Genetic analysis of translation initiation in bacteria: An initiator tRNA-centric view.

Translation of messenger RNA (mRNA) in bacteria occurs in the steps of initiation, elongation, termination, and ribosome recycling. The initiation step comprises multiple stages and uses a special transfer RNA (tRNA) called initiator tRNA (i-tRNA), which is first aminoacylated and then formylated using methionine and N10 -formyl-tetrahydrofolate (N10 -fTHF), respectively. Both methionine and N10 -fTHF are produced via one-carbon metabolism, linking translation initiation with active cellular metabolism. The fidelity of i-tRNA binding to the ribosomal peptidyl-site (P-site) is attributed to the structural features in its acceptor stem, and the highly conserved three consecutive G-C base pairs (3GC pairs) in the anticodon stem. The acceptor stem region is important in formylation of the amino acid attached to i-tRNA and in its initial binding to the P-site. And, the 3GC pairs are crucial in transiting the i-tRNA through various stages of initiation. We utilized the feature of 3GC pairs to investigate the nuanced layers of scrutiny that ensure fidelity of translation initiation through i-tRNA abundance and its interactions with the components of the translation apparatus. We discuss the importance of i-tRNA in the final stages of ribosome maturation, as also the roles of the Shine-Dalgarno sequence, ribosome heterogeneity, initiation factors, ribosome recycling factor, and coevolution of the translation apparatus in orchestrating a delicate balance between the fidelity of initiation and/or its leakiness to generate proteome plasticity in cells to confer growth fitness advantages in response to the dynamic nutritional states.

Use of DNA image cytometry in conducting oral cancer screening in rural India.

OBJECTIVES: Oral cancer screening can assist in the early detection of oral potentially malignant lesions (OPMLs) and prevention of oral cancers. It can be challenging for clinicians to differentiate OPMLs from benign conditions. Adjunct screening tools such as fluorescence visualisation (FV) and DNA image cytometry (DNA-ICM) have shown success in identifying OPMLs in high-risk clinics. For the first time we aimed to assess these technologies in Indian rural settings and evaluate if these tools helped clinicians identify high-risk lesions during screening. METHODS: Dental students and residents screened participants in five screening camps held in villages outside of Hyderabad, India, using extraoral, intraoral, and FV examinations. Lesion and normal tissue brushings were collected for DNA-ICM analysis and cytology. RESULTS: Of the 1116 participants screened, 184 lesions were observed in 152 participants. Based on white light examination (WLE), 45 lesions were recommended for biopsy. Thirty-five were completed on site; 25 (71%) were diagnosed with low-grade dysplasias (17 mild, 8 moderate) and the remaining 10 showed no signs of dysplasia. FV loss was noted in all but one dysplastic lesion and showed a sensitivity of 96% and specificity of 17%. Cytology combined with DNA-ICM had a sensitivity of 64% and specificity of 86% in detecting dysplasia. CONCLUSION: DNA-ICM combined with cytology identified the majority of dysplastic lesions and identified additional lesions, which were not considered high-risk during WLE and biopsy on site. Efforts to follow-up with these participants are ongoing. FV identified most high-risk lesions but added limited value over WLE.

Systematic evolution of initiation factor 3 and the ribosomal protein uS12 optimizes Escherichia coli growth with an unconventional initiator tRNA.

The anticodon stem of initiator tRNA (i-tRNA) possesses the characteristic three consecutive GC base pairs (G29:C41, G30:C40, and G31:C39 abbreviated as GC/GC/GC or 3GC pairs) crucial to commencing translation. To understand the importance of this highly conserved element, we isolated two fast-growing suppressors of Escherichia coli sustained solely on an unconventional i-tRNA (i-tRNAcg/GC/cg ) having cg/GC/cg sequence instead of the conventional GC/GC/GC. Both suppressors have the common mutation of V93A in initiation factor 3 (IF3), and additional mutations of either V32L (Sup-1) or H76L (Sup-2) in small subunit ribosomal protein 12 (uS12). The V93A mutation in IF3 was necessary for relaxed fidelity of i-tRNA selection to sustain on i-tRNAcg/GC/cg though with a retarded growth. Subsequent mutations in uS12 salvaged the retarded growth by enhancing the fidelity of translation. The H76L mutation in uS12 showed better fidelity of i-tRNA selection. However, the V32L mutation compensated for the deficient fidelity of i-tRNA selection by ensuring an efficient fidelity check by ribosome recycling factor (RRF). We reveal unique genetic networks between uS12, IF3 and i-tRNA in initiation and between uS12, elongation factor-G (EF-G), RRF, and Pth (peptidyl-tRNA hydrolase) which, taken together, govern the fidelity of translation in bacteria.

Predicting Progression of Low-Grade Oral Dysplasia Using Brushing-Based DNA Ploidy and Chromatin Organization Analysis.

Most oral cancers arise from oral potentially malignant lesions, which show varying grades of dysplasia. Risk of progression increases with increasing grade of dysplasia; however, risk prediction among oral low-grade dysplasia (LGD), that is, mild and moderate dysplasia can be challenging as only 5%-15% transform. Moreover, grading of dysplasia is subjective and varies with the area of the lesion being biopsied. To date, no biomarkers or tools are used clinically to triage oral LGDs. This study uses a combination of DNA ploidy and chromatin organization (CO) scores from cells obtained from lesion brushings to identify oral LGDs at high-risk of progression. A total of 130 lesion brushings from patients with oral LGDs were selected of which 16 (12.3%) lesions progressed to severe dysplasia or cancer. DNA ploidy and CO scores were analyzed from nuclear features measured by our in-house DNA image cytometry (DNA-ICM) system and used to classify brushings into low-risk and high-risk. A total of 57 samples were classified as high-risk of which 13 were progressors. High-risk DNA brushing was significant for progression (P = 0.001) and grade of dysplasia (P = 0.004). Multivariate analysis showed high-risk DNA brushing showed 5.1- to 8-fold increased risk of progression, a stronger predictor than dysplasia grading and lesion clinical features. DNA-ICM can serve as a non-invasive, high-throughput tool to identify high-risk lesions several years before transformation. This will help clinicians focus on such lesions whereas low-risk lesions may be spared from unnecessary biopsies.Prevention Relevance: DNA ploidy and chromatin organization of cells collected from oral potentially malignant lesions (OPMLs) can identify lesions at high-risk of progression several years prior. This non-invasive test would enable clinicians to triage high-risk (OPMLs) for closer follow-up while low-risk lesions can undergo less frequent biopsies reducing burden on healthcare resources.

Intraoral Photography Recommendations for Remote Risk Assessment and Monitoring of Oral Mucosal Lesions.

Oral cancer is a global health issue with substantial morbidity and a high mortality rate mainly because of late-stage diagnosis. Cancerous lesions are often preceded by potentially malignant lesions that may be detected during routine dental examinations. Not only is the oral cavity easily accessible for screening, but the clinical risk factors of the disease are also known. However, patients may not always be able to access screening services or receive follow-up for diagnosed lesions. In these circumstances, intraoral photos are crucial for timely triage, risk assessment, and monitoring of oral lesions. Further, photos form an integral part of a patient's records, facilitate patient education and communication between health care providers, and provide important information during the referral process. To ensure that intraoral photos are of good quality and standardised there is a need to establish recommendations regarding intraoral photography in oral mucosal screening. This article recommends methods to help health professionals and patients obtain interpretable intraoral photographs. Suggestions to achieve ideal lighting, mirror placement, camera angle, and retraction have been discussed. These recommendations are adaptable to easily available smartphone or point-and-shoot cameras and may be further used to develop future teledentistry platforms.

A mutation in the ribosomal protein uS12 reveals novel functions of its universally conserved PNSA loop.

The ribosomal protein uS12 is conserved across all domains of life. Recently, a heterozygous spontaneous mutation in human uS12 (corresponding to R49K mutation immediately downstream of the universally conserved 44 PNSA47 loop in Escherichia coli uS12) was identified for causing ribosomopathy, highlighting the importance of the PNSA loop. To investigate the effects of a similar mutation in the absence of any wild-type alleles, we mutated the rpsL gene (encoding uS12) in E. coli. Consistent with its pathology (in humans), we were unable to generate the R49K mutation in E. coli in the absence of a support plasmid. However, we were able to generate the L48K mutation in its immediate vicinity. The L48K mutation resulted in a cold sensitive phenotype and ribosome biogenesis defect in the strain. We show that the L48K mutation impacts the steps of initiation and elongation. Furthermore, the genetic interactions of the L48K mutation with RRF and Pth suggest a novel role of the PNSA loop in ribosome recycling. Our studies reveal new functions of the PNSA loop in uS12, which has so far been studied in the context of translation elongation.

The role of DNA image cytometry in screening oral potentially malignant lesions using brushings: A systematic review.

It is believed that the majority of oral cancers develop from oral potentially malignant lesions (OPML). Though they can be easily detected during screening, risk stratification is difficult. During screening clinicians often find it difficult to distinguish OPMLs from benign lesions, and predicting OPML at risk of malignant transformation is particularly challenging. DNA aneuploidy has been known to be a marker of malignancy in a number of sites including the oral cavity. We performed a systematic review to evaluate the effectiveness of DNA-ICM using brushings in differentiating OPMLs from benign/inflammatory lesions during screening and in predicting malignant transformation. MEDLINE, Pubmed, EMBASE electronic databases were systematically searched using a combination of keywords and subject headings. A total of 11 articles satisfied our inclusion criteria. These studies reported a wide range of sensitivity (16-96.4%) and specificity (90-100%) due to the differences in study design, definitions of high risk or low risk lesions and DNA-ICM protocol used. No long-term longitudinal studies were identified to assess the role of DNA-ICM using brushings in predicting malignant transformation. No studies evaluated the role of DNA-ICM in community screening settings. A number of studies combined DNA-ICM with other techniques like cytology or argyrophilic nucleolar organizer region counts leading to improved test results. In spite of DNA aneuploidy being accepted as a marker of malignancy, there is limited evidence of DNA-ICM using brushings being successful as an adjunct oral cancer screening tool. Longitudinal studies and large community screening studies need to be undertaken to draw stronger conclusion.

Development of mCherry tagged UdgX as a highly sensitive molecular probe for specific detection of uracils in DNA.

Uracil is not always a mistakenly occurring base in DNA. Uracils in DNA genomes are known to be important in the life cycles of Bacillus subtilis phages (PBS1/2) and the malarial parasite, Plasmodium falciparum; and have been implicated in the development of fruit fly and antibody maturation in B-lymphocytes. Availability of a sensitive, specific and robust technique for the detection uracils in genes/genomes is essential to understand its varied biological roles. Mycobacterium smegmatis UdgX (MsmUdgX), identified and characterised in our laboratory, forms covalent complexes with the uracil sites in DNA in a specific manner. MsmUdgX cleaves the glycosidic bond between uracil and the deoxyribose sugar in DNA to produce uracilate and oxocarbenium ions. The oxocarbenium ion is then captured into a covalent complex by the nucleophilic attack of a histidine side chain of MsmUdgX. Here, we describe the use of a fusion protein, mCherry tagged MsmUdgX (mChUdgX), which combines the property of MsmUdgX to covalently and specifically bind the uracil sites in the genome, with the sensitivity of fluorescent detection of mCherry as a reporter. We show that both the purified mChUdgX and the Escherichia coli cell-extracts overexpressing mChUdgX provide high sensitivity and specificity of detecting uracils in DNA.