Phosphatidylinositol-4,5-bisphosphate was proposed to be an important regulator of large dense-core vesicle exocytosis from neuroendocrine tissues. Here, we have examined the kinetics of secretion in chromaffin cells from mice lacking phosphatidylinositol phosphate kinase type I␥, the major neuronal phosphatidylinositol-4-phosphate 5-kinase. Absence of this enzyme caused a reduction of the readily releasable vesicle pool and its refilling rate, with a small increase in morphologically docked vesicles, indicating a defect in vesicle priming. Furthermore, amperometry revealed a delay in fusion pore expansion. These results provide direct genetic evidence for a key role of phosphatidylinositol-4,5-bisphosphate synthesis in the regulation of large dense-core vesicle fusion dynamics.exocytosis ͉ fusion pore ͉ granule ͉ phosphatidylinositol-4,5-bisphosphate ͉ secretion S trong evidence has implicated phosphoinositides (PIs) in the regulation of membrane traffic, including exocytosis (1-4). A first clue suggesting a direct role of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P 2 ] in secretion came from its requirement for Ca 2ϩ -dependent exocytosis in broken chromaffin cells, independent from its phospholipase C (PLC)-mediated cleavage (5). Subsequently, a PI(4)P 5-kinase activity was implicated as a critical factor in the Ca 2ϩ -activated secretion of large dense-core vesicles (LDCVs) from permeabilized PC12 cells, a chromaffin cell line (6). In addition, the molecular characterization of the exocytic machinery revealed that several proteins that directly or indirectly participate in the priming and fusion of neurosecretory vesicles contain PI(4,5)P 2 -binding domains (1, 4, 7). For example, synaptotagmin, a putative Ca 2ϩ sensor in the exocytosis of synaptic vesicles and LDCVs, binds PI(4,5)P 2 by means of C2 domains, and in vitro assays showed that this interaction is important in membrane fusion (8-10). Ca 2ϩ -dependent activator for secretion (CAPS), which is required for Ca 2ϩ -evoked LDCV secretion (11), binds PI(4,5)P 2 by means of a PH domain (12). Moreover, manipulations that artificially mask available PI(4,5)P 2 , such as overexpression of a PI(4,5)P 2 -binding module, the PH domain of PLC␦1, inhibits LDCV exocytosis (13,14). Disruption of the function of Arf6, a positive regulator of PI(4,5)P 2 synthesis by means of its action on type I phosphoinositide-4-phosphate (PIP) kinases (15, 16), impairs neuroendocrine secretion (14, 17); conversely, stimulation of Arf6 enhances secretion (18). Diacylglycerol, a metabolic product of PI(4,5)P 2 , is also implicated in synaptic vesicle and LDCV fusion with the plasma membrane (PM). Diacylglycerol, whose levels can be regulated not only by PLC-mediated cleavage of PI(4,5)P 2 but also by alternative metabolic pathways, binds to Munc13, and this interaction plays an essential role in the priming reaction of exocytosis (19)(20)(21)(22).PI(4,5)P 2 can be generated by type I and type II PIP kinases, which function as PI(4)P 5-kinases and PI(5)P 4-kinases, respectively ...

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BackgroundOutbreaks of acute Chagas disease associated with oral transmission are easily detected nowadays with trained health personnel in areas of low endemicity, or in which the vector transmission has been interrupted. Given the biological and genetic diversity of Trypanosoma cruzi, the high morbidity, mortality, and the observed therapeutic failure, new characteristics of these outbreaks need to be addressed at different levels, both in Trypanosoma cruzi as in patient response. The aim of this work was to evaluate the patient’s features involved in six outbreaks of acute Chagas disease which occurred in Santander, Colombia, and the characteristics of Trypanosoma cruzi clones isolated from these patients, to establish the potential relationship between the etiologic agent features with host behavior.MethodsThe clinical, pathological and epidemiological aspects of outbreaks were analyzed. In addition, Trypanosoma cruzi clones were biologically characterized both in vitro and in vivo, and the susceptibility to the classical trypanocidal drugs nifurtimox and benznidazole was evaluated. Trypanosoma cruzi clones were genotyped by means of mini-exon intergenic spacer and cytochrome b genes sequencing.ResultsAll clones were DTU I, and based on the mini-exon intergenic spacer, belong to two genotypes: G2 related with sub-urban, and G11 with rural outbreaks. Girón outbreak clones with higher susceptibility to drugs presented G2 genotype and C/T transition in Cyt b. The outbreaks affected mainly young population (±25.9 years), and the mortality rate was 10 %. The cardiac tissue showed intense inflammatory infiltrate, myocardial necrosis and abundant amastigote nests. However, although the gastrointestinal tissue was congestive, no inflammation or parasites were observed.ConclusionsAlthough all clones belong to DTU I, two intra-DTU genotypes were found with the sequencing of the mini-exon intergenic spacer, however there is no strict correlation between genetic groups, the cycles of the parasite or the clinical forms of the disease. Trypanosoma cruzi clones from Girón with higher sensitivity to nifurtimox presented a particular G2 genotype and C/T transition in Cyt b. When the diagnosis was early, the patients responded well to antichagasic treatment, which highlights the importance of diagnosis and treatment early to prevent fatal outcomes associated with these acute episodes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1218-2) contains supplementary material, which is available to authorized users.

Introduction: Benznidazole (BZL) and Nifurtimox (NFX) are the pharmacological treatment for acute phase Chagas Disease (CD); however, therapy resistance and residual mortality development remain important unresolved issues. Posaconazole (POS) has shown a trypanocidal effect in vivo and in vitro. Thus, this study aimed at comparing the T. Cruzi parasitic load-reducing effect of the combination of BZL+POS against that of monotherapy with either, during acute phase CD, in an experimental murine model. Methods: Nineteen Wistar rats were randomly allocated to four groups and inoculated with the trypomastigotes of T. cruzi strain´s JChVcl1. The rats were administered anti-parasites from day 20-29 post-infection. The Pizzi and Brener method was used for parasitemia measurement. Longitudinal data analysis for the continuous outcome of repeated measures was performed using parasitemia as the outcome measured at days 20, 22, 24, 27, and 29 post-infection. Results: All four groups had similar parasitic loads (p=0.143) prior to therapy initiation. Among the three treatment groups, the BZL+POS (n=5) group showed the highest mean parasitic load reduction (p=0.000) compared with the control group. Likewise, the BZL+POS group rats showed an earlier therapeutic effect and were the only ones without parasites in their myocardial samples. Conclusions: Treatment of acute phase CD with BZL+POS was more efficacious at parasitemia and myocardial injury reduction, compared with monotherapy with either.

Counting spatially and temporally overlapping events in image sequences and estimating their shape-size and duration features are important issues in some applications. We propose a stochastic model, a particular case of the nonisotropic 3D Boolean model, for performing this analysis: the temporal Boolean model. Some probabilistic properties are derived and a methodology for parameter estimation from time-lapse image sequences is proposed using an explicit treatment of the temporal dimension. We estimate the mean number of germs per unit area and time, the mean grain size and the duration distribution. A wide simulation study in order to assess the proposed estimators showed promising results. The model was applied on biological image sequences of in-vivo cells in order to estimate new parameters such as the mean number and duration distribution of endocytic events. Our results show that the proposed temporal Boolean model is effective for obtaining information about dynamic processes which exhibit short-lived, but spatially and temporally overlapping events.

The study of central corneal endothelium morphology is important in Ophthalmology. Some of the pathologies that could compromise endothelial cell morphology are trauma, cataract, surgery, use of contact lenses, corneal dystrophies or degenerations. The quantitative analysis of cell shape and cellular pattern is more sensitive in detecting subtle changes in endothelial morphology than cell density measurement or cell area analysis. In this paper, the morphology of the central cornea, the most important area from the point of view of vision, is studied through an associated bivariate spatial point pattern: the centroids of the cells and the triple points, that is, the points where three different cells converge. Nine different summary descriptors (widely used in the statistical analysis of spatial point patterns) have been used: the empty space distribution function; the nearest neighbour distribution function and Ripley's K-function for each type of point separately (centroids and triple points), plus the corresponding three versions of these functions in the bivariate case. A control sample with similar age and cell density and no known abnormality is associated to each patient. The above descriptors are calculated for the patient and the controls. Each descriptive of the patient is compared with the corresponding descriptors from the controls by means of a graphical analysis and a formal test. Some patients presenting different pathologies are analysed in detail. Endothelia considered morphologically abnormal by visual inspection, which were not detected by hexagonality or density analysis, could be distinguished from control endothelia by these new descriptors.