Novel Zebrafish Patient-Derived Tumor Xenograft Methodology for Evaluating Efficacy of Immune-Stimulating BCG Therapy in Urinary Bladder Cancer.

BACKGROUND: Bacillus Calmette-Guérin (BCG) immunotherapy is the standard-of-care adjuvant therapy for non-muscle-invasive bladder cancer in patients at considerable risk of disease recurrence. Although its exact mechanism of action is unknown, BCG significantly reduces this risk in responding patients but is mainly associated with toxic side-effects in those facing treatment resistance. Methods that allow the identification of BCG responders are, therefore, urgently needed. METHODS: Fluorescently labelled UM-UC-3 cells and dissociated patient tumor samples were used to establish zebrafish tumor xenograft (ZTX) models. Changes in the relative primary tumor size and cell dissemination to the tail were evaluated via fluorescence microscopy at three days post-implantation. The data were compared to the treatment outcomes of the corresponding patients. Toxicity was evaluated based on gross morphological evaluation of the treated zebrafish larvae. RESULTS: BCG-induced toxicity was avoided by removing the water-soluble fraction of the BCG formulation prior to use. BCG treatment via co-injection with the tumor cells resulted in significant and dose-dependent primary tumor size regression. Heat-inactivation of BCG decreased this effect, while intravenous BCG injections were ineffective. ZTX models were successfully established for six of six patients based on TUR-B biopsies. In two of these models, significant tumor regression was observed, which, in both cases, corresponded to the treatment response in the patients. CONCLUSIONS: The observed BCG-related anti-tumor effect indicates that ZTX models might predict the BCG response and thereby improve treatment planning. More experiments and clinical studies are needed, however, to elucidate the BCG mechanism and estimate the predictive value.

Lysine in Combination With Estradiol Promote Dissemination of Estrogen Receptor Positive Breast Cancer via Upregulation of U2AF1 and RPN2 Proteins.

The majority of estrogen receptor positive (ER+) breast cancer (BC) maintain the ER at metastatic sites. Despite anti-estrogen therapy, almost 30% of ER+ BC patients relapse. Thus, new therapeutic targets for ER+ BC are needed. Amino acids (AAs) may affect the metastatic capacity by affecting inflammatory cells. Essential AAs (EAAs) cannot be produced by human cells and might therefore be targetable as therapeutics. Here we sampled extracellular EAAs in vivo by microdialysis in human BC. Mass spectrometry-based proteomics was used to identify proteins affected after EAA and estradiol (E2) exposure to BC cells. Proteins relevant for patient survival were identified, knocked down in BC cells, and metastatic capability was determined in vivo in the transgenic zebrafish model. We found that lysine was the most utilized EAA in human ER+BC in vivo. In zebrafish, lysine in presence of E2 increased neutrophil-dependent dissemination of ER+ BC cells via upregulation of U2AF1 and RPN2 proteins, which both correlated with poor prognosis of ER+ BC patients in clinical databases. Knockdown of U2AF1 and RPN2 decreased the expression of several cell-adhesion molecules resulting in diminished dissemination. Dietary lysine or its related metabolic pathways may be useful therapeutic targets in ER+ BC.

Mineralogical evidence of unfavorable geochemical interactions between urban runoff pollutants and filter materials in low-impact systems.

We report on the geochemical interactions between a synthetic urban runoff (SUR) and the minerals of materials used in a multi-layered column filter (soil, sand, gravel, and tezontle) at the laboratory scale, which mimicked an unvegetated low-impact development (LID) system. After five 8 h infiltration cycles using the SUR, the average concentrations of Pb and Mn decreased slightly at the column outlet, as did HCO3-, SO42-, and Na+, whereas Mg increased and Cl-, Ca2+, and K+ were only detected at the outlet. The filter materials were comprised of silicates, Mn-bearing oxides (hausmannite and manganite), carbonates (calcite), chlorides (sylvite), and sulfates (anglesite, lanarkite, barite, and epsomite). PHREEQC modeling allowed the identification of the geochemical processes that occurred in the filter. The results showed the removal capacity of the filter materials through the formation of secondary minerals such as rhodochrosite (MnCO3) and the over-saturation of anglesite (PbSO4), and also showed that they may mobilize ions from the upper to the interior layers (as Mg2+ from epsomite, MgSO4·7H2O, and Ba2+ from barite, BaSO4). We highlight the importance of knowing the geological nature of filter materials used in LID systems because they may lead to the geogenic mobilization of toxic contaminants to the environment.

Synchronized tissue-scale vasculogenesis and ubiquitous lateral sprouting underlie the unique architecture of the choriocapillaris.

The choriocapillaris is an exceptionally high density, two-dimensional, sheet-like capillary network, characterized by the highest exchange rate of nutrients for waste products per area in the organism. These unique morphological and physiological features are critical for supporting the extreme metabolic requirements of the outer retina needed for vision. The developmental mechanisms and processes responsible for generating this unique vascular network remain, however, poorly understood. Here we take advantage of the zebrafish as a model organism for gaining novel insights into the cellular dynamics and molecular signaling mechanisms involved in the development of the choriocapillaris. We show for the first time that zebrafish have a choriocapillaris highly similar to that in mammals, and that it is initially formed by a novel process of synchronized vasculogenesis occurring simultaneously across the entire outer retina. This initial vascular network expands by un-inhibited sprouting angiogenesis whereby all endothelial cells adopt tip-cell characteristics, a process which is sustained throughout embryonic and early post-natal development, even after the choriocapillaris becomes perfused. Ubiquitous sprouting was maintained by continuous VEGF-VEGFR2 signaling in endothelial cells delaying maturation until immediately before stages where vision becomes important for survival, leading to the unparalleled high density and lobular structure of this vasculature. Sprouting was throughout development limited to two dimensions by Bruch's membrane and the sclera at the anterior and posterior surfaces respectively. These novel cellular and molecular mechanisms underlying choriocapillaris development were recapitulated in mice. In conclusion, our findings reveal novel mechanisms underlying the development of the choriocapillaris during zebrafish and mouse development. These results may explain the uniquely high density and sheet-like organization of this vasculature.

Effect of photocatalytic Fe2O3 nanoparticles on urban runoff pollutant removal by permeable concrete.

Permeable pavements are an efficient urban runoff (UR) management solution that also improve water quality. In this work, a photocatalytic layer of Fe2O3 nanoparticles (NP) was incorporated into permeable concrete to evaluate its impact on the removal of several microbiological (Escherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila, and Enterococcus faecalis) and physicochemical (N-NH4+, N-NO3-, phenol, PO43-, Fe, Mn, and Pb) pollutants. First, permeable concrete samples were created with sufficient compressive strength and hydraulic conductivity for light traffic. The test samples were then coated with a mixture containing either 3% or 5% Fe2O3 NP by cement weight. Control samples were prepared without NP. Scanning electron microscopy, energy dispersive spectroscopy, and X-ray photoelectron spectroscopy analyses showed that the nanoparticles remained unaltered on the concrete's surface. Synthetic URs simulating the microbiological or physicochemical composition of real UR were applied to the samples to evaluate their pollutant removal efficiencies. The depollution performances of the test (with 3% and 5% Fe2O3 NP) and control samples were statistically compared. The test samples (3% NP, 5% NP, and the controls) significantly modified (p < 0.05) most of the measured variables (i.e., the concentrations of E. coli, A. hydrophila, PO43-, Fe, Mn, and Pb) in the synthetic URs. Unexpectedly, the test samples (with 3% or 5% Fe2O3 NP) did not significantly remove (p > 0.05) some pollutants prone to oxidation, such as phenol or ammonium. However, the 5% NP sample significantly enhanced Mn removal. In general, the decontamination performances of the concrete samples with Fe2O3 NP were not influenced by the nanoparticles; thus, they did not appear to add value to the generated permeable concrete. Nevertheless, our results indicate the considerable benefits of implementing permeable concrete to improve the quality of UR.

Enhancement of the biogas and biofertilizer production from Opuntia heliabravoana Scheinvar.

Waste Opuntia is an abundant source of biomass to produce biogas and biofertilizer in a small and commercial scale. This crop has a high biomass yield, wide adaptation to diverse climatic zones, rapid growth, and low input requirements. This study aimed to evaluate the combined effect of adjusting C/N ratio and an alkaline pretreatment (AP) of waste Opuntia heliabravoana Scheinvar in the production of biogas and biofertilizer in anaerobic reactors. AP bioreactors produced more biogas than the control (C, without the combined effect of AP); besides, in this process, it was not necessary to use additional water due to the high content of water that is present in the tissue of this crop. On the other hand, both biofertilizers (C and AP) had enssential microbial groups that help to enhance plant nutrition as S-reducers, S-oxidizers, amylolytic, cellulolytic bacteria, anaerobic S-mineralizers, cellulolytic fungi, and P-solubilizers. Also, the AP treatment to help to increase 1.5:1 total nitrogen (TN) concentration decreased the pathogenic microorganisms in the biofertilizer compared to the C treatment. For this reason, Opuntia spp. is a good substrate for production of biogas and biofertilizer with essential nutrients for many crops in area with water scarcity.

Adipocytes Promote Early Steps of Breast Cancer Cell Dissemination via Interleukin-8.

Fat is a major tissue component in human breast cancer (BC). Whether breast adipocytes (BAd) affect early stages of BC metastasis is yet unknown. BC progression is dependent on angiogenesis and inflammation, and interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF) are key regulators of these events. Here, we show that BAd increased the dissemination of estrogen receptor positive BC cells (BCC) in vivo in the zebrafish model of metastasis, while dissemination of the more aggressive and metastatic BCC such as estrogen receptor negative was unaffected. While anti-VEGF and anti-IL-8 exhibited equal inhibition of angiogenesis at the primary tumor site, anti-IL-8 reduced BCC dissemination whereas anti-VEGF had minor effects on this early metastatic event. Mechanistically, overexpression of cell-adhesion molecules in BCC and neutrophils via IL-8 increased the dissemination of BCC. Importantly, the extracellular in vivo levels of IL-8 were 40-fold higher than those of VEGF in human BC. Our results suggest that IL-8 is a clinical relevant and promising therapeutic target for human BC.

Estradiol Promotes Breast Cancer Cell Migration via Recruitment and Activation of Neutrophils.

Estradiol (E2) plays a key role in breast cancer progression. Most breast cancer recurrences express the estrogen receptor (ER), but nearly 50% of patients are resistant to antiestrogen therapy. Novel therapeutic targets of ER-positive breast cancers are needed. Protumoral neutrophils expressing the lymphocyte function-associated antigen 1 (LFA-1) integrin may mediate cancer metastasis, and TGFß1 is the major chemoattractant for neutrophils. The role of E2 in neutrophil-ER+ breast cancer cell interactions is unknown. We studied this in vivo using murine breast cancers in immunocompetent mice and human breast cancers in nude mice. Cell dissemination was evaluated in a zebrafish model, and microdialysis of breast cancer patients was performed. In vitro studies were done with mammosphere cultures of breast cancer cells and human neutrophils. We found that E2 increased the number of LFA-1+ neutrophils recruited to the invasive edge of mouse tumors, increased TGFß1 secretion and promoted neutrophil infiltration in mammospheres, and induced overexpression of LFA-1 in neutrophils. In zebrafish, in the presence of E2, neutrophils increased dissemination of ER+ breast cancer cells via LFA-1 and TGFß1, thus causing noninvasive cancer cells to be highly metastatic. Time-lapse imaging in zebrafish revealed close interactions of neutrophils with cancer cells, which drove breast cancer metastasis. We also found that extracellular TGFß1 was overproduced in human breast cancer tissue compared with adjacent normal breast tissue. Thus, E2 can regulate immune/cancer cell interactions in tumor microenvironments. Our results indicate that extracellular TGFß1 is a relevant target in human breast cancer. Cancer Immunol Res; 5(3); 234-47. ©2017 AACR.

Effectiveness of rabbit manure biofertilizer in barley crop yield.

The quality of biofertilizers is usually assessed only in terms of the amount of nutrients that they supply to the crops and their lack of viable pathogens and phytotoxicity. The goal of this study was to determine the effectiveness of a liquid biofertilizer obtained from rabbit manure in terms of presence of pathogens, phytotoxicity, and its effect on the grain yield and other agronomic traits of barley (Hordeum vulgare L.). Environmental effects of the biofertilizer were also evaluated by following its influence on selected soil parameters. We applied the biofertilizer at five combinations of doses and timings each and in two application modes (foliar or direct soil application) within a randomized complete block design with three replicates and using a chemical fertilizer as control. The agronomic traits evaluated were plant height, root length, dry weight, and number of leaves and stems at three growth stages: tillering, jointing, and flowering. The effectiveness of the biofertilizer was significantly modified by the mode of application, the growth stage of the crop, and the dose of biofertilizer applied. The results showed that the foliar application of the biofertilizer at the tillering stage produced the highest increase in grain yield (59.7 %, p < 0.10). The use of the biofertilizer caused significant changes in soil, particularly concerning pH, EC, Ca, Zn, Mg, and Mn. It is our view that the production and use of biofertilizers are a reliable alternative to deal with a solid waste problem while food security is increased.

Quality of urban runoff in wet and dry seasons: a case study in a semi-arid zone.

Urban runoff (UR) is a promising new resource that may alleviate growing tensions in numerous arid and semi-arid regions of the world. However, it is precisely in these zones that the available UR quality characteristics are scarcer. This work aims to evaluate a wide set of parameters to establish a detailed approach to both the quality of UR in a midsized city in Central Mexico and the feasibility of using UR to recharge aquifers. UR from an institutional land use site was sampled during wet and dry seasons and assessed for suspended solids, organic matter, nutrients, microorganisms, metals, and persistent organic chemicals (i.e., polycyclic aromatic hydrocarbons, PAH). The results were analyzed using multivariate statistical methods to identify relationships among the variables, the sampling sites and the seasons. The soil erosion and the leaching of materials due to the water flow through vegetated areas were identified as the most influencing factor on the quality of the site runoff in both dry and wet seasons. Additionally, data were more heterogeneous during the dry season, and higher pollutant concentrations were found both during the dry season and in more pervious zones. We consider UR a promising water source for recharging aquifers in arid and semi-arid zones if a program is implemented that can integrate an adequate runoff treatment system, soil protection, and other non-structural measures.

Evaluation of natural materials as exogenous carbon sources for biological treatment of low carbon-to-nitrogen wastewater.

In the bacterial processes involved in the mitigation of nitrogen pollution, an adequately high carbon-to-nitrogen (C : N) ratio is key to sustain denitrification. We evaluated three natural materials (woodchips, barley grains, and peanut shells) as carbon sources for low C : N wastewater. The amount of organic matter released from these materials to aqueous media was evaluated, as well as their pollution swapping potential by measuring the release of total Kjeldahl nitrogen, N-NH4 (+), NO2 (-), and NO3 (-), and total phosphorous. Barley grains yielded the highest amount of organic matter, which also showed to be the most easily biodegradable. Woodchips and peanut shells released carbon rather steadily and so they would not require frequent replenishment from biological reactors. These materials produced eluates with lower concentrations of nutrients than the leachates from barley grains. However, as woodchips yielded lower amounts of suspended solids, they constitute an adequate exogenous source for the biological treatment of carbon-deficient effluents.

Cadmium Removal from Aqueous Systems Using Opuntia albicarpa L. Scheinvar as Biosorbent.

The aim of this research was to investigate the use of a natural adsorbent like nopal (Opuntia albicarpa L. Scheinvar) for removing cadmium from aqueous solutions with low concentrations of this metal. Two treatments were applied to the cladodes: a dehydration to get dehydrated nopal (DHN) and heating up to 90°C to obtain a thermally treated nopal (TN). After examining the effect of various pH values (2-7), the capacity of each biosorbent was examined in batch sorption tests at different dosages (0, 500, 1000, 1500, 2000, and 3000 mg L(-1)). The results indicated that adsorption of cadmium to biomass of DHN and TN was highly dependent on pH and biosorbent dosage. The best removal of cadmium (53.3%, corresponding to q e of 0.155 mg g(-1)) was obtained at pH 4.0 by using the TN sorbent. Infrared and Raman spectra confirmed that cadmium removal occurred via adsorption to -OH functional groups.

Resistance and inactivation kinetics of bacterial strains isolated from the non-chlorinated and chlorinated effluents of a WWTP.

The microbiological quality of water from a wastewater treatment plant that uses sodium hypochlorite as a disinfectant was assessed. Mesophilic aerobic bacteria were not removed efficiently. This fact allowed for the isolation of several bacterial strains from the effluents. Molecular identification indicated that the strains were related to Aeromonas hydrophila, Escherichia coli (three strains), Enterobacter cloacae, Kluyvera cryocrescens (three strains), Kluyvera intermedia, Citrobacter freundii (two strains), Bacillus sp. and Enterobacter sp. The first five strains, which were isolated from the non-chlorinated effluent, were used to test resistance to chlorine disinfection using three sets of variables: disinfectant concentration (8, 20 and 30 mg·L(-1)), contact time (0, 15 and 30 min) and water temperature (20, 25 and 30 °C). The results demonstrated that the strains have independent responses to experimental conditions and that the most efficient treatment was an 8 mg·L(-1) dose of disinfectant at a temperature of 20 °C for 30 min. The other eight strains, which were isolated from the chlorinated effluent, were used to analyze inactivation kinetics using the disinfectant at a dose of 15 mg·L(-1) with various retention times (0, 10, 20, 30, 60 and 90 min). The results indicated that during the inactivation process, there was no relationship between removal percentage and retention time and that the strains have no common response to the treatments.

Novel fusion protein derived from vasostatin 30 and vasoinhibin II-14.1 potently inhibits coronary endothelial cell proliferation.

Angiogenesis has been considered an important target for cancer therapy. The inhibition of angiogenesis represents a promising strategy for anti-cancer treatment, tumor growth inhibition, and metastasis. Vasostatin 30 (Vs30), and the 14.1 kDa vasoinhibin (Vi-II-14.1) are two peptides with remarkable anti-tumor and anti-angiogenic effect. The aim of this study was to produce a novel fusion protein between Vs30 and Vi-II-14.1, denominated VS_VI, to obtain a new protein with higher biological activity. The protein fusion genes were cloned into a T7 promoter-based vector, expressed in Escherichia coli BL21-SI and purified by affinity column chromatography. In vitro assays showed that the recombinant fusion protein inhibited rat coronary endothelial cell proliferation at 65.5 % at 10 nM, whereas recombinant Vs30 and Vi-II-14.1 inhibited at 33 and 50.5 % respectively, at the same concentration. The results showed that VS_VI is significantly more active than the Vs30 and Vi-II-14.1 separately. In addition, a practical classification of the vasoinhibins based on the peptide origin and theoretical molecular weight is proposed. This is the first study to produce a new fusion protein derived from Vs30 and Vi-II-14.1, both of them proposed as promising therapeutic agents.

Chlorine disinfection of Pseudomonas aeruginosa, total coliforms, Escherichia coli and Enterococcus faecalis: revisiting reclaimed water regulations.

Pathogenic organisms can be transmitted orally through drinking water or through skin and mucosae by both direct and indirect contact, and their presence in water thus has a negative impact on public health. In wastewater treatment plants (WWTP), water is disinfected to inactivate pathogens. The quantification of several microbial indicators in aquatic systems is required to estimate the biological quality of such systems. So far, coliform bacteria have been used as traditional indicators world-wide. This study has assessed the resistance of total coliforms, Escherichia coli, Pseudomonas aeruginosa and Enterococcus faecalis to three dosages of sodium hypochlorite (NaClO) at two exposure times. The bacteria were isolated from secondary effluents of a WWTP located in Hidalgo, Mexico. The results show that the number of colony-forming units of all studied bacterial types decreased when both the NaClO concentration and exposure times increased. However, they were not eliminated. The inclusion of the species Pseudomonas aeruginosa in regulations for treated wastewater quality as a new indicator is highly recommended due to its importance as an opportunistic pathogen. The detection of this species along with the traditional organisms could be particulary significant for reclaimed water to be used with direct human contact.

Standardization of activated sludge for biodegradation tests.

Activated sludges are an inoculum source commonly used in biodegradation studies, as wastewater treatment facilities constitute an entry point to the environment for many chemicals. In this paper, the main issues relating to the use of activated sludge in biodegradability tests are presented. Special attention is also devoted to discussing the factors affecting both the activity of the microbial communities and the test results. After a short survey of the state of the art of microbiology of activated sludge, the paper focuses on the methods used to reduce the variations in the diversity, quality and quantity of these communities. Finally, use of surrogates as reference materials in biodegradability tests is discussed.

Model-based design of different fedbatch strategies for phenol degradation in acclimatized activated sludge cultures.

Microbial degradation of phenol was studied using batch and fedbatch cultures of acclimatized activated sludge under a wide range of phenol (0-793 mg l(-1)) and biomass (0.74-6.7 g l(-1)) initial concentrations. As cell growth continued after total phenol removal, the production and later consumption of a main metabolic intermediate was considered the step governing the biodegradation kinetics. A model that takes explicitly into account the kinetics of the intermediate was developed by introducing a specific growth rate model associated with its consumption and the incorporation of a dual-substrate inhibitory effect on phenol degradation. Biomass growth and phenol removal were adequately predicted in all the cultures. Moreover, the model-based design of the fedbatch feeding strategies allowed driving separately the phenol degradation under substrate-limitation and substrate-inhibition modes. A sensitivity analysis was also performed in order to establish the importance of the parameters in the accuracy of model predictions.

Manganese accumulation in plants of the mining zone of Hidalgo, Mexico.

Soil, sediments, water and plant samples from the mining zone of Molango were collected and analyzed and Mn-tolerant and Mn-accumulator plants were identified. Soil and sediments presented moderately alkaline and reducing conditions, a normal electrical conductivity, a sandy texture and medium-to-high cation exchange capacities. These properties favored the presence of Mn(2+), which is the form most easily assimilated by plants, and the total Mn concentration (11,637-106,104 mg kg(-1) dried weight, DW) was at phytotoxic level. Water was also an important Mn source. Equisetum hyemale and Telypteris kunthii survived in the presence of such Mn concentrations using an exclusion strategy, while Cnidoscolus multilobus, Platanus mexicana, Solanum diversifolium, Asclepius curassavica L. and Pluchea sympitifolia employed an accumulation strategy. These plants could be useful to re-vegetate and stabilize Mn tailings in order to decrease the erosion effects.

A method for measuring the anoxic biodegradability under denitrifying conditions.

A test for assessing the anoxic biodegradability of organic compounds under denitrifying conditions is proposed. The method is based on the recovery and quantification of the CO2 produced, which is evidence of complete biodegradation of the test compound (added as the sole carbon source). The tests were carried out in a mineral medium, with nitrate as electron acceptor. Whole lake sediments, sediment extracts and a commercial inoculum were assayed as a possible inoculum source by means of glucose biodegradability tests. It was found that the sediment extracts constitute a suitable and environmentally-relevant inoculum source, since they add non-significant amounts of carbon to the tests. Two xenobiotic compounds, namely, aniline and phenol, were tested in the aforementioned conditions as well as in a standard aerobic biodegradability test. Both aniline and phenol attained a biodegradation level higher than 60% in a short time period (<28 days) and thus can be considered as readily biodegradable in denitrifying environments. Nevertheless, the kinetics obtained in the anoxic test were slower than in aerobic conditions, and even suggested the accumulation of intermediate metabolites in the case of phenol. The results of this study indicate that the fate of xenobiotic compounds under anoxic conditions differs from that observed in an oxic environment, and therefore it should be considered by standard biodegradability testing procedures.

Inocula from activated sludge for ready biodegradability testing: homogenization by preconditioning.

The use of activated sludge as inoculum source in ready biodegradability tests (RBT) suffers from several drawbacks related to the heterogeneity of these communities. In this work, the ability of a 7-day aeration period in a mineral medium to homogenize the characteristics of various activated sludges, as suggested by some RBT, was studied. The biodegradation potential of three activated sludge supernatants obtained from different wastewater treatment plants was assessed in terms of cultivable cell density, dehydrogenasic activity and a profile of hydrolytic enzymes. After the preconditioning, the homogenization of these characteristics in the supernatants was observed, as well as a decrease. When preconditioned inocula were used in acetate RBT, the biodegradation kinetics were homogenized. However, some preconditioned supernatants lost their ability to degrade an easily-assimilable xenobiotic compound (aniline) during the observation period, showing the effect of inoculum preconditioning on the behavior of complex bacterial communities, specialist populations (e.g. aniline degraders) being more sensitive than generalist populations (e.g. acetate degraders). These results show that preconditioning cannot be an optional inoculum pretreatment in RBT, and emphasize the importance of further studies focusing on inoculum homogenization.