Development of nature-based sustainable passive technologies for treating and disinfecting municipal wastewater: Experiences from constructed wetlands and slow sand filter.

There is an urgent need to develop low-cost technology for effective wastewater treatment and its further disinfection to the level that makes it economically useful. This work has designed and evaluated the various types of constructed wetlands (CWs) followed by a slow sand filter (SSF) for wastewater treatment and disinfection. The studied CWs were, CWs with gravels (CW-G), free water surface-CW (FWS-CWs), and CWs integrated microbial fuel cell (MFC) with granular graphite (CW-MFC-GG) planted with Canna indica plant species. These CWs were operated as secondary wastewater treatment technologies followed by SSF for disinfection purposes. The highest total coliform removal was observed in the combination of CW-MFC-GG-SSF which achieved a final concentration of 172 CFU/100 mL, whereas faecal coliform removal was 100 % with the combinations of CW-G-SSF and CW-MFC-GG-SSF, achieving 0 CFU/100 mL in the effluent. In contrast, FWS-SSF achieved the lowest total and faecal coliform removal attaining a final concentration of 542 CFU/100 mL and 240 CFU/100 mL, respectively. Furthermore, E. coli were detected as negative/absent in CW-G-SSF and CW-MFC-GG-SSF, while it was positive for FWS-SSF. In addition, the highest turbidity removal was achieved in CW-MFC-GG and SSF combination of 92.75 % from the municipal wastewater influent turbidity of 82.8 NTU. Furthermore, in terms of overall treatment performance of CW-G-SSF and CW-MFC-GG-SSF, these systems were able to treat 72.7 ± 5.5 % and 67.0 ± 2.4 % of COD and 92.3 % and 87.6 % of phosphate, respectively. Additionally, CW-MFC-GG also exhibited a power density of 85.71 mA/m3 and a current density of 25.71 mW/m3 with 700 Ω of internal resistance. Thus, CW-G and CW-MFC-GG followed by SSF could be a promising solution for enhanced disinfection and wastewater treatment.

Pharmacokinetic characterization of fluorocoxib D, a cyclooxygenase-2-targeted optical imaging agent for detection of cancer.

SIGNIFICANCE: Fluorocoxib D, N-[(rhodamin-X-yl)but-4-yl]-2-[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetamide, is a water-soluble optical imaging agent to detect cyclooxygenase-2 (COX-2)-expressing cancer cells. AIM: We evaluated the pharmacokinetic and safety properties of fluorocoxib D and its ability to detect cancer cells in vitro and in vivo. APPROACH: Pharmacokinetic parameters of fluorocoxib D were assessed from plasma collected at designated time points after intravenous administration of 1 mg / kg fluorocoxib D in six research dogs using a high-performance liquid chromatography analysis. Safety of fluorocoxib D was assessed for 3 days after its administration using physical assessment, complete blood count, serum chemistry profile, and complete urinalysis in six research dogs. The ability of fluorocoxib D to detect COX-2-expressing cancer cells was performed using human 5637 cells in vitro and during rhinoscopy evaluation of specific fluorocoxib D uptake by canine cancer cells in vivo. RESULTS: No evidence of toxicity and no clinically relevant adverse events were noted in dogs. Peak concentration of fluorocoxib D (114.8 ± 50.5 ng / ml) was detected in plasma collected at 0.5 h after its administration. Pretreatment of celecoxib blocked specific uptake of fluorocoxib D in COX-2-expressing human 5637 cancer cells. Fluorocoxib D uptake was detected in histology-confirmed COX-2-expressing head and neck cancer during rhinoscopy in a client-owned dog in vivo. Specific tumor-to-normal tissue ratio of detected fluorocoxib D signal was in an average of 3.7 ± 0.9 using Image J analysis. CONCLUSIONS: Our results suggest that fluorocoxib D is a safe optical imaging agent used for detection of COX-2-expressing cancers and their margins during image-guided minimally invasive biopsy and surgical procedures.

Detection of tyrosine kinase inhibitors-induced COX-2 expression in bladder cancer by fluorocoxib A.

Among challenges of targeted therapies is the activation of alternative pro-survival signaling pathways in cancer cells, resulting in an acquired drug resistance. Cyclooxygenase-2 (COX-2) is overexpressed in bladder cancer cells, making it an attractive molecular target for the detection and treatment of cancer. Fluorocoxib A is an optical imaging agent that selectively targets COX-2. In this study, we evaluated the ability of fluorocoxib A to monitor the responses of bladder cancer to targeted therapies in vivo. The effects of several tyrosine kinase inhibitors (TKIs: axitinib, AB1010, toceranib, imatinib, erlotinib, gefitinib, imatinib, sorafenib, vandetanib, SP600125, UO126, and AZD 5438) on COX-2 expression were validated in ten human and canine bladder cancer cell lines (J82, RT4, T24, UM-UC-3, 5637, SW780, TCCSUP, K9TCC#1Lillie, K9TCC#2Dakota, K9TCC#5Lilly) in vitro. The effects of TKIs on bladder cancer in vivo were evaluated using the COX-2-expressing K9TCC#5Lilly xenograft mouse model and detected by fluorocoxib A. The increased COX-2 expression was detected by all tested TKIs in at least one of the tested COX-2-expressing bladder cancer cell lines (5637, SW780, TCCSUP, K9TCC#1Lillie, K9TCC#2Dakota, and K9TCC#5Lilly) in vitro. In addition, fluorocoxib A uptake correlated with the AB1010- and imatinib-induced COX-2 expression in the K9TCC#5Lilly xenografts in vivo. In conclusion, these results indicate that fluorocoxib A could be used for the monitoring the early responses to targeted therapies in COX-2-expressing bladder cancer.

Platelet-rich plasma affects the proliferation of canine bone marrow-derived mesenchymal stromal cells in vitro.

BACKGROUND: Reported efficacy of platelet-rich plasma (PRP) in regenerative medicine is contradictory. We validated the effects of PRP on proliferation of canine bone marrow-derived multipotent mesenchymal stromal cells (K9BMMSCs) in vitro. PRP was extracted from blood of six dogs with osteoarthritis. K9BMMSCs were established from bone marrow and characterized for CD90 and CD19 expression by immunocytochemistry. Effects of PRP concentrations on viability of matching autologous K9BMMSCs were validated using MTS assay. RESULTS: Positive CD90 and negative CD19 expression confirmed MSC origin. PRP at 40% volume/volume concentration increased, while PRP at 80 and 100% v/v concentrations suppressed viability of tested K9BMMSCs. CONCLUSION: PRP concentration plays an important role in K9BMMSCs viability, which could affect tissue repairs in vivo.

WT1 regulates cyclin A1 expression in K562 cells.

The restricted expression of Wilms tumor 1 (WT1) and cyclin A1 (CCNA1) in normal tissues, as opposed to their abnormal expression in leukemia demonstrates the applicability of WT1 and CCNA1 as cancer antigens for immunotherapy, and as markers for prognosis and relapse. In this study, the WT1 and CCNA1 mRNA levels were found to be elevated in bone marrow samples from pediatric acute promyelocytic leukemia (APL or AML­M3) patients, and to be quite varied in pediatric acute lymphocytic leukemia (ALL) patients, compared to non­leukemic bone marrow controls. Consistent with the observed upregulation of both WT1 and CCNA1 in APL, WT1 overexpression elevated the CCNA1 mRNA levels in K562 leukemia cells. Treatment with curcumin decreased the WT1 levels in K562 cells, and also decreased CCNA1 protein expression. The examination of the CCNA1 promoter identified potential canonical WT1 binding sites within the 3­kb region upstream of the transcription start site. Chromatin immunoprecipitation and luciferase reporter assays confirmed WT1 binding and the activation of the CCNA1 promoter. Furthermore, the GC­rich core CCNA1 promoter region provided additional non­canonical WT1 activation sites, as revealed by promoter assays. The importance of the GC­rich core region of the CCNA1 promoter was confirmed by treating the K562 cells with mithramycin A, which blocks the binding of zinc finger transcription factors to GC­rich sequences. Mithramycin A subsequently suppressed both CCNA1 promoter activity and protein expression in the K562 cells. Taken together, the data from the WT1 overexpression, and curcumin and mithramycin A treatment experiments, as well as those from chromatin binding assays, along with inferences from patient RNA analyses, establish a plausible link between WT1 and CCNA1, and support the functional significance of an elevated WT1 expression in leukemia, which may also affect CCNA1 expression.

Mutations of p53 decrease sensitivity to the anthracycline treatments in bladder cancer cells.

Due to doxorubicin (Dox) cardiotoxicity, the next generation of novel non-cardiotoxic anthracyclines, including AD 312 and AD 198, were synthesized and validated. In this study, we assessed the efficacy and mechanisms of anthracyclines-induced apoptosis and inhibition of cell viability in human bladder cancer cells expressing wild-type (wt) p53 (RT4 and SW780) and mutated (mt) p53 (UM-UC-3, 5637, T-24, J82, and TCCSUP) protein. Anthracyclines inhibited cell viability in tested TCC cells, but were less effective in mt-p53 TCC cells, especially in the drug-resistant J82 and TCCSUP cells. Anthracyclines upregulated the expression of wt p53 protein in RT4 and SW780 cells, but had no effect on expression of mt p53 protein in UM-UC-3, 5637, T-24, J82, and TCCSUP cells. The anthracyclines activated caspase 3/7 and cleavage of PARP in wt-p53 RT4 and SW780 cells, and mt-p53 5637, UM-UC-3, and T-24, but not in mt-p53 J82 and TCCSUP cells. The anthracyclines-induced cleavage of PARP was blocked by p53 siRNA in wt-p53 RT4 cells. Co-treatment of AD 198 with PRIMA-1 significantly inhibited cell viability of mt-p53 J82 cells, but had no effect in wt-p53 RT4 cells. AD 198 blocked c-myc expression in mt-p53 UM-UC-3, 5637, T-24, and J82 cells, however no expression of c-myc was detected in wt-p53 RT4 and SW780 cells. In conclusion, our results demonstrated that the anthracycline-induced resistance in bladder cancer cells positively correlated with TP53 mutations in the tetramerization domain in J82 and TCCSUP cells. Further, AD 312 and AD 198 are promising chemotherapeutic drugs for bladder cancer, especially in combination with PRIMA-1.

Aligned nanofiber material supports cell growth and increases osteogenesis in canine adipose-derived mesenchymal stem cells in vitro.

Tissue engineering shows great promise for the treatment of degenerative diseases, including bone repair. Polymer nanofibers provide a three-dimensional (3-D) scaffold for attachment and growth of mesenchymal stem cells. Increasing evidence supports that fiber alignment on scaffolds plays a major role in the viability and differentiation of stem cells. We compared the cell viability of canine adipose tissue-derived mesenchymal stem cells (cADMSCs) cultured in the aligned- (NanoAligned™) and random- (NanoECM™) oriented polycaprolactone (PCL) nanofiber-coated plates to control polystyrene tissue culture plates using a proliferation assay. Ability of the plates to induce differentiation of cADMSCs into osteocytes, adipocytes, and neurons was evaluated based on expression of the osteocyte markers, COL1A1 and osterix; adipocyte markers PPARγ2 and LPL; and neuronal marker nestin using RT-PCR. Proliferation results demonstrated that aligned-oriented PCL nanofiber-coated plates were more suitable substrate for cADMSCs after 7 days in culture compared to random-oriented PCL nanofiber-coated or control plates. Additionally, we demonstrated that both 3-D PCL nanofiber-coated plates were a better scaffold for cADMSCs differentiation into osteocytes compared to control plates. In conclusion, our results confirm that PCL nanofiber is a suitable tissue engineering material for use in regenerative medicine for canine patients in vivo. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1780-1788, 2018.

Evaluation of simultaneous nutrient and COD removal with polyhydroxybutyrate (PHB) accumulation using mixed microbial consortia under anoxic condition and their bioinformatics analysis.

Simultaneous nitrate-N, phosphate and COD removal was evaluated from synthetic waste water using mixed microbial consortia in an anoxic environment under various initial carbon load (ICL) in a batch scale reactor system. Within 6 hours of incubation, enriched DNPAOs (Denitrifying Polyphosphate Accumulating Microorganisms) were able to remove maximum COD (87%) at 2 g/L of ICL whereas maximum nitrate-N (97%) and phosphate (87%) removal along with PHB accumulation (49 mg/L) was achieved at 8 g/L of ICL. Exhaustion of nitrate-N, beyond 6 hours of incubation, had a detrimental effect on COD and phosphate removal rate. Fresh supply of nitrate-N to the reaction medium, beyond 6 hours, helped revive the removal rates of both COD and phosphate. Therefore, it was apparent that in spite of a high carbon load, maximum COD and nutrient removal can be maintained, with adequate nitrate-N availability. Denitrifying condition in the medium was evident from an increasing pH trend. PHB accumulation by the mixed culture was directly proportional to ICL; however the time taken for accumulation at higher ICL was more. Unlike conventional EBPR, PHB depletion did not support phosphate accumulation in this case. The unique aspect of all the batch studies were PHB accumulation was observed along with phosphate uptake and nitrate reduction under anoxic conditions. Bioinformatics analysis followed by pyrosequencing of the mixed culture DNA from the seed sludge revealed the dominance of denitrifying population, such as Corynebacterium, Rhodocyclus and Paraccocus (Alphaproteobacteria and Betaproteobacteria). Rarefaction curve indicated complete bacterial population and corresponding number of OTUs through sequence analysis. Chao1 and Shannon index (H') was used to study the diversity of sampling. "UCI95" and "LCI95" indicated 95% confidence level of upper and lower values of Chao1 for each distance. Values of Chao1 index supported the results of rarefaction curve.

Diversity of marine bacteria producing beta-glucosidase inhibitors.

BACKGROUND: Beta-glucosidase inhibitors are being extensively studied for use as anti-diabetics, anti-obesity and anti-tumour compounds. So far, these compounds have been reported in large numbers from plants, mushrooms, algae and fungi. There are very few reports of such inhibitors from bacteria in the open literature, particularly marine bacteria; although the best known inhibitor deoxynojirimycin was isolated from bacilli and actinomycete. Through this study, we tried to discover the diversity of microbial associates of marine sponge and sediment producing ß-glucosidase inhibitors. RESULTS: We found 41 (22.7%) out of 181 bacteria, produced such inhibitors. The inhibitors are abundant in bacterial associates of marine sponge Aka coralliphaga. When these bacteria were phylogenetically analyzed, it was found that marine bacteria producing glucosidase inhibitors belong to the phylum Firmicutes (23), Actinobacteria (9), Proteobacteria (7) and Bacteroidetes (1). CONCLUSION: A significant number of marine bacteria belonging to a wide range of bacterial taxa were found to produce ß-glucosidase inhibitors. These compounds are abundantly present in bacteria of the phylum Firmicutes followed by the phylum Actinobacteria. The results nurture a hope of finding new compounds, which can inhibit glucosidases, in the bacterial domain of marine organisms. Thus, marine microbial cells can be utilized as producers of pharmacologically essential enzyme inhibitors.

The Wilms' tumor gene (WT1) regulates E-cadherin expression and migration of prostate cancer cells.

BACKGROUND: One key step in the development of prostate cancer (PCa) metastasis is the loss of E-cadherin expression associated with increased cellular motility and tumor invasion. This loss of E-cadherin expression is also required during normal embryogenesis and similar transcriptional repressors have been identified in both processes. We have previously reported the presence of one such transcription factor, WT1 in high Gleason grade prostate tumor tissues, and its absence in non-neoplastic or benign prostatic hyperplasia tissues. RESULTS: To better understand the effect of WT1 on E-cadherin expression and migration of PCa cells we quantified WT1 and E-cadherin mRNA levels in normal prostate epithelial and PCa cell lines with varying migratory potential. In WT1 transfected cells E-cadherin transcript levels were decreased, while they were increased in siWT1-RNA transfected PCa cells, suggesting that elevated WT1 expression was sufficient to dampen E-cadherin levels and potentially enhance migratory ability. To delineate the mechanism of WT1-mediated repression of E-cadherin, potential WT1 binding sites were tested in vitro and in vivo binding of WT1 to the E-cadherin promoter in the chromatin of LNCaP and PC3 cells was assessed by Chromatin Immunoprecipitation. The effect of WT1 binding was measured in reporter assays; in PC3 and DU145 cells WT1 decreased the activity of the proximal E-cadherin promoter. Using site-directed mutagenesis, a newly identified WT1 binding site located 146 bp from the transcription start site was shown to be required for this repression by WT1. Transwell migration and wound healing assays revealed that in LNCaP cells with low migratory potential, over-expression of WT1 was sufficient to enhance migration, conversely, in the highly migratory PC3 cells silencing of WT1 decreased migration. CONCLUSIONS: These findings suggested that WT1 expression in high grade prostate cancer may contribute to migration and metastasis. Thus, in prostate cancer WT1 may function as a novel oncogene facilitating development of the lethal metastatic phenotype.