Impact and control of methicillin-resistant Staphylococcus aureus (MRSA) in long-term care facilities

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a growing concern in long-term care facilities (LTCF). Epidemiologic studies performed in our area have shown high rates of MRSA colonization in elderly residents, and very high rates in some centres. However, the clinical impact of nasal MRSA colonization is often slight. Prevention of MRSA transmission in LTCF includes the application of simple preventive measures with proven effectiveness, such as hand washing, adequately covering of infected wounds, and proper use of antibiotics, without limiting the activities of colonized residents or carrying out active surveillance.     

Detailed architecture of a DNA translocating machine: the high-resolution structure of the bacteriophage phi29 connector particle.

The three-dimensional crystal structure of the bacteriophage phi29 connector has been solved and refined to 2.1A resolution. This 422 kDa oligomeric protein connects the head of the phage to its tail and translocates the DNA into the prohead during packaging. Each monomer has an elongated shape and is composed of a central, mainly alpha-helical domain that includes a three-helix bundle, a distal alpha/beta domain and a proximal six-stranded SH3-like domain. The protomers assemble into a 12-mer, propeller-like, super-structure with a 35 A wide central channel. The surface of the channel is mainly electronegative, but it includes two lysine rings 20 A apart. On the external surface of the particle a hydrophobic belt extends to the concave area below the SH3-like domain, which forms a crown that retains the particle in the head. The lipophilic belt contacts the non-matching symmetry vertex of the capsid and forms a bearing for the connector rotation. The structure suggests a translocation mechanism in which the longitudinal displacement of the DNA along its axis is coupled to connector spinning.     

Combined effects of seed and soil quality drive seedling performance of a late‐successional canopy tree in a tropical forest

Habitat loss and fragmentation affect the structure and functioning of forested ecosystems worldwide, yet we lack an understanding of how species respond to environmental changes. Here, we examined reproductive success and seedling performance of Poulsenia armata (Moraceae) in continuous and fragmented forests of Los Tuxtlas, southern Mexico. We further investigated how maternal habitat and soil conditions manifested in the seedling stage. We determined seed quality and seedling performance by combining isotopic analyses in seed quality with field observations of P. armata fruit production and a common‐‐garden experiment. Soil conditions in forest fragments negatively impacted P. armata reproductive success. Trees of P. armata in forest fragments were smaller in size and produced fewer fruits and smaller seeds with lower quality compared with trees from the continuous forest. The combined effects of maternal habitat and soil conditions determined seedling survival and growth of this tropical tree. Notably, seedlings had restricted plasticity for biomass allocation to roots, limiting the capacity of fragmented populations to compensate for the initial low N content in seeds. Trees in forest fragments at Los Tuxtlas produced offspring competitively inferior and potentially less resilient than counterparts in continuous forest, jeopardizing future persistence of this late‐successional tree species.     

Improved surveillance for early detection of a potential invasive species: the alien Rose-ringed parakeet <Emphasis Type="Italic">Psittacula krameri</Emphasis> in Australia

The Rose-ringed parakeet Psittacula krameri is the most widely introduced parrot in the world, and is an important agricultural pest and competitor with native wildlife. In Australia, it is classified as an ‘extreme threat’, yet captive individuals frequently escape into the wild. The distribution and frequency of incursions are currently unknown, as are the potential impacts of the species in Australia. This lack of critical ecological information greatly limits effective biosecurity surveillance and decision-making efforts. We compiled a unique dataset, which combined passive surveillance sources from government and online resources, for all available information on parakeet detections at-large in Australia. We investigated whether geographic variables successfully predicted parakeet incursions, and used species distribution models to assess the potential distribution and economic impacts on agricultural assets. We recorded 864 incursions for the period 1999–2013; mostly escaped birds reported to missing animal websites. Escapes were reported most frequently within, or around, large cities. Incursions were best predicted by factors related to human presence and activity, such as global human footprint and intensive land uses. We recommend surveillance of high (predicted) establishment areas adjacent to cities where a feral parakeet population could most affect horticultural production. Novel passive surveillance datasets combined with species distribution models can be used to identify the regions where potential invasive species are most likely to establish. Subsequently, active surveillance can be targeted to the areas of highest predicted potential risk. We recommend an integrated approach that includes outreach programs involving local communities, as well as traditional biosecurity surveillance, for detecting new incursions.     

Effects of water stress on respiration in soybean leaves

The effect of water stress on respiration and mitochondrial electron transport has been studied in soybean (Glycine max) leaves, using the oxygen-isotope-fractionation technique. Treatments with three levels of water stress were applied by irrigation to replace 100%, 50%, and 0% of daily water use by transpiration. The levels of water stress were characterized in terms of light-saturated stomatal conductance (g(s)): well irrigated (g(s) > 0.2 mol H(2)O m(-2) s(-1)), mildly water stressed (g(s) between 0.1 and 0.2 mol H(2)O m(-2) s(-1)), and severely water stressed (g(s) < 0.1 mol H(2)O m(-2) s(-1)). Although net photosynthesis decreased by 40% and 70% under mild and severe water stress, respectively, the total respiratory oxygen uptake (V(t)) was not significantly different at any water-stress level. However, severe water stress caused a significant shift of electrons from the cytochrome to the alternative pathway. The electron partitioning through the alternative pathway increased from 10% to 12% under well-watered or mild water-stress conditions to near 40% under severe water stress. Consequently, the calculated rate of mitochondrial ATP synthesis decreased by 32% under severe water stress. Unlike many other stresses, water stress did not affect the levels of mitochondrial alternative oxidase protein. This suggests a biochemical regulation (other than protein synthesis) that causes this mitochondrial electron shift.     

Brain-derived neurotrophic factor modulates dopaminergic deficits in a transgenic mouse model of Huntington's disease

Dysfunction of dopaminergic neurons may contribute to motor impairment in Huntington's disease. Here, we study the role of brain-derived neurotrophic factor (BDNF) in alterations of the nigrostriatal system associated with transgenics carrying mutant huntingtin. Using huntingtin-BDNF+/- double-mutant mice, we analyzed the effects of reducing the levels of BDNF expression in a model of Huntington's disease (R6/1). When compared with R6/1 mice, these mice exhibit an increased number of aggregates in the substantia nigra pars compacta. In addition, reduction of BDNF expression exacerbates the dopaminergic neuronal dysfunction seen in mutant huntingtin mice, such as the decrease in retrograde labelling of dopaminergic neurons and striatal dopamine content. However, mutant huntingtin mice with normal or lowered BDNF expression show the same decrease in the anterograde transport, number of dopaminergic neurons and nigral volume. In addition, reduced BDNF expression causes decreased dopamine receptor expression in mutant huntingtin mice. Examination of changes in locomotor activity induced by dopamine receptor agonists revealed that, in comparison with R6/1 mice, the double mutant mice exhibit lower activity in response to amphetamine, but not to apomorphine. In conclusion, these findings demonstrate that the decreased BDNF expression observed in Huntington's disease exacerbates dopaminergic neuronal dysfunction, which may participate in the motor disturbances associated with this neurodegenerative disorder.     

Peptide binding induces large scale changes in inter-domain mobility in human Pin1

Pin1 is a peptidyl-prolyl cis/trans isomerase (PPIase) essential for cell cycle regulation. Pin1-catalyzed peptidyl-prolyl isomerization provides a key conformational switch to activate phosphorylation sites with the common phospho-Ser/Thr-Pro sequence motif. This motif is ubiquitously exploited in cellular response to a variety of signals. Pin1 is able to bind phospho-Ser/Thr-Pro-containing sequences at two different sites that compete for the same substrate. One binding site is located within the N-terminal WW domain, which is essential for protein targeting and localization. The other binding site is located in the C-terminal catalytic domain, which is structural homologous to the FK506-binding protein (FKBP) class of PPIases. A flexible linker of 12 residues connects the WW and catalytic domain. To characterize the structure and dynamics of full-length Pin1 in solution, high resolution NMR methods have been used to map the nature of interactions between the two domains of Pin1. In addition, the influence of target peptides on domain interactions has been investigated. The studies reveal a dynamic picture of the domain interactions. 15N spin relaxation data, differential chemical shift mapping, and residual dipolar coupling data indicate that Pin1 can either behave as two independent domains connected by the flexible linker or as a single intact domain with some amount of hinge bending motion depending on the sequence of the bound peptide. The functional importance of the modulation of relative domain flexibility in light of the multitude of interaction partners of Pin1 is discussed.     

CARD15/NOD2 mutational analysis and genotype-phenotype correlation in 612 patients with inflammatory bowel disease

CARD15/NOD2 encodes a protein involved in bacterial recognition by monocytes. Mutations in CARD15 have recently been found in patients with Crohn disease (CD), a chronic inflammatory condition of the digestive tract. Here, we report the mutational analyses of CARD15 in 453 patients with CD, including 166 sporadic and 287 familial cases, 159 patients with ulcerative colitis (UC), and 103 healthy control subjects. Of 67 sequence variations identified, 9 had an allele frequency >5% in patients with CD. Six of them were considered to be polymorphisms, and three (R702W, G908R, and 1007fs) were confirmed to be independently associated with susceptibility to CD. Also considered as potential disease-causing mutations (DCMs) were 27 rare additional mutations. The three main variants (R702W, G908R, and 1007fs) represented 32%, 18%, and 31%, respectively, of the total CD mutations, whereas the total of the 27 rare mutations represented 19% of DCMs. Altogether, 93% of the mutations were located in the distal third of the gene. No mutations were found to be associated with UC. In contrast, 50% of patients with CD carried at least one DCM, including 17% who had a double mutation. This observation confirmed the gene-dosage effect in CD. The patients with double-dose mutations were characterized by a younger age at onset (16.9 years vs. 19.8 years; P=.01), a more frequent stricturing phenotype (53% vs. 28%; P=.00003; odds ratio 2.92), and a less frequent colonic involvement (43% vs. 62%; P=.003; odds ratio 0.44) than were seen in those patients who had no mutation. The severity of the disease and extraintestinal manifestations were not different for any of the CARD15 genotypes. The proportion of familial and sporadic cases and the proportion of patients with smoking habits were similar in the groups of patients with CD with or without mutation. These findings provide tools for a DNA-based test of susceptibility and for genetic counseling in inflammatory bowel disease.     

Ultracentrifugation micromethod for preparation of small experimental animal lipoproteins

Sequential flotation ultracentrifugation is commonly used in the preparation of plasma lipoproteins. However, protocols often require prolonged centrifugation time (48-72 h) and large plasma volumes (2-20 ml), which makes them unsuitable for studies on small laboratory animals. Although analytical techniques such as FPLC have often small sample requirements, further fraction analysis is often limited to the small fraction volume obtained. A sequential ultracentrifugation micromethod is described to obtain rat lipoprotein fractions from 400 microl of plasma in a cumulative centrifugation time of 7.5 h. Fraction volumes were determined and densities were adjusted to those of rat plasma lipoproteins. Polyacrylamide gel electrophoresis and enzymatic measurements of triglycerides, total cholesterol, and phospholipids were used to assess the purity of the lipoprotein fractions. The results were compared with those obtained from a classical sequential ultracentrifugation protocol. The micromethod presented here can be further adapted to other experimental animal species with little modifications.     

The structure of human prokallikrein 6 reveals a novel activation mechanism for the kallikrein family

Zyme/protease M/neurosin/human kallikrein 6 (hK6) is a member of the human kallikrein family of trypsin-like serine proteinases and was originally identified as being down-regulated in metastatic breast and ovarian tumors when compared with corresponding primary tumors. Recent evidence suggests that hK6 may serve as a circulating tumor marker in ovarian cancers. In addition, it was described in the brain of Parkinson's disease and Alzheimer's disease patients, where it is implicated in amyloid precursor protein processing. It is thus a biomarker for these diseases. To examine the mechanism of activation of hK6, we have solved the structure of its proform, the first of a human kallikrein family member. The proenzyme displays a fold that exhibits chimeric features between those of trypsinogen and other family members. It lacks the characteristic "kallikrein loop" and forms the six disulfide bridges of trypsin. Pro-hK6 displays a completely closed specificity pocket and a unique conformation of the regions involved in structural rearrangements upon proteolytic cleavage activation. This points to a novel activation mechanism, which could be extrapolated to other human kallikreins.