The myriad roles of cyclic AMP in microbial pathogens: from signal to sword

All organisms must sense and respond to their external environments, and this signal transduction often involves second messengers such as cyclic nucleotides. One such nucleotide is cyclic AMP, a universal second messenger that is used by diverse forms of life, including mammals, fungi, protozoa and bacteria. In this review, we discuss the many roles of cAMP in bacterial, fungal and protozoan pathogens and its contributions to microbial pathogenesis. These roles include the coordination of intracellular processes, such as virulence gene expression, with extracellular signals from the environment, and the manipulation of host immunity by increasing cAMP levels in host cells during infection.     

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.     

Alpha4beta7/MAdCAM-1 interactions play an essential role in transitioning cryptopatches into isolated lymphoid follicles and a nonessential role in cryptopatch formation

The alpha(4) integrins alpha(4)beta(7) and alpha(4)beta(1), and their ligands mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) and VCAM-1, have diverse functions, including roles in the formation of secondary lymphoid tissues at early time points during the colonization and clustering of the fetal lymphoid tissue inducer (LTi) cells and at later time points during the recruitment of lymphocytes. In this study, we evaluated the role of alpha(4) integrins in the development of a recently appreciated class of intestinal lymphoid tissues, isolated lymphoid follicles (ILFs). We observed that diverse ILF cellular populations express alpha(4)beta(7) and alpha(4)beta(1), including the LTi-like cells and lymphocytes, while ILF stromal cells and vessels within ILFs express VCAM-1 and MAdCAM-1, respectively. Evaluation of adult and neonatal beta(7)(-/-) mice and adult and neonatal mice given blocking Abs to alpha(4)beta(7), MAdCAM-1, or VCAM-1 did not identify a role for alpha(4) integrins in cryptopatch (CP) development; however, these studies demonstrated that alpha(4)beta(7) and MAdCAM-1 are required for the transitioning of CP into lymphoid tissues containing lymphocytes or ILFs. Competitive bone marrow transfers demonstrated that beta(7)(-/-) LTi-like cells had a reduced but not significantly impaired ability to localize to CP. Bone marrow transfers and adoptive transfers of B lymphocytes revealed that beta(7) expression by B lymphocytes was essential for their entry into the developing ILFs. These findings demonstrate an essential role for alpha(4)beta(7)/MAdCAM-1 in ILF development corresponding to the influx of beta(7)-expressing lymphocytes and a nonessential role for beta(7)-localizing LTi-like cells to the small intestine.     

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).     

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.