Structural basis of trehalose recognition by the mycobacterial LpqY-SugABC transporter.

The Mycobacterium tuberculosis (Mtb) LpqY-SugABC ATP-binding cassette transporter is a recycling system that imports trehalose released during remodeling of the Mtb cell-envelope. As this process is essential for the virulence of the Mtb pathogen, it may represent an important target for tuberculosis drug and diagnostic development, but the transporter specificity and molecular determinants of substrate recognition are unknown. To address this, we have determined the structural and biochemical basis of how mycobacteria transport trehalose using a combination of crystallography, saturation transfer difference NMR, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the synthesis of trehalose analogs. This analysis pinpoints key residues of the LpqY substrate binding lipoprotein that dictate substrate-specific recognition and has revealed which disaccharide modifications are tolerated. These findings provide critical insights into how the essential Mtb LpqY-SugABC transporter reuses trehalose and modified analogs and specifies a framework that can be exploited for the design of new antitubercular agents and/or diagnostic tools.

Pediatric Resident and Program Director Views on Climate Change and Health Curricula: A Multi-Institution Study.

PURPOSE: The American Academy of Pediatrics emphasized in a 2007 policy statement the importance of educating trainees on the impacts of climate change on children's health, yet few studies have evaluated trainee knowledge and attitudes about climate change-related health effects in children. This multi-institution study assessed pediatric resident and program director (1) knowledge/attitudes on climate change and health, (2) perspectives on the importance of incorporating climate and health content into pediatric graduate medical education, and (3) preferred topics/activities to include in climate and health curricula. METHOD: This mixed-methods study employed an anonymous cross-sectional survey of pediatric residents and residency program directors from Association of Pediatric Program Directors (APPD) Longitudinal Educational Assessment Research Network (LEARN)-affiliated programs. Multivariable regression models and factor analyses were used to examine associations among resident demographics and resident knowledge, attitudes, and interest in a climate change curriculum. A conventional content analysis was conducted for the open-ended responses. RESULTS: Eighteen programs participated in the study with all program directors (100% response rate) and 663 residents (average response rate per program, 53%; overall response rate, 42%) completing respective surveys. Of the program directors, only 3 (17%) felt very or moderately knowledgeable about the association between climate change and health impacts. The majority of residents (n=423, 64%) agreed/strongly agreed that physicians should discuss global warming/climate change and its health effects with patients/families, while only 138 residents (21%) agreed/strongly agreed that they were comfortable talking with patients and families about these issues. Most residents (n=498, 76%) and program directors (n=15, 83%) agreed/strongly agreed that a climate change curriculum should be incorporated into their pediatrics training program. CONCLUSIONS: Pediatric residents and program directors support curricula that prepare future pediatricians to address the impact of climate change on children's health; however, few programs currently offer specific training, despite identified needs.

The mycobacterial desaturase DesA2 is associated with mycolic acid biosynthesis.

Mycolic acids are critical for the survival and virulence of Mycobacterium tuberculosis, the causative agent of tuberculosis. Double bond formation in the merochain of mycolic acids remains poorly understood, though we have previously shown desA1, encoding an aerobic desaturase, is involved in mycolic acid desaturation. Here we show that a second desaturase encoded by desA2 is also involved in mycolate biosynthesis. DesA2 is essential for growth of the fast-growing Mycobacterium smegmatis in laboratory media. Conditional depletion of DesA2 led to a decrease in mycolic acid biosynthesis and loss of mycobacterial viability. Additionally, DesA2-depleted cells also accumulated fatty acids of chain lengths C19-C24. The complete loss of mycolate biosynthesis following DesA2 depletion, and the absence of any monoenoic derivatives (found to accumulate on depletion of DesA1) suggests an early role for DesA2 in the mycolic acid biosynthesis machinery, highlighting its potential as a drug target.

Interrogation of the Pathogen Box reveals small molecule ligands against the mycobacterial trehalose transporter LpqY-SugABC.

Tuberculosis, caused by Mycobacterium tuberculosis, claims ∼1.5 million lives annually. Effective chemotherapy is essential to control TB, however the emergence of drug-resistant strains of TB have seriously threatened global attempts to control and eradicate this deadly pathogen. Trehalose recycling via the LpqY-SugABC importer is essential for the virulence and survival of Mtb and inhibiting or hijacking this transport system is an attractive approach for the development of novel anti-tubercular and diagnostic agents. Therefore, we interrogated the drug-like compounds in the open-source Medicines for Malaria Pathogen Box and successfully identified seven compounds from the TB, kinetoplastids and reference compound disease sets that recognise LpqY. The molecules have diverse chemical scaffolds, are not specific trehalose analogues, and may be used as novel templates to facilitate the development of therapeutics that kill Mtb with a novel mechanism of action via the mycobacterial trehalose LpqY-SugABC transport system.

Peer Crowd Identification and Adolescent Health Behaviors: Results From a Statewide Representative Study.

PURPOSE: Peer crowds are macro-level subcultures that share similarities across geographic areas. Over the past decade, dozens of studies have explored the association between adolescent peer crowds and risk behaviors, and how they can inform public health efforts. However, despite the interest, researchers have not yet reported on crowd size and risk levels from a representative sample, making it difficult for practitioners to apply peer crowd science to interventions. The current study reports findings from the first statewide representative sample of adolescent peer crowd identification and health behaviors. METHODS: Weighted data were analyzed from the 2015 Virginia Youth Survey of Health Behaviors ( n = 4,367). Peer crowds were measured via the I-Base Survey™, a photo-based peer crowd survey instrument. Frequencies and confidence intervals of select behaviors including tobacco use, substance use, nutrition, physical activity, and violence were examined to identify high- and low-risk crowds. Logistic regression was used to calculate adjusted odds ratios for each crowd and behavior. RESULTS: Risky behaviors clustered in two peer crowds. Hip Hop crowd identification was associated with substance use, violence, and some depression and suicidal behaviors. Alternative crowd identification was associated with increased risk for some substance use behaviors, depression and suicide, bullying, physical inactivity, and obesity. Mainstream and, to a lesser extent, Popular, identities were associated with decreased risk for most behaviors. CONCLUSIONS: Findings from the first representative study of peer crowds and adolescent behavior identify two high-risk groups, providing critical insights for practitioners seeking to maximize public health interventions by targeting high-risk crowds.

A GFP-strategy for efficient recombinant protein overexpression and purification in Mycobacterium smegmatis.

One of the major obstacles to obtaining a complete structural and functional understanding of proteins encoded by the Mycobacterium tuberculosis (Mtb) pathogen is due to significant difficulties in producing recombinant mycobacterial proteins. Recent advances that have utilised the closely related Mycobacterium smegmatis species as a native host have been effective. Here we have developed a method for the rapid screening of both protein production and purification strategies of mycobacterial proteins in whole M. smegmatis cells following green fluorescent protein (GFP) fluorescence as an indicator. We have adapted the inducible T7-promoter based pYUB1062 shuttle vector by the addition of a tobacco etch virus (TEV) cleavable C-terminal GFP enabling the target protein to be produced as a GFP-fusion with a poly-histidine tag for affinity purification. We illustrate the advantages of a fluorescent monitoring approach with the production and purification of the mycobacterial N-acetylglucosamine-6-phosphate deacetylase (NagA)-GFP fusion protein. The GFP system described here will accelerate the production of mycobacterial proteins that can be used to understand the molecular mechanisms of Mtb proteins and facilitate drug discovery efforts.