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Nanobodies are becoming increasingly popular as tools for manipulating and visualising proteins in vivo. The ability to control nanobody/antigen interactions using light could provide precise spatiotemporal control over protein function. We develop a general approach to engineer photo-activatable nanobodies using photocaged amino acids that are introduced into the target binding interface by genetic code expansion. Guided by computational alanine scanning and molecular dynamics simulations, we tune nanobody/target binding affinity to eliminate binding before uncaging. Upon photo-activation using 365 nm light, binding is restored. We use this approach to generate improved photocaged variants of two anti-GFP nanobodies that function robustly when directly expressed in a complex intracellular environment together with their antigen. We apply them to control subcellular protein localisation in the nematode worm Caenorhabditis elegans. Our approach applies predictions derived from computational modelling directly in a living animal and demonstrates the importance of accounting for in vivo effects on protein-protein interactions.

Genetic code expansion in multicellular organisms is currently limited to the use of repurposed amber stop codons. Here we introduce a system for the use of quadruplet codons to direct incorporation of non-canonical amino acids in vivo in an animal, the nematode worm Caenorhabditis elegans. We develop hybrid pyrrolysyl tRNA variants to incorporate non-canonical amino acids in response to the quadruplet codon UAGA. We demonstrate the efficiency of the quadruplet decoding system by incorporating photocaged amino acids into two proteins widely used as genetic tools. We use photocaged lysine to express photocaged Cre recombinase for the optical control of gene expression and photocaged cysteine to express photo-activatable caspase for light inducible cell ablation. Our approach will facilitate the routine adoption of quadruplet decoding for genetic code expansion in eukaryotic cells and multicellular organisms.

Pikionis’s project for the mostly-residential town of Exonì (1950-1954) was supposed to be built on the slopes of Mount Imettos, designed as a response to the pressing housing crisis in post-war Greece. For the project’s promoters – sociologists, historians, architects, anthropologists and artists – Exonì would reconcile the values of the past with those of the present, both with respect to urban and architectural aspects as well as the socio-economic expectations of Greece in the 1950s.

Nanobodies are becoming increasingly popular as tools for manipulating and visualising proteins in vivo. The ability to control nanobody/antigen interactions using light could provide precise spatiotemporal control over protein function. We develop a general approach to engineer photo-activatable nanobodies using photocaged amino acids that are introduced into the target binding interface by genetic code expansion. Guided by computational alanine scanning and molecular-dynamics simulations, we tune nanobody/target binding affinity to eliminate binding before uncaging. Upon photo-activation, binding is restored. We use this approach to generate improved photocaged variants of two anti-GFP nanobodies. These variants exhibit photo-activatable binding triggered by illumination with 365nm light. We demonstrate that the photocaged nanobodies we have created are highly robust and function in a complex cellular environment. We apply them to control subcellular protein localisation in the nematode worm C. elegans. Our approach provides a rare example of computationally designed proteins being directly applied in living animals and demonstrates the importance of accounting for in vivo effects on protein-protein interactions.

Two thirds of the 302 neurons in C. elegans form bilaterally symmetric pairs in its physical connectome, and similar gross morphological symmetries are seen in the nervous systems of many other animals. A central question is whether and how this morphological symmetry is broken to produce functional asymmetry. Addressing this question, in all but two cases, has been impossible because no promoters are known that can direct gene expression to a single cell within a symmetric pair. Here we develop an efficient genetic code expansion system in C. elegans and use this system to create a photo-activatable version of Cre recombinase. Using this system, we target single neurons within a bilaterally symmetric pair (PLMR and PLML) with a laser. This turns on Cre and thereby switches on expression of an optogenetic channel in a single cell. We hereby overcome the limitation that these neurons cannot be targeted by genetic means. Our approach enables the generation of large numbers of animals for downstream experiments. By globally illuminating groups of freely moving animals to stimulate the targeted neurons that express an optogenetic channel we dissect the individual contributions of PLMR and PLML to the C. elegans touch response. Our results reveal that the individual neurons make asymmetric contributions to this behaviour, and suggest distinct roles for PLMR and PLML in the habituation to repeated stimulation. Our results demonstrate how genetic code expansion and optical targeting can be combined to break the symmetry of neuron pairs in C. elegans and thereby dissect the contributions of individual neurons to behaviour.

To investigate the prevalence of obesity in adults of a large region of Central Greece.The target group was adults aged 18 to 79 years who were residents of the region of Thessaly for at least one year. A sample of 852 individuals stratified for sex and age were included. Each subject underwent a thorough physical examination and body mass index (BMI) was calculated from body weight and height. Waist and hip circumferences as well as body fat content were additionally measured.Mean (SD) BMI for the total population was 27.5+/-5.5 and was significantly higher in males than in females (28.2+/-4.4 vs. 26.9+/-6.2, p0.001). The overall prevalence of obesity was 26.6% distributed equally between men (27.8%) and women (25.6%), whereas prevalence of overweight was 39.4% with male predominance (50.8% vs. 29.3%, p0.001). Morbid obesity (MO) was found in 3.5% with female predominance. The prevalence of central obesity, using waist circumference cut-off points (102cm for men,88cm for women), was comparable in males (40.4%) and females (35.3%). There was a positive association between obesity, central obesity, and age. The prevalence of overweight (19.5%) and obesity (9.4%) in the age-range of 18-29 years almost doubled in the next decade of age and attained the highest value, respectively, in the age-range of 50 to 59 (48.2%), and of 60 to 70 years group (38.9%).The rates of overweight and obesity in the population of Thessaly are relatively high with overweight being more prominent in males than in females, whereas MO was higher in females compared to males.

Apolipoprotein E (ApoE), and especially its epsilon4 isoform, is considered a risk factor predisposing to coronary heart disease. We hypothesized that the absence of epsilon4 allele offers a better chance for longer life. So we compared the prevalence of ApoE genotypes in 80 healthy aged individuals (HAI) (>80 years) and 391 Greek adults (median age 43 years) with ApoE genotype distribution consistent with the Hardy-Weinberg equilibrium (chi(2) = 5.93, p >.05). ApoE genotypes were comparable in both groups with the exception of E3/3 and E3/4, which were significantly higher (87.50% vs 75.99%, p =.025) and lower (5.00% vs 13.19%, p =.036), respectively, in HAI. The epsilon2 and epsilon3 allele frequencies were not different between the groups. The epsilon4 allele was significantly less frequent in HAI compared to controls (3.1% vs 8.58%, p =.020). Our results indicate an unfavorable effect of epsilon4 allele on longevity that may be attenuated by environmental and/or other genetic factors.

Cardiovascular risk factors seem to be affected by androgens, which exert their action through the androgen receptor (AR). Androgenic action correlates inversely with a polymorphic CAG repeat region in the AR gene encoding for glutamine residues the length of which appears to influence high density lipoprotein (HDL) cholesterol levels. The aim of the study was to investigate the possible association between AR gene polymorphism and serum sex steroids and lipids. 170 healthy males, aged 22-59 years (mean 42 years), were included in the study. Anthropometrical as well as sociometrical parameters were recorded. Body fat content (BFC) (% fat mass) was measured by bioelectrical impedance. Serum lipids and total and free testosterone (T) and estradiol (E(2)) levels were measured in each subject. AR gene CAG repeats length was determined. No significant correlation was found between the length of AR gene polyglutamine tract and the levels of gonadal steroids (total and free T, total and free E(2)) or to the lipid levels (Triglycerides, total, HDL and LDL cholesterol). In addition, serum lipid levels were not significantly different in the lower compared to higher half of CAG repeats length distribution. On multiple regression analysis BFC was found to predict HDL-cholesterol and triglycerides were found to show, respectively, significant negative and positive correlation with body fat content. In conclusion, AR gene polymorphism may not predict sex steroid levels in healthy males. Possible impact of CAG repeats length on lipids profile has not been established.

Two thirds of the 302 neurons in C. elegans form bilaterally symmetric pairs in its physical connectome, and similar gross morphological symmetries are seen in the nervous systems of many other animals. A central question is whether and how this morphological symmetry is broken to produce functional asymmetry. Addressing this question, in all but two cases, has been impossible because no promoters are known that can direct gene expression to a single cell within a symmetric pair. Here we develop an efficient genetic code expansion system in C. elegans and use this system to create a photo-activatable version of Cre recombinase. Using this system, we target single neurons within a bilaterally symmetric pair (PLMR and PLML) with a laser. This turns on Cre and thereby switches on expression of an optogenetic channel in a single cell. We hereby overcome the limitation that these neurons cannot be targeted by genetic means. Our approach enables the generation of large numbers of animals for downstream experiments. By globally illuminating groups of freely moving animals to stimulate the targeted neurons that express an optogenetic channel we dissect the individual contributions of PLMR and PLML to the C. elegans touch response. Our results reveal that the individual neurons make asymmetric contributions to this behaviour, and suggest distinct roles for PLMR and PLML in the habituation to repeated stimulation. Our results demonstrate how genetic code expansion and optical targeting can be combined to break the symmetry of neuron pairs in C. elegans and thereby dissect the contributions of individual neurons to behaviour.