Architectures and complex functions of tandem riboswitches.

Riboswitch architectures that involve the binding of a single ligand to a single RNA aptamer domain result in ordinary dose-response curves that require approximately a 100-fold change in ligand concentration to cover nearly the full dynamic range for gene regulation. However, by using multiple riboswitches or aptamer domains in tandem, these ligand-sensing structures can produce additional, complex gene control outcomes. In the current study, we have computationally searched for tandem riboswitch architectures in bacteria to provide a more complete understanding of the diverse biological and biochemical functions of gene control elements that are made exclusively of RNA. Numerous different arrangements of tandem homologous riboswitch architectures are exploited by bacteria to create more 'digital' gene control devices, which operate over a narrower ligand concentration range. Also, two heterologous riboswitch aptamers are sometimes employed to create two-input Boolean logic gates with various types of genetic outputs. These findings illustrate the sophisticated genetic decisions that can be made by using molecular sensors and switches based only on RNA.

Employing a ZTP Riboswitch to Detect Bacterial Folate Biosynthesis Inhibitors in a Small Molecule High-Throughput Screen.

Various riboswitch classes are being discovered that precisely monitor the status of important biological processes, including metabolic pathway function, signaling for physiological adaptations, and responses to toxic agents. Biochemical components for some of these processes might make excellent targets for the development of novel antibacterial molecules, which can be broadly sought by using phenotypic drug discovery (PDD) methods. However, PDD data do not normally provide clues regarding the target for each hit compound. We have developed and validated a robust fluorescent reporter system based on a ZTP riboswitch that identifies numerous folate biosynthesis inhibitors with high sensitivity and precision. The utility of the riboswitch-based PDD strategy was evaluated using Escherichia coli bacteria by conducting a 128 310-compound high-throughput screen, which identified 78 sulfanilamide derivatives among the many initial hits. Similarly, representatives of other riboswitch classes could be employed to rapidly match antibacterial hits with the biological processes they target.

Creating Evidence-Based Youth Mental Health Policy in Sub-Saharan Africa: A Description of the Integrated Approach to Addressing the Issue of Youth Depression in Malawi and Tanzania.

Addressing depression in young people is a health-care policy need in sub-Saharan Africa. There exists poor mental health literacy, high levels of stigma, and weak capacity at the community level to address this health-care need. These challenges are significant barriers to accessing mental health care for depression, soon to be the largest single contributor to the global burden of disease. We here describe an innovative approach that addresses these issues simultaneously while concurrently strengthening key mental health components in existing education and health-care systems as successfully applied in Malawi and replicated in Tanzania. Improving the pathway to care for young people with depression requires the following: improving mental health literacy (MHL) of communities, youth, and teachers; enhancing case identification and linking schools to community health clinics; improving the capacity of community health-care providers to identify, diagnose, and effectively treat depression in youth. Funded by Grand Challenges Canada, we developed and applied a program called "An Integrated Approach to Addressing the Challenge of Depression Among the Youth in Malawi and Tanzania" (IACD). This was an example of, a horizontally integrated pathway to care model designed to be applied in low-resource settings. The model is designed to 1) improve awareness/knowledge of mental health and mental disorders (especially depression) in communities; 2) enhance mental health literacy among youth and teachers within schools; 3) enhance capacity for teachers to identify students with possible depression; 4) create linkages between schools and community health clinics for improved access to mental health care for youth identified with possible depression; and 5) enhance the capacity of community-based health-care providers to identify, diagnose, and effectively treat youth with depression. With the use of interactive, youth-informed weekly radio programs, mental health curriculum training for teachers and peer educators in secondary schools, and a clinical competency training program for community-based health workers, the innovation created a "hub and spoke" model for improving mental health care for young people. Positive results obtained in Malawi and replicated in Tanzania suggest that this approach may provide an effective and potentially sustainable framework for enhancing youth mental health care, thus providing a policy ready framework that can be considered for application in sub-Saharan Africa.

Control of Nasal Tip Position: Quantitative Assessment of Columellar Strut versus Caudal Septal Extension Graft.

BACKGROUND: Control and maintenance of nasal tip position are critical in rhinoplasty. Two frequent methods of exerting tip control are columellar strut and caudal septal extension graft. However, no quantitative data exist comparing the two methods over time. The purpose of this study was to analyze maintenance of tip projection and rotation following either columellar strut or septal extension graft. METHODS: A retrospective cohort study of patients undergoing rhinoplasty was reviewed. Three-dimensional photogrammetric evaluation of patients with either columellar strut or septal extension graft to increase tip projection was performed. Anthropometric points were analyzed in a blinded fashion. Outcome variables were tip projection, nasal length, the Goode ratio, and tip rotation. Results were stratified based on technique and compared statistically. RESULTS: One hundred six patients were included. Overall, 66 percent were female, with an average age of 34.5 years. A columellar strut was used in 42 percent of cases (n = 45), and a septal extension graft was used in 57 percent (n = 61). Analysis showed greater maintenance of tip rotation over time with the septal extension graft compared with the columellar strut [-1.01 percent change (p = not significant versus -5.08 percent change (p = 0.009)]. Tip projection, nasal length, and the Goode ratio decreased over time for both groups, but the differences were not statistically different. CONCLUSIONS: Nasal tip projection and rotation appear to decrease from the immediate postoperative position. In this study, both septal extension graft and columellar strut exhibit similar changes in tip projection with time; however, septal extension graft is better able to preserve tip rotation compared with the columellar strut. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

Rare variants of the FMN riboswitch class in Clostridium difficile and other bacteria exhibit altered ligand specificity.

Many bacteria use flavin mononucleotide (FMN) riboswitches to control the expression of genes responsible for the biosynthesis and transport of this enzyme cofactor or its precursor, riboflavin. Rare variants of FMN riboswitches found in strains of Clostridium difficile and some other bacteria typically control the expression of proteins annotated as transporters, including multidrug efflux pumps. These RNAs no longer recognize FMN, and differ from the original riboswitch consensus sequence at nucleotide positions normally involved in binding of the ribityl and phosphate moieties of the cofactor. Representatives of one of the two variant subtypes were found to bind the FMN precursor riboflavin and the FMN degradation products lumiflavin and lumichrome. Although the biologically relevant ligand sensed by these variant FMN riboswitches remains uncertain, our findings suggest that many strains of C. difficile might use rare riboswitches to sense flavin degradation products and activate transporters for their detoxification.

Challenges of ligand identification for the second wave of orphan riboswitch candidates.

Orphan riboswitch candidates are noncoding RNA motifs whose representatives are believed to function as genetic regulatory elements, but whose target ligands have yet to be identified. The study of certain orphans, particularly classes that have resisted experimental validation for many years, has led to the discovery of important biological pathways and processes once their ligands were identified. Previously, we highlighted details for four of the most common and intriguing orphan riboswitch candidates. This facilitated the validation of riboswitches for the signaling molecules c-di-AMP, ZTP, and ppGpp, the metal ion Mn2+, and the metabolites guanidine and PRPP. Such studies also yield useful linkages between the ligands sensed by the riboswitches and numerous biochemical pathways. In the current report, we describe the known characteristics of 30 distinct classes of orphan riboswitch candidates - some of which have remained unsolved for over a decade. We also discuss the prospects for uncovering novel biological insights via focused studies on these RNAs. Lastly, we make recommendations for experimental objectives along the path to finding ligands for these mysterious RNAs.

Detection of 224 candidate structured RNAs by comparative analysis of specific subsets of intergenic regions.

The discovery of structured non-coding RNAs (ncRNAs) in bacteria can reveal new facets of biology and biochemistry. Comparative genomics analyses executed by powerful computer algorithms have successfully been used to uncover many novel bacterial ncRNA classes in recent years. However, this general search strategy favors the discovery of more common ncRNA classes, whereas progressively rarer classes are correspondingly more difficult to identify. In the current study, we confront this problem by devising several methods to select subsets of intergenic regions that can concentrate these rare RNA classes, thereby increasing the probability that comparative sequence analysis approaches will reveal their existence. By implementing these methods, we discovered 224 novel ncRNA classes, which include ROOL RNA, an RNA class averaging 581 nt and present in multiple phyla, several highly conserved and widespread ncRNA classes with properties that suggest sophisticated biochemical functions and a multitude of putative cis-regulatory RNA classes involved in a variety of biological processes. We expect that further research on these newly found RNA classes will reveal additional aspects of novel biology, and allow for greater insights into the biochemistry performed by ncRNAs.

Clinic outcomes of the Pathway to Care Model: A cross-sectional survey of adolescent depression in Malawi.

BACKGROUND: Depression is one of the leading contributors to the global burden of disease and often has an onset during adolescence. While effective treatments are available, many low-income countries, such as Malawi, lack appropriately trained health providers in community health settings, and this limits access to effective mental healthcare for young people with depression. To address this need, a Canadian-developed youth depression Pathway to Care Model, linking school-based mental health literacy interventions to training of community healthcare providers, was adapted for use in Malawi and successfully applied. METHODS: A sample of healthcare providers (N = 25) from community health clinics (N = 9) were trained in the use of comprehensive, systematic clinical interventions, addressing the identification, diagnosis, and treatment of depression in youth who had been referred from schools where mental health literacy interventions had been implemented. Referral outcomes were obtained using a standardised clinical record form. RESULTS: Over 120 clinical outcome forms were available for analysis. Seventy percent of youth referred by their teachers were diagnosed with depression. Most youth diagnosed with depression identified physical symptoms as their primary difficulty. Available standardised outcome measures applied by clinicians indicated that, overall, youth showed positive outcomes as a result of treatment. CONCLUSIONS: Community healthcare providers in Malawi were trained in the identification, diagnosis, and treatment of youth depression. When this training was applied in usual clinical care to youth referred from schools, it led to generally favourable clinical outcomes. To our knowledge, this is the first demonstration of a clinically feasible intervention that results in positive outcomes for young people with depression in Malawi, and it may provide a useful model to replicate elsewhere in sub-Saharan Africa.

The Effect of Nasal Tip Rotation on Upper Lip Length.

Background: Increasing the nasolabial angle (NLA) with tip rotation generates the appearance of a lengthened lower facial third. In particular, the upper lip show seems increased following elevation of the nasal tip. Objectives: The purpose of this study is to quantify the impact of tip rotation on upper lip length (ULL), and to establish a predictable correlation between the two. Methods: A retrospective cohort study of rhinoplasty patients with increased tip rotation, using either caudal septal extension graft (CSEG) or columellar strut graft (CS), was performed. Three-dimensional photos were obtained and analyzed anthropometrically and used to measure the ULL and NLA. The deltas between NLA and ULL at the various time points, was then compared using linear regression with P < .05 recognized as statistically significant. Results: One-hundred and fifty patients were identified and 88 patients met inclusion criteria. CS and CSEG were used in 40% (n = 36), and 60% (n = 52), respectively. Three-dimensional assessment showed that as the NLA positively correlated with the ULL in both cohorts. The CSEG group created a greater NLA and ULL compared to the CS cohort. Both NLA and ULL decreased over time, but remained statistically increased as compared with preoperative measurements. For every one degree of NLA increase, the ULL increases by 0.05 mm. Conclusions: Increasing nasal tip rotation in rhinoplasty results in greater upper lip show. Both CS and CSEG can effectively increase tip rotation and ULL. A predictable correlation of nearly 0.05 mm of ULL for every 1 degree of tip rotation is shown. Level of Evidence: 4.

Addressing Adolescent Depression in Tanzania: Positive Primary Care Workforce Outcomes Using a Training Cascade Model.

BACKGROUND: This is a report on the outcomes of a training program for community clinic healthcare providers in identification, diagnosis, and treatment of adolescent Depression in Tanzania using a training cascade model. METHODS: Lead trainers adapted a Canadian certified adolescent Depression program for use in Tanzania to train clinic healthcare providers in the identification, diagnosis, and treatment of Depression in young people. As part of this training program, the knowledge, attitudes, and a number of other outcomes pertaining to healthcare providers and healthcare practice were assessed. RESULTS: The program significantly, substantially, and sustainably improved provider knowledge and confidence. Further, healthcare providers' personal help-seeking efficacy also significantly increased as well as the clinicians' reported number of adolescent patients identified, diagnosed, and treated for Depression. CONCLUSION: To our knowledge, this is the first study reporting positive outcomes of a training program addressing adolescent Depression in Tanzanian community clinics. These results suggest that the application of this training cascade approach may be a feasible model for developing the capacity of healthcare providers to address youth Depression in a low-income, low-resource setting.

A school mental health literacy curriculum resource training approach: effects on Tanzanian teachers' mental health knowledge, stigma and help-seeking efficacy.

BACKGROUND: Mental health literacy (MHL) is foundational for mental health promotion, prevention, stigma reduction, and care; School supported information pertaining to MHL in sub-Saharan Africa is extremely limited, including in Tanzania. Successful application of a school MHL curriculum resource may be an effective way to increase teacher MHL and therefore help to improve mental health outcomes for students. METHODS: Secondary school teachers in Tanzania were trained on the African Guide (AG) a school MHL curriculum resource culturally adapted from a Canadian MHL resource (The Guide) for use in Africa. Teacher training workshops on the classroom application of the AG were used to evaluate its impact on mental health literacy in a sample of Tanzanian Secondary school teachers. Pre-post training assessment of participant knowledge and attitudes was conducted. Help-seeking efficacy for teachers themselves and their interventions for students, friends, family members and peers were determined. RESULTS: Paired t test (n = 37) results demonstrate highly significant improvements in teacher's overall knowledge (p < 0.001; d = 1.14), including mental health knowledge, (p < 0.001; d = 1.14) and curriculum specific knowledge (p < 0.01; d = 0.63). Teachers' stigma against mental illness decreased significantly following the training (p < 0.001; d = 0.61). Independent t tests comparing the paired sample against unpaired sample also demonstrated significant differences between the groups for teacher's overall knowledge (p < 0.001). Teachers also reported high rates (greater than ¾ of the sample) of positive help-seeking efficacy for themselves as well as for their students, friends, family members and peers. As a result of the training, the number of students teachers identified for potential mental health care totaled over 200. CONCLUSIONS: These positive results, when taken together with other research, suggest that the use of a classroom-based resource (the AG) that integrates MHL into existing school curriculum through training teachers may be an effective and sustainable way to increase the MHL (improved knowledge, decreased stigma and positive help-seeking efficacy) of teachers in Tanzania. As this study replicated the results of a previous intervention in Malawi, consideration could be given to scaling up this intervention in both countries and applying this resource and approach in other countries in East Africa.

Thermoresponsive, in situ cross-linkable hydrogels based on N-isopropylacrylamide: fabrication, characterization and mesenchymal stem cell encapsulation.

Hydrogels that solidify in response to a dual, physical and chemical, mechanism upon temperature increase were fabricated and characterized. The hydrogels were based on N-isopropylacrylamide, which renders them thermoresponsive, and contained covalently cross-linkable moieties in the macromers. The effects of the macromer end group, acrylate or methacrylate, and the fabrication conditions on the degradative and swelling properties of the hydrogels were investigated. The hydrogels exhibited higher swelling below their lower critical solution temperature (LCST). When immersed in cell culture medium at physiological temperature, which was above their LCST, hydrogels showed constant swelling and no degradation over 8 weeks, with the methacrylated hydrogels showing greater swelling than their acrylated analogs. In addition, hydrogels immersed in cell culture medium under the same conditions showed lower swelling compared with phosphate-buffered saline. The interplay between chemical cross-linking and thermally induced phase separation affected the swelling characteristics of the hydrogels in different media. Mesenchymal stem cells encapsulated in the hydrogels in vitro were viable over 3 weeks and markers of osteogenic differentiation were detected when the cells were cultured with osteogenic supplements. Hydrogel mineralization in the absence of cells was observed in cell culture medium with the addition of fetal bovine serum and ß-glycerol phosphate. The results suggest that these hydrogels may be suitable as carriers for cell delivery in tissue engineering.