode a poly(A)-binding protein (PABP) that is constitutively expressed and is essential for viability. While it is unclear why overexpression of the fabM caused sporulation, we showed that this activity required other known early developmental regulators including brlAβ, flbA, flbB, flbC, and fluG. We propose that fabM is an example of a gene that is not only required for growth, but also has specific functions early in development that assist developmental induction, presumably by allowing translation of specific mRNAs like brlA.     

The Saccharomyces Cerevisiae Spt8 Gene Encodes a Very Acidic Protein That Is Functionally Related to Spt3 and Tata-Binding Protein

Mutations in the Saccharomyces cerevisiae SPT8 gene were previously isolated as suppressors of retrotransposon insertion mutations in the 5' regions of the HIS4 and LYS2 genes. Mutations in SPT8 confer phenotypes similar to those caused by particular mutations in SPT15, which encodes the TATA-binding protein (TBP). These phenotypes are also similar to those caused by mutations in the SPT3 gene, which encodes a protein that directly interacts with TBP. We have now cloned and sequenced the SPT8 gene and have shown that it encodes a predicted protein of 602 amino acids with an extremely acidic amino terminus. In addition, the predicted SPT8 amino acid sequence contains one copy of a sequence motif found in multiple copies in a number of other eukaryotic proteins, including the β subunit of heterotrimeric G proteins. To investigate further the relationship between SPT8, SPT3 and TBP, we have analyzed the effect of an spt8 null mutation in combination with different spt3 and spt15 mutations. This genetic analysis has shown that an spt8 deletion mutation is suppressed by particular spt3 alleles. Taken together with previous results, these data suggest that the SPT8 protein is required, directly or indirectly, for TBP function at particular promoters and that the role of SPT8 may be to promote a functional interaction between SPT3 and TBP.     

Body Size Is a Significant Predictor of Congruency in Species Richness Patterns: A Meta-Analysis of Aquatic Studies

Biodiversity losses over the next century are predicted to result in alterations of ecosystem functions that are on par with other major drivers of global change. Given the seriousness of this issue, there is a need to effectively monitor global biodiversity. Because performing biodiversity censuses of all taxonomic groups is prohibitively costly, indicator groups have been studied to estimate the biodiversity of different taxonomic groups. Quantifying cross-taxon congruence is a method of evaluating the assumption that the diversity of one taxonomic group can be used to predict the diversity of another. To improve the predictive ability of cross-taxon congruence in aquatic ecosystems, we evaluated whether body size, measured as the ratio of average body length between organismal groups, is a significant predictor of their cross-taxon biodiversity congruence. To test this hypothesis, we searched the published literature and screened for studies that used species richness correlations as their metric of cross-taxon congruence. We extracted 96 correlation coefficients from 16 studies, which encompassed 784 inland water bodies. With these correlation coefficients, we conducted a categorical meta-analysis, grouping data based on the body size ratio of organisms. Our results showed that cross-taxon congruence is variable among sites and between different groups (r values ranging between −0.53 to 0.88). In addition, our quantitative meta-analysis demonstrated that organisms most similar in body size showed stronger species richness correlations than organisms which differed increasingly in size (r_adj² = 0.94, p = 0.02). We propose that future studies applying biodiversity indicators in aquatic ecosystems consider functional traits such as body size, so as to increase their success at predicting the biodiversity of taxonomic groups where cost-effective conservation tools are needed.     

Allatostatin-C/AstC-R2 Is a Novel Pathway to Modulate the Circadian Activity Pattern in Drosophila

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Predatory Functional Morphology in Raptors: Interdigital Variation in Talon Size Is Related to Prey Restraint and Immobilisation Technique

Despite the ubiquity of raptors in terrestrial ecosystems, many aspects of their predatory behaviour remain poorly understood. Surprisingly little is known about the morphology of raptor talons and how they are employed during feeding behaviour. Talon size variation among digits can be used to distinguish families of raptors and is related to different techniques of prey restraint and immobilisation. The hypertrophied talons on digits (D) I and II in Accipitridae have evolved primarily to restrain large struggling prey while they are immobilised by dismemberment. Falconidae have only modest talons on each digit and only slightly enlarged D-I and II. For immobilisation, Falconini rely more strongly on strike impact and breaking the necks of their prey, having evolved a ‘tooth’ on the beak to aid in doing so. Pandionidae have enlarged, highly recurved talons on each digit, an adaptation for piscivory, convergently seen to a lesser extent in fishing eagles. Strigiformes bear enlarged talons with comparatively low curvature on each digit, part of a suite of adaptations to increase constriction efficiency by maximising grip strength, indicative of specialisation on small prey. Restraint and immobilisation strategy change as prey increase in size. Small prey are restrained by containment within the foot and immobilised by constriction and beak attacks. Large prey are restrained by pinning under the bodyweight of the raptor, maintaining grip with the talons, and immobilised by dismemberment (Accipitridae), or severing the spinal cord (Falconini). Within all raptors, physical attributes of the feet trade off against each other to attain great strength, but it is the variable means by which this is achieved that distinguishes them ecologically. Our findings show that interdigital talon morphology varies consistently among raptor families, and that this is directly correlative with variation in their typical prey capture and restraint strategy.     

Acclimation of Leaf Nitrogen to Vertical Light Gradient at Anthesis in Wheat Is a Whole-Plant Process That Scales with the Size of the Canopy

Vertical leaf nitrogen (N) gradient within a canopy is classically considered as a key adaptation to the local light environment that would tend to maximize canopy photosynthesis. We studied the vertical leaf N gradient with respect to the light gradient for wheat (Triticum aestivum) canopies with the aims of quantifying its modulation by crop N status and genetic variability and analyzing its ecophysiological determinants. The vertical distribution of leaf N and light was analyzed at anthesis for 16 cultivars grown in the field in two consecutive seasons under two levels of N. The N extinction coefficient with respect to light (b) varied with N supply and cultivar. Interestingly, a scaling relationship was observed between b and the size of the canopy for all the cultivars in the different environmental conditions. The scaling coefficient of the b-green area index relationship differed among cultivars, suggesting that cultivars could be more or less adapted to low-productivity environments. We conclude that the acclimation of the leaf N gradient to the light gradient is a whole-plant process that depends on canopy size. This study demonstrates that modeling leaf N distribution and canopy expansion based on the assumption that leaf N distribution parallels that of the light is inappropriate. We provide a robust relationship accounting for vertical leaf N gradient with respect to vertical light gradient as a function of canopy size.In cereals, as in many crop species, nitrogen (N) nutrition is a major determinant in the elaboration of grain yield and quality (Lemaire and Millard, 1999; Lawlor, 2002; Hikosaka, 2005). N is involved in both meristematic and photosynthetic activities, with consequences on plant architecture and carbon acquisition and in fine on grain yield and protein concentration. Beside the total amount of N absorbed by the crop, the allocation of N among plant organs plays a key role in determining crop productivity and quality (Grindlay, 1997; Dreccer et al., 1998; Hikosaka, 2005).Light interception and leaf N content are the two main factors governing carbon assimilation at the leaf scale (Evans, 1989). For various species, both light and leaf N attenuate with cumulative leaf area index counted from the top of the canopy (Field, 1983; Hirose and Werger, 1987). Leaf N vertical gradients have been regarded as an adaptive response to the local light environment, maximizing canopy photosynthesis and N utilization efficiency (Hirose and Werger, 1987; Hikosaka et al., 1994; Drouet and Bonhomme, 1999), as N is largely contained in the assimilatory enzyme Rubisco. Theoretical studies indicated that leaf N maximizes canopy photosynthesis when it parallels the light gradient (i.e. when the light [K_L] and N [K_N] extinction coefficients are equal), considering that the leaf N gradient is “optimal” in accordance with the “optimization theory” (Field, 1983; Hirose and Werger, 1987; Anten et al., 1995b).Factors other than the photosynthetic photon flux density (PPFD) might be responsible for the observed leaf N distribution. For instance, the acropetal gradients of leaf age (Hikosaka et al., 1994; Hikosaka, 2005) and light composition (Rousseaux et al., 1999) are known to strengthen the leaf N gradient. However, the impact of each of these factors has been shown to be much less than that of the PPFD gradient (Werger and Hirose, 1991; Pons and de Jong-van Berkel, 2004), although for the grass species Brachypodium pinnatum other factors than light might be involved (Pons et al., 1993). At the molecular level, the process could be driven by the import of compounds such as cytokinins transported in the transpiration stream (Pons et al., 2001; Boonman et al., 2007). Although the actual N distribution usually follows the light gradient, in all studies it is less steep than the calculated optimal N profile maximizing canopy photosynthesis (Pons et al., 1989; Yin et al., 2003). Possible reasons for this discrepancy have been discussed in detail by Kull (2002). Sink-source relations and in particular the demand for N could modulate the light-leaf N relationship (Dreccer et al., 1998), but conflicting results have been reported regarding the effect of N availability on the light-leaf N relationship. While some authors found no effect of N availability (Sinclair and Shiraiwa, 1993; Milroy et al., 2001), others found that the N gradient relative to light (i.e. K_L/K_N) was steeper under low N (Hikosaka et al., 1994; Grindlay et al., 1995; Lötscher et al., 2003) or that the response of the light-leaf N relationship to N availability depended on the developmental stage (Dreccer et al., 2000). Interspecific differences in the light-leaf N relationship have also been reported and were related to differences in phenotypic plasticity (Aerts, 1996) or plant architecture (leaf stature and branching pattern; Anten et al., 1995a; Lötscher et al., 2003).Since canopy photosynthesis is dependent upon the leaf N gradient, it has been suggested that the pattern of leaf N distribution could be responsible for part of the genetic variability associated with the negative correlation between grain yield and protein concentration reported for various crop species (Dreccer et al., 1998). In wheat (Triticum aestivum), N accumulated before anthesis contributes 30% to 70% of grain N (Mi et al., 2000; Kichey et al., 2007). The efficiency of N translocation from the lower to the upper leaves may increase with the steepness of the N gradient, with only a negligible effect on canopy carbon gain integrated over the whole grain-filling period. This hypothesis is consistent with experimental studies based on a range of genotypes showing that, at a given grain yield level, grain protein concentration is positively related to the efficiency of N translocation either from the lower to the upper leaves (Wang et al., 2005) or from the leaves to the grains (Monaghan et al., 2001; Jukanti and Fischer, 2008). Only a few studies have investigated the intraspecific variability of the light-N relationship at the intraspecific level (Shiraiwa and Sinclair, 1993; Bindraban, 1999; Bertheloot et al., 2008; van Oosterom et al., 2010). For wheat, published analyses of the genetic variability of the light-leaf N relationship were limited to only two to five genotypes, and no genetic differences were found (Bindraban, 1999; Bertheloot et al., 2008).This paper focuses on the genetic variability of the vertical leaf N gradient with respect to light for wheat. Three main issues were investigated. What is the effect of N supply on the vertical distribution of leaf N? Does the distribution of leaf N with respect to light differ among genotypes? If the adjustment of leaf N to the light gradient varies with both the genotype and N supply, could this genetic and environmental variability have a unique ecophysiological determinant (driving variable)?These questions were addressed using 16 genotypes (Supplemental Table S1) covering a wide range of variation for N use efficiency (i.e. grain dry mass yield per unit of available mineral N from the soil and fertilizer), for grain protein concentration (Le Gouis et al., 2000; Foulkes et al., 2006; Gaju et al., 2011) and for the deviation from the negative correlation between grain yield and protein concentration (Oury et al., 2003). The 16 genotypes were grown in the field under two conditions of N supply (N− and N+ for low- and high-N treatments, respectively) in order to modulate crop N status at Clermont-Ferrand (CF) in France in two consecutive seasons (experiments CF07 and CF08). In addition, four of the 16 cultivars representing the variability observed for N utilization and N uptake efficiency were grown in the field under two conditions of N supply at Sutton Bonington (SB) in the United Kingdom in one season (experiment SB07). The distribution of leaf N was analyzed at anthesis. The first reason for this is that the distribution of both light and leaf N within the canopy is relatively stable from this phenological stage until almost the end of grain filling (Bertheloot et al., 2008). Whereas the canopy green area index (GAI) decreases dramatically during the grain-filling period, the structure of the canopy affecting light interception does not change significantly during that period. Both the vertical light and N distributions down the canopy are unchanged during most of the grain-filling period; therefore, the K_N-to-K_L ratio is constant during that period (Bertheloot et al., 2008). Similarly, Archontoulis et al. (2011) showed that K_N-to-K_L ratio is not modified during the vegetative and reproductive stages for field-gown sunflower (Helianthus annuus) crops. Therefore, as most of the final grain yield results from carbon assimilated after anthesis (Bidinger et al., 1977; Gebbing and Schnyder, 1999), the N distribution at anthesis is very relevant in terms of carbon assimilation and grain yield in wheat. A second reason is that the number and potential size of grains are determined around anthesis, which therefore appears as a critical stage in the formation of grain yield. A better understanding of the ecophysiological determinants of leaf N gradient at this phenological stage could consequently be crucial for improving wheat productivity and quality (Dreccer et al., 1998).     

Microheterogeneity in 16S Ribosomal DNA-Defined Bacterial Populations from a Stratified Planktonic Environment Is Related to Temporal Changes and to Ecological Adaptations

Temporal changes of the bacterioplankton from a meromictic lake (Lake Vilar, Banyoles, Spain) were analyzed with four culture-independent techniques: epifluorescence microscopy, PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting, fluorescence in situ whole-cell hybridization and flow cytometry sorting. Microscopically, blooms of one cyanobacterium (Synechococcus sp.-like), one green sulfur bacterium (Chlorobium phaeobacteroides-like), and one purple sulfur bacterium (Thiocystis minor-like) were observed at different depths and times. DGGE retrieved these populations and, additionally, populations related to the Cytophaga-Flavobacterium-Bacteroides phylum as predominant community members. The analyses of partial 16S ribosomal DNA sequences from the DGGE fingerprints (550 bp analyzed) revealed higher genetic diversity than expected from microscopic observation for most of these groups. Thus, the sequences of two Synechococcus spp. (both had a similarity of 97% to Synechococcus sp. strain PCC6307 in 16S rRNA), two Thiocystis spp. (similarities to Thiocystis minor of 93 and 94%, respectively), and three Cytophaga spp. (similarities to Cytophaga fermentans of 88 and 89% and to Cytophaga sp. of 93%, respectively) were obtained. The two populations of Synechococcus exhibited different pigment compositions and temporal distributions and their 16S rRNA sequences were 97.3% similar. The two Thiocystis populations differed neither in pigment composition nor in morphology, but their 16S rRNA sequences were only 92.3% similar and they also showed different distributions over time. Finally, two of the Cytophaga spp. showed 96.2% similarity between the 16S rRNA sequences, but one of them was found to be mostly attached to particles and only in winter. Thus, the identity of the main populations changed over time, but the function of the microbial guilds was maintained. Our data showed that temporal shifts in the identity of the predominant population is a new explanation for the environmental 16S rRNA microdiversity retrieved from microbial assemblages and support the hypothesis that clusters of closely related 16S rRNA environmental sequences may actually represent numerous closely related, yet ecologically distinct, populations.     

Aortopulmonary Collateral Flow Is Related to Pulmonary Artery Size and Affects Ventricular Dimensions in Patients after the Fontan Procedure

Background

Aortopulmonary collaterals (APCs) are frequently found in patients with a single-ventricle (SV) circulation. However, knowledge about the clinical significance of the systemic-to-pulmonary shunt flow in patients after the modified Fontan procedure and its potential causes is limited. Accordingly, the aim of our study was to detect and quantify APC flow using cardiovascular magnetic resonance (CMR) and assess its impact on SV volume and function as well as to evaluate the role of the size of the pulmonary arteries in regard to the development of APCs.

Methods

60 patients (mean age 13.3 ± 6.8 years) after the Fontan procedure without patent tunnel fenestration underwent CMR as part of their routine clinical assessment that included ventricular functional analysis and flow measurements in the inferior vena cava (IVC), superior vena cava (SVC) and ascending aorta (Ao). APC flow was quantified using the systemic flow estimator: (Ao) - (IVC + SVC). Pulmonary artery index (Nakata index) was calculated as RPA + LPA area/body surface area using contrast enhanced MR angiography. The patient cohort was divided into two groups according to the median APC flow: group 1 < 0.495 l/min/m² and group 2 > 0.495 l/min/m².

Results

Group 1 patients had significant smaller SV enddiastolic (71 ± 16 vs 87 ± 25 ml/m²; p=0.004) and endsystolic volumes (29 ± 11 vs 40 ± 21 ml/m²; p=0.02) whereas ejection fraction (59 ± 9 vs 56 ± 13%; p=0.38) differed not significantly. Interestingly, pulmonary artery size showed a significant inverse correlation with APC flow (r=-0.50, p=0.002).

Conclusions

Volume load due to APC flow in Fontan patients affected SV dimensions, but did not result in an impairment of SV function. APC flow was related to small pulmonary artery size, suggesting that small pulmonary arteries represent a potential stimulus for the development of APCs.     

Adaptive (TINT) Changes in the Tumor Bearing Organ Are Related to Prostate Tumor Size and Aggressiveness

In order to grow, tumors need to induce supportive alterations in the tumor-bearing organ, by us named tumor instructed normal tissue (TINT) changes. We now examined if the nature and magnitude of these responses were related to tumor size and aggressiveness. Three different Dunning rat prostate tumor cells were implanted into the prostate of immune-competent rats; 1) fast growing and metastatic MatLyLu tumor cells 2) fast growing and poorly metastatic AT-1 tumor cells, and 3) slow growing and non-metastatic G tumor cells. All tumor types induced increases in macrophage, mast cell and vascular densities and in vascular cell-proliferation in the tumor-bearing prostate lobe compared to controls. These increases occurred in parallel with tumor growth. The most pronounced and rapid responses were seen in the prostate tissue surrounding MatLyLu tumors. They were, also when small, particularly effective in attracting macrophages and stimulating growth of not only micro-vessels but also small arteries and veins compared to the less aggressive AT-1 and G tumors. The nature and magnitude of tumor-induced changes in the tumor-bearing organ are related to tumor size but also to tumor aggressiveness. These findings, supported by previous observation in patient samples, suggest that one additional way to evaluate prostate tumor aggressiveness could be to monitor its effect on adjacent tissues.     

Mutation in Parkinson Disease-Associated,G-Protein-Coupled Receptor 37 (GPR37/PaelR) Is Related to Autism Spectrum Disorder

Little is known about the molecular pathogenesis of Autism spectrum disorder (ASD), a neurodevelopmental disorder. Here we identified two mutations in the G-protein-coupled receptor 37 gene (GPR37) localized on chromosome 7q31–33, called the AUTS1 region, of ASD patients; 1585–1587 ttc del (Del312F) in one Japanese patient and G2324A (R558Q) in one Caucasian patient. The Del312F was located in the conserved transmembrane domain, and the R558Q was located in a conserved region just distal to the last transmembrane domain. In addition, a potential ASD-related GPR37 variant, T589M, was found in 7 affected Caucasian men from five different families. Our results suggested that some alleles in GPR37 were related to the deleterious effect of ASD. GPR37 is associated with the dopamine transporter to modulate dopamine uptake, and regulates behavioral responses to dopaminergic drugs. Thus, dopaminergic neurons may be involved in the ASD. However, we also detected the Del321F mutation in the patient''s unaffected father and R558Q in not only an affected brother but also an unaffected mother. The identification of unaffected parents that carried the mutated alleles suggested that the manifestation of ASD was also influenced by factors other than these mutations, including endoplasmic reticulum stress of the mutated proteins or gender. Our study will provide the new insight into the molecular pathogenesis of ASD.     

A Lower Olfactory Capacity Is Related to Higher Circulating Concentrations of Endocannabinoid 2-Arachidonoylglycerol and Higher Body Mass Index in Women

The endocannabinoid (eCB) system can promote food intake by increasing odor detection in mice. The eCB system is over-active in human obesity. Our aim is to measure circulating eCB concentrations and olfactory capacity in a human sample that includes people with obesity and explore the possible interaction between olfaction, obesity and the eCB system. The study sample was made up of 161 females with five groups of body mass index sub-categories ranging from under-weight to morbidly obese. We assessed olfactory capacity with the “Sniffin´Sticks” test, which measures olfactory threshold-discrimination-identification (TDI) capacity. We measured plasma concentrations of the eCBs 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine or anandamide (AEA), and several eCB-related compounds, 2-acylglycerols and N-acylethanolamines. 2-AG and other 2-acylglycerols fasting plasma circulating plasma concentrations were higher in obese and morbidly obese subjects. AEA and other N-acylethanolamine circulating concentrations were lower in under-weight subjects. Olfactory TDI scores were lower in obese and morbidly obese subjects. Lower TDI scores were independently associated with higher 2-AG fasting plasma circulating concentrations, higher %body fat, and higher body mass index, after controlling for age, smoking, menstruation, and use of contraceptives. Our results show that obese subjects have a lower olfactory capacity than non-obese ones and that elevated fasting plasma circulating 2-AG concentrations in obesity are linked to a lower olfactory capacity. In agreement with previous studies we show that eCBs AEA and 2-AG, and their respective congeners have a distinct profile in relation to body mass index. The present report is the first study in humans in which olfactory capacity and circulating eCB concentrations have been measured in the same subjects.