Search

Background: Pharmacological management of chronic neuropathic pain (CNP) still represents a major clinical challenge. Collective harnessing of both the scientific evidence base and clinical experience (of clinicians and patients) can play a key role in informing treatment pathways and contribute to the debate on specific treatments (e.g., cannabinoids). A group of expert clinicians (pain specialists and psychiatrists), scientists, and patient representatives convened to assess the relative benefit-safety balance of 12 pharmacological treatments, including orally administered cannabinoids/cannabis-based medicinal products, for the treatment of CNP in adults. Methods: A decision conference provided the process of creating a multicriteria decision analysis (MCDA) model, in which the group collectively scored the drugs on 17 effect criteria relevant to benefits and safety and then weighted the criteria for their clinical relevance. Findings: Cannabis-based medicinal products consisting of tetrahydrocannabinol/cannabidiol (THC/CBD), in a 1:1 ratio, achieved the highest overall score, 79 (out of 100), followed by CBD dominant at 75, then THC dominant at 72. Duloxetine and the gabapentinoids scored in the 60s, amitriptyline, tramadol, and ibuprofen in the 50s, methadone and oxycodone in the 40s, and morphine and fentanyl in the 30s. Sensitivity analyses showed that even if the pain reduction and quality-of-life scores for THC/CBD and THC are halved, their benefit-safety balances remain better than those of the noncannabinoid drugs. Interpretation: The benefit-safety profiles for cannabinoids were higher than for other commonly used medications for CNP largely because they contribute more to quality of life and have a more favorable side effect profile. The results also reflect the shortcomings of alternative pharmacological treatments with respect to safety and mitigation of neuropathic pain symptoms. Further high-quality clinical trials and systematic comprehensive capture of clinical experience with cannabinoids is warranted. These results demonstrate once again the complexity and multimodal mechanisms underlying the clinical experience and impact of chronic pain.

The link between environment, alteration in DNA methylation and cancer has been well established in humans; yet, it is under-studied in unsequenced non-model organisms. The occurrence of liver tumors in the flatfish dab collected at certain UK sampling sites exceeds 20%, yet the causative agents and the molecular mechanisms of tumor formation are not known, especially regarding the balance between epigenetic and genetic factors. Methylated DNA Immunoprecipitation (MeDIP) combined with de novo high-throughput DNA sequencing were used to investigate DNA methylation changes in dab hepatocellular adenoma tumors for the first time in an unsequenced species. Novel custom-made dab gene expression arrays were designed and used to determine the relationship between DNA methylation and gene expression. In addition, the confirmatory techniques of bisulfite sequencing PCR (BSP) and RT-PCR were applied. Genes involved in pathways related to cancer, including apoptosis, wnt/β-catenin signaling and genomic and non-genomic estrogen responses, were altered both in methylation and transcription. Global methylation was statistically significantly 1.8-fold reduced in hepatocellular adenoma and non-cancerous surrounding tissues compared with liver from non-cancer bearing dab. Based on the identified changes and chemical exposure data, our study supports the epigenetic model of cancer. We hypothesize that chronic exposure to a mixture of environmental contaminants contributes to a global hypomethylation followed by further epigenetic and genomic changes. The findings suggest a link between environment, epigenetics and cancer in fish tumors in the wild and show the utility of this methodology for studies in non-model organisms.

The sole, Solea senegalensis, is a common flatfish of Atlantic and Mediterranean waters with a high potential for aquaculture. However, its cultivation is hampered by high sensitivity to different stresses and several infectious diseases. Improving protection from pathogens and stressors is thus a key step in reaching a standardized production. Fish were exposed to lipopolysaccharide (LPS), a mimetic of bacterial infections, and copper sulphate (CuSO(4)), used in aquaculture to control algae and outbreaks of infectious diseases. We employed a European flounder cDNA microarray to determine the transcriptomic responses of Senegalese sole to these exposures. Microarray analyses showed that many genes were altered in expression following both LPS and copper treatments in comparison to vehicle controls. Gene ontology analysis highlighted copper-specific induction of genes related to cellular adhesion and cell signalling, LPS-specific induction of genes related to the immune response, and a common induction of genes related to unfolded protein binding, intracellular transport/secretion and proteasome. Additionally transcripts for glutathione-S-transferases were down-regulated by LPS, and those for digestive enzymes were down-regulated by both treatments. We selected nine changing genes for absolute quantification of transcript copy numbers by real-time RT-PCR to validate microarray differential expression and to assess inter-individual variability in individual fishes. The quantitative RT-PCR data correlated highly with the microarray results. Overall, data reported provide novel insights into the molecular pathways that could mediate the immune and heavy metal stress responses in Senegalese sole and thus might have biotechnological applications in the culture of this important fish species.

We evaluated the effects of two putative non-genotoxic hepatic carcinogens, hexabromocyclododecane (HBCD) and 17-beta oestradiol (E(2)) on global and CpG promoter DNA methylation in both primary human hepatocytes and hepatocellular carcinoma (HepG2) cells. The mRNA gene expression levels of genes involved particularly in cell cycle were also evaluated and potential correlation with DNA methylation status examined. HBCD at 0.03 and 0.3 ng/mL did not produce statistically significant differences in global genomic methylation. However, E(2) (0.1 ng/mL) significantly lowered global DNA methylation levels in HepG2 cells by approximately 65% (P

The GENIPOL cDNA microarray, comprising some 14 000 elements representing 3336 unique expressed sequence tag clusters, was developed for studies on pollutant chemical effects on hepatic gene expression in the sentinel flatfish species Platichthys flesus, the European flounder. This array derived from probes obtained by targeted cloning of xenobiotic metabolising and responsive genes, subtractive suppressive hybridization of pollutant-treated fish and subtracted normalized cDNA libraries of treated fish has been successfully utilized for diagnosing effects of prototypical pollutants, endocrine disruptors and analysis of environmental effects. In the latter context, it has been recommended by the International Council for Exploration of the Sea that the array is tested in European monitoring programmes for assessment of the biological effects of contaminants. Sequence homology is sufficiently great between P. flesus and other pleuronectid flatfish to enable satisfactory use with other species, including American winter flounder and Japanese flounder, plaice, Atlantic halibut and sole. Elucidation of the effects of pollutants requires knowledge of other affectors of gene expression, and in this context the observed effects of a bacterial infection have been studied. The ‘infection’ was experimentally applied by challenge with a vaccine containing Aeromonas salmonicida, the bacterial agent causing furunculosis in flounder and salmonids. These results show that, despite its currently limited gene coverage, application of this array is not confined to toxicology.

Grapefruit growers in the tropics require information about existing and new citrus cultivars with high productivity potential. The objective of this study was to determine the growth, yield, and fruit quality performance of seven pigmented and four white grapefruit cultivars under the dry tropic conditions of Colima, Mexico. The trees were budded on sour orange (Citrus aurantium L.) rootstock and planted at a distance of 8 × 4 m. ‘Oroblanco’ and ‘Marsh Gardner’ white-fleshed grapefruit cultivars and ‘Chandler’, a pink-fleshed pummelo, were the largest trees with the greatest height (5.0–5.6 m), canopy diameter (6.2–6.3 m), trunk diameter (21.9–23.3 cm), and canopy volume (109–123 m3). Lower height (4.3–4.8 m) and canopy volume (73–96 m3), but with similar canopy diameter to the previously mentioned cultivars, were recorded for the remaining pigmented cultivars. ‘Chandler’ pummelo and four pigmented grapefruit cultivars (‘Shambar’, ‘Rio Red’, ‘Ray Ruby’, and ‘Redblush #3’) had yearly productions of 34.8, 34.9, 34.1, 32.7, and 30.6 ton ha−1, respectively. The most productive white grapefruit cultivar was ‘Marsh Gardner’ (30.5 ton ha−1). Grapefruit cultivars having the largest fruit size showed a higher inverse relationship between fruit weight and yield than those with small fruit. Most genotypes had higher values of fruit weight, juice content, and maturity index than those required by the local market. The most promising grapefruit cultivars based on their acceptable growth, yield superior to 30 ton ha−1, and acceptable fruit color were ‘Rio Red’, ‘Shambar’, ‘Ray Ruby’, and ‘Redblush #3’.

Lysergic acid diethylamide (LSD) is a non-selective serotonin-receptor agonist that was first synthesized in 1938 and identified as (potently) psychoactive in 1943. Psychedelics have been used by indigenous cultures for millennia [1]; however, because of LSD’s unique potency and the timing of its discovery (coinciding with a period of major discovery in psychopharmacology), it is generally regarded as the quintessential contemporary psychedelic [2]. LSD has profound modulatory effects on consciousness and was used extensively in psychological research and psychiatric practice in the 1950s and 1960s [3]. In spite of this, however, there have been no modern human imaging studies of its acute effects on the brain. Here we studied the effects of LSD on intrinsic functional connectivity within the human brain using fMRI. High-level association cortices (partially overlapping with the default-mode, salience, and frontoparietal attention networks) and the thalamus showed increased global connectivity under the drug. The cortical areas showing increased global connectivity overlapped significantly with a map of serotonin 2A (5-HT2A) receptor densities (the key site of action of psychedelic drugs [4]). LSD also increased global integration by inflating the level of communication between normally distinct brain networks. The increase in global connectivity observed under LSD correlated with subjective reports of “ego dissolution.” The present results provide the first evidence that LSD selectively expands global connectivity in the brain, compromising the brain’s modular and “rich-club” organization and, simultaneously, the perceptual boundaries between the self and the environment.

The induction of hepatic cytochrome P450 1A (CYP 1A) is an important step in the response to contaminants such as polycyclic aromatic hydrocarbons (PAHs), and has been used as a biomarker of exposure in fish. Several consensus response elements have been identified, including eight potential xenobiotic response elements (XREs) in the promoter region of the European flounder cytochrome P450 1A gene. However not all of these sequences are necessarily active. To help elucidate the molecular regulation of this important gene, site directed mutagenesis and dual-luciferase reporter gene assays were employed to characterize the consensus transcription factor binding sites of the CYP 1A 5′ flanking region. Mutation of response elements situated −1103, −859, −709 and −172 bases upstream of the transcription start site reduced the induction to 2.75, 1.51, 3.25 and 3.05 fold, respectively, compared with the full-length promoter (4.0-fold induction) on exposure to the PAH 3-methylcholanthrene (3MC) (1.0 μM). These results indicate that four out of eight different XREs are functional in the control of CYP 1A in the flounder. The activity of these response elements adds to the evidence for considerable diversity in vertebrate CYP 1A regulation.

Microbial biofilms improve the quality and palatability of coarse particulate organic matter (CPOM) for freshwater invertebrates, but small-scale interactions between CPOM-associated biofilms and meiofauna are poorly understood. The aim of this work was to investigate whether the composition of biofilms attached to leaf litter influenced development (from nauplius to adult) and reproduction (embryonic developmental time and number of broods, eggs and nauplii per female) of the meiofaunal copepod Bryocamptus zschokkei. Biofilm composition was varied by allowing growth on different species of leaf (beech, Fagus sylvatica vs. oak, Quercus robur) and for different periods of time (beech leaves conditioned for 2 vs. 6 weeks). Despite significant differences in biofilm composition between oak and beech leaves (greater microbial colonisation and bacterial densities on the former), there was no significant difference in the development or reproduction of B. zschokkei fed on these biofilms. There were, however, significant differences in copepod reproduction between leaves conditioned for different times. Hatching success was significantly higher on leaves conditioned for 6 compared with 2 weeks, reflecting increased diatom densities on the former and suggesting that micro-algae may be an important food source to ovigerous females. Conversely, copepodid development was significantly slower on leaves conditioned for 6 compared with 2 weeks, reflecting the significantly lower bacterial densities on the latter; bacteria are likely to be a major component of the diet of smaller copepod life stages. These data suggest that small-scale changes in the composition of microbial biofilms can have a significant and complex influence on the life-history characteristics of B. zschokkei, and such meiofauna-detritus interactions may have important implications for the trophic dynamics of streams.

Luminex Single Antigen Beads Assay (SAB) are currently the most widely used means to identify HLA antibodies in sera from sensitized transplant candidates. Although these assays greatly enhance solid organ transplantation practices in terms of transplantig highly sensitized patients and the ability to perform virtual (electronic) crossmatch, issues concerning identifying unusual patterns or reactivity in some patients have emerged. This unusual pattern can be seen in male non-sensitized patients, patients with underlying auto-immune diseases, and normally sensitized patients. In order to investigate this pattern further, our lab utilized two primary testing platform. The One Lambda SAB Class I and Class II, was utilized as our primary testing patform. The LifeCodes SAB Class I and Class II was used as the secondary testing platform. Patterns of unusual reactivity were also examined by performing a flow cytometry crossmatch with surrogate donors. As control group, sera from 40 sensitized and non-sensitized patients were utilized to perfom the initial comparative study. Our results showed the HLA antibodies pattern of reactivity were identical between the two platforms. For patients with unusual HLA antibodies patterns, there was minimal correlation between the two platforms. False positive patterns identified by One Lambda SAB assays, were re-tested with LifeCodes SAB assays and results either in none or completely different false positive reactive patterns (see graph below). The absence of correlation between the two testing platforms in terms of false positive patterns might suggest that the patterns of reactivity might be directed to non-HLA IgG. In order to address that, we performed total IgG purification from two sera with this false positive pattern utilizing Protein A magnetic beads. SAB assay performed on purified IgG fraction eluted from the beads resulted in significant increase in the MFI values of these samples without any effect on the reactivity. In conclusion, interefence in SAB assays could be due to IgG fraction. Additional studies utilizing further blocking might be needed to eliminate this interfering patterns. Download : Download high-res image (237KB) Download : Download full-size image

The tungsten alloy of 91% tungsten, 6% nickel and 3% cobalt (WNC 91-6-3) induces rhabdomyosarcoma when implanted into a rat thigh muscle. To investigate whether this effect is species-specific human HSkMc primary muscle cells were exposed to WNC 91-6-3 particles and responses were compared with those from a rat skeletal muscle cell line (L6-C11). Toxicity was assessed by the adenylate kinase assay and microscopy, DNA damage by the Comet assay. Caspase 3 enzyme activity was measured and oligonucleotide microarrays were used for transcriptional profiling. WNC 91-6-3 particles caused toxicity in cells adjacent to the particles and also increased DNA strand breaks. Inhibition of caspase 3 by WNC 91-6-3 occurred in rat but not in human cells. In both rat and human cells, the transcriptional response to WNC 91-6-3 showed repression of transcripts encoding muscle-specific proteins with induction of glycolysis, hypoxia, stress responses and transcripts associated with DNA damage and cell death. In human cells, genes encoding metallothioneins were also induced, together with genes related to angiogenesis, dysregulation of apoptosis and proliferation consistent with pre-neoplastic changes. An alloy containing iron, WNF 97-2-1, which is non-carcinogenic in vivo in rats, did not show these transcriptional changes in vitro in either species while the corresponding cobalt-containing alloy, WNC 97-2-1 elicited similar responses to WNC 91-6-3. Tungsten alloys containing both nickel and cobalt therefore have the potential to be carcinogenic in man and in vitro assays coupled with transcriptomics can be used to identify alloys, which may lead to tumour formation, by dysregulation of biochemical processes.

Background While estrogens protect against coronary artery disease in women, it is unclear whether they influence cardiovascular function in men. The present report describes coronary vascular abnormalities and the lipoprotein profile of a male patient with estrogen insensitivity caused by a disruptive mutation in the estrogen-receptor gene. Methods and Results Stress thallium scintigraphy, echocardiography, and electron-beam computed tomography (CT) scanning of the coronary arteries and detailed lipoprotein analysis were performed. Electron-beam CT scanning of the coronary arteries showed calcium in the left anterior descending artery. Lipoprotein analysis showed relatively low levels of total (130 mg/dL), LDL (97 mg/dL), and HDL (34 mg/dL) cholesterol; apolipoprotein A-I (91.7 mg/dL;); and lipoprotein(a) (4.1 nmol/L), but normal levels of triglycerides (97 mg/dL) and pre-β-1-HDL cholesterol (61 μg/mL). Conclusions The absence of functional estrogen receptors may be a novel risk factor for coronary artery disease in men.

The role of glia as a contributing factor to motor neuron (MN) death in amyotrophic lateral sclerosis (ALS) is becoming increasingly appreciated. However, most studies implicating astrocytes have focused solely on models of ALS caused by superoxide dismutase 1 (SOD1) mutations. The goal of our study was to determine whether astrocytes contribute to wild-type MN death in the case of ALS caused by mutations in tar-DNA binding protein 43 (TDP-43). Since it is currently unknown how TDP-43 mutations cause disease, we derived astrocytes for study from both gain and loss of function mouse models of TDP-43. Astrocytes overexpressing mutant TDP-43(A315T) as well as astrocytes lacking TDP-43 were morphologically indistinguishable from wild-type astrocytes in vitro. Furthermore, astrocytes with these TDP-43 alterations did not cause the death of wild-type MNs in co-culture. To investigate the in vivo effects of TDP-43 alterations in astrocytes, glial-restricted precursors were transplanted to the wild-type rat spinal cord where they differentiated into astrocytes and interacted with host MNs. Astrocytes with TDP-43 alterations did not cause host wild-type MN damage although they were capable of engrafting and interacting with host MNs with the same efficiency as wild-type astrocytes. These data indicate that astrocytes do not adopt the same toxic phenotype as mutant SOD1 astrocytes when TDP-43 is mutated or expression levels are modified. Our study reinforces the heterogeneity in ALS disease mechanisms and highlights the potential for future screening subsets of ALS patients prior to treatment with cell type-directed therapies.

Interactions between epigenome and the environment in biology and in disease are of fundamental importance. The incidence of hepatocellular adenomas in flatfish exceeds 20% in some environments forming a unique opportunity to study environmental tumorigenesis of general relevance to cancer in humans. We report the novel finding of marked DNA methylation and metabolite concentration changes in histopathologically normal tissue distal to tumors in fish liver. A multi-"omics" discovery approach led to targeted and quantitative gene transcription analyses and metabolite analyses of hepatocellular adenomas and histologically normal liver tissue in the same fish. We discovered a remarkable and consistent global DNA hypomethylation, modification of DNA methylation and gene transcription, and disruption of one-carbon metabolism in distal tissue compared to livers of non-tumor-bearing fish. The mechanism of this disruption is linked not to depletion of S-adenosylmethionine, as is often a feature of mammalian tumors, but to a decrease in choline and elevated S-adenosylhomocysteine, a potent inhibitor of DNA methyltransferase. This novel feature of normal-appearing tissue of tumor-bearing fish helps to understand the unprecedentedly high incidence of tumors in fish sampled from the field and adds weight to the controversial epigenetic progenitor model of tumorigenesis. With further studies, the modifications may offer opportunities as biomarkers of exposure to environmental factors influencing disease.

For comparative purposes, the toxicity of cadmium, hexavalent chromium and copper to marine fish larvae (Cyprinodon variegatus) and copepods (Tisbe battagliai) has been evaluated. Toxicity to fish larvae was measured in terms of survival and growth over 7 days, whilst toxicity to copepods was assessed in terms of survival and reproduction after 8 days exposure. For fish larvae, 96 h LC50 values (based on mean measured concentrations of total metal ion) were 1.23 mg Cd/litre, 31.6 mg Cr6+/litre and >0.22 mg Cu/litre. Subchronic values (SChVs) for larval fish survival and growth after 7 days were 0.75 mg Cd/litre, 24.0 mg Cr6+/litre and 0.16 mg Cu/litre. For copepod nauplii and adults, 96 h LC50 values were as follows: 0.46 mg Cd/litre and 0.34 mg Cd/litre, respectively; 1.60 Cr6+/litre, and 5.9 mg Cr6+/litre respectively; and 0.064 mg Cu/litre and 0.088 mg Cu/litre, respectively. SChVs for naupliar survival and adult survival or reproduction after 8 days were 0.024 mg Cd/litre, 0.42 mg Cr6+/litre and 0.008 mg Cu/litre.

At selected sites around the UK, the offshore sentinel flatfish species dab Limanda limanda are found to contain elevated levels of macroscopic liver tumors. Previous proteomic and metabolomic studies have demonstrated that differences exist between tumor and non-tumor tissues; however, these differing features were not identified, and little is known about the changes at the gene expression level, or whether prognostic markers are present and can be identified. A flounder Platichthys flesus custom cDNA microarray and RT-PCR were used to investigate hepatic mRNA expression in the histologically confirmed tumors, hepatocellular adenoma (HA) and hepatocellular carcinoma (HC) from dab, and in adjacent normal tissue from the same fish. Differences in gene expression were observed between tumor and normal tissues, and between tumor types. A class-prediction approach using 50 transcripts revealed sufficient group-specific expression profiles to allow segregation of samples dependent on their tumor type or the sex of the host. Vitellogenins were found to display the greatest induction (up to 500-fold induction) in some HC tumors from female fish and in both HA and HC tumors from males. To the best of our knowledge, this is the first report of the association of vitellogenin expression with tumors of wild fish.

To determine if pacing might prevent syncope in cardioinhibitory 'Malignant Vasovagal Syndrome' (also known as 'Neurally-Mediated Bradycardia/Hypotension'), a study of dual chamber pacing during head-up 60 degrees tilt was undertaken. Paired invasive tilts were performed in 10 patients who had a history of recurrent syncope, normal routine investigations including electrophysiological study and prior tilt-induced vasovagal syncope. Vasovagal reactions of identical severity were produced by prolonged 60 degrees head-up tilt on consecutive days in seven out of 10 patients. On day 2, without pacing, seven patients had tilt-induced vasovagal reactions and six became syncopal during the reaction. On day 3, with temporary DVI pacing with rate hysteresis, seven patients had tilt-induced vasovagal reactions and 1 patient was syncopal. Syncope was aborted in the other five patients. DVI pacing significantly improved cardiac index (CI) (one +/- 0.2 to 1.6 +/- 0.3 L/min/m2, P less than 0.01) and mean arterial blood pressure (MABP) (30 +/- 11 to 48 +/- 12 mmHg, P less than 0.01) during vasovagal reactions on day 3 compared with day 2. The mean period of time that patients could tolerate in the tilted position after the onset of the tilt-induced vasovagal reaction was significantly prolonged by pacing from 0.9 +/- 1.2 to 3.2 +/- 1.6 min (P less than 0.01). Dual chamber pacing may abort syncope in 85% of patients with cardioinhibitory malignant vasovagal syndrome. Pacing may prolong consciousness sufficiently during a vasovagal reaction to allow injury to be avoided.

The three-spined stickleback (Gasterosteus aculeatus) is ideally suited to laboratory studies, while its wide distribution in the northern hemisphere gives it great potential as a sentinel organism. In the setting of a UK-wide collaboration (Fish Toxicogenomics) we have developed a microarray for transcriptomic analysis of chemical responses in populations of G. aculeatus under laboratory and field conditions. Although several EST libraries are available for this species none are from chemical-exposed fish and thus unlikely to include a full set of pollutant-responsive genes. To harvest such transcripts cDNA libraries were produced from liver of chemical-exposed mature males. Two normalised full-length libraries were generated by different methods: (1) partial subtraction of polyA+ RNA against solid-phase cDNA using magnetic bead technology; (2) degradation of double stranded cDNA formed by abundant transcripts. To enrich for pollutant-responsive genes a subtracted EST library was also generated. For each library approximately 1.5K clones were sequenced and characterised using Blast2GO. All libraries contained pollutant-responsive transcripts not previously available while additionally the subtracted library was generally enriched approximately 1.2-10-fold for transcripts expected to be induced in response to the pollutants.

Structure-activity relationships have been investigated for inhibition of DNA-dependent protein kinase (DNA-PK) and ATM kinase by a series of pyran-2-ones, pyran-4-ones, thiopyran-4-ones, and pyridin-4-ones. A wide range of IC50 values were observed for pyranones and thiopyranones substituted at the 6-position, with the 3- and 5-positions proving intolerant to substitution. Related pyran-2-ones, pyran-4-ones, and thiopyran-4-ones showed similar IC50 values against DNA-PK, whereas the pyridin-4-one system proved, in general, ineffective at inhibiting DNA-PK. Extended libraries exploring the 6-position of 2-morpholino-pyran-4-ones and 2-morpholino-thiopyrano-4-ones identified the first highly potent and selective ATM inhibitor 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (151C; ATM; IC50=13 nM) and revealed constrained SARs for ATM inhibition compared with DNA-PK. One of the most potent DNA-PK inhibitors identified, 2-(4-methoxyphenyl)-6-(morpholin-4-yl)pyran-4-one (16; DNA-PK; IC50=220 nM) effectively sensitized HeLa cells to the topoisomerase II inhibitor etoposide in vitro.

Using an available cross-species cDNA microarray is advantageous for examining multigene expression patterns in non-model organisms, saving the need for construction of species-specific arrays. The aim of the present study was to estimate relative efficiency of cross-species hybridizations across bony fishes, using bioinformatics tools. The methodology may serve also as a model for similar evaluations in other taxa. The theoretical evaluation was done by substituting comparative whole-transcriptome sequence similarity information into the thermodynamic hybridization equation. Complementary DNA sequence assemblages of nine fish species belonging to common families or suborders and distributed across the bony fish taxonomic branch were selected for transcriptome-wise comparisons. Actual cross-species hybridizations among fish of different taxonomic distances were used to validate and eventually to calibrate the theoretically computed relative efficiencies.

Biomarker: CYP 1A gene expression. Exposure/effect represented: 3-methylcholanthrene (3MC) / induction of CYP 1A gene expression. Tissue/biological material/sample size: PLHC-1 cells (Poeciliopsis lucida) Method of analysis: CYP 1A induction was measured as the normalized firefly to Renilla luciferase ratio. The firefly and Renilla luciferase activity was measured using a luminometer (Tecan, Weymouth, UK) and the Dual-Luciferase Assay kit (Promega). Firefly luciferase expression was normalized to that of Renilla, and data were expressed as mean fold induction over DMSO controls. The DNA-binding abilities of the wild-type and mutated MRE fragments were compared using the gel shift assayImpact on outcome (including dose-response): Treatment with the prototypical PAH 3-methylcholanthrene (3MC) (1.0 microM) produced a dose-dependent response at the CYP 1A promoter, whereas treatment with cadmium (0-1.0 microM) produced little transcriptional activity at either the wild-type or mutated promoter. Cotreatment with cadmium (1.0 microM) and 3MC (1.0 microM) reduced induction at this promoter to 1.83-fold compared to 3MC treatment alone (4.0-fold induction). Mutation of the MRE site resulted in abolishment of this cadmium-related loss of 3MC-dependent activity. Furthermore, a retarded band was observed in the EMSA when the MRE was used as a probe and incubated with liver nuclear protein from flounder treated with cadmium. KEYWORDS - CLASIFFICATION: analysis;Animals;Aryl Hydrocarbon Hydroxylases;Biological Markers;biomarkers of exposure & effect: method development & validation;biosynthesis;Cadmium;Cell Line,Tumor;Cyprinidae;drug effects;Environmental Monitoring;enzymology;Flounder;Gene Expression Regulation,Enzymologic;Genes,Reporter;genetics;hydrocarbons;Liver;method development & validation;methods;Methylcholanthrene;Mutagenesis,Site-Directed;Mutation;Promoter Regions (Genetics);Research;Response Elements;toxicity;Transfection;validation;Water;Water Pollutants;Water Pollutants,Chemical; Cytochrome P450 1A (CYP 1A) is a member of a multigene family of xenobiotic metabolizing enzymes. CYP 1A is highly inducible by numerous environmental contaminants including polycyclic aromatic hydrocarbons (PAHs) and is widely used in biomonitoring studies. Therefore, understanding the regulation of this gene is important for accurate interpretation of biomarker data. We describe here the functional role of a metal response element (MRE) in the European flounder CYP 1A promoter region. To help elucidate the potential role of this MRE, reporter gene constructs, with or without site-directed mutagenesis, were used in conjunction with a dual-luciferase assay. The electrophoretic mobility shift assay (EMSA) was also used to investigate potential protein binding at this MRE site. Treatment with the prototypical PAH 3-methylcholanthrene (3MC) (1.0 microM) produced a dose-dependent response at the CYP 1A promoter, whereas treatment with cadmium (0-1.0 microM) produced little transcriptional activity at either the wild-type or mutated promoter. Cotreatment with cadmium (1.0 microM) and 3MC (1.0 microM) reduced induction at this promoter to 1.83-fold compared to 3MC treatment alone (4.0-fold induction). Mutation of the MRE site resulted in abolishment of this cadmium-related loss of 3MC-dependent activity. Furthermore, a retarded band was observed in the EMSA when the MRE was used as a probe and incubated with liver nuclear protein from flounder treated with cadmium. The results not only add to knowledge of the diversity in vertebrate CYP 1A regulation but also raise the complexity of interpretation of CYP 1A induction in monitoring studies that involve mixtures of PAHs and metals.

Forecasting the effects of stressors on the dynamics of natural populations requires assessment of the joint effects of a stressor and population density on the population response. The effects can be depicted as a contour map in which the population response, here assessed by population growth rate, varies with stress and density in the same way that the height of land above sea level varies with latitude and longitude. We present the first complete map of this type using as our model Folsomia candida exposed to five different concentrations of the widespread anthelmintic veterinary medicine ivermectin in replicated microcosm experiments lasting 49 days. The concentrations of ivermectin in yeast were 0.0, 6.8, 28.8, 66.4, and 210.0 mg/L wet weight. Increasing density and chemical concentration both significantly reduced the population growth rate of Folsomia candida, in part through effects on food consumption and fecundity. The interaction between density and ivermectin concentration was "less-than-additive," implying that at high density populations were able to compensate for the effects of the chemical. This result demonstrates that regulatory protocols carried out at low density (as in most past experiments) may seriously overestimate effects in the field, where densities are locally high and populations are resource limited (e.g., in feces of livestock treated with ivermectin).

Background— Inflammation after myocardial infarction (MI) heralds worse left ventricular (LV) function and clinical outcomes. However, whether inflammation affects LV function by extending myonecrosis and/or altering LV remodeling remains unknown. We hypothesized that cytotoxic aldehydes generated during oxidative stress may adversely affect remodeling and infarct size. One theoretical source of reactive aldehydes is oxidation of common α-amino acids by myeloperoxidase (MPO) released by leukocytes. However, a role for MPO in formation of aldehydes in vivo and the functional consequences of MPO-generated oxidants in ischemia/reperfusion models of MI have not been established. Methods and Results— In studies with cell types found in vascular tissue, MPO-oxidation products of glycine (formaldehyde) and threonine (acrolein) were the most cytotoxic. Mass spectrometry studies of myocardial tissue from murine models of acute MI (both chronic left anterior descending coronary artery ligation and ischemia/reperfusion injury) confirmed that MPO serves as a major enzymatic source in the generation of these cytotoxic aldehydes. Interestingly, although MPO-null mice experienced 35.1% ( P P Conclusions— The present data separate inflammatory effects on infarct size and LV remodeling and demonstrate that MPO-generated oxidants do not significantly affect tissue necrosis after MI but rather have a profound adverse effect on LV remodeling and function.

Concensus primers designed to CYP1A-conserved regions were used to amplify a 1.3 kb probe from flounder genomic DNA via polymerase chain reaction (PCR). A 14-kb clone was isolated from a flounder genomic library constructed in λFIXII. Of this clone, 8 kb was sequenced, including 3 kb of upstream sequence. The predicted amino acid sequence showed closest similarity to plaice CYP1A1 (98%). Gene structure conformed to the seven exons and six introns common to previous CYP1A sequences, but intron lengths were not conserved. Concensus sequences corresponding to xenobiotic and other response elements as well as TATA, CAAT and GC boxes were identified. Upstream sequence (3.5 kb) including the first exon and intron up to the putative start codon were amplified via PCR and inserted upstream of the luciferase gene in a pGL3 reporter gene construct. The HepG2 mammalian hepatoma cell line was transiently co-transfected with the flounder CYP1A reporter gene construct and the pRL-CMV internal control construct. The maximal induction upon exposure to 100nM 3-MC was 4.4-fold in comparison with carrier-treated cells. Use of deletion constructs resulted in loss of inducibility.

This study examined the potential utility of monitoring right ventricular dP/dt as a means of detecting imminent vasovagal syncope. To assess this possibility, continuous right ventricular pressure measurements were recorded in nine patients during induction of syncope by head-up tilt testing. Findings indicated that arterial pressure tended to fall prior to drop in heart rate. RV pressure exhibited a significant, but very small, fall during tilt. DP/dt max increased by 15-20% during tilt, then fell by a median of 30% from maximum value beginning about 2 minutes prior to syncope. Thus, further investigation of dP/dt as a potential marker of impending vasovagal syncope is warranted.

Aim Analysis of the single antigen bead (SAB) assay is frequently complicated by the presence of recurrent background patterns (BP), likely due to non-HLA antibodies cross-reacting with cryptic epitopes on denatured HLA molecules. We investigated whether this BP are less frequently detected by an assay with reduced amount of denatured antigens (iBeads). Methods Ninety-six serum samples from The University of Michigan Transplant Center tested between 4/2012 and 4/2013 were analyzed comparing iBeads with SAB. iBeads was considered successful when the BP present in SAB was absent in this assay. Individual pattern frequency was examined as well. Results Side-by-side comparison revealed that for 60% (n=58) of the samples, iBeads did not detect the non-specific BP shown by SAB assay. The same BP were encountered with both assays in 34% (n=33) of the samples, and were partially reduced in 6% (n=5) in iBeads. Certain BP were frequently undetected by iBeads. Among them, the Broad Cw pattern was the most frequent pattern seen by SAB (n=19) and undetected by iBeads in 95% (n=18). The BP A1/36, A68 (A∗68:02), and B8 were not detected by iBeads in 100% (n=7), whereas the pattern involving B45 was still found on iBeads. Other commonly detected patterns, such as B44, B76, B82, Cw1,12,15, Cw17 and others, were detected in some samples by iBeads and not in others.[Fig. 1] Conclusions iBeads assay performance appears to be better than the SAB assay on samples with non-HLA antibodies cross-reacting with cryptic epitopes of denatured HLA molecules. Persistent BP may be in relation to the concentration of interference or other samples characteristics as yet unknown. [figure1]

The use of khat ( Catha edulis) has been associated with a large number of physiological and societal harms, leading to calls for it to be controlled in the UK. The evidence of these harms is often equivocal, limited by confounding factors, or entirely anecdotal: high-powered, quality-controlled studies are lacking. Regardless, the body of relevant literature indicates that the once socially-regulated use of khat has been eroded. Some individuals have developed excessive consumption patterns, either using khat daily or in binge-sessions, though daily consumption is not necessarily problematic per se. The majority of users seem to use khat in moderation, where the associated harms appear low. For excessive users, harms associated with khat are greater, particularly relating to mental health. Social harms also seem to be largely related to excessive khat use rather than khat use itself. Even in cases of excessive khat use, however, causal relationships between chewing and harms have not been described. More research is required to establish the role of khat in liver disease, coronary problems, cancers of the digestive tract and incidents of domestic violence. Studies should consider the likeliness that certain users are more vulnerable to developing patterns of excessive khat use due to an interwoven set of factors such as social health determinants and pre- and post-migration experiences.

Aim We have previously reported the development of very high resolution (VHR) HLA genotyping, designed to resolve the majority of both the “common and well documented alleles” and the null alleles using the 454 GS FLX Sequencing System and Conexio genotyping software. Amplicon library construction was performed using 22 PCR fusion primers, containing genome target specific sequence with 454 sequencing adaptors. The VHR assay performed well but required extensive handling of individual amplicons. To simplify the workflow, we prepared the amplicon libraries by three alternative procedures. Methods PCR amplicons were generated from 10 cell lines for: HLA-A/B exons 1-5, HLA-C exons 1-7 and some intronic class I regions; DPA1, DPB1, DQA1 exon 2; and DQB1, DRB exons 2 and 3. Amplification required 22 pairs of fusion primers (14 primer pairs in the GS GType HLA primers plates from 454, plus 8 amplicons in a third plate) with 10 Multiplex Identifiers (MIDs), or 22 primer pairs each containing, at the 5′end, a universal sequence which matched the 3′ end of a set of primers that contained 10 MIDs and the 454 sequencing adaptors. Amplicons derived from fusion primers were purified, quantified, diluted to a standard concentration, then pooled OR pooled prior to the other operations. Amplicons generated by the four primer system on the Fluidigm Access Array™ were handled by the latter procedure. Pools of amplicons were sequenced on the GS FLX and genotyped with Conexio software. Results All three procedures resulted in comparable genotyping (concordance up to >98%). Pooling amplicons immediately following the genomic PCR gave a reduction in the number of pipetting steps necessary to produce an amplicon library by ∼80% and ∼90% using fusion primers and the four primer system, respectively. Conclusions Pooling amplicons early in the 454 library preparation procedure greatly simplifies the workflow of the 22 primer VHR HLA genotyping assay for fusion primers as well as the 4 primer system. Holcomb: Roche: Employee. Hoglund: Roche: Employee. Williams: Roche: Grant Research. Erlich: Roche: Employee.

Aim Genotyping of DRB1 and DRB3/4/5 is performed in our lab using generic DRB PCR primers, followed by sequencing on the 454 GS FLX and use of Conexio Genomics software. During genotyping homozygous cell lines, we detected additional sequences at about 7% of the read depth of the reported true allele. These artifacts appeared to be PCR crossovers between the DRB1 and DRB3/4/5 sequences. The crossovers usually had one or more mismatches with the IMGT database but occasionally matched a very rare allele (e.g. DRB1 ∗ 03:42). We performed a study to determine if these sequences were in vitro artifacts and to determine if some rare DRB alleles in the IMGT database might, in fact, be in vitro artifacts that had been submitted. Methods To test for in vitro artifact formation during late cycles of genomic PCR, we amplified the DRB loci from 21 homozygous cells lines under both our standard PCR conditions (35 cycles) and a reduced number of cycles (28), followed by 454 sequencing. Using the same PCR conditions, we also sequenced the DRB loci of four samples, each of which had previously been identified as having a novel DRB allele that was subsequently submitted to the IMGT database. Results For all homozygous 21 cell lines, amplification for 35 cycles gave low abundance sequences as possible “second alleles.” In 36% of the cases, these artifactual sequences corresponded to named alleles. Such sequences were not detected with amplification for 28 cycles. In the case of 4 samples with rare alleles previously submitted to the IMGT, we found that 3 were true alleles. The fourth, while not a crossover product, contained a sequencing error which was revealed by our 454 HLA genotyping assay. Conclusions Formation of in vitro crossover products of the DRB loci can occur in late cycles of PCR. PCR conditions have been identified that minimize generation of these in vitro artifacts. Clonal sequencing on the 454 GS FLX is a valuable method for revealing both these artifacts and traditional sequencing errors.

Aim The contribution of MICA antibodies to AMR and poor transplant outcomes have been described by multiple reports. In order to implement the MICA antibody assay screening in our transplant algorithm we evaluated the MICA sensitization status in our patient population. Methods We collected MICA antibody results from a group of 205 previously transplanted patients: 168 kidney transplants, 28 heart recipients and 9 lungs recipients. As a control we collected MICA antibody results from a group of 100 patients waiting for the first renal transplant. Antibody MICA detection was initially screened with LABScreen ® Mixed (One Lambda), and positive results were confirmed by MICA LABScreen ® singe antigen assay (One Lambda). Results As expected, the frequency of anti MICA antibody was higher in the previously transplanted patients when compared with the control group. We found anti-MICA in 21 (10.2%) transplanted patients vs. 3 (3%) in the control group. When we divided the transplanted group by organ type, we found anti-MICA antibodies in 10.1% of the kidney-transplanted patients, 14% of the heart-transplanted patients and 0% of the lung-transplanted patients. See Table 1 . Conclusions The frequency of anti-MICA antibodies was considerably higher in the group of previously transplanted patients. MICA typing information and Anti-MICA antibody results will help in the selection and management of patients receiving a kidney from a living donor, and improve the post-transplant management of patients receiving organs from deceased donors.

Cellular abnormalities are not limited to motor neurons in amyotrophic lateral sclerosis (ALS). There are numerous observations of astrocyte dysfunction in both humans with ALS and in SOD1(G93A) rodents, a widely studied ALS model. The present study therapeutically targeted astrocyte replacement in this model via transplantation of human Glial-Restricted Progenitors (hGRPs), lineage-restricted progenitors derived from human fetal neural tissue. Our previous findings demonstrated that transplantation of rodent-derived GRPs into cervical spinal cord ventral gray matter (in order to target therapy to diaphragmatic function) resulted in therapeutic efficacy in the SOD1(G93A) rat. Those findings demonstrated the feasibility and efficacy of transplantation-based astrocyte replacement for ALS, and also show that targeted multi-segmental cell delivery to cervical spinal cord is a promising therapeutic strategy, particularly because of its relevance to addressing respiratory compromise associated with ALS. The present study investigated the safety and in vivo survival, distribution, differentiation, and potential efficacy of hGRPs in the SOD1(G93A) mouse. hGRP transplants robustly survived and migrated in both gray and white matter and differentiated into astrocytes in SOD1(G93A) mice spinal cord, despite ongoing disease progression. However, cervical spinal cord transplants did not result in motor neuron protection or any therapeutic benefits on functional outcome measures. This study provides an in vivo characterization of this glial progenitor cell and provides a foundation for understanding their capacity for survival, integration within host tissues, differentiation into glial subtypes, migration, and lack of toxicity or tumor formation.

The USDA-ARS National Clonal Germplasm Repository for Citrus is establishing a virus-free clonal collection of 830 citrus germplasm accessions at its Riverside, California facility with the objective of reducing genetic vulnerability of citrus and providing virus-free clonal germplasm to researchers worldwide. Accessions in the collection are evaluated for trueness-to-type using leaf isozymes and are characterized for 58 descriptors. Research is expanding into RFLPs for development of a usable genomic map and cultivar-specific probes. Citrus seed, pollen and buds are being investigated for extended preservation under low temperatures and cryostorage. Preliminary data is promising but indicates considerable variability between cultivars. In-vitro culture of excised embryo tissue is being investigated. A comprehensive pathogen detection program is underway. Infected accessions receive shoot-tip micrografting and thermotherapy treatments to eliminate pathogens. Accessions are maintained as potted trees in greenhouse or aphid-proof screenhouses. A complete computer based record is maintained for each accession on site and in the USDA-ARS GRIN database.

Summary results of laboratory investigations into potential chronic effects of industrial effiuent discharges are presented and discussed. The sheepshead minnow (Cyprinodon variegatus) and the benthic copepod (Tisbe battagliai) were selected as test species. Toxicity tests were conducted on newly hatched (approximately 24 hours old) sheepshead minnow larvae. Survival and growth (as dry weight) effects were measured over 7 days. Two different stages of the copepod life cycle were tested: effects on adult female survival and reproduction were measured over 9 days, and naupliar survival over 7 days. The results were incorporated into an existing monitoring programme of the effiuent disposal area in the North Sea. Predicted effiuent dilutions in the disposal area would exceed one million times within 8 hours. This dilution is 18 and 100 times greater than the 7 day lowest no observed effect concentration (NOEC) for copepod and sheep shead minnow respectively.