Lactate oxidation is linked to energy conservation and to oxygen detoxification via a putative terminal cytochrome oxidase in Methanosarcina acetivorans.

The marine archaeon Methanosarcina acetivorans contains a putative NAD + -independent d-lactate dehydrogenase (D-iLDH/glycolate oxidase) encoded by the MA4631 gene, belonging to the FAD-oxidase C superfamily. Nucleotide sequences similar to MA4631 gene, were identified in other methanogens and Firmicutes with >90 and 35-40% identity, respectively. Therefore, the lactate metabolism in M. acetivorans is reported here. Cells subjected to intermittent pulses of oxygen (air-adapted; AA-Ma cells) consumed lactate only in combination with acetate, increasing methane production and biomass yield. In AA-Ma cells incubated with d-lactate plus [14C]-l-lactate, the radioactive label was found in methane, CO2 and glycogen, indicating that lactate metabolism fed both methanogenesis and gluconeogenesis. Moreover, d-lactate oxidation was coupled to O2-consumption which was sensitive to HQNO; also, AA-Ma cells showed high transcript levels of gene dld and those encoding subunits A (MA1006) and B (MA1007) of a putative cytochrome bd quinol oxidase, compared to anaerobic control cells. An E. coli mutant deficient in dld complemented with the MA4631 gene, grew with d-lactate as carbon source and showed membrane-bound d-lactate:quinone oxidoreductase activity. The product of the MA4631 gene is a FAD-containing monomer showing activity of iLDH with preference to d-lactate. The results suggested that air adapted M. acetivorans is able to co-metabolize lactate and acetate with associated oxygen consumption by triggering the transcription and synthesis of the D-iLDH and a putative cytochrome bd: methanophenazine (quinol) oxidoreductase. Biomass generation and O2 consumption, suggest a potentially new oxygen detoxification mechanism coupled to energy conservation in this methanogen.

Isolation and Identification of Arsenic-Resistant Extremophilic Bacteria from the Crater-Lake Volcano "El Chichon", Mexico.

The crater lake at "El Chichón" volcano is an extreme acid-thermal environment with high concentrations of heavy metals. In this study, two bacterial strains with the ability to resist high concentrations of arsenic (As) were isolated from water samples from the crater lake. Staphylococcus ARSC1-P and Stenotrophomonas ARSC2-V isolates were identified by use of the 16S rDNA gene. Staphylococcus ARSC1-P was able to grow in 400 mM of arsenate [As(V)] under oxic and anoxic conditions. The IC50 values were 36 and 382 mM for oxic and anoxic conditions, respectively. For its part, Stenotrophomonas ARSC2-V showed IC50 values of 110 mM and 2.15 for As(V) and arsenite [As(III)], respectively. Arsenic accumulated intracellularly in both species [11-25 nmol As × mg cellular prot-1 in cells cultured in 50 mM As(V)]. The present study shows evidence of microbes that can potentially be a resource for the bio-treatment of arsenic in contaminated sites, which highlights the importance of the "El Chichón" volcano as a source of bacterial strains that are adaptable to extreme conditions.

Mitochondrial oxidative stress contributes to diastolic dysfunction through impaired mitochondrial dynamics.

Diastolic dysfunction (DD) underlies heart failure with preserved ejection fraction (HFpEF), a clinical syndrome associated with aging that is becoming more prevalent. Despite extensive clinical studies, no effective treatment exists for HFpEF. Recent findings suggest that oxidative stress contributes to the pathophysiology of DD, but molecular mechanisms underpinning redox-sensitive cardiac remodeling in DD remain obscure. Using transgenic mice with mitochondria-targeted NOX4 overexpression (Nox4TG618) as a model, we demonstrate that NOX4-dependent mitochondrial oxidative stress induces DD in mice as measured by increased E/E', isovolumic relaxation time, Tau Glantz and reduced dP/dtmin while EF is preserved. In Nox4TG618 mice, fragmentation of cardiomyocyte mitochondria, increased DRP1 phosphorylation, decreased expression of MFN2, and a higher percentage of apoptotic cells in the myocardium are associated with lower ATP-driven and maximal mitochondrial oxygen consumption rates, a decrease in respiratory reserve, and a decrease in citrate synthase and Complex I activities. Transgenic mice have an increased concentration of TGFß and osteopontin in LV lysates, as well as MCP-1 in plasma, which correlates with a higher percentage of LV myocardial periostin- and ACTA2-positive cells compared with wild-type mice. Accordingly, the levels of ECM as measured by Picrosirius Red staining as well as interstitial deposition of collagen I are elevated in the myocardium of Nox4TG618 mice. The LV tissue of Nox4TG618 mice also exhibited increased ICaL current, calpain 2 expression, and altered/disrupted Z-disc structure. As it pertains to human pathology, similar changes were found in samples of LV from patients with DD. Finally, treatment with GKT137831, a specific NOX1 and NOX4 inhibitor, or overexpression of mCAT attenuated myocardial fibrosis and prevented DD in the Nox4TG618 mice. Together, our results indicate that mitochondrial oxidative stress contributes to DD by causing mitochondrial dysfunction, impaired mitochondrial dynamics, increased synthesis of pro-inflammatory and pro-fibrotic cytokines, activation of fibroblasts, and the accumulation of extracellular matrix, which leads to interstitial fibrosis and passive stiffness of the myocardium. Further, mitochondrial oxidative stress increases cardiomyocyte Ca2+ influx, which worsens CM relaxation and raises the LV filling pressure in conjunction with structural proteolytic damage.

Simplification of networks by conserving path diversity and minimisation of the search information.

Alternative paths in a network play an important role in its functionality as they can maintain the information flow under node/link failures. In this paper we explore the navigation of a network taking into account the alternative paths and in particular how can we describe this navigation in a concise way. Our approach is to simplify the network by aggregating into groups the nodes that do not contribute to alternative paths. We refer to these groups as super-nodes, and describe the post-aggregation network with super-nodes as the skeleton network. We present a method to describe with the least amount of information the paths in the super-nodes and skeleton network. Applying our method to several real networks we observed that there is scaling behaviour between the information required to describe all the paths in a network and the minimal information to describe the paths of its skeleton. We show how from this scaling we can evaluate the information of the paths for large networks with less computational cost.

Bioinspired snapping-claw apparatus to study hydrodynamic cavitation effects on the corrosion of metallic samples.

A creative low-cost and compact mechanical device that mimics the rapid closure of the pistol shrimp claw was used to conduct electrochemical experiments, in order to study the effects of hydrodynamic cavitation on the corrosion of aluminum and steel samples. Current-time curves show significant changes associated with local variations in dissolved O2 concentration, cavitation-induced erosion, and changes in the nature of the surface corrosion products.

Stability analysis of silver nanoparticle suspensions by cyclic voltammetry.

We report the application of cyclic voltammetry and absorption spectroscopy to the characterization and study of the stability of silver colloids in water. The samples are prepared via chemical reduction and the reactions are catalyzed by irradiation with white light. The electrochemical response is related to the characteristic sample surface plasmon resonance (SPR) in the UV-visible absorption spectra. Cyclic voltammetry shows a characteristic reduction peak whose position is specific to each analyzed sample. Optical analysis of a colloid precursor during a 12 h time span, under low-power white-light irradiation, shows that nanoparticles undergo change in size and surface state (absorption bands splitting and inversion) to attain the "stable" colloidal form. While the absorption spectrum bands of the precursor return almost periodically to similar positions, the cyclic voltammogram characteristic reduction peak is displaced as a function of time. Finally, we follow the SPR changes of one "stable" colloid being subjected to electrolysis, heating, and sunlight irradiation, for environmental remediation purposes. Sunlight exposure produces the most significant SPR intensity drop, but the electrochemical technique shows itself promising as well.

Estimating degree-degree correlation and network cores from the connectivity of high-degree nodes in complex networks.

Many of the structural characteristics of a network depend on the connectivity with and within the hubs. These dependencies can be related to the degree of a node and the number of links that a node shares with nodes of higher degree. In here we revise and present new results showing how to construct network ensembles which give a good approximation to the degree-degree correlations, and hence to the projections of this correlation like the assortativity coefficient or the average neighbours degree. We present a new bound for the structural cut-off degree based on the connectivity within the hubs. Also we show that the connections with and within the hubs can be used to define different networks cores. Two of these cores are related to the spectral properties and walks of length one and two which contain at least on hub node, and they are related to the eigenvector centrality. We introduce a new centrality measured based on the connectivity with the hubs. In addition, as the ensembles and cores are related by the connectivity of the hubs, we show several examples how changes in the hubs linkage effects the degree-degree correlations and core properties.

FruBPase II and ADP-PFK1 are involved in the modulation of carbon flow in the metabolism of carbohydrates in Methanosarcina acetivorans.

To enhance our understanding of the control of archaeal carbon central metabolism, a detailed analysis of the regulation mechanisms of both fructose1,6-bisphosphatase (FruBPase) and ADP-phosphofructokinase-1 (ADP-PFK1) was carried out in the methanogen Methanosarcina acetivorans. No correlations were found among the transcript levels of the MA_1152 and MA_3563 (frubpase type II and pfk1) genes, the FruBPase and ADP-PFK1 activities, and their protein contents. The kinetics of the recombinant FruBPase II and ADP-PFK1 were hyperbolic and showed simple mixed-type inhibition by AMP and ATP, respectively. Under physiological metabolite concentrations, the FruBPase II and ADP-PFK1 activities were strongly modulated by their inhibitors. To assess whether these enzymes were also regulated by a phosphorylation/dephosphorylation process, the recombinant enzymes and cytosolic-enriched fractions were incubated in the presence of commercial protein phosphatase or protein kinase. De-phosphorylation of ADP-PFK1 slightly decreased its activity (i.e. Vmax) and did not change its kinetic parameters and oligomeric state. Thus, the data indicated a predominant metabolic regulation of both FruBPase and ADP-PFK1 activities by adenine nucleotides and suggested high degrees of control on the respective pathway fluxes.

The contact angle of nanofluids as thermophysical property.

Droplet volume and temperature affect contact angle significantly. Phase change heat transfer processes of nanofluids - suspensions containing nanometre-sized particles - can only be modelled properly by understanding these effects. The approach proposed here considers the limiting contact angle of a droplet asymptotically approaching zero-volume as a thermophysical property to characterise nanofluids positioned on a certain substrate under a certain atmosphere. Graphene oxide, alumina, and gold nanoparticles are suspended in deionised water. Within the framework of a round robin test carried out by nine independent European institutes the contact angle of these suspensions on a stainless steel solid substrate is measured with high accuracy. No dependence of nanofluids contact angle of sessile droplets on the measurement device is found. However, the measurements reveal clear differences of the contact angle of nanofluids compared to the pure base fluid. Physically founded correlations of the contact angle in dependency of droplet temperature and volume are obtained from the data. Extrapolating these functions to zero droplet volume delivers the searched limiting contact angle depending only on the temperature. It is for the first time, that this specific parameter, is understood as a characteristic material property of nanofluid droplets placed on a certain substrate under a certain atmosphere. Together with the surface tension it provides the foundation of proper modelling phase change heat transfer processes of nanofluids.

Synthesis, characterization and biocompatibility of cadmium sulfide nanoparticles capped with dextrin for in vivo and in vitro imaging application.

BACKGROUND: The safe use in biomedicine of semiconductor nanoparticles, also known as quantum dots (QDs), requires a detailed understanding of the biocompatibility and toxicity of QDs in human beings. The biological characteristics and physicochemical properties of QDs entail new challenges regarding the management of potential adverse health effects following exposure. At certain concentrations, the synthesis of semiconductor nanoparticles of CdS using dextrin as capping agent, at certain concentration, to reduce their toxicity and improves their biocompatibility. RESULTS: This study successfully synthesized and characterized biocompatible dextrin-coated cadmium sulfide nanoparticles (CdS-Dx/QDs). The results show that CdS-Dx/QDs are cytotoxic at high concentrations (>2 µg/mL) in HepG2 and HEK293 cells. At low concentrations (<1 µg/mL), CdS-Dx/QDs were not toxic to HepG2 or HeLa cells. CdS-Dx nanoparticles only induced cell death by apoptosis in HEK293 cells at 1 µg/mL concentrations. The in vitro results showed that the cells efficiently took up the CdS-Dx/QDs and this resulted in strong fluorescence. The subcellular localization of CdS-Dx/QDs were usually small and apparently unique in the cytoplasm in HeLa cells but, in the case of HEK293 cells it were more abundant and found in cytoplasm and the nucleus. Animals treated with 100 µg/kg of CdS-Dx/QDs and sacrificed at 3, 7 and 18 h showed a differential distribution in their organs. Intense fluorescence was detected in lung and kidney, with moderate fluorescence detected in liver, spleen and brain. The biocompatibility and toxicity of CdS-Dx/QDs in animals treated daily with 100 µg/kg for 1 week showed the highest level of fluorescence in kidney, liver and brain. Less fluorescence was detected in lung and spleen. There was also evident presence of fluorescence in testis. The histopathological and biochemical analyses showed that CdS-Dx/QDs were non-toxic for rodents. CONCLUSIONS: The in vitro and in vivo studies confirmed the effective cellular uptake and even distribution pattern of CdS-Dx/QDs in tissues. CdS-Dx/QDs were biocompatible with tissues from rodents. The CdS-Dx/QDs used in this study can be potentially used in bio-imaging applications.

Contribution of the residual body in the spatial organization of Toxoplasma gondii tachyzoites within the parasitophorous vacuole.

Toxoplasma gondii proliferates and organizes within a parasitophorous vacuole in rosettes around a residual body and is surrounded by a membranous nanotubular network whose function remains unclear. Here, we characterized structure and function of the residual body in intracellular tachyzoites of the RH strain. Our data showed the residual body as a body limited by a membrane formed during proliferation of tachyzoites probably through the secretion of components and a pinching event of the membrane at the posterior end. It contributes in the intravacuolar parasite organization by the membrane connection between the tachyzoites posterior end and the residual body membrane to give place to the rosette conformation. Radial distribution of parasites in rosettes favors an efficient exteriorization. Absence of the network and presence of atypical residual bodies in a ΔGRA2-HXGPRT knock-out mutant affected the intravacuolar organization of tachyzoites and their exteriorization.

Tratamiento de una lesión endoperiodontal tipo III (combinada o verdadera): reporte de un caso / Treatment of type III periodontal-endodontic lesion (combined or true): a case report

Se presenta un caso clínico de una lesión endoperiodontal tipo III (combinada o verdadera) en un paciente de sexo femenino de 41 años de edad sin antecedentes sistémicos. La paciente fue derivada del Curso de Especialización en Periodoncia de la Escuela de Graduados de la Universidad de Chile debido a una lesión periapical en la pieza 3.6. El pronóstico en este tipo de lesiones es dudoso, ya que es necesario que se efectúe el tratamiento endodóntico así como el periodontal, y el resultado recae más en el tratamiento periodontal. La pieza fue tratada endodónticamente dejando medicamento intraconducto a base de Clorhexidina al 2 por ciento en gel por 7 días. Una vez obturado el diente se citó a la paciente 3 meses después para un control radiográfico. Actualmente el diente está totalmente asintomático, sin movilidad y con señales de reparación.

We report a case of a type III periodontal-endodontic lesion (combined or true) in a 41-year-old female patient without systemic history. The patient was transferred from the Specialization Course in Periodontology at the Graduate School of the University of Chile due to a periapical lesion in the tooth 3.6. The prognosis for this type of lesion is uncertain, since it is necessary to perform endodontic and periodontal treatment, and the result depends more on the periodontal treatment. The piece was treated endodontically leaving intracanal medication based on 2 percent Chlorhexidine gel for 7 days. Once the tooth obturated, we gave the patient an appointment 3 months later for a control radiography. Currently, the tooth is completely asymptomatic, without mobility and with signs of repair.

Effects of lead intoxication on intercellular junctions and biochemical alterations of the renal proximal tubule cells.

Lead intoxication is a worldwide health problem which frequently affects the kidney. In this work, we studied the effects of chronic lead intoxication (500 ppm of Pb in drinking water during seven months) on the structure, function and biochemical properties of rat proximal tubule cells. Lead-exposed animals showed increased lead concentration in kidney, reduction of calcium and amino acids uptake, oxidative damage and glucosuria, proteinuria, hematuria and reduced urinary pH. These biochemical and physiological alterations were related to striking morphological modifications in the structure of tubule epithelial cells and in the morphology of their mitochondria, nuclei, lysosomes, basal and apical membranes. Interestingly, in addition to the nuclei, inclusion bodies were found in the cytoplasm and in mitochondria. The epithelial cell structure modifications included an early loss of the apical microvillae, followed by a decrement of the luminal space and the respective apposition and proximity of apical membranes, resulting in the formation of atypical intercellular contacts and adhesion structures. Similar but less marked alterations were observed in subacute lead intoxication as well. Our work contributes in the understanding of the physiopathology of lead intoxication on the structure of renal tubular epithelial cell-cell contacts in vivo.

Analysis of neuronal proliferation, migration and differentiation in the postnatal brain using equine infectious anemia virus-based lentiviral vectors.

Ongoing neurogenesis in discrete sectors of the adult central nervous system depends on the mitotic activity of an elusive population of adult stem cells. The existence of adult neural stem cells provides an alternative approach to transplantation of embryonic stem cells in cell-based therapies. Owing to the limited intrinsic fate of adult stem cells and inhibitory nature of the adult brain for neurogenesis, accommodation for circuit replacement in the brain will require genetic and epigenetic manipulation. Here, we show that a replication-incompetent Equine Infectious Anemia Virus (EIAV) is highly suitable for stable and persistent gene transfer to adult neural stem cells. The transduced regions were free of long-lasting neuroimmune responses to EIAV. Transduction in the subventricular zone was specific to the stem cell niche, but spared the progeny of adult neural stem cells that includes transit amplifying progenitors (TAPs) and migrating neuroblasts. With time, EIAV-transduced stem cells passed on the transgene to TAPs and migrating neuroblasts, which ultimately differentiated into neurons in the olfactory bulbs. We show that EIAV is highly suitable for discovery and assessment of mechanisms that regulate proliferation, migration and differentiation in the postnatal brain.

Induction and regulation of conoid extrusion in Toxoplasma gondii.

Cell invasion by the intracellular parasite Toxoplasma gondii occurs through an active process that involves dynamic events, such as gliding motility and conoid extrusion, followed by a sequential secretion from specialized secretory organelles. Increase of intracellular Ca(2+) by ionophores induces conoid extrusion, although in an irreversible way, thus limiting the characterization of the regulatory pathways. In this report we studied the effect of different activating conoid conditions to characterize the regulatory mechanisms involved. Exposure of tachyzoites to ethanol, a well-known activator of microneme secretion through the increase of intracellular Ca(2+), induced conoid extrusion without affecting parasite viability nor its in vitro invasive capability, in a process that could be completely reverted and repeatedly reactivated. A temporal relationship between conoid extrusion and microneme secretion was here studied. Under this condition, signal transduction pathways and the precise role of the parasite cytoskeleton were characterized. Our results indicate that phospholipase C, Ca(2+) released through channels sensitive to inositol-3-phosphate and ryanodine, as well as myosin together with actin filaments, but not microtubules, all participate in conoid extrusion. Specific inhibitors for serine-threonine kinases blocked conoid extrusion; in contrast, calmodulin inhibitors did not affect the induction. A regulatory model for conoid activation is here proposed.

Entamoeba histolytica: monoclonal antibody against the beta1 integrin-like molecule (140 kDa) inhibits cell adhesion to extracellular matrix components.

We describe a monoclonal antibody (3C10) against the beta1 integrin-like molecule which immunoprecipitates two polypeptides of 140 and 155 kDa from detergent-soluble extract of Entamoeba histolytica. The 140-kDa polypeptide has been described as a beta subunit of the amoebic fibronectin receptor as it is recognized by an anti-integrin beta1 (human) monoclonal antibody in immunoblot assay. The receptor molecules were localized with the 3C10 monoclonal antibody in intracellular and surface membranes of E. histolytica trophozoites by immunofluorescence and immunogold labeling methods. Significant inhibitions of cell adhesion on extracellular matrix proteins such as fibronectin (56%) (P < 0.001) and collagen (50%) (P < 0.001) and partial inhibition on laminin (23%) (P > 0.1) were achieved by the monoclonal antibody.

Biochemical and histochemical analysis of 71 kDa dystrophin isoform (Dp71f) in rat brain.

Dp71 is a member of the dystrophin family and the most abundant dmd gene product in the brain. In the present study, we focused on a short dystrophin transcript named Dp71f, which is alternatively spliced when exon 78 is absent The topographic localization of this protein in the encephalon has not been properly described yet, nor its cellular or subcellular localization, and even less its functions. Dp71f was found to be a cytoplasmic 70 kDa protein and localized in all encephalon regions studied. Double labeling using specific markers for various cell types confirmed Dp71f distribution in the cytoplasm of all cell types studied. Labeling was more conspicuous near the nucleus and diminished towards the periphery of cells. In some cases, we observed cells that were positive for actin and Dp71f in regions corresponding to lamellipodia-like structures. Dp71f and Dp71d isoforms were differently distributed. Our study is the first specific and unambiguous description of the topography and cellular localization patterns of Dp71f in brain, suggesting that Dp71f is a ubiquitous protein.

Current concepts: the use of immunoperoxidase techniques in mohs micrographic surgery.

Mohs micrographic surgery is used for the removal of certain malignant tumors, both ensuring complete excision by histologic examination of margins as well as minimizing normal tissue loss. Recently, several investigators have incorporated the use of immunoperoxidase techniques to aid in the removal of selected high-risk carcinomas, sarcomas, and melanomas. We describe the basic principles of immunoperoxidase and review recent articles in which immunoperoxidase was used as an adjunct to routine hematoxylin-eosin staining in Mohs micrographic surgery. Additionally, we show examples of selected tumors comparing routine hematoxylin-eosin stains and immunoperoxidase. We believe the use of immunoperoxidase can be of significant value in the removal of certain high-risk tumors. In particular, this technique is useful in "unmasking" malignant cells in areas of dense inflammation, identification of some cases of perineural invasion, identification of pagetoid spread in carcinomas and melanomas, and finally in helping to identify subtle margins in dermatofibrosarcoma protuberans.

Early elicitor induction in members of a novel multigene family coding for highly related RING-H2 proteins in Arabidopsis thaliana.

We describe the identification and structural characterization of a novel family of Arabidopsis genes related to ATL2 which encode a variant of the RING zinc finger domain, known as RING-H2. Analysis of genes selected by us and of sequences from Arabidopsis stored in databases permitted the prediction of several RING-H2 proteins that contain highly homologous RING domains. The ATL gene family is represented by fifteen sequences that contain, in addition to the RING, a transmembrane domain which is located in most of them towards the N-terminal end. Transgenic Arabidopsis seedlings carrying the ATL2 promoter fused to the GUS reporter gene revealed that the expression of ATL2 is rapidly induced after exposure to chitin or inactivated crude cellulase preparations. Rapid induction of transcript accumulation of another member of the ATL family was also observed under the same conditions. These results suggest that some ATLs may be involved in the early stages of the defense response triggered in plants in response to pathogen attack.