Structural basis for c-di-GMP-mediated inside-out signaling controlling periplasmic proteolysis

The bacterial second messenger bis-(3'-5') cyclic dimeric guanosine monophosphate (c-di-GMP) has emerged as a central regulator for biofilm formation. Increased cellular c-di-GMP levels lead to stable cell attachment, which in Pseudomonas fluorescens requires the transmembrane receptor LapD. LapD exhibits a conserved and widely used modular architecture containing a HAMP domain and degenerate diguanylate cyclase and phosphodiesterase domains. c-di-GMP binding to the LapD degenerate phosphodiesterase domain is communicated via the HAMP relay to the periplasmic domain, triggering sequestration of the protease LapG, thus preventing cleavage of the surface adhesin LapA. Here, we elucidate the molecular mechanism of autoinhibition and activation of LapD based on structure-function analyses and crystal structures of the entire periplasmic domain and the intracellular signaling unit in two different states. In the absence of c-di-GMP, the intracellular module assumes an inactive conformation. Binding of c-di-GMP to the phosphodiesterase domain disrupts the inactive state, permitting the formation of a trans-subunit dimer interface between adjacent phosphodiesterase domains via interactions conserved in c-di-GMP-degrading enzymes. Efficient mechanical coupling of the conformational changes across the membrane is realized through an extensively domain-swapped, unique periplasmic fold. Our structural and functional analyses identified a conserved system for the regulation of periplasmic proteases in a wide variety of bacteria, including many free-living and pathogenic species.     

Screening for bacterial-fungal associations in a south-eastern US salt marsh using pre-established fungal monocultures

Both bacteria and fungi play critical roles in decomposition processes in many natural environments, yet only rarely have they been studied as an integrated community. We examined whether physical associations exist between individual bacterial and fungal species that co-occur on decaying smooth cordgrass, Spartina alterniflora, in a south-eastern US salt marsh. Fungal-pervaded decaying Spartina was used as "bait" for potential bacterial associates. The bundles (infiltrated with one of three dominant fungal members of the decomposer assemblage, or an autoclaved control) were placed in a salt marsh and collected biweekly for 6 weeks during the first experiment (late summer 2002), and weekly for 3 weeks during the second experiment (early summer 2003). Terminal-restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes was used to track colonization by bacterial taxa in association with the established fungal species. T-RFLP analysis of 18S-to-28S internal transcribed spacer (ITS) regions was used to monitor changes in fungal communities once bundles had been placed in the field. Results from both years were nearly identical, and showed that invasion by fungi other than the bait species was slow, resulting in a virtual fungal monoculture for several weeks into the experiments. Surprisingly, bacterial communities were unaffected by the identity of the fungal bait. Regardless of the fungal species, and even in the absence of prior fungal colonization, bacterial 16S rRNA profiles were remarkably similar. These results suggest that few species-specific associations, either positive or negative, exist between bacterial and fungal members of the Spartina decomposer community during initial colonization.     

Fingerprint formation

Fingerprints (epidermal ridges) have been used as a means of identifications for more than 2000 years. They have also been extensively studied scientifically by anthropologists and biologists. However, despite all the empirical and experimental knowledge, no widely accepted explanation for the development of epidermal ridges on fingers, palms and soles has yet emerged. In this article we argue that fingerprint patterns are created as the result of a buckling instability in the basal cell layer of the fetal epidermis. Analysis of the well-known von Karman equations informs us that the buckling direction is perpendicular to the direction of greatest stress in the basal layer. We propose that this stress is induced by resistance of furrows and creases to the differential growth of the basal layer and regression of the volar pads during the time of ridge formation. These ideas have been tested by computer experiments. The results are in close harmony with observations. Specifically, they are consistent with the well-known observation that the pattern type is related to the geometry of the fingertip surface when fingerprint patterns are formed.     

Polygonal planforms and phyllotaxis on plants

We demonstrate how phyllotaxis (the arrangement of leaves on plants) and the ribbed, hexagonal, or parallelogram planforms on plants can be understood as the energy-minimizing buckling pattern of a compressed sheet (the plant's tunica) on an elastic foundation. The key idea is that the elastic energy is minimized by configurations consisting of special triads of periodic deformations. We study the conditions that lead to continuous or discontinuous transitions between patterns, state testable predictions, and suggest experiments to test the theory.     

In situ formation of blends by photopolymerization of poly(ethylene glycol) dimethacrylate and polylactide

Blends of cross-linked poly(ethylene glycol) dimethacrylate (PEGDMA) and poly(d,l-lactide) (PLA) were prepared by mixing photoactive PEGDMA (molecular mass: 875 g/mol) and PLA, and subsequently photopolymerizing the mixture with visible light. The effects of PLA molecular mass and mass fraction on the rheological properties of the PEGDMA/PLA mixtures, and on the degree of methacrylate vinyl conversion (DC), as well as blend miscibility, microstructure, mechanical properties, in vitro swelling behavior, and cell responses were studied. PLA-2K (molecular mass: 2096 g/mol) and PLA-63K (molecular mass: 63 000 g/mol) formed miscible and partially miscible blends with cross-linked PEGDMA, respectively. The addition of the PLA-2K did not affect the immediate or post-cure (>24 h) DC of the PEGDMA upon photopolymerization. However, the addition of PLA-63K decreased the immediate DC of the PEGDMA, which can be increased through extending the curing time or post-curing period. Compared to the cross-linked neat PEGDMA and PLA-2K/PEGDMA blends, PLA-63K/PEGDMA blends were significantly stronger, stiffer, and tougher. Both types of blends and the cross-linked PEGDMA swelled when soaked in a phosphate buffered saline (PBS) solution. The attachment and spreading of MCT3-E1 cells increased with increasing PLA-63K content in the blends. The facile and rapid formation of PEGDMA/PLA blends by photopolymerization represents a simple and efficient approach to a class of biomaterials with a broad spectrum of properties.     

Affinity purification of streptavidin using tobacco mosaic virus particles as purification tags

A new protein affinity purification system has been developed. Recombinant tobacco mosaic virus (TMV) was used as an affinity matrix for isolation and purification of the given protein of interest. In model experiments, streptavidin-specific heptapeptide sequence TLIAHPQ was inserted into TMV coat protein near the C end. This oligopeptide did not interfere significantly with viral replication, assembly, and movement. Recombinant TMV functioned as an epitope tag recognizing streptavidin in plant protein extracts. Plant protein extracts containing streptavidin were incubated with recombinant TMV virions. Affinity complexes of viral particles with the protein of interest were collected by centrifugation. Recombinant TMV-streptavidin complex was dissociated with 0.2M acetic acid, pH 4.6, and was passed through membrane filter Nanosep 300K by centrifugation. The filtrate contained pure streptavidin. Recombinant TMV was left on the filter. TMV particles collected from the filter could be used for at least two more purification cycles. The streptavidin-specific recombinant TMV system was applied successfully for purification of streptavidin from Streptomyces avidinii. The authors believe that the TMV-based affinity system can also be used for the purification of other proteins.     

The potent colon carcinogen, 1,2-dimethylhydrazine induces mutations primarily in the colon

1,2-Dimethylhydrazine (DMH) is a potent colon carcinogen that is commonly used as an initiator in studies of the effects of diet on colon cancer. Previous studies have shown that although this compound produces multiple tumors in the colons in most individuals of every species tested, it is, at best, marginally mutagenic in the bone marrow (micronuclei) and small intestine (Dlb-1 mutations). Here we report its mutagenicity in the primary target tissue, the colonic epithelium, by means of the Mutatrade markMouse cII assay, an assay for intragenic mutations in a lambda shuttle vector that is integrated into the genome of these mice. Animals were treated with 0, 10, 20, or 30 mg/ml of DMH, either as a single injection or as multiple weekly injections, and mutations were measured in both the small intestine and colon. In the small intestine, there was an increase in mutant frequency following a single injection of DMH, but this was significant only at 30 mg/kg [induced mutant frequency (MF) = 18 x 10(-5) mutants/plaque]. In the colon, following a single treatment of DMH, there was a significant increase in mutant frequency at doses of 20 and 30 mg/kg (induced MF = 17 x 10(-5) and 23 x 10(-5) mutants/plaque, respectively). Following ten injections of 20 mg/kg of DMH, there was a greater than ten-fold increase in mutations in the colon (MF = 275 x 10(-5) mutants/plaque) than the small intestine (MF = 25 x 10(-5) mutants/plaque). These results show that DMH, under the conditions typically used for dietary studies, induces large numbers of mutations in the tissue in which it induces most cancers.     

Plastid stromules are induced by stress treatments acting through abscisic acid

Stromules are highly dynamic stroma-filled tubules that extend from the surface of all plastid types in all multi-cellular plants examined to date. The stromule frequency (percentage of plastids with stromules) has generally been regarded as characteristic of the cell and tissue type. However, the present study shows that various stress treatments, including drought and salt stress, are able to induce stromule formation in the epidermal cells of tobacco hypocotyls and the root hairs of wheat seedlings. Application of abscisic acid (ABA) to tobacco and wheat seedlings induced stromule formation very effectively, and application of abamine, a specific inhibitor of ABA synthesis, prevented stromule induction by mannitol. Stromule induction by ABA was dependent on cytosolic protein synthesis, but not plastid protein synthesis. Stromules were more abundant in dark-grown seedlings than in light-grown seedlings, and the stromule frequency was increased by transfer of light-grown seedlings to the dark and decreased by illumination of dark-grown seedlings. Stromule formation was sensitive to red and far-red light, but not to blue light. Stromules were induced by treatment with ACC (1-aminocyclopropane-1-carboxylic acid), the first committed ethylene precursor, and by treatment with methyl jasmonate, but disappeared upon treatment of seedlings with salicylate. These observations indicate that abiotic, and most probably biotic, stresses are able to induce the formation of stromules in tobacco and wheat seedlings.     

Children who acquire HIV infection perinatally are at higher risk of early death than those acquiring infection through breastmilk: a meta-analysis

BACKGROUND: Assumptions about survival of HIV-infected children in Africa without antiretroviral therapy need to be updated to inform ongoing UNAIDS modelling of paediatric HIV epidemics among children. Improved estimates of infant survival by timing of HIV-infection (perinatally or postnatally) are thus needed. METHODOLOGY/PRINCIPAL FINDINGS: A pooled analysis was conducted of individual data of all available intervention cohorts and randomized trials on prevention of HIV mother-to-child transmission in Africa. Studies were right-censored at the time of infant antiretroviral initiation. Overall mortality rate per 1000 child-years of follow-up was calculated by selected maternal and infant characteristics. The Kaplan-Meier method was used to estimate survival curves by child's HIV infection status and timing of HIV infection. Individual data from 12 studies were pooled, with 12,112 children of HIV-infected women. Mortality rates per 1,000 child-years follow-up were 39.3 and 381.6 for HIV-uninfected and infected children respectively. One year after acquisition of HIV infection, an estimated 26% postnatally and 52% perinatally infected children would have died; and 4% uninfected children by age 1 year. Mortality was independently associated with maternal death (adjusted hazard ratio 2.2, 95%CI 1.6-3.0), maternal CD4<350 cells/ml (1.4, 1.1-1.7), postnatal (3.1, 2.1-4.1) or peri-partum HIV-infection (12.4, 10.1-15.3). CONCLUSIONS/RESULTS: These results update previous work and inform future UNAIDS modelling by providing survival estimates for HIV-infected untreated African children by timing of infection. We highlight the urgent need for the prevention of peri-partum and postnatal transmission and timely assessment of HIV infection in infants to initiate antiretroviral care and support for HIV-infected children.