Gene silencing in the nucleus: mechanisms and new phenomena

Silencing of homologous genes by exogenously introduced dsRNA was first observed in C. elegans. Endogenous small RNAs (siRNAs, piRNAs, miRNAs) mediate regulation of expression of genes post-transcriptionally or at the level of transcription, when argonaute proteins complex with small RNAs to target genomic loci for chromatin modifications in a sequence-specific manner, in the nucleus. There have been previous reports of regulation of expression by targeting mRNA for silencing in the cytoplasm. We identified a region in the genome, flp-17 locus that is amenable to nuclear silencing mechanisms in a wildtype animal. C. elegans exhibits strong anti-foreign genome silencing in their germline as defense against invading viral or transposon DNA. This activity is extended to transgene DNA, resulting in its silencing in the germline. Mos1 is a foreign element, a transposon from Drosophila that is heterologously inserted in C. elegans genome. Trangenes are integrated in the chromosomal DNA by Mos1 based Single Copy insertion technology where homologous regions flanking the gene of interest promotes recombination and thus its integration at a specific mos site on the worm genome. In our experiments we observed robust silencing in the somatic cells of transgenes that were intended to be integrated in ttTi5605 mos site on C. elegans genome by homologous recombination. The silencing phenomenon involves epigenetic mechanisms. We hypothesize that over several generations, the worm has “learned” that mos is a foreign element and when transgene is integrated at that site, it is amenable to silencing by the epigenetic machinery. Furthermore, we identified a previously undescribed mutation (yy14) in eri-6 gene and show evidences pointing to a role in silencing of transgenes in somatic tissues. We observed an increase in trans-spliced mRNA from eri- 6/7 genes in yy14 mutants. Our model reasons out that increase in trans-spliced mRNA results will lead to upregulation of small RNAs (26G RNAs) that efficiently function in the silencing of our transgene, which needs further verification. In addition, ABC transporters in C. elegans have been previously shown to be required for efficient RNAi. haf-2, haf-6 and haf-9 mutants also exhibit defects in transposon mobilization. Chromatin modification by epigenetic machinery prevents mobilization of transposable elements. This includes RNAi effectors like siRNAs, Dicer and argonaute proteins. Thus, a previous study in the lab, highlights a link between ABC transporters and RNAi mechanisms in C. elegans. It is important to characterize the dimerization pattern of half ABC transporter proteins to gain insights on their functions and precise roles in RNAi

Systematic comparison of bacterial feeding strains for increased yield of Caenorhabditis elegans males by RNA interference-induced non-disjunction

AbstractRare Caenorhabditis elegans males arise when sex chromosome non-disjunction occurs during meiosis in self-fertilizing hermaphrodites. Non-disjunction is a relatively rare event, and males are typically observed at a frequency of less than one in five hundred wild-type animals. Males are required for genetic crosses and phenotypic analysis, yet current methods to generate large numbers of males can be cumbersome. Here, we identify RNAi reagents (dsRNA-expressing bacteria) with improved effectiveness for eliciting males. Specifically, we used RNAi to systematically reduce the expression of over two hundred genes with meiotic chromosome segregation functions, and we identified a set of RNAi reagents that robustly and reproducibly elicited male progeny

The Burden of Pediatric Critical Illness Among Pediatric Oncology Patients in Low- and Middle-income Countries: A Systematic Review and Meta-analysis

Background: Pediatric oncology patients have increased risk for critical illness; outcomes are well described in high-income countries (HICs); however, data is limited for low- and middle-income countries (LMICs). Methods: We systematically searched PubMed, EMBASE, Web of Science, CINAHL and Global Health databases for articles in 6 languages describing mortality in children with cancer admitted to intensive care units (ICUs) in LMICs. Two investigators independently assessed eligibility, data quality, and extracted data. We pooled ICU mortality estimates using random effect models. Results: Of 3641 studies identified, 22 studies were included, covering 4803 ICU admissions. Overall pooled mortality was 30.3 % [95 % Confidence-interval (CI) 21.7–40.6 %]. Mechanical ventilation [odds ratio (OR) 12.2, 95 %CI:6.2–24.0, p-value<0.001] and vasoactive infusions [OR 6.3 95 %CI:3.3–11.9, p-value<0.001] were associated with ICU mortality. Conclusions: ICU mortality among pediatric oncology patients in LMICs is similar to that in HICs, however, this review likely underestimates true mortality due to underrepresentation of studies from low-income countries

Randomized Clinical Trial of High-Dose Rifampicin With or Without Levofloxacin Versus Standard of Care for Pediatric Tuberculous Meningitis: The TBM-KIDS Trial

Background. Pediatric tuberculous meningitis (TBM) commonly causes death or disability. In adults, high-dose rifampicin may reduce mortality. The role of fluoroquinolones remains unclear. There have been no antimicrobial treatment trials for pediatric TBM. Methods. TBM-KIDS was a phase 2 open-label randomized trial among children with TBM in India and Malawi. Participants received isoniazid and pyrazinamide plus: (i) high-dose rifampicin (30 mg/kg) and ethambutol (R30HZE, arm 1); (ii) high-dose rifampicin and levofloxacin (R30HZL, arm 2); or (iii) standard-dose rifampicin and ethambutol (R15HZE, arm 3) for 8 weeks, followed by 10 months of standard treatment. Functional and neurocognitive outcomes were measured longitudinally using Modified Rankin Scale (MRS) and Mullen Scales of Early Learning (MSEL). Results. Of 2487 children prescreened, 79 were screened and 37 enrolled. Median age was 72 months; 49%, 43%, and 8% had stage I, II, and III disease, respectively. Grade 3 or higher adverse events occurred in 58%, 55%, and 36% of children in arms 1, 2, and 3, with 1 death (arm 1) and 6 early treatment discontinuations (4 in arm 1, 1 each in arms 2 and 3). By week 8, all children recovered to MRS score of 0 or 1. Average MSEL scores were significantly better in arm 1 than arm 3 in fine motor, receptive language, and expressive language domains (P < .01). Conclusions. In a pediatric TBM trial, functional outcomes were excellent overall. The trend toward higher frequency of adverse events but better neurocognitive outcomes in children receiving high-dose rifampicin requires confirmation in a larger trial. Clinical Trials Registration. NCT02958709

Fragment Finder 2.0: a computing server to identify structurally similar fragments

Fragment Finder 2.0 is a web-based interactive computing server which can be used to retrieve structurally similar protein fragments from 25 and 90% nonredundant data sets. The computing server identifies structurally similar fragments using the protein backbone C alpha angles. In addition, the identified fragments can be superimposed using either of the two structural superposition programs, STAMP and PROFIT, provided in the server. The freely available Java plug-in Jmol has been interfaced with the server for the visualization of the query and superposed fragments. The server is the updated version of a previously developed search engine and employs an in-house-developed fast pattern matching algorithm. This server can be accessed freely over the World Wide Web through the URL http://cluster.physics.iisc.ernet.in/ff/

Familial Alzheimer mutations stabilize synaptotoxic γ-secretase-substrate complexes

Summary: Mutations that cause familial Alzheimer’s disease (FAD) are found in amyloid precursor protein (APP) and presenilin, the catalytic component of γ-secretase, that together produce amyloid β-peptide (Aβ). Nevertheless, whether Aβ is the primary disease driver remains controversial. We report here that FAD mutations disrupt initial proteolytic events in the multistep processing of APP substrate C99 by γ-secretase. Cryoelectron microscopy reveals that a substrate mimetic traps γ-secretase during the transition state, and this structure aligns with activated enzyme-substrate complex captured by molecular dynamics simulations. In silico simulations and in cellulo fluorescence microscopy support stabilization of enzyme-substrate complexes by FAD mutations. Neuronal expression of C99 and/or presenilin-1 in Caenorhabditis elegans leads to synaptic loss only with FAD-mutant transgenes. Designed mutations that stabilize the enzyme-substrate complex and block Aβ production likewise led to synaptic loss. Collectively, these findings implicate the stalled process—not the products—of γ-secretase cleavage of substrates in FAD pathogenesis