Short-term effect of reclaimed wastewater quality gradient on soil microbiome during irrigation.

To investigate the effect of wastewater (WW) treatment on soil bacterial communities, water of different quality was used to irrigate eight lettuces per tank: raw municipal wastewater (RWW), WW treated with an aerated constructed wetland (CWW) and WW treated with a membrane bioreactor (MBW), and tap water (TW). The physicochemical and microbiological characteristics (quality indicators) of these water types were characterized, and the water and soil bacterial communities were monitored by quantitative PCR (qPCR) and 16S rRNA gene sequencing. Despite marked differences in microbial load and diversity of waters, soil communities remained remarkably stable after irrigation. Microbial biomass was increased only in soils irrigated with RWW. At the end of the irrigation period (day 84), soil and water shared a large fraction of their bacterial communities, from 43 % to 70 %, depending on the water quality, indicating a transfer of bacterial communities from water to soil. Overall, the relative abundance of Proteobacteria and Acidobacteria was increased and that of Actinobacteria was decreased in soils irrigated with MBW, CWW and even more with RWW. Multivariate ordination clearly separated soils in three groups: soils irrigated with the cleanest water (TW), with treated WW (MBW and CWW), and with untreated WW (RWW). Nitrifying, denitrifying, and nitrogen-fixing bacteria were quantified by qPCR targeting amoA, narG, and nifH, respectively. Nitrifying bacteria were the most affected by the water quality, as indicated by amoA copy number increase in RWW-irrigated soil and decrease in CWW-irrigated soil. Overall, the abundance of all three genes was positively influenced by RWW treatment. In conclusion, the 84 days of irrigation influenced the soil microbial communities, and the impact depended on the quality of the used water.

Brewery wastewater treatment using MBR coupled with nanofiltration or electrodialysis: biomass acclimation and treatment efficiency.

Breweries release significant amounts of wastewater loaded with various organic and mineral materials. Prior studies of membrane bioreactor (MBR) wastewater treatment have been conducted with very little interest granted to the conditions of biomass acclimation. This study displays biomass behavior during brewery wastewater treatment by an aerobic MBR. In addition, nanofiltration and electrodialysis have been studied as potential post-treatment to decrease mineral concentrations and permit further water reuse for agriculture. An anoxic/aerobic laboratory MBR, associated with a flat sulfonated polyether membrane was used for synthetic brewery wastewater treatment. Biomass acclimation was performed using a feeding substrate. Organic concentrations in the MBR influent varied from 700 mg COD/L to 10,600 mg COD/L (COD: chemical oxygen demand) for 110 days. The results indicate a good acclimation to effluent with high salts and organic matter loads. Steady evolution of biomass concentration and activities was achieved after 90 days of operation. A reduction of COD of around 95% was obtained with MBR and up to 99% with nanofiltration post-treatment for the reconstructed brewery effluent with an organic loading rate of 7 g COD/L·d and a solid and hydraulic retention time of 30 days and 36 hours. A good reduction of the salt content was also recorded primarily with the nanofiltration and electrodialysis processes.

Cost minimization in a full-scale conventional wastewater treatment plant: associated costs of biological energy consumption versus sludge production.

Energy consumption and sludge production minimization represent rising challenges for wastewater treatment plants (WWTPs). The goal of this study is to investigate how energy is consumed throughout the whole plant and how operating conditions affect this energy demand. A WWTP based on the activated sludge process was selected as a case study. Simulations were performed using a pre-compiled model implemented in GPS-X simulation software. Model validation was carried out by comparing experimental and modeling data of the dynamic behavior of the mixed liquor suspended solids (MLSS) concentration and nitrogen compounds concentration, energy consumption for aeration, mixing and sludge treatment and annual sludge production over a three year exercise. In this plant, the energy required for bioreactor aeration was calculated at approximately 44% of the total energy demand. A cost optimization strategy was applied by varying the MLSS concentrations (from 1 to 8 gTSS/L) while recording energy consumption, sludge production and effluent quality. An increase of MLSS led to an increase of the oxygen requirement for biomass aeration, but it also reduced total sludge production. Results permit identification of a key MLSS concentration allowing identification of the best compromise between levels of treatment required, biological energy demand and sludge production while minimizing the overall costs.

Low specificity of 2 tetanus rapid tests in Cambodia.

OBJECTIVES: Rapid testing for tetanus on serum or blood allows for an immediate evaluation of individual protection against tetanus in developed countries, using a "single step" immunochromatographic technique using tetanus toxoid. The specificity of these tests, compared to the reference method for tetanus, mouse serum neutralization testing, has however never been assessed in these countries, due to the difficulty to perform serum neutralization titration in mice, because of animal testing bioethical regulations. POPULATION AND METHODS: A collection of sera from adult volunteers in Cambodia, living in rural environment, was tested for tetanus antibodies by ELISA in France, and by mouse serum neutralization in Vietnam. This allowed estimating the sensitivity and specificity of 2 rapid tetanus tests, available on the market: TQS™ and Tetanotop™. RESULTS: The sensitivity of these tests was adequate, compared to mice serum neutralization test, for a test threshold of 0.01 IU/mL, (100% for TQS™, 91% for Tetanotop™), but their specificity was very low (1% for TQS™ and 13% for Tetanotop™). CONCLUSION: The results prove that these rapid tests for the assessment of individual protection against tetanus should not be used in the adult rural Cambodian population.

New urban wastewater treatment with autotrophic membrane bioreactor at low chemical oxygen demand/N substrate ratio.

The potential for total nitrogen removal from municipal wastewater has been evaluated in an autotrophic membrane bioreactor running with a low chemical oxygen demand (COD)/N ratio to simulate its combination with an upstream physicochemical process that retains a large proportion of organic matter. The tests were conducted in a laboratory scale submerged membrane bioreactor loaded with a synthetic influent. Nitrogen loading rate was 0.16 kgN-NH4+.m(-3).d(-1) and sodium acetate was added as a carbon source. Results have shown that nitrogen elimination can reach 85% for a COD/N ratio of 5, with COD removal exceeding 97%. However, a COD/N ratio of 3.5 was found to be the limiting factor for successfully reaching the overall target value of 10 mgN.L(-1) in the effluent. Nevertheless, low COD/N ratios make it possible to work with low total suspended solid concentrations in the bioreactor, which greatly facilitates membrane fouling control by a simple aeration and backwashing strategy.

Dynamic modeling of biodegradation and volatilization of hazardous aromatic substances in aerobic bioreactor.

The aerobic biological process is one of the best technologies available for removing hazardous organic substances from industrial wastewaters. But in the case of volatile organic compounds (benzene, toluene, ethylbenzene, p-xylene, naphthalene), volatilization can contribute significantly to their removal from the liquid phase. One major issue is to predict the competition between volatilization and biodegradation in biological process depending on the target molecule. The aim of this study was to develop an integrated dynamic model to evaluate the influence of operating conditions, kinetic parameters and physical properties of the molecule on the main pathways (biodegradation and volatilization) for the removal of Volatile Organic Compounds (VOC). After a comparison with experimental data, sensitivity studies were carried out in order to optimize the aerated biological process. Acclimatized biomass growth is limited by volatilization, which reduces the bioavailability of the substrate. Moreover, the amount of biodegraded substrate is directly proportional to the amount of active biomass stabilized in the process. Model outputs predict that biodegradation is enhanced at high SRT for molecules with low H and with a high growth rate population. Air flow rate should be optimized to meet the oxygen demand and to minimize VOC stripping. Finally, the feeding strategy was found to be the most influential operating parameter that should be adjusted in order to enhance VOC biodegradation and to limit their volatilization in sequencing batch reactors (SBR).

Expression of GCIP in transgenic mice decreases susceptibility to chemical hepatocarcinogenesis.

Transcription factors with helix-loop-helix (HLH) motif play critical roles in controlling the expression of genes involved in lineage commitment, cell fate determination, proliferation, and tumorigenesis. To examine whether the newly identified HLH protein GCIP/CCNDBP1 modulates cell fate determination and plays a role in hepatocyte growth, proliferation, and hepatocarcinogenesis, we generated transgenic mice with human GCIP gene driven by a liver-specific albumin promoter. We demonstrated that in GCIP transgenic mice, the overall liver growth and regeneration occurred normally after liver injury induced by carbon tetrachloride (CCl4). In the diethylnitrosamine (DEN)-induced mouse hepatocarcinogenesis, we demonstrated that overexpression of GCIP in mouse liver suppressed DEN-induced hepatocarcinogenesis at an early stage of tumor development. The number of hepatic adenomas at 24 weeks was significantly lower or not detected in GCIP transgenic male mice compared to the control mice under the same treatment. Although GCIP has little inhibition on the number of hepatic tumors at later stages (40 weeks), hepatocellular tumors in GCIP transgenic mice are smaller and well-differentiated compared to the poorly differentiated tumors in wild-type mice. Furthermore, we demonstrate that GCIP functions as a transcriptional suppressor, regulates the expression of cyclin D1, and inhibits anchorage-independent cell growth and colony formation in HepG2 cells, suggesting a significant role of GCIP in tumor initiation and development.

Mechanism of Kex2p inhibition by its proregion.

Many proteases are produced as zymogens bearing an N-terminal proregion acting both as intramolecular chaperone and as enzyme inhibitor. We studied here the inhibition mechanism of the yeast proprotein convertase Kex2p by its proregion. A recombinant secreted and soluble form of Kex2p was produced in Pichia pastoris and its enzymatic properties toward a fluorogenic synthetic peptide were characterized. Recombinant Escherichia coli-produced Kex2p proregion specifically and potently inhibited the enzyme, with an IC(50) of 160 nM. Exploration of the inhibition mechanism revealed that the proregion behaved as a mixed inhibitor.

Bile acid feeding increased proliferative activity and apical bile acid transporter expression in both small and large rat cholangiocytes.

Bile acids (BA) enter cholangiocytes by the Na(+)-dependent apical BA transporter (ABAT). By this mechanism, taurocholate (TC) and taurolithocholate (TLC) increase cholangiocyte proliferation. No in vivo studies exist regarding the anatomical sites involved in BA-regulation of cholangiocyte growth. Specific cholangiocyte subpopulations participate in BA-regulated proliferation. Proliferation was assessed in liver sections by determining the number of proliferating cellular nuclear antigen (PCNA)-positive cholangiocytes and cytokeratin-19 (CK-19)-positive ducts. We isolated small and large cholangiocytes from rats fed for 1 week TC, TLC, or BA control diet and determined PCNA and ABAT expression and BA transport activity. We evaluated if TC and TLC induction of ABAT expression was dependent on activation of PKC alpha. DNA replication was active only in large normal cholangiocytes. TC and TLC feeding increased proliferation of large cholangiocytes, induced the de novo activation of proliferation of small cholangiocytes, overexpression of ABAT and BA transport activity in large cholangiocytes, and de novo expression of ABAT and BA transport activity in small cholangiocytes. BA-stimulated ABAT expression was dependent on PKC activation in cholangiocytes. TC and TLC stimulate proliferation of small and large cholangiocytes associated with PKC-dependent up-regulation of ABAT.

Regulation of cholangiocyte bicarbonate secretion.

The objective of this review article is to discuss the role of secretin and its receptor in the regulation of the secretory activity of intrahepatic bile duct epithelial cells (i.e., cholangiocytes). After a brief overview of cholangiocyte functions, we provide an historical background for the role of secretin and its receptor in the regulation of ductal secretion. We review the newly developed experimental in vivo and in vitro tools, which lead to understanding of the mechanisms of secretin regulation of cholangiocyte functions. After a description of the intracellular mechanisms by which secretin stimulates ductal secretion, we discuss the heterogeneous responses of different-sized intrahepatic bile ducts to gastrointestinal hormones. Furthermore, we outline the role of a number of cooperative factors (e.g., nerves, alkaline phosphatase, gastrointestinal hormones, neuropeptides, and bile acids) in the regulation of secretin-stimulated ductal secretion. Finally, we discuss other factors that may also play an important role in the regulation of secretin-stimulated ductal secretion.

Regression of cholangiocyte proliferation after cessation of ANIT feeding is coupled with increased apoptosis.

Cholangiocyte proliferation and loss through apoptosis occur in cholestatic liver diseases. Our aim was to determine the mechanisms of apoptosis in an animal model of ductal hyperplasia. Rats were fed alpha-naphthylisothiocyanate (ANIT) for 2 wk and subsequently fed normal chow for 1, 2, and 4 wk. Proliferation was assessed in sections by morphometry and in small and large cholangiocytes by proliferating cellular nuclear antigen immunoblots and measurement of cAMP levels. Apoptosis and reactive oxygen species (ROS) levels were also assessed. ANIT feeding increased small and large cholangiocyte proliferation and apoptosis. Cessation of ANIT feeding was associated with decreased proliferation and a further increase in apoptosis in small and large cholangiocytes. Cholangiocytes from ANIT-fed rats or exposed to ANIT in vitro showed increased apoptosis and ROS generation. ANIT-induced duct injury results in enhanced proliferation and apoptosis in small and large cholangiocytes. The mechanism of ANIT-induced apoptosis may be due to ROS generation induced directly by ANIT. Our model has implications for understanding the pathophysiology of cholangiopathies (characterized by the coexistence of cholangiocyte apoptosis and proliferation).

Gastrin inhibits cholangiocarcinoma growth through increased apoptosis by activation of Ca2+-dependent protein kinase C-alpha.

BACKGROUND/AIMS: We determined the role of gastrin in the regulation of cholangiocarcinoma growth. METHODS: We evaluated for the functional presence of cholecystokinin (CCK)-B/gastrin receptors in the cholangiocarcinoma cell lines, Mz-ChA-1, HuH-28 and TFK-1. We determined the effect of gastrin on the growth of Mz-ChA-1, HuH-28 and TFK-1 cells. We evaluated the effect of gastrin on growth and apoptosis of Mz-ChA-1 in the absence or presence of inhibitors for CCK-A (L-364, 718) and CCK-B/gastrin (L-365, 260) receptors, the intracellular Ca2+ chelator (BAPTA/AM), and the protein kinase C (PKC)-alpha inhibitor, H7. We evaluated if gastrin effects on Mz-ChA-1 growth and apoptosis are associated with membrane translocation of PKC-alpha. RESULTS: Gastrin inhibited DNA synthesis of Mz-ChA-1, HuH-28 and TFK-1 cells in a dose- and time-dependent fashion. The antiproliferative effect of gastrin on Mz-ChA-1 cells was inhibited by L-365, 260, H7 and BAPTA/AM but not L-364, 718. Gastrin induced membrane translocation of PKC-alpha. The inhibition of growth of Mz-ChA-1 cells by gastrin was associated with increased apoptosis through a PKC-dependent mechanism. CONCLUSIONS: Gastrin inhibits the growth of Mz-ChA-1, HuH-28 and TFK-1 cells. Gastrin inhibits growth and induces apoptosis in Mz-ChA-1 cells through the Ca2+-dependent PKC-alpha. The data suggest a therapeutic role for gastrin in the modulation of cholangiocarcinoma growth.

Regulation of cholangiocyte proliferation.

Intrahepatic bile duct epithelial cells (i.e., cholangiocytes) are the target cells of chronic cholestatic liver diseases (i.e., cholangiopathies), which makes these cells of great interest to clinical hepatologists. This review will focus on "typical" cholangiocyte proliferation, whereas "atypical" (extension of cholangiocyte proliferation into parenchyma), and premalignant "oval" cell proliferation are reviewed elsewhere. The bile duct ligated (BDL) rat model, where most of the known mechanisms of cholangiocyte proliferation have been illustrated, was the first and remains the prototype animal model for "typical" cholangiocyte proliferation. Following a short overview of cholangiocyte functions, we briefly discuss the: (i) in vivo models [i.e., BDL (Fig. 1 and 4), chronic alpha-naphthylisothiocyanate (ANIT) or bile acid feeding (Fig. 2), acute carbon tetrachloride (CCl4) feeding and partial hepatectomy; and (ii) in vitro experimental tools [e.g., purified cholangiocytes and isolated intrahepatic bile duct units (IBDU)] that are key to the understanding of the mechanisms of "typical" cholangiocyte growth. In the second part of the review, we discuss a number of potential factors or conditions [e.g., gastrointestinal hormones, nerves, estrogens, blood supply, and growth factors] as well as the intracellular mechanisms [e.g., adenosine 3',5'-monophosphate (cAMP), and protein kinase C (PKC)] that may regulate "typical" cholangiocyte hyperplasia.

Interleukin-5 inhibition of biliary cell chloride currents and bile flow.

Recent studies have detected significant elevations of interleukin (IL)-5 mRNA in the liver parenchyma of patients with both primary biliary cirrhosis and acute rejection after liver transplantation. In both of these disorders, intrahepatic biliary epithelial cells (BECs) are the targets of injury. We hypothesized that BECs may themselves express IL-5 receptors that may modulate key biliary functions. RNAs coding for IL-5alpha and -beta receptors were amplified by RT/PCR from a biliary cell line derived from a human cholangiocarcinoma (Mz-ChA-1) and verified by DNA sequencing. IL-5 receptor distribution was detected immunocytochemically on Mz-ChA-1 cells, immortalized murine BEC, bile duct-ligated rat liver, and isolated cholangiocytes. Patch-clamp studies on Mz-ChA-1 cells showed that IL-5 inhibits 5'-N-ethylcarboxamidoadenosine-stimulated chloride currents. Additional functional studies showed that IL-5 inhibits secretin-induced bile flow. We conclude that BECs express IL-5 receptors and that IL-5 modulates BEC chloride currents and fluid secretion. Since IL-5 has previously been associated with cholestatic liver disease, we speculate that IL-5 may contribute to liver injury through its effects on biliary secretion.

The Kex2p proregion is essential for the biosynthesis of an active enzyme and requires a C-terminal basic residue for its function.

The Saccharomyces cerevisiae prohormone-processing enzyme Kex2p is biosynthesized as an inactive precursor extended by its N-terminal proregion. Here we show that deletion of the proregion renders Kex2p inactive both in vivo and in vitro. Absence of the proregion impaired glycosylation and stability and resulted in the retention of the enzyme in the endoplasmic reticulum. These phenotypes were partially complemented by expression of the proregion in trans. Trans complementation was specific to Kex2p proregion because expression of any of the seven mammalian prohormone convertase propeptides had no effect. These data are consistent with a model whereby Kex2p proregion functions as an intramolecular chaperone and indicate that covalent linkage to the protein is not an absolute requirement for proregion function. Furthermore, extensive mutagenesis revealed that, in addition to their function as proteolytic recognition sites, C-terminal basic residues play an active role in proregion-dependent Kex2p activation.

Gastrin inhibits cholangiocyte growth in bile duct-ligated rats by interaction with cholecystokinin-B/Gastrin receptors via D-myo-inositol 1,4,5-triphosphate-, Ca(2+)-, and protein kinase C alpha-dependent mechanisms.

We studied the role of gastrin in regulating cholangiocyte proliferation induced by bile duct ligation (BDL). In purified cholangiocytes, we evaluated (1) for the presence of cholecystokinin-B (CCK-B)/gastrin receptors, (2) the effect of gastrin on D-myo-Inositol 1,4,5-triphosphate (IP(3)) levels, and (3) the effect of gastrin on DNA synthesis and adenosine 3', 5'-monophosphate (cAMP) levels in the absence or presence of CCK-A (L-364,718) and CCK-B/gastrin (L-365,260) receptor inhibitors, 1, 2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis(acetxymethyl ester) (BAPTA/AM; an intracellular Ca(2+) chelator), and 2 protein kinase C (PKC) inhibitors, 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H7) and staurosporin. To evaluate if gastrin effects on cholangiocyte proliferation are mediated by the isoform PKCalpha, we evaluated (1) for the presence of PKCalpha in cholangiocytes and (2) the effect of gastrin on the PKCalpha protein expression in a triton-soluble (containing cytoplasm + membrane) and a triton-insoluble (containing cytoskeleton) fraction. To evaluate the effects of gastrin in vivo, immediately following BDL, gastrin or bovine serum albumin (BSA) was infused by minipumps for 7 days to rats and we measured cholangiocyte growth and cAMP levels. We found CCK-B/gastrin receptors on cholangiocytes. Gastrin increased IP(3) levels. Gastrin inhibited DNA synthesis and cAMP synthesis in cholangiocytes. Gastrin effects on cholangiocyte functions were blocked by L-365,260, BAPTA/AM, H7, and staurosporin but not by L-364,718. Gastrin induced translocation of PKCalpha from cholangiocyte cytoskeleton to membrane. In vivo, gastrin decreased cholangiocyte growth and cAMP synthesis compared with controls. We concluded that gastrin inhibits cholangiocyte growth in BDL rats by interacting with CCK-B/gastrin receptors through a signal transduction pathway involving IP(3), Ca(2+), and PKCalpha.