Versatility of MicroRNA Biogenesis

MicroRNAs (miRNAs) are short single-stranded RNA molecules that regulate gene expression. MiRNAs originate from large primary (pri) and precursor (pre) transcripts that undergo various processing steps along their biogenesis pathway till they reach their mature and functional form. It is not clear, however, whether all miRNAs are processed similarly. Here we show that the ratio between pre-miRNA and mature miRNA forms varies between different miRNAs. Moreover, over-expression of several factors involved in miRNA biogenesis, including Exportin-5, Drosha, NF90a, NF45 and KSRP, displayed bidirectional effects on pre/mature miRNA ratios, suggesting their intricate biogenesis sensitivity. In an attempt to identify additional factors that might explain the versatility in miRNA biogenesis we have analyzed the contribution of two hnRNP family members, hnRNPH1 and hnRNPR. Knock-down or over-expression of these genes suggested that hnRNPR inhibits, whereas hnRNPH1 facilitates, miRNA processing. Overall, our results emphasize that miRNA biogenesis is versatile

Adsorption and reaction of CO on (Pd–)Al2O3 and (Pd–)ZrO2: vibrational spectroscopy of carbonate formation

γ-Alumina is widely used as an oxide support in catalysis, and palladium nanoparticles supported by alumina represent one of the most frequently used dispersed metals. The surface sites of the catalysts are often probed via FTIR spectroscopy upon CO adsorption, which may result in the formation of surface carbonate species. We have examined this process in detail utilizing FTIR to monitor carbonate formation on γ-alumina and zirconia upon exposure to isotopically labelled and unlabelled CO and CO2. The same was carried out for well-defined Pd nanoparticles supported on Al2O3 or ZrO2. A water gas shift reaction of CO with surface hydroxyls was detected, which requires surface defect sites and adjacent OH groups. Furthermore, we have studied the effect of Cl synthesis residues, leading to strongly reduced carbonate formation and changes in the OH region (isolated OH groups were partly replaced or were even absent). To corroborate this finding, samples were deliberately poisoned with Cl to an extent comparable to that of synthesis residues, as confirmed by Auger electron spectroscopy. For catalysts prepared from Cl-containing precursors a new CO band at 2164 cm−1 was observed in the carbonyl region, which was ascribed to Pd interacting with Cl. Finally, the FTIR measurements were complemented by quantification of the amount of carbonates formed via chemisorption, which provides a tool to determine the concentration of reactive defect sites on the alumina surface

Unique and conserved MicroRNAs in wheat chromosome 5D revealed by next-generation sequencing

MicroRNAs are a class of short, non-coding, single-stranded RNAs that act as post-transcriptional regulators in gene expression. miRNA analysis of Triticum aestivum chromosome 5D was performed on 454 GS FLX Titanium sequences of flow sorted chromosome 5D with a total of 3,208,630 good quality reads representing 1.34x and 1.61x coverage of the short (5DS) and long (5DL) arms of the chromosome respectively. In silico and structural analyses revealed a total of 55 miRNAs; 48 and 42 miRNAs were found to be present on 5DL and 5DS respectively, of which 35 were common to both chromosome arms, while 13 miRNAs were specific to 5DL and 7 miRNAs were specific to 5DS. In total, 14 of the predicted miRNAs were identified in wheat for the first time. Representation (the copy number of each miRNA) was also found to be higher in 5DL (1,949) compared to 5DS (1,191). Targets were predicted for each miRNA, while expression analysis gave evidence of expression for 6 out of 55 miRNAs. Occurrences of the same miRNAs were also found in Brachypodium distachyon and Oryza sativa genome sequences to identify syntenic miRNA coding sequences. Based on this analysis, two other miRNAs: miR1133 and miR167 were detected in B. distachyon syntenic region of wheat 5DS. Five of the predicted miRNA coding regions (miR6220, miR5070, miR169, miR5085, miR2118) were experimentally verified to be located to the 5D chromosome and three of them : miR2118, miR169 and miR5085, were shown to be 5D specific. Furthermore miR2118 was shown to be expressed in Chinese Spring adult leaves. miRNA genes identified in this study will expand our understanding of gene regulation in bread wheat

Perturbation of the Dimer Interface of Triosephosphate Isomerase and its Effect on Trypanosoma cruzi

Most of the enzymes of parasites have their counterpart in the host. Throughout evolution, the three-dimensional architecture of enzymes and their catalytic sites are highly conserved. Thus, identifying molecules that act exclusively on the active sites of the enzymes from parasites is a difficult task. However, it is documented that the majority of enzymes consist of various subunits, and that conservation in the interface of the subunits is lower than in the catalytic site. Indeed, we found that there are significant differences in the interface between the two subunits of triosephosphate isomerase from Homo sapiens and Trypanosoma cruzi (TcTIM), which causes Chagas disease in the American continent. In the search for agents that specifically inhibit TcTIM, we found that 2,2′-dithioaniline (DTDA) is far more effective in inactivating TcTIM than the human enzyme, and that its detrimental effect is due to perturbation of the dimer interface. Remarkably, DTDA prevented the growth of Escherichia coli cells that had TcTIM instead of their own TIM and killed T. cruzi epimastigotes in culture. Thus, this study highlights a new approach base of targeting molecular interfaces of dimers

Unequal allelic expression of wild-type and mutated β-myosin in familial hypertrophic cardiomyopathy

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease, which in about 30% of the patients is caused by missense mutations in one allele of the β-myosin heavy chain (β-MHC) gene (MYH7). To address potential molecular mechanisms underlying the family-specific prognosis, we determined the relative expression of mutant versus wild-type MYH7-mRNA. We found a hitherto unknown mutation-dependent unequal expression of mutant to wild-type MYH7-mRNA, which is paralleled by similar unequal expression of β-MHC at the protein level. Relative abundance of mutated versus wild-type MYH7-mRNA was determined by a specific restriction digest approach and by real-time PCR (RT-qPCR). Fourteen samples from M. soleus and myocardium of 12 genotyped and clinically well-characterized FHC patients were analyzed. The fraction of mutated MYH7-mRNA in five patients with mutation R723G averaged to 66 and 68% of total MYH7-mRNA in soleus and myocardium, respectively. For mutations I736T, R719W and V606M, fractions of mutated MYH7-mRNA in M. soleus were 39, 57 and 29%, respectively. For all mutations, unequal abundance was similar at the protein level. Importantly, fractions of mutated transcripts were comparable among siblings, in younger relatives and unrelated carriers of the same mutation. Hence, the extent of unequal expression of mutated versus wild-type transcript and protein is characteristic for each mutation, implying cis-acting regulatory mechanisms. Bioinformatics suggest mRNA stability or splicing effectors to be affected by certain mutations. Intriguingly, we observed a correlation between disease expression and fraction of mutated mRNA and protein. This strongly suggests that mutation-specific allelic imbalance represents a new pathogenic factor for FHC

Accuracy of cardiovascular magnetic resonance in myocarditis: comparison of MR and histological findings in an animal model

Background: Because Endomyocardial Biopsy has low sensitivity of about 20%, it can be performed near to myocardium that presented as Late Gadolinium Enhancement (LGE) in cardiovascular magnetic resonance (CMR). However the important issue of comparing topography of CMR and histological findings has not yet been investigated. Thus the current study was performed using an animal model of myocarditis. Results: In 10 male Lewis rats Experimental Autoimmune myocarditis was induced, 10 rats served as control. On day 21 animals were examined by CMR to compare topographic distribution of LGE to histological inflammation. Sensitivity, specificity, positive and negative predictive values for LGE in diagnosing myocarditis were determined for each segment of myocardium. Latter diagnostic values varied widely depending on topographic distribution of LGE and inflammation as well as on the used CMR sequence. Sensitivity of LGE was up to 76% (left lateral myocardium) and positive predictive values were up to 85% (left lateral myocardium), whereas sensitivity and positive predictive value dropped to 0 - 33% (left inferior myocardium). Conclusions: Topographic distribution of LGE and histological inflammation seem to influence sensitivity, specifity, positive and negative predictive values. Nevertheless, positive predictive value for LGE of up to 85% indicates that Endomyocardial Biopsy should be performed "MR-guided". LGE seems to have greater sensitivity than Endomyocardial Biopsy for the diagnosis of myocarditis

Socio-economic status is inversely related to bed net use in Gabon

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Electron Tomography Reveals the Steps in Filovirus Budding

The filoviruses, Marburg and Ebola, are non-segmented negative-strand RNA viruses causing severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. The sequence of events that leads to release of filovirus particles from cells is poorly understood. Two contrasting mechanisms have been proposed, one proceeding via a “submarine-like” budding with the helical nucleocapsid emerging parallel to the plasma membrane, and the other via perpendicular “rocket-like” protrusion. Here we have infected cells with Marburg virus under BSL-4 containment conditions, and reconstructed the sequence of steps in the budding process in three dimensions using electron tomography of plastic-embedded cells. We find that highly infectious filamentous particles are released at early stages in infection. Budding proceeds via lateral association of intracellular nucleocapsid along its whole length with the plasma membrane, followed by rapid envelopment initiated at one end of the nucleocapsid, leading to a protruding intermediate. Scission results in local membrane instability at the rear of the virus. After prolonged infection, increased vesiculation of the plasma membrane correlates with changes in shape and infectivity of released viruses. Our observations demonstrate a cellular determinant of virus shape. They reconcile the contrasting models of filovirus budding and allow us to describe the sequence of events taking place during budding and release of Marburg virus. We propose that this represents a general sequence of events also followed by other filamentous and rod-shaped viruses