Expanding the Knowledge of the Geographic Distribution of Trypanosoma cruzi TcII and TcV/TcVI Genotypes in the Brazilian Amazon

Trypanosoma cruzi infection is a complex sylvatic enzooty involving a wide range of animal species. Six discrete typing units (DTUs) of T. cruzi, named TcI to TcVI, are currently recognized. One unanswered question concerning the epidemiology of T. cruzi is the distribution pattern of TcII and hybrid DTUs in nature, including their virtual absence in the Brazilian Amazon, the current endemic area of Chagas disease in Brazil. Herein, we characterized biological samples that were collected in previous epizootiological studies carried out in the Amazon Basin in Brazil. We performed T. cruzi genotyping using four polymorphic genes to identify T. cruzi DTUs: mini-exon, 1f8, histone 3 and gp72. This analysis was conducted in the following biological samples: (i) two T. cruzi isolates obtained by culturing of stools from the triatomine species Rhodnius picttipes and (ii) five serum samples from dogs in which trypomastigotes were observed during fresh blood examination. We report for the first time the presence of TcII and hybrid DTUs (TcV/TcVI) in the Amazon region in mixed infections with TcI. Furthermore, sequencing of the constitutive gene, gp72, demonstrated diversity in TcII even within the same forest fragment. These data show that TcII is distributed in the five main Brazilian biomes and is likely more prevalent than currently described. It is very probable that there is no biological or ecological barrier to the transmission and establishment of any DTU in any biome in Brazil.     

Persistence of Asthmatic Response after Ammonium Persulfate-Induced Occupational Asthma in Mice

Introduction

Since persulfate salts are an important cause of occupational asthma (OA), we aimed to study the persistence of respiratory symptoms after a single exposure to ammonium persulfate (AP) in AP-sensitized mice.

Material and Methods

BALB/c mice received dermal applications of AP or dimethylsulfoxide (DMSO) on days 1 and 8. On day 15, they received a single nasal instillation of AP or saline. Airway hyperresponsiveness (AHR) was assessed using methacholine provocation, while pulmonary inflammation was evaluated in bronchoalveolar lavage (BAL), and total serum immunoglobulin E (IgE), IgG1 and IgG2a were measured in blood at 1, 4, 8, 24 hours and 4, 8, 15 days after the single exposure to the causal agent. Histological studies of lungs were assessed.

Results

AP-treated mice showed a sustained increase in AHR, lasting up to 4 days after the challenge. There was a significant increase in the percentage of neutrophils 8 hours after the challenge, which persisted for 24 hours in AP-treated mice. The extent of airway inflammation was also seen in the histological analysis of the lungs from challenged mice. Slight increases in total serum IgE 4 days after the challenge were found, while IgG gradually increased further 4 to 15 days after the AP challenge in AP-sensitized mice.

Conclusions

In AP-sensitized mice, an Ig-independent response is induced after AP challenge. AHR appears immediately, but airway neutrophil inflammation appears later. This response decreases in time; at early stages only respiratory and inflammatory responses decrease, but later on immunological response decreases as well.     

Spatial arrangement of kin affects recruitment success in young male red grouse

Models have shown that population cycles might be driven by time lags resulting from positive feedback between kin structure and population change, coupled with negative feedback between density and population change. One such model operates through kin favouritism facilitating the recruitment of young cock red grouse. We investigated whether recruitment by young cocks depended on the presence and spatial arrangement of elder relatives in the territorial population. We used molecular genetic estimates of relatedness, and checked for effects of covariates including natal territory size, hatching date, body size, parasite burdens and local density. Philopatric recruitment by cock red grouse led to the formation of clusters of contiguous territories owned by kin. The probability that an individual young cock would establish a territory increased with the number of kin in his father's cluster. This pattern might have been due to genetic quality determining both recruitment success and the size of the paternal cluster. If so, there should have been a positive correlation between a young cock's probability of recruitment and the number of his relatives in the population, irrespective of their spatial distribution. This did not occur and so the effect of cluster size is unlikely to have been confounded by genetic quality. The only morphological measure correlated with recruitment success was supraorbital comb size. The results are consistent with the prediction that kin tolerance affects recruitment but were at the level of the individual within years, rather than the population among years. Hence an experimental test of the kin favouritism hypothesis for population cycles, by manipulation of relatedness in populations among years, is now required.     

Determination of stavudine in human plasma and urine by high-performance liquid chromatography using a reduced sample volume

Sensitive high-performance liquid chromatographic assays have been developed for the quantification of stavudine (2′,3′-didehydro-3′-deoxythymidine, d4T) in human plasma and urine. The methods are linear over the concentration ranges 0.025–25 and 2–150 μg/ml in plasma and urine, respectively. An aliquot of 200 μl of plasma was extracted with solid-phase extraction using Oasis^® cartridges, while urine samples were simply diluted 1/100 with HPLC water. The analytical column, mobile phase, instrumentation and chromatographic conditions are the same for both methods. The methods have been validated separately, and stability tests under various conditions have been performed. The detection limit is 12 ng/ml in plasma for a sample size of 200 μl. The bioanalytical assay has been used in a pharmacokinetic study of pregnant women and their newborns.     

An old drug and different ways to treat cutaneous leishmaniasis: Intralesional and intramuscular meglumine antimoniate in a reference center,Rio de Janeiro,Brazil

BackgroundTreatment of cutaneous leishmaniasis (CL) remains challenging since the drugs currently used are quite toxic, thus contributing to lethality unrelated to the disease itself but to adverse events (AE). The main objective was to evaluate different treatment regimens with meglumine antimoniate (MA), in a reference center in Rio de Janeiro, Brazil.MethodologyA historical cohort of 592 patients that underwent physical and laboratory examination were enrolled between 2000 and 2017. The outcome measures of effectiveness were epithelialization and complete healing of cutaneous lesions. AE were graded using a standardized scale. Three groups were evaluated: Standard regimen (SR): intramuscular (IM) MA 10–20 mg Sb⁵⁺/kg/day during 20 days (n = 46); Alternative regimen (AR): IM MA 5 mg Sb⁵⁺/kg/day during 30 days (n = 456); Intralesional route (IL): MA infiltration in the lesion(s) through subcutaneous injections (n = 90). Statistical analysis was performed through Fisher exact and Pearson Chi-square tests, Kruskal-Wallis, Kaplan-Meier and log-rank tests.ResultsSR, AR and IL showed efficacy of 95.3%, 84.3% and 75.9%, with abandonment rate of 6.5%, 2.4% and 3.4%, respectively. IL patients had more comorbidities (58.9%; p = 0.001), were mostly over 50 years of age (55.6%), and had an evolution time longer than 2 months (65.6%; p = 0.02). Time for epithelialization and complete healing were similar in IL and IM MA groups (p = 0.9 and p = 0.5; respectively). Total AE and moderate to severe AE that frequently led to treatment interruption were more common in SR group, while AR and IL showed less toxicity.Conclusions/SignificanceAR and IL showed less toxicity and may be good options especially in CL cases with comorbidities, although SR treatment was more effective. IL treatment was an effective and safe strategy, and it may be used as first therapy option as well as a rescue scheme in patients initially treated with other drugs.     

Phylogeographic structure and postglacial evolutionary history of water voles (Arvicola terrestris) in the United Kingdom

The phylogeographic pattern of mitochondrial DNA variation in water voles (Arvicola terrestris) from 57 localities across the United Kingdom and representative samples from Spain, France, Switzerland and Finland was determined from sequence variation in the central portion of the control region. Twenty-seven different haplotypes were resolved which formed two distinct phylogenetic clades. This major division separated haplotypes found in Scotland from those found in England and Wales. Nested clade analysis of haplotypes indicated that such a division was a consequence of allopatric fragmentation. The haplotypes found in Switzerland, France and Spain clustered with Scottish haplotypes, whereas the haplotype from Finland clustered with the English/Welsh haplotypes. These patterns indicate that contemporary Scottish populations are derived from an Iberian glacial refugium, whereas English and Welsh populations are derived from an eastern European refugium. As such, the postglacial recolonization of the United Kingdom must have involved two colonization events, either in different localities with no subsequent contact, or as two waves separated over time, with the second wave of colonizers displacing the first. An analysis of molecular variance (AMOVA) identified significant population genetic divergence within both the major clades, indicative of restricted gene flow and regional population isolation. The implications of both phylogeographical and population genetic structure are discussed in context with the conservation of water voles in Britain.     

Protein-Based Classifier to Predict Conversion from Clinically Isolated Syndrome to Multiple Sclerosis

Multiple sclerosis is an inflammatory, demyelinating, and neurodegenerative disease of the central nervous system. In most patients, the disease initiates with an episode of neurological disturbance referred to as clinically isolated syndrome, but not all patients with this syndrome develop multiple sclerosis over time, and currently, there is no clinical test that can conclusively establish whether a patient with a clinically isolated syndrome will eventually develop clinically defined multiple sclerosis. Here, we took advantage of the capabilities of targeted mass spectrometry to establish a diagnostic molecular classifier with high sensitivity and specificity able to differentiate between clinically isolated syndrome patients with a high and a low risk of developing multiple sclerosis. Based on the combination of abundances of proteins chitinase 3-like 1 and ala-β-his-dipeptidase in cerebrospinal fluid, we built a statistical model able to assign to each patient a precise probability of conversion to clinically defined multiple sclerosis. Our results are of special relevance for patients affected by multiple sclerosis as early treatment can prevent brain damage and slow down the disease progression.Multiple sclerosis is an inflammatory, demyelinating, and neurodegenerative disease of the central nervous system, and although the etiology of the disease is not fully understood, it is probably caused by the interaction of a complex genetic architecture and environmental factors. Multiple sclerosis affects over 2 million people worldwide, and it is typically diagnosed between ages 20 and 40, thus making a significant impact on public health and its economy (1).In most patients, the disease initiates with an episode of neurological disturbance referred to as clinically isolated syndrome. However, not all patients with this syndrome develop multiple sclerosis over time (2), and currently, the magnetic resonance imaging (MRI) abnormalities and the presence of IgG oligoclonal bands in cerebrospinal fluid (CSF) are used as predictors for later conversion to clinically definite multiple sclerosis (CDMS)¹ (3–5). Although such abnormalities are considered important factors that influence the likelihood of developing CDMS, there is currently no clinical test that can conclusively establish whether a patient with a clinically isolated syndrome will eventually develop CDMS.The lack of diagnostic and prognostic biomarkers is a common problem for many diseases lacking a complete etiology, which is the case for most neurological disorders related to the central nervous system such as Parkinson''s and Alzheimer''s diseases, schizophrenia, and multiple sclerosis. In the particular case of multiple sclerosis, early treatment of patients with a clinically isolated syndrome can prevent brain damage and slow down the disease progression (6). Therefore, the availability of a diagnostic test in the initial stages of the disease is not only desirable but also of extreme relevance to attenuate the degenerative effects of the disease.Biomarker validation has traditionally been dominated by enzyme linked immuno-sorbent assays (ELISA), but recent advances in proteomics techniques have enabled the measurement of a subset of selected proteins over a large dynamic concentration range in multiple samples. Targeted mass spectrometry has thus become the method of choice when quantifying simultaneously a panel of proteins across many different biological samples (7–9). In particular, selected reaction monitoring (SRM) is the gold standard targeted mass spectrometry method for protein quantification due to its high precision, reliability, and throughput (10–13). This targeted mass spectrometry method is performed on triple quadrupole instruments, in which a predefined peptide precursor ion is first isolated, and then selected fragment ions arising from its collisional dissociation are measured over time. Each pair of precursor and fragment ion is called a transition, and multiple transitions can be coordinately measured and used to conclusively identify and quantify a peptide in a clinical complex sample.In a previous study, we used a screening mass spectrometric approach to discover potential markers for multiple sclerosis conversion in patients that initially presented a clinical isolated syndrome (14). In that discovery phase, quantitative mass spectrometry with iTRAQ labeling was used to measure protein abundances in pooled CSF samples from patients presenting a clinical isolated syndrome that either remained normal (CIS) or had eventually converted to clinically definite multiple sclerosis (CDMS) (n = 60). In the initial screening, several proteins exhibited significant differences in abundance when comparing these two groups of patients. The abundance change in one of the altered proteins, chitinase 3-like 1 (CH3L1), was confirmed by ELISA in CSF of individual patients, whereas for others, such as semaphorin 7A (SEM7A) and ala-β-his-dipeptidase (CNDP1), their abundance changes were confirmed by targeted mass spectrometry in follow-up studies with independent cohorts (15). Moreover, the levels of CH3L1 were associated with brain MRI abnormalities and disability progression during the follow-up period, as well as with shorter time to conversion to clinically definite multiple sclerosis (14).We now set out to establish a diagnostic protein classifier with high sensitivity and specificity able to differentiate between patients with a clinically isolated syndrome that have either a high or a low risk of developing clinically definite multiple sclerosis over time. For this purpose, CSF samples from an independent patient cohort from the one used in the discovery study were collected, and a set of preselected protein biomarker candidates were systematically quantified by targeted mass spectrometry (SRM) and evaluated for their classification power. Out of this study, we established a protein classifier based on the combination of abundances of proteins chitinase 3-like 1 and ala-β-his-dipeptidase, which is able to differentiate with high sensitivity and specificity between patients with a clinically isolated syndrome that have either a high or low risk of developing clinically definite multiple sclerosis. Moreover, the statistical model built around this protein classifier enables clinicians to easily assign to each patient a precise probability of conversion to clinically definite multiple sclerosis (Fig. 1).Open in a separate windowFig. 1.General workflow used in the present study. Initially, protein candidates identified in our previous discovery studies—together with several proteins described by other groups—were selected and quantified by targeted mass spectrometry (SRM) in a relatively large cohort individual patients. Protein quantities were then evaluated by their capability of classifying patients with clinical isolated syndrome, and thus, the best prognostic protein combination was identified.     

Characterization of the proteolytic system present in Vasconcellea quercifolia latex

Vasconcellea quercifolia (Caricaceae) latex contains several cysteine endopeptidases with high proteolytic activity. Cysteine endopeptidases are the main active compounds used by the plant as a defense mechanism. A proteolytic preparation from V. quercifolia (“oak leaved papaya”) latex was purified by cation exchange chromatography. From SDS-PAGE and blotting of the selected fractions, the N-terminal amino acid sequences of polypeptides were determined by Edman’s degradation. The analysis by peptide mass fingerprinting (PMF) of the enzymes allowed their characterization and confirmed the presence of seven different cysteine proteinases in the latex of V. quercifolia. Moreover, the comparison between the tryptic maps with those deposited in databases using the MASCOT tool showed that none of the isolated proteases matched with another plant protease. Notably, a propeptidase was detected in the plant latex, which is being the first report in this sense. Furthermore, the cDNA of one of the cysteine proteases that is expressed in the latex of V. quercifolia was cloned and sequenced. The consensus sequence was aligned using the ClustalX web server, which allowed detecting a high degree of identity with cysteine proteases of the Caricaceae family and establishing the evolutionary relationship between them. We also observed a high conservation degree for those amino acid residues which are essential for the catalytic activity and tridimensional structure of the plant proteases belonging to the subfamily C1A. The PMF analysis strongly suggests that the sequence obtained corresponds to the VQ-III peptidase.     

Thyroid Status Is a Key Regulator of Both Flux and Efficiency of Oxidative Phosphorylation in Rat Hepatocytes

Thyroid status is crucial in energy homeostasis, but despite extensive studies the actual mechanism by which it regulates mitochondrial respiration and ATP synthesis is still unclear. We studied oxidative phosphorylation in both intact liver cells and isolated mitochondria from in vivo models of severe not life threatening hyper- and hypothyroidism. Thyroid status correlated with cellular and mitochondrial oxygen consumption rates as well as with maximal mitochondrial ATP production. Addition of a protonophoric uncoupler, 2,4-dinitrophenol, to hepatocytes did not mimic the cellular energetic change linked to hyperthyroidism. Mitochondrial content of cytochrome oxidase, ATP synthase, phosphate and adenine nucleotide carriers were increased in hyperthyroidism and decreased in hypothyroidism as compared to controls. As a result of these complex changes, the maximal rate of ATP synthesis increased in hyperthyroidism despite a decrease in ATP/O ratio, while in hypothyroidism ATP/O ratio increased but did not compensate for the flux limitation of oxidative phosphorylation. We conclude that energy homeostasis depends on a compromise between rate and efficiency, which is mainly regulated by thyroid hormones.