Mutations of Thr169 affect substrate specificity of pyranose 2-oxidase from Trametes multicolor

Site-directed mutagenesis was used to enhance the catalytic activity of pyranose 2-oxidase (P2Ox) from Trametes multicolor with different substrates. To this end, threonine at position 169 was replaced by glycine, alanine and serine, respectively. Using oxygen as electron acceptor the mutant T169G was equally active with d-glucose and d-galactose, whereas wild-type recombinant P2Ox only showed 5.2% relative activity with the latter substrate. When d-galactose was used as electron donor in saturating concentrations, T169G showed a 4.5-fold increase in its catalytic efficiency k_cat/K_M for the alternative electron acceptor 1,4-benzoquinone and a nine-fold increased k_cat/K_M value with the ferricenium ion compared with wt recP2Ox. Variant T169S showed an increase in its catalytic efficiency both with 1,4-benzoquinone (3.7 times) as well as with the ferricenium ion (1.4 times) when d-glucose was the substrate.     

Flow cytometric estimation of ‘labile iron pool’ in human white blood cells reveals a positive association with ageing

A small part of cellular iron, usually called ‘labile iron pool’ (LIP), is not securely stored and has the potential to catalyse the formation of highly reactive oxygen species. The present work estimated LIP levels in human white cells by using the analytical power of flow cytometry. The method relies essentially on already established principles but has the added value of monitoring LIP in different subpopulations of human blood cells concurrently in a single sample. Examination of 41 apparently healthy individuals revealed a positive correlation between LIP levels and the age of the donors (r=0.656, 0.572 and 0.702 for granulocytes, lymphocytes and monocytes, respectively, p<0.0001), indicating that cells of older individuals are prone to oxidations in conditions of oxidative stress. It is suggested that LIP estimation may represent a valuable tool in examinations searching for links between iron and a variety of oxidative stress-related pathological conditions.     

A molecular phylogenetic analysis of Speyeria and its implications for the management of the threatened Speyeria zerene hippolyta

The genetic structure of lineages can provide important information for delineating “evolutionarily significant units” (ESUs) for conservation, and for planning actions to protect and restore taxa threatened with extinction. Speyeria zerene hippolyta, the Oregon silverspot butterfly, is a U.S.A. federally threatened subspecies that is the focus of considerable conservation effort, but whose evolutionary relationships with other Speyeria taxa are not well-understood. We conducted a genetic analysis of nine Speyeria species and 25 subspecies from western U.S.A., using both mitochondrial and nuclear markers. Our goal was to determine whether such data supported (a) S. z. hippolyta’s designation as an ESU, and (b) the current morphologically-based taxonomy of Speyeria spp. Our data for S. z. hippolyta were equivocal; while nuclear markers resolved all these individuals into a single clade, mtDNA data suggested the existence of two clades. Aside from S. cybele, which was consistently supported as monophyletic, our data provided little support for most of the species currently recognized for western U.S. Speyeria, including S. zerene, and even less for the many subspecies designations. These genetic findings stand in contrast to the morphological differences recognized by experts, and suggest a relatively recent origin for many of these taxa. Two of 66 individuals screened for Wolbachia infection tested positive for this symbiont. Our results provide no persuasive evidence that S. z. hippolyta should lose its status as an ESU, but they have important implications for ongoing management actions such as population augmentation.     

Global Analysis of Apicomplexan Protein S‐Acyl Transferases Reveals an Enzyme Essential for Invasion

The advent of techniques to study palmitoylation on a whole proteome scale has revealed that it is an important reversible modification that plays a role in regulating multiple biological processes. Palmitoylation can control the affinity of a protein for lipid membranes, which allows it to impact protein trafficking, stability, folding, signalling and interactions. The publication of the palmitome of the schizont stage of Plasmodium falciparum implicated a role for palmitoylation in host cell invasion, protein export and organelle biogenesis. However, nothing is known so far about the repertoire of protein S‐acyl transferases (PATs) that catalyse this modification in Apicomplexa. We undertook a comprehensive analysis of the repertoire of Asp‐His‐His‐Cys cysteine‐rich domain (DHHC‐CRD) PAT family in Toxoplasma gondii and Plasmodium berghei by assessing their localization and essentiality. Unlike functional redundancies reported in other eukaryotes, some apicomplexan‐specific DHHCs are essential for parasite growth, and several are targeted to organelles unique to this phylum. Of particular interest is DHHC7, which localizes to rhoptry organelles in all parasites tested, including the major human pathogen P. falciparum. TgDHHC7 interferes with the localization of the rhoptry palmitoylated protein TgARO and affects the apical positioning of the rhoptry organelles. This PAT has a major impact on T. gondii host cell invasion, but not on the parasite's ability to egress.     

A simulation model of the Devils Hole pupfish population using monthly length-frequency distributions

The Devils Hole pupfish, Cyprinodon diabolis, is a federally-endangered fish that is endemic to Devils Hole, a discontiguous part of Death Valley National Park in Nye County, Nevada. Due to its status, Devils Hole pupfish monitoring must be non-obtrusive and thereby exclude techniques that require handling fish. Due to a recent decline in pupfish abundance, Devils Hole pupfish managers have expressed a need for a model that describes population dynamics. This population model would be used to identify vulnerable life history stage(s) and inform management actions. We constructed a set of individual-based simulation models designed to explore effects of population processes and evaluate assumptions. We developed a baseline model, whose output best resembled both observed length-frequency data and predicted intra-annual abundance patterns. We then ran simulations with 5 % increases in egg-larval, juvenile, and adult survival rates to better understand Devils Hole pupfish life history, thereby helping identify vulnerable life history stages that should become the target of management actions. Simulation models with temporally constant adult, juvenile, and egg-larval survival rates were able to reproduce observed length-frequency distributions and predicted intra-annual population patterns. In particular, models with monthly adult and juvenile survival rates of 80 % and an egg-larval survival rate of 4.7 % replicated patterns in observed data. Population growth was most affected by 5 % increases in egg-larval survival, whereas adult and juvenile survival rates had similar but lesser effects on population growth. Outputs from the model were used to assess factors suspected of influencing Devils Hole pupfish population decline.     

Reliability and Validity of Instruments for Assessing Perinatal Depression in African Settings: Systematic Review and Meta-Analysis

Background

A major barrier to improving perinatal mental health in Africa is the lack of locally validated tools for identifying probable cases of perinatal depression or for measuring changes in depression symptom severity. We systematically reviewed the evidence on the reliability and validity of instruments to assess perinatal depression in African settings.

Methods and Findings

Of 1,027 records identified through searching 7 electronic databases, we reviewed 126 full-text reports. We included 25 unique studies, which were disseminated in 26 journal articles and 1 doctoral dissertation. These enrolled 12,544 women living in nine different North and sub-Saharan African countries. Only three studies (12%) used instruments developed specifically for use in a given cultural setting. Most studies provided evidence of criterion-related validity (20 [80%]) or reliability (15 [60%]), while fewer studies provided evidence of construct validity, content validity, or internal structure. The Edinburgh postnatal depression scale (EPDS), assessed in 16 studies (64%), was the most frequently used instrument in our sample. Ten studies estimated the internal consistency of the EPDS (median estimated coefficient alpha, 0.84; interquartile range, 0.71-0.87). For the 14 studies that estimated sensitivity and specificity for the EPDS, we constructed 2 x 2 tables for each cut-off score. Using a bivariate random-effects model, we estimated a pooled sensitivity of 0.94 (95% confidence interval [CI], 0.68-0.99) and a pooled specificity of 0.77 (95% CI, 0.59-0.88) at a cut-off score of ≥9, with higher cut-off scores yielding greater specificity at the cost of lower sensitivity.

Conclusions

The EPDS can reliably and validly measure perinatal depression symptom severity or screen for probable postnatal depression in African countries, but more validation studies on other instruments are needed. In addition, more qualitative research is needed to adequately characterize local understandings of perinatal depression-like syndromes in different African contexts.     

Curcumin Intake Affects miRNA Signature in Murine Melanoma with mmu-miR-205-5p Most Significantly Altered

Melanoma is the most aggressive form of skin cancer with estimated 48,000 deaths per year worldwide. The polyphenol curcumin derived from the plant Curcuma longa is well known for its anti-inflammatory and anti-cancerogenic properties. Accordingly, dietary intake of this compound may be suitable for melanoma prevention. However, how this compound affects basic cellular mechanisms in developing melanoma still remains elusive. Therefore, the aim of this study was to investigate for the first time the impact of oral curcumin administration on the miRNA signature of engrafting melanoma. For this purpose, the effects of a 4% curcumin diet were tested on melanoma, which were established by injection of murine B78H1 cells in the flank of C57BL/6 mice. Curcumin diet or standard chow (control) was administered two weeks prior to injection of tumor cells until termination of the experiment. High throughput chip-based array analysis was deployed to detect alterations in the miRNA signature of the tumors. Curcumin treatment significantly reduced the growth of the flank tumors. Furthermore the miRNA expression signature in tumors was substantially altered by curcumin intake with mmu-miR-205-5p over 100 times higher expressed when compared to controls. The expression levels of identified key miRNAs in the tumor samples were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). A comparable expression pattern of these miRNAs was also detected in other curcumin-treated melanoma cell lines under in vitro conditions. Putative targets of curcumin-induced up-regulated miRNAs were enriched in ‘o-glycan biosynthesis’, ‘endoplasmatic reticulum protein processing’ and different cancer-related pathways. Western Blot analyses revealed that of these targets anti-apoptotic B-cell CLL/lymphoma 2 (Bcl-2) and proliferating cell nuclear antigen (PCNA) were significantly down-regulated in curcumin-treated tumors. These findings demonstrate a profound alteration of the miRNA expression signature in engrafting curcumin-treated melanoma with mmu-miR-205-5p being up-regulated most significantly.     

Personal and Household Hygiene,Environmental Contamination,and Health in Undergraduate Residence Halls in New York City, 2011

Background

While several studies have documented the importance of hand washing in the university setting, the added role of environmental hygiene remains poorly understood. The purpose of this study was to characterize the personal and environmental hygiene habits of college students, define the determinants of hygiene in this population, and assess the relationship between reported hygiene behaviors, environmental contamination, and health status.

Methods

501 undergraduate students completed a previously validated survey assessing baseline demographics, hygiene habits, determinants of hygiene, and health status. Sixty survey respondents had microbiological samples taken from eight standardized surfaces in their dormitory environment. Bacterial contamination was assessed using standard quantitative bacterial culture techniques. Additional culturing for coagulase-positive Staphylococcus and coliforms was performed using selective agar.

Results

While the vast majority of study participants (n = 461, 92%) believed that hand washing was important for infection prevention, there was a large amount of variation in reported personal hygiene practices. More women than men reported consistent hand washing before preparing food (p = .002) and after using the toilet (p = .001). Environmental hygiene showed similar variability although 73.3% (n = 367) of subjects reported dormitory cleaning at least once per month. Contamination of certain surfaces was common, with at least one third of all bookshelves, desks, refrigerator handles, toilet handles, and bathroom door handles positive for >10 CFU of bacteria per 4 cm² area. Coagulase-positive Staphylococcus was detected in three participants'' rooms (5%) and coliforms were present in six students'' rooms (10%). Surface contamination with any bacteria did not vary by frequency of cleaning or frequency of illness (p>.05).

Conclusions

Our results suggest that surface contamination, while prevalent, is unrelated to reported hygiene or health in the university setting. Further research into environmental reservoirs of infectious diseases may delineate whether surface decontamination is an effective target of hygiene interventions in this population.     

Red-Backed Vole Brain Promotes Highly Efficient In Vitro Amplification of Abnormal Prion Protein from Macaque and Human Brains Infected with Variant Creutzfeldt-Jakob Disease Agent

Rapid antemortem tests to detect individuals with transmissible spongiform encephalopathies (TSE) would contribute to public health. We investigated a technique known as protein misfolding cyclic amplification (PMCA) to amplify abnormal prion protein (PrP^TSE) from highly diluted variant Creutzfeldt-Jakob disease (vCJD)-infected human and macaque brain homogenates, seeking to improve the rapid detection of PrP^TSE in tissues and blood. Macaque vCJD PrP^TSE did not amplify using normal macaque brain homogenate as substrate (intraspecies PMCA). Next, we tested interspecies PMCA with normal brain homogenate of the southern red-backed vole (RBV), a close relative of the bank vole, seeded with macaque vCJD PrP^TSE. The RBV has a natural polymorphism at residue 170 of the PrP-encoding gene (N/N, S/S, and S/N). We investigated the effect of this polymorphism on amplification of human and macaque vCJD PrP^TSE. Meadow vole brain (170N/N PrP genotype) was also included in the panel of substrates tested. Both humans and macaques have the same 170S/S PrP genotype. Macaque PrP^TSE was best amplified with RBV 170S/S brain, although 170N/N and 170S/N were also competent substrates, while meadow vole brain was a poor substrate. In contrast, human PrP^TSE demonstrated a striking narrow selectivity for PMCA substrate and was successfully amplified only with RBV 170S/S brain. These observations suggest that macaque PrP^TSE was more permissive than human PrP^TSE in selecting the competent RBV substrate. RBV 170S/S brain was used to assess the sensitivity of PMCA with PrP^TSE from brains of humans and macaques with vCJD. PrP^TSE signals were reproducibly detected by Western blot in dilutions through 10^-12 of vCJD-infected 10% brain homogenates. This is the first report showing PrP^TSE from vCJD-infected human and macaque brains efficiently amplified with RBV brain as the substrate. Based on our estimates, PMCA showed a sensitivity that might be sufficient to detect PrP^TSE in vCJD-infected human and macaque blood.     

The 1.6 ? Crystal Structure of Pyranose Dehydrogenase from Agaricus meleagris Rationalizes Substrate Specificity and Reveals a Flavin Intermediate

Pyranose dehydrogenases (PDHs) are extracellular flavin-dependent oxidoreductases secreted by litter-decomposing fungi with a role in natural recycling of plant matter. All major monosaccharides in lignocellulose are oxidized by PDH at comparable yields and efficiencies. Oxidation takes place as single-oxidation or sequential double-oxidation reactions of the carbohydrates, resulting in sugar derivatives oxidized primarily at C2, C3 or C2/3 with the concomitant reduction of the flavin. A suitable electron acceptor then reoxidizes the reduced flavin. Whereas oxygen is a poor electron acceptor for PDH, several alternative acceptors, e.g., quinone compounds, naturally present during lignocellulose degradation, can be used. We have determined the 1.6-Å crystal structure of PDH from Agaricus meleagris. Interestingly, the flavin ring in PDH is modified by a covalent mono- or di-atomic species at the C(4a) position. Under normal conditions, PDH is not oxidized by oxygen; however, the related enzyme pyranose 2-oxidase (P2O) activates oxygen by a mechanism that proceeds via a covalent flavin C(4a)-hydroperoxide intermediate. Although the flavin C(4a) adduct is common in monooxygenases, it is unusual for flavoprotein oxidases, and it has been proposed that formation of the intermediate would be unfavorable in these oxidases. Thus, the flavin adduct in PDH not only shows that the adduct can be favorably accommodated in the active site, but also provides important details regarding the structural, spatial and physicochemical requirements for formation of this flavin intermediate in related oxidases. Extensive in silico modeling of carbohydrates in the PDH active site allowed us to rationalize the previously reported patterns of substrate specificity and regioselectivity. To evaluate the regioselectivity of D-glucose oxidation, reduction experiments were performed using fluorinated glucose. PDH was rapidly reduced by 3-fluorinated glucose, which has the C2 position accessible for oxidation, whereas 2-fluorinated glucose performed poorly (C3 accessible), indicating that the glucose C2 position is the primary site of attack.