The branched chain amino acids (BCAAs) modulate the development of the intra-cellular stages of Trypanosoma cruzi.

The life cycle of Trypanosoma cruzi, the etiological agent of Chagas disease, involves different forms of the parasite, which alternates between insect and vertebrate hosts. One critical process in the parasite's life cycle is metacyclogenesis, in which the replicative non-infective forms present in the insect midgut differentiate into non-dividing vertebrate-infective forms. It is known that proline (Pro) is important for this process and that leucine (Leu) and isoleucine (Ile) can act as inhibitors of metacyclogenesis. In this study, we investigated further the role of branched-chain amino acids (BCAAs) as negative modulators of parasite differentiation and infection capability in vitro. We found that BCAAs can down-regulate metacyclogenesis, inhibiting Pro-dependent differentiation. Furthermore, we evaluated the ability of all three BCAAs to influence the differentiation of intracellular stages and found that they could modulate the release of trypomastigotes from infected host cells. These findings suggest that BCAAs may have an important role in the complex life cycle of T. cruzi. Thus, enzymes of their metabolism and other interacting proteins could be potential targets for the development of new therapeutic strategies for Chagas disease.

Branched chain amino acids catabolism as a source of new drug targets in pathogenic protists.

Leucine, isoleucine, and valine, collectively termed Branched Chain Amino Acids (BCAA), are hydrophobic amino acids (AAs) and are essential for most eukaryotes since in these organisms they cannot be biosynthesized and must be supplied by the diet. These AAs are structurally relevant for muscle cells and, of course, important for the protein synthesis process. The metabolism of BCAA and its participation in different biological processes in mammals have been relatively well described. However, for other organisms as pathogenic parasites, the literature is really scarce. Here we review the BCAA catabolism, compile evidence on their relevance for pathogenic eukaryotes with special emphasis on kinetoplastids and highlight unique aspects of this underrated pathway.

Nitrogen uptake and macronutrients distribution in mango (Mangifera indica L. cv. Keitt) trees.

We assessed the effects of N fertigation regime on nutrient uptake and distribution in leaves and fruit of mango cv. Keitt grown in a lysimeter for four years. We applied three treatments: N1 - no N fertilization (less than 2 mg/L in the tap water); N2 - 10 mg/L N; and N3 - 20 mg/L N. Deficient N conditions (N1) resulted in low vegetation and fruit yield, high fruit:leaf ratio, high photosynthetic activity, high leaf P and K concentrations, as well as high sugar content and low acidity in the fruit. Excess N concentration (N3) enhanced vegetative growth and reduced fruit yield and gas exchange. The calculated annual nitrogen uptake heavily depended on the nitrogen supply, being highest for the N2 treatment (196 g/tree) as compared with the N1 (25 g/tree) or N3 (185 g/tree) treatments. Fruits were a major N sink being 82% (in N1), 26% (in N2), and 5% (in N3) of the total annual N supplied. The N accumulation rate in the fruit of the N1 and N2 treatment were above the N quantities supplied via fertigation, suggesting that N reserve in the vegetative tissues supplied the fruit's high N demand. These findings highlight the link between mango's N requirements and fruit yield, as well as the risks of excessive N fertilization.

Immunological differences between primary and metastatic breast cancer.

Background: Little is known about how the immune microenvironment of breast cancer evolves during disease progression. Patients and methods: We compared tumor infiltrating lymphocyte (TIL) count, programmed death-ligand 1 (PD-L1) protein expression by immunohistochemistry and mRNA levels of 730 immune-related genes using Nanostring technology in primary and metastatic cancer samples. Results: TIL counts and PD-L1 positivity were significantly lower in metastases. Immune cell metagenes corresponding to CD8, T-helper, T-reg, Cytotoxic T, Dendritic and Mastoid cells, and expression of 13 of 29 immuno-oncology therapeutic targets in clinical development including PD1, PD-L1, and CTLA4 were significantly lower in metastases. There was also coordinated down regulation of chemoattractant ligand/receptor pairs (CCL19/CCR7, CXCL9/CXCR3, IL15/IL15R), interferon regulated genes (STAT1, IRF-1,-4,-7, IFI-27,-35), granzyme/granulysin, MHC class I and immune proteasome (PSMB-8,-9,-10) expression in metastases. Immunotherapy response predictive signatures were also lower. The expression of macrophage markers (CD163, CCL2/CCR2, CSF1/CSFR1, CXCR4/CXCL12), protumorigenic toll-like receptor pathway genes (CD14/TLR-1,-2,-4,-5,-6/MyD88), HLA-E, ecto-nuclease CD73/NT5E and inhibitory complement receptors (CD-59,-55,-46) remained high in metastases and represent potential therapeutic targets. Conclusions: Metastatic breast cancers are immunologically more inert than the corresponding primary tumors but some immune-oncology targets and macrophage and angiogenesis signatures show preserved expression and suggest therapeutic combinations for clinical testing.

The active transport of histidine and its role in ATP production in Trypanosoma cruzi.

Trypanosoma cruzi, the aetiological agent of Chagas's disease, metabolizes glucose, and after its exhaustion, degrades amino acids as energy source. Here, we investigate histidine uptake and its participation in energy metabolism. No putative genes for the histidine biosynthetic pathway have been identified in genome databases of T. cruzi, suggesting that its uptake from extracellular medium is a requirement for the viability of the parasite. From this assumption, we characterized the uptake of histidine in T. cruzi, showing that this amino acid is incorporated through a single and saturable active system. We also show that histidine can be completely oxidised to CO2. This finding, together with the fact that genes encoding the putative enzymes for the histidine - glutamate degradation pathway were annotated, led us to infer its participation in the energy metabolism of the parasite. Here, we show that His is capable of restoring cell viability after long-term starvation. We confirm that as an energy source, His provides electrons to the electron transport chain, maintaining mitochondrial inner membrane potential and O2 consumption in a very efficient manner. Additionally, ATP biosynthesis from oxidative phosphorylation was found when His was the only oxidisable metabolite present, showing that this amino acid is involved in bioenergetics and parasite persistence within its invertebrate host.

Application of magnetically induced hyperthermia in the model protozoan Crithidia fasciculata as a potential therapy against parasitic infections.

BACKGROUND: Magnetic hyperthermia is currently a clinical therapy approved in the European Union for treatment of tumor cells, and uses magnetic nanoparticles (MNPs) under time-varying magnetic fields (TVMFs). The same basic principle seems promising against trypanosomatids causing Chagas disease and sleeping sickness, given that the therapeutic drugs available have severe side effects and that there are drug-resistant strains. However, no applications of this strategy against protozoan-induced diseases have been reported so far. In the present study, Crithidia fasciculata, a widely used model for therapeutic strategies against pathogenic trypanosomatids, was targeted with Fe(3)O(4) MNPs in order to provoke cell death remotely using TVMFs. METHODS: Iron oxide MNPs with average diameters of approximately 30 nm were synthesized by precipitation of FeSO(4) in basic medium. The MNPs were added to C. fasciculata choanomastigotes in the exponential phase and incubated overnight, removing excess MNPs using a DEAE-cellulose resin column. The amount of MNPs uploaded per cell was determined by magnetic measurement. The cells bearing MNPs were submitted to TVMFs using a homemade AC field applicator (f = 249 kHz, H = 13 kA/m), and the temperature variation during the experiments was measured. Scanning electron microscopy was used to assess morphological changes after the TVMF experiments. Cell viability was analyzed using an MTT colorimetric assay and flow cytometry. RESULTS: MNPs were incorporated into the cells, with no noticeable cytotoxicity. When a TVMF was applied to cells bearing MNPs, massive cell death was induced via a nonapoptotic mechanism. No effects were observed by applying TVMF to control cells not loaded with MNPs. No macroscopic rise in temperature was observed in the extracellular medium during the experiments. CONCLUSION: As a proof of principle, these data indicate that intracellular hyperthermia is a suitable technology to induce death of protozoan parasites bearing MNPs. These findings expand the possibilities for new therapeutic strategies combating parasitic infection.

pH-dependent mineral release and surface properties of cornstraw biochar: agronomic implications.

Surface charge and pH-dependent nutrient release properties of cornstraw biochar were examined to elucidate its potential agronomic benefits. Kinetics of element release was characterized by rapid H(+) consumption and rapid, pH-dependent P, Ca, and Mg release, followed by zero-order H(+) consumption and mineral dissolution reactions. Initial K release was not pH-dependent, nor was it followed by a zero-order reaction at any pH. Rapid and constant rate P releases were significant, having the potential to substitute substantial proportions of P fertilizer. K releases were also significant and may replace conventional K fertilizers, however, not long-term plant demand. The cation exchange capacity (CEC) of the biochar leached with a mild acidic solution increased linearly from 179 to 888 mmol(c) (kg C)(-1) over a pH range of 4-8, while the anion exchange capacity of 154 mmol(c) (kg C)(-1) was constant over the same pH range. Since native soil organic constituents have much higher CEC values (average 2800 mmol(c) (kg C)(-1) at pH 7), improved soil fertility as a result of enhanced cation retention by the biochar probably will be favorable only in sandy and low organic matter soils, unless surface oxidation during aging significantly increases its CEC.

Breast cancer in young women (YBC): prevalence of BRCA1/2 mutations and risk of secondary malignancies across diverse racial groups.

BACKGROUND: Despite significant differences in age of onset and incidence of breast cancer between Caucasian (CA), African-American (AA) and Korean (KO) women, little is known about differences in BRCA1/2 mutations in these populations. The purpose of this study is to evaluate the prevalence of BRCA1/2 mutations and the association between BRCA1/2 mutation status and secondary malignancies among young women with breast cancer in these three racially diverse groups. METHODS: Patients presenting to our breast cancer follow-up clinics selected solely on having a known breast cancer diagnosis at a young age (YBC defined as age <45 years at diagnosis) were invited to participate in this study. A total of 333 eligible women, 166 CA, 66 AA and 101 KO underwent complete sequencing of BRCA1/2 genes. Family history (FH) was classified as negative, moderate or strong. BRCA1/2 status was classified as wild type (WT), variant of uncertain significance (VUS) or deleterious (DEL). RESULTS: DEL across these three racially diverse populations of YBC were nearly identical: CA 17%, AA 14% and KO 14%. The type of DEL differed with AA having more frequent mutations in BRCA2, compared with CA and KO. VUS were predominantly in BRCA2 and AA had markedly higher frequency of VUS (38%) compared with CA (10%) and KO (12%). At 10-year follow-up from the time of initial diagnosis of breast cancer, the risk of secondary malignancies was similar among WT (14%) and VUS (16%), but markedly higher among DEL (39%). CONCLUSIONS: In these YBC, the frequency of DEL in BRCA1/2 is remarkably similar among the racially diverse groups at 14%-17%. VUS is more common in AA, but aligns closely with WT in risk of second cancers, age of onset and FH.

Racial differences in the incidence of BRCA1 and BRCA2 mutations in a cohort of early onset breast cancer patients: African American compared to white women.

PURPOSE: To evaluate the frequency and distribution of BRCA1 and BRCA2 mutations in a cohort of young women with breast cancer and to compare the distribution of mutations as a function of race. METHODS: After IRB approved informed consent, 170 white women and 30 African American women with known breast cancer diagnosed at a young age (45 years or less) underwent complete sequencing of the BRCA1 and BRCA2 genes. Each cohort represented approximately 40% of women of the same ethnic background aged 45 years or younger in a breast cancer database. RESULTS: Of the 200 patients tested, 131 (65%) had wild type mutations, 34 (17%) had deleterious mutations, and 35 (18%) had variants of uncertain significance. There were no significant differences between the white and African American cohorts regarding the percentage of deleterious mutations (17% v 17%). However, most African American patients had mutations in BRCA2 (4/5, 80%), while most mutations in the white cohort were in BRCA1 (20/29, 69%). In addition, 46% of the African American women had variants of uncertain significance, compared to only 12% of the white cohort. CONCLUSIONS: Young African American women with breast cancer have a similar frequency of deleterious mutations as white women, but have a significantly higher frequency of variants of uncertain significance. Review of these variants revealed that the majority were unlikely to be associated with disease risk or were likely to be polymorphisms. The implications for genetic testing and counselling in young women with breast cancer are discussed.

Differential effects of cholesterol on acyl chain order in erythrocyte membranes as a function of depth from the surface. An electron paramagnetic resonance (EPR) spin label study.

The purpose of this work is to analyze the effects of cholesterol modulation on acyl chain ordering in the membrane of human erythrocytes as a function of depth from the surface. Partial cholesterol depletion was achieved by incubation of erythrocytes with liposomes containing saturated phospholipids, or with methyl-beta-cyclodextrin (MbetaCD). Cholesterol enrichment was achieved by incubation with liposomes formed by phospholipids/cholesterol, or with the complex MbetaCD/cholesterol. Acyl chain order was studied with electron paramagnetic resonance spectroscopy (EPR) using spin labels that sense the lipid bilayer at different depths. It is shown that the increase in cholesterol stiffens acyl chains but decreases the interaction among lipid headgroups, while cholesterol depletion causes the opposite behavior. It is likely that the observed cholesterol effects are related to those stabilizing the cholesterol-rich detergent-insoluble membrane domains (rafts), recently shown to exist in erythrocytes.

Trypanosoma cruzi: identification of a galactose-binding protein that binds to cell surface of human erythrocytes and is involved in cell invasion by the parasite.

Trypanosoma cruzi must invade mammalian host cells to replicate and complete its life cycle. Almost all nucleated mammalian cells can be invaded by the parasite following a receptor-ligand recognition as an early prerequisite. In this work, we describe a 67-kDa lectin-like glycoprotein that binds to desialylated human erythrocyte membranes in a galactose-dependent way. This protein is present on the parasite surface in both infective and non-infective stages of T. cruzi. More interestingly, we demonstrate by lectin-immuno-histochemistry assays that the 67kDa protein is involved in the recognition of host-cell receptors in mouse cardiac tissue and human cardiac aortic endothelium and mammary artery tissue. Moreover, antibodies against the 67kDa glycoprotein inhibit in vitro host-cell invasion by 63%. These data suggest that the 67kDa glycoprotein in vivo is needed for host-cell invasion by T. cruzi.

Antibody maturation in Trypanosoma cruzi-infected rats.

The study of antibody avidity changes during infection has improved the understanding of the pathologic processes involved in several infectious diseases. In some infections, like toxoplasmosis, this information is being used for diagnostic purposes. Results of the evolution of antibody avidity for different specific antigens in Trypanosome cruzi-infected rats are presented. A Western blotting technique, combined with avidity analysis to identify antigens that elicit high-avidity antibodies, is suggested. In this system, antibodies showed high avidity values only during the chronic phase of infection and only in relation to antibodies against 21-, 33-, 41-, 42-, 56-, 58-, 66-, and 72-kDa antigens. Finally, a 97-kDa T. cruzi antigen, which was recognized by high-avidity antibodies and occurred in noninfected rats, was identified. These results allow us to evaluate the different antigens in chagasic infection. Our results show that with the correct choice of antigen it is possible to detect differences in maturation of antibodies and to discriminate, in an experimental model, between recent (acute) and chronic infections.

Evidence from sentence priming for an atypical language organisation in the brain of dyslexic males.

The present study used an experimental paradigm that combined sentence priming, lexical decision, and visual hemifield stimulation to investigate the relationship between dyslexia and linguistic processing by the two hemispheres. It was predicted that left hemisphere (LH) superiority for word recognition in a constraining versus a neutral sentence context would be reduced, or even reversed in dyslexic as compared to normal participants. Because both dyslexia and LH superiority for language processing are considered to be more prevalent for males than for females, it was further expected that this decrease, or reversal, in LH superiority for word recognition in a sentence context would be more pronounced for males with dyslexia than for females with dyslexia. A total of 88 children, half of whom were females, participated in the study: 44 children were diagnosed as dyslexic and 44 were normal readers. The results generally confirmed the hypothesis and provided strong support for a reversal in hemispheric asymmetry in males with dyslexia. Implications for sentence processing of the excessive involvement of the right hemisphere (RH) in the dyslexia of males are discussed and the lexical-decision-sentence-priming paradigm is recommended as a promising means of exploring the issues related to these implications.

Trypanosoma cruzi: specific detection of parasites by PCR in infected humans and vectors using a set of primers (BP1/BP2) targeted to a nuclear DNA sequence.

In the present work we evaluate Trypanosoma cruzi DNA detection by PCR using the nuclear oligonucleotides BP1/BP2 as primers. These primers are targeted to the 5' and 3' ends of the coding region for the flagellar protein F29. An amplification product of BP1/BP2 is a DNA band 692 bp long. Titration assays were performed to evaluate the minimum amount of parasite DNA that can be detected by this assay, resulting in 10 fg (equivalent to about 1/20 of the genome). The assay was also performed using T. cruzi DNA from different strains, clones, and human-derived isolates obtaining, in all cases, amplification products. No DNA amplification was observed when the PCR was performed using DNA from Leishmania braziliensis, but when T. rangeli DNA was used, a 615-bp-long fragment was amplified. Under appropriate gel conditions T. cruzi and T. rangeli DNA amplicons could be differentiated. When both conventional xenodiagnosis and PCR detection of parasite DNA in the feces of insect vectors fed with blood from infected patients were compared, 10 of 20 samples were positive by both techniques. However, 2 other samples with positive serology were also positive by PCR. When PCR was performed on blood samples from infected and uninfected individuals, 62 of 65 serologically positive human samples amplified the BP1/BP2 692-bp T. cruzi DNA fragment (sensitivity >95%). The 3 negative samples were positive when Southern blot hybridization was performed using the radiolabeled PCR amplification product as probe (sensitivity 100%).

Postdialysis fatigue.

To clarify the demographic and clinicolaboratory features of postdialysis fatigue (PDF), we enrolled 85 patients on maintenance hemodialysis in a cross-sectional study using validated questionnaires and chart review. Forty-three patients complained of fatigue after dialysis. On formal testing using the Kidney Disease Questionnaire, the PDF group had statistically greater severity of fatigue and somatic complaints than the group of patients without subjective fatigue (P = 0.03 and 0.04, respectively). On a scale measuring intensity of fatigue (1 = least to 5 = worst), the PDF group average was 3.4 +/- 1.2. PDF subjects reported that 80% +/- 25% of dialysis treatments were followed by fatigue symptoms. In 28 (65%) of patients, the symptoms started with the first dialysis treatment. They reported needing an average of 4.8 hours of rest or sleep to overcome the fatigue symptoms (range, 0 to 24 hours). There were no significant differences between patients with and without PDF in the following parameters: age; sex; type of renal disease; presence of diabetes mellitus, heart disease (congestive, ischemic), or chronic obstructive lung disease; blood pressure response to dialysis; type or adequacy of dialysis regimen; hematocrit; electrolytes; blood urea nitrogen; creatinine; cholesterol; albumin; parathyroid hormone; ejection fraction; and use of antihistamines, benzodiazepines, and narcotics. In the fatigue group, there was significantly greater use of antihypertensive medications known to have fatigue as a side effect (P = 0.007). Depression was more common in the fatigue group by Beck Depression score (11.6 +/- 8.0 v 7.8 +/- 6.3; P = 0.02). We conclude that (1) postdialysis fatigue is a common, often incapacitating symptom in patients on chronic extracorporeal dialysis; (2) no routinely measured parameter of clinical or dialytic function appears to predict postdialysis fatigue; and (3) depression is highly associated with postdialysis fatigue, but the cause-effect relationship is unclear.

Analysis, reanalysis, and self-analysis.

This paper describes a training analysis which required a reanalysis. An evaluation of the circumstances leading to the need for further psychoanalysis for the practicing analyst is detailed. A discussion of the value and necessity for self-analysis following analysis is presented. The description of a specific technique for conducting a self-analysis is rendered.

Maternal plasma and amniotic fluid 17 beta-estradiol, progesterone and cortisol concentrations in women with successfully and unsuccessfully treated preterm labor.

Maternal plasma and amniotic fluid (AF) were obtained for measurement of 17 beta-estradiol, progesterone and cortisol concentrations from 40 patients with preterm labor and intact membranes at 28-32 weeks of gestation: 20 delivered preterm and the remaining 20 patients responded to tocolytic treatment and delivered at term. Maternal plasma and AF concentrations of these hormones were measured with specific commercially available radioimmunoassay kits. Maternal plasma and AF 17 beta-estradiol concentrations were significantly higher in women who delivered preterm than in those who delivered at term, 8.0 ng/ml vs 3.5 ng/ml and 0.85 ng/ml vs. 0.6 ng/ml, respectively. No significant differences were found between groups in maternal plasma and AF progesterone concentrations. Maternal plasma cortisol concentrations were higher in the preterm delivery group than in the term group (235 ng/ml vs. 55 ng/ml, respectively). No significant differences were found in AF cortisol concentrations between groups.

Poly(methylene blue)-modified thick-film gold electrodes for the electrocatalytic oxidation of NADH and their application in glucose biosensors.

Electropolymerization of the phenothiazine derivative methylene blue (MB) on screen-printed, thick-film gold electrodes leads to electrocatalytically active and conducting layers of poly(methylene blue) (PMB) in intimate and stable contact with the electrode surface. The catalytic properties of the PMB films allow anodic oxidation of NADH at potentials as low as +200 mV vs. the saturated calomel electrode (SCE) reducing interferences from cooxidizable species as well as minimizing electrode fouling by enabling a simultaneous two-electron transfer mechanism. Dehydrogenase-based biosensors employing PMB-modified thick-film electrodes are obtained either by entrapment of the enzyme into the PMB layer itself or by laminating an enzyme membrane made of an aqueous poly(vinylacetate) dispersion over the PMB-modified electrode. Both methods are used to fabricate glucose biosensors which can be operated at low overpotentials, i.e. +200 mV vs. SCE.

Recruitment of lymphocytes during cutaneous delayed hypersensitivity in nonhuman primates is dependent on E-selectin and vascular cell adhesion molecule 1.

Previous investigations of cutaneous delayed hypersensitivity (DHR) in humans and animals have demonstrated that lymphocyte recruitment from blood is temporally and spatially associated with the de novo, asynchronous expression of both vascular cell adhesion molecule 1 (VCAM-1) and E-selectin on dermal endothelium. In this study, DHR was induced in rhesus monkeys sensitized against tuberculin in order to investigate the contribution of E-selectin and VCAM-1 in lymphocyte recruitment to skin. Intravenous infusions of neutralizing doses of F(ab')2 fragments of murine antibodies to either E-selectin or VCAM-1 during the early inductive phases of DHR showed that murine IgG localized to dermal endothelium at the site of DHR in a pattern kinetically similar to the expression of each endothelial adhesion protein. Most importantly, the relative numbers of lymphocytes localized to the inflammatory site were significantly reduced in DHR modified with infusions of antibodies to either VCAM-1 or E-selectin, while the numbers of lymphocytes recruited to skin in the animal given F(ab')2 fragments of an irrelevant murine monoclonal antibody of the same isotype and at the same dose were not changed. Moreover, in individual animals, the relative inhibition achieved with a particular antibody was proportional to the magnitude of expression of the targeted adhesion protein. Therefore, both VCAM-1 and E-selectin are functionally relevant in the genesis of cutaneous DHR, and each appears to contribute to lymphocyte recruitment in relation to its relative degree of expression in any one particular animal.