Techno-economic assessment of a novel algal-membrane system versus conventional wastewater treatment and advanced potable reuse processes: Part II.

This study developed a comprehensive techno-economic assessment (TEA) framework to evaluate an innovative algae resource recovery and near zero-liquid discharge potable reuse system (i.e., the main system) in comparison with a conventional potable water reuse system (i.e., the benchmark system). The TEA study aims to estimate the levelized costs of water of individual units and integrated processes including secondary wastewater treatment, advanced water purification for potable reuse, and sludge treatment. This would provide decision-makers valuable information regarding the capital and operational costs of the innovative main system versus a typical potable water reuse treatment train, along with possible routes of cost optimization and improvements for the design of full-scale facilities. The main system consists of (i) a novel algal-based wastewater treatment coupled with a dual forward osmosis and seawater reverse osmosis (Algal FO-SWRO) membranes system for potable water reuse and hydrothermal liquefaction (HTL) to produce bioenergy and subsequent nutrients extraction from the harvested algal biomass. The benchmark system includes (ii) an advanced water purification facility (AWPF) that consists of a conventional activated sludge biological treatment (CAS), microfiltration (MF), brackish water reverse osmosis (BWRO), ultraviolet/advanced oxidation process (UV-AOP), and granular activated carbon (GAC), with anaerobic digestion for sludge treatment. Capital expenditures (CAPEX) and operational expenditures (OPEX) were calculated for each unit of both systems (i.e., sub-systems). Based on a 76% overall water recovery designed for the benchmark system, the water cost was estimated at $2.03/m3. The highest costs in the benchmark system were found on the CAS and the anaerobic digester, with the UV-AOP combined with GAC for hydrogen peroxide (H2O2) quenching as the driving factor in the increased costs of the system. The cost of the main system, based on an overall 88% water recovery, was estimated to be $1.97/m3, with costs mostly driven by the FO and SWRO membranes. With further cost reduction and optimization for FO membranes such as membrane cost, water recovery, and flux, the main system can provide a much more economically viable alternative in its application than a typical benchmark system.

Life cycle energy use and greenhouse gas emissions for a novel algal-osmosis membrane system versus conventional advanced potable water reuse processes: Part I.

This study applied a life cycle assessment (LCA) methodology for a comparative environmental analysis between an innovative algae resource recovery and near zero-liquid discharge potable reuse system (i.e., the main system) versus a conventional potable reuse system (i.e., the benchmark system) through energy use and greenhouse gas (GHG) emissions. The objective of this study is to demonstrate that pilot-scale data coupled with LCA would provide valuable information for system optimization, integration, and improvements for the design of environmentally sustainable full-scale systems. This study also provides decision-makers valuable information regarding the energy demand and environmental impact of this innovative main system compared to a typical tried-and-true system for potable water reuse. The main system consists of a novel algal-based wastewater treatment coupled with a dual forward osmosis and seawater reverse osmosis (Algal FO-SWRO) membranes system for potable water recovery and hydrothermal liquefaction (HTL) to recover biofuels and valuable nutrients from the harvested algal biomass. The benchmark system refers to the current industry standard technologies for potable water reuse and waste management including a secondary biological treatment, microfiltration (MF), brackish water reverse osmosis (BWRO), ultraviolet/advanced oxidation process (UV-AOP), and granular activated carbon (GAC), as well as anaerobic digestion for sludge treatment. Respective energy and GHG emissions of both systems were normalized and compared considering 1 m3 of water recovered. Based on an overall water recovery of 76% designed for the benchmark system, the energy consumption totaled 4.83 kWh/m3, and the system was estimated to generate 2.42 kg of CO2 equivalent/m3 with most of the emissions coming from the biological treatment. The main system, based on an overall water recovery of 88%, was estimated to consume 4.76 kWh/m3 and emit 1.49 kg of CO2 eq/m3. The main system has high environmental resilience and can recover bioenergy and nutrients from wastewater with zero waste disposal. With the application of energy recovery devices for the HTL and the SWRO, increase in water recovery of the FO membrane, and replacement of the SWRO membrane with BWRO, the main system provides an energy-competitive and environmentally positive alternative with an energy demand of 2.57 kWh/m3 and low GHG emissions of 0.94 kg CO2 eq/m3.

Impacts of seasonality and operating conditions on algal-dual osmosis membrane system for potable water reuse: Part 2.

This study investigated the impact of seasonal variation and operating conditions on recovery of potable quality water from municipal wastewater effluent using an integrated algal treatment process with a dual forward osmosis (FO)-reverse osmosis (RO) membrane system. Pilot study of the algal process treating primary effluent validated the technical viability and seasonal performance during warm weather (May to October, 25-55 °C) using an extremophilic algal strain Galdieria sulphuraria, and during cold weather (November to April, 4-17 °C) using polyculture strains of algae and bacteria. Algal effluents from both seasons were used as the feed solution for the laboratory FO-RO study. In addition, pilot-scale FO-RO experiments were conducted to compare the system performance during treatment of algal effluent and secondary effluent from the conventional treatment facility. At 90% water recovery, the FO-RO achieved over 90% overall rejection of major ions and organic matter using the bench-scale system and over 99% rejection of all contaminants in pilot-scale studies. Detailed water quality analysis indicated that the product water from the integrated system met both the primary and secondary drinking water standards. This study demonstrated that the FO-RO system can be engineered as a viable alternative to treat algal effluent and secondary effluent for potable water reuse independent of seasonal variations and operating conditions.

Impacts of seasonality and operating conditions on water quality of algal versus conventional wastewater treatment: Part 1.

Municipal wastewater is a reliable source from which water, renewable energy, and nutrients could be recovered for beneficial use. Our previous efforts have documented that an innovative algal-based wastewater treatment (WWT) system could recover energy and nutrients from wastewater while having a lower energy footprint than conventional WWT processes. As a biological treatment process, the algal WWT can be affected by algal species, operating conditions, and meteorological factors. This study aimed to identify suitable algal cultures to treat municipal wastewater during warm and cold weather. The algal system achieved the secondary effluent discharge standards for biochemical oxygen demand and nutrients within 2-3 days during warm weather (May to October, 25-55 °C) using an extremophilic algal strain Galdieria sulphuraria; and within 1-2 days in winter (November to April, 4-17 °C) using polyculture strains of algae with bacteria. The impact of seasonal variation and operating conditions on the water quality of pilot-scale algal bioreactors was compared with a full-scale conventional WWT system. The treatment performance of the algal system (NH4-N: 1.3 ± 1.25 mg/L in winter and not detected in summer and conventional system; PO4-P: 0.89 ± 0.6 mg/L in winter, 0.02 ± 0.03 mg/L in summer and, 5.93 ± 1.32 mg/L in conventional system) was comparable or better than that of the conventional WWT in nutrients removal and other contaminants were below the discharge standards. This study indicates that the algal system can be engineered for reliable wastewater treatment independent of seasonal variations.

Patients' ability to read and understand dosing instructions of their own medicines - a cross sectional study in a hospital and community pharmacy setting.

BACKGROUND: Poor communication of medicines information to patients may cause medication errors. We assessed the completeness and readability of dosing instructions provided by pharmacists on dispensing labels and knowledge among patients on dosing instructions of their medicines. METHODS: A cross sectional study was conducted in a selected teaching hospital, and a community pharmacy, among 800 patients selected through a systematic sampling method, during a period of 2 months. Completeness of dosing instructions were assessed against a checklist. Patients were asked to read dosing instructions to assess readability. Patient knowledge on dosing instructions were determined through a questionnaire. Completeness, readability and knowledge were scored out of 10 for each dispensing label. RESULTS: A total of 1200 and 1372 dispensing labels were assessed in the hospital and community settings respectively. The median score out of 10, for completeness, readability and patient knowledge of dosing instructions were 6.7, 8.3 and 7.5 respectively in the hospital, and 7.5, 8.0 and 7.5 respectively in the community. Only a few dispensing labels had the route of administration (hospital, 0.5%; community, 0.8%) and the duration of treatment written (hospital, 0.25%; community, 0.65%) on them. Name (hospital, 48%; community, 27.3%) and strength (hospital, 40.2%; community, 36.6%) of medicines on dispensing labels were frequently misread. In both settings, readability scores significantly differed with education level (P < 0.001). CONCLUSIONS: Some important dosing instructions were missing in dispensing labels. Readability of dosing instructions by patients was also not 100% and differed by their education level. Pharmacists did not adhere to a standard procedure in providing dosing instructions leading to communication gaps with patients. Hence we recommend the development of a standard procedure to provide complete, clear and simple dosing instructions to patients, and continuous training for pharmacists on proper communication of dosing instructions to patients.

Targeting the KIT activating switch control pocket: a novel mechanism to inhibit neoplastic mast cell proliferation and mast cell activation.

Activating mutations in the receptor tyrosine kinase KIT, most notably KIT D816V, are commonly observed in patients with systemic mastocytosis. Thus, inhibition of KIT has been a major focus for treatment of this disorder. Here we investigated a novel approach to such inhibition. Utilizing rational drug design, we targeted the switch pocket (SP) of KIT, which regulates its catalytic conformation. Two SP inhibitors thus identified, DP-2976 and DP-4851, were examined for effects on neoplastic mast cell proliferation and mast cell activation. Autophosphorylation of both wild-type and, where also examined, KIT D816V activation was blocked by these compounds in transfected 293T cells, HMC 1.1 and 1.2 human mast cell lines, and in CD34(+)-derived human mast cells activated by stem cell factor (SCF). Both inhibitors induced apoptosis in the neoplastic mast cell lines and reduced survival of primary bone marrow mast cells from patients with mastocytosis. Moreover, the SP inhibitors more selectively blocked SCF potentiation of FcɛRI-mediated degranulation. Overall, SP inhibitors represent an innovative mechanism of KIT inhibition whose dual suppression of KIT D816V neoplastic mast cell proliferation and SCF-enhanced mast cell activation may provide significant therapeutic benefits.

Expression and stabilization of galactose oxidase in Escherichia coli by directed evolution.

We have used directed evolution methods to express a fungal enzyme, galactose oxidase (GOase), in functional form in Escherichia coli. The evolved enzymes retain the activity and substrate specificity of the native fungal oxidase, but are more thermostable, are expressed at a much higher level (up to 10.8 mg/l of purified GOase), and have reduced negative charge compared to wild type, all properties which are expected to facilitate applications and further evolution of the enzyme. Spectroscopic characterization of the recombinant enzymes reveals a tyrosyl radical of comparable stability to the native GOase from Fusarium.

Functional expression of horseradish peroxidase in Saccharomyces cerevisiae and Pichia pastoris.

The ability to engineer proteins by directed evolution requires functional expression of the target polypeptide in a recombinant host suitable for construction and screening libraries of enzyme variants. Bacteria and yeast are preferred, but eukaryotic proteins often fail to express in active form in these cells. We have attempted to resolve this problem by identifying mutations in the target gene that facilitate its functional expression in a given recombinant host. Here we examined expression of HRP in Saccharomyces cerevisiae. Through three rounds of directed evolution by random point mutagenesis and screening, we obtained a 40-fold increase in total HRP activity in the S.cerevisiae culture supernatant compared with wild-type, as measured on ABTS ¿2, 2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (260 units/l/OD(600)). Genes from wild-type and two high-activity clones were expressed in Pichia pastoris, where the total ABTS activity reached 600 units/l/OD(600) in shake flasks. The mutants show up to 5.4-fold higher specific activity towards ABTS and 2.3-fold higher specific activity towards guaiacol.

Functional contributions of the FcepsilonRIalpha and FepsilonRIgamma subunit domains in FcepsilonRI-mediated signaling in mast cells.

The functional contributions of the alpha and gamma subunit domains of the high affinity receptor for IgE (Fcepsilon-RI) were determined following chimeric receptor aggregation. Chimeric receptors of the extracellular (EC) and cytoplasmic tail (CT) domains of FcepsilonRI and the IL-2R p55 subunit (I) were constructed and stably expressed in RBL-2H3 cells. Signaling (inositol phosphate production, tyrosine phosphorylation, Ca2+ mobilization, and secretion of histamine and arachidonic acid metabolites) via alpha/gamma/gamma or I/gamma/gamma was similar to the native rat receptor, and both were shown to associate with endogenous FcepsilonRIbeta and FcepsilonRIgamma subunits. Therefore, the contributions of the EC domains could not be evaluated. The chimeras alpha/I/gamma and I/I/gamma were found to be single polypeptide chains, as they did not associate with beta and gamma. Signaling via alpha/I/gamma resulted in the appearance of biochemical events common to the native receptor. Cross-linking I/I/gamma elicited histamine release, [14C]arachidonic acid metabolites, tyrosine phosphorylation, Ca2+ mobilization, and only inositol trisphosphate production, which were not of a similar magnitude to the native FcepsilonRI. No biochemical events were elicited by cross-linking alpha/I/I or I/I/I. These results demonstrate that both the FcepsilonRIalpha EC domain and the FcepsilonRIgamma CT domain are essential for the FcepsilonRI signaling process, and that while FcepsilonRIIgamma CT plays a critical role in FepsilonRI signaling, the EC domain of FcepsilonRIalpha has a major contribution in signaling, as well as a role in modulating the magnitude of the biochemical events.

Direct gene delivery to synovium. An evaluation of potential vectors in vitro and in vivo.

OBJECTIVE: To assess the abilities of various vectors to transfer genes to the synovial lining of joints. METHODS: Vectors derived from retrovirus, adenovirus, and herpes simplex virus as well as cationic liposomes and naked plasmid DNA were evaluated. Each construct contained the lac Z marker gene; and one retroviral construct, and one plasmid also contained a gene encoding human interleukin-1 receptor antagonist. Gene expression was under the control of the human cytomegalovirus promoter in all vectors except the retrovirus, where the endogenous 5' long terminal repeat was retained as the promoter. Cultures of rabbit synovial fibroblasts were exposed to these vectors and stained with X-gal to identify lac Z+ cells. Vectors were then injected directly into rabbits' knee joints, and gene transfer and expression were assessed by X-gal staining and polymerase chain reaction (PCR). RESULTS: Adenovirus was a highly effective vector both in vitro and in vivo, with lac Z gene expression persisting for at least 28 days. However, an inflammatory response was noted in vivo. Cells infected in vitro and in vivo with herpes simplex virus also expressed the lac Z gene at high levels, but expression was limited by cytotoxicity. Retroviruses, in contrast, were effective only under in vitro conditions, permitting cell division. Liposomes gave variable in vitro results; when injected into joints in vivo, gene expression was low and was detectable for only a few days, even though a PCR signal persisted for at least 28 days. Unexpectedly, plasmid DNA was captured by the synoviocytes and expressed transiently following intraarticular injection. CONCLUSION: None of the vectors was ideal for in vivo gene delivery to synovium, although adenovirus was clearly the most effective of those tested. Retroviruses, although poor vectors for in vivo gene delivery, are well suited for ex vivo gene transfer to the synovial lining of joints.

Functional characterization of the signal transduction events mediated by Fc epsilon RI alpha and gamma chimeric receptors.

Chimeric receptors containing the Fc epsilon RI alpha and gamma subunit domains were constructed, stably transfected into RBL-2H3 cells, and characterized for the biochemical events which are elicited upon receptor aggregation. Chimeric receptors containing the extracellular (EC) domain of the human Fc epsilon RI alpha subunit, or the EC domain of the p55 subunit of the interleukin-2 receptor were fused to the human Fc epsilon RI gamma subunit transmembrane and cytoplasmic (CT) domains or only the CT domain. The chimeras generated included alpha/gamma/gamma, I/gamma/gamma, alpha/I/gamma or I/I/gamma. The results indicate that both the Fc epsilon RI alpha EC domain and the Fc epsilon RI alpha CT domain are essential for signalling.

Intraarticular expression of biologically active interleukin 1-receptor-antagonist protein by ex vivo gene transfer.

Gene therapy offers a radical different approach to the treatment of arthritis. Here we have demonstrated that two marker genes (lacZ and neo) and cDNA coding for a potentially therapeutic protein (human interleukin 1-receptor-antagonist protein; IRAP or IL-1ra) can be delivered, by ex vivo techniques, to the synovial lining of joints; intraarticular expression of IRAP inhibited intraarticular responses to interleukin 1. To achieve this, lapine synoviocytes were first transduced in culture by retroviral infection. The genetically modified synovial cells were then transplanted by intraarticular injection into the knee joints of rabbits, where they efficiently colonized the synovium. Assay of joint lavages confirmed the in vivo expression of biologically active human IRAP. With allografted cells, IRAP expression was lost by 12 days after transfer. In contrast, autografted synoviocytes continued to express IRAP for approximately 5 weeks. Knee joints expressing human IRAP were protected from the leukocytosis that otherwise follows the intraarticular injection of recombinant human interleukin 1 beta. Thus, we report the intraarticular expression and activity of a potentially therapeutic protein by gene-transfer technology; these experiments demonstrate the feasibility of treating arthritis and other joint disorders with gene therapy.

Gene transfer to synoviocytes: prospects for gene treatment of arthritis.

Joints are difficult organs to target therapeutically. Intravenous, intramuscular, and oral routes of drug delivery provide poor access to the joint, and expose the body systemically to the therapeutic agent. Although intraarticular injection provides direct access to the joint, most injected materials have a short intraarticular half-life. We propose to circumvent these problems by introducing into the synovium gene(s) coding for proteins with antiarthritic properties. Two methods of gene delivery to synovium are under development. In the direct approach, in situ transduction of synoviocytes follows the injection of suitable vectors into the joint. In the indirect approach, synovium is removed from the joint, its synoviocytes are isolated, and the cells transduced in vitro. Genetically modified cells are subsequently transplanted back into the synovium. Using retroviral vectors, we have been able to express the lacZ and neo genes in lapine synovial fibroblasts in vitro. Following neoselection, all cells became LacZ+. Neo-selected cells carrying the lacZ marker gene were transplanted back into the knees of recipient rabbits to examine the persistence and expression of these genes in vivo. Islands of LacZ+, transplanted cells persisted in the recipient joints for at least 3 months. Furthermore, Neo+ cells could be grown from synovia recovered from these joints. Initial attempts to use retroviruses for the direct, in situ transduction of synovium have failed, probably because synoviocytes in the normal synovium are mitotically inactive. Present efforts are directed towards further development of our techniques for transferring genes to joints, and using these techniques to antagonize the intraarticular actions of interleukin-1.(ABSTRACT TRUNCATED AT 250 WORDS)

The synovial activation of chondrocytes: evidence for complex cytokine interactions involving a possible novel factor.

Preparations of lapine synovial 'chondrocyte activating factors' (CAF) were analyzed for the presence of individual cytokines which modulate the production of neutral metalloproteinases (NMPs) and prostaglandin E2 (PGE2) by articular chondrocytes. A combination of different biochemical analyses suggested that synovial fibroblasts secrete IL-1 alpha, which activated chondrocytes directly, bFGF, which potentiated the activity of IL-1, and TGF-beta 1, which produced a bivalent response. TGF-beta 1 suppressed NMP synthesis by chondrocytes, but enhanced PGE2 synthesis. The IL-1 receptor antagonist protein (IRAP) eliminated chondrocyte activation by IL-1, but only partially inhibited activation by CAF. Thus, CAF may contain a cytokine in addition to IL-1 which activates chondrocytes. This putative additional factor was more thermosensitive than IL-1, and had an apparent molecular weight of approx. 20,000 when estimated by size exclusion chromatography. Of a variety of purified cytokines tested for their ability to induce NMPs in chondrocytes, only IL-1 was active. This favours the possibility that the activity which resists suppression by IRAP reflects the presence of a novel cytokine.

Synovial activation of chondrocytes: evidence for complex cytokine interactions.

Synoviocytes secrete factors which induce the synthesis of neutral metalloproteinases (NMP) and prostaglandin E2 (PGE2) by chondrocytes in a response called "chondrocyte activation". We analyzed synovial chondrocyte activating factors (CAF) for the presence of cytokines which modulated the NMP production by articular chondrocytes. These studies suggested the presence of several other cytokines in addition to interleukin-1 (IL-1). Both resting and activated synoviocytes contained mRNA for basic fibroblast growth factor (bFGF) which is a synergist for IL-1 induced NMP production, and secreted bFGF into their culture media. They also expressed mRNA for transforming growth factor beta (TGF beta) which inhibits IL-1 induced NMP production. These cells also produce tumor necrosis factor alpha (TNF alpha) and trace amounts of interleukin-6 (IL-6). In addition to these there is evidence for a synovial activator of chondrocytes which is distinct from IL-1. Since a number of recombinant cytokines including TNF alpha, IL-6 and bFGF failed to activate chondrocytes, this could be a novel cytokine.

Chondrocyte activation by interleukin-1: analysis of the synergistic properties of fibroblast growth factor and phorbol myristate acetate.

Following activation, monolayers of lapine articular chondrocytes secreted into their culture media large amounts of prostaglandin E2 (PGE2) and the neutral metalloproteinases collagenase and gelatinase. Partially purified preparations of synovial "chondrocyte activating factors" (CAF), which contain interleukin-1 (IL-1), generally proved stronger activators of chondrocytes than recombinant, human, IL-1 alpha (rHIL-1 alpha) or IL-1 beta (rHIL-1 beta). The presence of synergistic cytokines within the synovial material provides one possible explanation of this discrepancy. As first reported by K. Phadke (1987, Biochem. Biophys. Res. Commun. 142, 448-453) fibroblast growth factor (FGF) synergized with rHIL-1 in promoting the synthesis of neutral metalloproteinases. In our hands FGF alone did not induce neutral metalloproteinases and increased PGE2 synthesis only modestly. However, at doses from 1 ng/ml to 1 microgram/ml, FGF progressively enhanced the synthesis of PGE2, collagenase, and gelatinase by chondrocytes responding to rHIL-1. Acidic and basic FGF synergized equally well with both rHIL-1 alpha and rHIL-1 beta. Phorbol myristate acetate (PMA), but not the Ca2+-ionophore A23187, could substitute for FGF as a synergist. PMA alone was a poor inducer of collagenase or gelatinase but, unlike FGF, it greatly enhanced the synthesis of PGE2 by chondrocytes. Dot-blot analyses with a cDNA probe to collagenase mRNA confirmed that partially purified synovial CAF induced collagenase mRNA more effectively than rHIL-1, with rHIL-1 alpha being superior to rHIL-1 beta in this regard. The synergistic effects of both FGF and PMA upon IL-1-mediated collagenase induction were associated with increased abundance of collagenase mRNA.

Chondrocyte activation by interleukin-1: synergism with fibroblast growth factor and phorbol myristate acetate.

Exposure to synovial factors or purified interleukin-1 (IL-1) induces the production of prostaglandin E2 (PGE2) and the neutral proteinases (NP) collagenase, gelatinase and stromelysin by lapine articular chondrocytes. Having frequently found our partially purified synovial preparations to elicit this process of chondrocyte activation more strongly than recombinant IL-1, Phadke's report of synergism between IL-1 and fibroblast growth factor (FGF) intrigued us. In our hands, basic FGF (1 ng/ml-1 micrograms/ml) did not activate chondrocytes but, in a dose-dependent manner, enhanced the production of PGE2 and NP by chondrocytes exposed to IL-1 alpha or IL-1 beta (1-10 U/ml). Further examination determined that the basic FGF was a better synergist than acidic FGF. In view of reports that FGF activates protein kinase C, we tested whether phorbol myristate acetate (PMA) could substitute for FGF as a synergist. Not only did it do so, but PMA alone (0.1 ng/ml-100 ng/ml), unlike FGF, provoked the production of PGE2 by chondrocytes. The Ca2+ ionophore A23187 could not substitute for FGF in enhancing induction of the NP. Using a cDNA probe, we confirmed that the synergistic effects of both FGF and PMA upon IL-1 mediated collagenase induction, were associated with an increased abundance of collagenase mRNA.

Induction of collagenase mRNA in lapine articular chondrocytes by synovial factors and interleukin-1.

The cDNA probe H-9, originally constructed to recognize a portion of the mRNA for lapine synovial collagenase, also hybridized with a RNA of the same size (approximately 2.0 kb) isolated from activated lapine articular chondrocytes. Primary, monolayer cultures of lapine articular chondrocytes did not contain detectable amounts of this RNA, nor did they secrete measurable amounts of collagenase into their culture media. Following exposure to synovial factors, the chondrocytes contained high levels of collagenase mRNA, while their conditioned media had considerable collagenolytic activity. Collagenase mRNA started to appear in chondrocytes 3-5 h after treatment with the synovial material. Maximum levels occurred after 12-24 h. Recombinant human interleukin-1 also induced the appearance of this mRNA. We conclude that chondrocyte collagenase is likely to be the same gene product as synovial collagenase, and that its regulation by lapine articular chondrocytes probably occurs at a pretranslational level.