Enriched microbial communities for ammonium and nitrite removal from recirculating aquaculture systems.

The aim of this study was the enrichment of high-performance microbial communities in biofilters for removal of ammonium and nitrite from aquaculture water. Ammonium oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) were enriched from different environmental water samples. The microbial communities with higher ammonium and nitrite removal activity were selected and adapted to different temperatures [9 °C, 15 °C, room temperature (25 °C), and 30 °C]. The expression of genes involved in nitrification including ammonia monooxygenase (AMO) and nitrite oxidoreductase (NXR) were measured in temperature-adapted AOB and NOB microbiomes. The microbial species present in the selected microbiomes were identified via 16s rRNA sequencing. The microbial communities containing Nitrosomonas oligotropha and Nitrobacter winogradskyi showed the highest ammonium and nitrite removal activity at all temperatures used for adaptation. Furthermore, the microbial communities do not contain any pathogenic bacteria. They also exhibited the highest expression of AMO and NXR genes. Using the enriched microbial communities, we achieved a 288% and 181% improvement in ammonium and nitrite removal over the commonly used communities in biofilters at 9 °C, respectively. These results suggest that the selected microbiomes allowed for a significant improvement of water quality in a recirculating aquaculture system (RAS).

Abatement of water nutrient load in a fish culture system using the aquatic trophic levels.

Integrated aquatic systems are used to decrease the nutrient loads of effluents negating the negative environmental impacts of aquacultural systems. Some of these systems have a separate algae compartment requiring high maintenance. An integrated culture system was set up with different trophic levels: algae, zooplankton, and fish. The algal tank was in-line with the fish and zooplankton components to minimize the maintenance required for the algae. A control flow-through system was also set up without the algae and zooplankton compartments. The systems were run for 6 weeks, and water temperature, pH, dissolved oxygen, NO3 , NO2 , NH4 , and PO4 concentrations were measured. A removal rate was determined for each water parameter and the densities of the algae and zooplankton species were measured in each compartment of the integrated system. The concentrations of most nutrients in the integrated system were similar to those of the control system. The density of algae increased during the first 3 weeks and remained almost stable until the end of the experiment. There was an inverse relationship between the densities of two zooplankton suggesting compensatory effects on the control of the algal bloom. The integrated system improved water quality with minimal algal culture maintenance, water exchange, and no fish mortality. PRACTITIONER POINTS: An integrated system could effectively reduce the nutrient load of water. Water replacement in the integrated system was significantly lower than that of a flow through system. The inline plankton culture tanks decreased greatly the maintenance of the system.

Toxicity of polystyrene microplastics on juvenile Oncorhynchus mykiss (rainbow trout) after individual and combined exposure with chlorpyrifos .

Microplastic (MP) sorption and transfer of chemical contaminants has been widely reported, yet few studies have investigated combined effects of contaminant-loaded MPs on organisms. This study examined effects of pristine or chlorpyrifos (CPF)-loaded polystyrene (PS) fragments on histopathological and histomorphometrical biomarkers in rainbow trout (Onchorhynchus mykiss) . In laboratory, O. mykiss were exposed for 96 h to pristine PS-MPs concentrations (30 or 300 µg/L), concentrations of CPF alone (2 or 6 µg/L), and the same concentrations of CPF in the presence of PS-MPs in aquaria . Results showed the highest histopathological alterations in both CPF concentrations and when combined with PS-MPs in fish gills. Alternatively, high histopathological lesions including massive necrosis, infiltration of inflammatory cells, and shed of villi tips were observed in fish gut in high CPF concentrations combined with high PS-MP concentrations of (6 µg/L CPF+300 µg/L PS- MPs). Individual CPF and PS-MP concentrations or combined together showed significant changes in histomorphometrical biomarkers in fish gills, gut and skin. Findings highlight that pristine PS-MPs cause toxicity and increase adverse effects of CPF in O. mykiss, especially in gill tissue. We present evidence that pristine short-term exposure to even low concentrations of PS-MPs has a significant impact on biomarker responses in O. mykiss.

Time, pattern, and outcome of medulloblastoma relapse and their association with tumour biology at diagnosis and therapy: a multicentre cohort study.

BACKGROUND: Disease relapse occurs in around 30% of children with medulloblastoma, and is almost universally fatal. We aimed to establish whether the clinical and molecular characteristics of the disease at diagnosis are associated with the nature of relapse and subsequent disease course, and whether these associations could inform clinical management. METHODS: In this multicentre cohort study we comprehensively surveyed the clinical features of medulloblastoma relapse (time to relapse, pattern of relapse, time from relapse to death, and overall outcome) in centrally reviewed patients who relapsed following standard upfront therapies, from 16 UK Children's Cancer and Leukaemia Group institutions and four collaborating centres. We compared these relapse-associated features with clinical and molecular features at diagnosis, including established and recently described molecular features, prognostic factors, and treatment at diagnosis and relapse. FINDINGS: 247 patients (175 [71%] boys and 72 [29%] girls) with medulloblastoma relapse (median year of diagnosis 2000 [IQR 1995-2006]) were included in this study. 17 patients were later excluded from further analyses because they did not meet the age and treatment criteria for inclusion. Patients who received upfront craniospinal irradiation (irradiated group; 178 [72%] patients) had a more prolonged time to relapse compared with patients who did not receive upfront craniospinal irradiation (non-irradiated group; 52 [21%] patients; p<0·0001). In the non-irradiated group, craniospinal irradiation at relapse (hazard ratio [HR] 0·27, 95% CI 0·11-0·68) and desmoplastic/nodular histology (0·23, 0·07-0·77) were associated with prolonged time to death after relapse, MYC amplification was associated with a reduced overall survival (23·52, 4·85-114·05), and re-resection at relapse was associated with longer overall survival (0·17, 0·05-0·57). In the irradiated group, patients with MBGroup3 tumours relapsed significantly more quickly than did patients with MBGroup4 tumours (median 1·34 [0·99-1·89] years vs 2·04 [1·39-3·42 years; p=0·0043). Distant disease was prevalent in patients with MBGroup3 (23 [92%] of 25 patients) and MBGroup4 (56 [90%] of 62 patients) tumour relapses. Patients with distantly-relapsed MBGroup3 and MBGroup4 displayed both nodular and diffuse patterns of disease whereas isolated nodular relapses were rare in distantly-relapsed MBSHH (1 [8%] of 12 distantly-relapsed MBSHH were nodular alone compared with 26 [34%] of 77 distantly-relapsed MBGroup3 and MBGroup4). In MBGroup3 and MBGroup4, nodular disease was associated with a prolonged survival after relapse (HR 0·42, 0·21-0·81). Investigation of second-generation MBGroup3 and MBGroup4 molecular subtypes refined our understanding of heterogeneous relapse characteristics. Subtype VIII had prolonged time to relapse and subtype II had a rapid time from relapse to death. Subtypes II, III, and VIII developed a significantly higher incidence of distant disease at relapse whereas subtypes V and VII did not (equivalent rates to diagnosis). INTERPRETATION: This study suggests that the nature and outcome of medulloblastoma relapse are biology and therapy-dependent, providing translational opportunities for improved disease management through biology-directed disease surveillance, post-relapse prognostication, and risk-stratified selection of second-line treatment strategies. FUNDING: Cancer Research UK, Action Medical Research, The Tom Grahame Trust, The JGW Patterson Foundation, Star for Harris, The Institute of Child Health - Newcastle University - Institute of Child Health High-Risk Childhood Brain Tumour Network (co-funded by The Brain Tumour Charity, Great Ormond Street Children's Charity, and Children with Cancer UK).

Efficient Noisy Sound-Event Mixture Classification Using Adaptive-Sparse Complex-Valued Matrix Factorization and OvsO SVM.

This paper proposes a solution for events classification from a sole noisy mixture that consist of two major steps: a sound-event separation and a sound-event classification. The traditional complex nonnegative matrix factorization (CMF) is extended by cooperation with the optimal adaptive L1 sparsity to decompose a noisy single-channel mixture. The proposed adaptive L1 sparsity CMF algorithm encodes the spectra pattern and estimates the phase of the original signals in time-frequency representation. Their features enhance the temporal decomposition process efficiently. The support vector machine (SVM) based one versus one (OvsO) strategy was applied with a mean supervector to categorize the demixed sound into the matching sound-event class. The first step of the multi-class MSVM method is to segment the separated signal into blocks by sliding demixed signals, then encoding the three features of each block. Mel frequency cepstral coefficients, short-time energy, and short-time zero-crossing rate are learned with multi sound-event classes by the SVM based OvsO method. The mean supervector is encoded from the obtained features. The proposed method has been evaluated with both separation and classification scenarios using real-world single recorded signals and compared with the state-of-the-art separation method. Experimental results confirmed that the proposed method outperformed the state-of-the-art methods.

The growth performance, antioxidant capacity, immunological responses, and the resistance against Aeromonas hydrophila in Nile tilapia (Oreochromis niloticus) fed Pistacia vera hulls derived polysaccharide.

The present study aimed at evaluating the supplementation of dietary Pistacia vera hulls derived polysaccharide (PHDP) at varying levels (0, 2.5, 5, and 10 g/kg diet) on the growth rate, digestive enzyme activity, immune response, and antioxidative capacity of Nile tilapia. After the feeding trial (60 days), fish were injected intraperitoneally with Aeromonas hydrophila, and the cumulative mortality was calculated for 10 days. The final body weight, weight gain, specific growth rate, survival rate, amylase activity, red blood cells, hemoglobin, serum total protein, and serum lysozyme activity (LZM) were significantly higher in fish fed PHDP at 5 and 10 g/kg than fish fed 0 and 2.5 g/kg diet (P ≤ 0.05). However, the feed conversion ratio and glucose levels were significantly decreased by 5 and 10 g PHDP/kg diet. Further, the protease digestive activity, serum alkaline phosphatase, and serum alternative complement (ACH50) had higher values in fish fed 5 g PHDP/kg diet than tilapia fed 0 g PHDP/kg diet (P ≤ 0.05). The serum protease and immunoglobulin (Ig) presented the highest values in fish fed 10 g PHDP/kg diet followed by fish fed 5 g PHDP/kg diet (P ≤ 0.05). The LZM, ACH50, and Ig in the mucus samples were significantly higher in fish fed 5 or 10 g PHDP/kg diet than fish fed 0 g PHDP/kg diet (P ≤ 0.05). The levels of superoxide dismutase and catalase in the liver tissue was significantly higher in fish fed 5 or 10 g PHDP/kg diet than fish fed 2.5 g PHDP/kg diet (P ≤ 0.05). On the other hand, malondialdehyde concentration was significantly lower in tilapia fed 5 and 10 g PHDP/kg diet than tilapia fed 0 and 2.5 g PHDP/kg diet (P ≤ 0.05). The lowest mortality rate and the highest relative percentage survival were in fish fed 5 g/kg followed by 10 g level after 10 days of A. hydrophila injection. Accordingly, dietary PHDP at 5-10 g/kg is recommended to improve the growth performance, antioxidative capacity, immune response, and resistance against A. hydrophila in Nile tilapia.

Effects of dietary Lactobacillus plantarum (KC426951) in biofloc and stagnant-renewal culture systems on growth performance, mucosal parameters, and serum innate responses of Nile tilapia Oreochromis niloticus.

Effects of dietary Lactobacillus plantarum (KC426951) on growth and innate responses of Nile tilapia Oreochromis niloticus were evaluated in biofloc technology system and stagnant-renewal culture system (SRCS). The 90-day-long experiment contained four treatments: SRCS without probiotic (T1), SRCS with probiotic (T2), biofloc without probiotic (T3), and biofloc with probiotic (T4). The administration dose of probiotic was 2 × 108 CFU kg-1 diet. At the end of experiment, the mean final weights, specific growth rates, feed conversion ratios, and total biomass were significantly (P < 0.05) better in BFT treatments, with no significant effect of probiotic on these parameters in both culture systems. Meanwhile, skin mucosal parameters including total protein (TP), lysozyme (LYZ), alkaline phosphatase (ALP), and protease (PRO) activity were significantly enhanced following probiotic supplementation. T4 treatment displayed a significantly higher LYZ and ALP activity in mucus versus other treatments. Also, serum alternative complement activity was significantly heightened in probiotic-supplemented fish. Superoxide dismutase activity in T4 was detected higher than that of SRCS groups. The results of the current study demonstrated the enhancement of some mucosal and serum innate responses of Nile tilapia in both culture systems upon L. plantarum (KC426951) supplementation.

Cold-Resistant Heterotrophic Ammonium and Nitrite-Removing Bacteria Improve Aquaculture Conditions of Rainbow Trout (Oncorhynchus mykiss).

The aim of this study was isolation and characterization of heterotrophic bacteria capable of ammonium and nitrite removal at 15 °C (optimal temperature for growing rainbow trout Oncorhynchus mykiss). Environmental isolates were grown in liquid media containing ammonium or nitrite, and best strains in terms of growth and ammonium or nitrite removal were identified via 16S rRNA sequencing. Dyadobacter sp. (no. 68) and Janthinobacterium sp. (no. 100) were selected for optimal adaptation to growth at 15 °C and best ammonium and nitrite removal (P < 0.05), respectively. A heterotrophic ammonium and nitrite removal (HAN) microbial complex, containing selected strains, was prepared and applied in a trout culture system. After 10 days, the effect of microbial HAN complex was investigated in terms of ammonium and nitrite removal, as well as stress and immune indices present in the plasma of cultivated trout. Compared to a standard cultivation setup, addition of the HAN complex had a clear beneficial effect on keeping the un-ionized ammonia and nitrite level below prescribed standards (P < 0.05). This resulted in reduction of stress and immune reactions of cultivated fish (P < 0.05), leading to an augmentation of final weight and survival. Application of the selected microbial complex resulted in a significant improvement of the aquaculture ecosystem.

DNA damage, acetylcholinesterase activity, and hematological responses in rainbow trout exposed to the organophosphate malathion.

Effects of sub-lethal concentrations (0 (control), 0.009, 0.014, and 0.023 ppm) of the organophosphate insecticide "malathion" to rainbow trout (Oncorhynchus mykiss) after the determination of LC50-96 h value (0.093 ppm) were evaluated. Changes in biomarkers of neurotoxicity (acetylcholinesterase (AChE) activity), genotoxicity (DNA damage), and hematological parameters (red (RBC) and white (WBC) blood cell count, hemoglobin (Hb), hematocrit (Hct), mean cell hemoglobin (MCH), mean cell volume (MCV), and mean cell hemoglobin concentration (MCHC)) were assessed for a 15-day exposure. A significant time- and dose-dependent reduction in AChE activities of gill, muscle, brain, and liver tissues was found. However, the AChE activity was less affected by malathion concentration than by exposure time. DNA damage of erythrocytes at different malathion concentrations increased by increasing the experimental time up to the fourth day. A decrease in the count of WBC, RBC, and Hct and an increase in the number of MCH and MCV were observed by increasing malathion exposure dose and time (p < 0.05). An increase in the malathion concentration and exposure time significantly resulted in a decrease in Hb and an increase in MCHC. A significant improvement in AChE activity; DNA damage; and RBC, Hb, Hct, MCV, and MCH indices was detected during a 30-day recovery period, but the WBC count changed insignificantly. The recovery pattern based on 100% water exchange with clean water could be a successful strategy to improve the biomarker responses of rainbow trout habituating in contaminated aquatic environments.

Effects of chronic lead and cadmium exposure on the oriental river prawn (Macrobrachium nipponense) in laboratory conditions.

During the past decade, the total population and overall reproductive performance of the oriental river prawn (Macrobrachium nipponense) in the Anzali international wetland and its basin rivers has been severely reduced. This seems to be caused by an increase in heavy metal pollution from industrial wastewaters. We investigated the effects of chronic levels of lead (Pb) and cadmium (Cd) on reproductive success, hemato-immunology, crustacean hyperglycemic hormone (cHH), Pb/Cd bioaccumulation and histopathology of hepatopancreas of oriental river prawn. The present study was separately carried out using four chronic treatments for Pb (0 (control), 0.024, 0.072 and 0.12 µM/l) and Cd (0 (control), 0.026, 0.053 and 0.08 µM/l) in experimental tanks during 60 days. The broodstock were fed by commercial shrimp diet, ad libitum, twice a day. The results revealed that reproductive activities ceased when Pb levels were 0.072 and 0.12 µM/l and also when Cd levels reached 0.08 µM/l. At 0.024 µM/l Pb, 0.026 and 0.053 µM/l Cd, the prawns showed initial reproductive activity but after spawning, the egg-clutches were found detached from the pleopods of the brood females. Hemato-immunology results showed that by increasing the Pb and Cd concentrations, large granular hemocyte (LGH) and cHH values also increased. However, small granular hemocyte (SGH) count was inversely affected. A significant increase in mortality (P < 0.05) was observed as both Pb and Cd concentrations increased. Histological observations are indicative of increased necrosis, lumen size, melanization, vacuolation and abnormal lumen shape, as the heavy metals concentrations increased. In conclusion, obtained results can help to elucidate effects of Pb and Cd on freshwater decapod crustaceans reproductive performance and related physiological parameters.

Droplet vitrification versus straw cryopreservation for spermatozoa banking in Persian sturgeon (Acipenser persicus) from metabolite point of view.

Persian sturgeon (Acipenser persicus), a commercially valuable and critically endangered fish species has been suffering considerable declines in populations in the nature due to over-fishing, habitat destruction and marine pollution during past decades. Since there were no achievements in artificial reproduction programs, genetic resource banking such as gametes and embryo cryopreservation can be a good strategy however, reported resulting gamete qualities were considerably low. In the present study, the metabolome content of Persian sturgeon spermatozoa was investigated during common straw cryopreservation and novel droplet vitrification by the use of 1H NMR (Nuclear Magnetic Resonance) spectroscopy. Univariate (ANOVA) and multivariate (PCA) analysis showed significant differences in the metabolic profiles between cryopreserved and fresh spermatozoa samples. Adenine, creatine, creatine phosphate, glucose, guanidoacetate, lactate, N, N-dimethylglycine, and glycine levels showed no significant differences between these two cryopreservation techniques suggesting these metabolites and their corresponding enzymes and chemical pathways are so vulnerable to the temperature changes and even higher cooling rate in droplet vitrification could not conserve them. However, significant differences were found in acetate, creatinine, betaine, ß-alanine and trimethylamine N-oxide suggesting better efficiency of droplet vitrification in protection of some metabolites associated to spermatozoa energetics, redox balance and hypoxia compensation compared to straw cryopreservation.

1H NMR metabolic profiling of the cryopreserved spermatozoa of the wild endangered Persian sturgeon (Acipenser persicus) with the use of beta-cyclodextrin as an external cryoprotectant.

Persian sturgeon (Acipenser persicus) is an endangered species and genetic resource banking such as gametes and embryo preservation could be one of the most pursued conservation approaches. In this study, deleterious effects of the traditional cryopreservation technique and the effect of different doses of 2-hydroxypropyl-beta-cyclodextrin (HßCD) on thawed spermatozoa quality (motility duration and percentage) of Persian sturgeon (Acipenser persicus) were investigated from metabolic aspects of view. For cryopreserving, semen was diluted with Tris-HCl (100 mM) extenders containing 0, 5, 10, and 15 mM of HßCD in a ratio of 1:1 (semen/extenders). Semen-extenders were filled into 0.5-mL straws and were frozen with the vapor of liquid nitrogen, and then immersed into liquid nitrogen. Cryopreserved spermatozoa were thawed in water baths in 15 s. Two treatments with the highest and the lowest motility percentages (0 and 10 mM of HßCD) were chosen to reveal the extremes of the metabolites change range and were objected to 1H NMR spectroscopy. Univariate (ANOVA) and multivariate (PCA) analysis of the obtained metabolic profiles showed significant changes (P < 0.05) in metabolites. The use of 10 mM of HßCD was completely successful in the preservation of creatinine, glucose, guanidoacetate, O-phosphocholine, and N, N-dimethylglycine and probably their corresponding biochemical pathways, but it failed to preserve lactate, carnitine, betain, ß-alanin, and trimethylamine N-oxide. It was also partially successful in preserving acetate, creatine, creatine phosphate, and glycine, all suggesting how HßCD can be effective as a cryoprotectant.

Characterization of Gamma-Irradiated Rosmarinus officinalis L. (Rosemary).

OBJECTIVES: Rosmarinus officinalis L., a member of the family Lamiaceae, is regarded as the spice with the highest antioxidant activity. MATERIALS AND METHODS: In this study, the transmission electron microscopy, X-ray diffraction, and fourier transform infrared spectroscopy (FTIR) physicochemical characteristics of the nanostructure of gamma-irradiated rosemary were investigated. RESULTS: The particle size distribution of the gamma-irradiated rosemary prepared under irradiation at 30 kGy in a Cobalt-60 irradiator exhibited a very narrow size distribution with average size of 70 nm. The results showed that irradiated (30 kGy) and crude rosemary had similar patterns of FTIR spectra, typical of phenol compound, without any notable changes in the key bands and functional groups status. Rosemary irradiated with 50 kGy and 10 kGy showed the highest and lowest crystallinity, respectively. Rosemary crystallinity of irradiated samples was lower compared with the nonirradiated sample. CONCLUSION: Therefore, 30 kGy can be optimum for the synthesis of nanoparticles, average size of 70 nm, with low crystallinity and without any notable change in key bands compared to nonirradiated samples.

Effects of the prebiotic mannan-oligosaccharide on the stress response of feed deprived zebrafish (Danio rerio).

Feed deprivation has deleterious effects on fish behavior and stress physiology which may susceptible them to disease outbreak. Functional ingredients in diets may substantially impact the physiology and stress responses of host organisms. Here, we hypothesized that the administration of a dietary prebiotic might attenuate the negative influences of feed deprivation on the behavioral profile of anxiety and physiological responses to stress in zebrafish (Danio rerio). Fish were fed with either basal or mannan-oligosaccharide supplemented (0.4% MOS/kg diet) diets, once per day (normal-control: CN, and normal-prebiotic: PN) or once every other day (starved-control: CS, and starved-prebiotic: PS) for 8weeks. Afterwards, fish were subjected to a novel tank test to measure anxiety. Fish from the CS treatment exhibited more pronounced bottom-dwelling behavior than the other treatments. The number of transitions from the bottom to the top third of the novel tank was significantly higher in PN fish than the CS specimens. No significant differences were found between the CN and PS treatments in all of the anxiety behaviors. CS fish showed higher baseline cortisol levels than the other treatments, which was in line with higher expression of CRH gene in fish subjected to this treatment. Cortisol levels and CRH gene expression of the subjects were also measured after induction of two routine aquaculture stressors. CN and PS fish exhibited similar patterns of cortisol responses at most of the sampling times after stress, and PN specimens showed a significantly lower concentration of cortisol than the other treatments in most cases. Expression of the CRH gene was higher in feed deprived fish immediately after stress induction. Overall, the results show that feed deprivation in some cases influenced anxiety-like behaviors and elevated stress response in zebrafish juveniles; however, the addition of MOS to the diet helped deprived fish exhibit behaviors more typical of normally fed animals.

Novel molecular subgroups for clinical classification and outcome prediction in childhood medulloblastoma: a cohort study.

BACKGROUND: International consensus recognises four medulloblastoma molecular subgroups: WNT (MBWNT), SHH (MBSHH), group 3 (MBGrp3), and group 4 (MBGrp4), each defined by their characteristic genome-wide transcriptomic and DNA methylomic profiles. These subgroups have distinct clinicopathological and molecular features, and underpin current disease subclassification and initial subgroup-directed therapies that are underway in clinical trials. However, substantial biological heterogeneity and differences in survival are apparent within each subgroup, which remain to be resolved. We aimed to investigate whether additional molecular subgroups exist within childhood medulloblastoma and whether these could be used to improve disease subclassification and prognosis predictions. METHODS: In this retrospective cohort study, we assessed 428 primary medulloblastoma samples collected from UK Children's Cancer and Leukaemia Group (CCLG) treatment centres (UK), collaborating European institutions, and the UKCCSG-SIOP-PNET3 European clinical trial. An independent validation cohort (n=276) of archival tumour samples was also analysed. We analysed samples from patients with childhood medulloblastoma who were aged 0-16 years at diagnosis, and had central review of pathology and comprehensive clinical data. We did comprehensive molecular profiling, including DNA methylation microarray analysis, and did unsupervised class discovery of test and validation cohorts to identify consensus primary molecular subgroups and characterise their clinical and biological significance. We modelled survival of patients aged 3-16 years in patients (n=215) who had craniospinal irradiation and had been treated with a curative intent. FINDINGS: Seven robust and reproducible primary molecular subgroups of childhood medulloblastoma were identified. MBWNT remained unchanged and each remaining consensus subgroup was split in two. MBSHH was split into age-dependent subgroups corresponding to infant (<4·3 years; MBSHH-Infant; n=65) and childhood patients (≥4·3 years; MBSHH-Child; n=38). MBGrp3 and MBGrp4 were each split into high-risk (MBGrp3-HR [n=65] and MBGrp4-HR [n=85]) and low-risk (MBGrp3-LR [n=50] and MBGrp4-LR [n=73]) subgroups. These biological subgroups were validated in the independent cohort. We identified features of the seven subgroups that were predictive of outcome. Cross-validated subgroup-dependent survival models, incorporating these novel subgroups along with secondary clinicopathological and molecular features and established disease risk-factors, outperformed existing disease risk-stratification schemes. These subgroup-dependent models stratified patients into four clinical risk groups for 5-year progression-free survival: favourable risk (54 [25%] of 215 patients; 91% survival [95% CI 82-100]); standard risk (50 [23%] patients; 81% survival [70-94]); high-risk (82 [38%] patients; 42% survival [31-56]); and very high-risk (29 [13%] patients; 28% survival [14-56]). INTERPRETATION: The discovery of seven novel, clinically significant subgroups improves disease risk-stratification and could inform treatment decisions. These data provide a new foundation for future research and clinical investigations. FUNDING: Cancer Research UK, The Tom Grahame Trust, Star for Harris, Action Medical Research, SPARKS, The JGW Patterson Foundation, The INSTINCT network (co-funded by The Brain Tumour Charity, Great Ormond Street Children's Charity, and Children with Cancer UK).

Metabolic changes in droplet vitrified semen of wild endangered Persian sturgeon Acipenser persicus (Borodin, 1997).

Comparative quantitative metabolite profiling can be used for better understanding of cell functions and dysfunctions in particular circumstances such as sperm banking which is an important approach for cryopreservation of endangered species. Cryopreservation techniques have some deleterious effects on spermatozoa which put the obtained results in controversy. Therefore, in the present study, quantitative 1H NMR (Nuclear Magnetic Resonance) based metabolite profiling was conducted to evaluate metabolite changes related to energetics and some other detected metabolites in vitrified semen of critically endangered wild Acipenser persicus. The semen was diluted with extenders containing 0, 5, 10, and 15 µM of fish antifreeze protein (AFP) type III as a cryoprotectant. Semen-extenders were vitrified and stored for two days. Based on post-thaw motility duration and motility percentage assessments, two treatments with 10 µM and 0 µM of AFP had the highest and the lowest motility percentages respectively and they were objected to 1H NMR spectroscopy investigations in order to reveal the extremes of the metabolites dynamic range. Univariate (ANOVA) and multivariate (PCA) analysis of the resulting metabolic profiles indicated significant changes (P > 0.05) in metabolites. The level of some metabolites including acetate, adenine, creatine, creatine phosphate, lactate, betaine, sarcosine, ß-alanine and trimethylamine N-oxide significantly decreased in vitrified semen while some others such as creatinine, guanidinoacetate, N, N-dimethylglycine, and glycine significantly increased. There were also significant differences between vitrified treatments in levels of creatine, creatine phosphate, creatinine, glucose, guanidinoacetate, lactate, N, N-dimethylglycine, and glycine, suggesting how fish AFP type III can be effective as a cryoprotectant.

Influence of supplemented diet with Pediococcus acidilactici on non-specific immunity and stress indicators in green terror (Aequidens rivulatus) during hypoxia.

Green terror fish were fed for two months with three types of feed including: control diet (C), diet containing 2% fish oil (O) and diet containing fish oil supplemented with 0.1% Pediococcus acidilactici bacteria (PA). At the end of the feeding period, 50 fish of different groups (n = 3) with an average weight of 4.28 g were transferred to 9 tank. Hypoxia tests were set out by sampling at three times including before hypoxia (BH) initiation, hypoxia stress spot (H) and starting mortality (SM). In fish submitted to the diet containing P. acidilactici bacteria, the immune indices of lysozyme activity (4.08, 4.19 and 4.85 µg/ml)], complement activity (2.65, 2.77 and 2.1 U/ml) and total immunoglobulin (10.05, 10.25 and 9.9 µg/ml) improved in all stages of sampling (BH, H and SM), respectively (p < 0.05). The positive effects of the bacteria application also extends for the stress indicators including: cortisol (0.175, 0.3 and 0.335 µM/ml), glucose (0.9, 1.25 and 0.6 µg/ml) and lactate (2.7, 3 and 3.35) µg/ml and plasma electrolytes consisting of Na(+) (178.5, 175.43 and 175.8 mmol/l) and Cl(-)(123.85, 119.30 and 118.43 mmol/l) in all sampling stages (BH, H and SM), respectively (p < 0.05). Put it all together, P. acidilactici, acting as a probiotic, helps reducing stress symptoms in green terror fish.

The effect of Pediococcus acidilactici bacteria used as probiotic supplement on the growth and non-specific immune responses of green terror, Aequidens rivulatus.

A 56-day feeding trial was conducted on a species of ornamental fish called green terror (Aequidens rivulatus) (0.388 ± 0.0021 g) to assess the effect of probiotic bacteria, Pediococcus acidilactici on the growth indices and innate immune response. The fish were randomly allocated into 9 oval tanks (120 l) at a density of 60 fish per tank. The experimental diets were comprised of the control (C), C complemented with fish oil (O) and the probiotic and fish oil (PA) and fed ad lib twice a day. The growth indices (specific growth rate (SGR), feed conversion ratio (FCR) and immunological indices of fish fed the diets including lysozyme activity, total immunoglobulin and alternative complement activity were measured. The Fish fed with the diet containing P. acidilactici (PA) displayed significantly (P < 0.05) higher final weight (3.25 ± 0.065 g), weight gain (830.94 ± 9.46%), SGR (3.53 ± 0.02%/day) and lower FCR (1.45 ± 0.011) compared to those of other experimental diets. Total immunoglobulin (10.05 ± 0.12 µg/ml), lysozyme activity (4.08 ± 0.85 µg/ml) and alternative complement activity (2.65 ± 0.12 U/ml) in the serum of PA fed fish showed significant compared to other treatments (P < 0.05). The results showed positive effects of P. acidilactici as a potent probiotic on growth indices and non-specific immune system of green terror.

Statistical source identification of major and trace elements in groundwater downward the tailings dam of Miduk Copper Complex, Kerman, Iran.

Identifying the possible sources of potential harmful metals in groundwater systems plays a crucial role in evaluating the potential risks to residents and local plant cover. An attempt was made to define the origin of Al, Cd, Cu, Fe, Mo, Ni, and Pb in groundwater using multivariate statistic approaches [principal component analysis (PCA), hierarchical cluster analysis], and tailings sequential extraction by the method of Tessier et al. The concentrations of studied elements were measured in 42 samples collected from 15 stations surrounding and downward the tailings dam of Miduk Copper Complex, central province of Kerman, Iran. According to the PCA results, confirmed by cluster dendrogram and metal content measurement of tailings sequential extracts, two components accounting for nearly 73% of the total variance, controlled the heavy metal variability and classified the possible source of groundwater contamination into two categories: (1) upper seepage which controls the variability of Cd, Cu, Fe, Ni, and Pb and (2) toe seepage of tailings dump affecting on Mo and Al concentration in downstream groundwater.

Seasonal fluctuation in heavy metal pollution in Iran's Siahroud River. A preliminary study.

BACKGROUND: The management of its available water resources has become a key issue for Iran. During the last few decades, the water quality of Siahroud River in the coastal plain of the Caspian Sea in Gilan Province in Northern Iran has significantly degraded. The scarcity of water has been compounded by rapid population growth and increasing pollution from fertilizers, pesticides, and municipal and industrial wastes. One of the sources of this degradation is the movement of heavy metals from the river's watershed into the various water systems supported by the river, including the water system for Rasht City. METHODS: To study the magnitude of heavy metal pollution in the Siahroud River, seven heavy metals including Zn, Cu, Pb, Cd, Mn, Fe, and Ni were measured in duplicate from replicated water samples collected over five consecutive seasons and analyzed by Atomic Absorption Spectrometry. In situ measurements of pH were taken with the samples and total organic carbon (TOC) was analyzed by IR gas measurement. RESULTS: The results demonstrated that four of the seven heavy metals including Pb, Fe, Cd, and Mn exceeded permissible safe levels as established by the United States Environmental Protection Agency. Multivariate factor analysis suggested that industrial land-use was the main contributing factor for the high levels of Fe and Mn in the Siahroud River, whereas, Cd was principly from agricultural activities in the watershed. The data also suggested that pH and TOC had an important role in the behaviour of Pb and Mn, and that the elevated levels of these two heavy metals in Siahroud River was the consequence of other anthropogenic sources. Only negligible levels of Zn, Cu and Ni were detected. Finally, all the sampling stations were subjected to cluster analysis. The results indicated that three different zones could be distinguished according to the levels of pollution. In addition, it was shown that the urban areas did not have a significant impact on the heavy metal pollution in the river. This observation stems from the fact that the data from the sampling stations before and after Rasht City were not significantly different. RECOMMENDATIONS: Preventive measures need to be undertaken in the land-use systems and watersheds of the Siahroud River to reduce the pollution levels of Pb, Cd, Mn and Fe.Cd, Mn and Fe.