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.

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.

Enhancement of symbioses between butterfly caterpillars and ants by vibrational communication.

Butterfly caterpillars produce calls that appear to play a role in maintaining symbiotic associations with ants. A survey of butterfly species from South and Central America, North America, Europe, Thailand, and Australia suggests that the ability for caterpillars to call has evolved independently at least three times, and that calling may be ubiquitous among ant-associated species. Because ants use substrate-borne sound in their communication systems, this study points to the possibility that the calls of one insect species have evolved to attract other, unrelated species.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Surgery of anorectal anomalies.

This article discusses the disparate views regarding sphincteric anatomy, both normal and abnormal, and describes preoperative procedures for and operative approaches to resolving anorectal anomalies.

The pathogenesis of gastroschisis and omphalocele.

Omphalocele and gastroschisis appear, by virtue of their differences in postnatal findings and embryological development, to be different entities. An hypothesis concerning the genesis of gastroschisis is presented which is consistent with observed embryologic sequential change and known teratogenic possibilities. The apparent increase in number of cases of gastroschisis and the frequency of omphalocele warrant further experimental study of their pathogenesis, preferably in primates.

Familial occurrence of thyroglossal duct cyst.

Persistence and dilation of the embryologic thyroglossal tract gives rise to the condition of thyroglossal duct cyst. Although these cysts have an embryologic origin, there is rarely a history of inheritance. A search of the literature shows only two family reports, which includes a total of nine patients. We report on a third family with thyroglossal duct cyst in two members. The patients were female siblings aged 2 and 6 years, both of whom underwent successful surgical excision of their thyroglossal duct cysts by modified Sistrunk's technique.

Posterior sagittal anorectoplasty.

From October 1980 to November 1981, 34 patients with anorectal anomalies have been operated upon by a sagittal midline approach. The skin incision extends from the sacrum to the perineum (ventral aspect of the anal dimple). The superficial and deep layers of the external sphincter are identified by electrostimulation and split, with the coccyx, in the midline. Ileo- and pubococcygeal portions of the levator dorsally and then the striated muscle complex of the external sphinctor, pubococcygeus and the presumed puborectalis are split ventrally to the urethra. In no cases has the ventral portion of the levators been separated from the thick ventral portion of the external sphincter, hence the term "striated muscle complex." When the terminal bowel is dilated (congenitally ectatic), the bowel is tailored prior to reconstruction of the sphinctors. The posterior sagittal approach provides an excellent exposure for evaluation and mobilization of the terminal bowel. It enables one to construct an anal canal, suture the bowel wall to the striated musculature and the mucosa to the skin, thereby reducing or avoiding the complications of prolapse and stenosis. In males with ectasia and a rectourethral fistula, transrectal closure of the mucosa at the fistula site, leaving the rectal longitudinal smooth muscle insertions on the prostatic capsule, avoids damage to the nerves and genital structures.

Posterior sagittal anorectoplasty: important technical considerations and new applications.

Posterior sagittal anorectoplasty (PSARP) is a new technique for the repair of high anorectal malformations. It is based upon complete exposure of the anorectal region by means of a median sagittal incision that runs from the sacrum to the anal dimple, cutting through all muscle structures behind the rectum. It was learned through this procedure that the external sphincter is a functionally useful prominent structure. No puborectalis sling, as such, could be identified. It was possible, however, to recognize a muscle continuity from the skin to the sacral insertion of the levator ani. Since it is impossible to pull the generally ectatic rectum through without destroying the muscle structures present, the rectum must be tapered to allow suturing the muscle behind it. In all these anomalies, the rectum and urethra (or vagina) are very closely joined, sharing a common wall, and their separation calls for extensive exposure. A number of technical details clarified in the course of applying the procedure in 54 patients are fully discussed. This approach also proved to be very successful in the management of two patients with rectocloacal fistula, two with rectal atresia and two with stenosis. Colostomy has been closed in 27 patients and fecal continence may be described as excellent, except in those patients with severe sacral anomalies, and unquestionably superior to that obtained by us with other techniques.

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.