Acute renal failure in children. Multicenter prospective cohort study in medium-complexity intensive care units from the Colombian southeast.

BACKGROUND: Acute kidney injury is frequent in critically ill children; however, it varies in causality and epidemiology according to the level of patient care complexity. A multicenter prospective cohort study was conducted in four medium-complexity pediatric intensive care units from the Colombian southeast aimed to estimate the clinical prognosis of patients with diagnosis of acute kidney injury. METHODS: We included children >28 days and <18 years of age, who were admitted with diagnosis of acute kidney injury classified by Kidney Disease Improving Global Outcomes (KDIGO), during the period from January to December 2017. Severe acute kidney injury was defined as stage 2 and stage 3 classifications. Maximum KDIGO was evaluated during the hospital stay and follow up. Length of hospital stay, use of mechanical ventilation and vasoactive drugs, use of renal replacement therapy, and mortality were assessed until discharge. RESULTS: Prevalence at admission of acute kidney injury was 5.2% (95%CI 4.3% to 6.2%). It was found that 71% of the patients had their maximum KDIGO on day one; an increment in the maximum stage of acute kidney injury increased the pediatric intensive care unit stay. Patients with maximum KDIGO 3 were associated with greater use of mechanical ventilation (47%), compared with maximum KDIGO 2 (37%) and maximum KDIGO 1 (16%). Eight patients with maximum KDIGO 2 and 14 with maximum KDIGO 3 required renal replacement therapy. Mortality was at 11.8% (95%CI 6.4% to 19.4%). CONCLUSION: Acute kidney injury, established and classified according to KDIGO as severe and its maximum stage, was associated with worse clinical outcomes; early therapeutic efforts should focus on preventing the progression to severe stages.

Antiprotozoan lead discovery by aligning dry and wet screening: prediction, synthesis, and biological assay of novel quinoxalinones.

Protozoan parasites have been one of the most significant public health problems for centuries and several human infections caused by them have massive global impact. Most of the current drugs used to treat these illnesses have been used for decades and have many limitations such as the emergence of drug resistance, severe side-effects, low-to-medium drug efficacy, administration routes, cost, etc. These drugs have been largely neglected as models for drug development because they are majorly used in countries with limited resources and as a consequence with scarce marketing possibilities. Nowadays, there is a pressing need to identify and develop new drug-based antiprotozoan therapies. In an effort to overcome this problem, the main purpose of this study is to develop a QSARs-based ensemble classifier for antiprotozoan drug-like entities from a heterogeneous compounds collection. Here, we use some of the TOMOCOMD-CARDD molecular descriptors and linear discriminant analysis (LDA) to derive individual linear classification functions in order to discriminate between antiprotozoan and non-antiprotozoan compounds as a way to enable the computational screening of virtual combinatorial datasets and/or drugs already approved. Firstly, we construct a wide-spectrum benchmark database comprising of 680 organic chemicals with great structural variability (254 of them antiprotozoan agents and 426 to drugs having other clinical uses). This series of compounds was processed by a k-means cluster analysis in order to design training and predicting sets. In total, seven discriminant functions were obtained, by using the whole set of atom-based linear indices. All the LDA-based QSAR models show accuracies above 85% in the training set and values of Matthews correlation coefficients (C) vary from 0.70 to 0.86. The external validation set shows rather-good global classifications of around 80% (92.05% for best equation). Later, we developed a multi-agent QSAR classification system, in which the individual QSAR outputs are the inputs of the aforementioned fusion approach. Finally, the fusion model was used for the identification of a novel generation of lead-like antiprotozoan compounds by using ligand-based virtual screening of 'available' small molecules (with synthetic feasibility) in our 'in-house' library. A new molecular subsystem (quinoxalinones) was then theoretically selected as a promising lead series, and its derivatives subsequently synthesized, structurally characterized, and experimentally assayed by using in vitro screening that took into consideration a battery of five parasite-based assays. The chemicals 11(12) and 16 are the most active (hits) against apicomplexa (sporozoa) and mastigophora (flagellata) subphylum parasites, respectively. Both compounds depicted good activity in every protozoan in vitro panel and they did not show unspecific cytotoxicity on the host cells. The described technical framework seems to be a promising QSAR-classifier tool for the molecular discovery and development of novel classes of broad-antiprotozoan-spectrum drugs, which may meet the dual challenges posed by drug-resistant parasites and the rapid progression of protozoan illnesses.

Haemostatic role of intermediate filaments in adhered platelets: importance of the membranous system stability.

The role of platelets in coagulation and the haemostatic process was initially suggested two centuries ago, and under appropriate physiological stimuli, these undergo abrupt morphological changes, attaching and spreading on damaged endothelium, preventing bleeding. During the adhesion process, platelet cytoskeleton reorganizes generating compartments in which actin filaments, microtubules, and associated proteins are arranged in characteristic patterns mediating crucial events, such as centralization of their organelles, secretion of granule contents, aggregation with one another to form a haemostatic plug, and retraction of these aggregates. However, the role of Intermediate filaments during the platelet adhesion process has not been explored. J. Cell. Biochem. 114: 2050-2060, 2013. © 2013 Wiley Periodicals, Inc.

A role for ß-dystroglycan in the organization and structure of the nucleus in myoblasts.

We recently characterized a nuclear import pathway for ß-dystroglycan; however, its nuclear role remains unknown. In this study, we demonstrate for the first time, the interaction of ß-dystroglycan with distinct proteins from different nuclear compartments, including the nuclear envelope (NE) (emerin and lamins A/C and B1), splicing speckles (SC35), Cajal bodies (p80-coilin), and nucleoli (Nopp140). Electron microscopy analysis revealed that ß-dystroglycan localized in the inner nuclear membrane, nucleoplasm, and nucleoli. Interestingly, downregulation of ß-dystroglycan resulted in both mislocalization and decreased expression of emerin and lamin B1, but not lamin A/C, as well in disorganization of nucleoli, Cajal bodies, and splicing speckles with the concomitant decrease in the levels of Nopp140, and p80-coilin, but not SC35. Quantitative reverse transcription PCR and cycloheximide-mediated protein arrest assays revealed that ß-dystroglycan deficiency did not change mRNA expression of NE proteins emerin and lamin B1 bud did alter their stability, accelerating protein turnover. Furthermore, knockdown of ß-dystroglycan disrupted NE-mediated processes including nuclear morphology and centrosome-nucleus linkage, which provides evidence that ß-dystroglycan association with NE proteins is biologically relevant. Unexpectedly, ß-dystroglycan-depleted cells exhibited multiple centrosomes, a characteristic of cancerous cells. Overall, these findings imply that ß-dystroglycan is a nuclear scaffolding protein involved in nuclear organization and NE structure and function, and that might be a contributor to the biogenesis of nuclear envelopathies.

Mammographic breast density patterns in asymptomatic mexican women.

Breast density (BD) is a risk factor for breast cancer. Aims. To describe BD patterns in asymptomatic Mexican women and the pathological mammographic findings. Methods and Material. Prospective, descriptive, and comparative study. Women answered a questionnaire and their mammograms were analyzed according to BI-RADS. Univariate (χ(2)) and conditional logistic regression analyses were performed. Results. In 300 women studied the BD patterns were fat 56.7% (170), fibroglandular 29% (87), heterogeneously dense 5.7% (17), and dense pattern 8.6% (26). Prevalence of fat pattern was significantly different in women under 50 years (37.6%, 44/117) and older than 50 (68.8%, 126/183). Patterns of high breast density (BD) (dense + heterogeneously dense) were observed in 25.6% (30/117) of women ≤50 years and 7.1% (13/183) of women >50. Asymmetry in BD was observed in 22% (66/300). Compression cone ruled out underlying disease in 56 cases. In the remaining 10, biopsy revealed one fibroadenoma, one complex cyst, and 6 invasive and 2 intraductal carcinomas. 2.6% (8/300) of patients had non-palpable carcinomas. Benign lesions were observed in 63.3% (190/300) of cases, vascular calcification in 150 cases (78.9%), and fat necrosis in 38 cases (20%). Conclusions. Mexican women have a low percentage of high-density patterns.

Neuronal differentiation modulates the dystrophin Dp71d binding to the nuclear matrix.

The function of dystrophin Dp71 in neuronal cells remains unknown. To approach this issue, we have selected the PC12 neuronal cell line. These cells express both a Dp71f cytoplasmic variant and a Dp71d nuclear isoform. In this study, we demonstrated by electron and confocal microscopy analyses of in situ nuclear matrices and Western blotting evaluation of cell extracts that Dp71d associates with the nuclear matrix. Interestingly, this binding is modulated during NGF-induced neuronal differentiation of PC12 cells with a twofold increment in the differentiated cells, compared to control cells. Also, distribution of Dp71d along the periphery of the nuclear matrix observed in the undifferentiated cells is replaced by intense fluorescent foci localized in the center of the nucleoskeletal structure. In summary, we revealed that Dp71d is a dynamic component of nuclear matrix that might participate in the nuclear modeling occurring during neuronal differentiation.

Subcellular localization and dynamics of a digalactolipid-like epitope in Toxoplasma gondii.

Toxoplasma gondii is a unicellular parasite characterized by unique extracellular and intracellular membrane compartments. The lipid composition of subcellular membranes has not been determined, limiting our understanding of lipid homeostasis, control, and trafficking, a series of processes involved in pathogenesis. In addition to a mitochondrion, Toxoplasma contains a plastid called the apicoplast. The occurrence of a plastid raised the question of the presence of chloroplast galactolipids. Using three independent rabbit and rat antibodies against digalactosyldiacylglycerol (DGDG) from plant chloroplasts, we detected a class of Toxoplasma lipids harboring a digalactolipid-like epitope (DGLE). Immunolabeling characterization supports the notion that the DGLE polar head is similar to that of DGDG. Mass spectrometry analyses indicated that dihexosyl lipids having various hydrophobic moieties (ceramide, diacylglycerol, and acylalkylglycerol) might react with anti-DGDG, but we cannot exclude the possibility that more complex dihexosyl-terminated lipids might also be immunolabeled. DGLE localization was analyzed by immunofluorescence and immunoelectron microscopy and confirmed by subcellular fractionation. No immunolabeling of the apicoplast could be observed. DGLE was scattered in pellicle membrane domains in extracellular tachyzoites and was relocalized to the anterior tip of the cell upon invasion in an actin-dependent manner, providing insights on a possible role in pathogenetic processes. DGLE was detected in other Apicomplexa (i.e., Neospora, Plasmodium, Babesia, and Cryptosporidium).

Functional domains of the Toxoplasma GRA2 protein in the formation of the membranous nanotubular network of the parasitophorous vacuole.

Amphipathic alpha-helices have been proposed as the general means used by soluble proteins to induce membrane tubulation. Previous studies had shown that the GRA2 dense granule protein of Toxoplasma gondii would be a crucial protein for the formation of the intravacuolar membranous nanotubular network (MNN) and that one of the functions of the MNN is to organise the parasites within the parasitophorous vacuole. GRA2 is a small protein (185 amino acids), predicted to contain three amphipathic alpha-helices (alpha1: 70-92; alpha2: 95-110 and alpha3: 119-139) when using the standard programs of secondary structure prediction. To investigate the respective contribution of each alpha-helix in the GRA2 functions, we used DeltaGRA2-HXGPRT knock-out complementation: eight truncated forms of GRA2 were expressed in the deleted recipient and the phenotypes of these mutants were analysed. This study showed that: (i) alpha3, when associated with the N-terminal region (NT) and the C-terminal region (CT), is sufficient to target the protein to the parasite posterior end and to induce formation of membranous vesicles within the vacuole. However, when associated only with CT, alpha3 is not sufficient to provide the hydrophobicity required for membrane association; (ii) the alpha1alpha2 region is alone not sufficient to induce membrane tubulation within the PV; and (iii) only one mutant, NT-alpha1alpha2alpha3, restores most of the biochemical and functional properties of GRA2, including traffic to the dense granules, secretion into the vacuole, association with vacuolar membranes, induction of the MNN formation and organisation of the parasites within the vacuole.

In-vitro model for the ultrastructural study of the formation of thrombi in human platelets.

Platelets are cell fragments with dynamic properties involved in clot formation after tissue damage. Platelet activation causes a change in shape, secretion of intracellular granules and aggregation with each other through the cytoskeleton components and biochemical changes. Platelet adhesion, considered as the major event in haemostasis, has been studied in several in-vitro and in-vivo models to evaluate the feasible thrombogenicity of some materials, the dynamics of specific receptors, as well as the effect of different buffers and inhibitors in this process. In spite of the numerous reports about platelet activation, to date there is no information available about the fine structure of the platelet-platelet and platelet-substrate interactions. In the present report we describe an in-vitro system that allows the visualization of these interactions: platelets are adhered to an inert substrate, and interactions with suspended platelets as a process to initiate the formation of thrombi was followed by ultramicrotomy and transmission electron microscopy.

Identification and purification of actin from the subpellicular network of Toxoplasma gondii tachyzoites.

Toxoplasma gondii infects cells through dynamic events dependent on actin. Although the presence of cortical actin has been widely suggested, visualisation and localisation of actin filaments has not been reported. The subpellicular cytoskeleton network is a recently described structure possibly involved in the dynamic events. Using non-ionic detergent extractions, the cortical cytoskeleton network was enriched and used for the isolation and identification of actin. Actin was detected by Western blots in extracts of cytoskeleton networks, and it was localised by gold staining in the network and in both the apical end and the posterior polar ring. Actin was isolated from subpellicular cytoskeleton extracts by binding to DNase I, and it polymerised in vitro as filaments that were gold-decorated by a monoclonal anti-actin antibody. Filaments bound the subfragment 1 of heavy meromyosin, although with atypical arrangements in comparison with the arrowheads observed in muscle actin filaments. Treatment with cytochalasin D and colchicine altered the structural organisation of the subpellicular network indicating the participation of actin filaments and microtubules in the maintenance of its structure. Actin filaments and microtubules, in the subpellicular network, participate reciprocally in the maintaining of the parasite's shape and the gliding motility.