Protocol for DNA extraction of Cryptosporidium spp. oocysts in fecal samples

Molecular characterization of Cryptosporidium spp.oocysts in clinical samples is useful for public health since it allows the study of sources of contamination as well as the transmission in different geographical regions. Although widely used in developed countries, in Brazil it is restricted to academic studies, mostly using commercial kits for the extraction of genomic DNA, or in collaboration with external reference centers, rendering the method expensive and limited. The study proposes the application of the modifications recently introduced in the method improving feasibility with lower cost. This method was efficient for clinical samples preserved at -20 °C for up to six years and the low number of oocysts may be overcomed by repetitions of extraction.

HTLV-1 Tax Specific CD8+ T Cells Express Low Levels of Tim-3 in HTLV-1 Infection: Implications for Progression to Neurological Complications

The T cell immunoglobulin mucin 3 (Tim-3) receptor is highly expressed on HIV-1-specific T cells, rendering them partially ""exhausted'' and unable to contribute to the effective immune mediated control of viral replication. To elucidate novel mechanisms contributing to the HTLV-1 neurological complex and its classic neurological presentation called HAM/TSP (HTLV-1 associated myelopathy/tropical spastic paraparesis), we investigated the expression of the Tim-3 receptor on CD8(+) T cells from a cohort of HTLV-1 seropositive asymptomatic and symptomatic patients. Patients diagnosed with HAM/TSP down-regulated Tim-3 expression on both CD8(+) and CD4(+) T cells compared to asymptomatic patients and HTLV-1 seronegative controls. HTLV-1 Tax-specific, HLA-A*02 restricted CD8(+) T cells among HAM/TSP individuals expressed markedly lower levels of Tim-3. We observed Tax expressing cells in both Tim-3(+) and Tim-3(-) fractions. Taken together, these data indicate that there is a systematic downregulation of Tim-3 levels on T cells in HTLV-1 infection, sustaining a profoundly highly active population of potentially pathogenic T cells that may allow for the development of HTLV-1 complications.

Identification of COL6A1 as a differentially expressed gene in human astrocytomas

Diffuse infiltrating gliomas are the most common tumors of the central nervous system. Gliomas are classified by the WHO according to their histopathological and clinical characteristics into four classes: grade I (pilocytic astrocytoma), grade II (diffuse astrocytoma), grade III (anaplastic astrocytoma), and grade IV (glioblastoma multiforme). Several genes have already been correlated with astrocytomas, but many others are yet to be uncovered. By analyzing the public SAGE data from 21 patients, comprising low malignant grade astrocytomas and glioblastomas, we found COL6A1 to be differentially expressed, confirming this finding by real time RT-PCR in 66 surgical samples. To the best of our knowledge, COL6A1 has never been described in gliomas. The expression of this gene has significantly different means when normal glia is compared with low-grade astrocytomas (grades I and II) and high-grade astrocytomas (grades III and IV), with a tendency to be greater in higher grade samples, thus rendering it a powerful tumor marker.

Shelf and estuarine transport mechanisms affecting the supply of competent larvae in a suite of brachyuran crabs with different life histories

Supply of competent larvae to the benthic habitat is a major determinant of population dynamics in coastal and estuarine invertebrates with an indirect life cycle. Larval delivery may depend not only on physical transport mechanisms, but also on larval behavior and physiological progress to the competent stage. Yet, the combined analysis of such factors has seldom been attempted. We used time-series analyses to examine tide- and wind-driven mechanisms responsible for the supply of crab megalopae to an estuarine river under a major marine influence in SW Spain, and monitored the vertical distribution of upstream moving megalopae, their net flux and competent state. The species Panopeus africanus (estuarine), Brachynotus sexdentatus (euryhaline) and Nepinnotheres pinnotheres (coastal) comprised 80% of the whole sample, and responded in a similar way to tide and wind forcing. Tidal range was positively correlated to supply, with maxima 0 to 1 d after spring tides, suggesting selective tidal stream transport. Despite being extensively subjected to upwelling, downwind drift under the effect of westerlies, not Ekman transport, explained residual supply variation at our sampling area. Once in the estuary, net flux and competence state matched the expected trends. Net upstream flux increased from B. sexdentatus to P. africanus, favoring transport to a sheltered coastal habitat (N. pinnotheres), or to the upper estuary (P. africanus). Competence state was highest in N. pinnotheres, intermediate in B. sexdentatus and lowest in P. africanus, as expected if larvae respond to cues from adequate benthic habitat. P. africanus megalopae were found close to the bottom, not above, rendering slower upstream transport than anticipated.

Squeeze flow as a tool for developing optimized gypsum plasters

This paper presents a rheological investigation of pure gypsum (PG) and a commercial gypsum-lime-filler plaster (CP) using the modified Vicat apparatus and squeeze flow method. The samples were tested at several different intervals after manual or mechanical mixing. The results confirmed squeeze flow to be more sensitive in determining fresh paste behavior than the modified Vicat apparatus. PG set faster when prepared in mechanical mixer than when manually mixed. Conversely, the CP composition presented longer setting when mixed mechanically. The study also included the analysis of two ready-to-use polymer-based products for leveling and rendering (drywall joint compound - DJC; acrylic putty - AP) measured by squeeze flow and compared to the commercial composition. (C) 2008 Elsevier Ltd. All rights reserved.

Impact of adenosine nucleotide translocase (ANT) proline isomerization on Ca(2+)-induced cysteine relative mobility/mitochondrial permeability transition pore

Mitochondrial membrane carriers containing proline and cysteine, such as adenine nucleotide translocase (ANT), are potential targets of cyclophilin D (CyP-D) and potential Ca(2+)-induced permeability transition pore (PTP) components or regulators; CyP-D, a mitochondrial peptidyl-prolyl cis-trans isomerase, is the probable target of the PTP inhibitor cyclosporine A (CsA). In the present study, the impact of proline isomerization (from trans to cis) on the mitochondrial membrane carriers containing proline and cysteine was addressed using ANT as model. For this purpose, two different approaches were used: (i) Molecular dynamic (MD) analysis of ANT-Cys(56) relative mobility and (ii) light scattering techniques employing rat liver isolated mitochondria to assess both Ca(2+)-induced ANT conformational change and mitochondrial swelling. ANT-Pro(61) isomerization increased ANT-Cys(56) relative mobility and, moreover, desensitized ANT to the prevention of this effect by ADP. In addition, Ca(2+) induced ANT ""c"" conformation and opened PTP; while the first effect was fully inhibited, the second was only attenuated by CsA or ADP. Atractyloside (ATR), in turn, stabilized Ca(2+)-induced ANT ""c"" conformation, rendering the ANT conformational change and PTP opening less sensitive to the inhibition by CsA or ADP. These results suggest that Ca(2+) induces the ANT ""c"" conformation, apparently associated with PTP opening, but requires the CyP-D peptidyl-prolyl cis-trans isomerase activity for sustaining both effects.

Structural complexes of human adenine phosphoribosyltransferase reveal novel features of the APRT catalytic mechanism

Adenine phosphoribosyltransferase (APRT) is an important enzyme component of the purine recycling pathway. Parasitic protozoa of the order Kinetoplastida are unable to synthesize purines de novo and use the salvage pathway for the synthesis of purine bases rendering this biosynthetic pathway an attractive target for antiparasitic drug design. The recombinant human adenine phosphoribosyltransferase (hAPRT) structure was resolved in the presence of AMP in the active site to 1.76 angstrom resolution and with the substrates PRPP and adenine simultaneously bound to the catalytic site to 1.83 angstrom resolution. An additional structure was solved containing one subunit of the dimer in the apo-form to 2.10 angstrom resolution. Comparisons of these three hAPRT structures with other `type I` PRTases revealed several important features of this class of enzymes. Our data indicate that the flexible loop structure adopts an open conformation before and after binding of both substrates adenine and PRPR Comparative analyses presented here provide structural evidence to propose the role of Glu 104 as the residue that abstracts the proton of adenine N9 atom before its nucleophilic attack on the PRPP anomeric carbon. This work leads to new insights to the understanding of the APRT catalytic mechanism.

Scalable Visualization of Galaxies, Oceans, and Brains

One of the challenges in scientific visualization is to generate software libraries suitable for the large-scale data emerging from tera-scale simulations and instruments. We describe the efforts currently under way at SDSC and NPACI to address these challenges. The scope of the SDSC project spans data handling, graphics, visualization, and scientific application domains. Components of the research focus on the following areas: intelligent data storage, layout and handling, using an associated “Floor-Plan” (meta data); performance optimization on parallel architectures; extension of SDSC’s scalable, parallel, direct volume renderer to allow perspective viewing; and interactive rendering of fractional images (“imagelets”), which facilitates the examination of large datasets. These concepts are coordinated within a data-visualization pipeline, which operates on component data blocks sized to fit within the available computing resources. A key feature of the scheme is that the meta data, which tag the data blocks, can be propagated and applied consistently. This is possible at the disk level, in distributing the computations across parallel processors; in “imagelet” composition; and in feature tagging. The work reflects the emerging challenges and opportunities presented by the ongoing progress in high-performance computing (HPC) and the deployment of the data, computational, and visualization Grids.

Chemical treatment of Escherichia coli: 3. Selective extraction of a recombinant protein from cytoplasmic inclusion bodies in intact cells

In previous parts of this study we developed procedures for the high-efficiency chemical extraction of soluble and insoluble protein from intact Escherichia coli cells. Although high yields were obtained, extraction of recombinant protein directly from cytoplasmic inclusion bodies led to low product purity due to coextraction of soluble contaminants. In this work, a two-stage procedure for the selective extraction of recombinant protein at high efficiency and high purity is reported. In the first stage, inclusion-body stability is promoted by the addition of 15 mM 2-hydroxyethyldisulfide (2-HEDS), also known as oxidized P-mercaptoethanol, to the permeabil ization buffer (6 M urea + 3 mM ethylenediaminetetra-acetate [EDTA]). 2-HEDS is an oxidizing agent believed to promote disulfide bond formation, rendering the inclusion body resistant to solubilization in 6 M urea. Contaminating proteins are separated from the inclusion-body fraction by centrifugation. in the second stage, disulfide bonds are readily eliminated by including reducing agent (20 mM dithiothreitol [DTT]) into the permeabilization buffer. Extraction using this selective two-stage process yielded an 81% (w/w) recovery of the recombinant protein Long-R-3-IGF-I from inclusion bodies located in the cytoplasm of intact E. coli, at a purity of 46% (w/w). This was comparable to that achieved by conventional extraction (mechanical disruption followed by centrifugation and solubilization). A pilot-scale procedure was also demonstrated using a stirred reactor and diafiltration. This is the first reported study that achieves both high extraction efficiency and selectivity by the chemical treatment of cytoplasmic inclusion bodies in intact bacterial cells. (C) 1999 John Wiley & Sons, Inc.

Colour vision in billfish

Members of the billfish family are highly visual predatory teleosts inhabiting the open ocean. Little is known about their visual abilities in detail, but past studies have indicated that these fishes were:ere likely to be monochromats. This study however, presents evidence of two anatomically distinct cone types in billfish. The cells are arranged in a regular mosaic pattern of single and twin cones as in many fishes, and this arrangement suggests that the different cone types also show different spectral sensitivity, which is the basis for colour vision. First measurements using microspectrophotometry (MSP) revealed a peak absorption of the rod pigment at 484 nm, indicating that MSP, despite technical difficulties, will be a decisive tool in proving colour vision in these offshore fishes. When hunting, billfish such as the sailfish flash bright blue bars on their sides. This colour reflects largely in ultraviolet (UV) light at 350 nm as revealed by spectrophotometric measurements. Billfish lenses block light of wavelengths below 400 nm, presumably rendering the animal blind to the UV component of its own body colour. Interestingly at least two prey species of billfish have lenses transmitting light in the UV waveband and are therefore likely to perceive a large fraction of the UV peak found in the blue bar of the sailfish. The possible biological significance of this finding is discussed.

Depression in women living with HIV: clinical and psychosocial correlates

The number of Brazilian women living with HIV has increased significantly in past years, rendering studies of their particular care demands including psychiatric issues. This study measures the prevalence of major depression, using the Structured Clinical Interview for DSM-IV Axis I Disorders, in a sample of 120 women living with HIV in treatment at a reference centre in So Paulo. Socio-demographic variables, HIV-related clinical and laboratory data, including CD4+ cell counts and HIV plasma viral loads, as well as psychosocial features (intimate relationships, disclosure of HIV serostatus, partner`s serostatus and patient`s emotional and financial support) were investigated as factors potentially associated with depression. The prevalence of major depression at the time of evaluation was 25.8% (95% CI 18.2-33.4%). Clinical status (p = 0.002), lack of emotional support (p = 0.02), use of antidepressants (p = 0.028) and length of time since HIV diagnosis (p = 0.05) were associated with major depression in univariate analysis. In multivariate multiple-regression model, HIV clinical status, lack of emotional support and higher plasma viral loads were associated with depression. Sixty per cent of the women have a major depression diagnosis during lifetime. We conclude that major depression is highly prevalent among women living with HIV, but it is still underdiagnosed and undertreated.

Dormancy release in Australian fire ephemeral seeds during burial increases germination response to smoke water or heat

Fire ephemerals are short-lived plants that primarily germinate after fire. Fresh and laboratory-stored seeds are difficult to germinate ex situ, even in response to fire-related cues such as heat and smoke. Seeds of eight Australian fire ephemeral species were buried in unburnt and recently burnt sites of natural bushland during autumn. Seeds were exhumed after 6 and 12 months and incubated in water and smoke water, either with or without a heat treatment at 70 degrees C for 1 h. Generally, germination did not increase after 6 months of burial, but after 12 months of burial germination was enhanced in seven of the eight species. Actinotus leucocephalus produced higher germination following 12 months of burial without any further treatment, and smoke water and heat further improved germination. The four Gyrostemonaceae species, Codonocarpus cotinifolius, Gyrostemon racemiger, Gyrostemon ramulosus and Tersonia cyathiflora, only germinated in the presence of smoke water, and their germination was enhanced by burial. Burial improved germination in response to a heat treatment in Grevillea scapigera and Alyogyne huegelii seeds, but did not enhance Alyogyne hakeifolia germination. During concurrent dry laboratory storage of seeds at 15 degrees C, only Actinotus leucocephalus produced increased germination in response to smoke water and heat over time. In summary, soil burial can alter the dormancy status of a number of Australian fire ephemeral seeds, rendering them more responsive to germination cues such as smoke water and heat. The requirement for a period of burial before seeds become responsive to smoke and/or heat would ensure that seeds persist in the soil until a subsequent fire, when there is an increase in nutrients available for growth and reduced competition from other plants.

Electrical burns: A retrospective analysis across a 5-year period

This study aims to review the experience, at an institution, with patients who suffered electrical burns and study the peculiar characteristics of this type of burn as well as its complications and epidemiological aspects. The study includes medical records of patients with electrical burns who were admitted to the Burn Unit of Hospital das Clinicas in Sao Paulo, Brazil, from November 2001 to October 2006. They were classified into four categories: high voltage (>= 1000 V), low voltage (<1000 V), `flash burn` (in which there is no electrical current flow through the body of the patient) and burns caused by lightning. The complications were more severe and common in the high-voltage group, while longer hospital stays and more complex surgical procedures due to the greater depth of burns were also observed in this group. High-voltage burns are mainly labour-/occupation-related. The majority of the patients were young men at the beginning of their professional lives. This factor generates an important socio-economic impact due to the high incidence of sequelae, resulting in amputations, rendering them unable to maintain their occupations. (C) 2009 Elsevier Ltd and ISBI. All rights reserved.

Optimizing the CAMCOG test in the screening for mild cognitive impairment and incipient dementia: saving time with relevant domains

Objective: To identify the CAMCOG sub-items that best contribute for the identification of patients with mild cognitive impairment (MCI) and incipient Alzheimer`s disease (AD) in clinical practice. Methods: Cross-sectional assessment of 272 older adults (98 MCI, 82 AD, and 92 controls) with a standardized neuropsychological battery and the CAMCOG schedule. Backward logistic regression analysis with diagnosis (MCI and controls) as dependent variable and the sub-items of the CAMCOG as independent variable was carried out to determine the CAMCOG sub-items that predicted the diagnosis of MCI. Results: Lower scores on Language, Memory, Praxis, and Calculation CAMCOG sub-items were significantly associated with the diagnosis of MCI. A composite score obtained by the sum of these scores significantly discriminated MCI patients from comparison groups. This reduced version of the CAMCOG showed similar diagnostic accuracy than the original schedule for the identification of patients with MCI as compared to controls (AUC = 0.80 +/- 0.03 for the reduced CAMCOG; AUC = 0.79 +/- 0.03 for the original CAMCOG). Conclusion: This reduced version of the CAMCOG had similar diagnostic properties as the original CAMCOG and was faster and easier to administer, rendering it more suitable for the screening of subtle cognitive deficits in general clinical practice. Copyright (C) 2010 John Wiley & Sons, Ltd.

CpG-ODN combined with Neospora caninum lysate, but not with excreted-secreted antigen, enhances protection against infection in mice

CpG oligodeoxynucleotides (ODN) have shown to be potent immunoadjuvants for several pathogens, but there is limited information concerning their use in immunization protocols against neosporosis. This study aimed to evaluate the potential of CpG-ODN combined with Neosporar lysate antigen (NLA) or excreted-secreted antigen (NcESA) to induce protective immune response against Neospora caninum infection in mice. C57BL/6 mice were vaccinated subcutaneously three times at 2-week intervals with NLA, NLA+CpG, NcESA, NcESA+CpG, CpG (adjuvant control) or PBS (infection control). Serological assays showed an increased specific IgG2a response in animals immunized with either antigen plus adjuvant and elevated levels of the IgG1 isotype in those vaccinated with antigens alone. Splenocyte proliferative responses upon antigen stimulation were higher in groups immunized with NLA OF NcESA combined with CpG, showing increased IL-12 levels. Also, mice vaccinated with NcESA or NcESA+CpG demonstrated higher IFN-gamma levels and IFN-gamma/IL-10 ratio. After lethal challenge, mice immunized with NLA+CpG or NLA had lower Morbidity score and body weight changes in comparison to other groups, and animals did not succumb during acute infection. In contrast, NcESA+CpG or NcESA groups exhibited the highest morbidity scores, body weight impairment and mortality rates, associated with greatest brain parasite burden and inflammation. In conclusion, CpG-ODN was able to induce a Th1-type humoral immune response with predominant IgG2a levels for either NLA or NcESA, but resulting in an effective Th1-driven cellular immune response and total Protection only when combined with NLA. Vaccination with NcESA alone or combined with CpG resulted in a strong cellular immune response associated with high levels of IFN-gamma and inflammation, rendering mice more susceptible to parasite challenge. (C) 2009 Elsevier Ltd. All rights reserved.