A new method for the improvement of data production in phytochemical analysis

Introduction – The use of the average analytical signal for the construction of curves by the least squares method (LSM) over the standard addition method (SAM) is widespread. It would be advantageous, however, to find a way to avoid intermediary averages, which are known to be the cause of significant increases in standard deviations (SD). Objective – To develop a protocol that uses all gathered data to create curves by LSM over SAM. To use Excel^® for the estimation of y = mx + b and R² rather than using LSM equations for the SD of m, x and b. Methodology – The level of lead (II) in the bark (cork) of Quercus suber Linnaeus was determined using differential pulse anodic stripping voltammetry (DPASV). Three current samples were taken for each of the four standard additions. These signals were combined for adjustment by LSM. The results were compared with those obtained after averaging the current for each addition, and the expression of uncertainty in the measurements determined. Results – The new method shows an expanded uncertainty of ± 0.3321 μg/g (nearly 1.42%). The difference between the results obtained by the new and the old method is 0.01 μg/g (23.41 and 23.40 μg/g). The limit of detection changed approximately from 4.8 to 4 μg/g and the relative SD approximately from 9 to 6%. Conclusion – The absence of intermediary averages in curves improved the determination of lead (II) in cork by DPASV. Estimation of SD only with LSM equations produced results that were significantly worse. The changes are large enough to transform an apparently internally non‐validated procedure (repeatability for precision) into an internally validated procedure. Copyright © 2009 John Wiley & Sons, Ltd.     

Intravoxel Incoherent Motion Diffusion Weighted MR Imaging at 3.0 T: Assessment of Steatohepatitis and Fibrosis Compared with Liver Biopsy in Type 2 Diabetic Patients

ObjectiveTo evaluate the capability of intravoxel incoherent motion (IVIM) diffusion-weighted imaging (DWI) to assess steatohepatitis and fibrosis determined by histopathology in type 2 diabetic patients.MethodsFifty-nine type 2 diabetic patients (49 women, 10 men; mean age, 54 ± 9 years) were submitted to liver biopsy for the evaluation of non-alcoholic fatty liver disease (NAFLD) and underwent DWI on a 3.0T MR system using 10 b values. Institutional approval and patient consent were obtained. Pure molecular-based (D), perfusion-related (D*), and vascular fraction (f) were calculated using a double exponential model and least squares curve fitting. D, D*, and f were compared between patients with and without steatohepatitis and between patients with and without fibrosis. The variables were compared by using the Ranksum test and Student t-test.ResultsSteatohepatitis was observed in 22 patients and fibrosis in 16 patients. A lower D median (0.70 s/mm² vs. 0.83 s/mm², p<0.05) and a lower D* median (34.39 s/mm² vs. 45.23 s/mm², p<0.05) were observed among those with steatohepatitis. A lower D median (0.70 s/mm² vs. 0.82 s/mm², p<0.05) and a lower D* median (35.01 s/mm² vs. 44.76 s/mm², p=0.05) were also observed among those with fibrosis.ConclusionIVIM-DWI has the potential to aid in the characterization of steatohepatitis and fibrosis.     

Comparison of Unplanned Intensive Care Unit Readmission Scores: A Prospective Cohort Study

PurposeEarly discharge from the intensive care unit (ICU) may constitute a strategy of resource consumption optimization; however, unplanned readmission of hospitalized patients to an ICU is associated with a worse outcome. We aimed to compare the effectiveness of the Stability and Workload Index for Transfer score (SWIFT), Sequential Organ Failure Assessment score (SOFA) and simplified Therapeutic Intervention Scoring System (TISS-28) in predicting unplanned ICU readmission or unexpected death in the first 48 hours after discharge from the ICU.MethodsWe conducted a prospective cohort study in a single tertiary hospital in southern Brazil. All adult patients admitted to the ICU for more than 24 hours from January 2008 to December 2009 were evaluated. SWIFT, SOFA and TISS-28 scores were calculated on the day of discharge from the ICU. A stepwise logistic regression was conducted to evaluate the effectiveness of these scores in predicting unplanned ICU readmission or unexpected death in the first 48 hours after discharge from the ICU. Moreover, we conducted a direct accuracy comparison among SWIFT, SOFA and TISS-28 scores.ResultsA total of 1,277 patients were discharged from the ICU during the study period. The rate of unplanned ICU readmission or unexpected death in the first 48 hours after discharge from the ICU was 15% (192 patients). In the multivariate analysis, age (P = 0.001), length of ICU stay (P = 0.01), cirrhosis (P = 0.03), SWIFT (P = 0.001), SOFA (P = 0.01) and TISS-28 (P<0.001) constituted predictors of unplanned ICU readmission or unexpected death. The SWIFT, SOFA and TISS-28 scores showed similar predictive accuracy (AUC values were 0.66, 0.65 and 0.74, respectively; P = 0.58).ConclusionsSWIFT, SOFA and TISS-28 on the day of discharge from the ICU have only moderate accuracy in predicting ICU readmission or death. The present study did not find any differences in accuracy among the three scores.     

Conformational Changes in a Hyperthermostable Glycoside Hydrolase: Enzymatic Activity Is a Consequence of the Loop Dynamics and Protonation Balance

Endo-β-1, 4-mannanase from Thermotoga petrophila (TpMan) is a modular hyperthermostable enzyme involved in the degradation of mannan-containing polysaccharides. The degradation of these polysaccharides represents a key step for several industrial applications. Here, as part of a continuing investigation of TpMan, the region corresponding to the GH5 domain (TpManGH5) was characterized as a function of pH and temperature. The results indicated that the enzymatic activity of the TpManGH5 is pH-dependent, with its optimum activity occurring at pH 6. At pH 8, the studies demonstrated that TpManGH5 is a molecule with a nearly spherical tightly packed core displaying negligible flexibility in solution, and with size and shape very similar to crystal structure. However, TpManGH5 experiences an increase in radius of gyration in acidic conditions suggesting expansion of the molecule. Furthermore, at acidic pH values, TpManGH5 showed a less globular shape, probably due to a loop region slightly more expanded and flexible in solution (residues Y88 to A105). In addition, molecular dynamics simulations indicated that conformational changes caused by pH variation did not change the core of the TpManGH5, which means that only the above mentioned loop region presents high degree of fluctuations. The results also suggested that conformational changes of the loop region may facilitate polysaccharide and enzyme interaction. Finally, at pH 6 the results indicated that TpManGH5 is slightly more flexible at 65°C when compared to the same enzyme at 20°C. The biophysical characterization presented here is well correlated with the enzymatic activity and provide new insight into the structural basis for the temperature and pH-dependent activity of the TpManGH5. Also, the data suggest a loop region that provides a starting point for a rational design of biotechnological desired features.     

Connective-Tissue Growth Factor (CTGF/CCN2) Induces Astrogenesis and Fibronectin Expression of Embryonic Neural Cells In Vitro

Connective-tissue growth factor (CTGF) is a modular secreted protein implicated in multiple cellular events such as chondrogenesis, skeletogenesis, angiogenesis and wound healing. CTGF contains four different structural modules. This modular organization is characteristic of members of the CCN family. The acronym was derived from the first three members discovered, cysteine-rich 61 (CYR61), CTGF and nephroblastoma overexpressed (NOV). CTGF is implicated as a mediator of important cell processes such as adhesion, migration, proliferation and differentiation. Extensive data have shown that CTGF interacts particularly with the TGFβ, WNT and MAPK signaling pathways. The capacity of CTGF to interact with different growth factors lends it an important role during early and late development, especially in the anterior region of the embryo. ctgf knockout mice have several cranio-facial defects, and the skeletal system is also greatly affected due to an impairment of the vascular-system development during chondrogenesis. This study, for the first time, indicated that CTGF is a potent inductor of gliogenesis during development. Our results showed that in vitro addition of recombinant CTGF protein to an embryonic mouse neural precursor cell culture increased the number of GFAP- and GFAP/Nestin-positive cells. Surprisingly, CTGF also increased the number of Sox2-positive cells. Moreover, this induction seemed not to involve cell proliferation. In addition, exogenous CTGF activated p44/42 but not p38 or JNK MAPK signaling, and increased the expression and deposition of the fibronectin extracellular matrix protein. Finally, CTGF was also able to induce GFAP as well as Nestin expression in a human malignant glioma stem cell line, suggesting a possible role in the differentiation process of gliomas. These results implicate ctgf as a key gene for astrogenesis during development, and suggest that its mechanism may involve activation of p44/42 MAPK signaling. Additionally, CTGF-induced differentiation of glioblastoma stem cells into a less-tumorigenic state could increase the chances of successful intervention, since differentiated cells are more vulnerable to cancer treatments.     

Understanding Low-Acuity Visits to the Pediatric Emergency Department

Background

Canadian pediatric emergency department visits are increasing, with a disproportionate increase in low-acuity visits locally (33% of volume in 2008-09, 41% in 2011-12). We sought to understand: 1) presentation patterns and resource implications; 2) parents’ perceptions and motivations; and 3) alternate health care options considered prior to presenting with low-acuity problems.

Methods

We conducted a prospective cohort study at our tertiary pediatric emergency department serving two provinces to explore differences between patients with and without a primary care provider. During four, 2-week study periods over 1 year, parents of low-acuity visits received an anonymous survey. Presentation times, interventions, diagnoses and dispositions were captured on a data collection form linked to the survey by study number.

Results

Parents completed 2,443 surveys (74.1% response rate), with survey-data collection form pairs available for 2,146 visits. Overall, 89.7% of respondents had a primary care provider; 68% were family physicians. Surprisingly, 40% of visits occurred during weekday office hours and 27.3% occurred within 4 hours of symptom onset; 67.5% of those early presenters were for injuries. Few parents sought care from their primary care provider (25%), health information line (20.7%), or urgent care clinic (18.5%); 36% reported that they believed their child’s problem required the emergency department. Forty-five percent required only a history, physical exam and reassurance; only 11% required an intervention not available in an office setting. Patients without a primary care provider were significantly more likely to present during weekday office hours (p = 0.003), have longer symptom duration (p<0.001), and not know of other options (p = 0.001).

Conclusions

Many parents seek pediatric emergency department care for low-acuity problems despite their child having a primary care provider. Ensuring timely access to these providers may help reduce pediatric emergency department overuse. Educational initiatives should inform parents about low-acuity problems and where appropriate care can/should be accessed.     

Serosurvey of Smooth Brucella,Leptospira spp. and Toxoplasma gondii in Free-Ranging Jaguars (Panthera onca) and Domestic Animals from Brazil

This study investigated the exposure of jaguar populations and domestic animals to smooth Brucella, Leptospira spp. and Toxoplasma gondii in the Cerrado, Pantanal and Amazon biomes of Brazil. Between February 2000 and January 2010, serum samples from 31 jaguars (Panthera onca), 1,245 cattle (Bos taurus), 168 domestic dogs (Canis lupus familiaris) and 29 domestic cats (Felis catus) were collected and analysed by rose bengal test for smooth Brucella, microscopic agglutination test for Leptospira spp. and modified agglutination test for T. gondii. Cattle populations from all sites (9.88%) were exposed to smooth Brucella, but only one jaguar from Cerrado was exposed to this agent. Jaguars captured in the Cerrado (60.0%) and in the Pantanal (45.5%) were seropositive for different serovars of Leptospira spp., cattle (72.18%) and domestic dogs (13.1%) from the three sites and one domestic cat from Pantanal were also seropositive for the agent. The most prevalent serotype of Leptospira spp. identified in jaguars from the Cerrado (Grippotyphosa) and the Pantanal (Pomona) biomes were distinct from those found in the domestic animals sampled. Jaguars (100%), domestic dogs (38.28%) and domestic cats (82.76%) from the three areas were exposed to T. gondii. Our results show that brucellosis and leptospirosis could have been transmitted to jaguars by domestic animals; and jaguars probably play an important role in the maintenance of T. gondii in nature.     

The hypothesis of sympatric speciation as the dominant generator of endemism in a global hotspot of biodiversity

Allopatric or sympatric speciation influence the degree to which closely related species coexist in different manners, altering the patterns of phylogenetic structure and turnover among and between communities. The objective of this study was to examine whether phylogenetic community structure and turnover in the Brazilian Atlantic Forest permit conclusions about the dominant process for the formation of extant angiosperm richness of tree species. Therefore, we analyzed phylogenetic community structure (MPD, MNTD) as well as taxonomic (Jaccard similarity) and phylogenetic turnover (betaMPD, betaMNTD) among and between 49 tree communities distributed among three different habitat types. Mean annual precipitation and mean annual temperature in each survey area were estimated. Phylogenetic community structure does not differ between habitat types, although MPD reduces with mean annual temperature. Jaccard similarity decreases and betaMNTD increases with spatial distance and environmental differences between study sites. Spatial distance explains the largest portions of variance in the data, indicating dispersal limitation and the spatial aggregation of recently formed taxa, as betaMNTD is related to more recent evolutionary events. betaMPD, that is related to deep evolutionary splits, shows no spatial or environmental pattern, indicating that older clades are equally distributed across the Brazilian Atlantic Forest. While similarity pattern indicates dispersal limitations, the spatial turnover of betaMNTD is consistent with a high degree of sympatric speciation generating extant diversity and endemism in the Brazilian Atlantic Forest. More comprehensive approaches are necessary to reduce spatial sampling bias, uncertainties regarding angiosperm diversification patterns and confirm sympatric speciation as the dominant generator for the formation of extant species diversity in the Brazilian Atlantic Forest.     

The effects of the C-terminal amidation of mastoparans on their biological actions and interactions with membrane-mimetic systems

Polycationic peptides may present their C-termini in either amidated or acidic form; however, the effects of these conformations on the mechanisms of interaction with the membranes in general were not properly investigated up to now. Protonectarina-MP mastoparan with an either amidated or acidic C-terminus was utilized to study their interactions with anionic and zwitterionic vesicles, using measurements of dye leakage and a combination of H/D exchange and mass spectrometry to monitor peptide–membrane interactions. Mast cell degranulation, hemolysis and antibiosis assays were also performed using these peptides, and the results were correlated with the structural properties of the peptides. The C-terminal amidation promotes the stabilization of the secondary structure of the peptide, with a relatively high content of helical conformations, permitting a deeper interaction with the phospholipid constituents of animal and bacterial cell membranes. The results suggested that at low concentrations Protonectarina-MP interacts with the membranes in a way that both terminal regions remain positioned outside the external surface of the membrane, while the α-carbon backbone becomes partially embedded in the membrane core and changing constantly the conformation, and causing membrane destabilization. The amidation of the C-terminal residue appears to be responsible for the stabilization of the peptide conformation in a secondary structure that is richer in α-helix content than its acidic congener. The helical, amphipathic conformation, in turn, allows a deeper peptide–membrane interaction, favoring both biological activities that depend on peptide structure recognition by the GPCRs (such as exocytosis) and those activities dependent on membrane perturbation (such as hemolysis and antibiosis).     

Effect of Oxidation on the Structure of Human Low- and High-Density Lipoproteins

This work presents a controlled study of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) structural changes due to in vitro oxidation with copper ions. The changes were studied by small-angle x-ray scattering (SAXS) and dynamic light scattering (DLS) techniques in the case of LDL and by SAXS, DLS, and Z-scan (ZS) techniques in the case of HDL. SAXS data were analyzed with a to our knowledge new deconvolution method. This method provides the electron density profile of the samples directly from the intensity scattering of the monomers. Results show that LDL particles oxidized for 18 h show significant structural changes when compared to nonoxidized particles. Changes were observed in the electrical density profile, in size polydispersity, and in the degree of flexibility of the APO-B protein on the particle. HDL optical results obtained with the ZS technique showed a decrease of the amplitude of the nonlinear optical signal as a function of oxidation time. In contrast to LDL results reported in the literature, the HDL ZS signal does not lead to a complete loss of nonlinear optical signal after 18 h of copper oxidation. Also, the SAXS results did not indicate significant structural changes due to oxidation of HDL particles, and DLS results showed that a small number of oligomers formed in the sample oxidized for 18 h. All experimental results for the HDL samples indicate that this lipoprotein is more resistant to the oxidation process than are LDL particles.