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1.  The Use of a Heterogeneously Controlled Mouse Population Reveals a Significant Correlation of Acute Phase Parasitemia with Mortality in Chagas Disease 
PLoS ONE  2014;9(3):e91640.
Chagas disease develops upon infection with the protozoan parasite Trypanosoma cruzi and undergoes an acute phase characterized by massive parasite replication and the presence of parasites in the blood. This condition is known as acute phase parasitemia. This initial stage may result in a cure, in the development of the chronic stages of the disease or in the death of the infected host. Despite intensive investigation related to the characterization of the acute and chronic phases of the disease, the cause-effect relationship of acute phase parasitemia to the outcome of the disease is still poorly understood. In this study, we artificially generated a heterogeneously controlled mouse population by intercrossing F1 mice obtained from a parental breeding of highly susceptible A/J with highly resistant C57BL/6 mouse strains. This F2 population was infected and used to assess the correlation of acute phase parasitemia with the longevity of the animals. We used nonparametric statistical analyses and found a significant association between parasitemia and mortality. If males and females were evaluated separately, we found that the former were more susceptible to death, although parasitemia was similar in males and females. In females, we found a strong negative correlation between parasitemia and longevity. In males, however, additional factors independent of parasitemia may favor mouse mortality during the development of the disease. The correlations of acute phase parasitemia with mortality reported in this study may facilitate an appropriate prognostic approach to the disease in humans. Moreover, these results illustrate the complexity of the mammalian genetic traits that regulate host resistance during Chagas disease.
doi:10.1371/journal.pone.0091640
PMCID: PMC3961278  PMID: 24651711
2.  Structural and Molecular Modeling Features of P2X Receptors 
Currently, adenosine 5′-triphosphate (ATP) is recognized as the extracellular messenger that acts through P2 receptors. P2 receptors are divided into two subtypes: P2Y metabotropic receptors and P2X ionotropic receptors, both of which are found in virtually all mammalian cell types studied. Due to the difficulty in studying membrane protein structures by X-ray crystallography or NMR techniques, there is little information about these structures available in the literature. Two structures of the P2X4 receptor in truncated form have been solved by crystallography. Molecular modeling has proven to be an excellent tool for studying ionotropic receptors. Recently, modeling studies carried out on P2X receptors have advanced our knowledge of the P2X receptor structure-function relationships. This review presents a brief history of ion channel structural studies and shows how modeling approaches can be used to address relevant questions about P2X receptors.
doi:10.3390/ijms15034531
PMCID: PMC3975412  PMID: 24637936
P2X7 receptor; ion channel activity; patch-clamp
3.  Severe maternal morbidity and maternal near miss in the extremes of reproductive age: results from a national cross- sectional multicenter study 
Background
The aim of this study was to assess severe maternal morbidity (SMM) and near miss (NM) cases among adolescent girls and women over 35 years of age in the Brazilian Network for Surveillance of Severe Maternal Morbidity, using a set of standard criteria, compared to pregnant women aged 20 to 34 years.
Methods
A cross-sectional multicenter study conducted in 27 referral obstetric units in Brazil. All pregnant women admitted to these centers during a one-year period of prospective surveillance were screened to identify cases of maternal death (MD), NM and other SMM. Indicators of maternal morbidity and mortality were evaluated for the three age groups. Sociodemographic, clinical and obstetric characteristics, gestational and perinatal outcomes, main causes of morbidity and delays in care were also compared. Two multiple analysis models were performed, to estimate the adjusted prevalence ratio for identified factors that were independently associated with the occurrence of severe maternal outcome (SMO = MNM + MD).
Results
Among SMM and MD cases identified, the proportion of adolescent girls and older women were 17% each. The risk of MNM or death was 25% higher among older women. Maternal near miss ratio and maternal mortality ratios increased with age, but these ratios were also higher among adolescents aged 10 to 14, although the absolute numbers were low. On multivariate analysis, younger age was not identified as an independent risk factor for SMO, while this was true for older age (PR 1.25; 1.07-1.45).
Conclusions
SMO was high among women below 14 years of age and increased with age in Brazilian pregnant women.
doi:10.1186/1471-2393-14-77
PMCID: PMC3975952  PMID: 24555831
Maternal near-miss; Obstetric complication; Maternal death; Maternal age; Extremes of reproductive age; Maternal morbidity
4.  Gastroprotective and Ulcer Healing Effects of Essential Oil of Hyptis martiusii Benth. (Lamiaceae) 
PLoS ONE  2014;9(1):e84400.
Hyptis martiusii Benth. is an aromatic plant found in abundance in northeastern Brazil that is used in ethnomedicine to treat gastric disorders. The aim of this study was to elucidate the mechanisms of action involved in the gastroprotection of the essential oil of Hyptis martiusii (EOHM) and to evaluate its healing capacity. Wistar rats were exposed to different protocols and subsequently were treated with 1% Tween-80 aqueous solution (negative control), pantoprazole, carbenoxolone, N-acetylcysteine (depending on the specificity of each model) or EOHM. The antisecretory activity (basal or stimulated) was determined using the pyloric ligature method. The gastroprotective action of nitric oxide and sulphydryl groups (–SH groups), as well as the quantification of adherent mucus and the levels of malondialdehyde and –SH groups in gastric mucosa, were evaluated using ethanol-induced gastric ulcer model. The healing ability was evaluated using the acetic acid-induced gastric ulcer model and histological and immunohistochemical analysis (HE, PAS and PCNA). EOHM (400 mg/kg) reduced the volume and acidity of gastric secretion stimulated by histamine and pentagastrin. The gastroprotective effect of EOHM involves the participation of endogenous sulfhydryl groups. EOHM increased mucus production (54.8%), reduced levels of MDA (72.5%) and prevented the depletion of –SH groups (73.8%) in the gastric mucosa. The treatment with EOHM reduced in 70.3% the gastric lesion area, promoting significant regeneration of the gastric mucosa, as confirmed by histological analysis and analysis of proliferating cell nuclear antigen. The results show that gastroprotective effect of EOHM is mediated by cytoprotective and antioxidant mechanisms and by their antisecretory activity, and suggest that the essential oil of Hyptis martiusii is a promising candidate for the treatment of gastric ulcers.
doi:10.1371/journal.pone.0084400
PMCID: PMC3893125  PMID: 24454726
5.  Conservative Treatment of Uterine Cervical Adenocarcinoma in Pregnancy 
Cancer of the cervix is the most common malignancy diagnosed during pregnancy, with an incidence of 1–10 cases per 10,000 pregnancies. The desire of patients to maintain pregnancy and subsequent fertility is a difficult target to be achieved and should be widely studied, since it depends on the stage of disease, gestational age at diagnosis, and the woman's desire to maintain pregnancy. We describe in this report the case of a pregnant woman with invasive cervical adenocarcinoma in stage IB1 (FIGO) initially treated with neoadjuvant chemotherapy, followed by radical surgery and cesarean section in the same surgical procedure.
doi:10.1155/2013/692017
PMCID: PMC3877580  PMID: 24455354
6.  Physiological Responses of Zostera marina and Cymodocea nodosa to Light-Limitation Stress 
PLoS ONE  2013;8(11):e81058.
The effects of light-limitation stress were investigated in natural stands of the seagrasses Zostera marina and Cymodocea nodosa in Ria Formosa coastal lagoon, southern Portugal. Three levels of light attenuation were imposed for 3 weeks in two adjacent meadows (2–3 m depth), each dominated by one species. The response of photosynthesis to light was determined with oxygen electrodes. Chlorophylls and carotenoids were determined by high-pressure liquid chromatography (HPLC). Soluble protein, carbohydrates, malondialdehyde and phenol contents were also analysed. Both species showed evident signs of photoacclimation. Their maximum photosynthetic rates were significantly reduced with shading. Ratios between specific light harvesting carotenoids and the epoxidation state of xanthophyll cycle carotenoids revealed significantly higher light harvesting efficiency of C. nodosa, a competitive advantage in a low light environment. The contents of both soluble sugars and starch were considerably lower in Z. marina plants, particularly in the rhizomes, decreasing even further with shading. The different carbohydrate energy storage strategies found between the two species clearly favour C. nodosa's resilience to light deprivation, a condition enhanced by its intrinsic arrangement of the pigment pool. On the other hand, Z. marina revealed a lower tolerance to light reduction, mostly due to a less plastic arrangement of the pigment pool and lower carbohydrate storage. Our findings indicate that Z. marina is close to a light-mediated ecophysiological threshold in Ria Formosa.
doi:10.1371/journal.pone.0081058
PMCID: PMC3842938  PMID: 24312260
7.  Hand, foot, and mouth syndrome in an immunocompetent adult: a case report 
BMC Research Notes  2013;6:441.
Background
Hand, foot, and mouth syndrome (HFMS) is a common acute illness. It is characterized by mild clinical symptoms including fever, blisters, and sores in the mouth and on the palms and soles following a 3- to 7-day incubation period. This syndrome is rarely seen in adults.
Case presentation
A 35-year-old male Caucasian patient had a history of multiple episodes of acute pharyngitis, hypertension, hypercholesterolemia, and occasional abdominal pain. He presented with polyarthralgia in the knees and hands and odynophagia, followed by fever, oral mucosal aphthous lesions, and vesicles on the palms and soles. Three weeks after presentation, he was admitted to the emergency room with acute myocarditis. The in-hospital evaluation revealed positive serology for coxsackie A9 (1:160), positive anti-transglutaminase and anti-gliadin antibodies, normal immunoglobulins, and human immunodeficiency virus negativity.
Conclusion
We herein describe a case of HFMS that was associated with coxsackie A9 infection complicated by acute myocarditis. Although an association between celiac disease and HFMS has not been described, this patient’s immunologic disruption could have favored the development of infection and ultimately HFMS.
doi:10.1186/1756-0500-6-441
PMCID: PMC3819262  PMID: 24180226
Hand, foot, and mouth syndrome; Myopericarditis; Coxsackie A9; Celiac disease
8.  Polymorphic Sites at the Immunoregulatory CTLA-4 Gene Are Associated with Chronic Chagas Disease and Its Clinical Manifestations 
PLoS ONE  2013;8(10):e78367.
Background
Chagas disease affects approximately 10 million people mainly in Latin America. The immune regulation by the host seems to be an essential factor for disease evolution, and immune system inhibitory molecules such as CTLA-4 and PD-1 favor the maintenance of peripheral tolerance. Considering that polymorphisms at the immunoregulatory CTLA-4 and PDCD1 genes may alter their inhibitory function, we investigated the association of alleles, genotypes and haplotypes of polymorphic sites observed at the CTLA-4 and PDCD1 genes with different clinical manifestations of chronic Chagas disease (indeterminate, cardiac, digestive and mixed).
Methods
The polymorphisms at the CTLA-4 (-1722T/C, -318C/T and +49A/G) and PDCD1 (PD-1.3G/A) genes were typed using TaqMan methodology in 277 chronic Chagas disease patients classified into four groups, according to clinical characteristics, and 326 non-infected controls.
Results
Our results showed that CTLA-4 -1722CC genotype (22%), -1722C allele (27%) and CTLA-4 TCG (8.6%), TCA (26%) and CCA (15%) haplotypes were strongly associated with the indeterminate form, while the CTLA-4 -318CT genotype (82%) and CTLA-4 -318T allele (47%) were found mainly in patients with the mixed form of the disease. The CTLA-4 TCG haplotype (10.2%) was associated with the digestive form. On the other hand, the PD-1.3G/A polymorphism was not associated with chronic Chagas disease and its clinical manifestations.
Conclusions
Here, we showed that alleles, genotypes and haplotypes reported to increase the expression of the regulatory molecule CTLA-4 were associated with the indeterminate form of the disease. Taken together, our data support the idea that polymorphic sites at immunoregulatory genes may influence the development of Chagas disease variants.
doi:10.1371/journal.pone.0078367
PMCID: PMC3813449  PMID: 24205212
9.  5-Lipoxygenase Activity Increases Susceptibility to Experimental Paracoccidioides brasiliensis Infection 
Infection and Immunity  2013;81(4):1256-1266.
Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the thermodimorphic fungus Paracoccidioides brasiliensis. Leukotrienes and lipoxins are lipid mediators produced after 5-lipoxygenase (5-LO) activation that exhibit pro- and anti-inflammatory roles, respectively. Here, we have investigated the contribution of 5-LO enzymatic activity in PCM using an experimental model of P. brasiliensis infection. B6.129 wild-type (B6.129) and 5-LO-deficient (5-LO−/−) mice were intravenously inoculated with a virulent strain of P. brasiliensis (Pb18), and the survival rate of the infected mice was investigated on different days after yeast infection. 5-LO−/− mice exhibited an increased survival rate associated with a decreased number of CFU. The resistance of 5-LO−/− during PCM was associated with augmented nitric oxide (NO) production and the formation of compact granulomas. In addition, the absence of 5-LO was associated with a diminished number of CD4+ CD25+ regulatory T cells, higher levels of gamma interferon and interleukin-12, and increased T-bet (a T-box transcription factor that directs Th1 lineage commitment) mRNA levels in the lungs. Taken together, our results show for the first time that 5-LO enzymatic activity increases susceptibility to P. brasiliensis, suggesting that this pathway may be a potential target for therapeutic intervention during PCM.
doi:10.1128/IAI.01209-12
PMCID: PMC3639621  PMID: 23381993
10.  Common Fusion Transcripts Identified in Colorectal Cancer Cell Lines by High-Throughput RNA Sequencing12 
Translational Oncology  2013;6(5):546-553.
Colorectal cancer (CRC) is the third most common cancer disease in the Western world, and about 40% of the patients die from this disease. The cancer cells are commonly genetically unstable, but only a few low-frequency recurrent fusion genes have so far been reported for this disease. In this study, we present a thorough search for novel fusion transcripts in CRC using high-throughput RNA sequencing. From altogether 220 million paired-end sequence reads from seven CRC cell lines, we identified 3391 candidate fused transcripts. By stringent requirements, we nominated 11 candidate fusion transcripts for further experimental validation, of which 10 were positive by reverse transcription-polymerase chain reaction and Sanger sequencing. Six were intrachromosomal fusion transcripts, and interestingly, three of these, AKAP13-PDE8A, COMMD10-AP3S1, and CTB-35F21.1-PSD2, were present in, respectively, 18, 18, and 20 of 21 analyzed cell lines and in, respectively, 18, 61, and 48 (17%-58%) of 106 primary cancer tissues. These three fusion transcripts were also detected in 2 to 4 of 14 normal colonic mucosa samples (14%–28%). Whole-genome sequencing identified a specific genomic breakpoint in COMMD10-AP3S1 and further indicates that both the COMMD10-AP3S1 and AKAP13-PDE8A fusion transcripts are due to genomic duplications in specific cell lines. In conclusion, we have identified AKAP13-PDE8A, COMMD10-AP3S1, and CTB-35F21.1-PSD2 as novel intrachromosomal fusion transcripts and the most highly recurring chimeric transcripts described for CRC to date. The functional and clinical relevance of these chimeric RNA molecules remains to be elucidated.
PMCID: PMC3799197  PMID: 24151535
11.  Toxoplasma gondii Soluble Tachyzoite Antigen Triggers Protective Mechanisms against Fatal Intestinal Pathology in Oral Infection of C57BL/6 Mice 
PLoS ONE  2013;8(9):e75138.
Toxoplasma gondii induces a potent IL-12 response early in infection that results in IFN-γ-dependent control of parasite growth. It was previously shown that T. gondii soluble tachyzoite antigen (STAg) injected 48 hr before intraperitoneal infection reduces lipoxin A4 and 5-lipoxygenase (5-LO)-dependent systemic IL-12 and IFN-γ production as well as hepatic immunopathology. This study investigated the ability of STAg-pretreatment to control the fatal intestinal pathology that develops in C57BL/6 mice orally infected with 100 T. gondii cysts. STAg-pretreatment prolonged the animals’ survival by decreasing tissue parasitism and pathology, mainly in the ilea. Protection was associated with decreases in the systemic IFN-γ levels and IFN-γ and TNF message levels in the ilea and with increased TGF-β production in this tissue, but protection was independent of 5-LO and IL-4. STAg-pretreatment decreased CD4+ T cell, NK cell, CD11b+ monocyte and CD11b+CD11c+ dendritic cell numbers in the lamina propria and increased CD8+ T cells in the intestinal epithelial compartment. In parallel, decreases were observed in iNOS and IL-17 expression in this organ. These results demonstrate that pretreatment with STAg can induce the recruitment of protective CD8+ T cells to the intraepithelial compartment and decrease proinflammatory immune mechanisms that promote intestinal pathology in T. gondii infection.
doi:10.1371/journal.pone.0075138
PMCID: PMC3782460  PMID: 24086456
12.  Repeated-Doses Toxicity Study of the Essential Oil of Hyptis martiusii Benth. (Lamiaceae) in Swiss Mice 
Hyptis martiusii Benth. (Lamiaceae) is found in abundance in Northeastern Brazil where it is used in traditional medicine to treat gastric disorders. Since there are no studies reporting the toxicity and safety profile of this species, we investigated repeated-doses toxicity of the essential oil of Hyptis martiusii (EOHM). Swiss mice of both sexes were orally treated with EOHM (100 and 500 mg/kg) for 30 days, and biochemical, hematological, and morphological parameters were determined. No toxicity signs or deaths were recorded during the treatment with EOHM. The body weight gain was not affected, but there was an occasional variation in water and food consumption among mice of both sexes treated with both doses. The hematological and biochemical profiles did not show significant differences except for a decrease in the MCV and an increase in albumin, but these variations are within the limits described for the species. The microscopic analysis showed changes in liver, kidneys, lungs, and spleen; however, these changes do not have clinical relevance since they varied among the groups, including the control group. The results indicate that the treatment of repeated-doses with the essential oil of Hyptis martiusii showed low toxicity in mice.
doi:10.1155/2013/856168
PMCID: PMC3784159  PMID: 24151521
13.  Non-peptidic Cruzain Inhibitors with Trypanocidal Activity Discovered by Virtual Screening and In Vitro Assay 
A multi-step cascade strategy using integrated ligand- and target-based virtual screening methods was developed to select a small number of compounds from the ZINC database to be evaluated for trypanocidal activity. Winnowing the database to 23 selected compounds, 12 non-covalent binding cruzain inhibitors with affinity values (Ki) in the low micromolar range (3–60 µM) acting through a competitive inhibition mechanism were identified. This mechanism has been confirmed by determining the binding mode of the cruzain inhibitor Nequimed176 through X-ray crystallographic studies. Cruzain, a validated therapeutic target for new chemotherapy for Chagas disease, also shares high similarity with the mammalian homolog cathepsin L. Because increased activity of cathepsin L is related to invasive properties and has been linked to metastatic cancer cells, cruzain inhibitors from the same library were assayed against it. Affinity values were in a similar range (4–80 µM), yielding poor selectivity towards cruzain but raising the possibility of investigating such inhibitors for their effect on cell proliferation. In order to select the most promising enzyme inhibitors retaining trypanocidal activity for structure-activity relationship (SAR) studies, the most potent cruzain inhibitors were assayed against T. cruzi-infected cells. Two compounds were found to have trypanocidal activity. Using compound Nequimed42 as precursor, an SAR was established in which the 2-acetamidothiophene-3-carboxamide group was identified as essential for enzyme and parasite inhibition activities. The IC50 value for compound Nequimed42 acting against the trypomastigote form of the Tulahuen lacZ strain was found to be 10.6±0.1 µM, tenfold lower than that obtained for benznidazole, which was taken as positive control. In addition, by employing the strategy of molecular simplification, a smaller compound derived from Nequimed42 with a ligand efficiency (LE) of 0.33 kcal mol−1 atom−1 (compound Nequimed176) is highlighted as a novel non-peptidic, non-covalent cruzain inhibitor as a trypanocidal agent candidate for optimization.
Author Summary
Chagas disease (American trypanosomiasis) is a parasitic infection that kills millions of mostly poverty-stricken people in Latin America. In recent years it has also spread to nonendemic countries – the United States, Canada, Europe, Australia and Japan – as a result of immigration. The only available drugs for its treatment were introduced more than forty years ago, have low efficacy, and cause various severe side effects. This dire public health situation has prompted us to search for new small molecules to act as drug candidates to treat Chagas disease. The T. cruzi enzyme cruzain, a key biological catalyst used by the protozoan to digest host proteins, is a validated drug target for Chagas disease. By combining in silico molecular design, X-ray crystallography and biological screening, we found a new class of non-covalent small molecules that inhibit cruzain in low micromolar concentrations.
doi:10.1371/journal.pntd.0002370
PMCID: PMC3750009  PMID: 23991231
14.  Caffeine Increases Anaerobic Work and Restores Cycling Performance following a Protocol Designed to Lower Endogenous Carbohydrate Availability 
PLoS ONE  2013;8(8):e72025.
The purpose this study was to examine the effects of caffeine ingestion on performance and energy expenditure (anaerobic and aerobic contribution) during a 4-km cycling time trial (TT) performed after a carbohydrate (CHO) availability-lowering exercise protocol. After preliminary and familiarization trials, seven amateur cyclists performed three 4-km cycling TT in a double-blind, randomized and crossover design. The trials were performed either after no previous exercise (CON), or after a CHO availability-lowering exercise protocol (DEP) performed in the previous evening, followed by either placebo (DEP-PLA) or 5 mg.kg−1 of caffeine intake (DEP-CAF) 1 hour before the trial. Performance was reduced (−2.1%) in DEP-PLA vs CON (421.0±12.3 vs 412.4±9.7 s). However, performance was restored in DEP-CAF (404.6±17.1 s) compared with DEP-PLA, while no differences were found between DEP-CAF and CON. The anaerobic contribution was increased in DEP-CAF compared with both DEP-PLA and CON (67.4±14.91, 47. 3±14.6 and 55.3±14.0 W, respectively), and this was more pronounced in the first 3 km of the trial. Similarly, total anaerobic work was higher in DEP-CAF than in the other conditions. The integrated electromyographic activity, plasma lactate concentration, oxygen uptake, aerobic contribution and total aerobic work were not different between the conditions. The reduction in performance associated with low CHO availability is reversed with caffeine ingestion due to a higher anaerobic contribution, suggesting that caffeine could access an anaerobic “reserve” that is not used under normal conditions.
doi:10.1371/journal.pone.0072025
PMCID: PMC3747083  PMID: 23977198
15.  Maternal and Perinatal Outcomes of Exercise in Pregnant Women with Chronic Hypertension and/or Previous Preeclampsia: A Randomized Controlled Trial 
ISRN Obstetrics and Gynecology  2013;2013:857047.
Objectives. To evaluate the association between physical exercise supervised in pregnant women with chronic hypertension and/or previous preeclampsia and maternal and neonatal outcomes. Method. Randomized controlled trial, which included 116 pregnant women with chronic hypertension and/or previous preeclampsia, considered risk of preeclampsia development. They were divided into two groups: study group that performed physical exercise with a stationary bicycle once a week, for 30 minutes; the intensity was controlled (heart rate 20% above resting values), under professional supervision and a control group that was not engaged in any physical exercise. The data was retrieved from medical charts. Significance level assumed was 5%. Results. Women from study group performed 9.24 ± 7.03 of physical exercise sessions. There were no differences between groups comparing type of delivery and maternal outcomes, including maternal morbidity and hospitalization in intensive unit care, and neonatal outcomes, including birth weight, adequacy of weight to gestational age, prematurity, Apgar scale at first and fifth minutes, hospitalization in intensive unit care, and neonatal morbidity. Conclusions. Physical exercise using a stationary bicycle in pregnant women with chronic hypertension and/or previous preeclampsia, once a week, under professional supervision, did not interfere in the delivery method and did not produce maternal and neonatal risks of the occurrence of morbidity. This trial is registered with ClinicalTrials.gov NCT01395342.
doi:10.1155/2013/857047
PMCID: PMC3753734  PMID: 23997960
16.  Disruption of Calcium Homeostasis in Cardiomyocytes Underlies Cardiac Structural and Functional Changes in Severe Sepsis 
PLoS ONE  2013;8(7):e68809.
Sepsis, a major cause of morbidity/mortality in intensive care units worldwide, is commonly associated with cardiac dysfunction, which worsens the prognosis dramatically for patients. Although in recent years the concept of septic cardiomyopathy has evolved, the importance of myocardial structural alterations in sepsis has not been fully explored. This study offers novel and mechanistic data to clarify subcellular events that occur in the pathogenesis of septic cardiomyopathy and myocardial dysfunction in severe sepsis. Cultured neonatal mice cardiomyocytes subjected to serum obtained from mice with severe sepsis presented striking increment of [Ca2+]i and calpain-1 levels associated with decreased expression of dystrophin and disruption and derangement of F-actin filaments and cytoplasmic bleb formation. Severe sepsis induced in mice led to an increased expression of calpain-1 in cardiomyocytes. Moreover, decreased myocardial amounts of dystrophin, sarcomeric actin, and myosin heavy chain were observed in septic hearts associated with depressed cardiac contractile dysfunction and a very low survival rate. Actin and myosin from the sarcomere are first disassembled by calpain and then ubiquitinated and degraded by proteasome or sequestered inside specialized vacuoles called autophagosomes, delivered to the lysosome for degradation forming autophagolysosomes. Verapamil and dantrolene prevented the increase of calpain-1 levels and preserved dystrophin, actin, and myosin loss/reduction as well cardiac contractile dysfunction associated with strikingly improved survival rate. These abnormal parameters emerge as therapeutic targets, which modulation may provide beneficial effects on future vascular outcomes and mortality in sepsis. Further studies are needed to shed light on this mechanism, mainly regarding specific calpain inhibitors.
doi:10.1371/journal.pone.0068809
PMCID: PMC3720843  PMID: 23935889
19.  Novel 5′ Fusion Partners of ETV1 and ETV4 in Prostate Cancer12 
Neoplasia (New York, N.Y.)  2013;15(7):720-726.
Gene fusions involving the erythroblast transformation-specific (ETS) transcription factors ERG, ETV1, ETV4, ETV5, and FLI1 are a common feature of prostate carcinomas (PCas). The most common upstream fusion partner described is the androgen-regulated prostate-specific gene TMPRSS2, most frequently with ERG, but additional 5′ fusion partners have been described. We performed 5′ rapid amplification of cDNA ends in 18 PCas with ETV1, ETV4, or ETV5 outlier expression to identify the 5′ fusion partners. We also evaluated the exon-level expression profile of these ETS genes in 14 cases. We identified and confirmed by fluorescent in situ hybridization (FISH) and reverse transcription-polymerase chain reaction the two novel chimeric genes OR51E2-ETV1 and UBTF-ETV4 in two PCas. OR51E2 encodes a G-protein-coupled receptor that is overexpressed in PCas, whereas UBTF is a ubiquitously expressed gene encoding an HMG-box DNA-binding protein involved in ribosome biogenesis. We additionally describe two novel gene fusion combinations of previously described genes, namely, SLC45A3-ETV4 and HERVK17-ETV4. Finally, we found one PCa with TMPRSS2-ETV1, one with C15orf21-ETV1, one with EST14-ETV1, and two with 14q133-q21.1-ETV1. In nine PCas (eight ETV1 and one ETV5), exhibiting ETS outlier expression and genomic rearrangement detected by FISH, no 5′ fusion partner was found. Our findings contribute significantly to characterize the heterogeneous group of ETS gene fusions and indicate that all genes described as 5′ fusion partners with one ETS gene can most likely be rearranged with any of the other ETS genes involved in prostate carcinogenesis.
PMCID: PMC3689235  PMID: 23814484
20.  Maternal morbidity and near miss associated with maternal age: the innovative approach of the 2006 Brazilian demographic health survey 
Clinics  2013;68(7):922-927.
OBJECTIVE:
To study the prevalence of potentially life-threatening maternal conditions and near miss in Brazil according to maternal age.
METHODS:
A secondary analysis of the 2006 Brazilian demographic health survey database using a validated questionnaire to evaluate maternal morbidity with a focus on age extremes. The study included 5,025 women with at least 1 live birth in the 5-year reference period preceding their interviews. Three age range periods were used: 15-19 years (younger age), 20-34 years (control), and 35-49 years (advanced maternal age). According to a pragmatic definition, any woman reporting eclampsia, hysterectomy, blood transfusion, or admission to the intensive care unit during her pregnancy/childbirth was considered a near-miss case. The associations between age and severe maternal morbidity were further assessed.
RESULTS:
For the 6,833 reported pregnancies, 73.7% of the women were 20-34 years old, 17.9% were of advanced maternal age, and only 8.4% were of younger age. More than 22% of the women had at least one of the complications appraised, and blood transfusion, which was more prevalent among the controls, was the only variable with a significant difference among the age groups. The overall rate of maternal near miss was 21.1 per 1000 live births. There was a trend of higher maternal near miss with increasing age. The only significant risk factor identified for maternal near miss was a lower literacy level among older women.
CONCLUSIONS:
There is a trend towards worse results with increasing age. The investigation of the determinants of maternal near miss at the community level using an innovative approach through a demographic health survey is an example suggested for under-resourced settings.
doi:10.6061/clinics/2013(07)06
PMCID: PMC3715023  PMID: 23917654
Morbidity; Obstetric Complication; Pregnancy in Adolescence; Maternal Age
21.  SAG2A protein from Toxoplasma gondii interacts with both innate and adaptive immune compartments of infected hosts 
Parasites & Vectors  2013;6:163.
Background
Toxoplasma gondii is an intracellular parasite that causes relevant clinical disease in humans and animals. Several studies have been performed in order to understand the interactions between proteins of the parasite and host cells. SAG2A is a 22 kDa protein that is mainly found in the surface of tachyzoites. In the present work, our aim was to correlate the predicted three-dimensional structure of this protein with the immune system of infected hosts.
Methods
To accomplish our goals, we performed in silico analysis of the amino acid sequence of SAG2A, correlating the predictions with in vitro stimulation of antigen presenting cells and serological assays.
Results
Structure modeling predicts that SAG2A protein possesses an unfolded C-terminal end, which varies its conformation within distinct strain types of T. gondii. This structure within the protein shelters a known B-cell immunodominant epitope, which presents low identity with its closest phyllogenetically related protein, an orthologue predicted in Neospora caninum. In agreement with the in silico observations, sera of known T. gondii infected mice and goats recognized recombinant SAG2A, whereas no serological cross-reactivity was observed with samples from N. caninum animals. Additionally, the C-terminal end of the protein was able to down-modulate pro-inflammatory responses of activated macrophages and dendritic cells.
Conclusions
Altogether, we demonstrate herein that recombinant SAG2A protein from T. gondii is immunologically relevant in the host-parasite interface and may be targeted in therapeutic and diagnostic procedures designed against the infection.
doi:10.1186/1756-3305-6-163
PMCID: PMC3706231  PMID: 23735002
Toxoplasmosis; Protein modeling; SAG2A; Monoclonal antibodies; Recombinant protein
22.  Dual effect of Lutzomyia longipalpis saliva on Leishmania braziliensis infection is mediated by distinct saliva-induced cellular recruitment into BALB/c mice ear 
BMC Microbiology  2013;13:102.
Background
Leishmania parasites are transmitted to their vertebrate hosts by infected Phlebotomine sand flies during the blood meal of the flies. Sand fly saliva is known to enhance Leishmania spp. infection, while pre-exposure to saliva protects mice against parasitic infections. In this study, we investigated the initial inflammatory leucocyte composition induced by one or three inocula of salivary gland extract (SGE) from Lutzomyia longipalpis in the presence or absence of Leishmania braziliensis.
Results
We demonstrated that inoculating SGE once (SGE-1X) or three times (SGE-3X), which represented a co-inoculation or a pre-exposure to saliva, respectively, resulted in different cellular infiltrate profiles. Whereas SGE-1X led to the recruitment of all leucocytes subtypes including CD4+ T cells, CD4+CD25+ T cells, dendritic cells, macrophages and neutrophils, the immune cell profile in the SGE-3X group differed dramatically, as CD4+ T cells, CD4+CD25+ T cells, dendritic cells, macrophages and neutrophils were decreased and CD8+ T cells were increased. The SGE-1X group did not show differences in the ear lesion size; however, the SGE-1X group harbored a higher number of parasites. On the other hand, the SGE-3X group demonstrated a protective effect against parasitic disease, as the parasite burden was lower even in the earlier stages of the infection, a period in which the SGE-1X group presented with larger and more severe lesions. These effects were also reflected in the cytokine profiles of both groups. Whereas the SGE-1X group presented with a substantial increase in IL-10 production, the SGE-3X group showed an increase in IFN-γ production in the draining lymph nodes. Analysis of the inflammatory cell populations present within the ear lesions, the SGE-1X group showed an increase in CD4+FOXP3+ cells, whereas the CD4+FOXP3+ population was reduced in the SGE-3X group. Moreover, CD4+ T cells and CD8+ T cells producing IFN-γ were highly detected in the ears of the SGE-3X mice prior to infection. In addition, upon treatment of SGE-3X mice with anti-IFN-γ monoclonal antibody, we observed a decrease in the protective effect of SGE-3X against L. braziliensis infection.
Conclusions
These results indicate that different inocula of Lutzomyia longipalpis salivary gland extract can markedly modify the cellular immune response, which is reflected in the pattern of susceptibility or resistance to Leishmania braziliensis infection.
doi:10.1186/1471-2180-13-102
PMCID: PMC3655920  PMID: 23656976
Phlebotomines saliva; Lutzomyia longipalpis saliva; Leishmania braziliensis; Inflammatory leucocytes; Cytokines; Immunoregulation
23.  Plasma Lipoproteins as Mediators of the Oxidative Stress Induced by UV Light in Human Skin: A Review of Biochemical and Biophysical Studies on Mechanisms of Apolipoprotein Alteration, Lipid Peroxidation, and Associated Skin Cell Responses 
There are numerous studies concerning the effect of UVB light on skin cells but fewer on other skin components such as the interstitial fluid. This review highlights high-density lipoprotein (HDL) and low-density lipoprotein (LDL) as important targets of UVB in interstitial fluid. Tryptophan residues are the sole apolipoprotein residues absorbing solar UVB. The UVB-induced one-electron oxidation of Trp produces •Trp and •O2− radicals which trigger lipid peroxidation. Immunoblots from buffered solutions or suction blister fluid reveal that propagation of photooxidative damage to other residues such as Tyr or disulfide bonds produces intra- and intermolecular bonds in apolipoproteins A-I, A-II, and B100. Partial repair of phenoxyl tyrosyl radicals (TyrO•) by α-tocopherol is observed with LDL and HDL on millisecond or second time scales, whereas limited repair of α-tocopherol by carotenoids occurs in only HDL. More effective repair of Tyr and α-tocopherol is observed with the flavonoid, quercetin, bound to serum albumin, but quercetin is less potent than new synthetic polyphenols in inhibiting LDL lipid peroxidation or restoring α-tocopherol. The systemic consequences of HDL and LDL oxidation and the activation and/or inhibition of signalling pathways by oxidized LDL and their ability to enhance transcription factor DNA binding activity are also reviewed.
doi:10.1155/2013/285825
PMCID: PMC3655670  PMID: 23738035
24.  Altered Expression of MGMT in High-Grade Gliomas Results from the Combined Effect of Epigenetic and Genetic Aberrations 
PLoS ONE  2013;8(3):e58206.
MGMT downregulation in high-grade gliomas (HGG) has been mostly attributed to aberrant promoter methylation and is associated with increased sensitivity to alkylating agent-based chemotherapy. However, HGG harboring 10q deletions also benefit from treatment with alkylating agents. Because the MGMT gene is mapped at 10q26, we hypothesized that both epigenetic and genetic alterations might affect its expression and predict response to chemotherapy. To test this hypothesis, promoter methylation and mRNA levels of MGMT were determined by quantitative methylation-specific PCR (qMSP) or methylation-specific multiplex ligation dependent probe amplification (MS-MLPA) and quantitative RT-PCR, respectively, in a retrospective series of 61 HGG. MGMT/chromosome 10 copy number variations were determined by FISH or MS-MLPA analysis. Molecular findings were correlated with clinical parameters to assess their predictive value. Overall, MGMT methylation ratios assessed by qMSP and MS-MLPA were inversely correlated with mRNA expression levels (best coefficient value obtained with MS-MLPA). By FISH analysis in 68.3% of the cases there was loss of 10q26.1 and in 15% of the cases polysomy was demonstrated; the latter displayed the highest levels of transcript. When genetic and epigenetic data were combined, cases with MGMT promoter methylation and MGMT loss depicted the lowest transcript levels, although an impact in response to alkylating agent chemotherapy was not apparent. Cooperation between epigenetic (promoter methylation) and genetic (monosomy, locus deletion) changes affecting MGMT in HGG is required for effective MGMT silencing. Hence, evaluation of copy number alterations might add relevant prognostic and predictive information concerning response to alkylating agent-based chemotherapy.
doi:10.1371/journal.pone.0058206
PMCID: PMC3594314  PMID: 23505468
25.  Batch production of a silk-elastin-like protein in E. coli BL21(DE3): key parameters for optimisation 
Background
Silk-elastin-like proteins (SELPs) combining the physicochemical and biological properties of silk and elastin have a high potential for use in the pharmaceutical, regenerative medicine and materials fields. Their development for use is however restrained by their production levels. Here we describe the batch production optimisation for a novel recently described SELP in the pET-E. coli BL21(DE3) expression system. Both a comprehensive empirical approach examining all process variables (media, induction time and period, temperature, pH, aeration and agitation) and a detailed characterisation of the bioprocess were carried out in an attempt to maximise production with this system.
Results
This study shows that maximum SELP volumetric production is achieved at 37°C using terrific broth at pH 6–7.5, a shake flask volume to medium volume ratio of 10:1 and an agitation speed of 200 rpm. Maximum induction is attained at the beginning of the stationary phase with 0.5 mM IPTG and an induction period of at least 4 hours. We show that the selection agents ampicillin and carbenicillin are rapidly degraded early in the cultivation and that plasmid stability decreases dramatically on induction. Furthermore, acetate accumulates during the bioprocess to levels which are shown to be inhibitory to the host cells. Using our optimised conditions, 500 mg/L of purified SELP was obtained.
Conclusions
We have identified the optimal conditions for the shake flask production of a novel SELP with the final production levels obtained being the highest reported to date. While this study is focused on SELPs, we believe that it could also be of general interest to any study where the pET (ampicillin selective marker)-E. coli BL21(DE3) expression system is used. In particular, we show that induction time is critical in this system with, in contrast to that which is generally believed, optimal production being obtained by induction at the beginning of the stationary phase. Furthermore, we believe that we are at or near the maximum productivity for the system used, with rapid degradation of the selective agent by plasmid encoded β-lactamase, plasmid instability on induction and high acetate production levels being the principal limiting factors for further improved production.
doi:10.1186/1475-2859-12-21
PMCID: PMC3623843  PMID: 23446293
Biopolymers; Silk-elastin like polymers; pET-E. coli BL21(DE3); Batch production

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