Sex chromosomes and associated rDNA form a heterochromatic network in the polytene nuclei of Bactrocera oleae (Diptera: Tephritidae)

The olive fruit fly, Bactrocera oleae, has a diploid set of 2n?=?12 chromosomes including a pair of sex chromosomes, XX in females and XY in males, but polytene nuclei show only five polytene chromosomes, obviously formed by five autosome pairs. Here we examined the fate of the sex chromosomes in the polytene complements of this species using fluorescence in situ hybridization (FISH) with the X and Y chromosome-derived probes, prepared by laser microdissection of the respective chromosomes from mitotic metaphases. Specificity of the probes was verified by FISH in preparations of mitotic chromosomes. In polytene nuclei, both probes hybridized strongly to a granular heterochromatic network, indicating thus underreplication of the sex chromosomes. The X chromosome probe (in both female and male nuclei) highlighted most of the granular mass, whereas the Y chromosome probe (in male nuclei) identified a small compact body of this heterochromatic network. Additional hybridization signals of the X probe were observed in the centromeric region of polytene chromosome II and in the telomeres of six polytene arms. We also examined distribution of the major ribosomal DNA (rDNA) using FISH with an 18S rDNA probe in both mitotic and polytene chromosome complements of B. oleae. In mitotic metaphases, the probe hybridized exclusively to the sex chromosomes. The probe signals localized a discrete rDNA site at the end of the short arm of the X chromosome, whereas they appeared dispersed over the entire dot-like Y chromosome. In polytene nuclei, the rDNA was found associated with the heterochromatic network representing the sex chromosomes. Only in nuclei with preserved nucleolar structure, the probe signals were scattered in the restricted area of the nucleolus. Thus, our study clearly shows that the granular heterochromatic network of polytene nuclei in B. oleae is formed by the underreplicated sex chromosomes and associated rDNA.     

Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET)

Abstract

Detrimental effects of ionizing radiation (IR) are correlated to the varying efficiency of IR to induce complex DNA damage. A double strand break (DSB) can be considered the simpler form of complex DNA damage. These types of damage can consist of DSBs, single strand breaks (SSBs) and/or non-DSB lesions such as base damages and apurinic/apyrimidinic (AP; abasic) sites in different combinations. Enthralling theoretical (Monte Carlo simulations) and experimental evidence suggests an increase in the complexity of DNA damage and therefore repair resistance with linear energy transfer (LET). In this study, we have measured the induction and processing of DSB and non-DSB oxidative clusters using adaptations of immunofluorescence. Specifically, we applied foci colocalization approaches as the most current methodologies for the in situ detection of clustered DNA lesions in a variety of human normal (FEP18-11-T1) and cancerous cell lines of varying repair efficiency (MCF7, HepG2, A549, MO59K/J) and radiation qualities of increasing LET, that is γ-, X-rays 0.3–1?keV/μm, α-particles 116?keV/μm and ³⁶Ar ions 270?keV/μm. Using γ-H2AX or 53BP1 foci staining as DSB probes, we calculated a DSB apparent rate of 5–16 DSBs/cell/Gy decreasing with LET. A similar trend was measured for non-DSB oxidized base lesions detected using antibodies against the human repair enzymes 8-oxoguanine-DNA glycosylase (OGG1) or AP endonuclease (APE1), that is damage foci as probes for oxidized purines or abasic sites, respectively. In addition, using colocalization parameters previously introduced by our groups, we detected an increasing clustering of damage for DSBs and non-DSBs. We also make correlations of damage complexity with the repair efficiency of each cell line and we discuss the biological importance of these new findings with regard to the severity of IR due to the complex nature of its DNA damage.     

Allelic Variation in Paralogs of GDP-l-Galactose Phosphorylase Is a Major Determinant of Vitamin C Concentrations in Apple Fruit

To identify the genetic factors underlying the regulation of fruit vitamin C (l-ascorbic acid [AsA]) concentrations, quantitative trait loci (QTL) studies were carried out in an F1 progeny derived from a cross between the apple (Malus × domestica) cultivars Telamon and Braeburn over three years. QTL were identified for AsA, glutathione, total antioxidant activity in both flesh and skin tissues, and various quality traits, including flesh browning. Four regions on chromosomes 10, 11, 16, and 17 contained stable fruit AsA-QTL clusters. Mapping of AsA metabolic genes identified colocations between orthologs of GDP-l-galactose phosphorylase (GGP), dehydroascorbate reductase (DHAR), and nucleobase-ascorbate transporter within these QTL clusters. Of particular interest are the three paralogs of MdGGP, which all colocated within AsA-QTL clusters. Allelic variants of MdGGP1 and MdGGP3 derived from the cultivar Braeburn parent were also consistently associated with higher fruit total AsA concentrations both within the mapping population (up to 10-fold) and across a range of commercial apple germplasm (up to 6-fold). Striking differences in the expression of the cv Braeburn MdGGP1 allele between fruit from high- and low-AsA genotypes clearly indicate a key role for MdGGP1 in the regulation of fruit AsA concentrations, and this MdGGP allele-specific single-nucleotide polymorphism marker represents an excellent candidate for directed breeding for enhanced fruit AsA concentrations. Interestingly, colocations were also found between MdDHAR3-3 and a stable QTL for browning in the cv Telamon parent, highlighting links between the redox status of the AsA pool and susceptibility to flesh browning.In plants, l-ascorbic acid (AsA; vitamin C) is essential for the detoxification of reactive oxygen species produced under stress or following exposure to pathogens. In addition to these antioxidant functions, AsA has been shown to be involved in a range of important cellular processes, including plant development and hormone signaling, cell cycle, cell expansion, senescence, and as a cofactor for a number of important enzymes (for review, see Davey et al., 2000; Smirnoff et al., 2001; Noctor, 2006). Fruit AsA concentrations have also been correlated with the maintenance of quality during postharvest storage (Davey and Keulemans, 2004; Davey et al., 2007) and have been linked to susceptibility to internal browning in both apple (Malus × domestica; Davey et al., 2006; Davey and Keulemans, 2009) and pear (Pyrus communis; Veltman et al., 1999; Franck et al., 2003). Finally, AsA is clearly an essential dietary component for humans, with a protective role proposed for many disorders and diseases (Diplock et al., 1998). Given its importance for all metabolically active tissues, there is widespread interest in unraveling the mechanisms underlying the genetic control of AsA concentrations in fruits as well as in how AsA interacts with other plant antioxidant pools.The concentration of AsA will be determined by the net rates of biosynthesis, recycling, degradation, and/or intercellular and intracellular transport, but the relative contribution of these various processes depends on several factors, including genetics, tissue type (Bulley et al., 2009), developmental stage (Hancock et al., 2007; Bulley et al., 2009; Ioannidi et al., 2009), and light intensity (Yabuta et al., 2007; Gautier et al., 2009). The biosynthesis of AsA proceeds via l-Gal (Wheeler et al., 1998), although conclusive evidence for all steps has only relatively recently become available (Conklin et al., 2006; Laing et al., 2007). Alternative biosynthetic routes involving uronic acids (Davey et al., 1999; Agius et al., 2003), l-gulose (Wolucka and Van Montagu, 2003), or myoinositol (Lorence et al., 2004) have been proposed in several plant species, including apple (Davey et al., 2004; Razavi et al., 2005; Fig. 1), but their physiological relevance and contribution to the AsA pool is still far from clear in most plant species, with the possible exception of strawberry (Fragaria × ananassa; Agius et al., 2003; Cruz-Rus et al., 2011; Zorrilla-Fontanesi et al., 2011).Open in a separate windowFigure 1.AsA biosynthetic and recycling pathways in plants: l-Gal pathway, reactions 1 to 9; l-gulose pathway, reactions 1 to 5 and 10 to 13; d-galacturonate pathway, reactions 14 to 16; myoinositol/glucuronate pathway, reactions 17 to 21; recycling pathway, reactions 22 to 27. Reactions with question marks are yet to be identified. Numbered reactions are as follows: 1, Glc-6-P isomerase; 2, Man-6-P isomerase (PMI; EC 5.3.1.8); 3, phosphomannomutase (PMM; EC 5.4.2.8); 4, GDP-d-Man pyrophosphorylase (GMP; EC 2.7.7.13); 5, GDP-d-Man 3′,5′-epimerase (GME; EC 5.1.3.18); 6, GDP-l-Gal-phosphorylase (VTC2 or GGP; EC 2.7.7.69); 7, l-Gal-1-P phosphatase (VTC4 or GPP; EC 3.1.3.25); 8, l-Gal dehydrogenase (GalDH; EC 1.1.1.48); 9, l-galactono-1,4-lactone dehydrogenase (GLDH; EC 1.3.2.3); 10, nucleotide pyrophosphatase or sugar-1-phosphate guanyltransferase; 11, sugar phosphatase; 12, sugar dehydrogenase; 13, l-gulono-1,4-lactone oxidase (EC 1.1.3.8); 14, d-galacturonate-1-phosphate uridyltransferase and d-galacturonate-1-phosphate phosphatase (possible); 15, d-galacturonate reductase (GalUR; EC 1.1.1.n9); 16, aldonolactonase; 17, myoinositol oxygenase (MIOX; EC 1.13.99.1); 18, d-glucuronate reductase (EC 1.1.1.19); 19, l-gulonolactonase; 20, d-glucuronate-1-phosphate uridyltransferase; 21, d-glucurono-1-phosphate phosphatase; 22, l-ascorbate peroxidase (APX; EC 1.11.1.11); 23, l-ascorbate oxidase (AO; EC 1.10.3.3); 24, monodehydroascorbate reductase (MDHAR; EC 1.6.5.4); 25, dehydroascorbate reductase (DHAR; EC 1.8.5.1); 26, GSH reductase (GR; EC 1.8.1.7). [See online article for color version of this figure.]As an antioxidant, AsA is able to accept electrons from a wide range of radical substrates, and in this process it becomes oxidized first to monodehydroascorbate and then to dehydroascorbate (DHA). These oxidized forms of AsA can be regenerated by the ascorbate-glutathione (GSH) cycle, so that GSH and the activities of GSH reductase, dehydroascorbate reductase (DHAR), and monodehydroascorbate reductase (MDHAR) maintain the size and redox status of the AsA pool (Noctor and Foyer, 1998; Fig. 1). Indeed, overexpression of an Arabidopsis (Arabidopsis thaliana) MDHAR (Eltayeb et al., 2007) and a wheat (Triticum aestivum) DHAR (Chen et al., 2003) have both been shown to increase foliar AsA concentrations in tobacco (Nicotiana tabacum). MDHAR activity has also been positively correlated with both AsA and fruit firmness in tomato (Solanum lycopersicum) after chilling stress (Stevens et al., 2008).Tissue AsA concentrations can also be maintained by intercellular transport, and there is evidence for the long-distance transport of AsA via the phloem from source (leaf) to sink (fruit) tissues (Franceschi and Tarlyn, 2002; Hancock et al., 2003). In apple, fruit AsA concentrations have been suggested to be partly dependent on the translocation of AsA from leaves (Li et al., 2009), but in black currant (Ribes nigrum), others concluded that the contribution of phloem AsA transport to fruit AsA concentrations was negligible (Hancock et al., 2007). While the actual mechanisms of long-distance transport of AsA have not been fully determined, attention has focused on the large family of Nucleobase-Ascorbate Transporters (NATs; de Koning and Diallinas, 2000), and NAT homologs have been found to be highly expressed in vascular tissues (Maurino et al., 2006).Genes involved in several of these mechanisms have been proposed to be key regulators of fruit AsA concentrations, including GDP-l-Gal phosphorylase (GGP) or vitamin c defective2 (VTC2) in kiwifruit (Actinidia deliciosa; Bulley et al., 2009, 2012) as well as GDP-Man-3,5-epimerase (GME; Gilbert et al., 2009), l-Gal-1-P-phosphatase (GPP or VTC4; Ioannidi et al., 2009), and MDHAR (Stevens et al., 2007) in tomato. However, apart from GGP (Bulley et al., 2012), overexpression of these structural genes has to date had limited success in altering the fruit AsA pool (Agius et al., 2003; Bulley et al., 2009; Haroldsen et al., 2011).In this work, we set out to identify potential genetic determinants of fruit AsA concentrations in apple fruit using a combination of molecular and genomic approaches. Initial quantitative trait loci (QTL) analyses of AsA concentrations (Davey et al., 2006) have been expanded to identify QTL for other antioxidants and fruit quality traits over three years, including results in 1 year comparing the concentrations of AsA in fruit and leaves. Alignments of the apple orthologs of genes involved in AsA biosynthesis, turnover, and transport against the whole genome sequence of cv Golden Delicious (Velasco et al., 2010) allowed us to identify candidate genes (CGs) colocating with stable QTL clusters. Using next-generation sequencing (RNA-Seq) data, polymorphic single-nucleotide polymorphism (SNP)-based markers were developed for these colocating CGs, and their positions on individual linkage groups were confirmed by linkage mapping in our mapping population. Finally, associations between allelic variants of these CGs and their expression levels in cultivars with contrasting AsA concentrations allowed us to develop allele-specific markers associated with high fruit AsA concentrations.     

CaMKII inhibition reduces arrhythmogenic Ca2+ events in subendocardial cryoinjured rat living myocardial slices

Spontaneous Ca²⁺ release (SCR) can cause triggered activity and initiate arrhythmias. Intrinsic transmural heterogeneities in Ca²⁺ handling and their propensity to disease remodeling may differentially modulate SCR throughout the left ventricular (LV) wall and cause transmural differences in arrhythmia susceptibility. Here, we aimed to dissect the effect of cardiac injury on SCR in different regions in the intact LV myocardium using cryoinjury on rat living myocardial slices (LMS). We studied SCR under proarrhythmic conditions using a fluorescent Ca²⁺ indicator and high-resolution imaging in LMS from the subendocardium (ENDO) and subepicardium (EPI). Cryoinjury caused structural remodeling, with loss in T-tubule density and an increased time of Ca²⁺ transients to peak after injury. In ENDO LMS, the Ca²⁺ transient amplitude and decay phase were reduced, while these were not affected in EPI LMS after cryoinjury. The frequency of spontaneous whole-slice contractions increased in ENDO LMS without affecting EPI LMS after injury. Cryoinjury caused an increase in foci that generates SCR in both ENDO and EPI LMS. In ENDO LMS, SCRs were more closely distributed and had reduced latencies after cryoinjury, whereas this was not affected in EPI LMS. Inhibition of CaMKII reduced the number, distribution, and latencies of SCR, as well as whole-slice contractions in ENDO LMS, but not in EPI LMS after cryoinjury. Furthermore, CaMKII inhibition did not affect the excitation–contraction coupling in cryoinjured ENDO or EPI LMS. In conclusion, we demonstrate increased arrhythmogenic susceptibility in the injured ENDO. Our findings show involvement of CaMKII and highlight the need for region-specific targeting in cardiac therapies.     

The Cost Effectiveness of Psychological and Pharmacological Interventions for Social Anxiety Disorder: A Model-Based Economic Analysis

Background

Social anxiety disorder is one of the most persistent and common anxiety disorders. Individually delivered psychological therapies are the most effective treatment options for adults with social anxiety disorder, but they are associated with high intervention costs. Therefore, the objective of this study was to assess the relative cost effectiveness of a variety of psychological and pharmacological interventions for adults with social anxiety disorder.

Methods

A decision-analytic model was constructed to compare costs and quality adjusted life years (QALYs) of 28 interventions for social anxiety disorder from the perspective of the British National Health Service and personal social services. Efficacy data were derived from a systematic review and network meta-analysis. Other model input parameters were based on published literature and national sources, supplemented by expert opinion.

Results

Individual cognitive therapy was the most cost-effective intervention for adults with social anxiety disorder, followed by generic individual cognitive behavioural therapy (CBT), phenelzine and book-based self-help without support. Other drugs, group-based psychological interventions and other individually delivered psychological interventions were less cost-effective. Results were influenced by limited evidence suggesting superiority of psychological interventions over drugs in retaining long-term effects. The analysis did not take into account side effects of drugs.

Conclusion

Various forms of individually delivered CBT appear to be the most cost-effective options for the treatment of adults with social anxiety disorder. Consideration of side effects of drugs would only strengthen this conclusion, as it would improve even further the cost effectiveness of individually delivered CBT relative to phenelzine, which was the next most cost-effective option, due to the serious side effects associated with phenelzine. Further research needs to determine more accurately the long-term comparative benefits and harms of psychological and pharmacological interventions for social anxiety disorder and establish their relative cost effectiveness with greater certainty.     

Genetic and cytogenetic analysis of the American cherry fruit fly, Rhagoletis cingulata (Diptera: Tephritidae)

The American eastern cherry fruit fly, Rhagoletis cingulata, a pest of cherries in the western hemisphere, invaded Europe in 1983, and since then dispersed to several European countries. Information on the genetics and cytogenetics of this pest is very scarce. The mitotic karyotype and detailed photographic maps of the salivary gland polytene chromosomes of R. cingulata are presented here. The mitotic metaphase complement consists of six pairs of chromosomes with the sex chromosomes being very small and similar in size. The analysis of the salivary gland polytene complement shows a total number of five long chromosomes (10 polytene arms), which correspond to the five autosomes of the mitotic nuclei and an extrachromosomal heterochromatic mass, which corresponds to the sex chromosomes. The banding patterns and the most characteristic features and prominent landmarks of each polytene chromosome are presented and discussed. Chromosomal homologies between R. cingulata, R. completa and R. cerasi are also proposed, based on the comparison of chromosome banding patterns. Furthermore, the detection and characterization of Wolbachia pipientis in the R. cingulata population studied is presented and the potential correlation with the asynaptic phenomena found in its polytene complement is discussed. In addition, 10 out of 24 microsatellite markers developed for other Rhagoletis species are cross-amplified, evaluated and proposed as useful markers for population and genetic studies in R. cingulata.     

Bone metabolism compensates for the delayed growth in small for gestational age neonates

The goal of the present study is to investigate the relationship between anthropometric and bone metabolism markers in a sample of neonates and their mothers. A sample of 20 SGA (small for the gestational age), AGA (appropriate for the gestational age) and LGA (large for the gestational age) term neonates and their 20 mothers was analyzed at birth and at exit. Elisa method was used to measure the OPG (Osteoprotegerin), RANK (Receptor activator of nuclear factor-kappaB), RANKL (Receptor activator of nuclear factor-kappaB Ligand), IGF-1 (Insulin-like growth factor 1), IGFBP3 (Insulin-like Growth Factor Binding Protein 3) and Leptin levels. Birth weight and length were positively correlated with RANKL, IGF-1 and IGFBP3 and negatively with the ratio OPG/RANKL. SGA neonates presented lower RANKL values and higher OPG/RANKL ratio while LGA neonates had higher RANK levels than AGA neonates. Positive association was shown between neonatal IGFBP3 and maternal IGF-1 values and between neonatal and maternal RANK values at birth and at exit. These results reveal a remarkable upregulation of OPG/RANKL ratio in SGA neonates, pointing out the role of bone turnover in compensating for the delayed neonatal growth.     

Solid phase microextraction gas chromatographic analysis of organophosphorus pesticides in biological samples

Headspace-solid phase microextraction (HS-SPME) was studied and optimised for the determination of four common organophosphorus pesticides (OPPs) in biological samples. Various parameters controlling SPME were studied: choice of SPME fiber, type and content of salt added, preheating and extraction time, desorption time, extraction temperature. Capillary gas chromatographic analysis with nitrogen phosphorus detection (GC-NPD) facilitates sensitive and selective detection of the OPPs: malathion, parathion, methyl parathion and diazinon. Fenitrothion was used as the internal standard. The method was applied to the determination of the pesticides in human biological specimens: whole blood, blood plasma, urine, cerebrospinal fluid, liver and kidney. Limits of detection ranged from 2 to 55 ng/ml depending on pesticide and type of specimen. The developed methodology overcomes limitations and obstacles of conventional methods such as the use of organic solvents, the formation of emulsions and the tedious-cumbersome procedures. The proposed protocol is seen as an attractive alternative to be used in routine toxicological analysis.