Mitochondria-originated redox signalling regulates KLF-1 to promote longevity in Caenorhabditis elegans.

Alternations of redox metabolism have been associated with the extension of lifespan in roundworm Caenorhabditis elegans, caused by moderate mitochondrial dysfunction, although the underlying signalling cascades are largely unknown. Previously, we identified transcriptional factor Krüppel-like factor-1 (KLF-1) as the main regulator of cytoprotective longevity-assurance pathways in the C. elegans long-lived mitochondrial mutants. Here, we show that KLF-1 translocation to the nucleus and the activation of the signalling cascade is dependent on the mitochondria-derived hydrogen peroxide (H2O2) produced during late developmental phases where aerobic respiration and somatic mitochondrial biogenesis peak. We further show that mitochondrial-inducible superoxide dismutase-3 (SOD-3), together with voltage-dependent anion channel-1 (VDAC-1), is required for the life-promoting H2O2 signalling that is further regulated by peroxiredoxin-3 (PRDX-3). Increased H2O2 release in the cytoplasm activates the p38 MAPK signalling cascade that induces KLF-1 translocation to the nucleus and the activation of transcription of C. elegans longevity-promoting genes, including cytoprotective cytochrome P450 oxidases. Taken together, our results underline the importance of redox-regulated signalling as the key regulator of longevity-inducing pathways in C. elegans, and position precisely timed mitochondria-derived H2O2 in the middle of it.

Identification of healthspan-promoting genes in Caenorhabditis elegans based on a human GWAS study.

To find drivers of healthy ageing, a genome-wide association study (GWAS) was performed in healthy and unhealthy older individuals. Healthy individuals were defined as free from cardiovascular disease, stroke, heart failure, major adverse cardiovascular event, diabetes, dementia, cancer, chronic obstructive pulmonary disease (COPD), asthma, rheumatism, Crohn's disease, malabsorption or kidney disease. Six single nucleotide polymorphisms (SNPs) with unknown function associated with ten human genes were identified as candidate healthspan markers. Thirteen homologous or closely related genes were selected in the model organism C. elegans for evaluating healthspan after targeted RNAi-mediated knockdown using pathogen resistance, muscle integrity, chemotaxis index and the activity of known longevity and stress response pathways as healthspan reporters. In addition, lifespan was monitored in the RNAi-treated nematodes. RNAi knockdown of yap-1, wwp-1, paxt-1 and several acdh genes resulted in heterogeneous phenotypes regarding muscle integrity, pathogen resistance, chemotactic behaviour, and lifespan. Based on these observations, we hypothesize that their human homologues WWC2, CDKN2AIP and ACADS may play a role in health maintenance in the elderly.

Accumulation of Glycogen and Upregulation of LEA-1 in C. elegans daf-2(e1370) Support Stress Resistance, Not Longevity.

DAF-16-dependent activation of a dauer-associated genetic program in the C. elegans insulin/IGF-1 daf-2(e1370) mutant leads to accumulation of large amounts of glycogen with concomitant upregulation of glycogen synthase, GSY-1. Glycogen is a major storage sugar in C. elegans that can be used as a short-term energy source for survival, and possibly as a reservoir for synthesis of a chemical chaperone trehalose. Its role in mitigating anoxia, osmotic and oxidative stress has been demonstrated previously. Furthermore, daf-2 mutants show increased abundance of the group 3 late embryogenesis abundant protein LEA-1, which has been found to act in synergy with trehalose to exert its protective role against desiccation and heat stress in vitro, and to be essential for desiccation tolerance in C. elegans dauer larvae. Here we demonstrate that accumulated glycogen is not required for daf-2 longevity, but specifically protects against hyperosmotic stress, and serves as an important energy source during starvation. Similarly, lea-1 does not act to support daf-2 longevity. Instead, it contributes to increased resistance of daf-2 mutants to heat, osmotic, and UV stress. In summary, our experimental results suggest that longevity and stress resistance can be uncoupled in IIS longevity mutants.

Prediction of biological age by morphological staging of sarcopenia in Caenorhabditis elegans.

Sarcopenia encompasses a progressive decline in muscle quantity and quality. Given its close association with ageing, it may represent a valuable healthspan marker. The commonalities with human muscle structure and facile visualization possibilities make Caenorhabditis elegans an attractive model for studying the relationship between sarcopenia and healthspan. However, classical visual assessment of muscle architecture is subjective and has low throughput. To resolve this, we have developed an image analysis pipeline for the quantification of muscle integrity in confocal microscopy images from a cohort of ageing myosin::GFP reporter worms. We extracted a variety of morphological descriptors and found a subset to scale linearly with age. This allowed establishing a linear model that predicts biological age from a morphological muscle signature. To validate the model, we evaluated muscle architecture in long-lived worms that are known to experience delayed sarcopenia by targeted knockdown of the daf-2 gene. We conclude that quantitative microscopy allows for staging sarcopenia in C. elegans and may foster the development of image-based screens in this model organism to identify modulators that mitigate age-related muscle frailty and thus improve healthspan.

Elevated Trehalose Levels in C. elegans daf-2 Mutants Increase Stress Resistance, Not Lifespan.

The C. elegans insulin/IGF-1 (insulin-like growth factor 1) signaling mutant daf-2 recapitulates the dauer metabolic signature-a shift towards lipid and carbohydrate accumulation-which may be linked to its longevity and stress resistance phenotypes. Trehalose, a disaccharide of glucose, is highly upregulated in daf­2 mutants and it has been linked to proteome stabilization and protection against heat, cold, desiccation, and hypoxia. Earlier studies suggested that elevated trehalose levels can explain up to 43% of the lifespan extension observed in daf-2 mutants. Here we demonstrate that trehalose accumulation is responsible for increased osmotolerance, and to some degree thermotolerance, rather than longevity in daf-2 mutants. This indicates that particular stress resistance phenotypes can be uncoupled from longevity.

Motor outcome after perinatal stroke and early prediction of unilateral spastic cerebral palsy.

BACKGROUND: Unilateral spastic cerebral palsy (USCP) occurs in 30%-68% of infants with perinatal stroke. Early detection of USCP is essential for referring infants to early intervention. The aims of this study were to report motor outcomes after perinatal stroke, and to determine the predictive value of the General Movements Assessment (GMA) and Hand Assessment for Infants (HAI) for detection of USCP. MATERIALS AND METHODS: This was a prospective observational study involving infants with perinatal stroke. GMA was conducted between 10 and 15 weeks post term-age (PTA). The HAI was performed between 3 and 5 months PTA. Motor outcome was collected between 12 and 36 months PTA. RESULTS: The sample consisted of 46 infants. Fifteen children (32.6%) were diagnosed with CP, two children with bilateral CP and 13 with USCP. Abnormal GMA had a sensitivity of 85% (95% confidence interval [CI] 55-98%) and a specificity of 52% (95% CI 33-71%) to predict USCP. When asymmetrically presented FMs were also considered as abnormal, sensitivity increased to 100%, hence the specificity declined to 43%. A HAI asymmetry index cut-off of 23, had both a sensitivity and a specificity of 100% to detect USCP. CONCLUSION: Using GMA and HAI can enable prediction of USCP before the age of 5 months in infants with perinatal stroke. Nevertheless, GMA must be interpreted with caution in this particular population. The HAI was found to be a very accurate screening tool for early detection of asymmetry and prediction of USCP.

DAF-16/FoxO in Caenorhabditis elegans and Its Role in Metabolic Remodeling.

DAF-16, the only forkhead box transcription factors class O (FoxO) homolog in Caenorhabditis elegans, integrates signals from upstream pathways to elicit transcriptional changes in many genes involved in aging, development, stress, metabolism, and immunity. The major regulator of DAF-16 activity is the insulin/insulin-like growth factor 1 (IGF-1) signaling (IIS) pathway, reduction of which leads to lifespan extension in worms, flies, mice, and humans. In C. elegans daf-2 mutants, reduced IIS leads to a heterochronic activation of a dauer survival program during adulthood. This program includes elevated antioxidant defense and a metabolic shift toward accumulation of carbohydrates (i.e., trehalose and glycogen) and triglycerides, and activation of the glyoxylate shunt, which could allow fat-to-carbohydrate conversion. The longevity of daf-2 mutants seems to be partially supported by endogenous trehalose, a nonreducing disaccharide that mammals cannot synthesize, which points toward considerable differences in downstream mechanisms by which IIS regulates aging in distinct groups.

The External Genitalia Score (EGS): A European Multicenter Validation Study.

CONTEXT: Standardized description of external genitalia is needed in the assessment of children with atypical genitalia. OBJECTIVES: To validate the External Genitalia Score (EGS), to present reference values for preterm and term babies up to 24 months and correlate obtained scores with anogenital distances (AGDs). DESIGN, SETTING: A European multicenter (n = 8) validation study was conducted from July 2016 to July 2018. PATIENTS AND METHODS: EGS is based on the external masculinization score but uses a gradual scale from female to male (range, 0-12) and terminology appropriate for both sexes. The reliability of EGS and AGDs was determined by the interclass correlation coefficient (ICC). Cross-sectional data were obtained in 686 term babies (0-24 months) and 181 preterm babies, and 111 babies with atypical genitalia. RESULTS: The ICC of EGS in typical and atypical genitalia is excellent and good, respectively. Median EGS (10th to 90th centile) in males < 28 weeks gestation is 10 (8.6-11.5); in males 28-32 weeks 11.5 (9.2-12); in males 33-36 weeks 11.5 (10.5-12) and in full-term males 12 (10.5-12). In all female babies, EGS is 0 (0-0). The mean (SD) lower/upper AGD ratio (AGDl/u) is 0.45 (0.1), with significant difference between AGDl/u in males 0.49 (0.1) and females 0.39 (0.1) and intermediate values in differences of sex development (DSDs) 0.43 (0.1). The AGDl/u correlates with EGS in males with typical genitalia and in atypical genitalia. CONCLUSIONS: EGS is a reliable and valid tool to describe external genitalia in premature and term babies up to 24 months. EGS correlates with AGDl/u in males. It facilitates standardized assessment, clinical decision-making and multicenter research.

The nutritional requirements of Caenorhabditis elegans.

Animals require sufficient intake of a variety of nutrients to support their development, somatic maintenance and reproduction. An adequate diet provides cell building blocks, chemical energy to drive cellular processes and essential nutrients that cannot be synthesised by the animal, or at least not in the required amounts. Dietary requirements of nematodes, including Caenorhabditis elegans have been extensively studied with the major aim to develop a chemically defined axenic medium that would support their growth and reproduction. At the same time, these studies helped elucidating important aspects of nutrition-related biochemistry and metabolism as well as the establishment of C. elegans as a powerful model in studying evolutionarily conserved pathways, and the influence of the diet on health.

Laser-induced remote release in vivo in C. elegans from novel silver nanoparticles-alginate hydrogel shells.

Non-destructive, controllable, remote light-induced release inside cells enables studying time- and space-specific processes in biology. In this work we demonstrate the remote release of tagged proteins in Caenorhabditis elegans (C. elegans) worms using a near-infrared laser light as a trigger from novel hydrogel shells functionalized with silver nanoparticles responsive to laser light. A new type of hydrogel shells was developed capable of withstanding prolonged storage in the lyophilized state to enable the uptake of the shell by worms, which takes place on an agar plate under standard culture conditions. Uptake of the shells by C. elegans was confirmed using confocal laser scanning microscopy, while release from alginate shells in C. elegans and the laser effect on the shells on a substrate in air was followed using fluorescence microscopy. In addition, Raman microscopy was used to track the localization of particles to avoid the influence of autofluorescence. Hierarchical cluster spectral analysis is used to extract information about the biochemical composition of an area of a nematode containing the hydrogel shells, whose Raman signal is enhanced by the SERS (Surface Enhanced Raman Scattering) effect due to hot spots formed by silver nanoparticles present in the shells. The in vivo release demonstrated here can be used to study intestinal microbiota and probiotic compounds as well as a possible future strategy for gene delivery in the worms, other insects and other organisms.

Gene Regulatory Variation in Drosophila melanogaster Renal Tissue.

Genetic variation influencing levels of gene expression is abundant in natural populations, and may exert its effects through complex mechanisms that depend on an organism's genetic background and the tissue in which expression is measured. We investigated natural variation in gene expression in the Malpighian tubules of three inbred Drosophila melanogaster strains and their F1 hybrids. One of the strains was from a population in the species' ancestral range (Zambia), while the other two were from a more recently derived population (Sweden). Although closely related, the two Swedish strains differed greatly in terms of their expression inheritance when hybridized with the Zambian strain, with one Swedish strain showing a large excess of genes with recessive expression inheritance, as well as a large number of genes with overdominant inheritance. Although most expression variation could be attributed to trans-regulation, there were ∼200 genes that showed allele-specific expression differences in each of the between-population hybrids, indicating that cis-regulation contributes as well. The cis-regulated genes were enriched with cytochrome P450 genes, and the upstream regions of six of these genes were incorporated into transgenic reporter gene constructs to test their effects on expression. Differential expression was observed for five of the six reporter genes in the Malpighian tubule, suggesting that a large proportion of cis-regulatory variation lies directly upstream of the affected gene. In most cases, the differential expression was specific to the Malpighian tubule or greater in this tissue than in the rest of the body, highlighting the importance of single-tissue studies of gene expression variation.

Perinatal/neonatal case presentation: unexpected severe respiratory insufficiency in a newborn with Holt-Oram Syndrome.

Holt-Oram syndrome is an autosomal dominant condition characterized by skeletal and cardiac defects. Pulmonary malformation is not reported to belong to the spectrum of this condition. We report a second case of a newborn with Holt-Oram syndrome who developed severe respiratory insufficiency shortly after birth. We discuss possible genetic links between abnormal pulmonary morphogenesis and Holt-Oram syndrome.