Chromosomal inversion polymorphisms are widespread across the species ranges of rough periwinkles (Littorina saxatilis and L. arcana).

Inversions are thought to play a key role in adaptation and speciation, suppressing recombination between diverging populations. Genes influencing adaptive traits cluster in inversions, and changes in inversion frequencies are associated with environmental differences. However, in many organisms, it is unclear if inversions are geographically and taxonomically widespread. The intertidal snail, Littorina saxatilis, is one such example. Strong associations between putative polymorphic inversions and phenotypic differences have been demonstrated between two ecotypes of L. saxatilis in Sweden and inferred elsewhere, but no direct evidence for inversion polymorphism currently exists across the species range. Using whole genome data from 107 snails, most inversion polymorphisms were found to be widespread across the species range. The frequencies of some inversion arrangements were significantly different among ecotypes, suggesting a parallel adaptive role. Many inversions were also polymorphic in the sister species, L. arcana, hinting at an ancient origin.

Differing associations between sex determination and sex-linked inversions in two ecotypes of Littorina saxatilis.

Sexual antagonism is a common hypothesis for driving the evolution of sex chromosomes, whereby recombination suppression is favored between sexually antagonistic loci and the sex-determining locus to maintain beneficial combinations of alleles. This results in the formation of a sex-determining region. Chromosomal inversions may contribute to recombination suppression but their precise role in sex chromosome evolution remains unclear. Because local adaptation is frequently facilitated through the suppression of recombination between adaptive loci by chromosomal inversions, there is potential for inversions that cover sex-determining regions to be involved in local adaptation as well, particularly if habitat variation creates environment-dependent sexual antagonism. With these processes in mind, we investigated sex determination in a well-studied example of local adaptation within a species: the intertidal snail, Littorina saxatilis. Using SNP data from a Swedish hybrid zone, we find novel evidence for a female-heterogametic sex determination system that is restricted to one ecotype. Our results suggest that four putative chromosomal inversions, two previously described and two newly discovered, span the putative sex chromosome pair. We determine their differing associations with sex, which suggest distinct strata of differing ages. The same inversions are found in the second ecotype but do not show any sex association. The striking disparity in inversion-sex associations between ecotypes that are connected by gene flow across a habitat transition that is just a few meters wide indicates a difference in selective regime that has produced a distinct barrier to the spread of the newly discovered sex-determining region between ecotypes. Such sex chromosome-environment interactions have not previously been uncovered in L. saxatilis and are known in few other organisms. A combination of both sex-specific selection and divergent natural selection is required to explain these highly unusual patterns.

Genetic architecture of repeated phenotypic divergence in Littorina saxatilis ecotype evolution.

Chromosomal inversions have been shown to play a major role in a local adaptation by suppressing recombination between alternative arrangements and maintaining beneficial allele combinations. However, so far, their importance relative to the remaining genome remains largely unknown. Understanding the genetic architecture of adaptation requires better estimates of how loci of different effect sizes contribute to phenotypic variation. Here, we used three Swedish islands where the marine snail Littorina saxatilis has repeatedly evolved into two distinct ecotypes along a habitat transition. We estimated the contribution of inversion polymorphisms to phenotypic divergence while controlling for polygenic effects in the remaining genome using a quantitative genetics framework. We confirmed the importance of inversions but showed that contributions of loci outside inversions are of similar magnitude, with variable proportions dependent on the trait and the population. Some inversions showed consistent effects across all sites, whereas others exhibited site-specific effects, indicating that the genomic basis for replicated phenotypic divergence is only partly shared. The contributions of sexual dimorphism as well as environmental factors to phenotypic variation were significant but minor compared to inversions and polygenic background. Overall, this integrated approach provides insight into the multiple mechanisms contributing to parallel phenotypic divergence.

Gene expression studies of plastic and evolutionary responses to global warming.

Phenotypic plasticity can be a rapid response for coping with global warming, yet may be insufficient to protect species from extinction. Evolutionary adaptation may reinforce adaptive or oppose maladaptive plastic responses. With advances in technology whole transcriptomes can provide us with an unprecedented overview of genes and functional processes underlying the interplay between plasticity and evolution. We advocate that insects provide ideal opportunities to study plasticity in non-adapted and thermally adapted populations to infer reaction norms across the whole transcriptome ('reactionomes'). This can advance our understanding of how the interplay between plasticity and evolution shapes responses to warming. So far, a limited number of studies suggest predominantly maladaptive plastic responses to novel environments that are reduced with time, but much more research is needed to infer general patterns.

Cystatin C relates to metabolism in healthy, pubertal adolescents.

INTRODUCTION: The cystatin C (CysC) serum level is a marker of glomerular filtration rate and depends on age, gender, and pubertal stage. We hypothesize that CysC might overall reflect energy homeostasis and be regulated by components of the endocrine system and metabolites in pubertal adolescents. METHODS: Serum CysC levels and further possible effector parameters in 5355 fasting, morning venous blood samples from 2035 healthy participants of the LIFE Child cohort study (age 8 to 18 years) were analyzed. Recruitment started in 2011, with probands followed up once a year. Linear univariate and stepwise multivariate regression analyses were performed. RESULTS: Annual growth rate, serum levels of thyroid hormones, parathyroid hormone, insulin-like growth factor 1, hemoglobin A1c (HbA1c), uric acid, and alkaline phosphatase show relevant and significant associations with CysC serum concentrations (p <0.001). Furthermore, male probands' CysC correlated with the body mass index and testosterone among other sexual hormones. Multivariate analyses revealed that uric acid and HbA1c are associated variables of CysC independent from gender (p <0.001). In males, alkaline phosphatase (p <0.001) is additionally significantly associated with CysC. Thyroid hormones show significant correlations only in multivariate analyses in females (p <0.001). CONCLUSIONS: The described associations strongly suggest an impact of children's metabolism on CysC serum levels. These alterations need to be considered in kidney diagnostics using CysC in adolescents. Additionally, further studies are needed on CysC in children.

Genetic variation for adaptive traits is associated with polymorphic inversions in Littorina saxatilis.

Chromosomal inversions have long been recognized for their role in local adaptation. By suppressing recombination in heterozygous individuals, they can maintain coadapted gene complexes and protect them from homogenizing effects of gene flow. However, to fully understand their importance for local adaptation we need to know their influence on phenotypes under divergent selection. For this, the marine snail Littorina saxatilis provides an ideal study system. Divergent ecotypes adapted to wave action and crab predation occur in close proximity on intertidal shores with gene flow between them. Here, we used F2 individuals obtained from crosses between the ecotypes to test for associations between genomic regions and traits distinguishing the Crab-/Wave-adapted ecotypes including size, shape, shell thickness, and behavior. We show that most of these traits are influenced by two previously detected inversion regions that are divergent between ecotypes. We thus gain a better understanding of one important underlying mechanism responsible for the rapid and repeated formation of ecotypes: divergent selection acting on inversions. We also found that some inversions contributed to more than one trait suggesting that they may contain several loci involved in adaptation, consistent with the hypothesis that suppression of recombination within inversions facilitates differentiation in the presence of gene flow.

Genetic variance in fitness and its cross-sex covariance predict adaptation during experimental evolution.

The additive genetic variation (VA ) of fitness in a population is of particular importance to quantify its adaptive potential and predict its response to rapid environmental change. Recent statistical advances in quantitative genetics and the use of new molecular tools have fostered great interest in estimating fitness VA in wild populations. However, the value of VA for fitness in predicting evolutionary changes over several generations remains mostly unknown. In our study, we addressed this question by combining classical quantitative genetics with experimental evolution in the model organism Tribolium castaneum (red flour beetle) in three new environmental conditions (Dry, Hot, Hot-Dry). We tested for potential constraints that might limit adaptation, including environmental and sex genetic antagonisms captured by negative genetic covariance between environments and female and male fitness, respectively. Observed fitness changes after 20 generations mainly matched our predictions. Given that body size is commonly used as a proxy for fitness, we also tested how this trait and its genetic variance (including nonadditive genetic variance) were impacted by environmental stress. In both traits, genetic variances were sex and condition dependent, but they differed in their variance composition, cross-sex and cross-environment genetic covariances, as well as in the environmental impact on VA .

Restoring ancestral phenotypes is a general pattern in gene expression evolution during adaptation to new environments in Tribolium castaneum.

Plasticity and evolution are two processes allowing populations to respond to environmental changes, but how both are related and impact each other remains controversial. We studied plastic and evolutionary responses in gene expression of Tribolium castaneum after exposure of the beetles to new environments that differed from ancestral conditions in temperature, humidity or both. Using experimental evolution with 10 replicated lines per condition, we were able to demonstrate adaptation after 20 generations. We measured whole-transcriptome gene expression with RNA-sequencing to infer evolutionary and plastic changes. We found more evidence for changes in mean expression (shift in the intercept of reaction norms) in adapted lines than for changes in plasticity (shifts in slopes). Plasticity was mainly preserved in selected lines and was responsible for a large part of the phenotypic divergence in expression between ancestral and new conditions. However, we found that genes with the largest evolutionary changes in expression also evolved reduced plasticity and often showed expression levels closer to the ancestral stage. Results obtained in the three different conditions were similar, suggesting that restoration of ancestral expression levels during adaptation is a general evolutionary pattern. With a larger sample in the most stressful condition, we were able to detect a positive correlation between the proportion of genes with reversion of the ancestral plastic response and mean fitness per selection line.

Additive and mostly adaptive plastic responses of gene expression to multiple stress in Tribolium castaneum.

Gene expression is known to be highly responsive to the environment and important for adjustment of metabolism but there is also growing evidence that differences in gene regulation contribute to species divergence and differences among locally adapted populations. However, most studies so far investigated populations when divergence had already occurred. Selection acting on expression levels at the onset of adaptation to an environmental change has not been characterized. Understanding the mechanisms is further complicated by the fact that environmental change is often multivariate, meaning that organisms are exposed to multiple stressors simultaneously with potentially interactive effects. Here we use a novel approach by combining fitness and whole-transcriptome data in a large-scale experiment to investigate responses to drought, heat and their combination in Tribolium castaneum. We found that fitness was reduced by both stressors and their combined effect was almost additive. Expression data showed that stressor responses were acting independently and did not interfere physiologically. Since we measured expression and fitness within the same individuals, we were able to estimate selection on gene expression levels. We found that variation in fitness can be attributed to gene expression variation and that selection pressures were environment dependent and opposite between control and stress conditions. We could further show that plastic responses of expression were largely adaptive, i.e. in the direction that should increase fitness.

[Diagnostic Precision of the Confocal Scanning Laser Ophthalmoscopy in the Large Optic Disc with Physiological Excavation - a Long-Term Study]. / Diagnostische Zuverlässigkeit der konfokalen Scanning-Laser-Ophthalmoskopie bei großen Papillen mit physiologischer Exkavation ­ eine Langzeitstudie.

We repeatedly examined 17 subjects with presumed bilateral physiological excavation labeled as pathological and/or borderline via HRT to verify the diagnosis of physiological cupping or to monitor the long-term progression into normal tension glaucoma. PATIENTS AND METHODS: 17 Subjects with presumed bilateral physiological cupping and large optic discs were included in this long-term follow-up study. All subjects underwent regular detailed ophthalmologic examinations, including intraocular pressure measurement via Goldmann applanation tonometry (GAT), retinal nerve fiber layer imaging via optical coherence tonometry (OCT) and visual field testing and optic disc imaging using the HRT. Glaucomatous progression was identified using the HRT's tools (stereometric trend analysis [STA] and topographic change analysis [TCA]). RESULTS: In the initial examination, all 17 subjects were classified as "pathological", by the HRT's Moorfield's Regression Analysis (MRA). Over the observation period of 9.2 ± 5 years, only 1 of the 17 subjects showed an ensured conversion to normal tension glaucoma with glaucomatous visual field defects. The remaining 16 subjects show no visual field defects to date. STA showed significant changes in 3 subjects alone, in 1 subject TCA showed a significant change alone, and in 1 subject both analyses showed a progressive change. CONCLUSION: After 9 years of regular examinations, 16 of the 17 subjects that were classified as "pathological" using MRA showed no glaucomatous visual field defects. In 5 out of these 16 subjects, progressive changes of the optic disc could be recorded via HRT. Therefore, the diagnostic precision of the HRT measurements seems to be limited in patients with large discs and physiological cupping.

Blood Pressure and Heart Rate Variability in Primary Open-Angle Glaucoma and Normal Tension Glaucoma.

PURPOSE: Vascular dysfunction and ischemia are believed to play an important part in the pathogenesis of glaucoma and especially of normal tension glaucoma (NTG). The aim of the present study was to analyze the blood pressure (BP) and heart rate variability patterns in patients with primary open-angle glaucoma (POAG) and NTG compared with controls. METHODS: In total, 37 patients with POAG, 27 patients with NTG, and 82 control subjects were included in a prospective clinical validation study. Continuous BP and heart rate were simultaneously recorded over 30 min (Glaucoscreen, aviant GmbH, Jena, Germany) under resting conditions. Time series of heart rate, systolic blood pressure (SBP) and diastolic blood pressure (DBP) were extracted and analyzed calculating univariate linear (time domain, frequency domain), nonlinear (symbolic dynamics), and bivariate (joint symbolic dynamics) indices. RESULTS: Overall, 12 parameters could be identified that were significantly different when comparing POAG patients and controls, whereas 80 parameters were significantly different in NTG patients compared with controls. The optimum set consisting of three indices showed a sensitivity of 81.5% at a specificity of 86.6% for NTG patients compared with a sensitivity of 62.2% at a specificity of 82.9% for POAG patients. CONCLUSIONS: Alterations in BP variability and coupling with heart rate suggest impaired patterns of autonomic cardiovascular regulation in glaucoma patients especially in patients with NTG.

Cystic masses of the lateral neck - Proposition of an algorithm for increased treatment efficiency.

Preoperative discrimination of solitary cervical branchial cleft cysts from cystic lymph node metastasis often is challenging. Surgical excision of the cystic formation and consecutive histopathological examination of tissue specimens are the only means resulting in the correct diagnosis. However, in case of malignancies surgery on the lateral neck prior to the definitive treatment is considered to negatively influence the patients' outcome. The rate of cystic lymph node metastasis in patients presenting with a lateral branchial cleft cyst, localization of the primary tumour and oncological outcome were investigated. Retrospective chart review of 131 patients presenting clinically with solitary lateral cervical cysts between. A malignant tumour was detected in 12 patients (9.2%). Malignant tumours were significantly more frequent in patients older than 40 years of age (22.0%; p = 0.0001). In patients older than 40 years of age with solitary lateral cervical cysts a malignancy should be presumed.

MDPV in forensic routine cases: Psychotic and aggressive behavior in relation to plasma concentrations.

The new psychoactive substance 3,4-methylenedioxypyrovalerone (MDPV) belongs to the group of synthetic cathinones and is purchased mainly as "research chemical" or "bath salt" on the illegal drug market, also in South Bavaria. MDPV was detected in blood and urine samples from 2010 on in 50 authentic routine cases in a forensic setting. Plasma concentrations in 46 cases with available blood specimens ranged from approximately 1.0 to 301µg/L (median 23.7; mean 47.9µg/L), detected by a fully validated LC-MS/MS method. Subjects aged between 16 and 54 years (median 36; mean 35 years) and reflected experienced chronic drug users. Accused offences were mainly violent crimes such as bodily harm, robberies, homicides and acts of resistance. A lot of subjects showed highly aggressive and violent behavior with endangerment of self and others and/or psychotic symptoms as confusion, hallucinations or paranoia. The risk for such behavior rises with MDPV plasma concentrations above as low as 30µg/L, whereby a time interval of 1.5h on average between incident and/or observation of impairment and blood sampling has to be taken into account. Comprehensive toxicological analysis proved poly-drug use in almost all cases including opiates/opioids, benzodiazepines and other sedatives, antidepressants and other stimulants, also other new psychoactive substances. Alcohol was detected only in three cases. Co-consumed benzodiazepines seem not be able to completely prevent psychotic effects despite their use as first-line treatment for patients with synthetic cathinone poisonings. The study demonstrates that relatively low plasma concentrations of MDPV could be associated with mental impairment which is relevant in the assessment of forensic cases.

High gene flow despite opposite chirality in hybrid zones between enantiomorphic door snails.

We studied differentiation and geneflow patterns between enantiomorphic door-snail species in two hybrid zones in the Bucegi Mountains (Romania) to investigate the effects of intrinsic barriers (complications in copulation) and extrinsic selection by environmental factors. A mitochondrial gene tree confirmed the historical separation of the examined populations into the dextral Alopia livida and the sinistral Alopia straminicollis in accordance with the morphological classification, but also indicated gene flow between the species. By contrast, a network based on amplified fragment length polymorphisms (AFLP) markers revealed local groups of populations as units independent of their species affiliation. Admixture analyses based on AFLP data showed that the genomes of most individuals in the hybrid zones are composed of parts of the genomes of both parental taxa. The introgression patterns of a notable fraction of the examined markers deviated from neutral introgression. However, the patterns of most non-neutral markers were not concordant between the two hybrid zones. There was also no concordance between non-neutral markers in the two genomic clines and markers that were correlated with environmental variables or markers that were correlated with the proportion of dextral individuals in the populations. Neither extrinsic selection by environmental factors nor intrinsic barriers resulting from positive frequency-dependent selection of the prevailing coiling direction were sufficient to maintain the distinctness of A. straminicollis and A. livida. Despite being historically separated units, we conclude that these taxa now merge where they come into contact.

Stereoselective Palladium-Catalyzed Decarboxylative γ-Arylation of Acyclic ß,γ-Unsaturated Carboxylic Acids.

Palladium-catalyzed γ-arylation of acyclic ß,γ-unsaturated carboxylic acids with various aryl iodides is reported. The cascade comprises a decarboxylative γ-palladation of ß,γ-unsaturated α,α'-disubstituted carboxylic acids and subsequent C(sp2)-C(sp3) bond formation to provide diaryl vinyl methanes in moderate to good yields and high E/Z-selectivities. Reaction with an enantiomerically pure acid revealed that the process occurs with high stereospecificity.

Presumable incipient hybrid speciation of door snails in previously glaciated areas in the Caucasus.

Homoploid hybrid speciation, speciation by hybridization without a change in chromosome number, may be the result of an encounter of closely related species in a habitat that is different from that usually occupied by these species. In the northwestern Caucasus the land snail species Micropontica caucasica and M. circassica form two distinct entities with little admixture at low and intermediate altitudes. However, at higher altitudes in the Lagonaki plateau, which were repeatedly glaciated, Micropontica populations with intermediate characters occur. Admixture analyses based on AFLP data demonstrated that the populations from the Lagonaki plateau are homoploid hybrids that now form a cluster separate from the parental species. The Lagonaki populations are characterized by a mtDNA haplotype clade that has been found in the parental species only once. The fixation of this haplotype clade in most hybrid populations suggests that these haplotypes are better adapted to the cooler conditions in high altitude habitats and have replaced the haplotypes of the parental species in a selective sweep. The fixation of a presumably adaptive mitochondrial haplotype clade in the Lagonaki populations is an important step towards speciation under the differential fitness species concept.

Evaluation of early anatomical changes following canaloplasty with anterior segment spectral-domain optical coherence tomography and ultrasound biomicroscopy.

PURPOSE: To analyse structural changes in conjunctiva, sclera and Schlemm's canal (SC) following canaloplasty with optical coherence tomography (AS-OCT) and ultrasound biomicroscopy (UBM). METHODS: Fifteen patients undergoing canaloplasty were included in this prospective study. AS-OCT images were acquired pre- and 1, 7, 30 and 90 days postoperatively. UBM was performed 3 months postoperatively. The surgical site was evaluated for the presence of SC, transscleral filtration, a scleral lake and the visibility of intra-Schlemm-sutures. The height and width of SC were measured at the 3 and 9 o'clock limbus position. RESULTS: After canaloplasty, SC was detectable with AS-OCT in 93% of the patients on day 1. The increase in height was higher than that in width (height: +369%, p = 0.0004, width: +152%, p = 0.002). IOP was negatively correlated to SC's width 1 week postoperatively (r = -0.63, p = 0.04) and to SC's height until 3 months (r = -0.66, p = 0.02) postoperatively. Using UBM, a reflection of the traction sutures indicated SC's position in all patients. Transscleral filtration was found in all patients using AS-OCT, demonstrating a peak 1 week postoperatively. At 3 months, a scleral lake could be visualized in 50% and 83% of patients using AS-OCT and UBM, respectively. CONCLUSIONS: AS-OCT offers a high resolution for imaging superficial conjunctival areas and SC after canaloplasty, whereas UBM is capable of detecting deeper structures such as scleral lakes or intra-canal-sutures. The results imply a correlation of the dilation of SC with the IOP-lowering effect and an early pronounced transscleral filtration following canaloplasty.

Identifying glaucoma patients by applying multivariate analyses of cardiovascular signals.

Glaucoma is a disease that damages the eye's optic nerve. However, the exact cause of this optic nerve damage is not yet fully understood. Besides the factors of age, genetics and others, such as obesity, medication and migraines, a vascular dysfunction is believed to be a significant factor leading to glaucoma. This study's objective was to investigate whether these vascular dysfunctions could be recognized by analyzing cardiovascular regulation in glaucoma patients. Linear and nonlinear methods were applied to the extracted heart rate (HR), and systolic/ diastolic blood pressure (DBP) time series to discriminate between 35 healthy controls and 20 glaucoma patients. The combination of indices from 30-min analysis of time domain (Renyi entropy of systolic blood pressure) and nonlinear dynamics (segmented Poincare plot analysis of DBP, high-resolution joint symbolic dynamics of DBP/ HR) were able to differentiate between controls and patients with a specificity and sensitivity of > 95%. Since changes in short-term blood pressure regulation patterns and heart-rate coupling are clear signs of a vascular dysfunction, this approach could be useful for providing an earlier diagnosis of glaucoma in clinical practice.

Blood Pressure and Heart Rate Variability to Detect Vascular Dysregulation in Glaucoma.

Purpose. To investigate blood pressure and heart rate variability in patients with primary open-angle glaucoma (POAG) to detect disturbed blood pressure regulation. Methods. Thirty-one patients with POAG (mean age 68 ± 10 years) and 48 control subjects (mean age 66 ± 10 years) were included in a prospective study. Continuous blood pressure and heart rate were simultaneously and noninvasively recorded over 30 min (Glaucoscreen, aviant GmbH, Jena, Germany). Data were analyzed calculating univariate linear (time domain and frequency domain), nonlinear (Symbolic Dynamics, SD) and bivariate (Joint Symbolic Dynamics, JSD) indices. Results. Using nonlinear methods, glaucoma patients were separated with more parameters compared to linear methods. In POAG, nonlinear univariate indices (pW113 and pW120_Sys) were increased while the indices pTH10_Sys and pTH11_Sys reflect a reduction of dominant patterns. Bivariate indices (JSDdia29, JSDdia50, and JSDdia52; coupling between heart rate and diastolic blood pressure) were increased in POAG. The optimum set consisting of six parameters (JSDdia29, JSDdia58, pTH9_Sys, pW231, pW110_Sys and pW120_Sys) revealed a sensitivity of 83.3% and specificity of 80.6%. Conclusions. Nonlinear uni- and bivariate indices of continuous recordings of blood pressure and heart rate are altered in glaucoma. Abnormal blood pressure variability suggests disturbed autonomic regulation in patients with glaucoma.