Absence of transitive and systemic pathways allows cell-specific and isoform-specific RNAi in Drosophila

RNA interference (RNAi) designates the multistep process by which double-stranded RNA induces the silencing of homologous endogenous genes. Some aspects of RNAi appear to be conserved throughout evolution, including the processing of trigger dsRNAs into small 21-23-bp siRNAs and their use to guide the degradation of complementary mRNAs. Two remarkable features of RNAi were uncovered in plants and Caenorhabditid elegans. First, RNA-dependent RNA polymerase activities allow the synthesis of siRNA complementary to sequences upstream of or downstream from the initial trigger region in the target mRNA, leading to a transitive RNAi with sequences that had not been initially targeted. Secondly, systemic RNAi may cause the targeting of gene silencing in one tissue to spread to other tissues. Using transgenes expressing dsRNA, we investigated whether transitive and systemic RNAi occur in DROSOPHILA: DsRNA-producing transgenes targeted RNAi to specific regions of alternative mRNA species of one gene without transitive effect directed to sequences downstream from or upstream of the initial trigger region. Moreover, specific expression of a dsRNA, using either cell-specific GAL4 drivers or random clonal activation of a GAL4 driver, mediated a cell-autonomous RNAi. Together, our results provide evidence that transitive and systemic aspects of RNAi are not conserved in Drosophila and demonstrate that dsRNA-producing transgenes allow powerful reverse genetic approaches to be conducted in this model organism, by knocking down gene functions at the resolution of a single-cell type and of a single isoform.     

The asymmetric spatial distribution of bacterial signal transduction proteins coordinates cell cycle events

The polar localization of signaling proteins that are essential for Caulobacter cell cycle control is temporally regulated. Here we provide evidence that phosphorylation of the essential response regulator, DivK, is required for both its function and its cell cycle-regulated localization. The asymmetric location of the DivJ and PleC histidine kinases and their antagonistic activities on the cellular concentration of phosphorylated DivK provide positional and temporal information for the ordered sequence of DivK localization during the cell cycle. DivJ activity on DivK affects its correct localization, which, in turn, is required for PleC function. Since DivJ and PleC regulate different cell cycle events, the interconnected function of these two histidine kinases through localization of a common response regulator provides a mechanism for coordinating cell cycle progression. Study of a DivK homolog in the morphologically symmetric bacterium Sinorhizobium meliloti suggests that this type of cell cycle mechanism is widespread in prokaryotes.     

Dispersal and fighting in male pollinating fig wasps

For more than two decades, it has been the dogma that the males of pollinating fig wasps do not fight and that they only mate in their native fig. Their extreme degree of local mating leads to highly female biased sex ratios that should eliminate the benefits of fighting and dispersal by males. Furthermore, males sharing a fig are often brothers, and fighting may be barred by kin selection. Therefore, theory supported the presumed absence of fighting and dispersal in pollinating fig wasp males. However, we report here that in pollinating fig wasps, fighting between brothers evolved at least four and possibly six time, and dispersal by males at least twice. This finding supports the idea that competition between relatives can cancel the ameliorating effects of relatedness. The explanation to this evolutionary puzzle, as well as the consequences of male dispersal and fighting, opens the doors to exciting new research.     

Improved gelatinase a selectivity by novel zinc binding groups containing galardin derivatives

The synthesis of several analogues of galardin, a MMP inhibitor, are presented with their in vitro inhibitory activity against MMP-1 and MMP-2. These compounds contain a distinct Zinc Binding Group (ZBG). Those having a 2-acylated-heterocycle as well as a 2-arylamide function do not exhibit a good inhibition/selectivity against the enzymes tested. On the contrary, those that are based on a hydrazide scaffold present potent selectivity for MMP-2 versus MMP-1.     

Upper Limb Evaluation and One-Year Follow Up of Non-Ambulant Patients with Spinal Muscular Atrophy: An Observational Multicenter Trial

Assessment of the upper limb strength in non-ambulant neuromuscular patients remains challenging. Although potential outcome measures have been reported, longitudinal data demonstrating sensitivity to clinical evolution in spinal muscular atrophy patients are critically lacking. Our study recruited 23 non-ambulant patients, 16 patients (males/females = 6/10; median age 15.4 years with a range from 10.7 to 31.1 years) with spinal muscular atrophy type II and 7 patients (males/females = 2/5; median age 19.9 years with a range from 8.3 to 29.9 years) with type III. The Brooke functional score was on median 3 with a range from 2 to 6. The average total vital capacity was 46%, and seven patients required non-invasive ventilation at night. Patients were assessed at baseline, 6 months, and 1 year using the Motor Function Measure and innovative devices MyoGrip, MyoPinch, and MoviPlate, which assess handgrip strength, key pinch strength, and hand/finger extension-flexion function, respectively. The study demonstrated the feasibility and reliability of these measures for all patients, and sensitivity to negative changes after the age of 14 years. The younger patients showed an increase of the distal force in the follow-up period. The distal force measurements and function were correlated to different functional scales. These data represent an important step in the process of validating these devices as potential outcome measures for future clinical trials.

Trial Registration

ClinicalTrials.gov NCT00993161     

Upper Limb Strength and Function Changes during a One-Year Follow-Up in Non-Ambulant Patients with Duchenne Muscular Dystrophy: An Observational Multicenter Trial

IntroductionUpper limb evaluation of patients with Duchenne Muscular Dystrophy is crucially important to evaluations of efficacy of new treatments in non-ambulant patients. In patients who have lost ambulation, there are few validated and informative outcome measures. In addition, longitudinal data demonstrating sensitivity to clinical evolution of outcome measures over short-term periods are lacking.ResultsOur study confirmed preliminary data previously reported regarding feasibility of use and of reliability of the MyoSet and the correlation at baseline between distal strength and clinical outcomes such as FVC, Brooke score, age, and duration since loss of ambulation. A significant correlation was observed between the distal upper limb strength and clinical variables. The sensitive dynamometers (MyoGrip and MyoPinch) and MoviPlate captured a 12-month change in non-ambulant Duchenne muscular dystrophy patients of all ages.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00993161","term_id":"NCT00993161"}}NCT00993161 {"type":"clinical-trial","attrs":{"text":"NCT00993161","term_id":"NCT00993161"}}NCT00993161     

Identification of a Novel Regulatory Mechanism of Nutrient Transport Controlled by TORC1-Npr1-Amu1/Par32

Fine-tuning the plasma-membrane permeability to essential nutrients is fundamental to cell growth optimization. Nutritional signals including nitrogen availability are integrated by the TORC1 complex which notably regulates arrestin-mediated endocytosis of amino-acid transporters. Ammonium is a ubiquitous compound playing key physiological roles in many, if not all, organisms. In yeast, it is a preferred nitrogen source transported by three Mep proteins which are orthologues of the mammalian Rhesus factors. By combining genetic, kinetic, biochemical and cell microscopy analyses, the current study reveals a novel mechanism enabling TORC1 to regulate the inherent activity of ammonium transport proteins, independently of arrestin-mediated endocytosis, identifying the still functional orphan Amu1/Par32 as a selective regulator intermediate. We show that, under poor nitrogen supply, the TORC1 effector kinase'' Npr1'' promotes phosphorylation of Amu1/Par32 which appears mainly cytosolic while ammonium transport proteins are active. Upon preferred nitrogen supplementation, like glutamine or ammonium addition, TORC1 upregulation enables Npr1 inhibition and Amu1/Par32 dephosphorylation. In these conditions, as in Npr1-lacking cells, hypophosphorylated Amu1/Par32 accumulates at the cell surface and mediates the inhibition of specific ammonium transport proteins. We show that the integrity of a conserved repeated motif of Amu1/Par32 is required for the interaction with these transport proteins. This study underscores the diversity of strategies enabling TORC1-Npr1 to selectively monitor cell permeability to nutrients by discriminating between transporters to be degraded or transiently inactivated and kept stable at the plasma membrane. This study further identifies the function of Amu1/Par32 in acute control of ammonium transport in response to variations in nitrogen availability.     

Overproduced <Emphasis Type="Italic">Brucella abortus</Emphasis> PdhS-mCherry forms soluble aggregates in <Emphasis Type="Italic">Escherichia coli</Emphasis>, partially associating with mobile foci of IbpA-YFP

Background  

When heterologous recombinant proteins are produced in Escherichia coli, they often precipitate to form insoluble aggregates of unfolded polypeptides called inclusion bodies. These structures are associated with chaperones like IbpA. However, there are reported cases of "non-classical" inclusion bodies in which proteins are soluble, folded and active.     

Polymorphism in a ferritin H gene from chromosome 6p

Summary This paper addresses the question of whether abnormalities in ferritin expression in the iron storage disease hemochromatosis (HC) involve major deletions or alterations in regions containing the two ferritin H genes that lie near the disease locus on chromosome 6p. We present evidence from analyses of Southern blots that neither gene is deleted in hemochromatosis. We also describe a polymorphism in one of the genes that we have previously shown to be a processed pseudogene. This polymorphism does not correlate with the presence of HC. The PIC value for this polymorphism was calculated as 0.49.