The interplay between folding-facilitating mechanisms in Trypanosoma cruzi endoplasmic reticulum

Lectin (calreticulin [CRT])-N-glycan-mediated quality control of glycoprotein folding is operative in trypanosomatid protozoa but protein-linked monoglucosylated N-glycans are exclusively formed in these microorganisms by UDP-Glc:glycoprotein glucosyltransferase (GT)-dependent glucosylation. The gene coding for this enzyme in the human pathogen Trypanosoma cruzi was identified and sequenced. Even though several of this parasite glycoproteins have been identified as essential components of differentiation and mammalian cell invasion processes, disruption of both GT-encoding alleles did not affect cell growth rate of epimastigote form parasites and only partially affected differentiation and mammalian cell invasion. The cellular content of one of the already identified T. cruzi glycoprotein virulence factors (cruzipain, a lysosomal proteinase) only showed a partial (5-20%) decrease in GT null mutants in spite of the fact that >90% of all cruzipain molecules interacted with CRT during their folding process in wild-type cells. Although extremely mild cell lysis and immunoprecipitation procedures were used, no CRT-cruzipain interaction was detected in GT null mutants but secretion of the proteinase was nevertheless delayed because of a lengthened interaction with Grp78/BiP probably caused by the detected induction of this chaperone in GT null mutants. This result provides a rationale for the absence of a more drastic consequence of GT absence. It was concluded that T. cruzi endoplasmic reticulum folding machinery presents an exquisite plasticity that allows the parasite to surmount the absence of the glycoprotein-specific folding facilitation mechanism.     

Nickel inhibits the slowly activating channels of radish vacuoles

The mechanism of inhibition by cytoplasmic nickel of slowly activating channels in radish vacuoles was investigated using the patch-clamp technique. The decrease in the macroscopic current induced by the presence of nickel in the cytoplasmic solution can be described by a Michaelis-Menten equation with an apparent dissociation constant of 0.45+/-0.03 mM. At the single-channel level, nickel moderately decreases the single-channel conductance, since the ratio between the chord conductance in the presence and in the absence of 1 mM cytosolic nickel is 0.89+/-0.06. Experiments performed to study the interaction between calcium, an activator of the channel, and nickel strongly suggest that these two ions bind to two distinct molecular sites. A simple mathematical model predicting the experimental observations is presented.     

Expression of truncated PrP targeted to Purkinje cells of PrP knockout mice causes Purkinje cell death and ataxia

PrP knockout mice with disruption of only the PrP-encoding region (Zürich I-type) remain healthy, whereas mice with deletions extending upstream of the PrP-encoding exon (Nagasaki-type) suffer Purkinje cell loss and ataxia, associated with ectopic expression of Doppel in brain, particularly in Purkinje cells. The phenotype is abrogated by co-expression of full-length PrP. Doppel is 25% similar to PrP, has the same globular fold, but lacks the flexible N-terminal tail. We now show that in Zürich I-type PrP-null mice, expression of N-terminally truncated PrP targeted to Purkinje cells also leads to Purkinje cell loss and ataxia, which are reversed by PrP. Doppel and truncated PrP probably cause Purkinje cell degeneration by the same mechanism.     

The Quantity and Turnover of Dead Wood in Permanent Forest Plots in Six Life Zones of Venezuela1

Dead wood can be an important component of the carbon pool in many forests, but few measurements have been made of this pool in tropical forests, To fill this gap, we determined the quantity of dead wood (downed and standing dead) in 25 long-term (up to 30 yr) permanent forest plots located in six different life zones of Venezuela. Downed wood was separated into fine (< 10 cm in diameter) and coarse (≥ 10 cm in diameter) classes, and three decomposition states (sound, intermediate, or rotten). The total quantity of dead wood, averaged by life zone, was lowest in the dry (2.43 Mg/ha), reached a peak in the moist (42.33 Mg/ha) and decreased slightly in the wet (34.50 Mg/ha) life zone. Most of the dead wood was in the standing dead category (about 42–76% of the total). The decomposition state of dead wood in all plots was mostly rotten (45%) or intermediate (44%); there was little sound wood (11%). Turnover rates of dead wood generally ranged between 0.03/yr to 0.52/yr with no clear trend with life zone. The large amount of dead wood in some plots was equivalent to about 20 percent or less of aboveground biomass, indicating that dead wood can represent a significant amount of carbon in these forests.     

SELECTIVE PERMEABILITY OF THE EXTRACELLULAR ENVELOPE OF THE MICROALGA SPONDYLOSIUM PANDURIFORME (CHLOROPHYCEAE) AS REVEALED BY ELECTRON PARAMAGNETIC RESONANCE

The aim of this work was to investigate the role of the polysaccharide sheath of the microalga Spondylosium panduriforme (Chlorophyceae, Desmidiaceae) in the selective permeability and transport of molecules into the interior of the cell. We have used the electron paramagnetic resonance (EPR) technique applied to a variety of spin labels of a hydrophobic nature with different substitutents on the ring (−OH, =O, −N=C=S, −NH₃⁺, and others). The spin label EPR signals were destroyed as a consequence of metabolic processes once the spin probes had entered the cells. The decay time of the EPR signal was regulated by the diffusion mechanism across the polysaccharide sheath, cell wall, and membrane. To discriminate the effect of the polysaccharide sheath from that of the cell wall and membrane, the polysaccharide sheath was removed by ultrasonic treatment. The decay times for the cells without capsule were faster than those for intact cells, and a possible mechanism of interaction involving hydrogen bonds between the spin labels and the −OH groups of the polysaccharide sheath is presented. These were expressed by their diffusion and friction coefficients as derived from Ficks' Second Law and the Einstein-Stokes equation and were summarized in terms of diffusion coefficients ( D ₁) for the capsule medium in the order: =O < −OH < −phe < −H < −N=C=S; and for cell wall and membrane ( D ₂): −OH < −H < =O < −NH₃⁺≅−phe < −N=C=S. For the friction coefficients ( f ₁ and f ₂), the order was inverted. These results suggest the capsule plays a role in selectivity as a result of polar interactions with the spin labels.     

Presence of guanine nucleotide-binding proteins in Catharanthus roseus transformed roots

Biochemical analysis revealed the presence of GTP-binding proteins (G-proteins) in Catharanthus roseus hairy root cultures. In a microsomal fraction, several proteins, with molecular masses of 17, 21, 38, 42, 65, and 79 kDa were substrates for ADP-ribosylation by cholera toxin. Antisera raised against a conserved amino-acid sequence (GTSNSGKSTIVKQMK) of mammalian G α subunits recognized three proteins of 42, 50, and 79 kDa. Incubation of nitrocellulose blots with [ α -³²P]-GTP also indicated the presence of several proteins (17, 21, 50, and 79 kDa) that could bind GTP. In this system, we previously identified a phosphatidylinositol 4,5-bisphosphate-phospholipase C (PLC, EC 3.1.4.11) activity. As the activation of PLC by G-proteins was described, we decided to see whether, in our system, G-protein activators, such as guanosine 5- o -(3-thiotriphosphate) (GTP Γ S) and sodium fluoride ions, were able to regulate PLC activity in C. roseus transformed roots. Our results show that these agents regulated PLC activity in an inhibitory fashion and that this effect is dose-dependent. GTP was ineffective in producing either stimulation or inhibition of PLC activity. Our results demonstrate that non-hydrolyzable guanine nucleotides and fluoride ions exert an inhibitory effect on membrane PLC activity. In summary, a set of proteins of 17, 21, 38, 42, 50, and 79 kDa present in C. roseus transformed roots possessed at least two of the three main characteristics of a GTP-binding protein, and one of these proteins may be involved in the regulation of PLC activity in C. roseus transformed roots.     

Costs and Epidemiological Changes of Chronic Diseases: Implications and Challenges for Health Systems

Background

The need to integrate economic and epidemiological aspects in the clinical perspective leads to a proposal for the analysis of health disparities and to an evaluation of the health services and of the new challenges which are now being faced by health system reforms in middle income countries.

Objective

To identify the epidemiological changes, the demand for health services and economic burden from chronic diseases (diabetes and hypertension) in a middle income county.

Methods

We conducted longitudinal analyses of costs and epidemiological changes for diabetes and hypertension in the Mexican health system. The study population included both the insured and uninsured populations. The cost-evaluation method was used, based on the instrumentation and consensus techniques. To estimate the epidemiological changes and financial consequences for 2014–2016, six models were constructed according to the Box-Jenkins technique, using confidence intervals of 95%, and the Box-Pierce test.

Results

Regarding epidemiological changes expected in both diseases for 2014 vs. 2016, an increase is expected, although results predict a greater increase for diabetes, 8–12% in all three studied institutions, (p < .05). Indeed, in the case of diabetes, the increase was 41469 cases for uninsured population (SSA) and 65737 for the insured population (IMSS and ISSSTE). On hypertension cases the increase was 38109 for uninsured vs 62895 for insured. Costs in US$ ranged from $699 to $748 for annual case management per patient in the case of diabetes, and from $485 to $622 in patients with hypertension. Comparing financial consequences of health services required by insured and uninsured populations, the greater increase (23%) will be for the insured population (p < .05). The financial requirements of both diseases will amount to 19.5% of the total budget for the uninsured and 12.5% for the insured population.

Conclusions

If the risk factors and the different health care models remain as they currently are, the economic impact of expected epidemiological changes on the social security system will be particularly strong. Another relevant challenge is the appearance of internal competition in the use and allocation of financial resources with programs for other chronic and infectious diseases.     

Plasma Free Hemoglobin and Microcirculatory Response to Fresh or Old Blood Transfusions in Sepsis

BackgroundFree hemoglobin (fHb) may induce vasoconstriction by scavenging nitric oxide. It may increase in older blood units due to storage lesions. This study evaluated whether old red blood cell transfusion increases plasma fHb in sepsis and how the microvascular response may be affected.MethodsThis is a secondary analysis of a randomized study. Twenty adult septic patients received either fresh or old (<10 or >15 days storage, respectively) RBC transfusions. fHb was measured in RBC units and in the plasma before and 1 hour after transfusion. Simultaneously, the sublingual microcirculation was assessed with sidestream-dark field imaging. The perfused boundary region was calculated as an index of glycocalyx damage. Tissue oxygen saturation (StO₂) and Hb index (THI) were measured with near-infrared spectroscopy and a vascular occlusion test was performed.ResultsSimilar fHb levels were found in the supernatant of fresh and old RBC units. Despite this, plasma fHb increased in the old RBC group after transfusion (from 0.125 [0.098–0.219] mg/mL to 0.238 [0.163–0.369] mg/mL, p = 0.006). The sublingual microcirculation was unaltered in both groups, while THI increased. The change in plasma fHb was inversely correlated with the changes in total vessel density (r = -0.57 [95% confidence interval -0.82, -0.16], p = 0.008), De Backer score (r = -0.63 [95% confidence interval -0.84, -0.25], p = 0.003) and THI (r = -0.72 [95% confidence interval -0.88, -0.39], p = 0.0003).ConclusionsOld RBC transfusion was associated with an increase in plasma fHb in septic patients. Increasing plasma fHb levels were associated with decreased microvascular density.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01584999","term_id":"NCT01584999"}}NCT01584999     

MAFCO: A Compression Tool for MAF Files

In the last decade, the cost of genomic sequencing has been decreasing so much that researchers all over the world accumulate huge amounts of data for present and future use. These genomic data need to be efficiently stored, because storage cost is not decreasing as fast as the cost of sequencing. In order to overcome this problem, the most popular general-purpose compression tool, gzip, is usually used. However, these tools were not specifically designed to compress this kind of data, and often fall short when the intention is to reduce the data size as much as possible. There are several compression algorithms available, even for genomic data, but very few have been designed to deal with Whole Genome Alignments, containing alignments between entire genomes of several species. In this paper, we present a lossless compression tool, MAFCO, specifically designed to compress MAF (Multiple Alignment Format) files. Compared to gzip, the proposed tool attains a compression gain from 34% to 57%, depending on the data set. When compared to a recent dedicated method, which is not compatible with some data sets, the compression gain of MAFCO is about 9%. Both source-code and binaries for several operating systems are freely available for non-commercial use at: http://bioinformatics.ua.pt/software/mafco.