Use of urinary gamma-glutamyl transferase (GGT) to monitor the pattern of proteinuria in dogs with leishmaniasis treated with N-methylglucamine antimoniate

The aim of this study was to assess if the coupled analysis of the urinary protein to creatinine (UPC) ratio and of the GGT/UC ratio (the ratio between urinary gamma-glutamyl transferase activity and urinary creatinine) may be used in treated leishmaniotic dogs to differentiate dogs with transient impairment of tubular function from dogs with persistent tubular damage. To this aim, 40 urine from 10 proteinuric and leishmaniotic dogs that at the first visit had high GGT/UC ratio, consistent with tubular damage, were collected and analyzed before treatments and 2, 4 and 6 weeks after treatment with N-methylglucamine antimoniate and allopurinol. Compared with pre-treatment values, at the end of the study period the UPC ratio decreased only in 5/10 dogs, which, however, were still proteinuric or borderline proteinuric. Conversely, the GGT/CU ratio decreased in 8/10 dogs and in 3 of them the values at the end of the study period were below the threshold consistent with tubular proteinuria. The GGT/UC values at 6 weeks was significantly lower than before treatment. However, transient increases were frequent for both the analytes. These results indicate that in most of the dogs that remain proteinuric after treatment, likely due to the persistent glomerular damage, the GGT/UC ratio tends to normalize. This suggests that in these dogs tubular proteinuria at admission depends on functional impairment of tubular cells likely due to the overflow of proteins from damaged glomeruli. However, tubular proteinuria occasionally persists, suggesting that tubulointerstitial damages persist even in dogs responsive to treatments

The CCA Anticodon Specifies Separate Functions Inside and Outside Translation in \u3cem\u3eBacillus cereus\u3c/em\u3e

Bacillus cereus 14579 encodes two tRNAs with the CCA anticodon, tRNATrp and tRNAOther. tRNATrp was separately aminoacylated by two enzymes, TrpRS1 and TrpRS2, which share only 34% similarity and display different catalytic capacities and specificities. TrpRS1 was 18-fold more proficient at aminoacylating tRNATrp with Trp, while TrpRS2 more efficiently utilizes the Trp analog 5-hydroxy Trp. tRNAOther was not aminoacylated by either TrpRS but instead by the combined activity of LysRS1 and LysRS2, which recognized sequence elements absent from tRNATrp. Polysomes were found to contain tRNATrp, consistent with its role in translation, but not tRNAOther suggesting a function outside protein synthesis. Regulation of the genes encoding TrpRS1 and TrpRS2 (trpS1 and trpS2) is dependent on riboswitch-mediated recognition of the CCA anticodon, and the role of tRNAOther in this process was investigated. Deletion of tRNAOther led to up to a 50 fold drop in trpS1 expression, which resulted in the loss of differential regulation of the trpS1 and trpS2 genes in stationary phase. These findings reveal that sequence-specific interactions with a tRNA anticodon can be confined to processes outside translation, suggesting a means by which such RNAs may evolve non-coding functions

Multiple Quality Control Pathways Limit Non-Protein Amino Acid Use by Yeast Cytoplasmic Phenylalanyl-tRNA Synthetase

Non-protein amino acids, particularly isomers of the proteinogenic amino acids, present a threat to proteome integrity if they are mistakenly inserted into proteins. Quality control during aminoacyl-tRNA synthesis reduces non-protein amino acid incorporation by both substrate discrimination and proofreading. For example phenylalanyl-tRNA synthetase (PheRS) proofreads the non-protein hydroxylated phenylalanine derivative m-Tyr after its attachment to tRNAPhe. We now show in Saccharomyces cerevisiae that PheRS misacylation of tRNAPhe with the more abundant Phe oxidation product o-Tyr is limited by kinetic discrimination against o-Tyr-AMP in the transfer step followed by o-Tyr-AMP release from the synthetic active site. This selective rejection of a non-protein aminoacyl-adenylate is in addition to known kinetic discrimination against certain non-cognates in the activation step as well as catalytic hydrolysis of mispaired aminoacyl-tRNAPhe species. We also report an unexpected resistance to cytotoxicity by a S. cerevisiae mutant with ablated post-transfer editing activity when supplemented with o-Tyr, cognate Phe, or Ala, the latter of which is not a substrate for activation by this enzyme. Our phenotypic, metabolomic, and kinetic analyses indicate at least three modes of discrimination against non-protein amino acids by S. cerevisiae PheRS and support a non-canonical role for SccytoPheRS post-transfer editing in response to amino acid stress

Divergence in Non-Cognate Amino Acid Recognition Between Class I and Class II Lysyl-tRNA Synthetases

Lysine insertion during coded protein synthesis requires lysyl-tRNALys, which is synthesized by lysyl-tRNA synthetase (LysRS). Two unrelated forms of LysRS are known: LysRS2, which is found in eukaryotes, most bacteria, and a few archaea, and LysRS1, which is found in most archaea and a few bacteria. To compare amino acid recognition between the two forms of LysRS, the effects of l-lysine analogues on aminoacylation were investigated. Both enzymes showed stereospecificity toward the l-enantiomer of lysine and discriminated against noncognate amino acids with different R-groups (arginine, ornithine). Lysine analogues containing substitutions at other positions were generally most effective as inhibitors of LysRS2. For example, theKi values for aminoacylation of S-(2-aminoethyl)-l-cysteine and l-lysinamide were over 180-fold lower with LysRS2 than with LysRS1. Of the other analogues tested, only γ-aminobutyric acid showed a significantly higherKi for LysRS2 than LysRS1. These data indicate that the lysine-binding site is more open in LysRS2 than in LysRS1, in agreement with previous structural studies. The physiological significance of divergent amino acid recognition was reflected by the in vivo resistance to growth inhibition imparted by LysRS1 against S-(2-aminoethyl)-l-cysteine and LysRS2 against γ-aminobutyric acid. These differences in resistance to naturally occurring noncognate amino acids suggest the distribution of LysRS1 and LysRS2 contributes to quality control during protein synthesis. In addition, the specific inhibition of LysRS1 indicates it is a potential drug target

Effects of industrial processing on pesticide multiresidues transfer from raw tomatoes to processed products

Pesticides are broadly used to improve food safety, although they can lead to adverse health effects on consumers. Various food processing approaches, at the industrial or domestic level, have been found to highly reduce the amount of pesticide residues in most food materials. In this work, samples of raw tomatoes were collected directly from the field and processed at the industrial level to produce purée, triple concentrated paste, fine pulp, and diced tomatoes. A multiresidue method based on a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged e Safe) sample preparation, followed by liquid chromatography‐tandem mass spectrometry analysis (LC‐MS/MS) for the assessment of 116 pesticides residues, was used. The analytical method has been validated according to SANTE indications. The recovery yields ranged from 75.5% to 115.3%, repeatability (RSDr) ranged from 3.4% to 18.3%, while reproducibility (RSDwR) ranged from 5.4% to 19.8%. The limit of quantifications (LOQs) ranged from 2.35 μg kg−1 for benthiavalicarb to 6.49 μg kg−1 for allethrin. A total of 159 raw tomato samples were collected from the field. The analysis showed the presence of 46 pesticides with azoxystrobin and chlorantraniliprole the most represented. On the other hand, all industrially processed samples showed values ≤ LOD, confirming that post‐harvest processes can lead to a decrease in pesticide residues from agricultural commodities

Effects of the functional Gpc-­B1 allele on soft durum wheat grain, milling, flour, dough, and breadmaking quality

Background and objectives: Utilization of durum wheat (Triticum turgidum subsp. durum) can be enhanced by increasing grain and flour protein content. One strategy to increase protein content is by introducing the functional Gpc-B1 allele from wild emmer (Triticum turgidum subsp. dicoccoides). Findings: Introduction of the functional Gpc-B1 allele into soft kernel durum increased grain and flour protein by 17 g/kg, increased dough strength as evidenced by SDS sedimentation volume and Mixograph dough mixing parameters, and increased straight-dough pan bread volume. When grown under arid conditions, high protein (151 g/kg) samples had decreased loaf volumes indicative of inelastic doughs. The functional Gpc-B1 allele was associated with decreased test weight, a small increase in SKCS hardness, and a modest increase in flour ash; otherwise, milling performance was not affected. Conclusions: Introgression of the Gpc-B1 functional allele from dicoccoides into durum wheat can improve dough strength and breadmaking quality. The effect tends to be consistent over environments but overall, Gpc-B1 made only a modest improvement in durum wheat breadmaking quality. Further studies with concomitant selection at other loci are needed to see the effects of Gpc-B1 among elite germplasm. Significance and novelty: Durum wheat production and consumption will increase as bread quality improves. The functional Gpc-B1 allele contributed to improved breadmaking quality. The present report is the first to examine the effect of this allele on breadmaking in durum wheat

Sodium-dodecylsulphate agarose gel electrophoresis (SDS-AGE) as a tool for monitoring the pattern of proteinuria in dogs with leishmaniasis

Dogs with leishmaniasis develop an immune-complex glomerulonephritis that suddenly induces functional or structural lesions in tubular cells. Leishmanicidal treatments should decrease immuno-complex formation and deposition. This can modify the composition of urinary proteins over time. The aim of this study was to assess whether sodium dodecyl sulphate-agarose gel electrophoresis (SDS-AGE), which differentiates urinary proteins based on their molecular weight (MW), may identify changes in the composition of urinary proteins associated with leishmanicidal treatments. Urine samples from 11 leishmaniotic dogs in IRIS stage I that were proteinuric (n = 10) or borderline proteinuric (n = 1) were collected before treatment and 2, 4 and 6 weeks after the beginning of treatment with meglumine antimoniate and allopurinol. The urinary protein to creatinine (UPC) ratio was measured just after collection and SDS-AGE was performed. Samples were classified as affected by glomerular or tubular proteinuria if at least two bands of MW higher or lower than that of albumin (69 kDa) were present\u37e mixed proteinuria was diagnosed when bands of MW higher and lower than that of albumin were present. All the dogs remained in IRIS stage I after treatment. Consistent with a previous report, the UPC ratio decreased in 6/11 dogs, remained unchanged in 2/11 dogs and increased in 3/11 dogs despite the amelioration of clinical signs, likely depending on the release of antigens, that form additional immune complexes after the death of the parasite. As expected, proteinuria before treatment was mixed in 7/11 cases, glomerular in 2/11 cases and tubular in 2/11 cases. In 3 dogs, mixed proteinuria persisted during the follow-up. In 4 dogs with mixed proteinuria, the samples collected after treatment became glomerular, tubular, or negative (despite a UPC ratio > 0.5)\u37e one dog with glomerular proteinuria and one dog with tubular proteinuria became negative and the others remained glomerular or tubular, respectively, after treatment. Pure tubular proteinuria or negative results in proteinuric dogs may depend on dilutional effects that do not allow detection of weak glomerular bands, on storage artifacts or on the presence of proteins from the genital tract. Apart from these artifacts, the pattern of proteinuria in dogs treated for leishmaniasis tends not to change over time. In conclusion, SDS-AGE may be influenced by preanalytical factors and does not provide reliable information during the follow-up of dogs treated for canine leishmaniasis

The Mechanism of β-N-methylamino-l-alanine Inhibition of tRNA Aminoacylation and Its Impact on Misincorporation

β-N-methylamino-l-alanine (BMAA) is a nonproteinogenic amino acid that has been associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Alzheimer\u27s disease (AD). BMAA has been found in human protein extracts; however, the mechanism by which it enters the proteome is still unclear. It has been suggested that BMAA is misincorporated at serine codons during protein synthesis, but direct evidence of its cotranslational incorporation is currently lacking. Here, using LC-MS–purified BMAA and several biochemical assays, we sought to determine whether any aminoacyl-tRNA synthetase (aaRS) utilizes BMAA as a substrate for aminoacylation. Despite BMAA\u27s previously predicted misincorporation at serine codons, following a screen for amino acid activation in ATP/PPi exchange assays, we observed that BMAA is not a substrate for human seryl-tRNA synthetase (SerRS). Instead, we observed that BMAA is a substrate for human alanyl-tRNA synthetase (AlaRS) and can form BMAA-tRNAAla by escaping from the intrinsic AlaRS proofreading activity. Furthermore, we found that BMAA inhibits both the cognate amino acid activation and the editing functions of AlaRS. Our results reveal that, in addition to being misincorporated during translation, BMAA may be able to disrupt the integrity of protein synthesis through multiple different mechanisms

A Pseudo-tRNA Modulates Antibiotic Resistance in \u3cem\u3eBacillus cereus\u3c/em\u3e

Bacterial genomic islands are often flanked by tRNA genes, which act as sites for the integration of foreign DNA into the host chromosome. For example, Bacillus cereus ATCC14579 contains a pathogenicity island flanked by a predicted pseudo-tRNA, tRNAOther, which does not function in translation. Deletion of tRNAOther led to significant changes in cell wall morphology and antibiotic resistance and was accompanied by changes in the expression of numerous genes involved in oxidative stress responses, several of which contain significant complementarities to sequences surrounding tRNAOther. This suggested that tRNAOther might be expressed as part of a larger RNA, and RACE analysis subsequently confirmed the existence of several RNA species that significantly extend both the 3′ and 5′-ends of tRNAOther. tRNAOther expression levels were found to be responsive to changes in extracellular iron concentration, consistent with the presence of three putative ferric uptake regulator (Fur) binding sites in the 5′ leader region of one of these larger RNAs. Taken together with previous data, this study now suggests that tRNAOther may function by providing a tRNA-like structural element within a larger regulatory RNA. These findings illustrate that while integration of genomic islands often leaves tRNA genes intact and functional, in other instances inactivation may generate tRNA-like elements that are then recruited to other functions in the cell