Exome sequencing reveals variants in known and novel candidate genes for severe sperm motility disorders.

STUDY QUESTION: What are the causative genetic variants in patients with male infertility due to severe sperm motility disorders? SUMMARY ANSWER: We identified high confidence disease-causing variants in multiple genes previously associated with severe sperm motility disorders in 10 out of 21 patients (48%) and variants in novel candidate genes in seven additional patients (33%). WHAT IS KNOWN ALREADY: Severe sperm motility disorders are a form of male infertility characterised by immotile sperm often in combination with a spectrum of structural abnormalities of the sperm flagellum that do not affect viability. Currently, depending on the clinical sub-categorisation, up to 50% of causality in patients with severe sperm motility disorders can be explained by pathogenic variants in at least 22 genes. STUDY DESIGN, SIZE, DURATION: We performed exome sequencing in 21 patients with severe sperm motility disorders from two different clinics. PARTICIPANTS/MATERIALS, SETTING, METHOD: Two groups of infertile men, one from Argentina (n = 9) and one from Australia (n = 12), with clinically defined severe sperm motility disorders (motility <5%) and normal morphology values of 0-4%, were included. All patients in the Argentine cohort were diagnosed with DFS-MMAF, based on light and transmission electron microscopy. Sperm ultrastructural information was not available for the Australian cohort. Exome sequencing was performed in all 21 patients and variants with an allele frequency of <1% in the gnomAD population were prioritised and interpreted. MAIN RESULTS AND ROLE OF CHANCE: In 10 of 21 patients (48%), we identified pathogenic variants in known sperm assembly genes: CFAP43 (3 patients); CFAP44 (2 patients), CFAP58 (1 patient), QRICH2 (2 patients), DNAH1 (1 patient) and DNAH6 (1 patient). The diagnostic rate did not differ markedly between the Argentinian and the Australian cohort (55% and 42%, respectively). Furthermore, we identified patients with variants in the novel human candidate sperm motility genes: DNAH12, DRC1, MDC1, PACRG, SSPL2C and TPTE2. One patient presented with variants in four candidate genes and it remains unclear which variants were responsible for the severe sperm motility defect in this patient. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: In this study, we described patients with either a homozygous or two heterozygous candidate pathogenic variants in genes linked to sperm motility disorders. Due to unavailability of parental DNA, we have not assessed the frequency of de novo or maternally inherited dominant variants and could not determine the parental origin of the mutations to establish in all cases that the mutations are present on both alleles. WIDER IMPLICATIONS OF THE FINDINGS: Our results confirm the likely causal role of variants in six known genes for sperm motility and we demonstrate that exome sequencing is an effective method to diagnose patients with severe sperm motility disorders (10/21 diagnosed; 48%). Furthermore, our analysis revealed six novel candidate genes for severe sperm motility disorders. Genome-wide sequencing of additional patient cohorts and re-analysis of exome data of currently unsolved cases may reveal additional variants in these novel candidate genes. STUDY FUNDING/COMPETING INTEREST(S): This project was supported in part by funding from the Australian National Health and Medical Research Council (APP1120356) to M.K.O.B., J.A.V. and R.I.M.L., The Netherlands Organisation for Scientific Research (918-15-667) to J.A.V., the Royal Society and Wolfson Foundation (WM160091) to J.A.V., as well as an Investigator Award in Science from the Wellcome Trust (209451) to J.A.V. and Grants from the National Research Council of Argentina (PIP 0900 and 4584) and ANPCyT (PICT 9591) to H.E.C. and a UUKi Rutherford Fund Fellowship awarded to B.J.H.

Both Palatable and Unpalatable Butterflies Use Bright Colors to Signal Difficulty of Capture to Predators.

Birds are able to recognize and learn to avoid attacking unpalatable, chemically defended butterflies after unpleasant experiences with them. It has also been suggested that birds learn to avoid prey that are efficient at escaping. This, however, remains poorly documented. Here, we argue that butterflies may utilize a variety of escape tactics against insectivorous birds and review evidence that birds avoid attacking butterflies that are hard to catch. We suggest that signaling difficulty of capture to predators is a widespread phenomenon in butterflies, and this ability may not be limited to palatable butterflies. The possibility that both palatable and unpalatable species signal difficulty of capture has not been fully explored, but helps explain the existence of aposematic coloration and escape mimicry in butterflies lacking defensive chemicals. This possibility may also change the role that putative Müllerian and Batesian mimics play in a variety of classical mimicry rings, thus opening new perspectives in the evolution of mimicry in butterflies.

A key to improved ion core confinement in the JET tokamak: ion stiffness mitigation due to combined plasma rotation and low magnetic shear.

New transport experiments on JET indicate that ion stiffness mitigation in the core of a rotating plasma, as described by Mantica et al. [Phys. Rev. Lett. 102, 175002 (2009)] results from the combined effect of high rotational shear and low magnetic shear. The observations have important implications for the understanding of improved ion core confinement in advanced tokamak scenarios. Simulations using quasilinear fluid and gyrofluid models show features of stiffness mitigation, while nonlinear gyrokinetic simulations do not. The JET experiments indicate that advanced tokamak scenarios in future devices will require sufficient rotational shear and the capability of q profile manipulation.

Vertical distribution, flight behaviour and evolution of wing morphology in Morpho butterflies.

1. Flight is a key innovation in the evolution of insects that is crucial to their dispersal, migration, territoriality, courtship and predator avoidance. Male butterflies have characteristic territoriality and courtship flight behaviours, and females use a characteristic flight behaviour when searching for host plants. This implies that selection acts on wing morphology to maximize flight performance for conducting important behaviours among sexes. 2. Butterflies in the genus Morpho are obvious components of neotropical forests, and many observations indicate that they show two broad categories of flight behaviour and flight height. Although species can be categorized as using gliding or flapping flight, and flying at either canopy or understorey height, the association of flight behaviour and flight height with wing shape evolution has never been explored. 3. Two clades within Morpho differ in flight behaviour and height. Males and females of one clade inhabit the forest understorey and use flapping flight, whereas in the other clade, males use gliding flight at canopy level and females use flapping flight in both canopy and understorey. 4. We used independent contrasts to answer whether wing shape is associated with flight behaviour and height. Given a single switch to canopy habitation and gliding flight, we compared contrasts for the node at which the switch to canopy flight occurred with the distribution of values in the two focal clades. We found significant changes in wing shape at the transition to canopy flight only in males, and no change in size for either sex. A second node within the canopy clade suggests that other factors may also be involved in wing shape evolution. Our results reinforce the hypothesis that natural selection acts differently on male and female butterfly wing shape and indicate that the transition to canopy flight cannot explain all wing shape diversity in Morpho. 5. This study provides a starting point for characterizing evolution of wing morphology in forest butterflies in the contexts of habitat selection and flight behaviour. Further, these observations suggest that exploring wing shape evolution for canopy and understorey species in other insects may help understand the effects of habitat destruction on biological diversity.

The effects of ants on the entomophagous butterfly caterpillar Feniseca tarquinius, and the putative role of chemical camouflage in the Feniseca-ant interaction.

Butterfly caterpillars in the lycaenid subfamily Miletinae are predators of ant-tended Homoptera, yet they lack specialized secretory and call-production organs crucial to ant association in other lycaenids. Here, we address the question of how miletine caterpillars have invaded the ant-Homoptera symbiosis through a study of the only New World miletine, Feniseca tarquinius, a predator of the wooly aphid Prociphilus tesselatus. Previous interpretations have suggested that F. tarquinius and other miletine caterpillars avoid ant aggression by concealing themselves under silken webs. In contrast, our field data indicate that F. tarquinius caterpillars are less likely to be concealed in the presence of the ants Camponotus pennsylvanicus and Formica obscuriventris than in the absence of ants, although caterpillar and ant behaviors vary between years. Chemical analysis and behavioral assays suggest that chemical camouflage, not physical concealment, is responsible for the ants' failure to detect and remove F. tarquinius caterpillars from aphid colonies. Analyses by gas chromatography indicate that the cuticular lipid composition of caterpillars are similar to that of their aphid prey, although it varies with prey species. Behavioral assays confirm that solvent extracts of F. tarquinius caterpillars and P. tesselatus aphids evoke similar behavioral responses in C. pennsylvanicus ants. Chemical camouflage is well known in social parasites of ants, but the present study represents one of a few documented cases where chemical deceit is important to interactions with ants outside the nest.

Discovery of novel p-arylthio cinnamides as antagonists of leukocyte function-associated antigen-1/intercellular adhesion molecule-1 interaction. 4. Structure-activity relationship of substituents on the benzene ring of the cinnamide.

We have shown that p-arylthio cinnamides can inhibit the interaction of LFA-1 and ICAM-1, which is involved in cell adhesion and the inflammatory process. We now show that 2,3-disubstitution on the aryl portion of the cinnamide results in enhanced activity over mono substitution on the ring. The best 2,3-substituents were chlorine and trifluoromethyl groups. Compounds 39 and 40 which contain two CF3 groups have IC(50) values of 0.5 and 0.1 nM, respectively, in inhibiting JY8 cells expressing LFA-1 on their surface, from adhering to ICAM-1. The structure-activity relationship (SAR) was examined using an NMR based model of the LFA-1 I domain/compound 31 complex. One of our compounds (38) was able to reduce cell migration in two different in vivo experiments.

Discovery of potent antagonists of leukocyte function-associated antigen-1/intercellular adhesion molecule-1 interaction. 3. Amide (C-ring) structure-activity relationship and improvement of overall properties of arylthio cinnamides.

The interaction of LFA-1 and ICAM-1 plays an important role in the cell adhesion process. On the basis of previously reported SAR and structural information on the binding of our p-arylthiocinnamide series to LFA-1, we have identified the cyclic amide (C-ring) as a site for modification. Improvement in potency and, more importantly, in the physical properties and pharmacokinetic profiles of the leading compounds resulted from this modification. One of the best compounds (11f) is also shown to reduce myocardial infarct size in rat.

Reduction of ion thermal diffusivity associated with the transition of the radial electric field in neutral-beam-heated plasmas in the large helical device.

Recent large helical device experiments revealed that the transition from ion root to electron root occurred for the first time in neutral-beam-heated discharges, where no nonthermal electrons exist. The measured values of the radial electric field were found to be in qualitative agreement with those estimated by neoclassical theory. A clear reduction of ion thermal diffusivity was observed after the mode transition from ion root to electron root as predicted by neoclassical theory when the neoclassical ion loss is more dominant than the anomalous ion loss.

Novel p-arylthio cinnamides as antagonists of leukocyte function-associated antigen-1/intracellular adhesion molecule-1 interaction. 2. Mechanism of inhibition and structure-based improvement of pharmaceutical properties.

The interaction between leukocyte function-associated antigen-1 (LFA-1) and intracellular adhesion molecule-1 (ICAM-1) has been implicated in inflammatory and immune diseases. Recently, a novel series of p-arylthio cinnamides has been described as potent antagonists of the LFA-1/ICAM-1 interaction. These compounds were found to bind to the I domain of LFA-1 using two-dimensional NMR spectroscopy of 15N-labeled LFA-1 I domain. On the basis of NOE studies between compound 1 and the I domain of LFA-1, a model of the complex was constructed. This model revealed that compound 1 does not directly inhibit ICAM-1 binding by interacting with the metal ion dependent adhesion site (MIDAS). Instead, it binds to the previously proposed I domain allosteric site (IDAS) of LFA-1 and likely modulates the activation of LFA-1 through its interaction with this regulatory site. A fragment-based NMR screening strategy was applied to identify small, more water-soluble ligands that bind to a specific region of the IDAS. When incorporated into the parent cinnamide template, the resulting analogues exhibited increased aqueous solubility and improved pharmacokinetic profiles in rats, demonstrating the power of this NMR-based screening approach for rapidly modifying high-affinity ligands.

Edge thermal transport barrier In LHD discharges

In LHD discharges a significant enhancement of the global energy confinement has been achieved for the first time in a helical device with an edge thermal barrier, which exhibits a sharp gradient at the edge of the temperature profile. Key features associated with the barrier are quite different from those seen in tokamaks: (i) almost no change in particle (including impurity) transport, (ii) a gradual formation of the barrier, (iii) a very high ratio of the edge temperature to the average temperature, and (iv) no edge relaxation phenomenon. These features are very attractive in applying the thermal barrier to future reactor grade devices.

Expression, purification, and characterization of human recombinant thrombopoietin in Chinese hamster ovary cells.

Thrombopoietin (TPO) is a primary regulator of megakaryocytopoiesis, a process through which megakaryocytes proliferate and mature into platelets. Recombinant human TPO (rhTPO) was expressed in Chinese hamster ovary (CHO) cells and purified from the culture medium. The cDNA encoding full-length TPO, including the native signal peptide sequence, was amplified by PCR from a human fetal liver cDNA library. The product was cloned into a mammalian expression vector under the control of the SV40 early promoter and enhancer. Secreted rhTPO was purified in three conventional chromatography steps. It migrates on SDS-PAGE as a broad band, characteristic of a heavily glycosylated protein, with an average molecular mass of 85 kDa. rhTPO expressed in CHO cells is biologically active in vitro as demonstrated by its ability to stimulate the proliferation of a megakaryocytic cell line and to trigger the JAK/STAT signal transduction pathway. rhTPO also shows activity in vivo as judged by the elevation of platelet count in treated mice.

Distribution of Ly-6C on lymphocyte subsets: I. Influence of allotype on T lymphocyte expression.

The expression patterns of the Ly-6C Ag were examined on splenic and thymic lymphocyte subsets of Ly-6.1 and Ly-6.2 strains of mice using the rat mAb 15.1. Ly-6C is expressed on subsets of CD4+ and CD8+ splenocytes, and a portion of NK cells. Within the splenic and lymph node CD4+ T cell compartment, Ly-6C expression is restricted to Ly-6.2 strains of mice, and is present on a subset of naive cells. Ly-6C is expressed on the majority of peripheral CD8+ T cells in both Ly-6.1 and Ly-6.2 strains, and is found primarily on the Ag-experienced subset. In the thymus, Ly-6C is present on subpopulations of CD4- CD8+, CD4- CD8-, and CD4+ CD8- cells. Ly-6C+ CD4- CD8+ thymocytes show a mature phenotype, while Ly-6C+ CD4- CD8- and Ly-6C+ CD4+ CD8- thymocytes appear to be part of the recently described NK1.1+ alphabeta TCR+ population. On account of the marked differences in Ly-6C expression on peripheral CD4+ T cells from Ly-6.1 and Ly-6.2 strains of mice, additional experiments were undertaken to assess Ly-6C expression in parental and Ly-6.1 x Ly-6.2 F1 mice. Neither phenotype dominated in the F1 offspring, with frequencies of Ly-6C+ CD4+ splenocytes falling in the intermediate range. Further experiments compared the staining patterns of the rat anti-pan Ly-6C (Ly-6.1 and Ly-6.2) Ab with a mouse anti-Ly-6.2 allotype specific Ab, with emphasis on both Ly-6.2 and Ly-6.1 x Ly-6.2 F1 mice. The results demonstrate the presence of lymphocytes that express the pan form of Ly-6C but not the form recognized by the alloantibody. This latter finding suggests the presence of more than one form of the Ly-6C Ag.

Four-dimensional imaging: computer visualization of 3D movements in living specimens.

The study of many biological processes requires the analysis of three-dimensional (3D) structures that change over time. Optical sectioning techniques can provide 3D data from living specimens; however, when 3D data are collected over a period of time, the quantity of image information produced leads to difficulties in interpretation. A computer-based system is described that permits the analysis and archiving of 3D image data taken over time. The system allows a user to roam through the full range of time points and focal planes in the data set. The user can animate images as an aid to visualization and can append multicolored labels and text notes to identified structures during data analysis. The system provides a valuable tool for the study of embryogenesis and cytoplasmic movements within cells and has considerable potential as an educational tool.

Morphing: a new graphics tool for animating confocal images.

The application of morphing to the display of developmental processes is described. This new graphics tool imparts the dynamic aspects of time-lapse microscopy to a series of fixed and stained images of a developing system. The technique is illustrated using confocal images of wing imaginal discs of Drosophila. It is anticipated that morphing can be applied not only to the display and analysis of developing systems but also to the elucidation of evolutionary relationships between species and to comparative anatomy.

New techniques to determine fluid status in hemodialyzed patients.

In this paper, several newly developed techniques for the estimation of the fluid status in hemodialysis patients were reviewed. Whereas echography of the inferior caval vein and the measurement of ANP and cGMP levels merely provide information about the intravascular volume, conductivity measurements are able to detect changes in the extracellular and intracellular compartments without being able to differentiate between the intravascular and interstitial fluid spaces. Echography of the inferior caval vein as a tool to assess over- and underhydration has been successfully validated against objective standards as right atrial pressure, total blood volume and the change in hemodynamic parameters during dialysis. Conductivity measurements were significantly related to vena cava measurements before and after dialysis. Whereas ANP levels were significantly related to the vena cava diameter before dialysis, in another group of patients, only a significant relation between the vena cava diameter and cGMP was observed in patients with normal left atrial hemodynamics, whereas they were not in patients with a dilated left atrium. Furthermore, in normovolemic patients with mitral insufficiency, ANP levels after dialysis remained increased compared to patients without mitral insufficiency, suggesting that, in addition to volume expansion, also altered left atrial hemodynamics influence the release of cGMP and ANP. Conductivity measurements and ANP before and after dialysis were not related, whereas only cGMP after dialysis was significantly related to conductivity measurements. ANP and cGMP were not related to the change in hemodynamic parameters during dialysis, questioning their reliability in the assessment of underhydration.

The influence of portoenterostomy with stoma on morbidity in pediatric patients with biliary atresia undergoing orthotopic liver transplantation.

A portoenterostomy (PE) procedure for extrahepatic biliary atresia (EHBA) is sometimes performed with a stoma in an attempt to reduce the incidence of acute cholangitis. The purpose of this study was to determine if the presence of a stoma increased the complication rate of patients undergoing orthotopic liver transplantation (OLT) for EHBA. The medical records of 42 consecutive patients with EHBA who underwent primary OLT between October 1988 and October 1991 were retrospectively reviewed. Three patients were excluded, since their grafts were lost within 3 days of OLT. The remaining 39 patients were divided into three groups: no PE (n = 7), PE without stoma (n = 23), and PE with stoma (n = 9). The mean age of the whole group was 19.62 +/- 24.37 months, with a range of 5 to 132 months. Mean weight was 9.62 kg, with a range of 4.2 to 41 kg. Survival at 3 and 12 months as well as number of retransplantations were similar among the three groups. However, at the time of OLT increased morbidity was observed, consisting of increased operative time and number of reoperations, whether or not the stoma had been closed prior to OLT.

Three-color immunofluorescence imaging of Drosophila embryos by laser scanning confocal microscopy.

We present a simple means for triple-labeling biological specimens by immunofluorescence using a laser scanning confocal microscope for imaging with a krypton/argon laser as a light source. Three separate images of fluorescein-, lissamine rhodamine- and cyanine-5-labeled antibodies are collected and subsequently merged to form the triple-labeled image, which is displayed at full-image resolution (24 bit) on a second image processing system. The technique is illustrated using immunofluorescence localization of three segmentation proteins in Drosophila embryos.

Congenital anomalies of the inferior vena cava: embryogenesis and MR features.

This article describes the MR appearances of the six most common congenital anomalies of the inferior vena cava. As a basis for understanding those anomalies, it describes the embryology of the inferior vena cava, based on an actual study of embryos and fetuses. The article takes a fresh look at the original research in this area, discusses the possible embryogenesis of the relevant anomalies, and describes different opinions on that subject, where different opinions exist.

Derivation of mixed phenotype cell lines with ras-myc- and raf-myc-containing retroviral vectors after infection of murine long-term bone marrow cultures.

Murine long-term bone marrow cultures were infected with retroviral vectors expressing the viral ras, raf, and myc oncogenes, alone and in combination. Stably transformed clonal cell lines were obtained after infection with ras-myc and raf-myc retroviruses but not by vectors expressing ras, myc, or raf alone. Northern blot analysis demonstrated that the two clonal cell lines expressed high levels of vector-specific transcripts. Phenotypic analysis of the cell lines by flow microfluorimetry and histochemical staining suggested that both cell lines expressed markers associated with cells of the megakaryocyte differentiation pathway. Histochemical staining demonstrated that these cell lines also expressed cytoplasmic enzymes associated with granulocytes and/or monocytes/macrophages. These cell lines, despite their clonal origin, are therefore of a mixed phenotype.