Ecological divergence and hybridization of Neotropical Leishmania parasites.

The tropical Andes are an important natural laboratory to understand speciation in many taxa. Here we examined the evolutionary history of parasites of the Leishmania braziliensis species complex based on whole-genome sequencing of 67 isolates from 47 localities in Peru. We first show the origin of Andean Leishmania as a clade of near-clonal lineages that diverged from admixed Amazonian ancestors, accompanied by a significant reduction in genome diversity and large structural variations implicated in host-parasite interactions. Within the Andean species, patterns of population structure were strongly associated with biogeographical origin. Molecular clock and ecological niche modeling suggested that the history of diversification of the Andean lineages is limited to the Late Pleistocene and intimately associated with habitat contractions driven by climate change. These results suggest that changes in forestation over the past 150,000 y have influenced speciation and diversity of these Neotropical parasites. Second, genome-scale analyses provided evidence of meiotic-like recombination between Andean and Amazonian Leishmania species, resulting in full-genome hybrids. The mitochondrial genome of these hybrids consisted of homogeneous uniparental maxicircles, but minicircles originated from both parental species. We further show that mitochondrial minicircles-but not maxicircles-show a similar evolutionary pattern to the nuclear genome, suggesting that compatibility between nuclear-encoded mitochondrial genes and minicircle-encoded guide RNA genes is essential to maintain efficient respiration. By comparing full nuclear and mitochondrial genome ancestries, our data expand our appreciation on the genetic consequences of diversification and hybridization in parasitic protozoa.

Chloride intracellular channel 4 (CLIC4) expression profile in the mouse medial prefrontal cortex and its regulation by ethanol.

BACKGROUND: Chloride intracellular channel 4 (CLIC4) is a multifunctional metamorphic protein for which a growing body of evidence supports a major role in the brain's molecular and behavioral responses to ethanol (EtOH). Although key to understanding the functional biology underlying this role, little is known about the cellular and subcellular expression patterns of CLIC4 in brain and how they are affected by EtOH. METHODS: We used qRT-PCR to assess Clic4 mRNA expression in the medial prefrontal cortex (mPFC) of C57BL/6J mice in the absence and presence of acute EtOH exposure. Two complementary immunohistochemical techniques were employed to assess the subcellular localization of the CLIC4 protein and its pattern of expression across brain cell types in the mPFC in the absence and presence of acute EtOH. RESULTS: Through immunohistochemical and stereological techniques, we show that CLIC4 protein is robustly expressed by oligodendrocytes (most abundant), microglia, and astrocytes, with minimal expression in neurons. Following acute EtOH exposure, we observed a rapid increase in Clic4 mRNA expression in female but not male mice and an overall increase in the number of oligodendrocytes and astrocytes expressing the CLIC4 protein. CONCLUSIONS: These findings suggest that Clic4 functions as an early response gene for acute EtOH in brain, which likely underlies its ability to modulate EtOH behavior. Our results also suggest that the role of CLIC4 in the brain's response to EtOH is mediated through oligodendrocytes.

GCSscore: an R package for differential gene expression analysis in Affymetrix/Thermo-Fisher whole transcriptome microarrays.

BACKGROUND: Despite the increasing use of RNAseq for transcriptome analysis, microarrays remain a widely-used methodology for genomic studies. The latest generation of Affymetrix/Thermo-Fisher microarrays, the ClariomD/XTA and ClariomS array, provide a sensitive and facile method for complex transcriptome expression analysis. However, existing methods of analysis for these high-density arrays do not leverage the statistical power contained in having multiple oligonucleotides representing each gene/exon, but rather summarize probes into a single expression value. We previously developed a methodology, the Sscore algorithm, for probe-level identification of differentially expressed genes (DEGs) between treatment and control samples with oligonucleotide microarrays. The Sscore algorithm was validated for sensitive detection of DEGs by comparison with existing methods. However, the prior version of the Sscore algorithm and a R-based implementation software, sscore, do not function with the latest generations of Affymetrix/Fisher microarrays due to changes in microarray design that eliminated probes previously used for estimation of non-specific binding. RESULTS: Here we describe the GCSscore algorithm, which utilizes the GC-content of a given oligonucleotide probe to estimate non-specific binding using antigenomic background probes found on new generations of arrays. We implemented this algorithm in an improved GCSscore R package for analysis of modern oligonucleotide microarrays. GCSscore has multiple methods for grouping individual probes on the ClariomD/XTA chips, providing the user with differential expression analysis at the gene-level and the exon-level. By utilizing the direct probe-level intensities, the GCSscore algorithm was able to detect DEGs under stringent statistical criteria for all Clariom-based arrays. We demonstrate that for older 3'-IVT arrays, GCSscore produced very similar differential gene expression analysis results compared to the original Sscore method. However, GCSscore functioned well for both the ClariomS and ClariomD/XTA newer microarrays and outperformed existing analysis approaches insofar as the number of DEGs and cognate biological functions identified. This was particularly striking for analysis of the highly complex ClariomD/XTA based arrays. CONCLUSIONS: The GCSscore package represents a powerful new application for analysis of the newest generation of oligonucleotide microarrays such as the ClariomS and ClariomD/XTA arrays produced by Affymetrix/Fisher.

Is Levodopa Response a Valid Indicator of Parkinson's Disease?

BACKGROUND: The clinical diagnosis of Parkinson's disease (PD) requires the presence of parkinsonism and supportive criteria that include a clear and dramatic beneficial response to dopaminergic therapy. Our aim was to test the diagnostic criterion of dopaminergic response by evaluating its association with pathologically confirmed diagnoses in a large population of parkinsonian patients. METHODS: We reviewed clinical data maintained in an electronic medical record from all patients with autopsy data who had been seen in the Movement Disorders Center at Washington University, St. Louis, between 1996 and 2018. All patients with parkinsonism who underwent postmortem neuropathologic examination were included in this analysis. RESULTS: There were 257 unique parkinsonian patients with autopsy-based diagnoses who had received dopaminergic therapy. Marked or moderate response to dopaminergic therapy occurred in 91.2% (166/182) of those with autopsy-confirmed PD, 52.0% (13/25) of those with autopsy-confirmed multiple systems atrophy, 44.4% (8/18) of those with autopsy-confirmed progressive supranuclear palsy, and 1 (1/8) with autopsy-confirmed corticobasal degeneration. Other diagnoses were responsible for the remaining 24 individuals, 9 of whom had a moderate response to dopaminergic therapy. CONCLUSION: A substantial response to dopaminergic therapy is frequent but not universal in PD. An absent response does not exclude PD. In other neurodegenerative disorders associated with parkinsonism, a prominent response may also be evident, but this occurs less frequently than in PD. © 2020 International Parkinson and Movement Disorder Society.

Phase determination in dual phase steels via HREBSD-based tetragonality mapping.

Electron Backscatter Diffraction (EBSD) is a widely used approach for characterising the microstructure of various materials. However, it is difficult to accurately distinguish similar (body centred cubic and body centred tetragonal, with small tetragonality) phases in steels using standard EBSD software. One method to tackle the problem of phase distinction is to measure the tetragonality of the phases, which can be done using simulated patterns and cross-correlation techniques to detect distortion away from a perfectly cubic crystal lattice. However, small errors in the determination of microscope geometry (the so-called pattern or projection centre) can cause significant errors in tetragonality measurement and lead to erroneous results. This paper utilises a new approach for accurate pattern centre determination via a strain minimisation routine across a large number of grains in dual phase steels. Tetragonality maps are then produced and used to identify phase and estimate local carbon content. The technique is implemented using both kinetically simulated and dynamically simulated patterns to determine their relative accuracy. Tetragonality maps, and subsequent phase maps, based on dynamically simulated patterns in a point-by-point and grain average comparison are found to consistently produce more precise and accurate results, with close to 90% accuracy for grain phase identification, when compared with an image-quality identification method. The error in tetragonality measurements appears to be of the order of 1%, thus producing a commensurate ∼0.2% error in carbon content estimation. Such an error makes the technique unsuitable for estimation of total carbon content of most commercial steels, which often have carbon levels below 0.1%. However, even in the DP steel for this study (0.1 wt.% carbon) it can be used to map carbon in regions with higher accumulation (such as in martensite with nonhomogeneous carbon content). LAY DESCRIPTION: Electron Backscatter Diffraction (EBSD) is a widely used approach for characterising the microstructure of various materials. However, it is difficult to accurately distinguish similar (BCC and BCT) phases in steels using standard EBSD software due to the small difference in crystal structure. One method to tackle the problem of phase distinction is to measure the tetragonality, or apparent 'strain' in the crystal lattice, of the phases. This can be done by comparing experimental EBSD patterns with simulated patterns via cross-correlation techniques, to detect distortion away from a perfectly cubic crystal lattice. However, small errors in the determination of microscope geometry (the so-called pattern or projection centre) can cause significant errors in tetragonality measurement and lead to erroneous results. This paper utilises a new approach for accurate pattern centre determination via a strain minimisation routine across a large number of grains in dual phase steels. Tetragonality maps are then produced and used to identify phase and estimate local carbon content. The technique is implemented using both simple kinetically simulated and more complex dynamically simulated patterns to determine their relative accuracy. Tetragonality maps, and subsequent phase maps, based on dynamically simulated patterns in a point-by-point and grain average comparison are found to consistently produce more precise and accurate results, with close to 90% accuracy for grain phase identification, when compared with an image-quality identification method. The error in tetragonality measurements appears to be of the order of 1%, thus producing a commensurate error in carbon content estimation. Such an error makes an estimate of total carbon content particularly unsuitable for low carbon steels; although maps of local carbon content may still be revealing. Application of the method developed in this paper will lead to better understanding of the complex microstructures of steels, and the potential to design microstructures that deliver higher strength and ductility for common applications, such as vehicle components.

Identifying a novel biological mechanism for alcohol addiction associated with circRNA networks acting as potential miRNA sponges.

Our lab and others have shown that chronic alcohol use leads to gene and miRNA expression changes across the mesocorticolimbic (MCL) system. Circular RNAs (circRNAs) are noncoding RNAs that form closed-loop structures and are reported to alter gene expression through miRNA sequestration, thus providing a potentially novel neurobiological mechanism for the development of alcohol dependence (AD). Genome-wide expression of circRNA was assessed in the nucleus accumbens (NAc) from 32 AD-matched cases/controls. Significant circRNAs (unadj. p ≤ 0.05) were identified via regression and clustered in circRNA networks via weighted gene co-expression network analysis (WGCNA). CircRNA interactions with previously generated mRNA and miRNA were detected via correlation and bioinformatic analyses. Significant circRNAs (N = 542) clustered in nine significant AD modules (FWER p ≤ 0.05), within which we identified 137 circRNA hubs. We detected 23 significant circRNA-miRNA-mRNA interactions (FDR ≤ 0.10). Among these, circRNA-406742 and miR-1200 significantly interact with the highest number of mRNA, including genes associated with neuronal functioning and alcohol addiction (HRAS, PRKCB, HOMER1, and PCLO). Finally, we integrate genotypic information that revealed 96 significant circRNA expression quantitative trait loci (eQTLs) (unadj. p ≤ 0.002) that showed significant enrichment within recent alcohol use disorder (AUD) and smoking genome-wide association study (GWAS). To our knowledge, this is the first study to examine the role of circRNA in the neuropathology of AD. We show that circRNAs impact mRNA expression by interacting with miRNA in the NAc of AD subjects. More importantly, we provide indirect evidence for the clinical importance of circRNA in the development of AUD by detecting a significant enrichment of our circRNA eQTLs among GWAS of substance abuse.

Assessing the Role of Long Noncoding RNA in Nucleus Accumbens in Subjects With Alcohol Dependence.

BACKGROUND: Long noncoding RNA (lncRNA) have been implicated in the etiology of alcohol use. Since lncRNA provide another layer of complexity to the transcriptome, assessing their expression in the brain is the first critical step toward understanding lncRNA functions in alcohol use and addiction. Thus, we sought to profile lncRNA expression in the nucleus accumbens (NAc) in a large postmortem alcohol brain sample. METHODS: LncRNA and protein-coding gene (PCG) expressions in the NAc from 41 subjects with alcohol dependence (AD) and 41 controls were assessed via a regression model. Weighted gene coexpression network analysis was used to identify lncRNA and PCG networks (i.e., modules) significantly correlated with AD. Within the significant modules, key network genes (i.e., hubs) were also identified. The lncRNA and PCG hubs were correlated via Pearson correlations to elucidate the potential biological functions of lncRNA. The lncRNA and PCG hubs were further integrated with GWAS data to identify expression quantitative trait loci (eQTL). RESULTS: At Bonferroni adj. p-value ≤ 0.05, we identified 19 lncRNA and 5 PCG significant modules, which were enriched for neuronal and immune-related processes. In these modules, we further identified 86 and 315 PCG and lncRNA hubs, respectively. At false discovery rate (FDR) of 10%, the correlation analyses between the lncRNA and PCG hubs revealed 3,125 positive and 1,860 negative correlations. Integration of hubs with genotype data identified 243 eQTLs affecting the expression of 39 and 204 PCG and lncRNA hubs, respectively. CONCLUSIONS: Our study identified lncRNA and gene networks significantly associated with AD in the NAc, coordinated lncRNA and mRNA coexpression changes, highlighting potentially regulatory functions for the lncRNA, and our genetic (cis-eQTL) analysis provides novel insights into the etiological mechanisms of AD.

Genome-Wide Meta-Analyses of FTND and TTFC Phenotypes.

INTRODUCTION: FTND (FagerstrÓ§m test for nicotine dependence) and TTFC (time to smoke first cigarette in the morning) are common measures of nicotine dependence (ND). However, genome-wide meta-analysis for these phenotypes has not been reported. METHODS: Genome-wide meta-analyses for FTND (N = 19,431) and TTFC (N = 18,567) phenotypes were conducted for adult smokers of European ancestry from 14 independent cohorts. RESULTS: We found that SORBS2 on 4q35 (p = 4.05 × 10-8), BG182718 on 11q22 (p = 1.02 × 10-8), and AA333164 on 14q21 (p = 4.11 × 10-9) were associated with TTFC phenotype. We attempted replication of leading candidates with independent samples (FTND, N = 7010 and TTFC, N = 10 061), however, due to limited power of the replication samples, the replication of these new loci did not reach significance. In gene-based analyses, COPB2 was found associated with FTND phenotype, and TFCP2L1, RELN, and INO80C were associated with TTFC phenotype. In pathway and network analyses, we found that the interconnected interactions among the endocytosis, regulation of actin cytoskeleton, axon guidance, MAPK signaling, and chemokine signaling pathways were involved in ND. CONCLUSIONS: Our analyses identified several promising candidates for both FTND and TTFC phenotypes, and further verification of these candidates was necessary. Candidates supported by both FTND and TTFC (CHRNA4, THSD7B, RBFOX1, and ZNF804A) were associated with addiction to alcohol, cocaine, and heroin, and were associated with autism and schizophrenia. We also identified novel pathways involved in cigarette smoking. The pathway interactions highlighted the importance of receptor recycling and internalization in ND. IMPLICATIONS: Understanding the genetic architecture of cigarette smoking and ND is critical to develop effective prevention and treatment. Our study identified novel candidates and biological pathways involved in FTND and TTFC phenotypes, and this will facilitate further investigation of these candidates and pathways.

Digital Image Correlation of Forescatter Detector Images for Simultaneous Strain and Orientation Mapping.

Improved plasticity models require simultaneous experimental local strain and microstructural evolution data. Microscopy tools, such as electron backscatter diffraction (EBSD), that can monitor transformation at the relevant length-scale, are often incompatible with digital image correlation (DIC) techniques required to determine local deformation. In this paper, the viability of forescatter detector (FSD) images as the basis for the DIC study is investigated. Standard FSD and an integrated EBSD/FSD approach (Pattern Region of Interest Analysis System: PRIAS™) are analyzed. Simultaneous strain and microstructure maps are obtained for tensile deformation of Q&P 1180 steel up to ~14% strain. Tests on an undeformed sample that is simply shifted indicate a standard deviation of error in strain of around 0.4% without additional complications from a deformed surface. The method resolves strain bands at ~2 µm spacing but does not provide significant sub-grain strain resolution. Similar resolution was obtained for mechanically polished and electropolished samples, despite electropolished surfaces presenting a smoother, simpler topography. While the resolution of the PRIAS approach depends upon the EBSD step size, the 80 nm step size used provides seemingly similar resolution as 8,000× (22.7 nm) FSD images. Surface feature evolution prevents DIC analysis across large strain steps (>6% strain), but restarting DIC, using an FSD reference image from an interim strain step, allows reasonable DIC across the stress­strain curve. Furthermore, the data are obtained easily and provide complementary information for EBSD analysis.

Detection of Immunoglobulin G1 Against rK39 Improves Monitoring of Treatment Outcomes in Visceral Leishmaniasis.

BACKGROUND: Visceral leishmaniasis (VL), caused by the Leishmania donovani complex, is a fatal, neglected tropical disease that is targeted for elimination in India, Nepal, and Bangladesh. Improved diagnostic tests are required for early case detection and for monitoring the outcomes of treatments. Previous investigations using Leishmania lysate antigen demonstrated that the immunoglobulin (Ig) G1 response is a potential indicator of a patient's clinical status after chemotherapy. METHODS: IgG1 or IgG enzyme-linked immunosorbent assays (ELISAs) with rK39 or lysate antigens and novel IgG1 rK39 rapid diagnostic tests (RDTs) were assessed with Indian VL serum samples from the following clinical groups: paired pre- and postchemotherapy (deemed cured); relapsed; other infectious diseases; and endemic, healthy controls. RESULTS: With paired pre- and post-treatment samples (n = 37 pairs), ELISAs with rK39- and IgG1-specific conjugates gave a far more discriminative decrease in post-treatment antibody responses when compared to IgG (P < .0001). Novel IgG1 rK39 RDTs provided strong evidence for decreased IgG1 responses in patients who had successful treatment (P < .0001). Furthermore, both IgG1 rK39 RDTs (n = 38) and ELISAs showed a highly significant difference in test outcomes between cured patients and those who relapsed (n = 23; P < .0001). RDTs were more sensitive than corresponding ELISAs. CONCLUSIONS: We present strong evidence for the use of IgG1 in monitoring treatment outcomes in VL, and the first use of an IgG1-based RDT using the rK39 antigen for the discrimination of post-treatment cure versus relapse in VL. Such an RDT may have a significant role in monitoring patients and in targeted control and elimination of this devastating disease.

Resurgence of Vaccine-Preventable Diseases in Venezuela as a Regional Public Health Threat in the Americas.

Venezuela's tumbling economy and authoritarian rule have precipitated an unprecedented humanitarian crisis. Hyperinflation rates now exceed 45,000%, and Venezuela's health system is in free fall. The country is experiencing a massive exodus of biomedical scientists and qualified healthcare professionals. Reemergence of arthropod-borne and vaccine-preventable diseases has sparked serious epidemics that also affect neighboring countries. In this article, we discuss the ongoing epidemics of measles and diphtheria in Venezuela and their disproportionate impact on indigenous populations. We also discuss the potential for reemergence of poliomyelitis and conclude that action to halt the spread of vaccine-preventable diseases within Venezuela is a matter of urgency for the country and the region. We further provide specific recommendations for addressing this crisis.

Beyond genome-wide significance: integrative approaches to the interpretation and extension of GWAS findings for alcohol use disorder.

Alcohol use disorder (AUD) is a heritable complex behavior. Due to the highly polygenic nature of AUD, identifying genetic variants that comprise this heritable variation has proved to be challenging. With the exception of functional variants in alcohol metabolizing genes (e.g. ADH1B and ALDH2), few other candidate loci have been confidently linked to AUD. Genome-wide association studies (GWAS) of AUD and other alcohol-related phenotypes have either produced few hits with genome-wide significance or have failed to replicate on further study. These issues reinforce the complex nature of the genetic underpinnings for AUD and suggest that both GWAS studies with larger samples and additional analysis approaches that better harness the nominally significant loci in existing GWAS are needed. Here, we review approaches of interest in the post-GWAS era, including in silico functional analyses; functional partitioning of single nucleotide polymorphism heritability; aggregation of signal into genes and gene networks; and validation of identified loci, genes and gene networks in postmortem brain tissue and across species. These integrative approaches hold promise to illuminate our understanding of the biological basis of AUD; however, we recognize that the main challenge continues to be the extremely polygenic nature of AUD, which necessitates large samples to identify multiple loci associated with AUD liability.

Severity of Chagasic Cardiomyopathy Is Associated With Response to a Novel Rapid Diagnostic Test for Trypanosoma cruzi TcII/V/VI.

Background: Trypanosoma cruzi causes Chagas disease in the Americas. The outcome of infection ranges from lifelong asymptomatic status to severe disease. Relationship between T. cruzi lineage (TcI-TcVI) infection history and prognosis is not understood. We previously described peptide-based lineage-specific enzyme-linked immunosorbent assay (ELISA) with trypomastigote small surface antigen (TSSA). Methods: A novel rapid diagnostic test (RDT; Chagas Sero K-SeT) that incorporates a peptide that corresponds to the TSSA II/V/VI common epitope was developed and validated by comparison with ELISA. Patients from Bolivia and Peru, including individuals with varying cardiac pathology, and matched mothers and neonates, were then tested using Chagas Sero K-SeT. Results: Chagas Sero K-SeT and ELISA results, with a Bolivian subset of cardiac patients, mothers, and neonates, were in accord. In adult chronic infections (n = 121), comparison of severity class A (no evidence of Chagas cardiomyopathy) with class B (electrocardiogram suggestive of Chagas cardiomyopathy) and class C/D (decreased left ventricular ejection fraction; moderate/severe Chagas cardiomyopathy) revealed a statistically significant increase in Chagas Sero K-SeT reactivity with increasing severity (χ2 for trend, 7.39; P = .007). In Peru, Chagas Sero K-SeT detected the sporadic TcII/V/VI infections. Conclusions: We developed a low cost RDT that can replace ELISA for identification of TSSA II/V/VI immunoglobulin G. Most importantly, we show that response to this RDT is associated with severity of Chagas cardiomyopathy and thus may have prognostic value. Repeated challenge with T. cruzi infection may both exacerbate disease progression and boost the immune response to the TSSApep-II/V/VI epitope.

A step towards intelligent EBSD microscopy: machine-learning prediction of twin activity in MgAZ31.

Although microscopy is often treated as a quasi-static exercise for obtaining a snapshot of events and structure, it is clear that a more dynamic approach, involving real-time decision making for guiding the investigation process, may provide deeper insights, more efficiently. On the other hand, many applications of machine learning involve the interpretation of local circumstances from experience gained over many observations; that is, machine learning potentially provides an ideal solution for more efficient microscopy. This paper explores the potential for informing the microscope's observation strategy while characterising critical events. In particular, the identification of regions likely to experience twin activity (twin interaction with grain boundary) in AZ31 magnesium is attempted, from only local information. EBSD-based observations in the neighbourhoods of twin activity are fed into a machine-learning environment to inform the future search for such events, and the accuracy of the resultant decisions is quantified relative to the number of prior observations. The potential for utilising different types of local information, and their resultant value in the prediction process, is also assessed. After applying an attribute selection filter, and various other machine-learning tools, a decision-tree model is able to classify likely neighbourhoods of twin activity with 85% accuracy. The resultant framework provides the first step towards an intelligent microscopy for efficient observation of stochastic events during in situ microscopy campaigns. LAY DESCRIPTION: One role of artificial intelligence is to predict future events after learning from many previous observations. In materials science, various phenomena (such as crack nucleation) are difficult to predict because they have been insufficiently observed. Furthermore, observation is difficult, precisely because their location cannot be predicted, leading to a chicken and egg conundrum. This paper applies machine learning to the search for twin nucleation sites in a magnesium alloy, in an attempt to guide the observation of twin nucleation events in a microscope based on previous observations. As more data is obtained, the accuracy of the location prediction will increase. In the current case, the machine-learning tool achieved 85% accuracy for predicting the location of twin interactions with grain boundaries after several thousand observations. The resultant framework provides the first step towards an intelligent microscopy for efficient observation of stochastic events during in situ microscopy campaigns.

A Whole Methylome Study of Ethanol Exposure in Brain and Blood: An Exploration of the Utility of Peripheral Blood as Proxy Tissue for Brain in Alcohol Methylation Studies.

BACKGROUND: Recent reviews have highlighted the potential use of blood-based methylation biomarkers as diagnostic and prognostic tools of current and future alcohol use and addiction. Due to the substantial overlap that often exists between methylation patterns across different tissues, including blood and brain, blood-based methylation may track methylation changes in brain; however, little work has explored the overlap in alcohol-related methylation in these tissues. METHODS: To study the effects of alcohol on the brain methylome and identify possible biomarkers of these changes in blood, we performed a methylome-wide association study in brain and blood from 40 male DBA/2J mice that received either an acute ethanol (EtOH) or saline intraperitoneal injection. To investigate all 22 million CpGs in the mouse genome, we enriched for the methylated genomic fraction using methyl-CpG binding domain (MBD) protein capture followed by next-generation sequencing (MBD-seq). We performed association tests in blood and brain separately followed by enrichment testing to determine whether there was overlapping alcohol-related methylation in the 2 tissues. RESULTS: The top result for brain was a CpG located in an intron of Ttc39b (p = 5.65 × 10-08 ), and for blood, the top result was located in Espnl (p = 5.11 × 10-08 ). Analyses implicated pathways involved in inflammation and neuronal differentiation, such as CXCR4, IL-7, and Wnt signaling. Enrichment tests indicated significant overlap among the top results in brain and blood. Pathway analyses of the overlapping genes converge on MAPKinase signaling (p = 5.6 × 10-05 ) which plays a central role in acute and chronic responses to alcohol and glutamate receptor pathways, which can regulate neuroplastic changes underlying addictive behavior. CONCLUSIONS: Overall, we have shown some methylation changes in brain and blood after acute EtOH administration and that the changes in blood partly mirror the changes in brain suggesting the potential for DNA methylation in blood to be biomarkers of alcohol use.

Molecular typing reveals the co-existence of two transmission cycles of American cutaneous leishmaniasis in the Andean Region of Venezuela with Lutzomyia migonei as the vector.

BACKGROUND The transmission routes for American cutaneous leishmaniasis (ACL) are in flux, so studies examining its transmission in humans, mammalian hosts, and sand fly vectors are urgently needed. OBJECTIVES The aim of this work was understand the epidemiological cycles of Leishmania spp., which causes ACL in the Andean Region of Venezuela, by identifying the Leishmania and the sand fly species involved in human and dog infections. METHODS Thirty-one biopsies from patients in Mérida and Táchira states with suspected ACL were studied by both parasitological tests (cultures and hamster inoculation) and a molecular test [Internal transcribed spacer 1 (ITS1) nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)]. We also conducted a survey to detect Leishmania infection in dogs (Immunifluorescence antibody test and ITS1 nested PCR-RFLP) and sand flies (ITS1 nested PCR-RFLP) from El Carrizal, a highly endemic focus of ACL in Venezuela. FINDINGS Three different Leishmania species were identified in the clinical samples from humans (Leishmania braziliensis, L. guyanensis, and L. mexicana) and dogs (L. guyanensis and L. mexicana). The predominant sand fly species found were those from the Verrucarum group (infected with L. mexicana) and Lutzomyia migonei (infected with L. guyanensis and L. mexicana). MAIN CONCLUSIONS We show that Lu. migonei may be the putative vector in two ACL epidemiological cycles, involving L. guyanensis and L. mexicana. We also report for the first time the presence of L. guyanensis in domestic animals.

Hosts and vectors of Trypanosoma cruzi discrete typing units in the Chagas disease endemic region of the Paraguayan Chaco.

Active Trypanosoma cruzi transmission persists in the Gran Chaco region, which is considered hyperendemic for Chagas disease. Understanding domestic and sylvatic transmission cycles and therefore the relationship between vectors and mammalian hosts is crucial to designing and implementing improved effective control strategies. Here we describe the species of triatomine vectors and the sylvatic mammal reservoirs of T. cruzi, in different localities of the Paraguayan and Bolivian Chaco. We identify the T. cruzi genotypes discrete typing units (DTUs) and provide a map of their geographical distribution. A total of 1044 triatomines and 138 sylvatic mammals were captured. Five per cent of the triatomines were microscopically positive for T. cruzi (55 Triatoma infestans from Paraguay and one sylvatic Triatoma guasayana from Bolivia) and 17 animals (12·3%) comprising eight of 28 (28·5%) Dasypus novemcinctus, four of 27 (14·8%) Euphractus sexcinctus, three of 64 (4·7%) Chaetophractus spp. and two of 14 (14·3%) Didelphis albiventris. The most common DTU infecting domestic triatomine bugs was TcV (64%), followed by TcVI (28%), TcII (6·5%) and TcIII (1·5%). TcIII was overwhelmingly associated with armadillo species. We confirm the primary role of T. infestans in domestic transmission, armadillo species as the principal sylvatic hosts of TcIII, and consider the potential risk of TcIII as an agent of Chagas disease in the Chaco.

Importation of Hybrid Human-Associated Trypanosoma cruzi Strains of Southern South American Origin, Colombia.

We report the characterization of Trypanosoma cruzi of southern South American origin among humans, domestic vectors, and peridomestic hosts in Colombia using high-resolution nuclear and mitochondrial genotyping. Expanding our understanding of the geographic range of lineage TcVI, which is associated with severe Chagas disease, will help clarify risk of human infection for improved disease control.

Nicotine Enhances the Hypnotic and Hypothermic Effects of Alcohol in the Mouse.

BACKGROUND: Ethanol (EtOH) and nicotine abuse are 2 leading causes of preventable mortality in the world, but little is known about the pharmacological mechanisms mediating co-abuse. Few studies have examined the interaction of the acute effects of EtOH and nicotine. Here, we examine the effects of nicotine administration on the duration of EtOH-induced loss of righting reflex (LORR) and characterize the nature of their pharmacological interactions in C57BL/6J mice. METHODS: We assessed the effects of EtOH and nicotine and the nature of their interaction in the LORR test using isobolographic analysis after acute injection in C57BL/6J male mice. Next, we examined the importance of receptor efficacy using nicotinic partial agonists varenicline and sazetidine. We evaluated the involvement of major nicotinic acetylcholine receptor (nAChR) subtypes using nicotinic antagonist mecamylamine and nicotinic α4- and α7-knockout mice. The selectivity of nicotine's actions on EtOH-induced LORR was examined by testing nicotine's effects on the hypnotic properties of ketamine and pentobarbital. We also assessed the development of tolerance after repeated nicotine exposure. Last, we assessed whether the effects of nicotine on EtOH-induced LORR extend to hypothermia and EtOH intake in the drinking in the dark (DID) paradigm. RESULTS: We found that acute nicotine injection enhances EtOH's hypnotic effects in a synergistic manner and that receptor efficacy plays an important role in this interaction. Furthermore, tolerance developed to the enhancement of EtOH's hypnotic effects by nicotine after repeated exposure of the drug. α4* and α7 nAChRs seem to play an important role in nicotine-EtOH interaction in the LORR test. In addition, the magnitude of EtOH-induced LORR enhancement by nicotine was more pronounced in C57BL/6J than DBA/2J mice. Furthermore, acute nicotine enhanced ketamine and pentobarbital hypnotic effects in the mouse. Finally, nicotine enhanced EtOH-induced hypothermia but decreased EtOH intake in the DID test. CONCLUSIONS: Our results demonstrate that nicotine synergistically enhances EtOH-induced LORR in the mouse.