ALFY-Controlled DVL3 Autophagy Regulates Wnt Signaling,Determining Human Brain Size

Primary microcephaly is a congenital neurodevelopmental disorder of reduced head circumference and brain volume, with fewer neurons in the cortex of the developing brain due to premature transition between symmetrical and asymmetrical cellular division of the neuronal stem cell layer during neurogenesis. We now show through linkage analysis and whole exome sequencing, that a dominant mutation in ALFY, encoding an autophagy scaffold protein, causes human primary microcephaly. We demonstrate the dominant effect of the mutation in drosophila: transgenic flies harboring the human mutant allele display small brain volume, recapitulating the disease phenotype. Moreover, eye-specific expression of human mutant ALFY causes rough eye phenotype. In molecular terms, we demonstrate that normally ALFY attenuates the canonical Wnt signaling pathway via autophagy-dependent removal specifically of aggregates of DVL3 and not of Dvl1 or Dvl2. Thus, autophagic attenuation of Wnt signaling through removal of Dvl3 aggregates by ALFY acts in determining human brain size.     

Fine structure of the ventral nerve centre and interspecific identification of individual neurons in the enigmatic Chaetognatha

The enigmatic arrow worms (Chaetognatha) are marine carnivores and among the most abundant planktonic organisms. Their phylogenetic position has been heavily debated for a long time. Most recent molecular studies still provide a diverging picture and suggest arrow worms to be some kind of basal protostomes. In an effort to understand the organization of the nervous system in this clade for a broad comparison with other Metazoa we analysed the ultrastructure of the ventral nerve centre in Spadella cephaloptera by transmission electron microscopy. We were able to identify six different types of neurons in the bilateral somata clusters by means of the cytoplasmic composition (regarding the structure of the neurite and soma including the shape and eu-/heterochromatin ratio within the nucleus) as well as the size and position of these neurons. Furthermore, our study provides new insights into the neuropil composition of the ventral nerve centre and several other fine structural features. Our second goal was to examine if individually identifiable neurons are present in the ventral nerve centres of four chaetognath species, Sagitta setosa, Sagitta enflata, Pterosagitta draco, and Spadella cephaloptera. For that purpose, we processed whole mount specimens of these species for immunolocalization of RFamide-related neuropeptides and analysed them with confocal laser-scanning microscopy. Our experiments provide evidence for the interspecific homology of individual neurons in the ventral nerve centres of these four chaetognath species suggesting that the potential to generate serially arranged neurons with individual identities is part of their ground pattern.     

The effect of administering equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) post artificial insemination on fertility of lactating dairy cows

The objective was to evaluate the effect of equine chorionic gonadotropin (eCG) and hCG post artificial insemination (AI) on fertility of lactating dairy cows. In Experiment 1, cows were either treated with eCG on Day 22 post AI (400 IU; n = 80) or left untreated (n = 84). On Day 29, pregnant cows were either treated with hCG (2500 IU; n = 32) or left untreated (n = 36). Pregnancy and progesterone were evaluated on Days 29 and 45. In Experiment 2, cows (n = 28) were either treated with eCG on Day 22 (n = 13) or left untreated (n = 15) and either treated with hCG on Day 29 (n = 14) or left untreated (n = 14). Blood sampling and ultrasonography were conducted between Days 22 and 45. In Experiment 3, cows were either treated with eCG on Day 22 post AI (n = 229) or left untreated (n = 241). Pregnancy was evaluated on Days 36 and 85. In Experiment 1, eCG on Day 22 increased (P < 0.02) the number of pregnant cows on Day 29 (50.0 vs. 33.3%) and on Day 45, the increase was higher (P < 0.01) in cows with timed AI (41.2 vs. 6.5%) than in cows AI at detected estrus (50.0 vs. 37.8%). Pregnancy losses were reduced by eCG and hCG, but increased in cows that did not receive eCG but were given hCG (P < 0.01). Treatment with hCG tended (P < 0.06) to increase progesterone in control cows, but not in cows treated with eCG. In Experiment 2, hCG increased (P < 0.01) the number of accessory CLs on Day 35 (28.5 vs. 0.0%) and tended (P < 0.07) to increase progesterone. In Experiment 3, eCG increased the number of pregnant cows (P < 0.05) on Days 36 and 85, but only in cows with low body condition (eCG = 45.6 and 43.5%; Control = 22.9 and 22.9%). In conclusion, eCG at 22 days post insemination increased fertility, primarily in cows with low body condition and reduced pregnancy losses when given 7 days before hCG; hCG induced accessory CLs and slightly increased progesterone, but hCG given in the absence of a prior eCG treatment reduced fertility.     

Dominance of the alpha1B-adrenergic receptor and its subcellular localization in human and TRAMP prostate cancer cell lines

The function and distribution of alpha1-adrenergic receptor (AR) subtypes in prostate cancer cells is well characterized. Previous studies have used RNA localization or low-avidity antibodies in tissue or cell lines to determine the alpha1-AR subtype and suggested that the alpha1A-AR is dominant. Two androgen-insensitive, human metastatic cancer cell lines DU145 and PC3 were used as well as the mouse TRAMP C1-C3 primary and clonal cell lines. The density of alpha1-ARs was determined by saturation binding and the distribution of the different alpha1-AR subtypes was examined by competition-binding experiments. In contrast to previous studies, the major alpha1-AR subtype in DU145, PC3 and all of the TRAMP cell lines is the alpha1B-AR. DU145 cells contained 100% of the alpha1B-AR subtype, whereas PC3 cells were composed of 21% alpha1 A-AR and 79% alpha1B-AR. TRAMP cell lines contained between 66% and 79% of the alpha1B-AR with minor fractions of the other two subtypes. Faster doubling time in the TRAMP cell lines correlated with decreasing alpha 1B-AR and increasing alpha1 A- and alpha1D-AR densities. Transfection with EGFP-tagged alpha1B-ARs revealed that localization was mainly intracellular, but the majority of the receptors translocated to the cell surface after extended preincubation (18 hr) with either agonist or antagonist. Localization was confirmed by ligand-binding studies and inositol phosphate assays where prolonged preincubation with either agonist and/or antagonist increased the density and function of alpha 1-ARs, suggesting that the native receptors were mostly intracellular and nonfunctional. Our studies indicate that alpha1B-ARs are the major alpha1-AR subtype expressed in DU145, PC3, and all TRAMP cell lines, but most of the receptor is localized in intracellular compartments in a nonfunctional state, which can be rescued upon prolonged incubation with any ligand.     

Allosterically coupled calcium and magnesium binding sites are unmasked by ryanodine receptor chimeras

We studied cation regulation of wild-type ryanodine receptor type 1 (_WTRyR1), type 3 (_WTRyR3), and RyR3/RyR1 chimeras (Ch) expressed in 1B5 dyspedic myotubes. Using [³H]ryanodine binding to sarcoplasmic reticulum (SR) membranes, Ca²⁺ titrations with _WTRyR3 and three chimeras show biphasic activation that is allosterically coupled to an attenuated inhibition relative to _WTRyR1. Chimeras show biphasic Mg²⁺ inhibition profiles at 3 and 10 μM Ca²⁺, no observable inhibition at 20 μM Ca²⁺ and monophasic inhibition at 100 μM Ca²⁺. Ca²⁺ imaging of intact myotubes expressing Ch-4 exhibit caffeine-induced Ca²⁺ transients with inhibition kinetics that are significantly slower than those expressing _WTRyR1 or _WTRyR3. Four new aspects of RyR regulation are evident: (1) high affinity (H) activation and low affinity (L) inhibition sites are allosterically coupled, (2) Ca²⁺ facilitates removal of the inherent Mg²⁺ block, (3) _WTRyR3 exhibits reduced cooperativity between H activation sites when compared to _WTRyR1, and (4) uncoupling of these sites in Ch-4 results in decreased rates of inactivation of caffeine-induced Ca²⁺ transients.     

Adenylate cyclase influences filamentous haemagglutinin-mediated attachment of Bordetella pertussis to epithelial alveolar cells

Attachment to epithelial cells in the respiratory tract is a key event in Bordetella pertussis colonization. Filamentous haemagglutinin (FHA) is an important virulence factor mediating adhesion to host cells. In this study, the relevance of the interaction between FHA and adenylate cyclase toxin (ACT) during bacterial attachment was investigated. Mutants lacking either FHA or ACT showed significantly decreased adherence to epithelial respiratory cells. The use of several ACT-specific monoclonal antibodies and antiserum showed that the decrease in attachment of strains lacking ACT expression could not be explained by the adhesin-like activity of ACT, or a change of any of the biological activities of ACT. Immunoblot analysis showed that the lack of ACT expression did not interfere with FHA localization. An heparin-inhibitable carbohydrate-binding site is crucial in the process of FHA-mediated bacterial binding to epithelial cells. In the presence of heparin attachment of wild-type B. pertussis, but not of the isogenic ACT defective mutant, to epithelial cells was significantly decreased. These results suggest that ACT enhances the adhesive functions of FHA, and modifies the performance of the FHA heparin-inhibitable carbohydrate binding site. We propose that the presence of ACT in the outer membrane of B. pertussis to play a role in the functionality of FHA.     

Intronic Variants in the NFKB1 Gene May Influence Hearing Forecast in Patients with Unilateral Sensorineural Hearing Loss in Meniere's Disease

Meniere''s disease is an episodic vestibular syndrome associated with sensorineural hearing loss (SNHL) and tinnitus. Patients with MD have an elevated prevalence of several autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis and psoriasis), which suggests a shared autoimmune background. Functional variants of several genes involved in the NF-κB pathway, such as REL, TNFAIP3, NFKB1 and TNIP1, have been associated with two or more immune-mediated diseases and allelic variations in the TLR10 gene may influence bilateral affectation and clinical course in MD. We have genotyped 716 cases of MD and 1628 controls by using the ImmunoChip, a high-density genotyping array containing 186 autoimmune loci, to explore the association of immune system related-loci with sporadic MD. Although no single nucleotide polymorphism (SNP) reached a genome-wide significant association (p<10⁻⁸), we selected allelic variants in the NF-kB pathway for further analyses to evaluate the impact of these SNPs in the clinical outcome of MD in our cohort. None of the selected SNPs increased susceptibility for MD in patients with uni or bilateral SNHL. However, two potential regulatory variants in the NFKB1 gene (rs3774937 and rs4648011) were associated with a faster hearing loss progression in patients with unilateral SNHL. So, individuals with unilateral MD carrying the C allele in rs3774937 or G allele in rs4648011 had a shorter mean time to reach hearing stage 3 (>40 dB HL) (log-rank test, corrected p values were p = 0.009 for rs3774937 and p = 0.003 for rs4648011, respectively). No variants influenced hearing in bilateral MD. Our data support that the allelic variants rs3774937 and rs4648011 can modify hearing outcome in patients with MD and unilateral SNHL.     

Efficient site-specific integration of large transgenes by an enhanced herpes simplex virus/adeno-associated virus hybrid amplicon vector

We previously demonstrated that a herpes simplex virus type 1 (HSV-1)/adeno-associated virus (AAV) hybrid amplicon vector constructed by inserting the sequences of regulatory protein (rep) and inverted terminal repeats of AAV into an HSV amplicon vector resulted in the enhanced stability of transgene expression compared to the original HSV-1 amplicon vector. However, problems related to the expression of Rep compromised its therapeutic applications. We report here a new HSV/AAV hybrid amplicon vector system that not only solved problems associated with Rep expression but also markedly improved the stable transduction efficiency of this vector. This new HSV/AAV vector is designed in a way that little or no Rep would be expressed in packaging cells, but it can be expressed in transduced cells if Cre recombinase is provided. Furthermore, Rep expression will be automatically suppressed as a consequence of Rep-mediated integration. Our results showed that the new hybrid amplicon vector yielded titers comparable to those of standard amplicon vectors. When Cre-expressing 293 cells were transduced, a low level of Rep expression was detected, and stable transduction was achieved in approximately 22% of transduced cells; of those cells, approximately 70% transduction was achieved by Rep-mediated site-specific integration. In the majority of the stably transduced cells, Rep expression was no longer observed. Our results also proved that this vector system is capable of efficiently accommodating and site-specifically integrating large transgenes, such as the full-length dystrophin expression cassette. Thus, the new HSV/AAV vector demonstrated unique advantages in safe and effective delivery of long-lasting transgene expression into human cells.     

Oocyte apoptosis is suppressed by disruption of the acid sphingomyelinase gene or by sphingosine-1-phosphate therapy

The time at which ovarian failure (menopause) occurs in females is determined by the size of the oocyte reserve provided at birth, as well as by the rate at which this endowment is depleted throughout post-natal life. Here we show that disruption of the gene for acid sphingomyelinase in female mice suppressed the normal apoptotic deletion of fetal oocytes, leading to neonatal ovarian hyperplasia. Ex vivo, oocytes lacking the gene for acid sphingomyelinase or wild-type oocytes treated with sphingosine-1-phosphate resisted developmental apoptosis and apoptosis induced by anti-cancer therapy, confirming cell autonomy of the death defect. Moreover, radiation-induced oocyte loss in adult wild-type female mice, the event that drives premature ovarian failure and infertility in female cancer patients, was completely prevented by in vivo therapy with sphingosine-1-phosphate. Thus, the sphingomyelin pathway regulates developmental death of oocytes, and sphingosine-1-phosphate provides a new approach to preserve ovarian function in vivo.     

Targeting molecular interactions essential for Plasmodium sexual reproduction

Malaria remains one of the most devastating infectious diseases, killing up to a million people every year. Whereas much progress has been made in understanding the life cycle of the parasite in the human host and in the mosquito vector, significant gaps of knowledge remain. Fertilization of malaria parasites, a process that takes place in the lumen of the mosquito midgut, is poorly understood and the molecular interactions (receptor–ligand) required for Plasmodium fertilization remain elusive. By use of a phage display library, we identified FG1 (Female Gamete peptide 1), a peptide that binds specifically to the surface of female Plasmodium berghei gametes. Importantly, FG1 but not a scrambled version of the peptide, strongly reduces P. berghei oocyst formation by interfering with fertilization. In addition, FG1 also inhibits P. falciparum oocyst formation suggesting that the peptide binds to a molecule on the surface of the female gamete whose structure is conserved. Identification of the molecular interactions disrupted by the FG1 peptide may lead to the development of novel malaria transmission‐blocking strategies.