De novo truncating mutations in AHDC1 in individuals with syndromic expressive language delay, hypotonia, and sleep apnea.

Clinical whole-exome sequencing (WES) for identification of mutations leading to Mendelian disease has been offered to the medical community since 2011. Clinically undiagnosed neurological disorders are the most frequent basis for test referral, and currently, approximately 25% of such cases are diagnosed at the molecular level. To date, there are approximately 4,000 "known" disease-associated loci, and many are associated with striking dysmorphic features, making genotype-phenotype correlations relatively straightforward. A significant fraction of cases, however, lack characteristic dysmorphism or clinical pathognomonic traits and are dependent upon molecular tests for definitive diagnoses. Further, many molecular diagnoses are guided by recent gene-disease association discoveries. Hence, there is a critical interplay between clinical testing and research leading to gene-disease association discovery. Here, we describe four probands, all of whom presented with hypotonia, intellectual disability, global developmental delay, and mildly dysmorphic facial features. Three of the four also had sleep apnea. Each was a simplex case without a remarkable family history. Using WES, we identified AHDC1 de novo truncating mutations that most likely cause this genetic syndrome.

Local signalling environments and human male infertility: what we can learn from mouse models.

Infertility is one of the most prevalent public health problems facing young adult males in today's society. A clear, treatable cause of infertility cannot be determined in a large number of these patients, and a growing body of evidence suggests that infertility in many of these men may be due to genetic causes. Studies using mouse knockout technology have been integral for examination of normal spermatogenesis and to identify proteins essential for this process, which in turn are candidate genes for human male infertility. Successful spermatogenesis depends on a delicate balance of local signalling factors, and this review focuses on the genes that encode these factors. Normal functioning of all testicular cell types is essential for fertility and might also be crucial to prevent germ cell oncogenesis. Analysis of these signalling processes in spermatogenesis using mouse models has provided investigators with an invaluable tool to effectively translate basic science research to the research of human disease and infertility.

Genetic evidence that SMAD2 is not required for gonadal tumor development in inhibin-deficient mice.

BACKGROUND: Inhibin is a tumor-suppressor and activin antagonist. Inhibin-deficient mice develop gonadal tumors and a cachexia wasting syndrome due to enhanced activin signaling. Because activins signal through SMAD2 and SMAD3 in vitro and loss of SMAD3 attenuates ovarian tumor development in inhibin-deficient females, we sought to determine the role of SMAD2 in the development of ovarian tumors originating from the granulosa cell lineage. METHODS: Using an inhibin alpha null mouse model and a conditional knockout strategy, double conditional knockout mice of Smad2 and inhibin alpha were generated in the current study. The survival rate and development of gonadal tumors and the accompanying cachexia wasting syndrome were monitored. RESULTS: Nearly identical to the controls, the Smad2 and inhibin alpha double knockout mice succumbed to weight loss, aggressive tumor progression, and death. Furthermore, elevated activin levels and activin-induced pathologies in the liver and stomach characteristic of inhibin deficiency were also observed in these mice. Our results indicate that SMAD2 ablation does not protect inhibin-deficient females from the development of ovarian tumors or the cachexia wasting syndrome. CONCLUSIONS: SMAD2 is not required for mediating tumorigenic signals of activin in ovarian tumor development caused by loss of inhibin.

Connecting the dots in the mechanism of action of Cucurbitacin E (CurE) - path analysis and steered molecular dynamics reveal the precise site of entry and the passage of CurE in filamentous actin.

Cucurbitacin E (CurE) modulates actin cytoskeleton by forming an irreversible covalent bond with Cys257 of actin. The reported binding conformation of CurE is deeply buried in the subdomain 4 of actin and is closely situated to the ATP-binding site. The entry and the path taken by CurE to reach this buried site remain a mystery. In this study, steered molecular dynamics (SMD) simulations were conducted to delineate the diffusion of CurE to its binding site. SMD simulations reveal that the distinctive entry site of CurE found in subdomain 4 is by itself a closed and compact region of two loops lying beside each other like a closed door and CurE induces it to open. From this point, CurE moves toward its binding site through a path facilitated by Thr188, Leu261, Ile267, Ile309, Tyr306. This study is also an insight into how CurE distinctly differentiates its tunnel to the binding site from the ATP-binding site. The conformational changes of CurE along the path to the binding site are surprisingly very minimal and closely resemble the attack conformation at the end of simulation. The study reveals that the little energy spent by the molecule is compensated by the enthalpic contribution to binding-free energy barrier making it undoubtedly the most preferred path of CurE. This study is the first of its kind in which the SMD was used to derive the complete and continuous translocation of one of the most potent phytochemicals, CurE through the binding site gorge of actin. [Formula: see text] Communicated by Ramaswamy H. Sarma.

Azadirachtin(A) distinctively modulates subdomain 2 of actin - novel mechanism to induce depolymerization revealed by molecular dynamics study.

Azadirachtin(A) (AZA), a potential insecticide from neem, binds to actin and induces depolymerization in Drosophila. AZA binds to the pocket same as that of Latrunculin A (LAT), but LAT inhibits actin polymerization by stiffening the actin structure and affects the ADP-ATP exchange. The mechanism by which AZA induces actin depolymerization is not clearly understood. Therefore, different computational experiments were conducted to delineate the precise mechanism of AZA-induced actin depolymerization. Molecular dynamics studies showed that AZA strongly interacted with subdomain 2 and destabilized the interactions between subdomain 2 of one actin and subdomains 1 and 4 of the adjacent actin, causing the separation of actin subunits. The separation was observed between subdomain 3 of subunit n and subdomain 4 of subunit n + 2. However, the specific triggering point for the separation of the subunits was the destabilization of direct interactions between subdomain 2 of subunit n (Arg39, Val45, Gly46 and Arg62) and subdomain 4 of subunit n + 2 (Asp286, Ile287, Asp288, Ile289, Asp244 and Lys291). These results reveal a unique mechanism of an actin filament modulator that induces depolymerization. This mechanism of AZA can be used to design similar molecules against mammalian actins for cancer therapy.

Docking, molecular dynamics and QM/MM studies to delineate the mode of binding of CucurbitacinE to F-actin.

CucurbitacinE (CurE) has been known to bind covalently to F-actin and inhibit depolymerization. However, the mode of binding of CurE to F-actin and the consequent changes in the F-actin dynamics have not been studied. Through quantum mechanical/molecular mechanical (QM/MM) and density function theory (DFT) simulations after the molecular dynamics (MD) simulations of the docked complex of F-actin and CurE, a detailed transition state (TS) model for the Michael reaction is proposed. The TS model shows nucleophilic attack of the sulphur of Cys257 at the ß-carbon of Michael Acceptor of CurE producing an enol intermediate that forms a covalent bond with CurE. The MD results show a clear difference between the structure of the F-actin in free form and F-actin complexed with CurE. CurE affects the conformation of the nucleotide binding pocket increasing the binding affinity between F-actin and ADP, which in turn could affect the nucleotide exchange. CurE binding also limits the correlated displacement of the relatively flexible domain 1 of F-actin causing the protein to retain a flat structure and to transform into a stable "tense" state. This structural transition could inhibit depolymerization of F-actin. In conclusion, CurE allosterically modulates ADP and stabilizes F-actin structure, thereby affecting nucleotide exchange and depolymerization of F-actin.

Effect of glass composition on the degradation properties and ion release characteristics of phosphate glass--polycaprolactone composites.

A series of polycaprolactone and ternary-based (Na(2)O)(0.55-x)(CaO)(x)(P(2)O(5))(0.45) glass composites were created, each containing 20% volume percentage of glass with various calcium compositions. A short-term degradation study was carried out to investigate the physical and ion release behaviour of these composites, utilising analytical techniques such as dynamical mechanical analysis, and ion chromatography. All the composites experienced significant loss of weight and stiffness throughout the study, with the 24 mol% calcium composites losing the greatest amount of weight and stiffness. The pH profile of the aqueous solutions in which the composites were placed were initially acidic, but began to neutralise mid-way through the study, with the 36 mol% solution achieving the most acidic conditions. The ion release behaviour mirrored the mass loss behaviour of the glass component of the composites. The cations (sodium and calcium ions) release was comparable with the initial stages of composite mass degradation, both of which exhibited almost immediate release when placed into solution. The 24 mol% composites underwent rapid rates of cation release, while the 36 mol% experienced the slowest rates of release. By contrast, anion (phosphates and polyphosphates) release showed a dissimilar trend, with rapid release of the P(2)O(7) and P(3)O(10) occurring during the first few hours in solution, whilst the P(3)O(9) structure released steadily during the first 48 h in solution. Finally, PO(4) release was at a constant rate over the duration of the study, releasing up to 300 ppm from the 32 and 36 mol% samples by the end of 200 h. To summarise, these results show that by combining phosphate glasses with biodegradable polymer, it is possible to create composites whose rate of degradation can be controlled to meet the needs of their end application.

Testicular cell adhesion molecule 1 (TCAM1) is not essential for fertility.

Testicular cell adhesion molecule 1 (Tcam1) is a testis-expressed gene that is evolutionarily conserved in most mammalian species. The putative location of TCAM1 on the cell surface makes it an attractive contraceptive target to study. We found that Tcam1 transcription is enriched in the adult testis, and in situ hybridization revealed that Tcam1 is expressed in pachytene to secondary spermatocytes. Immunofluorescence for TCAM1 protein showed strong expression along cell membranes of spermatocytes and weak localization to round spermatids. In light of this evidence, we hypothesized that TCAM1 interacts with an unknown receptor on the surface of Sertoli cells and that this interaction is important for germ cell-Sertoli cell interactions. However, Tcam1 knockout mice that we generated are fertile, and testis weights and sperm counts were not significantly altered. Therefore, we conclude that TCAM1 is not essential for male fertility or germ cell function in Mus musculus.

Absence of inhibin alpha and retinoblastoma protein leads to early sertoli cell dysfunction.

Sertoli cells, the support cells of mammalian spermatogenesis, are regulated by a number of nuclear factors and express retinoblastoma (RB) tumor suppressor protein. We hypothesized that RB is an important mediator of Sertoli cell tumorigenesis in inhibin alpha knockout (Inha KO) mice. In our previous mouse studies, we found that conditional knockout (cKO) of Rb in Sertoli cells caused progressive Sertoli cell dysfunction. Initially, loss of RB had no gross effect on Sertoli cell function as the mice were fertile with normal testis weights at 6 weeks of age, but by 10-14 weeks of age, mutant mice demonstrated severe Sertoli cell dysfunction and infertility. Although double knockout (dKO) of Rb and Inha did not result in exacerbation of the tumorigenic phenotype of Inha-null mice, we found that the dKO mice demonstrate an acceleration of Sertoli cell dysfunction compared to Rb cKO mice. Specifically, in contrast to Rb cKO mice, Inha/Rb dKO mice showed signs of Sertoli cell dysfunction as early as 4 weeks of age. These results demonstrate that RB is not essential for Sertoli cell tumorigenesis in Inha KO mice but that loss of Inha accelerates the infertility phenotype of Rb cKO mice.

Granulosa cell-expressed BMPR1A and BMPR1B have unique functions in regulating fertility but act redundantly to suppress ovarian tumor development.

Bone morphogenetic proteins (BMPs) have diverse roles in development and reproduction. Although several BMPs are produced by oocytes, thecal cells, and granulosa cells of developing follicles, the in vivo functions of most of these ligands are unknown. BMP signals are transduced by multiple type I and type II TGFbeta family receptors, and of the type I receptors, BMP receptor 1A (BMPR1A) and BMP receptor 1B (BMPR1B) are known to be expressed in rodent granulosa cells. Female mice homozygous null for Bmpr1b are sterile due to compromised cumulus expansion, but the function of BMPR1A in the ovary is unknown. To further decipher a role for BMP signaling in mouse granulosa cells, we deleted Bmpr1a in the granulosa cells of the ovary and found Bmpr1a conditional knockout females to be subfertile with reduced spontaneous ovulation. To explore the redundant functions of BMP receptor signaling in the ovary, we generated Bmpr1a Bmpr1b double-mutant mice, which developed granulosa cell tumors that have evidence of increased TGFbeta and hedgehog signaling. Thus, similar to SMAD1 and SMAD5, which have redundant roles in suppressing granulosa cell tumor development in mice, two type I BMP receptors, BMPR1A and BMPR1B, function together to prevent ovarian tumorigenesis. These studies support a role for a functional BMP signaling axis as a tumor suppressor pathway in the ovary, with BMPR1A and BMPR1B acting downstream of BMP ligands and upstream of BMP receptor SMADs.

Retinoblastoma protein plays multiple essential roles in the terminal differentiation of Sertoli cells.

Retinoblastoma protein (RB) plays crucial roles in cell cycle control and cellular differentiation. Specifically, RB impairs the G(1) to S phase transition by acting as a repressor of the E2F family of transcriptional activators while also contributing towards terminal differentiation by modulating the activity of tissue-specific transcription factors. To examine the role of RB in Sertoli cells, the androgen-dependent somatic support cell of the testis, we created a Sertoli cell-specific conditional knockout of Rb. Initially, loss of RB has no gross effect on Sertoli cell function because the mice are fertile with normal testis weights at 6 wk of age. However, by 10-14 wk of age, mutant mice demonstrate severe Sertoli cell dysfunction and infertility. We show that mutant mature Sertoli cells continue cycling with defective regulation of multiple E2F1- and androgen-regulated genes and concurrent activation of apoptotic and p53-regulated genes. The most striking defects in mature Sertoli cell function are increased permeability of the blood-testis barrier, impaired tissue remodeling, and defective germ cell-Sertoli cell interactions. Our results demonstrate that RB is essential for proper terminal differentiation of Sertoli cells.

Appetite for reproduction: dietary restriction, aging and the mammalian gonad.

The major physiologic theory of aging, the disposable soma theory, links dietary restriction (DR), also known as calorie or food restriction, to prolonged lifespan and makes specific predictions about the effects of aging and DR on reproduction. A recent study in BMC Biology profiling the effects of aging and DR on gonadal gene expression provides novel molecular evidence that has a significant impact on this theory of aging.