MRNIP is a replication fork protection factor.

The remodeling of stalled replication forks to form four-way DNA junctions is an important component of the replication stress response. Nascent DNA at the regressed arms of these reversed forks is protected by RAD51 and the tumor suppressors BRCA1/2, and when this function is compromised, stalled forks undergo pathological MRE11-dependent degradation, leading to chromosomal instability. However, the mechanisms regulating MRE11 functions at reversed forks are currently unclear. Here, we identify the MRE11-binding protein MRNIP as a novel fork protection factor that directly binds to MRE11 and specifically represses its exonuclease activity. The loss of MRNIP results in impaired replication fork progression, MRE11 exonuclease-dependent degradation of reversed forks, persistence of underreplicated genomic regions, chemosensitivity, and chromosome instability. Our findings identify MRNIP as a novel regulator of MRE11 at reversed forks and provide evidence that regulation of specific MRE11 nuclease activities ensures protection of nascent DNA and thereby genome integrity.

Impacts of municipal wastewater oxidative treatments: changes in metal physical speciation and bioavailability.

The environmental repercussions of the discharge of disinfected effluents are still poorly understood. This study assessed the impact of ozonation and UV oxidative treatment processes on metal forms - particulate, colloidal and permeable fractions - and bioavailability in disinfected wastewaters. In addition to wastewater analyses, mussels were placed in continuous flow-through aquaria and exposed for 4wk to wastewater, then metals in their tissues were analysed in parallel with exposure biomarkers. Metal size distribution was affected by oxidative processes; results showed that ozonation treatment generally increases the permeable fraction of some metals, particularly Cd and Cu, in treated waters, whereas UV treatment fosters the formation of permeable Zn. Ozone treatment of wastewater generally increased the bioavailability of specific metals. Metal bioaccumulation was in most cases significantly higher in mussels exposed to ozone-treated effluent compared to the UV treatment: 58%, 32%, 42% and 47% higher, respectively, for Ag, Cd, Cr and Cu. Physical metal speciation in these wastewaters comparatively measured the permeable fraction of metals to relate them to the bioaccumulation results for the exposed mussels. The levels of lipid peroxidation were significantly increased in gills but not in the digestive gland. The levels of metallothionein in the digestive gland were also significantly reduced suggest decreased input of particulate metals. Results of bioaccumulation in mussels suggested that metal bioavailability can be modified by the different oxidative processes. Despite this disadvantage, ozonation still represents a great choice of treatment considering the overall environmental benefits.

Ultra-high-performance liquid chromatography profiling method for chemical screening of proanthocyanidins in Czech hops.

Hops represent an important natural source of bioactive polyphenols, particularly proanthocyanidins, which can contribute to prevention of several civilization diseases, owing to their antioxidant and radical scavenging activity. We have developed a high-throughput ultra-high-performance liquid chromatography time-of-flight mass spectrometry profiling method, which can be used for monitoring of bioactive proanthocyanidins in hops. The method was applied for analysis of hops of four Czech varieties (Saaz, Sladek, Preminat and Agnus) from the 2011 crop (9 localities, 11 samples) and the 2012 crop (24 localities, 40 samples). Hop samples were extracted by acetone and the analytes were separated on the Acquity UPLC BEH Shield RP18 column. Partial validation of the method revealed a satisfactory intra-day repeatability of the method for retention times (relative standard deviation within 1.39%) as well as areas under the peaks (within 9.89%). Experimental data were evaluated using principal component analysis and cluster analysis. Significant amounts of di-, tri- and tetramer proanthocyanidins consisting of (epi)catechin and (epi)gallocatechin were found in the hop samples. The dependence of the proantocyanidin composition on both the variety and the growing locality was observed. Specifically, the traditional Saaz variety contained more frequently oligomers formed by (epi)catechin units only, whereas the varieties Premiant and Agnus produced oligomers consisting of (epi)catechin as well as (epi)gallocatechin units. The relative abundance of proanthocyanidins in studied hop varieties from the two crops, 2011 and 2012, did correspond to each other. In the further perspective, the method may also be used for prediction of qualitative marks or authenticity verification of hops.

Evidence of neuroendocrine disruption in freshwater mussels exposed to municipal wastewaters.

The purpose of this study was to test the hypothesis that exposure to municipal effluents can disrupt the neuroendocrine system in Elliptio complanata freshwater mussels. The capacity of ozonation to mitigate these effects was also examined. Mussels were exposed for 14 days to a continuous flow of increasing concentrations of the effluent before and after ozonation. Neuroendocrinal effects were examined by tracking changes in acetylcholinesterase, glutamate, gamma-aminobutyrate, serotonin, dopamine and their respective adenylcyclase activities in synapse membranes, monoamine oxidase and vitellogenin-like proteins. Oxidative stress and damage were examined by superoxide dismutase and lipid peroxidation, respectively, in the visceral tissues. The results revealed that the exposure of freshwater mussels increased the levels of vitellogenin-like proteins in both the primary-treated and ozonated effluents, dopamine and glutamate, and decreased the turnover of the neurostimulant acetylcholine. Moreover, these endpoints were significantly correlated with oxidative stress and damage. A canonical analysis of the responses revealed that dopamine and the neuroexcitatory neuromediators--acetylcholesterase and glutamate--were the endpoints more strongly related with oxidative stress and damage. Mussel morphology and estrogenic biomarkers (vitellogenin-like proteins, gonad lipid stores) were also significantly related, albeit to a lesser extent, to oxidative stress and damage. In general, ozone treatment was not sufficient to mitigate the observed neuroendocrinal effects in freshwater mussels. We conclude that the continuous exposure of freshwater mussels to municipal wastewater effluents leads to neuroendrocinal alterations and to oxidative stress.

Alterations in DNA metabolism in Elliptio complanata mussels after exposure to municipal effluents.

This study sought to examine the genotoxic potential in Elliptio complanata freshwater mussels exposed to a physically and chemically treated municipal effluent before and after ozone treatment. Mussels were continuously exposed to increasing concentrations of the effluents for 14 days. Genotoxicity was determined by tracking changes in key enzymes for purine and pyrimidine synthesis (dehydrofolate reductase and aspartate transcarbamoylase), catabolism of purines (xanthine oxido-reductase) and DNA strand-break levels as determined by the alkaline precipitation assay. Other biomarkers related to xenobiotic biotransformation (cytochrome P4503A and glutathione S-transferase activities), metal metabolism (labile zinc and redox state of metathioneins) and oxidative stress (superoxide dismutase activity) were also determined in the mussels. The data revealed that dehydrofolate reductase activity was reduced by the initial effluent and increased by the ozonated effluent. Aspartate transcarbamoylase activity was significantly induced only with the ozonated effluent. The levels of DNA strand breaks responded in a biphasic manner in mussels exposed to the physically and chemically treated effluent where an initial decrease was observed at a low effluent concentration (3% v/v) followed by an increase in DNA strand breaks at a higher effluent concentration (20%). This response pattern was lost in the ozonated effluent, where only a decrease in DNA breaks was found. Xanthine oxidoreductase activity was not significantly affected but did correlate significantly with dehydrofolate reductase activity. Multivariate factorial and canonical analyses revealed that oxidative stress and metal/xenobiotic metabolism markers were strongly correlated with DNA strand breaks in mussels, suggesting that the presence of metals (zinc) and planar hydroxylated hydrocarbons present in these effluents were strong contributors to the observed response. We conclude that municipal effluents contain a complex mixture of pollutants that could modulate DNA synthesis and repair mechanisms in mussels.

The effects of a primary-treated municipal effluent on the immune system of rainbow trout (Oncorhynchus mykiss): exposure duration and contribution of suspended particles.

Municipal sewage effluents are complex mixtures of contaminants known to disrupt both immune and endocrine functions in aquatic organisms. The present study sought to determine the impacts of municipal effluent on the immune systems of juvenile rainbow trout (Oncorhynchus mykiss), by exposing specimens to low concentrations (0.01%, 0.1%, 1% or 10%) of sewage effluent for periods of 28 or 90 days. The soluble and insoluble fractions of the effluent were also studied to assess the contribution of fractions rich in microorganisms and particles on fish immune systems. To this end, the trout were also exposed to soluble and insoluble fractions of the effluent for a period of 28 days. Immunocompetence was assessed by the following three parameters: phagocytosis, natural cytotoxic cells (NCC) and blastogenesis of lymphocytes under mitogen stimulation. Fish exposed to the 1% sewage effluent concentration for 28 days had enhanced phagocytic activity; at 90 days, phagocytic activity was reduced. T and B lymphocyte proliferation in fish from both groups was similarly stimulated. Phagocytosis and NCC activities were influenced more by the insoluble fraction than the soluble fraction of the effluent. Conversely, mitogen-stimulated T lymphocyte proliferation was enhanced in cells of fish exposed to the soluble fraction of the effluents, with a dampening effect on the insoluble (particulate) fraction of the effluent. In conclusion, the effects of the effluent and its fractions were higher at the cellular-mediated immunity level than at the acquired immunity level. Immunotoxicity data on the soluble fraction of the effluent were more closely associated to data on the unfractionated effluent, but the contribution of the particulate fraction could not be completely ignored for phagocytosis and B lymphocyte proliferation.

Effects of ozone, ultraviolet and peracetic acid disinfection of a primary-treated municipal effluent on the immune system of rainbow trout (Oncorhynchus mykiss).

Municipal sewage effluents are complex mixtures that are known to compromise the health condition of aquatic organisms. The aim of this study was to evaluate the impacts of various wastewater disinfection processes on the immune system of juvenile rainbow trout (Oncorhynchus mykiss). The trout were exposed to a primary-treated effluent for 28 days before and after one of each of the following treatments: ultraviolet (UV) radiation, ozonation and peracetic acid. Immune function was characterized in leucocytes from the anterior head kidney by the following three parameters: phagocytosis activity, natural cytotoxic cells (NCC) function and lymphocyte (B and T) proliferation assays. The results show that the fish mass to length ratio was significantly decreased for the primary-treated and all three disinfection processes. Exposure to the primary-treated effluent led to a significant increase in macrophage-related phagocytosis; the addition of a disinfection step was effective in removing this effect. Both unstimulated and mitogen-stimulated T lymphocyte proliferation in fish decreased dramatically in fish exposed to the ozonated effluent compared to fish exposed to either the primary-treated effluent or to aquarium water. Stimulation of T lymphocytes proliferation was observed with the peracetic acid treatment group. In conclusion, the disinfection strategy used can modify the immune system in fish at the level of T lymphocyte proliferation but was effective to remove the effects on phagocytosis activity.

Immunotoxic effects on freshwater mussels of a primary-treated wastewater before and after ozonation: a pilot plant study.

The immunotoxic potential of a primary-treated municipal effluent following enhanced disinfection by ozonation was studied in the freshwater mussel Elliptio complanata. Mussels were exposed to increasing concentrations (0%, 1%, 3%, 10%, and 20% v/v) of the effluent before and after ozone treatment (approximately 10 mg/L of purged O(3)) in a continuous flow-through laboratory for 7 weeks. Immunocompetence was appraised by measuring phagocytosis, cell viability (fluorescein retention), cell adherence to polystyrene micro-wells, cyclooxygenase (COX) activity and total nitrite levels in peripheral hemocytes from the hemolymphs. The results showed that phagocytosis was significantly inhibited by the primary-treated effluent at a threshold concentration of 1.7% v/v. Cell viability was also significantly reduced three-fold compared to controls at the same effluent threshold concentration, but hemocyte adherence was unchanged. COX activity was increased 1.3-fold at a threshold concentration of 14% v/v. Total nitrite levels were significantly increased 2.2-fold at a threshold concentration of 5.5% v/v. Ozone treatment of the effluent was not successful in removing phagocytic inhibition, but did completely remove cytotoxicity from hemocytes. Ozonation also reduced cell adherence at a threshold concentration of 1.7% v/v. The inflammatory properties of the effluent appeared to be accentuated by the ozone, as evidenced by an increase in COX activity, which reached 2.6-fold activity compared to controls, as compared to the 1.3-fold increase witnessed in the primary-treated effluent. Furthermore, total nitrite levels reached a two-fold induction at a threshold concentration of 1.7% v/v in the ozone-enhanced effluent compared to 5.5% v/v in the primary-treated effluent. In conclusion, ozonation of a primary-treated effluent successfully reduced microbial loading and completely removed cytotoxicity, but increased the inflammatory properties of the effluent. Investigations aimed at examining the impacts of sustained inflammation on the host's capacity to remove potentially pathogenic bacteria are recommended.

Neurotoxicological effects of a primary and ozonated treated wastewater on freshwater mussels exposed to an experimental flow-through system.

The neurotoxic potential of a primary-treated and ozonated municipal effluent was examined using feral freshwater Elliptio complanata mussels. Specimens were exposed to increasing concentrations (0, 1, 3, 10 and 20% v/v) of a primary-treated effluent before and after treatment with 10 mg/L of ozone in a mesocosm-type experiment for 30 days. A suite of biomarkers was used to assess the potential neurotoxic stress of the wastewaters on these benthic invertebrates: opiate binding sites, gamma-aminobutyric acid (GABA) metabolism, monoamines levels (serotonin, dopamine), monoamine oxidase, acetylcholinesterase and lipid peroxidation. Gametogenic activity was also determined by the gonado-somatic index and by vitellogenin-like proteins. The results show that the number of opiate binding sites increased slightly, especially after ozonation. GABA metabolism was generally reduced, suggesting higher glutamate stimulation than GABA dampening effects in mussel ganglia. This excitatory state was further confirmed by decreased acetylcholinesterase activity in gonadal tissues. The turnover of dopamine was enhanced with increased serotonin levels, but accompanied by reduced catabolism, as evidenced by decreased monoamine oxidase activity. Moreover, oxidative stress was increased, as determined by lipid peroxidation in the gonad (containing ganglia), which was significantly correlated with acetylcholinesterase activity and dopamine metabolism. The gonado-somatic index was significantly reduced with increased levels of vitellogenin-like proteins, again confirming the estrogenic action of these wastewaters. The data suggest that exposure to a primary-treated municipal effluent before and after ozonation leads to an excitotoxic syndrome implicating perturbations in GABA, dopamine and acetylcholine signaling. The increase in dopamine metabolism may be associated with the occurrence of opiate-like compounds (i.e. morphine) in the effluent. In general, ozonation reduced the severity of the responses, indicating that this disinfection strategy does not increase neurotoxicity to mussels.

Toxicological effects of primary-treated urban wastewaters, before and after ozone treatment, on freshwater mussels (Elliptio complanata).

This study examined the toxic potential of a primary-treated municipal effluent, before and after ozonation, in freshwater mussels. Animals were exposed to various concentrations (0, 1, 3, 10 and 20% v/v) of a primary-treated effluent and also after a treatment with ozone at 10 mg/L in continuous flow-through mode for seven weeks. A suite of biomarkers was used to assess the potential toxic effects of various contaminants typically present in municipal wastewaters: heavy metal metabolism (metallothioneins and labile zinc), cytochrome P4501A1 and 3A4, glutathione S-transferase activities (biotransformation of organic compounds), lipid peroxidation and xanthine oxidoreductase (oxygen radical scavenging), DNA damage, mitochondrial electron transport activity at various temperatures and gonad lipid levels (cellular energy allocation) and aspartate transcarbamoylase and dihydrofolate reductase (gonad activity). On the one hand, some biomarkers, including metallothioneins, labile zinc, glutathione S-transferase, cytochrome P4503A4 activity, dehydrofolate reductase and aspartate transcarbamoylase, were readily decreased. In contrast, these biomarkers, cytochrome P4501A1, gill lipid peroxidation, DNA strand breaks in gills and digestive gland, mitochondrial electron transport at high and low temperatures (temperature-dependent activity) and total gonad lipids, were readily increased. In general, ozone treatment reduced adverse effects by either decreasing the intensity of the toxic responses or increasing the threshold concentration. For gill lipid peroxidation, however, intensity was greater at a higher threshold concentration. Ozone treatment eliminated the temperature sensitivity of the mitochondrial electron transport system, indicating a loss of interaction between temperature and urban pollution in terms of energy expenditure in mussels. Ozone treatment could significantly decrease either the toxic potency or intensity of urban pollutants at the expense of increased oxidative stress in gills of freshwater mussels.

Dissection of the functions of the Saccharomyces cerevisiae RAD6 postreplicative repair group in mutagenesis and UV sensitivity.

The RAD6 postreplicative repair group participates in various processes of DNA metabolism. To elucidate the contribution of RAD6 to starvation-associated mutagenesis, which occurs in nongrowing cells cultivated under selective conditions, we analyzed the phenotype of strains expressing various alleles of the RAD6 gene and single and multiple mutants of the RAD6, RAD5, RAD18, REV3, and MMS2 genes from the RAD6 repair group. Our results show that the RAD6 repair pathway is also active in starving cells and its contribution to starvation-associated mutagenesis is similar to that of spontaneous mutagenesis. Epistatic analysis based on both spontaneous and starvation-associated mutagenesis and UV sensitivity showed that the RAD6 repair group consists of distinct repair pathways of different relative importance requiring, besides the presence of Rad6, also either Rad18 or Rad5 or both. We postulate the existence of four pathways: (1) nonmutagenic Rad5/Rad6/Rad18, (2) mutagenic Rad5/Rad6 /Rev3, (3) mutagenic Rad6/Rad18/Rev3, and (4) Rad6/Rad18/Rad30. Furthermore, we show that the high mutation rate observed in rad6 mutants is caused by a mutator different from Rev3. From our data and data previously published, we suggest a role for Rad6 in DNA repair and mutagenesis and propose a model for the RAD6 postreplicative repair group.

Removal of indicator bacteria, human enteric viruses, Giardia cysts, and Cryptosporidium oocysts at a large wastewater primary treatment facility.

Pathogens and fecal indicator bacteria occurrence and removal were studied for a period of 6 months at the Montreal Urban Community wastewater treatment facility. With a capacity of about 7.6 million cubic metres per day (two billion U.S. gallons per day), it is the largest primary physico-chemical treatment plant in America. The plant discharges a nondisinfected effluent containing about 20 mg/L of suspended matter and 0.5 mg/L of total phosphorus on the basis of average annual concentrations. BDO5 (annual mean) is 75 mg/L before treatment and 32 mg/L after treatment. Samples were collected for a period of 6 months, and they demonstrated that the plant was not efficient at removing indicator bacteria and the pathogens tested. Fecal coliforms were the most numerous of the indicator bacteria and their removal averaged 25%. Fecal streptococci removal was 29%, while Escherichia coli removal was 12%. In untreated sewage, fecal coliforms, E. coli, and human enteric viruses were more numerous in summer and early autumn. Fecal streptococci counts remained relatively similar throughout the period. Clostridium perfringens removal averaged 51%. Giardia cysts levels were not markedly different throughout the study period, and 76% of the cysts were removed by treatment. Cryptosporidium oocyst counts were erratic, probably due to the methods, and removal was 27%. Human enteric viruses were detected in all samples of raw and treated wastewater with no removal observed (0%). Overall, the plant did not perform well for the removal of fecal indicator bacteria, human enteric viruses, or parasite cysts. Supplementary treatment and disinfection were recommended to protect public health. Various alternatives are being evaluated.

[1000 laparoscopic cholecystectomies--evaluation of complications]. / Tisíc laparoskopických cholecystektomií--hodnocení komplikací.

At the Department of Surgery in Liberec since the middle of 1992 to the end of 1995 one thousand laparoscopic cholecystectomies were performed. Peroperative cholangiography is not used as a routine method. The percentage of conversions declined from 10% to 3% as the technical skill improved. The period taken up by the operation decreased from 140 minutes in the first operation to 15-30 minutes in non-complicated laparoscopic cholecystectomy. The most serious complication was a lesion of the choledochus, resolved by a Y-Roux anastomosis. The patients were discharged on the second or third day after operation and are followed up in the out-patient department.

[Laparoscopic suturing of perforated duodenal and prepyloric ulcers]. / Laparoskopická sutura perforovaného duodenálního a prepylorického vredu.

The authors submit an account of five patients with a perforated duodenal and prepyloric ulcer. All patients were treated by laparoscopy only. A laparoscopic suture of the perforated ulcer was made without omentoplasty with lavage and drainage. The patients recovered without complications. Early mobilisation and rapid convalescence are the greatest advantage of the minimal invasive approach in general. Laparoscopic treatment of a perforated ulcer is safe method when handled by an experienced laparoscopic surgeon.