Problems arising during translation of mRNAs lead to ribosome stalling and collisions that trigger a series of quality control events. However, the global cellular response to ribosome collisions has ...not been explored. Here, we uncover a function for ribosome collisions in signal transduction. Using translation elongation inhibitors and general cellular stress conditions, including amino acid starvation and UV irradiation, we show that ribosome collisions activate the stress-activated protein kinase (SAPK) and GCN2-mediated stress response pathways. We show that the MAPKKK ZAK functions as the sentinel for ribosome collisions and is required for immediate early activation of both SAPK (p38/JNK) and GCN2 signaling pathways. Selective ribosome profiling and biochemistry demonstrate that although ZAK generally associates with elongating ribosomes on polysomal mRNAs, it specifically auto-phosphorylates on the minimal unit of colliding ribosomes, the disome. Together, these results provide molecular insights into how perturbation of translational homeostasis regulates cell fate.
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•Ribosome collisions trigger the ribotoxic stress response•Ribosome collisions activate the GCN2-mediated integrated stress response•The MAPKKK ZAK⍺ functions as a sentinel for colliding ribosomes
Cellular stress, such as amino acid starvation and UV irradiation, causes widespread ribosome collisions. Colliding ribosomes serve as a platform that recruits ZAK⍺ and triggers two inter-related but distinct signaling pathways—the ribotoxic stress response and the integrated stress response—to regulate cell fate decisions.
Inhibitory codon pairs and poly(A) tracts within the translated mRNA cause ribosome stalling and reduce protein output. The molecular mechanisms that drive these stalling events, however, are still ...unknown. Here, we use a combination of in vitro biochemistry, ribosome profiling, and cryo‐EM to define molecular mechanisms that lead to these ribosome stalls. First, we use an in vitro reconstituted yeast translation system to demonstrate that inhibitory codon pairs slow elongation rates which are partially rescued by increased tRNA concentration or by an artificial tRNA not dependent on wobble base‐pairing. Ribosome profiling data extend these observations by revealing that paused ribosomes with empty A sites are enriched on these sequences. Cryo‐EM structures of stalled ribosomes provide a structural explanation for the observed effects by showing decoding‐incompatible conformations of mRNA in the A sites of all studied stall‐ and collision‐inducing sequences. Interestingly, in the case of poly(A) tracts, the inhibitory conformation of the mRNA in the A site involves a nucleotide stacking array. Together, these data demonstrate a novel mRNA‐induced mechanisms of translational stalling in eukaryotic ribosomes.
Synopsis
A combination of in vitro biochemistry, ribosome profiling, and cryo‐EM defines molecular events that lead to ribosome stalling at inhibitory codon combinations and poly(A) tracts, demonstrating novel mRNA‐induced mechanisms slowing translational elongation by eukaryotic ribosomes.
Inhibitory codon pairs and poly(A) tracts within mRNAs form decoding‐incompatible conformations in the decoding center of the ribosome.
Direct effects of these structures on decoding are documented by high‐resolution ribosome profiling, kinetic studies and detailed cryo‐EM structural analysis.
These data demonstrate a novel mRNA‐induced mechanism of translational stalling in eukaryotic ribosomes.
The cryo‐EM structure of poly(A)‐stalled disomes reveals a novel conformation for collided ribosomes that rationalizes translational frameshifting.
A combination of in vitro biochemistry, ribosome profiling, and cryo‐EM reveals that elongation‐slowing mRNA elements stall ribosomes in decoding‐incompatible RNA conformations in the ribosomal decoding center.
Ribosomes undergo substantial conformational changes during translation elongation to accommodate incoming aminoacyl-tRNAs and translocate along the mRNA template. We used multiple elongation ...inhibitors and chemical probing to define ribosome conformational states corresponding to differently sized ribosome-protected mRNA fragments (RPFs) generated by ribosome profiling. We show, using various genetic and environmental perturbations, that short 20–22 or classical 27–29 nucleotide RPFs correspond to ribosomes with open or occupied ribosomal A sites, respectively. These distinct states of translation elongation are readily discerned by ribosome profiling in all eukaryotes we tested, including fungi, worms, and mammals. This high-resolution ribosome profiling approach reveals mechanisms of translation-elongation arrest during distinct stress conditions. Hyperosmotic stress inhibits translocation through Rck2-dependent eEF2 phosphorylation, whereas oxidative stress traps ribosomes in a pre-translocation state, independent of Rck2-driven eEF2 phosphorylation. These results provide insights and approaches for defining the molecular events that impact translation elongation throughout biology.
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•High-resolution ribosome profiling resolves distinct ribosome functional states•Codon occupancy of decoding ribosomes is highly anti-correlated with tRNA abundance•Cellular stress conditions trigger distinct paths of elongation regulation
Cellular stress triggers the regulation of protein synthesis at the levels of initiation and elongation. Wu et al. developed high-resolution ribosome profiling methodology to resolve ribosome functional states during translation elongation and revealed distinct mechanisms of elongation arrest in response to various stress conditions.
The eukaryotic translation factor eIF5A, originally identified as an initiation factor, was later shown to promote translation elongation of iterated proline sequences. Using a combination of ...ribosome profiling and in vitro biochemistry, we report a much broader role for eIF5A in elongation and uncover a critical function for eIF5A in termination. Ribosome profiling of an eIF5A-depleted strain reveals a global elongation defect, with abundant ribosomes stalling at many sequences, not limited to proline stretches. Our data also show ribosome accumulation at stop codons and in the 3′ UTR, suggesting a global defect in termination in the absence of eIF5A. Using an in vitro reconstituted translation system, we find that eIF5A strongly promotes the translation of the stalling sequences identified by profiling and increases the rate of peptidyl-tRNA hydrolysis more than 17-fold. We conclude that eIF5A functions broadly in elongation and termination, rationalizing its high cellular abundance and essential nature.
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•Depletion of eIF5A leads to global translation elongation and termination defects•eIF5A alleviates stalling on many motifs besides polyproline tracts•eIF5A stimulates eRF1-mediated peptidyl-tRNA hydrolysis in translation termination
In this manuscript, Schuller et al. characterize the activity of the translation factor eIF5A. They show that eIF5A accelerates peptidyl transfer at most, if not all, sequences and promotes peptide release. These expanded roles for eIF5A help to explain its essential nature and high abundance in eukaryotes.
The gut microbiota plays a role in nonalcoholic fatty liver disease (NAFLD), but data about gut dysbiosis in Asians with NAFLD remains scarce. We analyzed the differences in fecal microbiota between ...adults with and without NAFLD. This cross-sectional study examined adults with histology-proven NAFLD (25 nonalcoholic fatty liver (NAFL) patients, 25 nonalcoholic steatohepatitis (NASH) patients, and 25 living liver donors (healthy controls)). The taxonomic composition of the gut microbiota was determined by 16S ribosomal RNA gene sequencing of stool samples. The NAFL and NASH groups showed lower total bacterial diversity and richness than the controls. NAFLD patients had higher levels of the phylum Bacteroidetes and lower levels of Firmicutes than controls. The genus
, family
, order
, and class
were less abundant in patients with NAFL or NASH than healthy individuals. The lipopolysaccharide biosynthesis pathway was differentially enriched in the NASH group. This study examined the largest number of Asian patients with biopsy-proven NAFL and NASH in terms of dysbiosis of the gut microbiota in NAFLD patients. NAFLD patients had higher levels of Bacteroidetes and lower levels of Firmicutes
These results are different from research from western countries and could provide different targets for therapies by region.
Resveratrol (
-3,5,4'-trihydroxystibene, RSV) is a kind of polyphenol which has anti-inflammatory, antioxidant, anti-allergy, and anti-cancer properties, as well as being a scavenger of free radicals ...and preventing cardiovascular diseases. However, it is quite unstable in light, heat, and other conditions, and decays easily due to environmental factors. For these reasons, this study used a new type of carrier, transfersome, to encapsulate RSV. Transfersome consists of phosphatidyl choline (PC) from a liposomal system and non-ionic edge activators (EA). EA are an important ingredient in the formulation of transfersome; they can enhance the flexibility of the lipid bimolecular membrane of transfersome. Due to its ultradeformability, it also allows drugs to penetrate the skin, even through the stratum corneum. We hope that this new encapsulation technique will improve the stability and enhance the permeability of RSV. Concluding all the tested parameters, the best production condition was 5% PC/EA (3:1) and 5% ethanol in distilled water, with an ultrasonic bath and stirring at 500 rpm, followed by high pressure homogenization. The optimal particle size was 40.13 ± 0.51 nm and the entrapment efficiency (EE) was 59.93 ± 0.99%. The results of antioxidant activity analysis showed that transfersomes were comparable to the RSV group (unencapsulated). During in vitro transdermal delivery analysis, after 6 h, D1-20(W) increased 27.59% by accumulation. Cell viability assay showed that the cytotoxicity of D3-80(W) was reduced by 34.45% compared with the same concentration of RSV. Therefore, we successfully prepared RSV transfersomes and also improved the stability, solubility, and safety of RSV.
The adaptation process for first-year medical students is an important problem because it significantly affects educational activities. The previous study showed that 63% of students had difficulties ...adapting to the learning process in their first year at medical school. Therefore, students need the most suitable learning style to support the educational process, such as Problem-based learning (PBL). This method can improve critical thinking skills, problem-solving and self-directed learning. Although PBL has been adopted in medical education, the effectiveness of PBL in first-year medical students is still not yet clear. The purpose of this meta-analysis is to verify whether the PBL approach has a positive effect in improving knowledge, problem-solving and self-directed learning in first-year medical students compared with the conventional method.
We searched PubMed, ScienceDirect, Cochrane, and Google Scholar databases until June 5, 2021. Search terms included problem-based learning, effectiveness, effectivity, and medical student. We excluded studies with the final-year medical student populations. All analyses in our study were carried out using Review Manager version 5.3 (RevMan Cochrane, London, UK).
Seven eligible studies (622 patients) were included. The pooled analysis demonstrated no significant difference between PBL with conventional learning method in critical thinking/knowledge assessment (p = 0.29), problem-solving aspect (p = 0.47), and self-directed learning aspect (p = 0.34).
The present study concluded that the PBL approach in first-year medical students appeared to be ineffective in improving critical thinking/knowledge, problem-solving, and self-directed compared with the conventional teaching method.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
It is discovered that the memory‐type behaviors of novel carbohydrate‐block‐polyisoprene (MH‐b‐PI) block copolymers‐based devices, including write‐once‐read‐many‐times, Flash, and ...dynamic‐random‐access‐memory, can be easily controlled by the self‐assembly nanostructures (vertical cylinder, horizontal cylinder, and order‐packed sphere), in which the MH and PI blocks, respectively, provide the charge‐trapping and stretchable function. With increasing the flexible PI block length, the stretchability of the designed copolymers can be significantly improved up to 100% without forming cracks. Thus, intrinsically stretchable resistive memory devices (polydimethylsiloxane(PDMS)/carbon nanotubes(CNTs)/MH‐b‐PI thin film/Al) using the MH‐b‐PI thin film as an active layer is successfully fabricated and that using the MH‐b‐PI12.6k under 100% strain exhibits an excellent ON/OFF current ratio of over 106 (reading at −1 V) with stable V
set around −2 V. Furthermore, the endurance characteristics can be maintained over 500 cycles upon 40% strain. This work establishes and represents a novel avenue for the design of green carbohydrate‐derived and stretchable memory materials.
Novel stretchable block copolymers, carbohydrate‐block‐polyisoprene (MH‐b‐PI), designed for developing fully stretchable resistive memory, are demonstrated. Diverse self‐assembly nanostructures with respect to the ratio of charge‐trapping MH to stretchable PI can exhibit different memory behaviors. The polymer with longer PI, MH‐b‐PI12.6k, shows an excellent ON/OFF ratio of over 106 upon 100% stretching and the endurance characteristics can be maintained over 500 cycles.
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