A bacterial index to estimate lake trophic level: National scale validation.

Lakes and their catchments have been subjected to centuries to millennia of exploitation by humans. Efficient monitoring methods are required to promote proactive protection and management. Traditional monitoring is time consuming and expensive, which limits the number of lakes monitored. Lake surface sediments provide a temporally integrated representation of environmental conditions and contain high microbial biomass. Based on these attributes, we hypothesized that bacteria associated with lake trophic states could be identified and used to develop an index that would not be confounded by non-nutrient stressor gradients. Metabarcoding (16S rRNA gene) was used to assess bacterial communities present in surface sediments from 259 non-saline lakes in New Zealand encompassing a range of trophic states from alpine microtrophic lakes to lowland hypertrophic lakes. A subset of lakes (n = 96) with monitoring data was used to identify indicator amplicon sequence variants (ASVs) associated with different trophic states. A total of 10,888 indicator taxa were identified and used to develop a Sediment Bacterial Trophic Index (SBTI), which signficantly correlated (r2 = 0.842, P < 0.001) with the Trophic Lake Index. The SBTI was then derived for the remaining 163 lakes, providing new knowledge of the trophic state of these unmonitored lakes. This new, robust DNA-based tool provides a rapid and cost-effective method that will allow a greater number of lakes to be monitored and more effectively managed in New Zealand and globally. The SBTI could also be applied in a paleolimnological context to investigate changes in trophic status over centuries to millennia.

Co-ingestion of Antioxidant Drinks With an Unhealthy Challenge Meal Fails to Prevent Post-prandial Endothelial Dysfunction: An Open-Label, Crossover Study in Older Overweight Volunteers.

Eating a high calorie meal is known to induce endothelial dysfunction and it is reported that consuming drinks rich in antioxidants may be protective against this. In this study we assessed the effects of three antioxidant drinks with considerable disparity in their antioxidant content on endothelial function. Seven apparently healthy overweight and older adults (BMI 25-35; mean age 57 ± 3 years; one male, six females) completed four trials in a randomized counterbalanced design. Water (control), orange juice, green tea, or red wine were consumed with a high calorie meal (>900 kcal). Endothelial function was measured by flow-mediated dilatation immediately before (fasted, baseline) and 2 h after the meal. Blood samples were also obtained for lipid and glucose analysis, plasma nitrite ( NO 2 - ) and oxidized low-density lipoprotein (ox-LDL). Participants returned after a minimum 3 days washout to complete the remaining arms of the study. The results found that the high calorie meal induced a substantial increase in triglycerides, but not cholesterol or glucose, at 2 h after meal ingestion. FMD was significantly reduced by ∼35% at this timepoint, but the effect was not attenuated by co-ingestion of any of the antioxidant drinks. Reduced FMD was mirrored by a reduction in NO 2 - , but ox-LDL was not increased at 2 h after the meal. None of the undertaken measures were influenced by the antioxidant drinks. We conclude that co-ingestion of none of our test antioxidant drinks protected against the substantial post-prandial endothelial dysfunction induced by an unhealthy meal challenge in our sample population at a 2 h timepoint.

A comparison of droplet digital polymerase chain reaction (PCR), quantitative PCR and metabarcoding for species-specific detection in environmental DNA.

Targeted species-specific and community-wide molecular diagnostics tools are being used with increasing frequency to detect invasive or rare species. Few studies have compared the sensitivity and specificity of these approaches. In the present study environmental DNA from 90 filtered seawater and 120 biofouling samples was analyzed with quantitative PCR (qPCR), droplet digital PCR (ddPCR) and metabarcoding targeting the cytochrome c oxidase I (COI) and 18S rRNA genes for the Mediterranean fanworm Sabella spallanzanii. The qPCR analyses detected S. spallanzanii in 53% of water and 85% of biofouling samples. Using ddPCR S. spallanzanii was detected in 61% of water of water and 95% of biofouling samples. There were strong relationships between COI copy numbers determined via qPCR and ddPCR (water R2  = 0.81, p < .001, biofouling R2  = 0.68, p < .001); however, qPCR copy numbers were on average 125-fold lower than those measured using ddPCR. Using metabarcoding there was higher detection in water samples when targeting the COI (40%) compared to 18S rRNA (5.4%). The difference was less pronounced in biofouling samples (25% COI, 29% 18S rRNA). Occupancy modelling showed that although the occupancy estimate was higher for biofouling samples (ψ = 1.0), higher probabilities of detection were derived for water samples. Detection probabilities of ddPCR (1.0) and qPCR (0.93) were nearly double metabarcoding (0.57 to 0.27 marker dependent). Studies that aim to detect specific invasive or rare species in environmental samples should consider using targeted approaches until a detailed understanding of how community and matrix complexity, and primer biases affect metabarcoding data.

Rapid and accurate identification by real-time PCR of biotoxin-producing dinoflagellates from the family gymnodiniaceae.

The identification of toxin-producing dinoflagellates for monitoring programmes and bio-compound discovery requires considerable taxonomic expertise. It can also be difficult to morphologically differentiate toxic and non-toxic species or strains. Various molecular methods have been used for dinoflagellate identification and detection, and this study describes the development of eight real-time polymerase chain reaction (PCR) assays targeting the large subunit ribosomal RNA (LSU rRNA) gene of species from the genera Gymnodinium, Karenia, Karlodinium, and Takayama. Assays proved to be highly specific and sensitive, and the assay for G. catenatum was further developed for quantification in response to a bloom in Manukau Harbour, New Zealand. The assay estimated cell densities from environmental samples as low as 0.07 cells per PCR reaction, which equated to three cells per litre. This assay not only enabled conclusive species identification but also detected the presence of cells below the limit of detection for light microscopy. This study demonstrates the usefulness of real-time PCR as a sensitive and rapid molecular technique for the detection and quantification of micro-algae from environmental samples.

Principal component and causal analysis of structural and acute in vitro toxicity data for nanoparticles.

Structure toxicity relationship analysis was conducted using principal component analysis (PCA) for a panel of nanoparticles that included dry powders of oxides of titanium, zinc, cerium and silicon, dry powders of silvers, suspensions of polystyrene latex beads and dry particles of carbon black, nanotubes and fullerene, as well as diesel exhaust particles. Acute in vitro toxicity was assessed by different measures of cell viability, apoptosis and necrosis, haemolytic effects and the impact on cell morphology, while structural properties were characterised by particle size and size distribution, surface area, morphology, metal content, reactivity, free radical generation and zeta potential. Different acute toxicity measures were processed using PCA that classified the particles and identified four materials with an acute toxicity profile: zinc oxide, polystyrene latex amine, nanotubes and nickel oxide. PCA and contribution plot analysis then focused on identifying the structural properties that could determine the acute cytotoxicity of these four materials. It was found that metal content was an explanatory variable for acute toxicity associated with zinc oxide and nickel oxide, while high aspect ratio appeared the most important feature in nanotubes. Particle charge was considered as a determinant for high toxicity of polystyrene latex amine.

Investigating diet as the source of tetrodotoxin in Pleurobranchaea maculata.

The origin of tetrodotoxin (TTX) is highly debated; researchers have postulated either an endogenous or exogenous source with the host accumulating TTX symbiotically or via food chain transmission. The aim of this study was to determine whether the grey side-gilled sea slug (Pleurobranchaea maculata) could obtain TTX from a dietary source, and to attempt to identify this source through environmental surveys. Eighteen non-toxic P. maculata were maintained in aquariums and twelve were fed a TTX-containing diet. Three P. maculata were harvested after 1 h, 24 h, 17 days and 39 days and TTX concentrations in their stomach, gonad, mantle and remaining tissue/fluids determined using liquid chromatography-mass spectrometry. Tetrodotoxin was detected in all organs/tissue after 1 h with an average uptake of 32%. This decreased throughout the experiment (21%, 15% and 9%, respectively). Benthic surveys at sites with dense populations of toxic P. maculata detected very low or no TTX in other organisms. This study demonstrates that P. maculata can accumulate TTX through their diet. However, based on the absence of an identifiable TTX source in the environment, in concert with the extremely high TTX concentrations and short life spans of P. maculata, it is unlikely to be the sole TTX source for this species.

A novel cutaneous Fatty Acid-binding protein-related signaling pathway leading to malignant progression in prostate cancer cells.

Cutaneous fatty acid-binding protein (C-FABP), a cancer promoter and metastasis inducer, is overexpressed in the majority of prostatic carcinomas. Investigation of molecular mechanisms involved in tumor-promoting activity of C-FABP has established that there is a fatty acid-initiated signaling pathway leading to malignant progression of prostatic cancer cells. Increased C-FABP expression plays an important role in this novel signaling pathway. Thus, when C-FABP expression is increased, excessive amounts of fatty acids are transported into the nucleus where they act as signaling molecules to stimulate their nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ). The activated PPARγ then modulates the expression of its downstream target regulatory genes, which eventually lead to enhanced tumor expansion and aggressiveness caused by an overgrowth of cells with reduced apoptosis and an increased angiogenesis.

Functional analysis of the ATM-p53-p21 pathway in the LRF CLL4 trial: blockade at the level of p21 is associated with short response duration.

PURPOSE: This study sought to establish whether functional analysis of the ATM-p53-p21 pathway adds to the information provided by currently available prognostic factors in patients with chronic lymphocytic leukemia (CLL) requiring frontline chemotherapy. EXPERIMENTAL DESIGN: Cryopreserved blood mononuclear cells from 278 patients entering the LRF CLL4 trial comparing chlorambucil, fludarabine, and fludarabine plus cyclophosphamide were analyzed for ATM-p53-p21 pathway defects using an ex vivo functional assay that uses ionizing radiation to activate ATM and flow cytometry to measure upregulation of p53 and p21 proteins. Clinical endpoints were compared between groups of patients defined by their pathway status. RESULTS: ATM-p53-p21 pathway defects of four different types (A, B, C, and D) were identified in 194 of 278 (70%) samples. The type A defect (high constitutive p53 expression combined with impaired p21 upregulation) and the type C defect (impaired p21 upregulation despite an intact p53 response) were each associated with short progression-free survival. The type A defect was associated with chemoresistance, whereas the type C defect was associated with early relapse. As expected, the type A defect was strongly associated with TP53 deletion/mutation. In contrast, the type C defect was not associated with any of the other prognostic factors examined, including TP53/ATM deletion, TP53 mutation, and IGHV mutational status. Detection of the type C defect added to the prognostic information provided by TP53/ATM deletion, TP53 mutation, and IGHV status. CONCLUSION: Our findings implicate blockade of the ATM-p53-p21 pathway at the level of p21 as a hitherto unrecognized determinant of early disease recurrence following successful cytoreduction.

Tetrodotoxin concentrations in Pleurobranchaea maculata: temporal, spatial and individual variability from New Zealand populations.

Tetrodotoxin (TTX) is a potent neurotoxin that has been identified in a range of phylogenetically unrelated marine and terrestrial organisms. Tetrodotoxin was recently detected in New Zealand in Pleurobranchaea maculata (the grey side-gilled sea slug). From June 2010 to June 2011 wild specimens were collected from 10 locations around New Zealand. At one site (Narrow Neck Beach, Auckland) up to 10 individuals were collected monthly for 6 months. Attempts were also made to rear P. maculata in captivity. Tetrodotoxin was detected in samples from eight of the ten sites. The highest average (368.7 mg kg⁻¹) and maximum (1414.0 mg kg⁻¹) concentrations were measured in samples from Illiomama Rock (Auckland). Of the toxic populations tested there was significant variability in TTX concentrations among individuals, with the highest difference (62 fold) measured at Illiomama Rock. Tetrodotoxin concentrations in samples from Narrow Neck Beach varied temporally, ranging from an average of 184 mg kg⁻¹ in June 2010 to 17.5 mg kg⁻¹ by December 2010. There was no correlation between TTX levels and mass. The highest levels correspond with the egg laying season (June-August) and this, in concert with the detection of high levels of TTX in eggs and early larval stages, suggests that TTX may have a defensive function in P. maculata. Only one larva was successfully reared to full maturation and no TTX was detected.

Photochemistry of trans-Cr(cyclam)(ONO)2+, a nitric oxide precursor.

Experimental and density functional theory (DFT) studies are described that are focused on outlining the reactivity of the known photochemical nitric oxide precursor trans-Cr(cyclam)(ONO)(2)(+) ("CrONO", cyclam = 1,4,8,11-tetrazacycltetradecane). Studies in both aerated and deaerated aqueous media are described as are the roles of both the oxidant O(2) and a reductant such as glutathione in trapping the apparent Cr(IV) photoreaction intermediate trans-Cr(cyclam)(O)(ONO)(+). Also reported and characterized structurally is the Cr(V) product of long-term photolysis in the absence of reducing agents, the trans-dioxo species [trans-Cr(cyclam)(O)(2)](ClO(4)). Photosensitization experiments indicate that at least a significant fraction of the reaction occurs from the lowest energy doublet excited state(s). Lastly, cell culture experiments demonstrate that CrONO has little or no acute toxicity either before or after photolysis.

Expression of cutaneous fatty acid-binding protein (C-FABP) in prostate cancer: potential prognostic marker and target for tumourigenicity-suppression.

C-FABP or E-FABP is a metastasis inducing gene over expressed in human prostate carcinomas. To study its prognostic significance, an archival set of prostate tissues was analysed immunohistochemically. Levels of both nuclear and cytoplasmic C-FABP expression in carcinoma cells were significantly higher than those in normal and BPH tissues and the increased C-FABP was significantly associated with a reduced patient survival time. To test the therapeutic potential of targeting C-FABP, a clone (Si-clone-2) of cells was established by interfering C-FABP expression in highly malignant PC-3M cells. Suppression of C-FABP in cancer cells significantly inhibited their proliferation and tumourigenicity in vitro. When Si-clone-2 cells were orthotopically implanted into the prostate gland of mouse, 2/13 mice produced primary tumours with an average size of 23+/-5 mg, and no metastasis was produced in any of the 13 animals. Whereas in the control group, all 14 mice produced primary tumours with an average size of 1450+/-370 mg and 9/14 (64.3%) produced metastasis. When inoculated subcutaneously, all 5 mice inoculated with control cells developed tumours from day 4, with an average size of 1471+/-544 mm(3) at 5 weeks after the inoculation; whereas Si-clone-2 cells produced no tumours in any of the 5 animals at any time-point, indicating the suppression occurred at the initiation stage. Our results suggest that C-FABP may be used as a potential prognostic marker to predict patient outcome and the increased C-FABP expression is a possible target to inhibit the malignant progression of prostate cancer cells.

Cryopreservation of economically valuable marine micro-algae in the classes Bacillariophyceae, Chlorophyceae, Cyanophyceae, Dinophyceae, Haptophyceae, Prasinophyceae, and Rhodophyceae.

The ability to routinely cryopreserve micro-algal species reduces costs associated with maintaining large culture collections and reduces the risks of losing particular strains or species through contamination and genetic drift. Cryopreservation is also a useful adjunct in aquaculture hatcheries for strains of micro-algae where the nutritional status may change as a result of continuous sub-culture. In this study, cryopreservation of isolates from seven micro-algal classes was investigated. Successful candidates included the marine dinoflagellates Amphidinium carterae, Amphidinium trulla, and Gymnodinium simplex, and the haptophytes Chrysochromulina simplex, Prymnesium parvum, Prymnesium parvum f. patelliferum, Isochrysis galbana, and Pavlova lutheri. Also successfully cryopreserved were the planktonic diatoms Chaetoceros calcitrans, Chaetoceros muelleri, Chaetoceros sp., and the benthic Nitzschia ovalis, the chlorophyte Chlamydomonas coccoides, the rhodophyte Porphyridium purpureum, the prasinophytes Tetraselmis chuii, and Tetraselmis suecica, and the cyanophytes Raphidiopsis sp., and Aphanizomenon flos-aquae. All species were successfully cryopreserved using 15% Me2SO.

Prognostic significance of osteopontin expression in human prostate cancer.

To test the hypothesis that expression of osteopontin (OPN), an integrin-binding glycoprotein, can independently predict the potential aggressiveness of prostate cancer, the status of OPN expression in benign and malignant prostate cancer cell lines and tissues was analysed by Western blot and immunohistochemistry. Amongst the four prostate cell lines analysed, the level of OPN expressed in the benign PNT-2 cells was set at 1, the relative level of OPN expressed in the weakly malignant cell line LNCaP was increased to 1.5. In the highly malignant cell lines Du-145 and PC-3, the level of OPN expression was further increased to 2.9 and 4.4, respectively. An increased expression of OPN was also observed in the prostate tissue samples. When the level of OPN in normal tissue was set at 1, its level in benign prostate hyperplasia (BPH) was similar at 0.99 +/- 0.2, whereas the OPN level in the highly malignant carcinoma tissue was greatly increased by nearly 6-fold to 5.9 +/- 0.3. Amongst the 116 cases examined immunocytochemically, of the 10 normal cases, 3 (30%) were unstained and 7 (70%) stained weakly positive (+). Amongst the 36 BPH samples, 32 (89%) stained weakly positive (+) and 4 (11%) were unstained (-). For the 70 carcinomas analysed, 31 (44%) stained strongly positive (+++), 20 (29%) stained moderately positive (++) and 19 (27%) stained weakly positive (+). These results showed that the level of OPN expressed between the normal and the BPH samples was not significantly different (Fisher's exact test, p = 0.16). However, in comparison to that in the BPH samples, the expression of OPN in the carcinoma tissues was significantly increased (Chi-square test, p < 0.0001). Kaplan-Meier survival analysis showed that the increased level of OPN expression was significantly (n = 70, p = 0.03) associated with reduced survival time of the patients. The OPN expression was increased with the increasing Gleason scores of the carcinomas (Chi-square test, p < 0.001). The results in our study support our hypothesis and suggest that the increased OPN level may be involved in the malignant transformation of prostate epithelial cells and OPN expression level is an important determinant for patient survival.

High-level expression of cutaneous fatty acid-binding protein in prostatic carcinomas and its effect on tumorigenicity.

The expression of cutaneous fatty acid-binding protein (C-FABP) in prostate tissues was examined by immunohistochemistry. Among the 76 cases, all seven (100%) normal tissues were unstained. Of the 35 benign prostatic hyperplasia (BPH), 25 (71.4%) specimens were unstained and 10 (28.6%) were stained positively. For the 34 prostatic carcinomas, the C-FABP expression was remarkably increased: 25 (73.5%) samples stained positively, and only nine (26.5%) were unstained. Transfection of a vector expressing an antisense C-FABP transcript into the PC-3M prostatic cancer cells yielded two transfectant lines: PC-3M-CFABP-1 and PC-3M-CFABP-3, producing, respectively, a 3.8- and a 6.9-fold reduction in C-FABP levels. Comparing with the control transfectants, the in vitro invasiveness of both PC-3M-CFABP-1 and PC-3M-CFABP-3 was significantly reduced. When tested in nude mouse, the average size of tumours produced by PC-3M-CFABP-1 and by PC-3M-CFABP-3 was reduced by 2.9- and 4.2-fold respectively, in comparison with that of tumours produced by the control transfectants. Analysis showed that the decreased vascular endothelial growth factor (VEGF) and microvessel densities in the tumours were associated with the reduced C-FABP. These data show that C-FABP is increased in prostatic carcinoma cells and suppression of its expression can significantly inhibit the tumorigenicity, probably by reducing the expression of VEGF.