Structural changes enable start codon recognition by the eukaryotic translation initiation complex.

During eukaryotic translation initiation, initiator tRNA does not insert fully into the P decoding site on the 40S ribosomal subunit. This conformation (POUT) is compatible with scanning mRNA for the AUG start codon. Base pairing with AUG is thought to promote isomerization to a more stable conformation (PIN) that arrests scanning and promotes dissociation of eIF1 from the 40S subunit. Here, we present a cryoEM reconstruction of a yeast preinitiation complex at 4.0 Å resolution with initiator tRNA in the PIN state, prior to eIF1 release. The structure reveals stabilization of the codon-anticodon duplex by the N-terminal tail of eIF1A, changes in the structure of eIF1 likely instrumental in its subsequent release, and changes in the conformation of eIF2. The mRNA traverses the entire mRNA cleft and makes connections to the regulatory domain of eIF2?, eIF1A, and ribosomal elements that allow recognition of context nucleotides surrounding the AUG codon.

A Small Study on Clostridioides difficile in Spinach Field Soil and the Chemical and Microbial Factors that may Influence Prevalence.

Clostridioides difficile is a human pathogen that is ubiquitous in soil. Despite increasing infection rates and evidence of foodborne transmission, there is limited data on prevalence in soil or which factors influence persistence. The aim of this study was to investigate the prevalence of these bacteria in soil from three different spinach fields and to examine the chemical composition (carbon, organic carbon, nitrogen, organic matter, minerals and pH) and microbiota to gain insight into the factors that may promote/inhibit C. difficile. The overall C. difficile prevalence (10%) was lower than expected (based on international studies) and a significantly (P < 0.05) higher prevalence was obtained in Field 3 (20%) as compared to Fields 1 and 2 (5% each). Analysis of the soil suggested that the pH as well as organic matter, calcium and phosphorus content directly and indirectly (via the microbiota) influenced the prevalence of C. difficile in adjacent fields, where other factors (eg. climate) are similar. Although further studies are required to validate our findings, the data provides the first step in developing potential soil based control strategies.

The effect of cold storage and cooking on the viability of Clostridioides difficile spores in consumer foods.

The increased detection of clinical cases of Clostridioides difficile coupled with the persistence of clostridial spores at various stages along the food chain suggest that this pathogen may be foodborne. This study examined C. difficile (ribotypes 078 and 126) spore viability in chicken breast, beef steak, spinach leaves and cottage cheese during refrigerated (4 °C) and frozen (-20 °C) storage with and without a subsequent sous vide mild cooking (60 °C, 1 h). Spore inactivation at 80 °C in phosphate buffer solution, beef and chicken were also investigated to provide D80°C values and determine if PBS was a suitable model system for real food matrices. There was no decrease in spore concentration after chilled or frozen storage and/or sous vide cooking at 60 °C. Non-log-linear thermal inactivation was observed for both C. difficile ribotypes at 80 °C in phosphate buffer solution (PBS), beef and chicken. The predicted PBS D80°C values of 5.72±[2.90, 8.55] min and 7.50±[6.61, 8.39] min for RT078 and RT126, respectively, were in agreement with the food matrices D80°C values of 5.65 min (95% CI range from 4.29 to 8.89 min) for RT078 and 7.35 min (95% CI range from 6.81 to 7.01 min) for RT126. It was concluded that C. difficile spores survive chilled and frozen storage and mild cooking at 60 °C but may be inactivated at 80 °C. Moreover thermal inactivation in PBS was representative of that observed in real food matrices (beef and chicken).

Wide mutational analysis to ascertain the functional roles of eL33 in ribosome biogenesis and translation initiation.

An extensive mutational analysis of RPL33A, encoding the yeast ribosomal protein L33A (eL33) allowed us to identify several novel rpl33a mutants with different translational phenotypes. Most of the rpl33a mutants are defective in the processing of 35S and 27S pre-rRNA precursors and the production of mature rRNAs, exhibiting reductions in the amounts of ribosomal subunits and altered polysome profiles. Some of the rpl33a mutants exhibit a Gcd- phenotype of constitutive derepression of GCN4 translation and strong slow growth phenotypes at several temperatures. Interestingly, some of the later mutants also show a detectable increase in the UUG/AUG translation initiation ratio that can be suppressed by eIF1 overexpression, suggesting a requirement for eL33 and a correct 60S/40S subunit ratio for the proper recognition of the AUG start codon. In addition to producing differential reductions in the rates of pre-rRNA maturation and perhaps in r-protein assembly, most of the point rpl33a mutations alter specific molecular interactions of eL33 with the rRNAs and other r-proteins in the 60S structure. Thus, rpl33a mutations cause distinctive effects on the abundance and/or functionality of 60S subunits, leading to more or less pronounced defects in the rates and fidelity of mRNA translation.

The prevalence of Clostridioides difficile on farms, in abattoirs and in retail foods in Ireland.

An increasing proportion of Clostridioides difficile infections (CDI) are community acquired. This study tested farm, abattoir and retail food samples for C. difficile, using peer reviewed culture and molecular methods. The contamination rate on beef, sheep and broiler farms ranged from 2/30 (7%) to 25/30 (83%) in faeces, soil and water samples, while concentrations ranged from 2.9 log10 cfu/ml to 8.4 log10 cfu/g. The prevalence and associated counts were much lower in abattoir samples. Although 26/60 were C. difficile positive by enrichment and PCR, only 6 samples yielded counts by direct plating (1.1 log10 cfu/cm2 to 5.1 log10 cfu/g). At retail, 9/240 samples were C. difficile positive, including corned beef (1), spinach leaves (2), iceberg lettuce, little gem lettuce, wild rocket, coleslaw, whole milk yogurt and cottage cheese (1 sample each), with counts of up to 6.8 log10 cfu/g. The tcdA, tcdB, cdtA, cdtB, tcdC and tcdR genes were detected in 41%, 99.2%, 33.6%, 32%, 46.7% and 31.1%, respectively, of the 122 C. difficile isolates obtained. It was concluded that although the prevalence of C. difficile decreased along the food chain, retail foods were still heavily contaminated. This pathogen may therefore be foodborne, perhaps necessitating dietary advice for potentially vulnerable patients.

Pressurized Extraction as an Opportunity to Recover Antioxidants from Orange Peels: Heat treatment and Nanoemulsion Design for Modulating Oxidative Stress.

Orange peel by-products generated in the food industry are an important source of value-added compounds that can be potentially reused. In the current research, the effect of oven-drying (50-70 °C) and freeze-drying on the bioactive compounds and antioxidant potential from Navelina, Salustriana, and Sanguina peel waste was investigated using pressurized extraction (ASE). Sixty volatile components were identified by ASE-GC-MS. The levels of terpene derivatives (sesquitenenes, alcohols, aldehydes, hydrocarbons, and esters) remained practically unaffected among fresh and freeze-dried orange peels, whereas drying at 70 °C caused significative decreases in Navelina, Salustriana, and Sanguina peels. Hesperidin and narirutin were the main flavonoids quantified by HPLC-MS. Freeze-dried Sanguina peels showed the highest levels of total-polyphenols (113.3 mg GAE·g-1), total flavonoids (39.0 mg QE·g-1), outstanding values of hesperedin (187.6 µg·g-1), phenol acids (16.54 mg·g-1 DW), and the greatest antioxidant values (DPPH•, FRAP, and ABTS•+ assays) in comparison with oven-dried samples and the other varieties. Nanotechnology approaches allowed the formulation of antioxidant-loaded nanoemulsions, stabilized with lecithin, starting from orange peel extracts. Those provided 70-80% of protection against oxidative UV-radiation, also decreasing the ROS levels into the Caco-2 cells. Overall, pressurized extracts from freeze-drying orange peel can be considered a good source of natural antioxidants that could be exploited in food applications for the development of new products of commercial interest.

Pressurized liquid extraction to obtain chia seeds oils extracts enriched in tocochromanols. Nanoemulsions approaches to preserve the antioxidant potential.

The objective of this study was to use accelerated-solvent-extraction to achieve antioxidant extracts from chia seeds oils, enriched in tocopherols and tocotrienols, namely tocochromanols. Nanotechnology applications have been also incorporated to develop an innovative formulation of chia seeds oil nanoemulsion that preserve its antioxidant potential after conditions of oxidative stress. Chia seeds oils proved to be a valuable source of tocochromanols, from 568.84 to 855.98 µg g-1, depending on the geographical provenance. Quantitative data obtained by LC-DAD-ESI-MS/MS showed outstanding levels of γ-Tocopherol, over 83%, followed far behind by Tocopherols-(α, ß, δ) and Tocotrienols-(α, ß, δ, γ)-tocotrienols. The characteristic tocochromanols fingerprint of chia seeds oils was positively correlated with the FRAP and DPPH antioxidant activity of the extracts (between 18.81 and 138.48 mg Trolox/g). Formulation of the Chia seeds oils as nanoemulsions did not compromised the antioxidant properties of fresh extracts. Interestingly, nanoemulsions retained about the 80% of the initial antioxidant capacity after UV-induced stress, where the non-emulsified oils displayed a remarkable reduction (50-60%) on its antioxidant capacity under the same conditions. These antioxidant chia seeds formulations can constitute a promising strategy to vectorizing vitamin E isomers, in order to be used for food fortification, natural additives and to increase the self-life of food products during packing.

Enhanced eIF1 binding to the 40S ribosome impedes conformational rearrangements of the preinitiation complex and elevates initiation accuracy.

In the current model of translation initiation by the scanning mechanism, eIF1 promotes an open conformation of the 40S subunit competent for rapidly loading the eIF2·GTP·Met-tRNAi ternary complex (TC) in a metastable conformation (POUT) capable of sampling triplets entering the P site while blocking accommodation of Met-tRNAi in the PIN state and preventing completion of GTP hydrolysis (Pi release) by the TC. All of these functions should be reversed by eIF1 dissociation from the preinitiation complex (PIC) on AUG recognition. We tested this model by selecting eIF1 Ssu(-) mutations that suppress the elevated UUG initiation and reduced rate of TC loading in vivo conferred by an eIF1 (Sui(-)) substitution that eliminates a direct contact of eIF1 with the 40S subunit. Importantly, several Ssu(-) substitutions increase eIF1 affinity for 40S subunits in vitro, and the strongest-binding variant (D61G), predicted to eliminate ionic repulsion with 18S rRNA, both reduces the rate of eIF1 dissociation and destabilizes the PIN state of TC binding in reconstituted PICs harboring Sui(-) variants of eIF5 or eIF2. These findings establish that eIF1 dissociation from the 40S subunit is required for the PIN mode of TC binding and AUG recognition and that increasing eIF1 affinity for the 40S subunit increases initiation accuracy in vivo. Our results further demonstrate that the GTPase-activating protein eIF5 and ß-subunit of eIF2 promote accuracy by controlling eIF1 dissociation and the stability of TC binding to the PIC, beyond their roles in regulating GTP hydrolysis by eIF2.

Eukaryotic translation initiation factor eIF5 promotes the accuracy of start codon recognition by regulating Pi release and conformational transitions of the preinitiation complex.

eIF5 is the GTPase activating protein (GAP) for the eIF2 · GTP · Met-tRNAi (Met) ternary complex with a critical role in initiation codon selection. Previous work suggested that the eIF5 mutation G31R/SUI5 elevates initiation at UUG codons by increasing GAP function. Subsequent work implicated eIF5 in rearrangement of the preinitiation complex (PIC) from an open, scanning conformation to a closed state at AUG codons, from which Pi is released from eIF2 · GDP · Pi. To identify eIF5 functions crucial for accurate initiation, we investigated the consequences of G31R on GTP hydrolysis and Pi release, and the effects of intragenic G31R suppressors on these reactions, and on the partitioning of PICs between open and closed states. eIF5-G31R altered regulation of Pi release, accelerating it at UUG while decreasing it at AUG codons, consistent with its ability to stabilize the closed complex at UUG. Suppressor G62S mitigates both defects of G31R, accounting for its efficient suppression of UUG initiation in G31R,G62S cells; however suppressor M18V impairs GTP hydrolysis with little effect on PIC conformation. The strong defect in GTP hydrolysis conferred by M18V likely explains its broad suppression of Sui(-) mutations in numerous factors. We conclude that both of eIF5's functions, regulating Pi release and stabilizing the closed PIC conformation, contribute to stringent AUG selection in vivo.

ß-Hairpin loop of eukaryotic initiation factor 1 (eIF1) mediates 40 S ribosome binding to regulate initiator tRNA(Met) recruitment and accuracy of AUG selection in vivo.

Recognition of the translation initiation codon is thought to require dissociation of eIF1 from the 40 S ribosomal subunit, enabling irreversible GTP hydrolysis (Pi release) by the eIF2·GTP·Met-tRNAi ternary complex (TC), rearrangement of the 40 S subunit to a closed conformation incompatible with scanning, and stable binding of Met-tRNAi to the P site. The crystal structure of a Tetrahymena 40 S·eIF1 complex revealed several basic amino acids in eIF1 contacting 18 S rRNA, and we tested the prediction that their counterparts in yeast eIF1 are required to prevent premature eIF1 dissociation from scanning ribosomes at non-AUG triplets. Supporting this idea, substituting Lys-60 in helix α1, or either Lys-37 or Arg-33 in ß-hairpin loop-1, impairs binding of yeast eIF1 to 40 S·eIF1A complexes in vitro, and it confers increased initiation at UUG codons (Sui(-) phenotype) or lethality, in a manner suppressed by overexpressing the mutant proteins or by an eIF1A mutation (17-21) known to impede eIF1 dissociation in vitro. The eIF1 Sui(-) mutations also derepress translation of GCN4 mRNA, indicating impaired ternary complex loading, and this Gcd(-) phenotype is likewise suppressed by eIF1 overexpression or the 17-21 mutation. These findings indicate that direct contacts of eIF1 with 18 S rRNA seen in the Tetrahymena 40 S·eIF1 complex are crucial in yeast to stabilize the open conformation of the 40 S subunit and are required for rapid TC loading and ribosomal scanning and to impede rearrangement to the closed complex at non-AUG codons. Finally, we implicate the unstructured N-terminal tail of eIF1 in blocking rearrangement to the closed conformation in the scanning preinitiation complex.