Fgf signaling is required for photoreceptor maintenance in the adult zebrafish retina

Fibroblast growth factors (Fgf) are secreted signaling molecules that have mitogenic, patterning, neurotrophic and angiogenic properties. Their importance during embryonic development in patterning and morphogenesis of the vertebrate eye is well known, but less is known about the role of Fgfs in the adult vertebrate retina. To address Fgf function in adult retina, we determined the spatial distribution of components of the Fgf signaling pathway in the adult zebrafish retina. We detected differential expression of Fgf receptors, ligands and downstream Fgf targets within specific retinal layers. Furthermore, we blocked Fgf signaling in the retina, by expressing a dominant negative variant of Fgf receptor 1 conditionally in transgenic animals. After blocking Fgf signaling we observe a fast and progressive photoreceptor degeneration and disorganization of retinal tissue, coupled with cell death in the outer nuclear layer. Following the degeneration of photoreceptors, a profound regeneration response is triggered that starts with proliferation in the inner nuclear layer. Ultimately, rod and cone photoreceptors are regenerated completely. Our study reveals the requirement of Fgf signaling to maintain photoreceptors and for proliferation during regeneration in the adult zebrafish retina.     

Excision of HIV-1 proviral DNA by recombinant cell permeable tre-recombinase

Over the previous years, comprehensive studies on antiretroviral drugs resulted in the successful introduction of highly active antiretroviral therapy (HAART) into clinical practice for treatment of HIV/AIDS. However, there is still need for new therapeutic approaches, since HAART cannot eradicate HIV-1 from the infected organism and, unfortunately, can be associated with long-term toxicity and the development of drug resistance. In contrast, novel gene therapy strategies may have the potential to reverse the infection by eradicating HIV-1. For example, expression of long terminal repeat (LTR)-specific recombinase (Tre-recombinase) has been shown to result in chromosomal excision of proviral DNA and, in consequence, in the eradication of HIV-1 from infected cell cultures. However, the delivery of Tre-recombinase currently depends on the genetic manipulation of target cells, a process that is complicating such therapeutic approaches and, thus, might be undesirable in a clinical setting. In this report we demonstrate that E.coli expressed Tre-recombinases, tagged either with the protein transduction domain (PTD) from the HIV-1 Tat trans-activator or the translocation motif (TLM) of the Hepatitis B virus PreS2 protein, were able to translocate efficiently into cells and showed significant recombination activity on HIV-1 LTR sequences. Tre activity was observed using episomal and stable integrated reporter constructs in transfected HeLa cells. Furthermore, the TLM-tagged enzyme was able to excise the full-length proviral DNA from chromosomal integration sites of HIV-1-infected HeLa and CEM-SS cells. The presented data confirm Tre-recombinase activity on integrated HIV-1 and provide the basis for the non-genetic transient application of engineered recombinases, which may be a valuable component of future HIV eradication strategies.     

Polyfunctional CD4(+) T Cell Responses to Immunodominant Epitopes Correlate with Disease Activity of Virulent Salmonella

Salmonella enterica serovars are intracellular bacteria capable of causing typhoid fever and gastroenteritis of significant morbidity and mortality worldwide. Current prophylactic and therapeutic treatment is hampered by the emergence of multidrug-resistant (MDR) strains of Salmonella, and vaccines provide only temporal and partial protection in vaccinees. To develop more effective Salmonella vaccines, it is important to understand the development of protective adaptive immunity to virulent Salmonella. Here we report the identification of novel CD4(+) T cell peptide epitopes, which are conserved among Salmonella serovars. Immunization of Salmonella-infected mice with these peptide epitopes reduces the burden of Salmonella disease. Furthermore, we show that distinct polyfunctional (interferon-γ(+), tumor necrosis factor(+), and interleukin-2(+)) Salmonella-specific CD4(+) T cell responses develop with respect to magnitude and kinetics. Moreover, we found that CD4(+) T cell responses against immunodominant epitopes are predictive for active Salmonella disease. Collectively, these data could contribute to improved diagnosis of Salmonella-related diseases and rational design of Salmonella vaccines.     

Molecular mechanisms of tolerance to cyanobacterial protease inhibitors revealed by clonal differences in Daphnia magna

Protease inhibitors of primary producers are a major food quality constraint for herbivores. In nutrient‐rich freshwater ecosystems, the interaction between primary producers and herbivores is mainly represented by Daphnia and cyanobacteria. Protease inhibitors have been found in many cyanobacterial blooms. These inhibitors have been shown (both in vitro and in situ) to inhibit the most important group of digestive proteases in the daphnid's gut, that is, trypsins and chymotrypsins. In this study, we fed four different Daphnia magna genotypes with the trypsin‐inhibitor‐containing cyanobacterial strain Microcystis aeruginosa PCC 7806 Mut. Upon exposure to dietary trypsin inhibitors, all D. magna genotypes showed increased gene expression of digestive trypsins and chymotrypsins. Exposure to dietary trypsin inhibitors resulted in increased activity of chymotrypsins and reduced activity of trypsin. Strong intraspecific differences in tolerance of the four D. magna genotypes to the dietary trypsin inhibitors were found. The degree of tolerance depended on the D. magna genotype. The genotypes' tolerance was positively correlated with the residual trypsin activity and the different IC₅₀ values of the trypsins. On the genetic level, the different trypsin loci varied between the D. magna genotypes. The two tolerant Daphnia genotypes that both originate from the same lake, which frequently produces cyanobacterial blooms, clustered in a neighbour‐joining phylogenetic tree based on the three trypsin loci. This suggests that the genetic variability of trypsin loci was an important cause for the observed intraspecific variability in tolerance to cyanobacterial trypsin inhibitors. Based on these findings, it is reasonable to assume that such genetic variability can also be found in natural populations and thus constitutes the basis for local adaptation of natural populations to dietary protease inhibitors.     

Patterns of Post-Glacial Genetic Differentiation in Marginal Populations of a Marine Microalga

This study investigates the genetic structure of an eukaryotic microorganism, the toxic dinoflagellate Alexandrium ostenfeldii, from the Baltic Sea, a geologically young and ecologically marginal brackish water estuary which is predicted to support evolution of distinct, genetically impoverished lineages of marine macroorganisms. Analyses of the internal transcribed spacer (ITS) sequences and Amplified Fragment Length Polymorphism (AFLP) of 84 A. ostenfeldii isolates from five different Baltic locations and multiple external sites revealed that Baltic A. ostenfeldii is phylogenetically differentiated from other lineages of the species and micro-geographically fragmented within the Baltic Sea. Significant genetic differentiation (F _ST) between northern and southern locations was correlated to geographical distance. However, instead of discrete genetic units or continuous genetic differentiation, the analysis of population structure suggests a complex and partially hierarchic pattern of genetic differentiation. The observed pattern suggests that initial colonization was followed by local differentiation and varying degrees of dispersal, most likely depending on local habitat conditions and prevailing current systems separating the Baltic Sea populations. Local subpopulations generally exhibited low levels of overall gene diversity. Association analysis suggests predominately asexual reproduction most likely accompanied by frequency shifts of clonal lineages during planktonic growth. Our results indicate that the general pattern of genetic differentiation and reduced genetic diversity of Baltic populations found in large organisms also applies to microscopic eukaryotic organisms.     

The role of DNA sensing and innate immune receptor TLR9 in otitis media

Otitis media (OM), a common infectious disease in children, is associated with bacterial middle ear (ME) infection. Toll-like receptors (TLRs) are important mediators of innate immune responses, and TLR9 specifically recognizes the unmethylated cytidine-phosphate-guanosine (CpG) motifs in bacterial DNA. Additional sensors of foreign DNA have recently been identified. The role of DNA sensing and TLR9 was investigated in a murine model of OM induced by non-typeable Haemophilus influenzae (NTHi). Expression of genes related to DNA-sensing pathways involved in innate immunity was assessed via DNA microarray, qPCR and immunohistochemistry. Middle ear responses to NTHi were examined in wild-type and TLR9(-/-) mice by histopathology and bacterial culture. Expression of TLR9 signaling genes was modestly up-regulated during OM, as was TLR9 protein in both ME mucosal cells and infiltrating leukocytes. However, genes known to be regulated by CpG DNA were dramatically up-regulated, as were genes involved in DNA sensing by DIA, Pol-III and AIM2. Toll-like receptor 9 deletion significantly prolonged the inflammatory response induced by NTHi in the ME and delayed bacterial clearance. The results suggest that DNA sensing via TLR9 plays a role in OM pathogenesis and recovery. Alternative forms of DNA sensing may also contribute to OM.     

SDCCAG8 obesity alleles and reduced weight loss after a lifestyle intervention in overweight children and adolescents

Genome-wide association analyses (GWAS) contributed to the detection of a number of single-nucleotide polymorphisms (SNPs) associated with obesity. However, little is known about the impact of the obesity-risk alleles on weight loss-related phenotypes after lifestyle interventions. A recent meta-analysis of GWAS reported five genomic loci near or in the genes FTO, MC4R, TMEM18, SDCCAG8, TNKS/MSRA that were associated with obesity in children and adolescents. Here, we analyzed the effect of the 10 SNPs representative of the five loci on measures of weight loss and cardiometabolic risk after a 1-year lifestyle intervention in 401 children and adolescents (mean age 10.74 years; 55.4% female; mean BMI 27.42 kg/m(2), mean BMI-standard deviation score (SDS) 2.37). For confirmation of one locus genotyping of three intronic SNPs in SDCCAG8 was performed in 626 obese adults who completed the 10-week hypoenergetic diet program. Intronic variants of SDCCAG8, which are associated with early onset obesity, are associated with reduced weight loss after a 1-year lifestyle intervention in overweight children and adolescents even after adjusting for age, sex, baseline measurement, or multiple testing (all P < 10(-6)). However, our results could not be confirmed in 626 obese adults undertaking a hypoenergetic diet intervention.     

Associations of Children’s Appetitive Traits with Weight and Dietary Behaviours in the Context of General Parenting

Background

Individual variations in child weight can be explained by genetic and behavioural susceptibility to obesity. Behavioural susceptibility can be expressed in appetite-related traits, e.g. food responsiveness. Research into such behavioural factors is important, as it can provide starting points for (preventive) interventions.

Objectives

To examine associations of children’s appetitive traits with weight and with fruit, snack and sugar-sweetened beverage intake, and to examine whether parenting style interacts with appetite in determining child weight/intake.

Methods

Data were used from 1275 children participating in the INPACT study in 2009–2010, with a mean age of 9 years in 2009. Their height and weight were measured to calculate body mass index (BMI). Parents completed a questionnaire to measure children’s appetitive traits, children’s dietary intake and parenting style. Child BMI z-scores, fruit, snack and sugar-sweetened beverage intake were regressed on appetitive traits. Moderation by parenting style was tested by adding interaction terms to the regression analyses.

Results

Food-approaching appetitive traits were positively, and food-avoidant appetitive traits were negatively related to child BMI z-scores and to child fruit intake. There were no or less consistent associations for snack and sugar-sweetened beverage intake. Authoritative parenting voided the negative association between food fussiness and fruit intake, while neglecting parenting strengthened the positive association between food-approaching appetitive traits and weight.

Conclusions

Early assessment of appetitive traits could be used to identify children at risk for overweight. As parenting style can moderate the associations between appetitive traits and weight/intake in a favourable way, parents are a promising target group for preventive interventions aimed at influencing the effect of appetitive traits on children.