A comparative analysis of immunohistochemistry and fluorescent in situ hybridization assay to detect anaplastic lymphoma kinase status in lung adenocarcinoma cases: A search for a testing algorithm.

INTRODUCTION: Testing for echinoderm microtubule-associated protein-like 4 (EML4) anaplastic lymphoma kinase (ALK) translocation by fluorescence in situ hybridization (FISH) is well established whereas the Food and Drug Administration (FDA) ALK immunohistochemical (IHC) test is relatively new. AIMS AND OBJECTIVE: The aim of this study is to compare FDA-approved ALK IHC test (D5F3 clone) with the standard ALK FISH test. MATERIALS AND METHODS: A validation and a test arm with 100 and 200 cases of Formalin-Fixed, Paraffin-embedded blocks of lung adenocarcinoma, respectively, comprised the material. All cases had ALK IHC test on automated Ventana Benchmark XT IHC slide stainer using anti-ALK D5F3 rabbit monoclonal primary antibody; when positive tumor cells (any percentage) showed strong granular cytoplasmic staining. For the FISH test, Vysis ALK Dual Color Break Apart Rearrangement Probe (Abbott Molecular Inc.,) was used to detect ALK gene 2p23 rearrangements; when positive the red and green signals were split two signal diameter apart and/or isolated 3'red signal were detected in more than 15% tumor cells. The ALK FISH results were available in all 100 validation cases and 64-test arm cases which formed the basis of this analysis. RESULTS: The ALK IHC test was positive in 16% cases; four discordant cases were ALK IHC positive but ALK FISH negative, but no case was ALK IHC negative and ALK FISH positive. There was 100% sensitivity, 90.5% specificity, and 93.75% accuracy. CONCLUSION: A negative ALK IHC result obviates the need for a FISH test barring those with a strong clinical profile, and a positive ALK IHC result is sufficient basis for the initiation of treatment.

Electricity production from municipal solid waste using microbial fuel cells.

The organic content of municipal solid waste has long been an attractive source of renewable energy, mainly as a solid fuel in waste-to-energy plants. This study focuses on the potential to use microbial fuel cells to convert municipal solid waste organics into energy using various operational conditions. The results showed that two-chamber microbial fuel cells with carbon felt and carbon felt allocation had a higher maximal power density (20.12 and 30.47 mW m(-2) for 1.5 and 4 L, respectively) than those of other electrode plate allocations. Most two-chamber microbial fuel cells (1.5 and 4 L) had a higher maximal power density than single-chamber ones with corresponding electrode plate allocations. Municipal solid waste with alkali hydrolysis pre-treatment and K3Fe(CN)6 as an electron acceptor improved the maximal power density to 1817.88 mW m(-2) (~0.49% coulomb efficiency, from 0.05-0.49%). The maximal power density from experiments using individual 1.5 and 4 L two-chamber microbial fuel cells, and serial and parallel connections of 1.5 and 4 L two-chamber microbial fuel cells, was found to be in the order of individual 4 L (30.47 mW m(-2)) > serial connection of 1.5 and 4 L (27.75) > individual 1.5 L (20.12) > parallel connection of 1.5 and 4 L (17.04) two-chamber microbial fuel cells . The power density using municipal solid waste microbial fuel cells was compared with information in the literature and discussed.

Effects of spiked metals on the MSW anaerobic digestion.

This study aimed to investigate the effects of eight metals on the anaerobic digestion of the organic fraction of municipal solid waste (OFMSW) in bioreactors. Anaerobic bioreactors containing 200 mL MSW mixed completely with 200 m L sludge seeding. Ca and K (0, 1000, 2000 and 6,000 mg L(-1)) and Cr, Ni, Zn, Co, Mo and W (0, 5, 50 and 100 mg L(-1)) of various dose were added to anaerobic bioreactors to examine their anaerobic digestion performance. Results showed that except K and Zn, Ca (~728 to ~1,461 mg L(-1)), Cr (~0.0022 to ~0.0212 mg L(-1)), Ni (~0.801 to ~5.362 mg L(-1)), Co (~0.148 to ~0.580 mg L(-1)), Mo (~0.044 to ~52.94 mg L(-1)) and W (~0.658 to ~40.39 mg L(-1)) had the potential to enhance the biogas production. On the other hand, except Mo and W, inhibitory concentrations IC(50) of Ca, K, Cr, Ni, Zn and Co were found to be ~3252, ~2097, ~0.124, ~7.239, ~0.482, ~8.625 mg L(-1), respectively. Eight spiked metals showed that they were adsorbed by MSW to a different extent resulting in different liquid metals levels and potential stimulation and inhibition on MSW anaerobic digestion. These results were discussed and compared to results from literature.

Integration of CNS survival and differentiation by HIF2α.

Hypoxia-inducible factor (HIF) 1α and HIF2α and the inhibitor of apoptosis survivin represent prominent markers of many human cancers. They are also widely expressed in various embryonic tissues, including the central nervous system; however, little is known about their functions in embryos. Here, we show that zebrafish HIF2α protects neural progenitor cells and neural differentiation processes by upregulating the survivin orthologues birc5a and birc5b during embryogenesis. Morpholino-mediated knockdown of hif2α reduced the transcription of birc5a and birc5b, induced p53-independent apoptosis and abrogated neural cell differentiation. Depletion of birc5a and birc5b recaptured the neural development defects that were observed in the hif2α morphants. The phenotypes induced by HIF2α depletion were largely rescued by ectopic birc5a and birc5b mRNAs, indicating that Birc5a and Birc5b act downstream of HIF2α. Chromatin immunoprecipitation assay revealed that HIF2α binds to birc5a and birc5b promoters directly to modulate their transcriptions. Knockdown of hif2α, birc5a or birc5b reduced the expression of the cdk inhibitors p27/cdkn1b and p57/cdkn1c and increased ccnd1/cyclin D1 transcription in the surviving neural progenitor cells. The reduction in elavl3/HuC expression and enhanced pcna, nestin, ascl1b and sox3 expression indicate that the surviving neural progenitor cells in hif2α morphants maintain a high proliferation rate without terminally differentiating. We propose that a subset of developmental defects attributed to HIF2α depletion is due in part to the loss of survivin activity.

Modeling biogas production from organic fraction of MSW co-digested with MSWI ashes in anaerobic bioreactors.

This study aims at investigating the effects of MSW incinerator fly ash (FA) and bottom ash (BA) on the anaerobic co-digestion of OFMSW with FA or BA. It also simulates the biogas production from various dosed and control bioreactors. Results showed that suitable ashes addition (FA/MSW 10 and 20 g L(-1) and BA/MSW 100 g L(-1)) could improve the MSW anaerobic digestion and enhance the biogas production rates. FA/MSW 20 g L(-1) bioreactor had the higher biogas production and rate implying the potential option for MSW anaerobic co-digestion. Modeling studies showed that exponential plot simulated better for FA/MSW 10 g L(-1) and control bioreactors while Gaussian plot was applicable for FA/MSW 20 g L(-1) one. Linear and exponential plot of descending limb both simulated better for BA/MSW 100 g L(-1) bioreactor. Modified Gompertz plot showed higher correlation of biogas accumulation than exponential rise to maximum plot for all bioreactors.

Novel modeling concept for evaluating the effects of cadmium and copper on heterotrophic growth and lysis rates in activated sludge process.

A new modeling concept to evaluate the effects of cadmium and copper on heterotrophic growth rate constant (mu(H)) and lysis rate constant (b(H)) in activated sludge was introduced. The oxygen uptake rate (OUR) was employed to measure the constants. The results indicated that the mu(H) value decreased from 4.52 to 3.26 d(-1) or by 28% when 0.7 mg L(-1) of cadmium was added. Contrarily the b(H) value increased from 0.31 to 0.35 d(-1) or by 11%. When adding 0.7 mg L(-1) of copper, the mu(H) value decreased to 2.80 d(-1) or by 38%. The b(H) value increased to 0.42 d(-1) or by 35%. After regression, the inhibitory effect was in a good agreement with non-competitive inhibition kinetic. The inhibition coefficient values for cadmium and copper were 1.82 and 1.21 mg L(-1), respectively. The relation between the b(H) values and heavy metal concentrations agreed with exponential type well. The heavy metal would enhance b(H) value. Using these data, a new kinetic model was established and used to simulate the degree of inhibition. It was evident that not only the inhibitory effect on mu(H) but also that the enhancement effect on b(H) should be considered when heavy metal presented.

Biostabilization assessment of MSW co-disposed with MSWI fly ash in anaerobic bioreactors.

Municipal solid waste incinerator (MSWI) fly ash has been examined for possible use as landfill interim cover. For this aim, three anaerobic bioreactors, 1.2m high and 0.2m in diameter, were used to assess the co-digestion or co-disposal performance of MSW and MSWI fly ash. Two bioreactors contained ratios of 10 and 20 g fly ash per liter of MSW (or 0.2 and 0.4 g g(-1) VS, that is, 0.2 and 0.4 g fly ash per gram volatile solids (VS) of MSW). The remaining bioreactor was used as control, without fly ash addition. The results showed that gas production rate was enhanced by the appropriate addition of MSWI fly ash, with a rate of approximately 6.5l day(-1)kg(-1)VS at peak production in the ash-added bioreactors, compared to approximately 4l day(-1)kg(-1)VS in control. Conductivity, alkali metals and VS in leachate were higher in the fly ash-added bioreactors compared to control. The results show that MSW decomposition was maintained throughout at near-neutral pH and might be improved by release of alkali and trace metals from fly ash. Heavy metals exerted no inhibitory effect on MSW digestion in all three bioreactors. These phenomena indicate that proper amounts of MSWI fly ash, co-disposed or co-digested with MSW, could facilitate bacterial activity, digestion efficiency and gas production rates.

Solubility of heavy metals added to MSW.

This paper aims to investigate the six heavy metal levels (Cd, Cr, Cu, Pb, Ni and Zn) in municipal solid waste (MSW) at different pHs. It intends to provide the baseline information of metals solubility in MSW co-disposed or co-digested with MSW incinerator ashes in landfill or anaerobic bioreactors or heavy metals contaminated in anaerobic digesters. One milliliter (equal to 1mg) of each metal was added to the 100ml MSW and the batch reactor test was carried out. The results showed that higher HNO3 and NaOH were consumed at extreme pH of 1 and 13 compared to those from pH 2 to 11 due to the comparably higher buffer capacity. Pb was found to have the least soluble level, highest metal adsorption (%) and highest partitioning Kd (lg(-1)) between pH 3 and 12. In contrast, Ni showed the highest soluble level, lowest metal adsorption (%) and lowest Kd (lg(-1)) between pH 4 and 12. Except Ni and Cr, other four metals seemed to show the amphibious properties as comparative higher solubility was found in the acidic and basic conditions.

Comparisons of grey and neural network prediction of industrial park wastewater effluent using influent quality and online monitoring parameters.

In this study, Grey model (GM) and artificial neural network (ANN) were employed to predict suspended solids (SSeff) and chemical oxygen demand (CODeff) in the effluent from a wastewater treatment plant in industrial park of Taiwan. When constructing model or predicting, the influent quality or online monitoring parameters were adopted as the input variables. ANN was also adopted for comparison. The results indicated that the minimum MAPEs of 16.13 and 9.85% for SSeff and CODeff could be achieved using GMs when online monitoring parameters were taken as the input variables. Although a good fitness could be achieved using ANN, they required a large quantity of data. Contrarily, GM only required a small amount of data (at least four data) and the prediction results were even better than those of ANN. Therefore, GM could be applied successfully in predicting effluent when the information was not sufficient. The results also indicated that these simple online monitoring parameters could be applied on prediction of effluent quality well.

Evaluating impact level of different factors in environmental impact assessment for incinerator plants using GM (1, N) model.

In this study, the impact levels in environmental impact assessment (EIA) reports of 10 incinerator plants were quantified and discussed. The relationship between the quantified impact levels and the plant scale factors of BeiTou, LiZe, BaLi, LuTsao, RenWu, PingTung, SiJhou and HsinChu were constructed, and the impact levels of the GangShan (GS) and YongKong (YK) plants were predicted using grey model GM (1, N). Finally, the effects of plant scale factors on impact levels were evaluated using grey model GM (1, N) too. According to the predicted results of GM, the relative errors of topography/geology/soil, air quality, hydrology/water quality, solid waste, noise, terrestrial fauna/flora, aquatic fauna/flora and traffic in the GS plant were 17%, 14%, 15%, 17%, 75%, 16%, 13%, and 37%, respectively. The relative errors of the same environmental items in the YK plant were 1%, 18%, 10%, 40%, 37%, 3%, 25% and 33%, respectively. According to GM (1, N), design capacity (DC) and heat value (HV) were the plant scale factors that affected the impact levels significantly in each environmental item, and thus were the most significant plant scale factors. GM (1, N) was effective in predicting the environmental impact and analyzing the reasonableness of the impact. If there is an EIA for a new incinerator plant to be reviewed in the future, the official committee of the Taiwan EPA could review the reasonableness of impact levels in EIA reports quickly.

Biotreatment of sulfate-rich wastewater in an anaerobic/micro-aerobic bioreactor system.

The focus of this study was on sulfate-rich wastewater treatment in a novel anaerobic/micro-aerobic bioreactor system. The system was composed of an upward-flow anaerobic sludge blanket (UASB) reactor and a floated bed micro-aerobic reactor, which was packed with elastic porous carriers and was controlled in a situation of dissolved oxygen below 0.5 mg l(-1). The floated bed micro-aerobic reactor was developed for accumulating a higher amount of biomass in carriers with a short hydraulic retention time (HRT) for biological oxidation of hydrogen sulfide to elemental sulfur. During long-term steady state operation, experimental results showed that an average of 70 +/- 6% of sulfate was transformed to hydrogen sulfide in UASB reactor. Moreover, the overwhelming majority of sulfide was oxidized to elemental sulfur and sulfate in micro-aerobic reactor; and the recirculation of effluent to UASB reactor reduced effectively the degree of inhibition caused by sulfate-rich wastewater. In UASB reactor, chemical oxygen demand (COD) removal efficiency increased with COD loading, in contrast, the performance of sulfate removal decreased with the increase in sulfate loading in a range of 1.0-1.75 kg SO4(2-) m(-3) d(-1). In micro-aerobic reactor, sulfide was removed almost completely under the operation of HRT 2.8 h. Furthermore, experimental results of continuous operations revealed that oxidation-reduction potential (ORP) was an adequate parameter for controlling biological oxidation of sulfide. When ORP was regulated in a lower range of -250 to -300 mV, the amount of regenerated sulfate was reduced significantly in micro-aerobic reactor.

The impacts of the AOC concentration on biofilm formation under higher shear force condition.

The logistic growth model was applied in the study to evaluate the impacts of assimilable organic carbon (AOC) concentration on the growth characteristics of biofilm and bulk bacteria under high flow velocity condition. The experimental results showed that there existed a growth and decline relation between biofilm and bulk bacteria at the low (0.05 mg/L) and medium (0.5 mg/L) AOC levels. Increasing the AOC concentration up to 1.0 mg/L, it resulted in high amounts of biofilm and bulk bacteria simultaneously. Although the carrying capacity of biofilm bacteria at the medium condition of AOC level was substantially reduced, the specific growth rate (GR) of biofilm bacteria was largest at this condition. It showed that the reduction of biofilm bacteria quantity did not represent the suppression of bacterial growth. The quantity of bulk water bacteria was obviously dependent with the quantity of biofilm bacteria and the increase of free bacteria with time in networks was mainly due to the growth and detachment of biofilm bacteria, not due to the growth of free bacteria themselves. The maximum growth rate of biofilm bacteria was increased upon increasing the AOC level. It indicated that the AOC level was an important factor affecting the growth of biofilm bacteria.

Microbial kinetic analysis of three different types of EBNR process.

The disadvantages of developed biological nutrient removal (BNR) processes (additional energy for liquid circulation and addition of external carbon substrate for denitrification in anoxic zones) were improved by reconfiguring the process into (1) an anaerobic zone followed by multiple stages of aerobic-anoxic zones (TNCU3 process) or (2) anaerobic, oxic, anoxic, oxic zones in sequence (TNCU2 process). These two pilot plants were operated at a recycling sludge ratio of 0.5 without internal recycle of nitrified supernatant. The sludge retention time was maintained at 10 d. The main objective of this study is to analyze the kinetics of different microorganisms in these two processes and A2O process by using the Activated Sludge Model No. 2d. The effective removal efficiency of carbon, total phosphorus and total nitrogen at 87-98%, 92-100% and 63-80%, respectively, were achieved in the testing runs. According to model simulations, the microbial kinetics in the TNCU3 and TNCU2 processes would be affected by different operations. When the step feeding strategy was adopted, the HRT was longer due to the less influent flowrate in the front stages and the microbes would grow in quantities by about 6% in the aerobic reactors. In the followed anoxic reactors, the microbes would decrease in quantities by about 12% due to the dilution effect. The dilution effects in TNCU3 and TNCU2 processes did not take place in A2O process because the recycling mixed liquid from the aerobic reactor to the anoxic reactor still contained particulate components. The XH, XPAO, and XAUT concentrations in the effluent of the last tank were lower when the step-feeding mode was adopted. The TNCU3 and TNCU2 processes could be operated efficiently without nitrified liquid circulation and addition of external carbon substrate for denitrification.

Sulphonation of dehydroepiandrosterone and neurosteroids: molecular cloning, expression, and functional characterization of a novel zebrafish SULT2 cytosolic sulphotransferase.

By searching the zebrafish EST (expressed-sequence tag) database, we have identified two partial cDNA clones encoding the 5' and 3' regions of a putative zebrafish sulphotransferase (ST). Using the reverse transcription-PCR technique, a full-length cDNA encoding this zebrafish ST was successfully cloned. Sequence analysis revealed that this novel zebrafish ST displays 44%, 43% and 40% amino acid identity with mouse SULT2B1, human SULT2B1b and human SULT2A1 ST respectively. This zebrafish ST therefore appears to belong to the SULT2 cytosolic ST gene family. Recombinant zebrafish ST, expressed using the pGEX-2TK prokaryotic expression system and purified from transformed Escherichia coli cells, migrated as a 34 kDa protein upon SDS/PAGE. Purified zebrafish ST displayed a strong sulphonating activity toward DHEA (dehydroepiandrosterone), with a optimum pH of 9.5. The enzyme also exhibited activities toward several neurosteroids with differential K(m) and V(max) values. A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 20 degrees C and 43 degrees C. Among ten different divalent metal cations tested, Fe2+ and Cd(2+ exhibited small, but significant, stimulatory effects, whereas Hg2+ and Cu2+ displayed considerably stronger inhibitory effects on the DHEA-sulphonating activity of the enzyme. These results constitute the first study on the molecular cloning, expression, and characterization of a zebrafish cytosolic SULT2 ST.

cDNA cloning, expression, and functional characterization of a zebrafish SULT1 cytosolic sulfotransferase.

Using the reverse transcriptase-polymerase chain reaction technique, a full-length cDNA encoding a novel zebrafish sulfotransferase was cloned and sequenced. Sequence analysis indicated that this zebrafish sulfotransferase belongs to the SULT1 cytosolic sulfotransferase gene family. The recombinant form of the zebrafish sulfotransferase, purified from Escherichia coli cells, displayed sulfating activities toward a number of endogenous compounds, in particular dopamine and thyroid hormones, in addition to xenobiotics including some flavonoids, isoflavonoids, and other phenolic compounds. The zebrafish sulfotransferase exhibited substrate dependence in pH optimum. In comparison with those determined with dopamine as substrate, the zebrafish sulfotransferase displayed much lower K(m) and higher V(max) with n-propyl gallate as substrate. A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 20 and 43 degrees C. Among 10 divalent metal cations tested, Hg(2+), Co(2+), Zn(2+), Cd(2+), Cu(2+), and Pb(2+) exhibited dramatic inhibitory effects on the activity of the zebrafish sulfotransferase.

Differential roles of human monoamine (M)-form and simple phenol (P)-form phenol sulfotransferases in drug metabolism.

Cytosolic sulfotransferases (STs) are traditionally known as Phase II drug-metabolizing or detoxifying enzymes that facilitate the removal of drugs and other xenobiotic compounds. In this study, we carried out a systematic investigation on the sulfation of drug compounds by two major human phenol STs (PSTs), the monoamine (M)-form and simple phenol (P)-form PSTs. Activity data obtained showed the differential substrate specificity of the two enzymes for the thirteen drug compounds tested. Kinetic studies revealed that the M-form PST displayed stereoselectivity for the chiral drug, isoproterenol. The effects of divalent metal cations on the activity of the M-form and P-form PSTs toward representative drug compounds were quantitatively evaluated. Results obtained indicated that the drug-sulfating activities of the two human PSTs were partially or completely inhibited or stimulated by the ten divalent metal cations tested at a 5 mM concentration. The two enzymes appeared to be less sensitive to the effects of physiologically more abundant metal cations such as Mg(2+) and Ca(2+), but more sensitive to the detrimental effects of other metal cations that may enter the body as environmental contaminants.

Sulfation of hydroxychlorobiphenyls. Molecular cloning, expression, and functional characterization of zebrafish SULT1 sulfotransferases.

As a first step toward developing a zebrafish model for investigating the role of sulfation in counteracting environmental estrogenic chemicals, we have embarked on the identification and characterization of cytosolic sulfotransferases (STs) in zebrafish. By searching the zebrafish expressed sequence tag database, we have identified two cDNA clones encoding putative cytosolic STs. These two zebrafish ST cDNAs were isolated and subjected to nucleotide sequencing. Sequence data revealed that the two zebrafish STs are highly homologous, being approximately 82% identical in their amino acid sequences. Both of them display approximately 50% amino acid sequence identity to human SULT1A1, rat SULT1A1, and mouse SULT1C1 ST. These two zebrafish STs therefore appear to belong to the SULT1 cytosolic ST gene family. Recombinant zebrafish STs (designated SULT1 STs 1 and 2), expressed using the pGEX-2TK prokaryotic expression system and purified from transformed Escherichia coli cells, migrated as approximately 35 kDa proteins on SDS/PAGE. Purified zebrafish SULT1 STs 1 and 2 displayed differential sulfating activities toward a number of endogenous compounds and xenobiotics including hydroxychlorobiphenyls. Kinetic constants of the two enzymes toward two representative hydroxychlorobiphenyls, 3-chloro-4-biphenylol and 3,3',5,5'-tetrachloro-4,4'-biphenyldiol, and 3,3',5-triiodo-l-thyronine were determined. A thermostability experiment revealed the two enzymes to be relatively stable over the range 20-43 degrees C. Among 10 different divalent metal cations tested, Co2+, Zn2+, Cd2+, and Pb2+ exhibited considerable inhibitory effects, while Hg2+ and Cu2+ rendered both enzymes virtually inactive.

Molecular cloning, expression, and functional characterization of a novel zebrafish cytosolic sulfotransferase.

By searching the zebrafish expressed sequence tag (EST) database, we have identified a cDNA clone encoding a putative zebrafish cytosolic sulfotransferase (ST). This cDNA was isolated and subjected to nucleotide sequencing. Analysis of the sequence data revealed that this novel zebrafish ST displays 32-35% amino acid sequence identity to members of all major cytosolic ST gene families. Therefore, this zebrafish ST, while belonging to the cytosolic ST gene superfamily, appears to be independent from all known constituent ST gene families. Recombinant zebrafish ST, expressed using the pET23c prokaryotic expression vector and purified from transformed Escherichia coli cells, migrated as a 34-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified zebrafish ST displayed sulfating activities toward dopamine and thyroid hormones (T(3) and T(4)), with a pH optimum spanning 7-9. The enzyme also exhibited activities toward a number of xenobiotics including some flavonoids, isoflavonoids, and other phenolic compounds. A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 20 and 48 degrees C. Among 10 divalent metal cations tested, Fe(++), Hg(++), Co(++), Zn(++), Cu(++), and Cd(++) exhibited dramatic inhibitory effects on the activity of the enzyme. These results constitute a first study on the cloning, expression, and characterization of a zebrafish cytosolic ST.