The role of Pnut and its functional domains in Drosophila spermatogenesis

Drosophila Pnut protein belongs to the family of septins, conservative GTPases participating in cytokinesis and many more other fundamental cellular processes. Because of their filamentous appearance, membrane association and functions, septins are considered as the fourth component of the cytoskeleton, along with actin, microtubules and intermediate filaments. However, septins are much less studied than the other cytoskeleton elements. We had previously demonstrated that deletion of the peanut (pnut) gene leads to mitotic abnormalities in somatic cells. The goal of this work was to study the role of pnut in Drosophila spermatogenesis. We designed a construct for pnut RNA interference allowing pnut expression to be suppressed ectopically. We analyzed the effect of pnut RNA interference on Drosophila spermatogenesis. The most sensitive to Pnut depletion were germ line cells at the earliest stages of spermatogenesis: the suppression of pnut expression at these stages leads to male sterility as a result of immotile sperm. Testes of those sterile males did not show any significant meiotic defects; axonemes and mitochondria were normal. We also analyzed the effect of mutations in Pnut conservative domains on Drosophila spermatogenesis. Mutations in the GTPase domain resulted in cyst elongation defects. Deletions of the C-terminal domain led to abnormal testis morphology. Both GTPase domain and C-terminal domain mutant males were sterile and produced immotile sperm. To summarize, we showed that Pnut participates in spermiogenesis, that is, late stages of spermatogenesis, when major morphological changes in spermatocytes occur

Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.

New Mutations in the 5′ Region of the Notch Gene Affect Drosophila melanogaster Oogenesis

The Notch pathway is an important and evolutionarily conserved signaling system involved in the development of multicellular organisms. Notch signaling plays an important role in the regulation of proliferation and differentiation of many cell types. In this study, we report new aspects of Notch gene participation in oogenesis using our previously generated mutations. The mutations consist of an insertion of an auxiliary element of a transgene construct into the first intron of the gene and a series of directed deletions within the 5′ regulatory region of Notch. We showed that some of these mutations affect Drosophila oogenesis. This insertion, either alone or in combination with the deletion of an insulator sequence, led to lower expression of Notch in the ovaries. As a result, the formation of egg chambers was disturbed in middle oogenesis. These abnormalities have not been described previously and imply one more function of Notch in oogenesis. It can be assumed that Notch is associated with not only follicular epithelium morphogenesis but also cellular mechanisms of oocyte growth

The complex role of transcription factor GAGA in germline death during Drosophila spermatogenesis: transcriptomic and bioinformatic analyses

The GAGA protein (also known as GAF) is a transcription factor encoded by the Trl gene in D. melanogaster. GAGA is involved in the regulation of transcription of many genes at all stages of fly development and life. Recently, we investigated the participation of GAGA in spermatogenesis and discovered that Trl mutants experience massive degradation of germline cells in the testes. Trl underexpression induces autophagic death of spermatocytes, thereby leading to reduced testis size. Here, we aimed to determine the role of the transcription factor GAGA in the regulation of ectopic germline cell death. We investigated how Trl underexpression affects gene expression in the testes. We identified 15,993 genes in three biological replicates of our RNA-seq analysis and compared transcript levels between hypomorphic TrlR85/Trl362 and Oregon testes. A total of 2,437 differentially expressed genes were found, including 1,686 upregulated and 751 downregulated genes. At the transcriptional level, we detected the development of cellular stress in the Trl-mutant testes: downregulation of the genes normally expressed in the testes (indicating slowed or abrogated spermatocyte differentiation) and increased expression of metabolic and proteolysis-related genes, including stress response long noncoding RNAs. Nonetheless, in the Flybase Gene Ontology lists of genes related to cell death, autophagy, or stress, there was no enrichment with GAGA-binding sites. Furthermore, we did not identify any specific GAGA-dependent cell death pathway that could regulate spermatocyte death. Thus, our data suggest that GAGA deficiency in male germline cells leads to an imbalance of metabolic processes, impaired mitochondrial function, and cell death due to cellular stress

Microtubule-mediated transport of the tumor-suppressor protein Merlin and its mutants

The neurofibromatosis type 2 (NF2) tumor-suppressor protein Merlin is a member of the ERM family of proteins that links the cytoskeleton to the plasma membrane. In humans, mutations in the NF2 gene cause neurofibromatosis type-2 (NF2), a cancer syndrome characterized by the development of tumors of the nervous system. Previous reports have suggested that the subcellular distribution of Merlin is critical to its function, and that several NF2 mutants that lack tumor-suppressor activity present improper localization. Here we used a Drosophila cell culture model to study the distribution and mechanism of intracellular transport of Merlin and its mutants. We found that Drosophila Merlin formed cytoplasmic particles that move bidirectionally along microtubules. A single NF2-causing amino acid substitution in the FERM domain dramatically inhibited Merlin particle movement. Surprisingly, the presence of this immotile Merlin mutant also inhibited trafficking of the WT protein. Analysis of the movement of WT protein using RNAi and pull-downs showed that Merlin particles are associated with and moved by microtubule motors (kinesin-1 and cytoplasmic dynein), and that binding of motors and movement is regulated by Merlin phosphorylation. Inhibition of Merlin transport by expression of the dominant-negative mutant or depletion of kinesin-1 results in increased nuclear accumulation of the transcriptional coactivator Yorkie. These results demonstrate the requirement of microtubule-dependent transport for Merlin function