Analysis of the C. elegans Argonaute family reveals that distinct Argonautes act sequentially during RNAi

Argonaute (AGO) proteins interact with small RNAs to mediate gene silencing. C. elegans contains 27 AGO genes, raising the question of what roles these genes play in RNAi and related gene-silencing pathways. Here we describe 31 deletion alleles representing all of the previously uncharacterized AGO genes. Analysis of single- and multiple-AGO mutant strains reveals functions in several pathways, including (1) chromosome segregation, (2) fertility, and (3) at least two separate steps in the RNAi pathway. We show that RDE-1 interacts with trigger-derived sense and antisense RNAs to initiate RNAi, while several other AGO proteins interact with amplified siRNAs to mediate downstream silencing. Overexpression of downstream AGOs enhances silencing, suggesting that these proteins are limiting for RNAi. Interestingly, these AGO proteins lack key residues required for mRNA cleavage. Our findings support a two-step model for RNAi, in which functionally and structurally distinct AGOs act sequentially to direct gene silencing.     

The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans

Double-stranded (ds) RNA induces potent gene silencing, termed RNA interference (RNAi). At an early step in RNAi, an RNaseIII-related enzyme, Dicer (DCR-1), processes long-trigger dsRNA into small interfering RNAs (siRNAs). DCR-1 is also required for processing endogenous regulatory RNAs called miRNAs, but how DCR-1 recognizes its endogenous and foreign substrates is not yet understood. Here we show that the C. elegans RNAi pathway gene, rde-4, encodes a dsRNA binding protein that interacts during RNAi with RNA identical to the trigger dsRNA. RDE-4 protein also interacts in vivo with DCR-1, RDE-1, and a conserved DExH-box helicase. Our findings suggest a model in which RDE-4 and RDE-1 function together to detect and retain foreign dsRNA and to present this dsRNA to DCR-1 for processing.     

Hsa-miR-584-5p as a novel candidate biomarker in Turkish men with severe coronary artery disease

Molecular Biology Reports - Coronary artery disease (CAD) is still the preliminary cause of mortality and morbidity in the developed world. Identification of novel predictive and therapeutic...     

A Method for Selectively Enriching Microbial DNA from Contaminating Vertebrate Host DNA

DNA samples derived from vertebrate skin, bodily cavities and body fluids contain both host and microbial DNA; the latter often present as a minor component. Consequently, DNA sequencing of a microbiome sample frequently yields reads originating from the microbe(s) of interest, but with a vast excess of host genome-derived reads. In this study, we used a methyl-CpG binding domain (MBD) to separate methylated host DNA from microbial DNA based on differences in CpG methylation density. MBD fused to the Fc region of a human antibody (MBD-Fc) binds strongly to protein A paramagnetic beads, forming an effective one-step enrichment complex that was used to remove human or fish host DNA from bacterial and protistan DNA for subsequent sequencing and analysis. We report enrichment of DNA samples from human saliva, human blood, a mock malaria-infected blood sample and a black molly fish. When reads were mapped to reference genomes, sequence reads aligning to host genomes decreased 50-fold, while bacterial and Plasmodium DNA sequences reads increased 8–11.5-fold. The Shannon-Wiener diversity index was calculated for 149 bacterial species in saliva before and after enrichment. Unenriched saliva had an index of 4.72, while the enriched sample had an index of 4.80. The similarity of these indices demonstrates that bacterial species diversity and relative phylotype abundance remain conserved in enriched samples. Enrichment using the MBD-Fc method holds promise for targeted microbiome sequence analysis across a broad range of sample types.     

Detection of primary clarithromycin resistance of Helicobacter pylori and association between cagA + status and clinical outcome

Helicobacter pylori was examined in 110 patients (82 (74.5) with gastritis, 18 (16.4) with duodenitis, six (5.5) with duodenal ulcer and gastroesophageal reflux, and four (3.6 %) with normal) with gastrointestinal problems living in rural area, no history of macrolide use, and detected by culture (71.8) or direct detection from gastric biopsies by PCR (82.7 %). Also, cagA gene was identified using PCR and was found positive in 68/91 (74.7 %) strains. The prevalence of clarithromycin-resistant H. pylori was investigated by two methods including PCR–RFLP (7.7 (A2142G 1.1 and A2143G 6.6 %)) and twofold agar dilution (8.9 %) to detect phenotypic and genotypic status simultaneously. Among all the H. pylori positive patients, eight (8.8 %) isolates were found to be resistant to clarithromycin by at least one of the AD and/or PCR–RFLP methods. H. pylori positive rates were significantly correlated with patients' sex, age, and endoscopic findings (p?=?0.040, <0.001 and <0.001, respectively). There were no differences in gender or endoscopic findings related to cagA ⁺ and cagA ^? patients. The gene of cagA was not significantly helpful in predicting the clinical outcome of H. pylori infection alone. In conclusion, we revealed that there was a low prevalence of primer clarithromycin resistance in patients living in rural area with no history of macrolide use. The prevalence of mutant strains among the macrolide-resistant H. pylori varies even geographically between close provinces.     

Aeropalynological spectrum of Hatay,Turkey, the eastern coast of the Mediterranean Sea

An aeropalynological study during the years 2014–2015 was performed in Hatay, which is a unique sociocultural and phytogeographical area located on the border of Turkey and Syria on the eastern coast of the Mediterranean. The sampling was performed by a Hirst-type volumetric sampler (Lanzoni VPPS 2000), and pollen grains of 54 taxa were identified, of which 83.21% of the annual sum belonged to woody taxa. The highest pollen concentration was recorded in February, of which a large amount came from the Cupressaceae/Taxaceae families. The diversity of the pollen reflected the vegetation of the area and plantations of the city center, but pollen grains from Euro-Siberian elements specific to Mount Amanos could not be recorded. Pollen types found at more than 3% of the annual pollen index and considered dominant pollen types were as follows: Cupressaceae/Taxaceae (50.86%), Olea europaea (12.67%), Moraceae (7.20%), Poaceae (5.99%), Quercus (5.35%), Urticaceae (3.79%) and Pinus (3.70%); almost all dominant pollen types in the city atmosphere were previously stated to be allergic. The main pollen season starting dates of common pollen types found were one or two weeks earlier than those of the surroundings. Many statistically significant correlations were found between daily pollen concentrations and daily meteorological parameters, e.g., Cupressaceae/Taxaceae Poaceae and Urticaceae pollen correlated negatively with mean temperature in both years, and in the hindermost two families daily pollen amounts significantly correlated with wind speed in the second year. Daily Olea europaea pollen concentration showed a significant negative correlation with the amount of total daily rainfall in the second year.     

High‐Performance Suspended Particle Devices Based on Copper‐Reduced Graphene Oxide Core–Shell Nanowire Electrodes

Smart windows are one of the key components of so‐called “green” buildings. These windows are based on an actively switchable electro‐optic material that is sandwiched between two transparent electrodes. Although great progress has been made in identifying the optimal materials for such active windows, there is still a great need to improve their key elements, especially the performance of the transparent electrodes. Here, a new suspended particle device (SPD), holding a great potential for smart window applications, which is built upon copper‐reduced graphene oxide (Cu‐rGO) core–shell nanowire (NW) films as a transparent conductive electrode is reported. With the wrapping of rGO, the Cu NW electrodes demonstrate both high optical transparency and electrical conductivity, as well as significantly improved stability under various testing conditions. The novel sandwich‐structured SPDs, based on these electrodes, show a large change in their optical transmittance (42%) between “on” and “off” states, impressively fast switching time and superior stability. These high performances are comparable to those of the SPDs based on indium tin oxide electrodes. These promising results pave the way for the electrodes to be an integral part of a variety of optoelectronic devices, including energy‐friendly and flexible electronics.     

Comparative analysis of human UCB and adipose tissue derived mesenchymal stem cells for their differentiation potential into brown and white adipocytes

The differentiation potential of umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) into brown and white adipocytes in comparison to Adipose tissue derived MSCs (AD-MSCs) were investigated in order to characterize their potency for future cell therapies. MSCs were isolated from ten UCB samples and six liposuction materials. MSCs were differentiated into white and brown adipocytes after characterization by flow cytometry. Differentiated adipocytes were stained with Oil Red O and hematoxylin/eosin. The UCP1 protein levels in brown adipocytes were investigated by immunofluoresence and western blot analysis. Cells that expressed mesenchymal stem cells markers (CD34?, CD45?, CD90+ and CD105+) were successfully isolated from UCB and adipose tissue. Oil Red O staining demonstrated that white and brown adipocytes obtained from AD-MSCs showed 85 and 61% of red pixels, while it was 3 and 1.9%, respectively for white and brown adipocytes obtained from UCB-MSCs. Fluorescence microscopy analysis showed strong uncoupling protein 1 (UCP1) signaling in brown adipocytes, especially which were obtained from AD-MSCs. Quantification of UCP1 protein amount showed 4- and 10.64-fold increase in UCP1 contents of brown adipocytes derived from UCB-MSCs and AD-MSCs, respectively in comparison to undifferentiated MSCs (P?<?0.004). UCB-MSCs showed only a little differentiation tendency into adipocytes means it is not an appropriate stem cell type to be differentiated into these cell types. In contrast, high differentiation efficiency of AD-MSCs into brown and white adipocytes make it appropriate stem cell type to use in future regenerative medicine of soft tissue disorders or fighting with obesity and its related disorders.