Availability of diagnostics and antifungals, and training in their use, will reduce deaths from advanced HIV disease (by up to 30%). 2 Mistaken diagnoses of pulmonary tuberculosis when actually the problem is a fungal lung infection will be averted. Implementation of these priorities will strengthen public health systems, support antimicrobial stewardship, 9 develop clinician skills, and appropriately diversify differential diagnosis. New approaches have to be explored, such as the implementation of artificial intelligence, to address the shortage of health-care workers in the Latin American and Caribbean region, Africa, and southeast Asia. We anticipate that the enhancement, innovation, and increased integration of fungal disease diagnosis and management within the health system will benefit not only those with fungal disease, but also improve the effectiveness, efficiency, and quality of the entire healthcare system.We declare no competing interests.

A system for testing the effects of specific codons on gene expression is described. Tandem test and control genes are contained in a transcription unit for bacteriophage T7 RNA polymerase in a multicopy plasmid, and nearly identical test and control mRNAs are generated from the primary transcript by RNase III cleavages.Their coding sequences, derived from T7 gene 9, are translated efficiently and have few low-usage codons of Escherichia coli. The upstream test gene contains a site for insertion of test codons, and the downstream control gene has a 45-codon deletion that allows test and control mRNAs and proteins to be separated by gel electrophoresis. Codons can be inserted among identical flanking codons after codon 13, 223, or 307 in codon test vectors pCT1, pCT2, and pCT3, respectively, the third site being six codons from the termination codon. The insertion of two to five consecutive AGG (low-usage) arginine codons selectively reduced the production of full-length test protein to extents that depended on the number of AGG codons, the site of insertion, and the amount of test mRNA. Production of aberrant proteins was also stimulated at high levels of mRNA. The effects occurred primarily at the translational level and were not produced by CGU (high-usage) arginine codons. Our results are consistent with the idea that sufficiently high levels of the AGG mRNA can cause essentially all of the tRNAAGG in the cell to become sequestered in translating peptidyl_tRNAAGG_mRNA-ribosome complexes stalled at the first of two consecutive AGG codons and that the approach of an upstream translating ribosome stimulates a stalled ribosome to frameshift, hop, or terminate translation.Most amino acids are encoded by more than one codon, and frequencies of use of synonymous codons tend to reflect the relative abundance of the tRNAs that recognize them (7,29). Synonymous codons may be translated at different rates (16,17) MATERUILS AND METHODSCodon test plasmids. Plasmids pCT1, pCT2, and pCT3 ( Fig. 1) were assembled from elements of pET vectors (13,24) and of T7 DNA (6). Elements were assembled by using a natural XbaI site (T'CTAGA, where the primer indicates the position of cleavage) between the 410 promoter and the slO translation initiation region for the gene 10 major capsid protein of T7, a natural NdeI site (CA'TATG) at the slO initiation codon, and newly introduced XbaI or NheI (G'CTAGC) sites at the ends of elements. Plasmids were assembled in modular fashion by fusions of compatible ends of DNA fragments from plasmids carrying individual elements or combinations of elements. Most of the fusions were between fragments that had one end at the PstI site in the on May 7, 2018 by guest

Homocysteine thiolactone is a product of an error-editing reaction, catalyzed by Escherrchiu colr and Saccharomyces cerevrsiae methionyl-tRNA synthetases, which prevents incorporation of homocysteine into tRNA and protein both in vitro and in vivo. Here, homocysteine thiolactone is also shown to be synthesized by cultured mammalian cells such as human cervical carcinoma (HeLa), mouse renal adenocarcinoma (RAG), and Chinese hamster ovary (CHO) cells labeled with ['5S]methionine, but not by normal human and mouse (Balblc 3T3) fibroblasts. A temperaturesensitive methionyl-tRNA synthetase mutant of CHO cells, Met-l, does not make the thiolactone at the non-permissive temperature. The data indicate that methionyl-tRNA synthetase is involved in synthesis of homocysteine thiolactone in CHO cells, thereby extending this important proofreading mechanism to mammalian cells.

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