Exploring genetic diversity, population structure, and phylogeography in Paracoccidioides species using AFLP markers.

Paracoccidioidomycosis (PCM) is a life-threatening systemic fungal infection acquired after inhalation of Paracoccidioides propagules from the environment. The main agents include members of the P. brasiliensis complex (phylogenetically-defined species S1, PS2, PS3, and PS4) and P. lutzii. DNA-sequencing of protein-coding loci (e.g., GP43, ARF, and TUB1) is the reference method for recognizing Paracoccidioides species due to a lack of robust phenotypic markers. Thus, developing new molecular markers that are informative and cost-effective is key to providing quality information to explore genetic diversity within Paracoccidioides. We report using new amplified fragment length polymorphism (AFLP) markers and mating-type analysis for genotyping Paracoccidioides species. The bioinformatic analysis generated 144 in silico AFLP profiles, highlighting two discriminatory primer pairs combinations (#1 EcoRI-AC/MseI-CT and #2 EcoRI-AT/MseI-CT). The combinations #1 and #2 were used in vitro to genotype 165 Paracoccidioides isolates recovered from across a vast area of South America. Considering the overall scored AFLP markers in vitro (67-87 fragments), the values of polymorphism information content (PIC = 0.3345-0.3456), marker index (MI = 0.0018), effective multiplex ratio (E = 44.6788-60.3818), resolving power (Rp = 22.3152-34.3152), discriminating power (D = 0.5183-0.5553), expected heterozygosity (H = 0.4247-0.4443), and mean heterozygosity (H avp  = 0.00002-0.00004), demonstrated the utility of AFLP markers to speciate Paracoccidioides and to dissect both deep and fine-scale genetic structures. Analysis of molecular variance (AMOVA) revealed that the total genetic variance (65-66 %) was due to variability among P. brasiliensis complex and P. lutzii (PhiPT = 0.651-0.658, P < 0.0001), supporting a highly structured population. Heterothallism was the exclusive mating strategy, and the distributions of MAT1-1 or MAT1-2 idiomorphs were not significantly skewed (1:1 ratio) for P. brasiliensis s. str. (χ2 = 1.025; P = 0.3113), P. venezuelensis (χ2 = 0.692; P = 0.4054), and P. lutzii (χ2 = 0.027; P = 0.8694), supporting random mating within each species. In contrast, skewed distributions were found for P. americana (χ2 = 8.909; P = 0.0028) and P. restrepiensis (χ2 = 4.571; P = 0.0325) with a preponderance of MAT1-1. Geographical distributions confirmed that P. americana, P. restrepiensis, and P. lutzii are more widespread than previously thought. P. brasiliensis s. str. is by far the most widely occurring lineage in Latin America countries, occurring in all regions of Brazil. Our new DNA fingerprint assay proved to be rapid, reproducible, and highly discriminatory, to give insights into the taxonomy, ecology, and epidemiology of Paracoccidioides species, guiding disease-control strategies to mitigate PCM.

Trends in the molecular epidemiology and population genetics of emerging Sporothrix species.

Sporothrix (Ophiostomatales) comprises species that are pathogenic to humans and other mammals as well as environmental fungi. Developments in molecular phylogeny have changed our perceptions about the epidemiology, host-association, and virulence of Sporothrix. The classical agent of sporotrichosis, Sporothrix schenckii, now comprises several species nested in a clinical clade with S. brasiliensis, S. globosa, and S. luriei. To gain a more precise view of outbreaks dynamics, structure, and origin of genetic variation within and among populations of Sporothrix, we applied three sets of discriminatory AFLP markers (#3 EcoRI-GA/MseI-TT, #5 EcoRI-GA/MseI-AG, and #6 EcoRI-TA/MseI-AA) and mating-type analysis to a large collection of human, animal and environmental isolates spanning the major endemic areas. A total of 451 polymorphic loci were amplified in vitro from 188 samples, and revealed high polymorphism information content (PIC = 0.1765-0.2253), marker index (MI = 0.0001-0.0002), effective multiplex ratio (E = 15.1720-23.5591), resolving power (Rp = 26.1075-40.2795), discriminating power (D = 0.9766-0.9879), expected heterozygosity (H = 0.1957-0.2588), and mean heterozygosity (Havp  = 0.000007-0.000009), demonstrating the effectiveness of AFLP markers to speciate Sporothrix. Analysis using the program structure indicated three genetic clusters matching S. brasiliensis (population 1), S. schenckii (population 2), and S. globosa (population 3), with the presence of patterns of admixture amongst all populations. AMOVA revealed highly structured clusters (PhiPT = 0.458-0.484, P < 0.0001), with roughly equivalent genetic variability within (46-48 %) and between (52-54 %) populations. Heterothallism was the exclusive mating strategy, and the distributions of MAT1-1 or MAT1-2 idiomorphs were not significantly skewed (1:1 ratio) for S. schenckii (χ2 = 2.522; P = 0.1122), supporting random mating. In contrast, skewed distributions were found for S. globosa (χ2 = 9.529; P = 0.0020) with a predominance of MAT1-1 isolates, and regional differences were highlighted for S. brasiliensis with the overwhelming occurrence of MAT1-2 in Rio de Janeiro (χ2 = 14.222; P = 0.0002) and Pernambuco (χ2 = 7.364; P = 0.0067), in comparison to a higher prevalence of MAT1-1 in the Rio Grande do Sul (χ2 = 7.364; P = 0.0067). Epidemiological trends reveal the geographic expansion of cat-transmitted sporotrichosis due to S. brasiliensis via founder effect. These data support Rio de Janeiro as the centre of origin that has led to the spread of this disease to other regions in Brazil. Our ability to reconstruct the source, spread, and evolution of the ongoing outbreaks from molecular data provides high-quality information for decision-making aimed at mitigating the progression of the disease. Other uses include surveillance, rapid diagnosis, case connectivity, and guiding access to appropriate antifungal treatment.

The global epidemiology of emerging Histoplasma species in recent years.

Histoplasmosis is a serious infectious disease in humans caused by Histoplasma spp. (Onygenales), whose natural reservoirs are thought to be soil enriched with bird and bat guano. The true global burden of histoplasmosis is underestimated and frequently the pulmonary manifestations are misdiagnosed as tuberculosis. Molecular data on epidemiology of Histoplasma are still scarce, even though there is increasing recognition of histoplasmosis in recent years in areas distant from the traditional endemic regions in the Americas. We used multi-locus sequence data from protein coding loci (ADP-ribosylation factor, H antigen precursor, and delta-9 fatty acid desaturase), DNA barcoding (ITS1/2+5.8s), AFLP markers and mating type analysis to determine the genetic diversity, population structure and recognise the existence of different phylogenetic species among 436 isolates of Histoplasma obtained globally. Our study describes new phylogenetic species and the molecular characteristics of Histoplasma lineages causing outbreaks with a high number of severe outcomes in Northeast Brazil between 2011 and 2015. Genetic diversity levels provide evidence for recombination, common ancestry and clustering of Brazilian isolates at different geographic scales with the emergence of LAm C, a new genotype assigned to a separate population cluster in Northeast Brazil that exhibited low diversity indicative of isolation. The global survey revealed that the high genetic variability among Brazilian isolates along with the presence of divergent cryptic species and/or genotypes may support the hypothesis of Brazil being the center of dispersion of Histoplasma in South America, possibly with the contribution of migratory hosts such as birds and bats. Outside Brazil, the predominant species depends on the region. We confirm that histoplasmosis has significantly broadened its area of occurrence, an important feature of emerging pathogens. From a practical point of view, our data point to the emergence of histoplasmosis caused by a plethora of genotypes, and will enable epidemiological analysis focused on understanding the processes that lead to histoplasmosis. Further, the description of this diversity opens avenues for comparative genomic studies, which will allow progress toward a consensus taxonomy, improve understanding of the presence of hybrids in natural populations of medically relevant fungi, test reproductive barriers and to explore the significance of this variation.

Multi-platform metabolomics analyses of a broad collection of fragrant and non-fragrant rice varieties reveals the high complexity of grain quality characteristics.

The quality of rice in terms not only of its nutritional value but also in terms of its aroma and flavour is becoming increasingly important in modern rice breeding where global targets are focused on both yield stability and grain quality. In the present paper we have exploited advanced, multi-platform metabolomics approaches to determine the biochemical differences in 31 rice varieties from a diverse range of genetic backgrounds and origin. All were grown under the specific local conditions for which they have been bred and all aspects of varietal identification and sample purity have been guaranteed by local experts from each country. Metabolomics analyses using 6 platforms have revealed the extent of biochemical differences (and similarities) between the chosen rice genotypes. Comparison of fragrant rice varieties showed a difference in the metabolic profiles of jasmine and basmati varieties. However with no consistent separation of the germplasm class. Storage of grains had a significant effect on the metabolome of both basmati and jasmine rice varieties but changes were different for the two rice types. This shows how metabolic changes may help prove a causal relationship with developing good quality in basmati rice or incurring quality loss in jasmine rice in aged grains. Such metabolomics approaches are leading to hypotheses on the potential links between grain quality attributes, biochemical composition and genotype in the context of breeding for improvement. With this knowledge we shall establish a stronger, evidence-based foundation upon which to build targeted strategies to support breeders in their quest for improved rice varieties.

Merits of random forests emerge in evaluation of chemometric classifiers by external validation.

Real-world applications will inevitably entail divergence between samples on which chemometric classifiers are trained and the unknowns requiring classification. This has long been recognized, but there is a shortage of empirical studies on which classifiers perform best in 'external validation' (EV), where the unknown samples are subject to sources of variation relative to the population used to train the classifier. Survey of 286 classification studies in analytical chemistry found only 6.6% that stated elements of variance between training and test samples. Instead, most tested classifiers using hold-outs or resampling (usually cross-validation) from the same population used in training. The present study evaluated a wide range of classifiers on NMR and mass spectra of plant and food materials, from four projects with different data properties (e.g., different numbers and prevalence of classes) and classification objectives. Use of cross-validation was found to be optimistic relative to EV on samples of different provenance to the training set (e.g., different genotypes, different growth conditions, different seasons of crop harvest). For classifier evaluations across the diverse tasks, we used ranks-based non-parametric comparisons, and permutation-based significance tests. Although latent variable methods (e.g., PLSDA) were used in 64% of the surveyed papers, they were among the less successful classifiers in EV, and orthogonal signal correction was counterproductive. Instead, the best EV performances were obtained with machine learning schemes that coped with the high dimensionality (914-1898 features). Random forests confirmed their resilience to high dimensionality, as best overall performers on the full data, despite being used in only 4.5% of the surveyed papers. Most other machine learning classifiers were improved by a feature selection filter (ReliefF), but still did not out-perform random forests.

Is there evidence of recent hepatitis E virus infection in English and North Welsh blood donors?

BACKGROUND AND OBJECTIVE: The risk of hepatitis E virus (HEV) to blood safety remains unknown in England. Reports of persistent HEV infection with serious disease sequelae indicate that transfusion transmitted HEV is not a trivial disease in immunosuppressed patients. MATERIALS AND METHODS: Samples from unselected blood donors and donors with a history of jaundice were tested for HEV antibody and RNA. RESULTS: Overall, 10% of the donor sera were anti-HEV IgG reactive. Four of the donor samples were anti-HEV IgM reactive but HEV RNA negative. CONCLUSION: There is evidence of probable recent HEV infections in donors with a predicted attack rate of 2.8%.

Intranasal sumatriptan in post-ECT headache: results of an open-label trial.

BACKGROUND: Significant headaches occur in up to 45% of patients receiving electroconvulsive therapy (ECT) as a result of treatment. Headaches may at times be severe and affect patient compliance with this treatment modality. The 5-HT(1B/1D) receptor agonist sumatriptan has been reported to be effective for post-ECT headache in several case reports. The aim of the present open-label study was to assess the efficacy and tolerability of intranasal sumatriptan for post-ECT headache. METHOD: Patients undergoing ECT who experienced moderate-to-severe post-ECT headache were enrolled in the study. Patients were asked to rate their headache severity and describe headache characteristics using a standard headache diary. Headaches rated as severe or moderate were treated with 20 mg of intranasal sumatriptan. Additional headache ratings were recorded at 0.25, 0.5, 1.0, 1.5, and 2 hours after sumatriptan administration and compared with baseline values. RESULTS: Eight female patients (ages 34-45 years old) participated in the trial and experienced a total of 13 post-ECT headaches, which were treated with intranasal sumatriptan. Of the headaches treated, six (46.2%) were described as severe and seven (53.8%) were characterized as moderate in severity. Twelve (92.3%) of the treated headaches responded by the 2 hour posttreatment time point and 11 (84.6%) had responded within 1 hour. Comparisons made at the 1- and 2-hour time point revealed a statistically significant improvement from baseline (p = 0.002). Of the 12 headaches that responded, 6 (50%) were reported as no pain and 5 (38.5%) were reported as only mild pain at 1 hour following treatment. At the 2-hour assessment, an additional headache, which had previously not responded, was rated at mild resulting in six (50%) headaches with complete resolution of pain and six (50%) with a decrease in pain symptoms from moderate or severe to mild. Overall, sumatriptan treatment was well tolerated, and no significant adverse effects or changes in vital signs were recorded. In no case was a second dose of sumatriptan given. The most common complaint was the taste of the medication (n = 4), which was not treatment limiting. No patient withdrew from the study due to an adverse event. CONCLUSION: Intranasal sumatriptan spray may be an effective, well-tolerated, and prompt treatment for patients experiencing moderate-to-severe post-ECT headache. Preventing post-ECT headache may contribute to patient compliance with the ECT treatment modality. Additionally, the known pharmacologic effects of sumatriptan and the generally positive results found in the present study suggest that ECT-induced headache is vascular in origin. Further placebo-controlled, double-blind studies are needed to confirm our open-label results.

Metabolite profiling of sesquiterpene lactones from Lactuca species. Major latex components are novel oxalate and sulfate conjugates of lactucin and its derivatives.

Wounding leaves or stems of Lactuca species releases a milky latex onto the plant surface. We have examined the constituents of latex from Lactuca sativa (lettuce) cv. Diana. The major components were shown to be novel 15-oxalyl and 8-sulfate conjugates of the guaianolide sesquiterpene lactones, lactucin, deoxylactucin, and lactucopicrin. The oxalates were unstable, reverting to the parent sesquiterpene lactone on hydrolysis. Oxalyl derivatives have been reported rarely from natural sources. The sulfates were stable and are the first reported sesquiterpene sulfates from plants. Unusual tannins based on 4-hydroxyphenylacetyl conjugates of glucose were also identified. Significant qualitative and quantitative variation was found in sesquiterpene lactone profiles in different lettuce varieties and in other Lactuca spp. The proportions of each conjugate in latex also changed depending on the stage of plant development. A similar profile was found in chicory, in which oxalyl conjugates were identified, but the 8-sulfate conjugates were notably absent. The presence of the constitutive sesquiterpene lactones was not correlated with resistance to pathogens but may have a significant bearing on the molecular basis of the bitter taste of lettuce and related species. The induced sesquiterpene lactone phytoalexin, lettucenin A, was found in the Lactuca spp. but not in chicory.

Anesthesia for electroconvulsive therapy: a review.

Anesthetic techniques have evolved to improve the comfort and safety of modern electroconvulsive therapy (ECT). The authors review the literature and discuss the selection, preparation, and management from an anesthetic perspective. Specifically, the management of medications preprocedure and coexisting diseases is discussed. A review of induction agents, muscle relaxants, and other medications utilized in ECT is included.

Gibberellin-photoaffinity labelling of two polypeptides in plant plasma membranes.

Two polypeptides of M(r) 68 kDa and 18 kDa were gibberellin (GA)-photoaffinity labelled in vitro in plasma membrane preparations from oat (Avena sativa L.) aleurone and from leaves and stems of wild-type and GA-sensitivity mutants of different species. Labelling of these polypeptides could be competed by biologically active, but not by inactive, GAs, indicating the likely biological significance of these interactions. On 2-dimensional gels the radiolabelled polypeptides were each resolved as one intensely labelled low abundance spot with a slightly lower pl form adjacent to it. There was a strong pH dependency for both labelling events, which correlated well with pH values at which GA are known to be most biologically active. A semi-dwarf GA-sensitivity mutant of sweet pea (Lathyrus odoratus L.), lb, showed reduced photoaffinity labelling of both polypeptides compared with the wild type, Lb. In the GA-insensitive Arabidopsis thaliana mutant gai, the level of labelling was the same as in wild type, GAI. This is the first report of GA-binding proteins in plant plasma membranes. Some preliminary sequence data are given for one of the labelled polypeptides. We discuss these mutants and consider their possible roles in GA perception or action.

Electroconvulsive therapy in a patient with a cerebellar meningioma.

We report a 66-year-old woman with a cerebellar meningioma who successfully received electroconvulsive therapy (ECT) for mania. No special modifications in technique were required. A growing literature supports the relative safety of ECT in patients with small, stable brain tumors.

Electroconvulsive therapy for treatment of intractable seizures. Initial findings in two children.

We treated two children with intractable epilepsy with electroconvulsive therapy (ECT) for seizure control. One child showed a change in seizure pattern with treatment, which at greater intensity was also effective in stopping nonconvulsive status epilepticus. The other child showed a decrease in spontaneous seizure frequency during short-term treatment. These findings suggest a possible role for ECT in the management of intractable epilepsy in children who are not candidates for epileptic surgery.

ECT for the treatment of Huntington's disease: a case study.

We report a 56-year-old man with Huntington's disease whose chorea substantially improved after treatment with electroconvulsive therapy (ECT). ECT should be considered for medically refractory cases of movement disorders, especially those with severe disability.

ECT for the treatment of mood disorders in cancer patients.

The authors review the literature and report three patients with cancer whose comorbid mood disorder was successfully treated with electroconvulsive therapy (ECT). They make recommendations for early consideration of ECT and aggressive treatment of depression in this high-risk population.