Biomineralization potential and biological properties of a new tantalum oxide (Ta2O5)-containing calcium silicate cement.

OBJECTIVE: The present study evaluated the biological effects and biomineralization potential of a new tantalum oxide (Ta2O5)-containing material designed for vital pulp therapy or perforation repair (NeoMTA 2), compared to NeoMTA Plus and Bio-C Repair. MATERIAL AND METHODS: Human dental pulp stem cells (hDPSCs) were exposed to different eluates from NeoMTA Plus, NeoMTA 2, and Bio-C Repair. Ion release from each material was determined using inductively coupled plasma-optical emission spectrometry (ICP-MS). The biological experiments performed were MTT assays, apoptosis/necrosis assays, adhesion assays, migration assays, morphology evaluation, and reactive oxygen species (ROS) production analysis. Biomineralization was assessed by Alizarin red S staining. Finally, osteo/odontogenic gene expression was determined by real-time quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR). Data were analyzed using one-way ANOVA followed by Tukey's multiple comparison test. RESULTS: NeoMTA 2 displayed a significantly higher calcium release compared to the other materials (p < 0.05). When hDPSCs were cultured in presence of the different material eluates, all groups exhibited similar hDPSC viability and migration rates when compared to untreated cells. Substantial cell attachment and spreading were observed in all materials' surfaces, without significant differences. hDPSCs treated with NeoMTA 2 displayed an upregulation of ALP, Col1A1, RUNX2 (p < 0.001), ON, and DSPP genes (p < 0.05), and showed the highest mineralization potential compared to other groups (p < 0.001). Finally, the more concentrated eluates from these materials, specially NeoMTA Plus and NeoMTA 2, promoted higher ROS production in hDPSCs compared to Bio-C Repair and control cells (p < 0.001), although these ROS levels did not result in increased cell death. CONCLUSIONS: The new tantalum oxide (Ta2O5)-containing material shows an adequate cytocompatibility and the ability to promote biomineralization without using chemical osteogenic inducers, showing great potential as a new material for vital pulp therapy. CLINICAL RELEVANCE: NeoMTA 2 seems to be a promising material for vital pulp therapy. Further studies considering its biocompatibility and biomineralization potential are necessary.

Cytocompatibility and bioactive properties of the new dual-curing resin-modified calcium silicate-based material for vital pulp therapy.

OBJECTIVE: The aim of the present study was to evaluate the in vitro biocompatibility of Theracal PT, Theracal LC, and MTA Angelus, considered as bioactive materials used for vital pulp treatment, on human dental pulp stem cells (hDPSCs). MATERIALS AND METHODS: Human dental pulp stem cells (hDPSCs) were isolated from third molars, and material eluates were prepared (undiluted, 1:2, and 1:4 ratios). The hDPSC cytotoxicity, adhesion, morphology, viability, and cell migration were assessed. The mineralization nodule formation was determined by Alizarin red S staining (ARS). The odonto/osteogenic differentiation potential was assessed by osteo/odontogenic marker expression real-time qPCR. The chemical composition and ion release of the vital pulp materials were determined by energy dispersive X-ray (EDX) and inductively coupled plasma-mass spectrometry (ICP-MS), respectively. Statistical differences were assessed by ANOVA and Tukey's test (p < 0.05). RESULTS: The three vital pulp materials showed variable levels of calcium, tungsten, silicon, and zirconium release and in their chemical composition. Cytocompatibility assays revealed higher hDPSC viability and migration rates when treated with Theracal PT than with Theracal LC. The lowest cell adhesion and spreading were observed in all Theracal LC-treated groups, whereas the highest were observed when treated with MTA. Theracal PT and MTA promoted the upregulation of DSPP and RUNX2 gene expression (p < 0.05). After 21 days, both MTA Angelus and Theracal PT-treated cells exhibited a significantly higher mineralized nodule formation than the negative control (p < 0.05). CONCLUSIONS: This study demonstrates the favorable in vitro cytocompatibility and bioactive properties of the recently introduced Theracal PT and the well-established MTA Angelus on hDPSCs, as opposed to Theracal LC. More studies, including in vivo animal testing are suggested before these new formulations might be used in the clinical setting. CLINICAL RELEVANCE: Theracal PT is a new material that could be clinically suitable for vital pulp therapy. Further studies considering its biocompatibility and bioactivity are necessary.

Microstructural composition, ion release, and bioactive potential of new premixed calcium silicate-based endodontic sealers indicated for warm vertical compaction technique.

OBJECTIVE: The aim of this study was to evaluate the microstructural composition, ion release, cytocompatibility, and mineralization potential of Bio-C Sealer ION+ (BCI) and EndoSequence BC Sealer HiFlow (BCHiF), compared with AH Plus (AHP), in contact with human periodontal ligament cells (hPDLCs). MATERIALS AND METHODS: The sealers' ionic composition and release were assessed using energy-dispersive spectroscopy (EDS) and inductively coupled plasma mass spectrometry (ICP-MS), respectively. For the biological assays, hPDLCs were isolated from third molars, and sealer extracts were prepared (undiluted, 1:2, and 1:4 ratios). An MTT assay, wound-healing assay, and cell morphology and adhesion analysis were performed. Activity-related gene expression was determined using RT-qPCR, and mineralization potential was assessed using Alizarin Red staining (ARS). Statistical analyses were performed using one-way ANOVA and Tukey's post hoc test (α < 0.05). RESULTS: The three sealers exhibited variable levels of silicon, calcium, zirconium, and tungsten release and in their composition. Both BCI and BCHiF groups showed positive results in cytocompatibility assays, unlike AHP. The BCHiF group showed an upregulation of CAP (p < 0.01), CEMP1, ALP, and RUNX2 (p < 0.001) compared with the negative control, while the BCI group showed an upregulation of CEMP1 (p < 0.01), CAP, and RUNX2 (p < 0.001). Both groups also exhibited a greater mineralization potential than the negative and positive controls (p < 0.001). CONCLUSIONS: The calcium silicate-based sealers considered in the present in vitro study exhibited a high calcium ion release, adequate cytocompatibility, upregulated osteo/cementogenic gene expression, and increased mineralized nodule formation in contact with hPDLCs. CLINICAL RELEVANCE: From a biological perspective, BCI and BCHiF could be clinically suitable for root canal filling.

High browsing skeletal adaptations in Spinophorosaurus reveal an evolutionary innovation in sauropod dinosaurs.

Sauropods were among the most diverse lineages of dinosaurs, with an ample geographic distribution throughout the Mesozoic. This evolutionary success is largely attributed to neck elongation and its impact on feeding efficiency. However, how neck elongation influenced exactly on feeding strategies is subject of debate. The process of mounting a nearly complete virtual skeleton of Spinophorosaurus nigerensis, from the Middle (?) Jurassic of Niger, has revealed several previously unknown osteological adaptations in this taxon. Wedged sacral and posterior dorsal vertebrae cause the presacral column to deflect antero-dorsally. This, together with elongated scapulae and humeri make the anterior region of the skeleton vertically lengthened. Also, elongated prezygapophyseal facets on the cervical vertebrae and a specialized first dorsal vertebra greatly increase the vertical range of motion of the neck. These characters support this early eusauropod as a more capable high browser than more basally branching sauropods. While limb proportions and zygapophyseal facets vary among Eusauropoda, the sacrum retained more than 10° of wedging in all Eusauropoda. This implied a functional constraint for sauropod species which evolved lower browsing feeding strategies: the antero-dorsal sloping caused by the sacrum had to be counteracted with further skeletal modifications, e.g. a ventrally curved mid to anterior presacral spine to hinder the dorsal slope of the whole presacral series caused by the wedged sacrum. This suggests that at least the last common ancestor of Eusauropoda developed high browsing capabilities, partially due to the modified wedged sacrum, likely a potential synapomorphy of the clade and key in the evolutionary history of the group.

Production of 1,3-propanediol from pure and crude glycerol using a UASB reactor with attached biomass in silicone support.

The 1,3-propanediol (1,3-PDO) yield and productivity from glycerol were studied over a 155-day period. A UASB reactor that also contained silicone support for biomass attachment was used to evaluate the optimal operational conditions and microbiota development. The highest average 1,3-PDO yield was 0.54 and 0.48 mol.mol-gly-1 when reactor pH was 5.0-5.5 and the applied loading rate was 18 and 20 g-gly.L-1.d-1 using the pure and crude substrate, respectively. The productivity was close to 7.5 g.L-1.d-1 for both substrates; therefore, the direct use of crude glycerol can be valorized in practice. Clostridium was the predominant genus for 1,3-PDO production and C. pasteurianum was dominant in the biofilm. Using crude glycerol, C. beijerinckii dropped strongly; some Clostridium population was then replaced by Klebsiella pneumoniae and Lactobacillus spp. The good process performance and the advances in the microbiota knowledge are steps forward to obtain a more cost-effective system in practice.

Inhibition of methanogenesis by chlorophenols: a kinetic approach.

Chlorophenols exert a crucial effect on the methanogenesis, considerably reducing both maximum methane potential and methanogenic rates. However, there is not enough information about the kinetic mechanism of chlorophenols toxicity on the methanogenesis, which is a key aspect for the control of the anaerobic digesters because of the sensitivity and the potential for energy recovery derived from methane release. The International Water Association-Anaerobic Digestion Model No. 1 (IWA-ADM1) can be adapted to a wide range of situations by updating or changing the equations in the model. The present study proposes a general kinetic model for methanogenesis. This model has been applied to predict the inhibition of methanogenesis by chlorophenols, and it can be used for updating the IWA-ADM1 when treating inhibitory compounds. The model was calibrated and validated using a wide broad of experimental sets of data of methane production by granular sludge in the presence of 2,4-dichlorophenol (24 DCP), 2,4,6-trichlorophenol (246TCP) and pentachlorophenol (PCP) in batch assays. A lag-phase of the effect of chlorophenols on the methanogenesis by non-adapted sludge was detected and modeled by the kinetic model proposed. In addition, the inhibitory effect of PCP was more pronounced on the acetoclastic methanogenesis than on the hydrogenotrophic one. Non-competitive and uncompetitive inhibition types were detected using 24 DCP and 246 TCP, whereas a suicide or irreversible inhibition type was observed in the case of PCP. Values of inhibition constants considerably varied depending on the chlorophenol used, between 45 mg24DCPL(-1), 41-51 mg246TCPL(-1) and 0.9-7.8 mgPCPL(-1). The higher toxicity of PCP is related with its hydrophobicity, which was determined by adsorption tests and using partition coefficients n-octanol/water. Modeling was accompanied by high statistical support in all cases, which confirmed the validation of the model proposed.

Anaerobic biodegradation of 2,4,6-trichlorophenol in expanded granular sludge bed and fluidized bed biofilm reactors bioaugmented with Desulfitobacterium spp.

The biodegradation of 2,4,6-trichlorophenol (246TCP) was studied using expanded granular sludge bed (EGSB) reactors and a fluidized bed biofilm reactor (FBBR) filled with activated carbon. One of the EGSB reactor and the FBBR were bioaugmented with Desulfitobacterium strains. 246TCP loading rate was gradually incremented from 10 to 250 mg L(-1) day(-1). The main pathway of dechlorination was in ortho-position, generating 4-chlorophenol and 2,4-dichlorophenol. The maintenance of both COD degradation efficiency (higher than 80%) and methanogenic efficiency (between 0.3 and 0.6 g CH4-COD g(-1) COD consumed) in EGSB reactor implies a great stability of the process. Through isotherm studies in FBBR, it could be deduced that around 52% of 246TCP was completely dechlorinated, whereas the adsorption involved around 16%. By means of FISH studies it was proved that the methanogenic Archaea community was maintained in the bioaugmented EGSB reactor, whereas in the FBBR this community was gradually developed until reaching stability. Desulfitobacterium community was also maintained in the reactors, although D. chlororespirans proportion rise in the FBBR at the higher 246TCP loading rates, implying that this species can withstand the 246TCP toxicity better than D. hafniense.

From Río Tinto to Mars: the terrestrial and extraterrestrial ecology of acidophiles.

The recent geomicrobiological characterization of Río Tinto, Iberian Pyrite Belt (IPB), has proven the importance of the iron cycle, not only in generating the extreme conditions of the habitat (low pH, high concentration of toxic heavy metals) but also in maintaining the high level of microbial diversity, both prokaryotic and eukaryotic, detected in the water column and the sediments. The extreme conditions of the Tinto basin are not the product of industrial contamination but the consequence of the presence of an underground bioreactor that obtains its energy from the massive sulfide minerals of the IPB. To test this hypothesis, a drilling project was carried out to intersect ground waters that interact with the mineral ore in order to provide evidence of subsurface microbial activities and the potential resources to support these activities. The oxidants that drive the system appear to come from the rock matrix, contradicting conventional acid mine drainage models. These resources need only groundwater to launch microbial metabolism. There are several similarities between the vast deposits of sulfates and iron oxides on Mars and the main sulfide-containing iron bioleaching products found in the Tinto. Firstly, the short-lived methane detected both in Mars' atmosphere and in the sediments and subsurface of the IPB and secondly, the abundance of iron, common to both. The physicochemical properties of iron make it a source of energy, a shield against radiation and oxidative stress as well as a natural pH controller. These similarities have led to Río Tinto's status as a Mars terrestrial analogue.

Effect of 2,4,6-trichlorophenol on the microbial activity of adapted anaerobic granular sludge bioaugmented with Desulfitobacterium strains.

The anaerobic degradation of 2,4,6-trichlorophenol (246TCP) has been studied in batch experiments. Granular sludges previously acclimated to 2,4-dichlorophenol (24DCP) and then adapted to at a load of 330 µM 246TCPd(-1) in two expanded granular sludge bed (EGSB) reactors were used. One of the reactors had been bioaugmented with Desulfitobacterium strains whereas the other served as control. 246TCP was tested at concentrations between 250 and 760 µM. The study focused on the fate of both fermentation products and chlorophenols derived from dechlorination of 246TCP. This compound mainly affected the biodegradation of acetate and propionate, which were inhibited at 246TCP concentrations above 380 µM. Lactate and ethanol were also accumulated at 760 µM 246TCP. Methanogenesis was strongly inhibited at 246TCP concentrations higher than 380 µM. A diauxic production of methane was observed, which can be described by a kinetic model in which acetoclastic methanogenesis was inhibited, whereas hydrogenotrophic methanogenesis was hardly affected by 246TCP. The similarity of the kinetic parameters obtained for the control and the bioaugmented sludges (K(i)=175-200 µM 246TCP and n=7) suggests that methanogenesis is not affected by the bioaugmentation. Moreover, the 246TCP dechlorination occurred mainly at ortho position, successively generating 24DCP and 4-chlorophenol (4CP), which was identified as final product. The bioaugmentation does not significantly improve the anaerobic biodegradation of 246TCP. It has been shown that the active biomass is capable of bioaccumulating 246TCP and products from dechlorination, which are subsequently excreted to the bulk medium when the biomass becomes active again. A kinetic model is proposed which simultaneously explains 246TCP and 24DCP reductive dechlorinations and includes the 246TCP bioaccumulation. The values of the kinetic parameters for 246TCP dechlorination were not affected by bioaugmentation (V(max)=5.3 and 5.1 µM h(-1) and K(s)=5.8 and 13.1 µM for control and bioaugmented sludges, respectively).

Composite anatomical-clinical-molecular prognostic model in non-small cell lung cancer.

The objective of the present study was to elaborate a survival model that integrates anatomic factors, according to the 2010 seventh edition of the tumour, node and metastasis (TNM) staging system, with clinical and molecular factors. Pathologic TNM descriptors (group A), clinical variables (group B), laboratory parameters (group C) and molecular markers (tissue microarrays; group D) were collected from 512 early-stage nonsmall cell lung cancer (NSCLC) patients with complete resection. A multivariate analysis stepped supervised learning classification algorithm was used. The prognostic performance by groups was: areas under the receiver operating characteristic curve (C-index): 0.67 (group A), 0.65 (Group B), 0.57 (group C) and 0.65 (group D). Considering all variables together selected for each of the four groups (integrated group) the C-index was 0.74 (95% CI 0.70-0.79), with statistically significant differences compared with each isolated group (from p = 0.006 to p < 0.001). Variables with the greatest prognostic discrimination were the presence of another ipsilobar nodule and tumour size > 3 cm, followed by other anatomical and clinical factors, and molecular expressions of phosphorylated mammalian target of rapamycin (phospho-mTOR), Ki67cell proliferation index and phosphorylated acetyl-coenzyme A carboxylase. This study on early-stage NSCLC shows the benefit from integrating pathological TNM, clinical and molecular factors into a composite prognostic model. The model of the integrated group classified patients with significantly higher accuracy compared to the TNM 2010 staging.

Cosmetic wastewater treatment by upflow anaerobic sludge blanket reactor.

Anaerobic treatment of pre-settled cosmetic wastewater in batch and continuous experiments has been investigated. Biodegradability tests showed high COD and solid removal efficiencies (about 70%), being the hydrolysis of solids the limiting step of the process. Continuous treatment was carried out in an upflow anaerobic sludge blanket reactor. High COD and TSS removal efficiencies (up to 95% and 85%, respectively) were achieved over a wide range of organic load rate (from 1.8 to 9.2g TCODL(-1)day(-1)). Methanogenesis inhibition was observed in batch assays, which can be predicted by means of a Haldane-based inhibition model. Both COD and solid removal were modelled by Monod and pseudo-first order models, respectively.

Comparison of UASB and EGSB performance on the anaerobic biodegradation of 2,4-dichlorophenol.

The anaerobic degradation of 2,4-dichlorophenol (2,4-DCP) in upflow anaerobic sludge blanket (UASB) and expanded granular sludge bed (EGSB) reactors using glucose as main carbon source was studied. The performance of both systems was compared in terms of 2,4-DCP and COD removal efficiencies, methane production, stability, granular sludge adaptability as well as reversion of the bacterial inhibition. Both organic and 2,4-DCP loading rates were incrementally varied through the experiments. With loading rates of 1.9 gCODL(-1)d(-1) and 100mg 2,4-DCP L(-1)d(-1), 75% and 84% removal efficiencies of this compound, accompanied by COD consumption efficiencies of 61% and 80% were achieved in the UASB and EGSB reactors, respectively. In these conditions, methane production reached 0.088 L CH(4)g(-1) COD in the EGSB reactor whereas in the UASB reactor was almost negligible. Decreasing the 2,4-DCP loading rate to 30 mgL(-1)d(-1) an improvement in the methane production was observed in both reactors (methanogenic activity of 0.148 and 0.192 L CH(4)g(-1) COD in UASB and EGSB reactors, respectively). Efficiency of dechlorination was improved in both reactors from around 30% to 80% by reducing to one-half the COD due to a decreasing of the 4-chlorophenol concentration accumulated in the effluents of both reactors. The dechlorination efficiency of the UASB reactor was dramatically inhibited at a 2,4-DCP feed concentration above around 210 mgL(-1) because of 2,4-DCP accumulation in the effluent. SEM studies revealed no significant morphological changes in the sludge granules.

Compared microbiology of granular sludge under autotrophic, mixotrophic and heterotrophic denitrification conditions.

Water contamination by nitrate is a wideworld extended phenomena. Biological autotrophic denitrification has a real potential to face this problem and presents less drawbacks than the most extended heterotrophic denitrification. Three bench-scale UASB reactors were operated under autotrophic (R1, H2S as electron donor), mixotrophic (R2, H2S plus p-cresol as electron donors) and heterotrophic (R3, p-cresol as electron donor) conditions using nitrate as terminal electron acceptor. 16S rDNA genetic libraries were built up to compare their microbial biodiversity. Six different bacteria phyla and three archaeal classes were observed. Proteobacteria was the main phyla in all reactors standing out the presence of denitrifiers. Microorganisms similar to Thiobacillus denitrificans and Acidovorax sp. performed the autotrophic denitification. These OTUs were displaced by chemoheterotrophic denitrifiers, especially by Limnobacter-like and Ottowia-like OTUs. Other phyla were Bacteroidetes, Chloroflexi, Firmicutes and Actinobacteria that--as well as Archaea members--were implicated in the degradation of organic matter, as substrate added as coming from endogenous sludge decay under autotrophic conditions. Archaea diversity remained low in all the reactors being Methanosaeta concilii the most abundant one.

Anaerobic biodegradation of 2,4,6-trichlorophenol by methanogenic granular sludge: role of co-substrates and methanogenic inhibition.

The influence of several co-substrates in the anaerobic biodegradation of 2,4,6-trichlorophenol (246TCP) by methanogenic granular sludge as well as in methanogenesis inhibition by 246TCP has been studied. 4 g-COD.L(-1) of lactate, sucrose, volatile fatty acids (VFA) acetate:propionate:butyrate 1:1:1, ethanol, methanol, yeast extract (YE), and 2 g-COD.L(-1) of formate and methylamine were tested. Two concentrations of 246TCP: 80 mg.L(-1) and 113 mg.L(-1) (this last corresponding to the EC(50) for acetotrophic methanogenesis) were tested. Three consecutive co-substrate and nutrient feedings were accomplished. 246TCP was added in the second feed, and the 246TCP removal rate increased considerably after the third feed. Accumulated metabolites after ortho-dechlorination, either 4-chlorophenol (4CP) (when methanol, ethanol or VFA were used as co-substrates) or 2,4-dichlorophenol (24DCP) (with lactate) avoided the complete dechlorination of 246TCP. With methylamine and formate this compound was degraded only partially. Monochlorophenols biodegradation was partially achieved with YE, but both 24DCP and 2,6-dichlorophenol (26DCP) were accumulated. In the presence of sucrose para-dechlorination was observed. 246TCP was better tolerated by methanogens when ethanol and methanol were added because of the highest specific methanogenic activity achieved with these co-substrates. Methanol and ethanol were the best co-substrates in the anaerobic biodegradation of 246TCP.

[Photodynamic therapy with verteporfin in choroidal neovascularization after refractive surgery]. / Terapia fotodinámica con verteporfina en neovascularización coroidea después de cirugía refractiva.

PURPOSE: To analyze the results obtained with Photodynamic Therapy (PDT) to treat subfoveal and juxtafoveal Choroidal Neovascularization (CNV) in patients with high myopia corrected by Laser-Assisted in situ Keratomileusis (LASIK) or by implanting a Phakic Intraocular Lens (PIOL). METHODS: We analyzed the results from 14 highly myopic eyes corrected by LASIK (seven cases) or by PIOL implantation (seven cases), which later developed CNV and were treated by PDT with verteporfin. RESULTS: Mean Best Corrected Visual Acuity (BCVA) after refractive surgery was 0.45 SD 0.17 (range, 0.2 to 0.8), with residual spherical equivalent (RSE) -0.5 SD 1.8 D (range, 1 to 5.5 D). After CNV appearance, BCVA was 0.10 SD 0.19 (range, 0.025 to 0.7). CNV was treated in all cases by PDT (mean, 2.0 SD 0.8 treatments). After CNV closure, the mean BCVA improved up to 0.22 SD 0.18 (range, 0.1 to 0.63) (RSE -1.4 SD 1.4 D, range, 0.5 to -4 D). Differences in RSE after refractive surgery and after PDT, and differences between BCVA after CNV appearance and final were not statistically significant (p=0.82 and p=0.06, respectively, Student's t test paired data). CONCLUSION: We consider that PDT is effective in achieving closure of CNV in myopic patients after refractive surgery without inducing changes in spherical equivalent.

Microbiological and structural aspects of granular sludge from autotrophic denitrifying reactors.

Denitrification is applied in the tertiary treatment of wastewater to reduce N-pollutants. Fluorescence in situ hybridisation (FISH), CARD (catalyzed reporter deposition)-FISH, cloning, and scanning electron microscopy (SEM) were applied to follow the evolution of the microbial composition and structure of granular sludge in autotrophic denitrifying bioreactors fed with nitrate and thiosulfate. With this goal, FISH oligonucleotide probes for the autotrophic denitrifiers, Thiobacillus denitrificans and Thiomicrospira denitrificans, were designed and their utility tested. CARD-FISH and cloning data showed that bacterial diversity changed with bioreactor operation time. After 110 days of operation, the abundance of Thiobacillus denitrificans cells increased considerably: from 1 to 35% of total DAPI-stained cells and from no isolated clones to 30% of the total positives clones. This fact strongly suggests that this microorganism played a dominant role in the autotrophic denitrification. The Archaeal diversity remained almost unchanged and it was mainly represented by Methanosaeta soehngenii. Scanning electron microscopy results indicated a considerable loss in the integrity of the sludge granules during the operation, with risk of sludge buoyancy.

[Corneal intra-epithelial neoplasia (CIN). Treatment by combined excision and antimetabolite therapy]. / Neoplasia intraepitelial corneal (CIN). Tratamiento mediante exéresis y antimetabolitos.

CASE REPORT: We present two clinical cases of corneal intra-epithelial neoplasia defined histopathologically. DISCUSSION: We sought to establish the importance of treatment with Mitomycin-C in two cases of corneal intra-epithelial neoplasia. The differential diagnosis was established based on the evolutionary character of the process, as well as specific histopathological aspects of these entities. In both cases, treatment with Mitomycin-C before, during and after surgery contributed to the decrease in the size of the lesion and to their cure without relapse.

Neoplasia intraepitelial corneal (CIN). Tratamiento mediante exéresis y antimetabolitos / Corneal intra-epithelial neoplasia (CIN). Treatment by combined excision and antimetabolite therapy

Caso clínico: Se presentan dos casos clínicos de neoplasia intrapitelial corneal diagnosticados mediante estudio histopatológico.Discusión: Se pretende establecer la importancia del tratamiento con Mitomicina C en dos casos de neoplasia intraepitelial corneal. Se establece el diagnóstico diferencial en base a los caracteres evolutivos del proceso, así como a los diferentes aspectos histopatológicos específicos de estas entidades. En ambos casos el tratamiento con Mitomicina C pre, intra y postoperatorio contribuyó a la disminución en el tamaño de la lesión y a su curación sin recidivas

Case Report: We present two clinical cases of corneal intra-epithelial neoplasia defined histopathologically. Discussion: We sought to establish the importance of treatment with Mitomycin-C in two cases of corneal intra-epithelial neoplasia. The differential diagnosis was established based on the evolutionary character of the process, as well as specific histopathological aspects of these entities. In both cases, treatment with Mitomycin-C before, during and after surgery contributed to the decrease in the size of the lesion and to their cure without relapse

Molecular ecology of anaerobic granular sludge grown at different conditions.

Qualitative and quantitative diversity of microorganisms present in anaerobic granular sludges fed with different substrates, as well as the structure of these granules have been studied using fluorescent 16S rRNA-targeted in situ hybridization and electron microscopy. The granules showed a multi-layered structure, in which both densely packed and loose micro-colonies, channels and holes could be observed. Only bacteria were found in the outer shell of the granules, while both archaea and bacteria were detected in the inner core. Although high cell density was found in the granules (more than 10(11) cells/gram, determined by DAPI-stain) only a low percentage of cells was able to hybridize with the rRNA-targeted probes. Significant quantitative and qualitative differences were observed in the composition of granules fed with different substrates (formate, acetate at high and low concentrations, propionate, sucrose, starch and peptone). Bacterial cells were mostly gram-positives. Active proteobacteria were scarce in the granules exposed to VFA. Syntrophobacteria became dominant in the propionate-grown biomass. Concerning methanogenic archaea, Methanosaeta was the predominant species using complex substrates or low acetate concentration fed granules, while Methanosarcina and members of Methanobacteriales were predominant in the granules grown at high concentration of acetate or formate, respectively. Other Methanomicrobiales and Methanococcales, have been detected in the anaerobic granular sludge in the conditions used in this work.