Metallodrug-protein interaction probed by synchrotron terahertz and neutron scattering spectroscopy.

This experimental work applied coherent synchrotron-radiation terahertz spectroscopy and inelastic neutron scattering to address two processes directly associated with the mode of action of metal-based anticancer agents that can severely undermine chemotherapeutic treatment: drug binding to human serum albumin, occurring during intravenous drug transport, and intracellular coordination to thiol-containing biomolecules (such as metallothioneins) associated with acquired drug resistance. Cisplatin and two dinuclear platinum (Pt)- and palladium (Pd)-polyamine agents developed by this research group, which have yielded promising results toward some types of human cancers, were investigated. Complementary synchrotron-radiation-terahertz and inelastic neutron scattering data revealed protein metalation, through S- and N-donor ligands from cysteine, methionine, and histidine residues. A clear impact of the Pt and Pd agents was evidenced, drug binding to albumin and metallothionein having been responsible for significant changes in the overall protein conformation, as well as for an increased flexibility and possible aggregation.

Engineering Corynebacterium glutamicum for the production of 2,3-butanediol.

BACKGROUND: 2,3-Butanediol is an important bulk chemical with a wide range of applications. In bacteria, this metabolite is synthesised from pyruvate via a three-step pathway involving α-acetolactate synthase, α-acetolactate decarboxylase and 2,3-butanediol dehydrogenase. Thus far, the best producers of 2,3-butanediol are pathogenic strains, hence, the development of more suitable organisms for industrial scale fermentation is needed. Herein, 2,3-butanediol production was engineered in the Generally Regarded As Safe (GRAS) organism Corynebacterium glutamicum. A two-stage fermentation process was implemented: first, cells were grown aerobically on acetate; in the subsequent production stage cells were used to convert glucose into 2,3-butanediol under non-growing and oxygen-limiting conditions. RESULTS: A gene cluster, encoding the 2,3-butanediol biosynthetic pathway of Lactococcus lactis, was assembled and expressed in background strains, C. glutamicum ΔldhA, C. glutamicum ΔaceEΔpqoΔldhA and C. glutamicum ΔaceEΔpqoΔldhAΔmdh, tailored to minimize pyruvate-consuming reactions, i.e., to prevent carbon loss in lactic, acetic and succinic acids. Producer strains were characterized in terms of activity of the relevant enzymes in the 2,3-butanediol forming pathway, growth, and production of 2,3-butanediol under oxygen-limited conditions. Productivity was maximized by manipulating the aeration rate in the production phase. The final strain, C. glutamicum ΔaceEΔpqoΔldhAΔmdh(pEKEx2-als,aldB,Ptuf butA), under optimized conditions produced 2,3-butanediol with a 0.66 mol mol(-1) yield on glucose, an overall productivity of 0.2 g L(-1) h(-1) and a titer of 6.3 g L(-1). CONCLUSIONS: We have successfully developed C. glutamicum into an efficient cell factory for 2,3-butanediol production. The use of the engineered strains as a basis for production of acetoin, a widespread food flavour, is proposed.

Carbon flux analysis by 13C nuclear magnetic resonance to determine the effect of CO2 on anaerobic succinate production by Corynebacterium glutamicum.

Wild-type Corynebacterium glutamicum produces a mixture of lactic, succinic, and acetic acids from glucose under oxygen deprivation. We investigated the effect of CO2 on the production of organic acids in a two-stage process: cells were grown aerobically in glucose, and subsequently, organic acid production by nongrowing cells was studied under anaerobic conditions. The presence of CO2 caused up to a 3-fold increase in the succinate yield (1 mol per mol of glucose) and about 2-fold increase in acetate, both at the expense of l-lactate production; moreover, dihydroxyacetone formation was abolished. The redistribution of carbon fluxes in response to CO2 was estimated by using (13)C-labeled glucose and (13)C nuclear magnetic resonance (NMR) analysis of the labeling patterns in end products. The flux analysis showed that 97% of succinate was produced via the reductive part of the tricarboxylic acid cycle, with the low activity of the oxidative branch being sufficient to provide the reducing equivalents needed for the redox balance. The flux via the pentose phosphate pathway was low (~5%) regardless of the presence or absence of CO2. Moreover, there was significant channeling of carbon to storage compounds (glycogen and trehalose) and concomitant catabolism of these reserves. The intracellular and extracellular pools of lactate and succinate were measured by in vivo NMR, and the stoichiometry (H(+):organic acid) of the respective exporters was calculated. This study shows that it is feasible to take advantage of natural cellular regulation mechanisms to obtain high yields of succinate with C. glutamicum without genetic manipulation.

Early nutritional intervention does not prevent long-term adverse events in women with breast cancer: A pilot study.

AIM: This study aims to evaluate the effect of early nutritional intervention on adverse clinical events in women with breast cancer undergoing neoadjuvant chemotherapy. DESIGN AND SETTINGS: This is a randomized clinical trial performed at the beginning of neoadjuvant chemotherapy for women with breast cancer treated at an oncology referral center (Brazil) and followed until the end of radiotherapy period, at least. Registered under ClinicalTrials.gov Identifier no. RBR-3SHHXS. METHODS: Participants were allocated to a control group - CG (nutritional guidance on healthy eating practices) or an intervention group - IC (nutritional guidance and individualized food plan). Chemotherapy toxicity (primary endpoint) was considered a precocious adverse clinical event and it was evaluated by self-reported gastrointestinal symptoms observed at any time during the first three cycles of treatment. Post-surgical complications, radiotherapy toxicity, and weight change were considered long-term adverse events. RESULTS: 34 women (19 in the IG and 15 in the CG) were evaluated. The early nutritional intervention was associated with low gastrointestinal chemotoxicity (nausea, vomiting, and constipation, p < 0.001, p < 0.048, and p < 0.024, respectively). However, there were no statically significant differences between both groups in the presence of long-term adverse events (radiotherapy toxicity-88.2% vs 76.9%, weight loss-21.1% vs 26.7% for IC and CG respectively, p > 0.05 for both). CONCLUSION: The early nutritional intervention was associated with a low frequency of precocious events, but not with long-term adverse events in women with breast cancer during treatment.

Accuracy of isolated nutrition indicators in diagnosing malnutrition and their prognostic value to predict death in patients with gastric and colorectal cancer: A prospective study.

BACKGROUND: The study aims to evaluate the accuracy of isolated nutrition indicators in diagnosing malnutrition in patients with gastric and colorectal cancer and their association with mortality. METHODS: Prospective cohort study involving patients with cancer (n = 178) attending a reference center of oncology at any point in the disease trajectory or treatment. Nutrition status was evaluated in a unique moment by body mass index (BMI), Patient-Generated Subjective Global Assessment (PG-SGA), handgrip strength (HGS), and calf circumference (CC). Kappa coefficient, accuracy, sensitivity, specificity, positive and negative predictive values, and area under the receiver operating characteristic curve (AUC) were calculated for each nutrition indicator (PG-SGA as the reference method). The Cox proportional hazards model was used to assess hazard ratio (HR) and CI of mortality. RESULTS: From the total patients, 11% were underweight, 48% were malnourished (PG-SGA B or C), 43% had low HGS, and 55% presented low CC. There were 46 deaths (25.8%). BMI, HGS, and CC showed poor and fair agreements (κ < 0.30 for all ) and poor accuracy (AUC < 0.70 for all) in identifying malnutrition by PG-SGA. After the adjustment for confounders (age, treatment performed, site, and stage of cancer), PG-SGA (HR, 2.9; 95% CI, 1.5-5.9) and low CC (HR, 2.4; 95% CI, 1.1-5.2) were independent predictors of mortality. CONCLUSION: The nutrition indicators are not accurate in diagnosing malnutrition, whereas PG-SGA and low CC could predict mortality in gastric and colorectal cancer patients. Thus, CC should be combined with PG-SGA in nutrition assessments.

Agreement between muscle mass assessments by computed tomography and calf circumference in patients with cancer: A cross-sectional study.

BACKGROUND & AIMS: Cancer influences body composition, including a loss of muscle mass (MM), associated with worse outcomes. The study aimed to evaluate the agreement between MM estimated by calf circumference (CC) and computed tomography (CT) image as a reference method. METHODS: A cross-sectional study including patients (>20 years) diagnosed with cancer attending a reference center of oncology. Spearman's correlation was performed to verify the correlation between CC and MM by CT, including skeletal muscle area - SMA and skeletal muscle index - SMI. ROC curves, Kappa coefficient, sensitivity, specificity, positive and negative predictive values were obtained. RESULTS: The study included 219 patients, age 62.9 ± 13.1 years (mean ± standard deviation). Low CC was observed in 43.8% of the patients, and 29.2% had low SMI. CC positively correlated with SMA (rho = 0.333) and SMI (rho = 0.329), and fair agreements (K = 0.268) were observed between CC and SMI, with higher and significant values for males (K = 0.332) and patients below 60 years (K = 0.419). The area under the curve (AUC) for low CC to identifying low SMI was equal to 0.685 (CI 95% 0.606-0.765). Low CC presented fair agreement to identify low SMI in the sample; however, the negative predictive value was almost 80% for all analyses. CONCLUSIONS: Low CC is not a surrogate for low SMI in patients with cancer, but it could be an alternative, non-invasive, easy-to-perform method to pre-screen patients with cancer with adequate SMI.

Engineering trehalose synthesis in Lactococcus lactis for improved stress tolerance.

Trehalose accumulation is a common cell defense strategy against a variety of stressful conditions. In particular, our team detected high levels of trehalose in Propionibacterium freudenreichii in response to acid stress, a result that led to the idea that endowing Lactococcus lactis with the capacity to synthesize trehalose could improve the acid tolerance of this organism. To this end, we took advantage of the endogenous genes involved in the trehalose catabolic pathway of L. lactis, i.e., trePP and pgmB, encoding trehalose 6-phosphate phosphorylase and ß-phosphoglucomutase, respectively, which enabled the synthesis of trehalose 6-phosphate. Given that L. lactis lacks trehalose 6-phosphate phosphatase, the respective gene, otsB, from the food-grade organism P. freudenreichii was used to provide the required activity. The trehalose yield was approximately 15% in resting cells and in mid-exponential-phase cells grown without pH control. The intracellular concentration of trehalose reached maximal values of approximately 170 mM, but at least 67% of the trehalose produced was found in the growth medium. The viability of mutant and control strains was examined after exposure to heat, cold or acid shock, and freeze-drying. The trehalose-producing strains showed improved tolerance (5- to 10-fold-higher survivability) to acid (pH 3) and cold shock (4°C); there was also a strong improvement in cell survival in response to heat shock (45°C), and no protection was rendered against dehydration. The insight provided by this work may help the design of food-grade strains optimized for the dairy industry as well as for oral drug delivery.

Low skeletal muscle radiodensity is the best predictor for short-term major surgical complications in gastrointestinal surgical cancer: A cohort study.

The aim of this study was to evaluate whether body composition, muscle function, and their association are predictive factors for short-term postoperative complications in patients with gastric and colorectal cancer. A prospective cohort study was conducted with patients undergoing resection of gastric and colorectal tumors. Nutritional status was assessed using Patient-Generated Subjective Global Assessment (PG-SGA) and anthropometric techniques. Low handgrip strength (HGS) was observed when <16kg for women, and <27kg for men. Computed tomography images were used to measure visceral adipose tissue, skeletal muscle index (SMI), and skeletal muscle radiodensity (SMD). Complications of grade II or above (according to Clavien-Dindo's classification) were considered in a follow-up period of up to 30 days after surgery. Major complications were defined when they reached grade III or above. A total of 84 patients were analyzed (57.1% female, 59.7 ± 12.6 years) and 19% were diagnosed with low HGS + low SMI or SMD. Postoperative complications occurred in 51.2%, and these patients presented significantly longer duration of surgery and hospital stay. Major complications were observed in 16.7% of the total number of patients. Binary logistic regression adjusted by age, sex, and tumor staging showed that low SMD, low HGS + low SMI or SMD, and obesity were independent risk factors for postoperative complications, but only low SMD was an independent risk factor for major postoperative complications. Low SMD is an independent risk factor for short-term major complications following surgery in patients with gastric and colorectal cancer.

Is perioperative fasting associated with complications, length of hospital stay and mortality among gastric and colorectal cancer patients? A cohort study.

BACKGROUND: During a surgical procedure, patients are often subjected to fasting for times that are more prolonged than the ideal, which may lead to complications. OBJECTIVE: To evaluate the duration of perioperative fasting and its association with postoperative complications, length of hospital stay (LOS) and mortality among gastric and colorectal cancer patients. DESIGN AND SETTING: Cohort study developed in a surgical oncology hospital in the city of Natal (Rio Grande do Norte, Brazil). METHODS: Patients aged over 18 years were included. The Clavien-Dindo surgical complication scale was used to evaluate occurrences of postoperative complications. LOS was defined as the number of days for which patients stayed in the hospital after surgery, or until the day of death. RESULTS: Seventy-seven patients participated (59.8 ± 11.8 years; 54.5% females; 70.1% with bowel tumor). The incidences of postoperative complications and death were 59.7% and 3.9%, respectively. The duration of perioperative fasting was 59.0 ± 21.4 hours, and it was higher among non-survivors and among patients with prolonged hospital stay (≥ 6 days). For each one-hour increase in the durations of perioperative and postoperative fasting, the odds of prolonged hospitalization increased by 12% (odds ratio, OR = 1.12; 95% confidence interval, CI 1.04-1.20) and 5% (OR = 1.05; 95% CI 1.02-1.08), respectively. CONCLUSION: Prolonged perioperative fasting, especially in the postoperative period, was observed in a sample of patients with gastric and colorectal cancer, and this was an independent predictor of LOS.

Low calf circumference is an independent predictor of mortality in cancer patients: A prospective cohort study.

OBJECTIVE: Loss of muscle mass is associated with worse outcomes in patients with cancer. The objective of this study was to evaluate the prognostic value of calf circumference (CC) and skeletal muscle index from computed tomography (CT) to predict mortality in patients with cancer. METHODS: A single-center prospective study was conducted with patients aged ≥20 y attending a reference center of oncology and who had recent abdominal CT images. Data were collected through a semistructured form and patients' records and included sociodemographic data (sex, age and ethnicity), clinical data (primary site and staging of tumor and treatments performed), anthropometric variables (body mass index and CC), and outcome (death). Low CC for men was considered to be ≤ 34 cm and for women ≤ 33 cm. Muscle mass was assessed by CT images at the level of L3. The Cox proportional hazard model adjusted for age, sex, and staging of disease was used. RESULTS: A total of 250 patients were evaluated, 52.8% female, with a median age of 63 y (interquartile ratio: 55-73). Normal body mass index was identified in 44.4%; 29.2% had low skeletal muscle index, and 46.4% had low CC. Death by any cause occurred in 16%, and only low CC was a significant predictor of mortality (hazard ratio = 3.01; confidence interval 1.52-5.98; P = 0.002). CONCLUSIONS: Low CC can predict risk of mortality in this cohort of patients. The findings suggest the use of CC as a simple, easy, cost-effective anthropometric measurement to quickly screen patients at risk of death who could benefit from targeted care to improve their prognosis.

Evaluation of the Enzymatic Arsenal Secreted by Myceliophthora thermophila During Growth on Sugarcane Bagasse With a Focus on LPMOs.

The high demand for energy and the increase of the greenhouse effect propel the necessity to develop new technologies to efficiently deconstruct the lignocellulosic materials into sugars monomers. Sugarcane bagasse is a rich polysaccharide residue from sugar and alcohol industries. The thermophilic fungus Myceliophthora thermophila (syn. Sporotrichum thermophilum) is an interesting model to study the enzymatic degradation of biomass. The genome of M. thermophila encodes an extensive repertoire of cellulolytic enzymes including 23 lytic polysaccharide monooxygenases (LPMOs) from the Auxiliary Activity family 9 (AA9), which are known to oxidatively cleave the ß-1,4 bonds and boost the cellulose conversion in a biorefinery context. To achieve a deeper understanding of the enzymatic capabilities of M. thermophila on sugarcane bagasse, we pretreated this lignocellulosic residue with different methods leading to solids with various cellulose/hemicellulose/lignin proportions and grew M. thermophila on these substrates. The secreted proteins were analyzed using proteomics taking advantage of two mass spectrometry methodologies. This approach unraveled the secretion of many CAZymes belonging to the Glycosyl Hydrolase (GH) and AA classes including several LPMOs that may contribute to the biomass degradation observed during fungal growth. Two AA9 LPMOs, called MtLPMO9B and MtLPMO9H, were selected from secretomic data and enzymatically characterized. Although MtLPMO9B and MtLPMO9H were both active on cellulose, they differed in terms of optimum temperatures and regioselectivity releasing either C1 or C1-C4 oxidized oligosaccharides, respectively. LPMO activities were also measured on sugarcane bagasse substrates with different levels of complexity. The boosting effect of these LPMOs on bagasse sugarcane saccharification by a Trichoderma reesei commercial cocktail was also observed. The partially delignified bagasse was the best substrate considering the oxidized oligosaccharides released and the acid treated bagasse was the best one in terms of saccharification boost.

Secretome of HepG2 cells infected with dengue virus: implications for pathogenesis.

Viral hemorrhagic fever is a clinical syndrome that poses serious global health threat. Among the causative agents, dengue virus (DV) has the highest incidence rate and its infection is the major cause of viral hemorrhagic fever in the world. Although the pathophysiological mechanisms of DV-induced diseases are not yet understood, it is well accepted that liver is a site of viral replication. In this study, we used proteomics to analyze infection of a hepatic cell lineage, HepG2, with DV, focusing on the secreted proteins. 1D-electrophoresis and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) were used, allowing the identification of a total of 107 proteins, among which 35 were found only in control secretome and 24 only in infected cells secretome. To validate these data, we performed 2D-eletrophoresis followed by MALDI-TOF/TOF, resulting in the identification of 20 proteins, 8 of them confirming LC-MS/MS results. We discuss the results obtained taking into account the proteins previously described in the secretome of HepG2 cells, proteins present in human plasma and proteins of interest for dengue pathogenesis. Altogether the data presented here provide clues for the progress in the understanding of the role of liver secretion in the progression of the disease.

Association Between Auditory and Visual Continuous Performance Tests in Students With ADHD.

OBJECTIVE: Continuous Performance Tests (CPTs) are known to measure inattention and impulsivity in students with ADHD. Many CPTs utilize a visual format. It is accepted that auditory tasks reflect attentional demand more closely in the classroom. Thus, the association between deficits found by auditory and visual CPTs needs to be studied. We hypothesized that impulsivity would be dependent on sensory modality and inattention would be a unitary cross-modal construct. METHOD: Forty-four students with ADHD performed two CPTs (visual and auditory). We analyzed correlations between the variables examined by the two tasks. RESULTS: There were strong correlations between variables measuring inattention. Correlations between auditory and visual measures of impulsivity were weak. CONCLUSION: Inattention is partially independent of modality. In contrast, response inhibition is modality-specific. Although ADHD is defined regardless of modality, hyperactive students may exhibit deficits in the auditory modality but not in the visual modality or vice versa.

Identification and characterization of a new member of snake venom thrombin inhibitors from Bothrops insularis using a proteomic approach.

Snake venom C-type lectin-like proteins (CLPs) are ubiquitously found in Viperidae snake venoms and differ from the C-type lectins as they display different biological activities but no carbohydrate-binding activity. Previous analysis of the transcriptome obtained from the Bothrops insularis venom gland showed the presence of two clusters homologous to bothrojaracin (BJC) chains alpha and beta. In an effort to identify a new BJC-like molecule, we used an approach associated with proteomic technologies to identify the presence of the expressed protein and then to purify and characterize a new thrombin inhibitor from B. insularis venom. We also constructed homology models of this protein and BJC, which were compared with other C-type lectin-like family members and revealed several conserved features of this intriguing snake venom toxin family.

Neuropsychiatric assessments in patients with multiple sclerosis in early phases and with low disability.

BACKGROUND: In the early phases of multiple sclerosis (MS), patients exhibit slight neuropsychiatric deficits that can only be detected using reliable tools. AIM: The present investigation aimed to examine neuropsychological performance in 35 patients with incipient MS. PATIENTS AND METHODS: For the MS group, the inclusion criteria included time of disease <3 years and low disability. The neuropsychological battery consisted of Rey Auditory Learning Test, Controlled Oral Word Association Test, Hooper Visual Organization Test, and Symbol Digit Modalities Test (SDMT). RESULTS: After correction for the educational level, no significant effect of MS on performance was found for all the tests except for the number of errors of the SDMT (NE-SDMT). Higher levels of education were associated with better performances in all tests, except for the NE-SDMT. MS patients made more errors than the controls. CONCLUSION: The effect on the NE-SDMT may reflect difficulties in the ability to inhibit inadequate responses. Patients may exhibit impulsive control disorders in incipient MS, independent of their educational level.

Use of genetically modified bacteria for drug delivery in humans: Revisiting the safety aspect.

The use of live, genetically modified bacteria as delivery vehicles for biologics is of considerable interest scientifically and has attracted significant commercial investment. We have pioneered the use of the commensal gut bacterium Bacteroides ovatus for the oral delivery of therapeutics to the gastrointestinal tract. Here we report on our investigations of the biological safety of engineered B. ovatus bacteria that includes the use of thymineless death as a containment strategy and the potential for the spread of transgenes in vivo in the mammalian gastrointestinal tract. We demonstrate the ability of GM-strains of Bacteroides to survive thymine starvation and overcome it through the exchange of genetic material. We also provide evidence for horizontal gene transfer in the mammalian gastrointestinal tract resulting in transgene-carrying wild type bacteria. These findings sound a strong note of caution on the employment of live genetically modified bacteria for the delivery of biologics.

What does handedness reveal about ADHD? An analysis based on CPT performance.

BACKGROUND: Attention-deficit/hyperactivity disorder (ADHD) is a developmental disorder. Continuous performance Tests (CPTs) aid the diagnosis. Handedness is linked to disabilities. OBJECTIVES: 1-To study the association between handedness and ADHD; 2-To verify the usefulness of the CPT in school settings; 3-To exam the relationship between handedness and CPT performance. METHOD: Each child was classified as right-consistent, left-consistent, or non-consistent. From the sample, 171 controls and 68 ADHDs fulfilled the inclusion criteria. The effect of handedness on the CPT was studied using a paired-sample that matched handedness by age, grade, gender, and ADHD. RESULTS: Left-handed students had a probability of suffering from ADHD 2.88 greater than right-handers. ANOVAs on standardized scores indicated that the ADHD students exhibited higher number of errors and higher variability of reaction times as compared to the controls. Discriminant analysis indicated that these CPT parameters could discriminate ADHD from controls. Repeated ANOVAs showed a significant effect of handedness on commission errors (CE) because left-handers made more CEs than right-handers. CONCLUSIONS: 1-The association between ADHD and handedness reflects that left-handers are less lateralized and have decreased interhemispheric connections; 2-The CPT can be used to measure different attention domains in school settings; 3- Left-handers have problems in the impulsive/hyperactivity domain.

Giant cell fibroma: a case report.

This article describes a case of giant cell fibroma in a 3-year-old boy, which is an uncommon age for this lesion. The cause of this pathology has not been determined. The lesion was excised by electro surgery and submitted to a histological exam, which confirmed the diagnosis. No recurrence was observed. Pediatric dentists should be capable to diagnose and treat this kind of lesion despite this lesion being an unusual pathology among children.

Proteomic analysis reveals differentially secreted proteins in the urine from patients with clear cell renal cell carcinoma.

OBJECTIVE: The aim of this study was to evaluate the differentially secreted protein profile in the urine from patients with clear cell renal cell carcinoma (ccRCC) using mass spectrometry-based methods. Urine composition can reflect kidney physiology and can be used to detect markers for renal diseases. Moreover, characterization of the secretome is likely to assist in the investigation of new drugs for biological targets and diagnose the ccRCC at an early stage. METHODS AND MATERIALS: Urine samples from patients were divided according to Fuhrman degree (FI-IV), which was associated with the cellular differentiation as good prognosis (GP) and poor prognosis (PP). Healthy individuals were used as the control group (CG). We used both qualitative and quantitative mass spectrometry-based analyses that involved the following approaches: 1-dimensional gel electrophoresis combined with liquid chromatography mass spectrometry in tandem (1DE LC-MS/MS), in-solution digestion combined with label-free 1-dimensional LC-MS(E) (1D LC-MS(E)), and bidimensional gel electrophoresis combined with matrix-assisted laser desorption/ionization time of flight in tandem (2DE MALDI-TOF/TOF) or combined with LC-MS/MS. RESULTS: All the strategies allowed the identification of 354 proteins from the CG, GP, and PP groups. Qualitative experiments using 1DE LC-MS/MS analysis detected different protein profiles, and 224 proteins were identified in all groups. The label-free MS(E) quantitative analysis identified 113 proteins and generated novel information on secreted protein profiles, including 49 up-secreted proteins in the urine from patients with ccRCC and 40 down-secreted proteins related to the CG. Proteins such as kininogen-1, uromodulin, apolipoprotein D, polyubiquitin, and CD59 glycoprotein were down secreted according to the groups CG>GP>PP. In contrast, apolipoprotein A, fibrinogen, and haptoglobin were up secreted in patient groups. The same expression profile observed for kininogen-1, apolipoprotein D, fibrinogen, and haptoglobin was corroborated by 2DE LC-MS/MS or 2DE MALDI-TOF/TOF analyses. These 2 strategies also showed 13 differentially secreted proteins among the 3 groups. CONCLUSIONS: The proteins kininogen-1, apolipoprotein D, fibrinogen, and haptoglobin presented similar quantitative protein profiles according to MS(E) and 2DE approaches. The latter proteins were up secreted and the former ones were down-regulated. The strategies used proved to be valuable in identifying proteins that were differentially secreted in urine from patients with RCC.

Identification of novel bradykinin-potentiating peptides and C-type natriuretic peptide from Lachesis muta venom.

The generation of expressed sequence tags (ESTs) from the pit-viper snake Lachesis muta venom glands allowed us to identify two cDNA isoforms which encode the precursors for bradykinin-potentiating peptides (BPPs) and a C-type natriuretic peptide (CNP). The sequence data derived from these cDNAs combined with the venom peptides identification using MALDI-TOF mass spectrometry analysis predicted that these molecules are the precursor protein isoforms that are further processed to produce five novel BPPs and a CNP. They were identified directly in crude venom using MALDI-TOF. The BPPs sequences were further confirmed by MALDI-TOF/TOF de novo sequencing, and an unusual BPP with a residue of tryptophan at the N-terminus (usually it is pyroglutamate) was identified. The putative processing steps required to form the mature BPPs and CNP seem to be similar to those proposed for the ones found in the venom of Bothrops jararaca and Glodyus blomhoffi.