Zygotic exposure to venlafaxine disrupts cortisol stress axis activity in multiple generations of zebrafish.

Ubiquitous use of antidepressants has resulted in increased concentrations of these pharmaceuticals in waterways receiving municipal wastewater effluent. Amongst these, venlafaxine, a selective serotonin and norepinephrine reuptake inhibitor, is commonly found at concentrations surpassing 1 ppb in surface waters. We recently showed that the deposition of venlafaxine in zebrafish (Danio rerio) embryos impacts neural development in the hypothalamus, suggesting the possibility of neuroendocrine disruptions due to this antidepressant. Here, we tested the hypothesis that early developmental exposure to venlafaxine disrupts the long-term functioning of the hypothalamus-pituitary-interrenal (HPI) axis in zebrafish. Embryos (1-4 cell stage) were injected with either 0, 1, or 10 ng venlafaxine, and the ontogeny of cortisol content, as well as changes in cortisol levels following a stressor in larvae and adults were assessed across 3 generations. Zygotic venlafaxine exposure did not affect the ontogeny of cortisol production, but there was a disruption in the cortisol response to stressor exposure, which was also evident in multiple generations. In the F0 generation, venlafaxine exposure did not affect cortisol levels in response to stressor exposure in larvae, but adult females, and not males, showed an attenuated cortisol response compared to control fish. This reduction in cortisol levels in the females was rescued by stimulation with adrenocorticotropic hormone, suggesting that the disruption was at the level of the hypothalamus-pituitary axis. Venlafaxine-mediated disruption in HPI axis functioning was also evident in the F1 and F2 generations, including impaired cortisol responses to a stressor in adult female and larval fish, respectively. Taken together, our results suggest that venlafaxine is an endocrine disruptor, and early developmental exposure to this antidepressant may have long-term and generational consequences on cortisol stress axis activity in zebrafish.

Emerging and Historical Contaminants Detected in Desert Rodents Collected Near a Low-Level Radioactive Waste Site.

In an effort to determine contaminant presence, concentrations, and movement from a low-level radioactive waste (LLRW) burial disposal site to ecosystems in the surrounding area, a study was developed to assess concentrations of per- and polyfluoroalkyl substances (PFAS), polychlorinated biphenyls (PCBs), and tritium. To complete this assessment small mammals, vegetation, soil, and insect samples were collected from areas within and adjacent to the Beatty, Nevada, LLRW site and from a reference area located approximately 3 km south of the LLRW site. Samples underwent analysis via liquid chromatography tandem mass spectrometry, gas chromatography mass spectrometry, or scintillation spectroscopy depending on the analyte of interest. Small mammal tissues showed maximum concentrations of over 1700 ng/g for PFAS, 1600 ng/g for PCBs, and 10 000 Bq/kg for tritium. The primary contaminants found in soil samples were PCBs, with maximum concentrations exceeding 25 ng/g. Trace amounts of PFAS were also detected in soils and insects. Only qualitative data were obtained from vegetation samples because of the complex matrix of the dominant plant species (creosote bush; Larrea tridentata [Sessé & Moc. ex DC.] Coville). Overall, these data indicate the presence of various anthropogenic contaminants in the ecosystem surrounding the LLRW area, but additional analyses are necessary to confirm the sources and migration pathways of PFAS and PCBs in this hyperarid environment. Environ Toxicol Chem 2021;40:727-734. © 2020 SETAC.

Plant Uptake of Per- and Polyfluoroalkyl Acids under a Maximum Bioavailability Scenario.

Although many studies have evaluated the fate of per- and polyfluoroalkyl acids (PFAAs) in aquatic environments, few have observed their fate in terrestrial environments. It has been proposed that ingestion could be a major PFAA exposure route for humans. We determined PFAA uptake in radish, carrot, and alfalfa under a maximum bioavailability scenario. Bioconcentration factors (BCFs) were determined in the edible tissue of radish (perfluorobutanesulfonate [PFBS] = 72; perfluorohexanesulfonate [PFHxS] = 13; perfluoroheptanoate [PFHpA] = 65; perfluorooctanoate [PFOA] = 18; perfluorooctanesulfonate [PFOS] = 2.9; and perfluorononanoate [PFNA] = 9.6), carrot (PFBS = 5.9; PFHxS = 1.1; PFHpA = 29; PFOA = 3.1; PFOS = 1; and PFNA = 1.4), and alfalfa (PFBS = 107; PFHxS = 12; PFHpA = 91; PFOA = 10; PFOS = 1.4; and PFNA = 1.7). Some of these PFAA BCFs are as much as 2 orders of magnitude higher than those measured previously in plants grown in biosolid-amended soils. Environ Toxicol Chem 2019;38:2497-2502. © 2019 SETAC.

Do naked mole rats accumulate a metabolic acidosis or an oxygen debt in severe hypoxia?

In severe hypoxia, most vertebrates increase anaerobic energy production, which results in the development of a metabolic acidosis and an O2 debt that must be repaid during reoxygenation. Naked mole rats (NMRs) are among the most hypoxia-tolerant mammals, capable of drastically reducing their metabolic rate in acute hypoxia while staying active and alert. We hypothesized that a key component of remaining active is an increased reliance on anaerobic metabolism during severe hypoxia. To test this hypothesis, we exposed NMRs to progressive reductions in inspired O2 (9-3% O2) followed by reoxygenation (21% O2) and measured breathing frequency, heart rate, behavioural activity, body temperature, metabolic rate, and also metabolic substrates and pH in blood and tissues. We found that NMRs exhibit robust metabolic rate depression in acute hypoxia, accompanied by declines in all physiological and behavioural variables examined. However, blood and tissue pH were unchanged, and tissue concentrations of ATP and phosphocreatine were maintained. NMRs increased their reliance on carbohydrates in hypoxia, and glucose was mobilized from the liver to the blood. Upon reoxygenation, NMRs entered into a coma-like state for ∼15-20 min, during which metabolic rate was negligible and body temperature remained suppressed. However, an imbalance in the time taken for the rates of O2 uptake (V̇O2 ) and CO2 production (V̇CO2 ) to return to normoxic levels during reoxygenation hint at the possibility that NMRs do utilize anaerobic metabolism during hypoxia but have a tissue and/or blood buffering capacity that masks typical markers of metabolic acidosis, and that the synthesis of glucose from lactate, rather than lactate oxidation, is prioritized during recovery.

Venlafaxine in Embryos Stimulates Neurogenesis and Disrupts Larval Behavior in Zebrafish.

Venlafaxine, a widely prescribed antidepressant, is a selective serotonin and norepinephrine reuptake inhibitor in humans, and this drug is prevalent in municipal wastewater effluents. While studies have shown that this drug affects juvenile fish behavior, little is known about the developmental impact on nontarget aquatic animals. We tested the hypothesis that venlafaxine deposition in the egg, mimicking maternal transfer of this antidepressant, disrupts developmental programming using zebrafish (Danio rerio) as a model. Embryos (1-4 cell stage) were microinjected with either 1 or 10 ng venlafaxine, which led to a rapid reduction (90%) of this drug in the embryo at hatch. There was a concomitant increase in the concentration of the major metabolite o-desmethylvenlafaxine during the same period. Embryonic exposure to venlafaxine accelerated early development, increased hatching rate and produced larger larvae at 5 days post fertilization. Also, there was an increase in neuronal birth in the hypothalamus, dorsal thalamus, posterior tuberculum, and the preoptic region, and this corresponded with a higher spatial expression of nrd4, a key marker of neurogenesis. The venlafaxine-exposed larvae were less active and covered shorter distance in a light and dark behavioral test compared to the controls. Overall, zygotic exposure to venlafaxine disrupts early development, including brain function, and compromises larval behavior, suggesting impact of this drug on developmental programming in zebrafish.

Microplastics in a freshwater environment receiving treated wastewater effluent.

Small plastic fragments (microplastics or solid particles <5 mm in size or "microbeads" used in personal care products and cosmetics) may ultimately find their way into aquatic environments. We studied the presence of microplastics (particle sizes 53-105 µm and 106-179 µm) spatially and temporally in 3 connected urban lakes being fed by treated wastewater effluent in Lubbock, Texas. These lakes also serve as drainage during storm events. Water samples from drainage playa wetlands within the city were also collected. Our interest was in determining the presence or absence of microplastics in a freshwater environment as well as the source apportionment between personal care products (via wastewater) and discarded plastics (via runoff). Results showed that average concentrations of microplastics in samples collected from lakes ranged from 0.79 ± 0.88 mg/L to 1.56 ± 1.64 mg/L for the 53-105 µm size fraction and from 0.31 ± 0.72 mg/L to 1.25 ± 1.98 mg/L for the 106-179 µm size fraction. For samples collected from playa wetlands, average microplastic concentrations ranged from 0.64 ± 0.92 mg/L to 5.51 ± 9.09 mg/L for the 53-105 µm size fraction and from nondetectable (ND) to 1.79 ± 3.04 mg/L for the 106-179 µm size fraction. Our results (based on comparison of microplastic masses) suggest that urban runoff also contributes microplastics to surface water in addition to the treated wastewater effluent (in this particular case). The present findings may assist in adopting additional monitoring efforts and provide information on the potential contribution of secondary microplastic input into aquatic environments. Integr Environ Assess Manag 2017;13:528-532. © 2017 SETAC.

Experiences of Pain and Expectations for Its Treatment Among Former Buruli Ulcer Patients.

Buruli ulcer (BU) is one of the 17 neglected tropical diseases for which the World Health Organization has adopted resolutions to improve treatment. BU was previously described as a relatively painless condition; however, recent research has indicated that some patients experience substantial pain. The objective of this study was to explore patients' experiences of pain and their expectations for its treatment. Semistructured interviews were conducted in a BU-endemic region of Ghana. Interviews were held with former BU patients (N = 20) and community controls (N = 19). Former patients were asked about BU-related pain and their expectations for its treatment. The interviews were conducted in October 2014, and were audiotaped, translated and transcribed into English, and then qualitatively analyzed. Of the 20 former BU patients interviewed, 19 (95%) reported experiencing pain, with patients reporting pain as a consequence of the ulcer and wound management. Some participants expressed pain through crying, whereas others did not openly express pain, sometimes because they feared the repercussions of doing so. Patients wanted to receive pain relief; however, many were unable to name a medication. Nonpharmaceutical options were cited as being an alternative. Many BU patients experience pain; however, former patients and community members alike appear to have a limited knowledge about available pain relief. A low-cost alternative to medication may be the use of nonpharmaceutical means for pain relief. Routine pain assessment may reduce patients' fear and unwillingness to express pain. Awareness of such issues will be valuable when implementing a BU pain relief guideline.

TARGIT-R (Retrospective): North American Experience with Intraoperative Radiation Using Low-Kilovoltage X-Rays for Breast Cancer.

BACKGROUND: Single-dose intraoperative radiotherapy (IORT) is an emerging treatment for women with early stage breast cancer. The objective of this study was to define the frequency of IORT use, patient selection, and outcomes of patients treated in North America. METHODS: A multi-institutional retrospective registry was created, and 19 institutions using low-kilovoltage IORT for the treatment of breast cancer entered data on patients treated at their institution before July 31, 2013. Patient selection, IORT treatment details, complications, and recurrences were analyzed. RESULTS: From 2007 to July 31, 2013, a total of 935 women were identified and treated with lumpectomy and IORT. A total of 822 patients had at least 6 months' follow-up documented and were included in the analysis. The number of IORT cases performed increased significantly over time (p < 0.001). The median patient age was 66.8 years. Most patients had disease that was <2 cm in size (90 %) and was estrogen positive (91 %); most patients had invasive ductal cancer (68 %). Of those who had a sentinel lymph node procedure performed, 89 % had negative sentinel lymph nodes. The types of IORT performed were primary IORT in 79 %, secondary IORT in 7 %, or planned boost in 14 %. Complications were low. At a median follow-up of 23.3 months, crude in-breast recurrence was 2.3 % for all patients treated. CONCLUSIONS: IORT use for the treatment of breast cancer is significantly increasing in North America, and physicians are selecting low-risk patients for this treatment option. Low complication and local recurrence rates support IORT as a treatment option for selected women with early stage breast cancer.

The effects of strain and ploidy on the physiological responses of rainbow trout (Oncorhynchus mykiss) to pH 9.5 exposure.

We characterized the physiological effects of exposure to pH9.5 on one domesticated and four wild strains of diploid and triploid juvenile rainbow trout (Oncorhynchus mykiss) over two consecutive years. In the first year, 35-70% of the individuals from the wild strains showed a loss of equilibrium (LOE) at 12 h exposure to pH9.5, with all fish from wild strains experiencing a LOE by 48 h. In contrast, <20% of the domesticated strain showed LOE over the 48 h exposure to pH9.5. In our second experiment, similar strain effects were observed, but far fewer fish showed LOE (≤50% in all strains) over 72 h at pH9.5. In both experiments, there was no effect of ploidy on time to LOE. In the fish that did not show LOE, high pH exposure resulted in significant increases in plasma, brain and muscle ammonia, with no effect of strain or ploidy on the extent of ammonia accumulation. Glutamine accumulated in the brain during high pH exposure, with a stoichiometric decrease in glutamate, but no differences were noted among strains or ploidies. Lactate also accumulated in the plasma to a similar extent in all trout strains and ploidies. Plasma chloride decreased at 24h exposure in all trout strains and ploidies, but recovered by 72 h. No change was observed in plasma sodium. Overall, our data suggest that the domesticated strain of trout is more tolerant of pH9.5 than the wild strains, but these differences in tolerance cannot be explained by our sub-lethal assessment of ammonia balance or ion regulation.

Assessing the validity and reproducibility of genome-scale predictions.

MOTIVATION: Validation and reproducibility of results is a central and pressing issue in genomics. Several recent embarrassing incidents involving the irreproducibility of high-profile studies have illustrated the importance of this issue and the need for rigorous methods for the assessment of reproducibility. RESULTS: Here, we describe an existing statistical model that is very well suited to this problem. We explain its utility for assessing the reproducibility of validation experiments, and apply it to a genome-scale study of adenosine deaminase acting on RNA (ADAR)-mediated RNA editing in Drosophila. We also introduce a statistical method for planning validation experiments that will obtain the tightest reproducibility confidence limits, which, for a fixed total number of experiments, returns the optimal number of replicates for the study. AVAILABILITY: Downloadable software and a web service for both the analysis of data from a reproducibility study and for the optimal design of these studies is provided at http://ccmbweb.ccv.brown.edu/reproducibility.html .

Paradoxical responses after start of antimicrobial treatment in Mycobacterium ulcerans infection.

BACKGROUND: Antimicrobial killing in mycobacterial infections may be accompanied by (transient) clinical deterioration, known as paradoxical reaction. To search for patterns reflecting such reactions in the treatment of Buruli ulcer (Mycobacterium ulcerans infection), the evolution of lesions of patients treated with antimicrobials was prospectively assessed. METHODS: The lesion size of participants of the BURULICO antimicrobial trial (with lesions ≤10 cm cross-sectional diameter) was assessed by careful palpation and recorded by serial acetate sheet tracings. Patients were treated with antimicrobials for 8 weeks. For the size analysis, participants whose treatment had failed, had skin grafting, or were coinfected with human immunodeficiency virus were excluded. For every time point, surface area was compared with the previous assessment. A generalized additive mixed model was used to study lesion evolution. Nonulcerative lesions were studied using digital images recording possible subsequent ulceration. RESULTS: Of 151 participants, 134 were included in the lesion size analysis. Peak paradoxical response occurred at week 8; >30% of participants showed an increase in lesion size as compared with the previous (week 6) assessment. Seventy-five of 90 (83%) of nonulcerative lesions ulcerated after start of treatment. Nine participants developed new lesions during or after treatment. All lesions subsequently healed. CONCLUSIONS: After start of antimicrobial treatment for Buruli ulcer, new or progressive ulceration is common before healing sets in. This paradoxical response, most prominent at the end of the 8-week antimicrobial treatment, should not be misinterpreted as failure to respond to treatment. Clinical Trials Registration. ClinicalTrials.gov, NCT00321178.

Antimicrobial treatment for early, limited Mycobacterium ulcerans infection: a randomised controlled trial.

BACKGROUND: Surgical debridement was the standard treatment for Mycobacterium ulcerans infection (Buruli ulcer disease) until WHO issued provisional guidelines in 2004 recommending treatment with antimicrobial drugs (streptomycin and rifampicin) in addition to surgery. These recommendations were based on observational studies and a small pilot study with microbiological endpoints. We investigated the efficacy of two regimens of antimicrobial treatment in early-stage M ulcerans infection. METHODS: In this parallel, open-label, randomised trial undertaken in two sites in Ghana, patients were eligible for enrolment if they were aged 5 years or older and had early (duration <6 months), limited (cross-sectional diameter <10 cm), M ulcerans infection confirmed by dry-reagent-based PCR. Eligible patients were randomly assigned to receive intramuscular streptomycin (15 mg/kg once daily) and oral rifampicin (10 mg/kg once daily) for 8 weeks (8-week streptomycin group; n=76) or streptomycin and rifampicin for 4 weeks followed by rifampicin and clarithromycin (7.5 mg/kg once daily), both orally, for 4 weeks (4-week streptomycin plus 4-week clarithromycin group; n=75). Randomisation was done by computer-generated minimisation for study site and type of lesion (ulceration or no ulceration). The randomly assigned allocation was sent from a central site by cell-phone text message to the study coordinator. The primary endpoint was lesion healing at 1 year after the start of treatment without lesion recurrence or extensive surgical debridement. Analysis was by intention-to-treat. This trial is registered with ClinicalTrials.gov, number NCT00321178. FINDINGS: Four patients were lost to follow-up (8-week streptomycin, one; 4-week streptomycin plus 4-week clarithromycin, three). Since these four participants had healed lesions at their last assessment, they were included in the analysis for the primary endpoint. 73 (96%) participants in the 8-week streptomycin group and 68 (91%) in the 4-week streptomycin plus 4-week clarithromycin group had healed lesions at 1 year (odds ratio 2.49, 95% CI 0.66 to infinity; p=0.16, one-sided Fisher's exact test). No participants had lesion recurrence at 1 year. Three participants had vestibulotoxic events (8-week streptomycin, one; 4-week streptomycin plus 4-week clarithromycin, two). One participant developed an injection abscess and two participants developed an abscess close to the initial lesion, which was incised and drained (all three participants were in the 4-week streptomycin plus 4-week clarithromycin group). INTERPRETATION: Antimycobacterial treatment for M ulcerans infection is effective in early, limited disease. 4 weeks of streptomycin and rifampicin followed by 4 weeks of rifampicin and clarithromycin has similar efficacy to 8 weeks of streptomycin and rifampicin; however, the number of injections of streptomycin can be reduced by switching to oral clarithromycin after 4 weeks. FUNDING: European Union (EU FP6 2003-INCO-Dev2-015476) and Buruli Ulcer Groningen Foundation.

High-throughput binding analysis determines the binding specificity of ASF/SF2 on alternatively spliced human pre-mRNAs.

High-throughput immunoprecipitation studies of transcription factors and splicing factors have revolutionized the fields of transcription and splicing. Recent location studies on Nova1/2 and Fox2 have identified a set of cellular targets of these splicing factors. One problem with identifying binding sites for splicing factors arises from the transient role of RNA in gene expression. The primary role of most splicing factors is to bind pre-mRNA co-transcriptionally and participate in the extremely rapid process of splice site selection and catalysis. Pre-mRNA is a labile species with a steady state level that is three orders of magnitude less abundant than mRNA. As many splicing factors also bind mRNA to some degree, these substrates tend to dominate the output of location studies. Here we present an in-vitro method for screening RNA protein interactions that circumvents these problems. We screen approximately 4000 alternatively spliced exons and the entire Hepatitis C genome for binding of ASF/SF2, the only splicing factor demonstrated to function as an oncogene. From the pre-mRNA sequences returned in this screen we discovered physiologically relevant ASF recognition element motifs. ASF binds two motifs: a C-rich and a purine rich motif. Comparisons with similar data derived from the hnRNP protein PTB reveals little overlap between strong PTB and ASF/SF2 sites. We illustrate how this method could be employed to screen disease alleles with the set of small molecules that have been shown to alter splicing in search for therapies for splicing diseases.

Next-generation SELEX identifies sequence and structural determinants of splicing factor binding in human pre-mRNA sequence.

Many splicing factors interact with both mRNA and pre-mRNA. The identification of these interactions has been greatly improved by the development of in vivo cross-linking immunoprecipitation. However, the output carries a strong sampling bias in favor of RNPs that form on more abundant RNA species like mRNA. We have developed a novel in vitro approach for surveying binding on pre-mRNA, without cross-linking or sampling bias. Briefly, this approach entails specifically designed oligonucleotide pools that tile through a pre-mRNA sequence. The pool is then partitioned into bound and unbound fractions, which are quantified by a two-color microarray. We applied this approach to locating splicing factor binding sites in and around approximately 4000 exons. We also quantified the effect of secondary structure on binding. The method is validated by the finding that U1snRNP binds at the 5' splice site (5'ss) with a specificity that is nearly identical to the splice donor motif. In agreement with prior reports, we also show that U1snRNP appears to have some affinity for intronic G triplets that are proximal to the 5'ss. Both U1snRNP and the polypyrimidine tract binding protein (PTB) avoid exonic binding, and the PTB binding map shows increased enrichment at the polypyrimidine tract. For PTB, we confirm polypyrimidine specificity and are also able to identify structural determinants of PTB binding. We detect multiple binding motifs enriched in the PTB bound fraction of oligonucleotides. These motif combinations augment binding in vitro and are also enriched in the vicinity of exons that have been determined to be in vivo targets of PTB.

Automated mapping of large-scale chromatin structure in ENCODE.

MOTIVATION: A recently developed DNaseI assay has given us our first genome-wide view of chromatin structure. In addition to cataloging DNaseI hypersensitive sites, these data allows us to more completely characterize overall features of chromatin accessibility. We employed a Bayesian hierarchical change-point model (CPM), a generalization of a hidden Markov Model (HMM), to characterize tiled microarray DNaseI sensitivity data available from the ENCODE project. RESULTS: Our analysis shows that the accessibility of chromatin to cleavage by DNaseI is well described by a four state model of local segments with each state described by a continuous mixture of Gaussian variables. The CPM produces a better fit to the observed data than the HMM. The large posterior probability for the four-state CPM suggests that the data falls naturally into four classes of regions, which we call major and minor DNaseI hypersensitive sites (DHSs), regions of intermediate sensitivity, and insensitive regions. These classes agree well with a model of chromatin in which local disruptions (DHSs) are concentrated within larger domains of intermediate sensitivity, the accessibility islands. The CPM assigns 92% of the bases within the ENCODE regions to the insensitive regions. The 5.8% of the bases that are in regions of intermediate sensitivity are clearly enriched in functional elements, including genes and activating histone modifications, while the remaining 2.2% of the bases in hypersensitive regions are very strongly enriched in these elements. AVAILABILITY: The CPM software is available upon request from the authors.

The Gibbs Centroid Sampler.

The Gibbs Centroid Sampler is a software package designed for locating conserved elements in biopolymer sequences. The Gibbs Centroid Sampler reports a centroid alignment, i.e. an alignment that has the minimum total distance to the set of samples chosen from the a posteriori probability distribution of transcription factor binding-site alignments. In so doing, it garners information from the full ensemble of solutions, rather than only the single most probable point that is the target of many motif-finding algorithms, including its predecessor, the Gibbs Recursive Sampler. Centroid estimators have been shown to yield substantial improvements, in both sensitivity and positive predictive values, to the prediction of RNA secondary structure and motif finding. The Gibbs Centroid Sampler, along with interactive tutorials, an online user manual, and information on downloading the software, is available at: http://bayesweb.wadsworth.org/gibbs/gibbs.html.

A phylogenetic Gibbs sampler that yields centroid solutions for cis-regulatory site prediction.

MOTIVATION: Identification of functionally conserved regulatory elements in sequence data from closely related organisms is becoming feasible, due to the rapid growth of public sequence databases. Closely related organisms are most likely to have common regulatory motifs; however, the recent speciation of such organisms results in the high degree of correlation in their genome sequences, confounding the detection of functional elements. Additionally, alignment algorithms that use optimization techniques are limited to the detection of a single alignment that may not be representative. Comparative-genomics studies must be able to address the phylogenetic correlation in the data and efficiently explore the alignment space, in order to make specific and biologically relevant predictions. RESULTS: We describe here a Gibbs sampler that employs a full phylogenetic model and reports an ensemble centroid solution. We describe regulatory motif detection using both simulated and real data, and demonstrate that this approach achieves improved specificity, sensitivity, and positive predictive value over non-phylogenetic algorithms, and over phylogenetic algorithms that report a maximum likelihood solution. AVAILABILITY: The software is freely available at http://bayesweb.wadsworth.org/gibbs/gibbs.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

In vitro and in vivo characterization of p-amino-phenethyl-m-trifluoromethylphenyl piperazine (PAPP), a novel serotonergic agonist with anthelmintic activity against Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus colubriformis.

The neurotransmitter serotonin (5-hydroxy tryptamine or 5HT) regulates key physiological processes in nematodes such as locomotion and feeding. PAPP (p-amino-phenethyl-m-trifluoromethylphenyl piperazine) is a known agonist of the 5-HT(1Hc) receptor of the barber pole worm, Haemonchus contortus. In this study, PAPP was highly active against L3-stage larvae of H. contortus and Trichostrongylus colubriformis in an in vitro larval migration assay, with EC50 values of 9.36 and 11.8 microM, respectively, that were comparable to levamisole (10.2 microM) and superior to pyrantel (55.39 microM). When administered orally or subcutaneously to nematode infected gerbils, PAPP provided >99% efficacy against H. contortus and >98% efficacy against Teladorsagia circumcincta at 100 mg/kg, comparable to levamisole at 10 mg/kg. Drug titration revealed significant activity down to 50 mg/kg against these two species. Spectrum was limited, however, with somewhat lower efficacy (83%) in T. colubriformis infected gerbils at 100 mg/kg. Oral delivery of hydrochloride, acetate and phosphate salts of PAPP to nematode infected gerbils did not result in an increase in either potency or spectrum. The finding that PAPP exhibits significant anthelmintic activity suggests that the nematode-specific serotonergic system is a viable target for future anthelmintic discovery.

Cytokine responses to stimulation of whole blood from patients with Buruli ulcer disease in Ghana.

Buruli ulcer disease (BUD), caused by Mycobacterium ulcerans, follows an indolent course of initial progression to ulceration accompanied by extensive tissue damage. It has been suggested that healing disease stages are accompanied by a protective immune response. We hypothesized that interleukin-4 (IL-4)- or IL-10-induced downregulation of Th-1 responses plays a key role in the progression of early BUD and that healing is accompanied by an augmented Th-1 response. Gamma interferon (IFN-gamma), IL-4, and IL-10 responses were measured after in vitro stimulation with phytohemagglutinin (PHA) and tuberculin purified protein derivative (PPD) of whole blood from 39 (23 early- and 16 late-stage) BUD patients and 39 healthy control subjects in Ghana. Additionally, 30 patients with active or treated tuberculosis (TB) serving as PPD-responsive positive controls were studied. Early-stage BUD patients produced significantly lower levels of IFN and IFN-gamma/IL-4 ratios compared to late-stage BUD patients after PHA stimulation. Compared to that of controls, IFN-gamma production after tuberculin stimulation was significantly higher in late-stage but not in early-stage BUD patients (P=0.009). IL-10 and IL-4 levels did not differ between BUD patients and controls, although active TB patients had significantly higher IL-10 production levels than did treated TB patients. Multivariate analysis showed no confounding factors. In conclusion, Th-1 down regulation in early BUD appears to reverse in later stages of BUD, although an association with IL-10 or IL-4 production does not emerge from our data. Here we show differences in Th-1-type cytokine production between early- and late-stage BUD that might reflect an improved immune defense over time.