Probing adenylation: using a fluorescently labelled ATP probe to directly label and immunoprecipitate VopS substrates

The bacterial effector VopS from Vibrio parahaemolyticus modifies host Rho GTPases to prevent downstream signalling, which leads to cell rounding and eventually apoptosis. While previous studies have used [α-(32)P] ATP for studying this enzyme, we sought to develop a non-radioactive chemical probe of VopS function. To guide these studies, the kinetic parameters were determined for a variety of nucleotides and the results indicated that the C6 position of adenosine was amenable to modification. Since Fl-ATP is a commercially available ATP analogue that is fluorescently tagged at the C6 position, we tested it as a VopS substrate, and the results show that VopS uses Fl-ATP to label Cdc42 in vitro and in MCF7 whole cell extracts. The utility of this probe was further demonstrated by immunoprecipitating Fl-ATP labeled Cdc42 as well as several novel substrate proteins. The proteins, which were identified by LC-MS/MS, include the small GTPases Rac1 and Cdc42 as well as several proteins that are potential VopS substrates and may be important for V. parahaemolyticus pathology. In total, these studies identify Fl-ATP as a valuable chemical probe of protein AMPylation.     

Sanitary Pad Interventions for Girls' Education in Ghana: A Pilot Study

Background

Increased education of girls in developing contexts is associated with a number of important positive health, social, and economic outcomes for a community. The event of menarche tends to coincide with girls'' transitions from primary to secondary education and may constitute a barrier for continued school attendance and performance. Following the MRC Framework for Complex Interventions, a pilot controlled study was conducted in Ghana to assess the role of sanitary pads in girls'' education.

Methods

A sample of 120 schoolgirls between the ages of 12 and 18 from four villages in Ghana participated in a non-randomized trial of sanitary pad provision with education. The trial had three levels of treatment: provision of pads with puberty education; puberty education alone; or control (no pads or education). The primary outcome was school attendance.

Results

After 3 months, providing pads with education significantly improved attendance among participants, (lambda 0.824, F = 3.760, p<.001). After 5 months, puberty education alone improved attendance to a similar level (M = 91.26, SD = 7.82) as sites where pads were provided with puberty education (Rural M = 89.74, SD = 9.34; Periurban M = 90.54, SD = 17.37), all of which were higher than control (M = 84.48, SD = 12.39). The total improvement through pads with education intervention after 5 months was a 9% increase in attendance. After 3 months, providing pads with education significantly improved attendance among participants. The changes in attendance at the end of the trial, after 5 months, were found to be significant by site over time. With puberty education alone resulting in a similar attendance level.

Conclusion

This pilot study demonstrated promising results of a low-cost, rapid-return intervention for girls'' education in a developing context. Given the considerable development needs of poorer countries and the potential of young women there, these results suggest that a large-scale cluster randomized trial is warranted.

Trial Registration

Pan African Clinical Trials Registry PACTR201202000361337     

Glutamyl Phosphate Is an Activated Intermediate in Actin Crosslinking by Actin Crosslinking Domain (ACD) Toxin

Actin Crosslinking Domain (ACD) is produced by several life-threatening Gram-negative pathogenic bacteria as part of larger toxins and delivered into the cytoplasm of eukaryotic host cells via Type I or Type VI secretion systems. Upon delivery, ACD disrupts the actin cytoskeleton by catalyzing intermolecular amide bond formation between E270 and K50 residues of actin, leading to the formation of polymerization-deficient actin oligomers. Ultimately, accumulation of the crosslinked oligomers results in structural and functional failure of the actin cytoskeleton in affected cells. In the present work, we advanced in our understanding of the ACD catalytic mechanism by discovering that the enzyme transfers the gamma-phosphoryl group of ATP to the E270 actin residue, resulting in the formation of an activated acyl phosphate intermediate. This intermediate is further hydrolyzed and the energy of hydrolysis is utilized for the formation of the amide bond between actin subunits. We also determined the pH optimum for the reaction and the kinetic parameters of ACD catalysis for its substrates, ATP and actin. ACD showed sigmoidal, non-Michaelis-Menten kinetics for actin (K_0.5 = 30 µM) reflecting involvement of two actin molecules in a single crosslinking event. We established that ACD can also utilize Mg²⁺-GTP to support crosslinking, but the kinetic parameters (K_M = 8 µM and 50 µM for ATP and GTP, respectively) suggest that ATP is the primary substrate of ACD in vivo. The optimal pH for ACD activity was in the range of 7.0–9.0. The elucidated kinetic mechanism of ACD toxicity adds to understanding of complex network of host-pathogen interactions.     

Development and cross-species testing of western bluebird (Sialia mexicana) microsatellite primers

Western and eastern bluebirds (Sialia mexicana and S. sialis) are socially monogamous passerines that engage in extra‐pair copulations. We obtained microsatellites from S. mexicana and optimized and characterized 15 microsatellite DNA loci in 60 individuals of this species. Primer pairs yielded an average of 13 alleles per locus in western bluebirds (range 3–35 alleles) with an average observed heterozygosity of 0.68 (range 0.27–0.88). All 15 loci also successfully amplified in S. sialis (n = 24), with an average of 11.5 alleles per locus (range 4–26) and an average observed heterozygosity of 0.59 (range 0.22–0.90).     

Influence of central and extremity circumferences on all-cause mortality in men and women

For a given level of adiposity, greater lower body circumferences appear to exert a protective effect on several disease outcomes including cardiovascular disease and diabetes; however, the independent associations between extremity circumferences and mortality have not been widely investigated. The purpose of this study was to determine the independent and shared influences of upper- and lower-body circumferences on the risk of mortality in a population-based sample of adults. The sample included 10,638 adults 20-69 years of age (5,012 men; 5,626 women) from the nationally representative 1981 Canada Fitness Survey (CFS), who were monitored for over 12 years for mortality. BMI was calculated from measured height and weight. Waist, hip, thigh, calf, and upper arm circumferences were measured using a flexible, nonelastic anthropometric tape. Sex-specific proportional hazards regression models were used to evaluate the relationship between standardized values (Z-scores) of extremity circumference measures, waist circumference (WC) and mortality. Age, smoking status, alcohol consumption, and leisure-time physical activity were collected by questionnaire and were included as covariates. During 131,563 person-years of follow-up, there were 340 deaths in men and 231 in women. After mutual adjustment, WC was positively associated with mortality whereas arm, thigh, and calf circumferences were significantly protective in men, and arm and thigh circumferences were protective in women. In conclusion, waist and extremity circumferences appear to have opposite, independent effects on mortality in this sample of Canadians. Independent of BMI and WC, men and women with larger extremity circumferences had a lower risk of mortality.     

Transparent adult zebrafish as a tool for in vivo transplantation analysis

The zebrafish is a useful model for understanding normal and cancer stem cells, but analysis has been limited to embryogenesis due to the opacity of the adult fish. To address this, we have created a transparent adult zebrafish in which we transplanted either hematopoietic stem/progenitor cells or tumor cells. In a hematopoiesis radiation recovery assay, transplantation of GFP-labeled marrow cells allowed for striking in vivo visual assessment of engraftment from 2 hr-5 weeks posttransplant. Using FACS analysis, both transparent and wild-type fish had equal engraftment, but this could only be visualized in the transparent recipient. In a tumor engraftment model, transplantation of RAS-melanoma cells allowed for visualization of tumor engraftment, proliferation, and distant metastases in as little as 5 days, which is not seen in wild-type recipients until 3 to 4 weeks. This transparent adult zebrafish serves as the ideal combination of both sensitivity and resolution for in vivo stem cell analyses.     

Innate and Learned Predator Recognition Mediated by Chemical Signals in Eurycea nana

Effective and efficient predator recognition and avoidance are essential for the persistence of prey populations, especially in habitats where non-native predators have been introduced. Predator recognition studies are commonly couched within a learned or innate dichotomous framework; however, characteristics of some systems or species could favor innate recognition combined with the ability to alter avoidance responses based on experience with predators. Eurycea nana is a fully aquatic salamander inhabiting a system with a diverse, yet temporally stable, community of native and non-native opportunistically foraging fish predators. To examine predator recognition, we examined avoidance responses (decreased activity) of predator-naïve (first-generation, captive-reared) and predator-experienced (recently collected) E. nana to the chemical cues of a native predator, a non-native predator, a non-predator, and a blank control. Both predator-naïve and predator-experienced E. nana significantly lowered activity in response to the native fish predator when compared with a blank control. Interestingly, predator-naïve E. nana decreased activity in response to the non-native fish predator while predator-experienced E. nana did not. These results indicate that while there is an innate component to predator recognition in E. nana , experience and risk assessment may also be important.