RNA-binding proteins in yeast mitochondria

This work focused on the further characterisation of Idhp and of the Krebs cycle enzymes citrate synthase 1 (Cit1p) and malate dehydrogenase 1 (Mdh1p) both of which have been identified as RNA-binding proteins without known RNA recognition motifs. Besides analysing their effects on mitochondrial translation and their organisation in protein complexes the work focused on the characterisation of the RNA-binding properties of recombinant Cit1p and Mdh1p: · Cit1p and Mdh1p play no essential role in mitochondrial protein synthesis. · Idhp is in a complex of molecular weight larger than the cytochrome c oxidase (250 kDa). · Cit1p and Mdh1p are in mitochondrial complexes smaller than 250 kDa. · 1000-fold molar excess of tRNA referring to COX2 leader RNA did not inhibit the RNA-binding of Cit1p and Mdh1p. · Cit1p and Mdh1p bind mitochondrial mRNAs (sense and antisense). The influence of cofactors and substrates on RNA-binding was analysed in order to reveal a possible link between the enzymatic function and the property of RNA-binding: · Acetyl-CoA and ATP inhibited the RNA-binding of Cit1p and Mdh1p at a concentration of 5 mM

Specialization of the Drosophila nuclear export family protein Nxf3 for piRNA precursor export.

The PIWI-interacting RNA (piRNA) pathway is a conserved small RNA-based immune system that protects animal germ cell genomes from the harmful effects of transposon mobilization. In Drosophila ovaries, most piRNAs originate from dual-strand clusters, which generate piRNAs from both genomic strands. Dual-strand clusters use noncanonical transcription mechanisms. Although transcribed by RNA polymerase II, cluster transcripts lack splicing signatures and poly(A) tails. mRNA processing is important for general mRNA export mediated by nuclear export factor 1 (Nxf1). Although UAP56, a component of the transcription and export complex, has been implicated in piRNA precursor export, it remains unknown how dual-strand cluster transcripts are specifically targeted for piRNA biogenesis by export from the nucleus to cytoplasmic processing centers. Here we report that dual-strand cluster transcript export requires CG13741/Bootlegger and the Drosophila nuclear export factor family protein Nxf3. Bootlegger is specifically recruited to piRNA clusters and in turn brings Nxf3. We found that Nxf3 specifically binds to piRNA precursors and is essential for their export to piRNA biogenesis sites, a process that is critical for germline transposon silencing. Our data shed light on how dual-strand clusters compensate for a lack of canonical features of mature mRNAs to be specifically exported via Nxf3, ensuring proper piRNA production

Magnetic Sample Environment for in situ SAXS WAXS Measurements on Magnetic Nanoparticles with Shape Anisotropy

A vacuum compatible magnetic sample environment has been developed and installed at the four crystal monochromator beamline of the Physikalisch Technische Bundesanstalt PTB at the synchrotron radiation facility BESSY II in Berlin, Germany. The design is based on a water cooled electromagnetic coil setup and is aimed to provide a magnetic flux density of up to 900 mT at the sample position. The magnetic field is applied in order to align or arrange magnetic nanoparticles which can then be measured using small angle X ray scattering SAXS and wide angle X ray scattering WAXS . This can be beneficial in the analysis of particles with arbitrary shape. The corresponding scattering patterns are collected as 2D images on vacuum compatible variants of the PILATUS 1M and PILATUS 100K detector

The metalloprotein YhcH is an anomerase providing N-acetylneuraminate aldolase with the open form of its substrate

N-acetylneuraminate (Neu5Ac), an abundant sugar present in glycans in vertebrates and some bacteria, can be used as an energy source by several prokaryotes, including Escherichia coli. In solution, more than 99% of Neu5Ac is in cyclic form (≈92% beta-anomer and ≈7% alpha-anomer), whereas <0.5% is in the open form. The aldolase that initiates Neu5Ac metabolism in E. coli, NanA, has been reported to act on the alphaanomer. Surprisingly, when we performed this reaction at pH 6 to minimize spontaneous anomerization, we found NanA and its human homolog NPL preferentially metabolize the open form of this substrate. We tested whether the E. coli Neu5Ac anomerase NanM could promote turnover, finding it stimulated the utilization of both beta and alpha-anomers by NanA in vitro. However, NanM is localized in the periplasmic space and cannot facilitate Neu5Ac metabolism by NanA in the cytoplasm in vivo. We discovered that YhcH, a cytoplasmic protein encoded by many Neu5Ac catabolic operons and belonging to a protein family of unknown function (DUF386), also facilitated Neu5Ac utilization by NanA and NPL and displayed Neu5Ac anomerase activity in vitro. YhcH contains Zn, and its accelerating effect on the aldolase reaction was inhibited by metal chelators. Remarkably, several transition metals accelerated Neu5Ac anomerization in the absence of enzyme. Experiments with E. coli mutants indicated that YhcH expression provides a selective advantage for growth on Neu5Ac. In conclusion, YhcH plays the unprecedented role of providing an aldolase with the preferred unstable open form of its substrate

Extending the Traditional Classroom Through Online Discussion: The Role of Student Motivation

Two studies addressed students' motivation and participation in an online discussion board which was part of a traditional lecture-based course. The discussion board represented an external communication resource to extend the learning activities beyond the classroom. Self-Determination Theory was used as the theoretical framework to investigate: (a) how students' participation in online discussion related to their intrinsic motivation and attitude toward the class; b) how students' intrinsic motivation changed over time; and c) what factors students noted as responsible for the changes in their motivation level. A total of 123 undergraduate students participated in online discussion as a normal part of their instructional technology class. The results showed that students' participation was related to their intrinsic motivation, but not to their computer/internet skills. Over time, students' intrinsic motivation for participating in online discussion dropped steadily. Student-reported reasons for the motivation changes are discussed.Yeshttps://us.sagepub.com/en-us/nam/manuscript-submission-guideline

Poly ionic liquid nanovesicles via polymerization induced self assembly and their stabilization of Cu nanoparticles for tailored CO2 electroreduction

Herein, we report a straightforward, scalable synthetic route towards poly ionic liquid PIL homopolymer nanovesicles NVs with a tunable particle size of 50 to 120 nm and a shell thickness of 15 to 60 nm via one step free radical polymerization induced self assembly. By increasing monomer concentration for polymerization, their nanoscopic morphology can evolve from hollow NVs to dense spheres, and finally to directional worms, in which a multilamellar packing of PIL chains occurred in all samples. The transformation mechanism of NVs internal morphology is studied in detail by coarse grained simulations, revealing a correlation between the PIL chain length and the shell thickness of NVs. To explore their potential applications, PIL NVs with varied shell thickness are in situ functionalized with ultra small 1 amp; 8764; 3 nm in size copper nanoparticles CuNPs and employed as electrocatalysts for CO2 electroreduction. The composite electrocatalysts exhibit a 2.5 fold enhancement in selectivity towards C1 products e.g., CH4 , compared to the pristine CuNPs. This enhancement is attributed to the strong electronic interactions between the CuNPs and the surface functionalities of PIL NVs. This study casts new aspects on using nanostructured PILs as new electrocatalyst supports in CO2 conversion to C1 product

Genetic and Chemical Evaluation of Trypanosoma brucei Oleate Desaturase as a Candidate Drug Target

Background: Trypanosomes can synthesize polyunsaturated fatty acids. Previously, we have shown that they possess stearoyl-CoA desaturase (SCD) and oleate desaturase (OD) to convert stearate (C18) into oleate (C18:1) and linoleate (C18:2), respectively. Here we examine if OD is essential to these parasites. Methodology: Cultured procyclic (insect-stage) form (PCF) and bloodstream-form (BSF) Trypanosoma brucei cells were treated with 12- and 13-thiastearic acid (12-TS and 13-TS), inhibitors of OD, and the expression of the enzyme was knocked down by RNA interference. The phenotype of these cells was studied. Principal Findings: Growth of PCF T. brucei was totally inhibited by 100 mM of 12-TS and 13-TS, with EC50 values of 4062 and 3062 mM, respectively. The BSF was more sensitive, with EC50 values of 763 and 261 mM, respectively. This growth phenotype was due to the inhibitory effect of thiastearates on OD and, to a lesser extent, on SCD. The enzyme inhibition caused a drop in total unsaturated fatty-acid level of the cells, with a slight increase in oleate but a drastic decrease in linoleate level, most probably affecting membrane fluidity. After knocking down OD expression in PCF, the linoleate content was notably reduced, whereas that of oleate drastically increased, maintaining the total unsaturated fatty-acid level unchanged. Interestingly, the growth phenotype of the RNAi-induced cells was similar to that found for thiastearate-treated trypanosomes, with the former cells growing twofold slower than the latter ones, indicating that the linoleate content itsel

Poly(ionic liquid) nanovesicles via polymerization induced self-assembly and their stabilization of Cu nanoparticles for tailored CO2 electroreduction

Herein, we report a straightforward, scalable synthetic route towards poly(ionic liquid) (PIL) homopolymer nanovesicles (NVs) with a tunable particle size of 50 to 120 nm and a shell thickness of 15 to 60 nm via one-step free radical polymerization induced self-assembly. By increasing monomer concentration for polymerization, their nanoscopic morphology can evolve from hollow NVs to dense spheres, and finally to directional worms, in which a multilamellar packing of PIL chains occurred in all samples. The transformation mechanism of NVs’ internal morphology is studied in detail by coarse-grained simulations, revealing a correlation between the PIL chain length and the shell thickness of NVs. To explore their potential applications, PIL NVs with varied shell thickness are in situ functionalized with ultra-small (1 ∼ 3 nm in size) copper nanoparticles (CuNPs) and employed as electrocatalysts for CO2 electroreduction. The composite electrocatalysts exhibit a 2.5-fold enhancement in selectivity towards C1 products (e.g., CH4), compared to the pristine CuNPs. This enhancement is attributed to the strong electronic interactions between the CuNPs and the surface functionalities of PIL NVs. This study casts new aspects on using nanostructured PILs as new electrocatalyst supports in CO2 conversion to C1 products