Cooperation of regulatory RNA and the RNA degradosome in transcript surveillance.

The ompD transcript, encoding an outer membrane porin in Salmonella, harbors a controlling element in its coding region that base-pairs imperfectly with a 'seed' region of the small regulatory RNA (sRNA) MicC. When tagged with the sRNA, the ompD mRNA is cleaved downstream of the pairing site by the conserved endoribonuclease RNase E, leading to transcript destruction. We observe that the sRNA-induced cleavage site is accessible to RNase E in vitro upon recruitment of ompD into the 30S translation pre-initiation complex (PIC) in the presence of the degradosome components. Evaluation of substrate accessibility suggests that the paused 30S PIC presents the mRNA for targeted recognition and degradation. Ribonuclease activity on PIC-bound ompD is critically dependent on the recruitment of RNase E into the multi-enzyme RNA degradosome, and our data suggest a process of substrate capture and handover to catalytic sites within the degradosome, in which sequential steps of seed matching and duplex remodelling contribute to cleavage efficiency. Our findings support a putative mechanism of surveillance at translation that potentially terminates gene expression efficiently and rapidly in response to signals provided by regulatory RNA.

Functions of unconventional mammalian translational GTPases GTPBP1 and GTPBP2.

GTP-binding protein 1 (GTPBP1) and GTPBP2 comprise a divergent group of translational GTPases with obscure functions, which are most closely related to eEF1A, eRF3, and Hbs1. Although recent reports implicated GTPBPs in mRNA surveillance and ribosome-associated quality control, how they perform these functions remains unknown. Here, we demonstrate that GTPBP1 possesses eEF1A-like elongation activity, delivering cognate aminoacyl-transfer RNA (aa-tRNA) to the ribosomal A site in a GTP-dependent manner. It also stimulates exosomal degradation of mRNAs in elongation complexes. The kinetics of GTPBP1-mediated elongation argues against its functioning in elongation per se but supports involvement in mRNA surveillance. Thus, GTP hydrolysis by GTPBP1 is not followed by rapid peptide bond formation, suggesting that after hydrolysis, GTPBP1 retains aa-tRNA, delaying its accommodation in the A site. In physiological settings, this would cause ribosome stalling, enabling GTPBP1 to elicit quality control programs; e.g., by recruiting the exosome. GTPBP1 can also deliver deacylated tRNA to the A site, indicating that it might function via interaction with deacylated tRNA, which accumulates during stresses. Although GTPBP2's binding to GTP was stimulated by Phe-tRNAPhe, suggesting that its function might also involve interaction with aa-tRNA, GTPBP2 lacked elongation activity and did not stimulate exosomal degradation, indicating that GTPBP1 and GTPBP2 have different functions.

Investigating age and ethnicity as novel high-risk phenotypes in mucinous ovarian cancer: retrospective study in a multi-ethnic population.

OBJECTIVES: Primary mucinous ovarian carcinoma represents 3% of ovarian cancers and is typically diagnosed early, yielding favorable outcomes. This study aims to identify risk factors, focussing on the impact of age and ethnicity on survival from primary mucinous ovarian cancer. METHODS: A retrospective observational study of patients treated at Sandwell and West Birmingham Hospitals NHS Trust and University Hospital Coventry and Warwickshire. Patients included were women aged ≥16 years, with primary mucinous ovarian cancer confirmed by specialist gynecological histopathologist and tumor immunohistochemistry, including cytokeratin-7, cytokeratin-20, and CDX2. Statistical analyses were performed using R integrated development environment, with survival assessed by Cox proportional hazards models and Kaplan-Meier plots. RESULTS: A total of 163 patients were analyzed; median age at diagnosis was 58 years (range 16-92), 145 (89%) were International Federation of Gynecology and Obstetrics stage I and 43 (26%) patients had infiltrative invasion. Women aged ≤45 years were more likely to have infiltrative invasion (RR=1.38, 95% CI 0.78 to 2.46), with increased risk of death associated with infiltrative invasion (HR=2.29, 95% CI 1.37 to 5.83). Compared with White counterparts, South Asian women were more likely to undergo fertility-sparing surgery (RR=3.52, 95% CI 1.48 to 8.32), and have infiltrative invasion (RR=1.25, 95% CI 0.60 to 2.58). South Asian women undergoing fertility-sparing surgery had worse prognosis than those undergoing traditional staging surgery (HR=2.20, 95% CI 0.39 to 13.14). In FIGO stage I disease, 59% South Asian and 37% White women received adjuvant chemotherapy (p=0.06). South Asian women exhibited a worse overall prognosis than White women (HR=2.07, 95% CI 0.86 to 4.36), particularly pronounced in those aged ≤45 years (HR=8.75, 95% CI 1.22 to 76.38). CONCLUSION: This study identified young age as a risk factor for diagnosis of infiltrative invasion. Fertility-sparing surgery in South Asian women is a risk factor for poorer prognosis. South Asian women exhibit poorer overall survival than their White counterparts.

Conformational rearrangements upon start codon recognition in human 48S translation initiation complex.

Selection of the translation start codon is a key step during protein synthesis in human cells. We obtained cryo-EM structures of human 48S initiation complexes and characterized the intermediates of codon recognition by kinetic methods using eIF1A as a reporter. Both approaches capture two distinct ribosome populations formed on an mRNA with a cognate AUG codon in the presence of eIF1, eIF1A, eIF2-GTP-Met-tRNAiMet and eIF3. The 'open' 40S subunit conformation differs from the human 48S scanning complex and represents an intermediate preceding the codon recognition step. The 'closed' form is similar to reported structures of complexes from yeast and mammals formed upon codon recognition, except for the orientation of eIF1A, which is unique in our structure. Kinetic experiments show how various initiation factors mediate the population distribution of open and closed conformations until 60S subunit docking. Our results provide insights into the timing and structure of human translation initiation intermediates and suggest the differences in the mechanisms of start codon selection between mammals and yeast.

Modulation of HIV-1 Gag/Gag-Pol frameshifting by tRNA abundance.

A hallmark of translation in human immunodeficiency virus type 1 (HIV-1) is a -1 programmed ribosome frameshifting event that produces the Gag-Pol fusion polyprotein. The constant Gag to Gag-Pol ratio is essential for the virion structure and infectivity. Here we show that the frameshifting efficiency is modulated by Leu-tRNALeu that reads the UUA codon at the mRNA slippery site. This tRNALeu isoacceptor is particularly rare in human cell lines derived from T-lymphocytes, the cells that are targeted by HIV-1. When UUA decoding is delayed, the frameshifting follows an alternative route, which maintains the Gag to Gag-Pol ratio constant. A second potential slippery site downstream of the first one is normally inefficient but can also support -1-frameshifting when altered by a compensatory resistance mutation in response to current antiviral drug therapy. Together these different regimes allow the virus to maintain a constant -1-frameshifting efficiency to ensure successful virus propagation.

Directional transition from initiation to elongation in bacterial translation.

The transition of the 30S initiation complex (IC) to the translating 70S ribosome after 50S subunit joining provides an important checkpoint for mRNA selection during translation in bacteria. Here, we study the timing and control of reactions that occur during 70S IC formation by rapid kinetic techniques, using a toolbox of fluorescence-labeled translation components. We present a kinetic model based on global fitting of time courses obtained with eight different reporters at increasing concentrations of 50S subunits. IF1 and IF3 together affect the kinetics of subunit joining, but do not alter the elemental rates of subsequent steps of 70S IC maturation. After 50S subunit joining, IF2-dependent reactions take place independent of the presence of IF1 or IF3. GTP hydrolysis triggers the efficient dissociation of fMet-tRNA(fMet) from IF2 and promotes the dissociation of IF2 and IF1 from the 70S IC, but does not affect IF3. The presence of non-hydrolyzable GTP analogs shifts the equilibrium towards a stable 70S-mRNA-IF1-IF2-fMet-tRNA(fMet) complex. Our kinetic analysis reveals the molecular choreography of the late stages in translation initiation.

Fit-for-purpose validation of a drug-tolerant immunogenicity assay for a human mAb drug in animal safety studies.

A modified biotin-drug extraction and acid dissociation (BEAD) immunogenicity assay was developed to detect anti-drug-antibodies (ADA) against the human anti-FXIIa monoclonal antibody (mAb) drug, Garadacimab (previously called CSL312). Multiple strategies were tested to optimize the signal-to-background (S/B) ratio, assay sensitivity and the drug tolerance. The modified BEAD assay was found to be highly drug tolerant (>500 µg/ml) with a sensitivity of 100 ng/ml, in line with current FDA regulatory guidelines. The assay was validated for use in a repeat-dose animal safety study and showed an acceptable intra-assay precision and robustness but a lower inter-assay precision. In-study sample analysis confirmed that the assay was fit-for-purpose (FFP) for the context-of-use (COU) in the nonclinical study and the results obtained were deemed meaningful.

Emotional well-being of dentists and the effect of lockdown during the COVID-19 pandemic: A nationwide study.

BACKGROUND: Lockdown in India might have adverse effects on the emotional health of the dental professionals; hence, the aim of the present study was to determine the impact of various factors on emotional well-being of dental professionals due to the effect of lockdown during COVID-19. MATERIALS AND METHODS: It was a cross-sectional, descriptive, questionnaire study conducted on dentists in India during April 2020. Sampling technique was convenient sampling, and sampling frame was all the dental professionals in India. The questionnaire was prepared online using Google Forms, and the link was generated which was shared among as many dentists accessible as possible, with the help of message, mails, and WhatsApp. The questionnaire consists of questions on Demographic details, practice, and emotional well-being. A total of 1844 dental professionals in 14 states in India responded. The statistical analysis was done using SPSS version no. 20. IBM SPSS version 20.0 (IBM; Armonk, New York USA) Descriptive statistics and multinomial regression test was applied. RESULTS: Majority of study participants (1096 [59.4%]) belonged to age group of 26-35 years. Emotional well-being score of 1206 (65.4%) dental professionals in the times of COVID-19 outbreak was poor with score between 26 and 78. It was assessed that males have significantly (P = 0.00**) (P < 0.001**) (**highly significant) poorer emotional well-being 0.085 (0.829-1.429) compared to females. Married dental professionals - 0.023 (0.011-1.221) had significantly better (P = 0.05*) emotional well-being than others. CONCLUSION: Emotional well-being score of majority of dental professionals in the times of COVID-19 outbreak was poor. Gender, marital status, socioeconomic status, years of experience, practicing in clinic, and average number of patients seen per month before lockdown have significant impact on emotional wellbeing of dental professionals.

Determinants of Tobacco Use among Children of a Rural Village in India: An Exploratory Qualitative Study.

BACKGROUND: Tobacco is one of the leading causes of preventable deaths. It is both a major social and health problem. According to National Sample Survey Organization of Government of India about 20 million children of ages 10-14 are estimated to be tobacco-addicted. There are grave consequences of tobacco both socially and also on health thus it is of utmost importance to understand the factors leading to its use and to plan strategies to reduce its intake. However, the health implications of this social issue in a rural context have not been explored. AIMS AND OBJECTIVE: this study makes an attempt to explore the health and social implications of tobacco usage by the children below the age of 14 years in hamlet. MATERIALS AND METHODS:   The present study employed a qualitative study design. Data was collected using focus group discussion and in-depth interview of key informants. Thematic analysis for exploring the explicit and implicit meanings within the data was done. The themes which emerged were knowledge about tobacco and the various products available, children and parents' tobacco use and habits, the health and social implication of tobacco use, reasons for tobacco use by the children. RESULTS: It was found tobacco use by the children was very common in the community. Parent, peer pressure, sibling pressure were found to be playing important role in the initiation of tobacco habit by the child. Further illiteracy and lack of awareness was also lead to tobacco use among children. CONCLUSION: The study identifies education and awareness of parents about the ill-effects of tobacco play an important role as parents act as role model for their children, thus equal stress should be laid in improving the parental habits. Even raising the prices of tobacco products can help in controlling this habit.
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Non-canonical Binding Site for Bacterial Initiation Factor 3 on the Large Ribosomal Subunit.

Canonical translation initiation in bacteria entails the assembly of the 30S initiation complex (IC), which binds the 50S subunit to form a 70S IC. IF3, a key initiation factor, is recruited to the 30S subunit at an early stage and is displaced from its primary binding site upon subunit joining. We employed four different FRET pairs to monitor IF3 relocation after 50S joining. IF3 moves away from the 30S subunit, IF1 and IF2, but can remain bound to the mature 70S IC. The secondary binding site is located on the 50S subunit in the vicinity of ribosomal protein L33. The interaction between IF3 and the 50S subunit is largely electrostatic with very high rates of IF3 binding and dissociation. The existence of the non-canonical binding site may help explain how IF3 participates in alternative initiation modes performed directly by the 70S ribosomes, such as initiation on leaderless mRNAs or re-initiation.

Impact of dental fear on oral health-related quality of life among school going and non-school going children in Udaipur city: A cross-sectional study.

AIM: To assess the impact of dental fear on different domains of oral health-related quality of life (OHRQoL) among school going and non-school going children in the Indian scenario. MATERIALS AND METHODS: The study sample consisted of 279 school children and 257 non-school going children thus making a total sample of 536 children. The sampling frame comprised of 12-15-year-old children attending two upper primary public schools and non-school going children working at shops or not working in Udaipur city, India. Information on dental fear and OHRQoL was obtained by personal interviews by a single trained and calibrated examiner through a structured questionnaire. Intercooled STATA version 9.2 was employed to perform statistical analysis. The level of significance was set at 5%. RESULTS: Mean dental fear scores among school going (35.41 [11.79]) and non-school going (47.59 [3.80]) children revealed that dental fear was significantly (P ≤ 0.05) higher among non-school going than among school going children. In school going children, the likelihood of having poor oral symptoms, functional limitation and poorer social and emotional well being were significantly (P ≤ 0.05) lesser as compared with non-school going children. CONCLUSIONS: Fear has a significant impact on different domains of OHRQoL, except emotional well being, among non-school going children.