Specificity in the biosynthesis of the universal tRNA nucleoside N6-threonylcarbamoyl adenosine (t6A)-TsaD is the gatekeeper.

N6-threonylcarbamoyl adenosine (t6A) is a nucleoside modification found in all kingdoms of life at position 37 of tRNAs decoding ANN codons, which functions in part to restrict translation initiation to AUG and suppress frameshifting at tandem ANN codons. In Bacteria the proteins TsaB, TsaC (or C2), TsaD, and TsaE, comprise the biosynthetic apparatus responsible for t6A formation. TsaC(C2) and TsaD harbor the relevant active sites, with TsaC(C2) catalyzing the formation of the intermediate threonylcarbamoyladenosine monophosphate (TC-AMP) from ATP, threonine, and CO2, and TsaD catalyzing the transfer of the threonylcarbamoyl moiety from TC-AMP to A37 of substrate tRNAs. Several related modified nucleosides, including hydroxynorvalylcarbamoyl adenosine (hn6A), have been identified in select organisms, but nothing is known about their biosynthesis. To better understand the mechanism and structural constraints on t6A formation, and to determine if related modified nucleosides are formed via parallel biosynthetic pathways or the t6A pathway, we carried out biochemical and biophysical investigations of the t6A systems from E. coli and T. maritima to address these questions. Using kinetic assays of TsaC(C2), tRNA modification assays, and NMR, our data demonstrate that TsaC(C2) exhibit relaxed substrate specificity, producing a variety of TC-AMP analogs that can differ in both the identity of the amino acid and nucleotide component, whereas TsaD displays more stringent specificity, but efficiently produces hn6A in E. coli and T. maritima tRNA. Thus, in organisms that contain modifications such as hn6A in their tRNA, we conclude that their origin is due to formation via the t6A pathway.

Is radiation therapy as a primary treatment modality for squamous cell carcinoma of the hand the best choice? Case series and review of the literature.

BACKGROUND: Squamous cell carcinoma (SCC) is the most common malignancy of the upper limb, and can pose a significant therapeutic challenge. Early treatment needs to be radical whilst maintaining function. METHODS: We describe two cases of upper limb squamous cell carcinoma treated with radiotherapy, review the literature on radiotherapy as a primary treatment modality, and discuss the specific difficulties treating SCC in the hand. RESULTS: Radiation therapy was inadequate in tumor clearance in both cases, with recurrence both extensive and distal to the initial focus. Moreover, both patients developed progressive functional loss related to pain, swelling, and stiffness. CONCLUSION: The evidence basis for radiation therapy as a primary modality is limited, although clearance rates are comparable to surgery. Both radiotherapy and surgery can be utilized to treat SCC. However, we make the case for the hand being especially susceptible to the unwanted side effects of radiotherapy.

NMR-based Structural Analysis of Threonylcarbamoyl-AMP Synthase and Its Substrate Interactions.

The hypermodified nucleoside N(6)-threonylcarbamoyladenosine (t(6)A37) is present in many distinct tRNA species and has been found in organisms in all domains of life. This post-transcriptional modification enhances translation fidelity by stabilizing the anticodon/codon interaction in the ribosomal decoding site. The biosynthetic pathway of t(6)A37 is complex and not well understood. In bacteria, the following four proteins have been discovered to be both required and sufficient for t(6)A37 modification: TsaC, TsaD, TsaB, and TsaE. Of these, TsaC and TsaD are members of universally conserved protein families. Although TsaC has been shown to catalyze the formation of L-threonylcarbamoyl-AMP, a key intermediate in the biosynthesis of t(6)A37, the details of the enzymatic mechanism remain unsolved. Therefore, the solution structure of Escherichia coli TsaC was characterized by NMR to further study the interactions with ATP and L-threonine, both substrates of TsaC in the biosynthesis of L-threonylcarbamoyl-AMP. Several conserved amino acids were identified that create a hydrophobic binding pocket for the adenine of ATP. Additionally, two residues were found to interact with L-threonine. Both binding sites are located in a deep cavity at the center of the protein. Models derived from the NMR data and molecular modeling reveal several sites with considerable conformational flexibility in TsaC that may be important for L-threonine recognition, ATP activation, and/or protein/protein interactions. These observations further the understanding of the enzymatic reaction catalyzed by TsaC, a threonylcarbamoyl-AMP synthase, and provide structure-based insight into the mechanism of t(6)A37 biosynthesis.

A role for the universal Kae1/Qri7/YgjD (COG0533) family in tRNA modification.

The YgjD/Kae1 family (COG0533) has been on the top-10 list of universally conserved proteins of unknown function for over 5 years. It has been linked to DNA maintenance in bacteria and mitochondria and transcription regulation and telomere homeostasis in eukaryotes, but its actual function has never been found. Based on a comparative genomic and structural analysis, we predicted this family was involved in the biosynthesis of N(6)-threonylcarbamoyl adenosine, a universal modification found at position 37 of tRNAs decoding ANN codons. This was confirmed as a yeast mutant lacking Kae1 is devoid of t(6)A. t(6)A(-) strains were also used to reveal that t(6)A has a critical role in initiation codon restriction to AUG and in restricting frameshifting at tandem ANN codons. We also showed that YaeZ, a YgjD paralog, is required for YgjD function in vivo in bacteria. This work lays the foundation for understanding the pleiotropic role of this universal protein family.

Biosynthesis of threonylcarbamoyl adenosine (t6A), a universal tRNA nucleoside.

The anticodon stem-loop (ASL) of transfer RNAs (tRNAs) drives decoding by interacting directly with the mRNA through codon/anticodon pairing. Chemically complex nucleoside modifications found in the ASL at positions 34 or 37 are known to be required for accurate decoding. Although over 100 distinct modifications have been structurally characterized in tRNAs, only a few are universally conserved, among them threonylcarbamoyl adenosine (t(6)A), found at position 37 in the anticodon loop of a subset of tRNA. Structural studies predict an important role for t(6)A in translational fidelity, and in vivo work supports this prediction. Although pioneering work in the 1970s identified the fundamental substrates for t(6)A biosynthesis, the enzymes responsible for its biosynthesis have remained an enigma. We report here the discovery that in bacteria four proteins (YgjD, YrdC, YjeE, and YeaZ) are both necessary and sufficient for t(6)A biosynthesis in vitro. Notably, YrdC and YgjD are members of universally conserved families that were ranked among the top 10 proteins of unknown function in need of functional characterization, while YeaZ and YjeE are specific to bacteria. This latter observation, coupled with the essentiality of all four proteins in bacteria, establishes this pathway as a compelling new target for antimicrobial development.

C.A.R.E.S: A mobile health program for alcohol risk reduction in community college students.

BACKGROUND: Compared to students at four-year residential colleges, Community College Students (CCS) are at greater risk for binge drinking and alcohol related risks, however few interventions have been developed specifically for the needs of CCS. METHODS AND DESIGN: This study design tests the efficacy of a smartphone app (CARES) compared to an existing online alcohol education (AE) program. CCS (n = 250) will be recruited using a nationwide social media campaign and randomly assigned to either arm, stratified by sex and age (over/under 21). Eligibility screening, consent and assessments are conducted online and both interventions are accessible by smartphone. Assessments will be conducted at baseline, 4- and 8- weeks, end of treatment (12-weeks) and at 6-month follow-up. The primary outcomes are heavy episodic drinking (past 2 weeks), and alcohol related problems Confidence in refusing alcohol, use of protective behavioral strategies and expectations regarding alcohol use. Demographics including age, sex, race/ethnicity, marital and parental status and employment will be analyzed as potential covariates. DISCUSSION: Community colleges serve approximately half of all US college students but frequently lack the resources to implement full-service alcohol prevention and education programs. The specific needs of CCS also differ from those of residential college students for whom most alcohol prevention programs have been developed. If proven efficacious, the CARES intervention may offer a scalable, easily disseminable program designed for the needs of community colleges and their students. ClinicalTrials.gov Registration: NCT03927482.

A text message delivered smoking cessation intervention: Design and rationale of the Text My Quit Study.

INTRODUCTION: Smoking cessation interventions delivered through mobile technologies offer promise as an effective intervention tool. However, most existing programs have not been empirically tested, were not developed with end-user participation, and/or do not address evidence-based cognitive and behavioral variables shown to enhance smoking cessation in clinical trials. In addition, many programs tested in research trials have required users to access the internet and/or a smartphone app to access all program features, limiting the potential reach of those programs. METHODS/DESIGN: This study is a randomized controlled trial testing the efficacy of the TMQ intervention for smoking cessation. All participants are randomly assigned to receive 12 weeks of either; (1) a tailored smoking-cessation intervention delivered 100% through text messaging (TMQ), or (2) non-smoking-related text messages serving as a control for contact and subject burden (Mojo). Assessments are conducted at baseline, 3- and 6-month follow-up. The primary outcome is prolonged abstinence using an intent-to-treat approach. To understand why TMQ may be more effective than Mojo, we will test several posited mechanisms of action (i.e., mediators) that may underlie intervention efficacy and will examine use of the TMQ integrated social support (ISS) network. At the end of treatment, semi-structured interviews will be conducted with TMQ participants. CONCLUSIONS: This study will provide a rigorous test of an innovative smoking cessation program delivered 100% through text messages. Use of mixed methodologies will provide the opportunity to enhance our understanding of the user's experience with TMQ and identify areas for future enhancement and/or expansion.

Management and outcomes in children with sinogenic intracranial abscesses.

OBJECTIVE: To report our experience of sinogenic intracranial abscesses in the paediatric population and to guide medical and surgical management. METHODS: All children with sinogenic intracranial abscesses presenting to a large university teaching hospital over a five-year period were included in the study. Data on clinical presentation, radiological findings, microbiology, medical and surgical management and follow-up were recorded and analysed. RESULTS: We identified 27 children aged 12.9 ± 3.4 years of which 56% were male. Fourteen (52%) children had extradural abscesses, nine (33%) subdural abscesses and four (15%) parenchymal abscesses. Early sinus drainage procedures were performed on 24 (89%) patients, and the same number required neurosurgical drainage. Streptococcus milleri was isolated in 18 (67%) cases. An initial conservative neurosurgical approach failed in 50% of cases where trialled, and was associated with longer length of stay (p = 0.025). In comparison to extradural abscesses, subdural abscesses were more likely to present with neurological deficits (p < 0.001) and reduced consciousness (p = 0.018), and required multiple neurosurgical procedures (p < 0.001), longer stays (p = 0.017), and had greater morbidity at six months (p = 0.017). A third of children had significant morbidity at six months, which included cognitive and behavioural problems (25%), residual hemiparesis (19%) and expressive dysphasia (7%). There were no mortalities. CONCLUSION: Sinusitis complicated by intracranial abscess remains a contemporary problem. We demonstrate good outcomes with an early combined rhinological and neurosurgical approach. S. milleri is identified as the causative organism in the majority of cases, and empirical antimicrobial treatments should reflect this.

Translating Behavioral Interventions Onto mHealth Platforms: Developing Text Message Interventions for Smoking and Alcohol.

The development of mHealth applications is often driven by the investigators and developers with relatively little input from the targeted population. User input is commonly limited to "like/dislike" post- intervention consumer satisfaction ratings or device or application specific user analytics such as usability. However, to produce successful mHealth applications with lasting effects on health behaviors it is crucial to obtain user input from the start of each project and throughout development. The aim of this tutorial is to illustrate how qualitative methods in an iterative process of development have been used in two separate behavior change interventions (targeting smoking and alcohol) delivered through mobile technologies (ie, text messaging). A series of focus groups were conducted to assist in translating a face-to-face smoking cessation intervention onto a text message (short message service, SMS) delivered format. Both focus groups and an advisory panel were used to shape the delivery and content of a text message delivered intervention for alcohol risk reduction. An in vivo method of constructing message content was used to develop text message content that was consistent with the notion of texting as "fingered speech". Formative research conducted with the target population using a participatory framework led to important changes in our approach to intervention structure, content development, and delivery. Using qualitative methods and an iterative approach that blends consumer-driven and investigator-driven aims can produce paradigm-shifting, novel intervention applications that maximize the likelihood of use by the target audience and their potential impact on health behaviors.

A Text Message Delivered Smoking Cessation Intervention: The Initial Trial of TXT-2-Quit: Randomized Controlled Trial.

BACKGROUND: Mobile technology offers the potential to deliver health-related interventions to individuals who would not otherwise present for in-person treatment. Text messaging (short message service, SMS), being the most ubiquitous form of mobile communication, is a promising method for reaching the most individuals. OBJECTIVE: The goal of the present study was to evaluate the feasibility and preliminary efficacy of a smoking cessation intervention program delivered through text messaging. METHODS: Adult participants (N=60, age range 18-52 years) took part in a single individual smoking cessation counseling session, and were then randomly assigned to receive either daily non-smoking related text messages (control condition) or the TXT-2-Quit (TXT) intervention. TXT consisted of automated smoking cessation messages tailored to individual's stage of smoking cessation, specialized messages provided on-demand based on user requests for additional support, and a peer-to-peer social support network. Generalized estimating equation analysis was used to assess the primary outcome (7-day point-prevalence abstinence) using a 2 (treatment groups)×3 (time points) repeated measures design across three time points: 8 weeks, 3 months, and 6 months. RESULTS: Smoking cessation results showed an overall significant group difference in 7-day point prevalence abstinence across all follow-up time points. Individuals given the TXT intervention, with higher odds of 7-day point prevalence abstinence for the TXT group compared to the Mojo group (OR=4.52, 95% CI=1.24, 16.53). However, individual comparisons at each time point did not show significant between-group differences, likely due to reduced statistical power. Intervention feasibility was greatly improved by switching from traditional face-to-face recruitment methods (4.7% yield) to an online/remote strategy (41.7% yield). CONCLUSIONS: Although this study was designed to develop and provide initial testing of the TXT-2-Quit system, these initial findings provide promising evidence that a text-based intervention can be successfully implemented with a diverse group of adult smokers. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01166464; http://clinicaltrials.gov/ct2/show/NCT01166464 (Archived by WebCite at http://www.webcitation.org/6IOE8XdE0).

Scoring function to predict solubility mutagenesis.

BACKGROUND: Mutagenesis is commonly used to engineer proteins with desirable properties not present in the wild type (WT) protein, such as increased or decreased stability, reactivity, or solubility. Experimentalists often have to choose a small subset of mutations from a large number of candidates to obtain the desired change, and computational techniques are invaluable to make the choices. While several such methods have been proposed to predict stability and reactivity mutagenesis, solubility has not received much attention. RESULTS: We use concepts from computational geometry to define a three body scoring function that predicts the change in protein solubility due to mutations. The scoring function captures both sequence and structure information. By exploring the literature, we have assembled a substantial database of 137 single- and multiple-point solubility mutations. Our database is the largest such collection with structural information known so far. We optimize the scoring function using linear programming (LP) methods to derive its weights based on training. Starting with default values of 1, we find weights in the range [0,2] so that predictions of increase or decrease in solubility are optimized. We compare the LP method to the standard machine learning techniques of support vector machines (SVM) and the Lasso. Using statistics for leave-one-out (LOO), 10-fold, and 3-fold cross validations (CV) for training and prediction, we demonstrate that the LP method performs the best overall. For the LOOCV, the LP method has an overall accuracy of 81%. AVAILABILITY: Executables of programs, tables of weights, and datasets of mutants are available from the following web page: http://www.wsu.edu/~kbala/OptSolMut.html.