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1.  Identification and Expression of Multiple CYP1-like and CYP3-like Genes in the Bivalve Mollusk Mytilus edulis 
Aquatic toxicology (Amsterdam, Netherlands)  2012;0:10.1016/j.aquatox.2012.11.017.
Various sequencing projects over the last several years have aided the discovery of previously uncharacterized invertebrate sequences, including new cytochrome P450 genes (CYPs). Here we present data on the identification and characterization of two CYP1-like and three CYP3-like genes from the bivalve mollusk Mytilus edulis, and assess their potential as biomarkers based on their responses to several known vertebrate aryl hydrocarbon receptor (AHR) agonists. Quantitative real-time PCR was used to measure CYP transcript levels in digestive gland, labial palps, adductor muscle, gill, foot, and different regions of the mantle. Levels of both CYP1-like genes were highest in digestive gland, whereas labial palps had the highest expression levels of the three CYP3-like genes followed by digestive gland and outer margin of the mantle. Mussels were exposed by injection to the AHR agonists, β-naphthoflavone (BNF; 25 μg.g−1), 3,3′,4,4′,5-polychlorinated biphenyl (PCB126; 2 μg.g−1), or 6-formylindolo[3,2-b]carbazole (FICZ; 0.1 μg.g−1), or to Aroclor 1254 (a mixture of PCBs; 50 μg.g−1) for 24 hours, followed by CYP expression analysis. There was no statistically significant change in expression of either of the CYP1-like genes after exposure to the various AHR agonists. The CYP3-like-1 gene was significantly up-regulated by BNF in gill tissues and the CYP3-like-2 gene was up-regulated in digestive gland by PCB126 and in gill tissue by BNF. These results suggest that distinct mechanisms of CYP gene activation could be present in M. edulis, although the importance of the CYP1-like and CYP3-like genes for xenobiotic and endogenous lipids biotransformation requires additional investigation.
PMCID: PMC3846546  PMID: 23277104
cytochrome P450; CYP; AHR; Mytilus edulis; blue mussel; pollution
2.  Activation of adenosine receptors improves renal antioxidant status in diabetic Wistar but not SHR rats 
Diabetes and hypertension independently contribute to renal injury, and the major mechanisms involved are increased reactive oxygen species (ROS) bioavailability and renin-angiotensin system (RAS) activation. We investigated the role of adenosine in controlling ROS production and RAS activation associated with renal dysfunction in hypertension and diabetes.
Fourteen days after induction of diabetes with streptozotocin in 12-week-old male Wistar and spontaneously hypertensive (SHR) rats, animals were treated during 7 days with 2-chloroadenosine (CADO group, 5 mg/kg/d), a stable analogue of adenosine, or underwent a sham operation procedure. At the end of the study (day 21), intra-arterial systolic blood pressure (SBP) was measured, and 24-h urine and plasma samples and renal tissue were collected.
CADO treatment decreased the plasma glucose concentration and glucose and protein excretion by more than 30% in both strains. CADO treatment decreased SBP in diabetic SHR rats (143 ± 8 versus 114 ± 4 mmHg, p < 0.05), but not in diabetic Wistar rats. The hypotensive effect of CADO was associated to a ∼70% increase in plasma angiotensinogen (AGT) concentration and a ∼50% decrease in urinary AGT excretion. CADO also caused a decrease in medullary and cortical hydrogen peroxide production of about 40%, which was associated with a proportional increase in glutathione peroxidase (GPx) activity in diabetic Wistar but not in diabetic SHR animals.
These results suggest that activation of adenosine receptors improves renal antioxidant capacity in diabetic Wistar but not SHR rats, although it improves glucose metabolism in both strains. Furthermore, activation of adenosine receptors does not seem to be directly influencing AGT production.
PMCID: PMC3916712  PMID: 24195577
Adenosine; angiotensinogen; hydrogen peroxide; hyperglycemia; kidney; systolic blood pressure
3.  Evaluation of fluoride release from experimental TiF4 and NaF varnishes in vitro  
Journal of Applied Oral Science  2014;22(2):138-143.
Fluoride varnishes play an important role in the prevention of dental caries, promoting the inhibition of demineralization and the increase of remineralization.
This study aimed to analyze the amount of fluoride released into water and artificial saliva from experimental TiF4 and NaF varnishes, with different concentrations, for 12 h.
Material and Methods
Fluoride varnishes were applied on acrylic blocks and then immersed in 10 ml of deionized water and artificial saliva in polystyrene bottles. The acrylic blocks were divided in seven groups (n=10): 1.55% TiF4 varnish (0.95% F, pH 1.0); 3.10% TiF4 varnish (1.90% F, pH 1.0); 3.10% and 4% TiF4 varnish (2.45% F, pH 1.0); 2.10% NaF varnish (0.95% F, pH 5.0); 4.20% NaF varnish (1.90% F, pH 5.0); 5.42% NaF varnish (2.45% F, pH 5.0) and control (no treatment, n=5). The fluoride release was analyzed after 1/2, 1, 3, 6, 9 and 12 h of exposure. The analysis was performed using an ion-specific electrode coupled to a potentiometer. Two-way ANOVA and Bonferroni's test were applied for the statistical analysis (p<0.05).
TiF4 varnishes released larger amounts of fluoride than NaF varnishes during the first 1/2 h, regardless of their concentration; 4% TiF4 varnish released more fluoride than NaF varnishes for the first 6 h. The peak of fluoride release occurred at 3 h. There was a better dose-response relationship among the varnishes exposed to water than to artificial saliva.
The 3.10% and 4% TiF4 -based varnishes have greater ability to release fluoride into water and artificial saliva compared to NaF varnish; however, more studies must be conducted to elucidate the mechanism of action of TiF4 varnish on tooth surface.
PMCID: PMC3956406  PMID: 24676585
Fluoride varnishes; Sodium fluoride; Titanium tetrafluoride
4.  Gem-Induced Cytoskeleton Remodeling Increases Cellular Migration of HTLV-1-Infected Cells, Formation of Infected-to-Target T-Cell Conjugates and Viral Transmission 
PLoS Pathogens  2014;10(2):e1003917.
Efficient HTLV-1 viral transmission occurs through cell-to-cell contacts. The Tax viral transcriptional activator protein facilitates this process. Using a comparative transcriptomic analysis, we recently identified a series of genes up-regulated in HTLV-1 Tax expressing T-lymphocytes. We focused our attention towards genes that are important for cytoskeleton dynamic and thus may possibly modulate cell-to-cell contacts. We first demonstrate that Gem, a member of the small GTP-binding proteins within the Ras superfamily, is expressed both at the RNA and protein levels in Tax-expressing cells and in HTLV-1-infected cell lines. Using a series of ChIP assays, we show that Tax recruits CREB and CREB Binding Protein (CBP) onto a c-AMP Responsive Element (CRE) present in the gem promoter. This CRE sequence is required to drive Tax-activated gem transcription. Since Gem is involved in cytoskeleton remodeling, we investigated its role in infected cells motility. We show that Gem co-localizes with F-actin and is involved both in T-cell spontaneous cell migration as well as chemotaxis in the presence of SDF-1/CXCL12. Importantly, gem knock-down in HTLV-1-infected cells decreases cell migration and conjugate formation. Finally, we demonstrate that Gem plays an important role in cell-to-cell viral transmission.
Author Summary
HTLV-1 was the first human oncoretrovirus to be discovered. Five to ten million people are infected, and 1–6% will develop either Adult T-cell Leukemia, or Tropical Spastic Paraparesis/HTLV-1 Associated Myelopathy (TSP/HAM). HTLV-1 infects primarily T-cells, but dendritic cells were also found to carry proviruses. Contrary to HIV-1, cell-free HTLV-1 viral particles are poorly infectious. Thus, efficient viral transmission relies on formation of virological synapses or formation and transfer of viral biofilm-like structures. The Tax viral transactivator plays a key role in both modes of transmission. Using transcriptomic analyses, we recently identified cellular genes that are deregulated following Tax expression in T-cells. We focused our attention on genes that are important for cell architecture and are thus likely to modulate cell-to-cell contacts and motility. We found that Gem was highly upregulated both at the RNA and protein levels in Tax-expressing cells and HTLV-1-infected cell lines. We further show that Tax binds cellular co-activators and transcription factor and activates transcription from the gem promoter. We demonstrated that Gem is involved in cellular migration of HTLV-1-infected cells. Importantly, gem knockdown decreases the rate of HTLV-1-infected cell migration and cell-to-cell conjugate formation. We also show that Gem plays an important role in HTLV-1 transmission through cell-to-cell contacts, the most efficient mode of viral infection.
PMCID: PMC3937318  PMID: 24586148
5.  Yeasts and hygienic-sanitary microbial indicators in water buffalo mozzarella produced and commercialized in Minas Gerais, Brazil 
Brazilian Journal of Microbiology  2014;44(3):701-707.
The aim of this work was to study the yeast populations and the main hygienic-sanitary microbial indicators in water buffalo mozzarella produced and commercialized in Minas Gerais, Brazil. Forty-two water buffalo mozzarella samples were purchased from retail outlets in Belo Horizonte. In addition, five samples of consecutive starter cultures, curd before acidification, acidified curd and mozzarella were collected at an industry in the city of Oliveira. Only three of the five water samples analyzed were suitable for consumption according to Brazilian sanitary standards. Four milk samples were highly contaminated with fecal coliforms, and did not meet the minimal hygienic-sanitary standards according to Brazilian regulations. Only one sample of buffalo muzzarela purchased from retail outlets exceeded the limit for coagulase-positive Staphylococcus. Eleven samples showed counts of thermotolerant coliforms higher than 5 × 103 CFU.g−1, but still lower than the maximum permitted by the Brazilian laws. Salmonella spp. and Listeria monocytogenes were not isolated. Debaryomyces hansenii, Candida lusitaniae and C. parapsilosis were the prevalent yeast species isolated from cheese. Among samples from the production stages, the acidified curd presented the highest numbers of yeasts, with C. catenulata being the most frequent species isolated. Some opportunistic yeast species such as C. guilliermondii, C. tropicalis, C. parapsilosis, C. lusitaniae, C. catenulata, C. rugosa and C. krusei occurred in the mozzarella cheese samples analyzed. The mozzarella cheese presented a low microbial load as compared to other cheese already studied, and the yeast biota included species typical of cheese and also opportunistic pathogens.
PMCID: PMC3910177  PMID: 24516436
water buffalo mozzarella; yeast diversity; opportunistic yeast; hygienic-sanitary microbial indicators
6.  Integrated analysis of microRNA and mRNA expression and association with HIF binding reveals the complexity of microRNA expression regulation under hypoxia 
Molecular Cancer  2014;13:28.
In mammalians, HIF is a master regulator of hypoxia gene expression through direct binding to DNA, while its role in microRNA expression regulation, critical in the hypoxia response, is not elucidated genome wide. Our aim is to investigate in depth the regulation of microRNA expression by hypoxia in the breast cancer cell line MCF-7, establish the relationship between microRNA expression and HIF binding sites, pri-miRNA transcription and microRNA processing gene expression.
MCF-7 cells were incubated at 1% Oxygen for 16, 32 and 48 h. SiRNA against HIF-1α and HIF-2α were performed as previously published. MicroRNA and mRNA expression were assessed using microRNA microarrays, small RNA sequencing, gene expression microarrays and Real time PCR. The Kraken pipeline was applied for microRNA-seq analysis along with Bioconductor packages. Microarray data was analysed using Limma (Bioconductor), ChIP-seq data were analysed using Gene Set Enrichment Analysis and multiple testing correction applied in all analyses.
Hypoxia time course microRNA sequencing data analysis identified 41 microRNAs significantly up- and 28 down-regulated, including hsa-miR-4521, hsa-miR-145-3p and hsa-miR-222-5p reported in conjunction with hypoxia for the first time. Integration of HIF-1α and HIF-2α ChIP-seq data with expression data showed overall association between binding sites and microRNA up-regulation, with hsa-miR-210-3p and microRNAs of miR-27a/23a/24-2 and miR-30b/30d clusters as predominant examples. Moreover the expression of hsa-miR-27a-3p and hsa-miR-24-3p was found positively associated to a hypoxia gene signature in breast cancer. Gene expression analysis showed no full coordination between pri-miRNA and microRNA expression, pointing towards additional levels of regulation. Several transcripts involved in microRNA processing were found regulated by hypoxia, of which DICER (down-regulated) and AGO4 (up-regulated) were HIF dependent. DICER expression was found inversely correlated to hypoxia in breast cancer.
Integrated analysis of microRNA, mRNA and ChIP-seq data in a model cell line supports the hypothesis that microRNA expression under hypoxia is regulated at transcriptional and post-transcriptional level, with the presence of HIF binding sites at microRNA genomic loci associated with up-regulation. The identification of hypoxia and HIF regulated microRNAs relevant for breast cancer is important for our understanding of disease development and design of therapeutic interventions.
PMCID: PMC3928101  PMID: 24517586
MicroRNA; Hypoxia; HIF; Transcription factor; Gene regulation
7.  Genome-Wide Saturation Mutagenesis of Burkholderia pseudomallei K96243 Predicts Essential Genes and Novel Targets for Antimicrobial Development 
mBio  2014;5(1):e00926-13.
Burkholderia pseudomallei is the causative agent of melioidosis, an often fatal infectious disease for which there is no vaccine. B. pseudomallei is listed as a tier 1 select agent, and as current therapeutic options are limited due to its natural resistance to most antibiotics, the development of new antimicrobial therapies is imperative. To identify drug targets and better understand the complex B. pseudomallei genome, we sought a genome-wide approach to identify lethal gene targets. As B. pseudomallei has an unusually large genome spread over two chromosomes, an extensive screen was required to achieve a comprehensive analysis. Here we describe transposon-directed insertion site sequencing (TraDIS) of a library of over 106 transposon insertion mutants, which provides the level of genome saturation required to identify essential genes. Using this technique, we have identified a set of 505 genes that are predicted to be essential in B. pseudomallei K96243. To validate our screen, three genes predicted to be essential, pyrH, accA, and sodB, and a gene predicted to be nonessential, bpss0370, were independently investigated through the generation of conditional mutants. The conditional mutants confirmed the TraDIS predictions, showing that we have generated a list of genes predicted to be essential and demonstrating that this technique can be used to analyze complex genomes and thus be more widely applied.
Burkholderia pseudomallei is a lethal human pathogen that is considered a potential bioterrorism threat and has limited treatment options due to an unusually high natural resistance to most antibiotics. We have identified a set of genes that are required for bacterial growth and thus are excellent candidates against which to develop potential novel antibiotics. To validate our approach, we constructed four mutants in which gene expression can be turned on and off conditionally to confirm that these genes are required for the bacteria to survive.
PMCID: PMC3950516  PMID: 24520057
8.  An Unusual Fat-Containing Presacral Tumor in an Elderly Patient 
Case Reports in Radiology  2014;2014:674365.
The authors present a case of a presacral myelolipoma diagnosed in an 84-year-old male patient with longstanding pelvic pain and past medical history of bladder cancer. Pelvic computed tomography (CT) revealed a well-encapsulated and lobulated presacral mass, with mixed fat and soft-tissue attenuation. Magnetic resonance (MR) imaging provided further confirmation of macroscopic intralesional fat and excluded either adjacent bone invasion or bladder cancer recurrence. A presacral myelolipoma was suspected based on imaging findings, with liposarcoma and teratoma having also been considered for the differential diagnosis. The histological confirmation of the tumor was only attained postoperatively. This case report alerts to the possible presacral location of myelolipomas, which should be considered for every fat-containing lesion detected in this region. The main clinical, imaging, and differential diagnoses of this entity are reviewed in this paper.
PMCID: PMC3933520  PMID: 24653850
9.  Influence of Laser Irradiation on Pits and Fissures: An In Situ Study 
Objective: The aim of this in situ study was to analyze the influence of the Er:YAG, Nd:YAG, and CO2 lasers on the enamel acid resistance of pits and fissures. Background data: The laser tissue interaction has been studied as a method of preventing occlusal caries. Methods: Thirteen volunteers wore palatal acrylic appliances containing human occlusal enamel blocks that were divided into four groups (G1, control; G2, Er:YAG; G3, Nd:YAG; G4, CO2). Each palatal acrylic appliance was used in the four studied groups and was used for 14 consecutive days. A sucrose solution was applied to the specimens six times per day. The specimens were then sectioned in half, and a microhardness test was applied. The other halves were analyzed using polarized light microscopy to measure the caries-like lesion areas, and a morphological analysis was conducted using a scanning electron microscope (SEM). Results: For the statistical analysis of the data obtained from the microhardness test (Knoop hardness number. [KHN]) (α=5%), Fisher's exact test was performed, and the group means were as follows: G1, 247±71; G2, 258±70; G3, 272±73; and G4, 298±56. The results demonstrated that the control group was significantly different from G3 and G4, which presented higher microhardness values. The Wilcoxon signed-rank test was used to analyze the data obtained from the caries-lesion area measurements (mm2) (α=5%) (G1, 0.01±1.08; G2, 0.13±0.18; G3, 0.05±0.17; and G4, 0.09±0.22). The results no showed significant differences among the groups in this analysis. Conclusions: Based on the results from the present study, it may be concluded that the CO2 and Nd:YAG lasers increased the enamel acid resistance in pits and fissures.
PMCID: PMC3565555  PMID: 23336742
10.  First Report of Carbapenem-Resistant Acinetobacter nosocomialis Isolates Harboring ISAba1-blaOXA-23 Genes in Latin America 
Journal of Clinical Microbiology  2013;51(8):2739-2741.
In recent years, different resistance genes have been found in Acinetobacter spp., especially in the species A. baumannii. We describe two isolates of carbapenem-resistant A. nosocomialis harboring ISAba1-blaOXA-23 and blaOXA-51 found in patients from the city of Porto Alegre, southern Brazil. To the best of the authors' knowledge, this is the first report of carbapenem-resistant A. nosocomialis in Latin America.
PMCID: PMC3719618  PMID: 23740725
11.  Molecular Epidemiology of Newcastle Disease in Mexico and the Potential Spillover of Viruses from Poultry into Wild Bird Species 
Applied and Environmental Microbiology  2013;79(16):4985-4992.
Newcastle disease, one of the most important health problems that affects the poultry industry around the world, is caused by virulent strains of Newcastle disease virus. Newcastle disease virus is considered to be endemic in several countries in the Americas, including Mexico. In order to control Newcastle disease outbreaks and spread, intensive vaccination programs, which include vaccines formulated with strains isolated at least 60 years ago, have been established. These vaccines are dissimilar in genotype to the virulent Newcastle disease viruses that had been circulating in Mexico until 2008. Here, 28 isolates obtained between 2008 and 2011 from different regions of Mexico from free-living wild birds, captive wild birds, and poultry were phylogenetically and biologically characterized in order to study the recent epidemiology of Newcastle disease viruses in Mexico. Here we demonstrate that, until recently, virulent viruses from genotype V continued to circulate and evolve in the country. All of the Newcastle disease viruses of low virulence, mostly isolated from nonvaccinated free-living wild birds and captive wild birds, were highly similar to LaSota (genotype II) and PHY-LMV42 (genotype I) vaccine strains. These findings, together with the discovery of two virulent viruses at the Mexican zoo, suggest that Newcastle disease viruses may be escaping from poultry into the environment.
PMCID: PMC3754691  PMID: 23770910
12.  A novel polyclonal antibody-based sandwich ELISA for detection of Plasmodium vivax developed from two lactate dehydrogenase protein segments 
Immunoassays for Plasmodium detection are, presently, most frequently based on monoclonal antibodies (MAbs); Polyclonal antibodies (PAbs), which are cheaper to develop and manufacture, are much less frequently used. In the present study we describe a sandwich ELISA assay which is capable of detecting P. vivax Lactate Dehydrogenase (LDH) in clinical blood samples, without cross reacting with those infected with P. falciparum.
Two recombinant proteins were produced from different regions of the P. vivax LDH gene. Two sandwich ELISA assay were then designed: One which uses mouse anti-LDH 1-43aa PAbs as primary antibodies (“Test 1”) and another which uses anti-LDH 35-305aa PAbs (“Test 2”) as the primary antibodies. Rabbit anti-LDH 1-43aa PAbs were used as capture antibodies in both ELISA assays. Blood samples taken from P. vivax and P. falciparum infected patients (confirmed by light microscopy) were analysed using both tests.
“Test 2” performed better at detecting microscopy-positive blood samples when compared to “Test 1”, identifying 131 of 154 positive samples (85%); 85 positives (55%) were identified using “test 1”. “Test 1” produced one false positive sample (from the 20 malaria-free control) blood samples; “test 2” produced none. Kappa coefficient analysis of the results produced a value of 0.267 when microscope-positive blood smears were compared with “test 1”, but 0.734 when microscope-positive blood smears were compared with the results from “test 2”. Positive predictive value (PPV) and negative predictive value (NPV) were observed to be 98% and 22% respectively, for “Test 1”, and 99% and 45%, for “test 2”. No cross reactivity was detected with P. falciparum positive blood samples (n = 15) with either test assay.
Both tests detected P. vivax infected blood and showed no evidence of cross-reacting with P. falciparum. Further studies will need to be conducted to establish the full potential of this technique for malaria diagnostics. As well as representing a promising new cost-effective novel technique for P. vivax diagnosis and research, the method for developing this assay also highlights the potential for PAb-based strategies for diagnostics in general.
PMCID: PMC3913629  PMID: 24475751
Malaria diagnosis; Lactate dehydrogenase; Recombinant protein; ELISA
13.  HIV Incidence in a Cohort of Women at Higher Risk in Beira, Mozambique: Prospective Study 2009–2012 
PLoS ONE  2014;9(1):e84979.
HIV is prevalent in Sofala Province, Mozambique. To inform future prevention research, we undertook a study in the provincial capital (Beira) to measure HIV incidence in women at higher risk of HIV and assess the feasibility of recruiting and retaining them as research participants.
Women age 18–35 were recruited from schools and places where women typically meet potential sexual partners. Eligibility criteria included HIV-seronegative status and self-report of at least 2 sexual partners in the last month. History of injection drug use was an exclusion criterion, but pregnancy was not. Participants were scheduled for monthly follow-up for 12 months, when they underwent face-to-face interviews, HIV counseling and testing, and pregnancy testing.
387 women were eligible and contributed follow-up data. Most were from 18–24 years old (median 21). Around one-third of participants (33.8%) reported at least one new sexual partner in the last month. Most women (65.5%) reported not using a modern method of contraception at baseline. Twenty-two women seroconverted for a prospective HIV incidence of 6.5 per 100 woman-years (WY; 95% confidence interval (CI): 4.1–9.9). Factors associated with HIV seroconversion in the multivariable analysis were: number of vaginal sex acts without using condoms with partners besides primary partner in the last 7 days (hazard ratio (HR) 1.7; 95% CI: 1.2–2.5) and using a form of contraception at baseline other than hormonal or condoms (vs. no method; HR 25.3; 95% CI: 2.5–253.5). The overall retention rate was 80.0% for the entire follow-up period.
We found a high HIV incidence in a cohort of young women reporting risky sexual behavior in Beira, Mozambique. HIV prevention programs should be strengthened. Regular HIV testing and condom use should be encouraged, particularly among younger women with multiple sexual partners.
PMCID: PMC3903474  PMID: 24475035
14.  Protective Action of Antioxidants on Hepatic Damage Induced by Griseofulvin 
The Scientific World Journal  2014;2014:982358.
Erythropoietic protoporphyria (EPP) is a disease associated with ferrochelatase deficiency and characterized by the accumulation of protoporphyrin IX (PROTO IX) in erythrocytes, liver, and skin. In some cases, a severe hepatic failure and cholestasis were observed. Griseofulvin (Gris) develops an experimental EPP with hepatic manifestations in mice such as PROTO IX accumulation followed by cellular damage as wells as necrotic and inflammatory processes. The antioxidant defense system was also altered. The aim was to evaluate the possible protective effect of different antioxidant compounds: trolox (Tx), ascorbic acid (Asc), the combination Tx and Asc, melatonin (Mel), and the polyphenols: ellagic acid, quercetin, chlorogenic acid, caffeic acid, gallic acid, and ferulic acid on liver damage and oxidative stress markers in a mouse model of EPP. Coadministration of Gris with Tx, Asc, and its combination, or Mel mainly affected heme biosynthetic pathway, resulting in a decrease in ALA-S activity which was increased by Gris, while the tested polyphenols exerted a protective effect on oxidative stress, decreasing lipid peroxidation and the activity of some antioxidant enzymes. In conclusion, antioxidant compounds can only protect partially against the liver damage induced by Gris, reducing oxidative stress or acting on heme regulation.
PMCID: PMC3913521  PMID: 24523661
15.  Neutrophil swarms require LTB4 and integrins at sites of cell death in vivo 
Nature  2013;498(7454):10.1038/nature12175.
Neutrophil recruitment from blood to extravascular sites of sterile or infectious tissue damage is a hallmark of early innate immune responses, and the molecular events leading to cell exit from the bloodstream have been well defined1,2. Once outside the vessel, individual neutrophils often show extremely coordinated chemotaxis and cluster formation reminiscent of the swarming behaviour of insects3–11. The molecular players that direct this response at the single-cell and population levels within the complexity of an inflamed tissue are unknown. Using two-photon intravital microscopy in mouse models of sterile injury and infection, we show a critical role for intercellular signal relay among neutrophils mediated by the lipid leukotriene B4, which acutely amplifies local cell death signals to enhance the radius of highly directed interstitial neutrophil recruitment. Integrin receptors are dispensable for long-distance migration12, but have a previously unappreciated role in maintaining dense cellular clusters when congregating neutrophils rearrange the collagenous fibre network of the dermis to form a collagen-free zone at the wound centre. In this newly formed environment, integrins, in concert with neutrophil-derived leukotriene B4 and other chemoattractants, promote local neutrophil interaction while forming a tight wound seal. This wound seal has borders that cease to grow in kinetic concert with late recruitment of monocytes and macrophages at the edge of the displaced collagen fibres. Together, these data provide an initial molecular map of the factors that contribute to neutrophil swarming in the extravascular space of a damaged tissue. They reveal how local events are propagated over large-range distances, and how auto-signalling produces coordinated, self-organized neutrophil-swarming behaviour that isolates the wound or infectious site from surrounding viable tissue.
PMCID: PMC3879961  PMID: 23708969
16.  Virus interference between H7N2 low pathogenic avian influenza virus and lentogenic Newcastle disease virus in experimental co-infections in chickens and turkeys 
Veterinary Research  2014;45(1):1.
Low pathogenicity avian influenza virus (LPAIV) and lentogenic Newcastle disease virus (lNDV) are commonly reported causes of respiratory disease in poultry worldwide with similar clinical and pathobiological presentation. Co-infections do occur but are not easily detected, and the impact of co-infections on pathobiology is unknown. In this study chickens and turkeys were infected with a lNDV vaccine strain (LaSota) and a H7N2 LPAIV (A/turkey/VA/SEP-67/2002) simultaneously or sequentially three days apart. No clinical signs were observed in chickens co-infected with the lNDV and LPAIV or in chickens infected with the viruses individually. However, the pattern of virus shed was different with co-infected chickens, which excreted lower titers of lNDV and LPAIV at 2 and 3 days post inoculation (dpi) and higher titers at subsequent time points. All turkeys inoculated with the LPAIV, whether or not they were exposed to lNDV, presented mild clinical signs. Co-infection effects were more pronounced in turkeys than in chickens with reduction in the number of birds shedding virus and in virus titers, especially when LPAIV was followed by lNDV. In conclusion, co-infection of chickens or turkeys with lNDV and LPAIV affected the replication dynamics of these viruses but did not affect clinical signs. The effect on virus replication was different depending on the species and on the time of infection. These results suggest that infection with a heterologous virus may result in temporary competition for cell receptors or competent cells for replication, most likely interferon-mediated, which decreases with time.
PMCID: PMC3890543  PMID: 24393488
17.  Pediatric lung transplantation: 10 years of experience 
Clinics  2014;69(Suppl 1):51-54.
Lung transplantation is a well-established treatment for advanced lung diseases. In children, the diseases that most commonly lead to the need for a transplantation are cystic fibrosis, pulmonary hypertension, and bronchiolitis. However, the number of pediatric lung transplantations being performed is low compared with the number of transplants performed in the adult age group. The objective of this study was to demonstrate our experience with pediatric lung transplants over a 10-year period in a program initially designed for adults.
PMCID: PMC3884157
Lung Transplantation; Pediatrics; Cystic Fibrosis; Bronchiectasis; Bronchiolitis
18.  Melanoma developed during pregnancy - A case report*  
Anais Brasileiros de Dermatologia  2014;89(1):157-159.
We describe a case of plantar interdigital cutaneous melanoma in a 22-year-old woman who reported changes in a pigmented lesion during pregnancy. Diagnosis was late and evolution unfavourable. The purpose of this report is to draw the attention of dermatologists to the need for careful regular examination of melanocytic lesions in pregnant women, not ignoring possible changes as always physiological.
PMCID: PMC3938370  PMID: 24626664
Melanoma; Neoplastic metastasis; Nevi and melanomas; Pigmented nevus; Pregnancy; Skin Neoplasm
19.  Novel features on the regulation by mitochondria of calcium and secretion transients in chromaffin cells challenged with acetylcholine at 37°C 
Physiological Reports  2013;1(7):e00182.
From experiments performed at room temperature, we know that the buffering of Ca2+ by mitochondria contributes to the shaping of the bulk cytosolic calcium transient ([Ca2+]c) and secretion transients of chromaffin cells stimulated with depolarizing pulses. We also know that the mitochondrial Ca2+ transporters and the release of catecholamine are faster at 37°C with respect to room temperature. Therefore, we planned this investigation to gain further insight into the contribution of mitochondrial Ca2+ buffering to the shaping of [Ca2+]c and catecholamine release transients, using some novel experimental conditions that have not been yet explored namely: (1) perifusion of bovine chromaffin cells (BCCs) with saline at 37°C and their repeated challenging with the physiological neurotransmitter acetylcholine (ACh); (2) separate blockade of mitochondrial Ca2+ uniporter (mCUP) with Ru360 or the mitochondrial Na+/Ca2+ exchanger (mNCX) with CGP37157; (3) full blockade of the mitochondrial Ca2+ cycling (mCC) by the simultaneous inhibition of the mCUP and the mNCX. Ru360 caused a pronounced delay of [Ca2+]c clearance and augmented secretion. In contrast, CGP37157 only caused a tiny delay of [Ca2+]c clearance and a mild decrease in secretion. The mCC resulting in continued Ca2+ uptake and its release back into the cytosol was interrupted by combined Ru360 + CGP37157 (Ru/CGP), the protonophore carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone, or combined oligomycin + rotenone (O/R); these three treatments caused a mild but sustained elevation of basal [Ca2+]c that, however, was not accompanied by a parallel increase in basal secretion. Nevertheless, all treatments caused a pronounced augmentation of ACh‐induced secretion, with minor changes of the ACh‐induced [Ca2+]c transients. Combined Ru/CGP did not alter the resting membrane potential in current‐clamped cells. Additionally, Ru/CGP did not increase basal [Ca2+]c near subplasmalemmal sites and caused a mild decrease in the size of the readily releasable vesicle pool. Our results provide new functional features in support of the view that in BCCs there are two subpopulations of mitochondria, M1 underneath the plasmalemma nearby exocytotic sites and M2 at the core cell nearby vesicle transport sites. While M1 serves to shape the ACh‐elicited exocytotic response through its efficient Ca2+ removal by the mCUP, M2 shapes the lower [Ca2+]c elevations required for new vesicle supply to the exocytotic machinery, from the large reserve vesicle pool at the cell core. The mCUP of the M1 pool seems to play a more prominent role in controlling the ACh responses, in comparison with the mNCX.
Regulation by mitochondria of exocytosis at 37°C.
PMCID: PMC3970745
Calcium homeostasis; catecholamines; CGP37157; chromaffin cells; exocytosis; FCCP; mitochondria; oligomycin; rotenone; Ru360
20.  Sulforaphane inhibits pancreatic cancer through disrupting Hsp90-p50Cdc37 complex and direct interactions with amino acids residues of Hsp90 
Sulforaphane [1-isothiocyanato-4-(methyl-sulfinyl) butane)], an isothiocyanate derived from cruciferous vegetables, has been shown to possess potent chemopreventive activity. We analyzed the effect of sulforaphane on the proliferation of pancreatic cancer cells. Sulforaphane inhibited pancreatic cancer cell growth in vitro with the IC50's around 10-15 μM and induced apoptosis. In pancreatic cancer xenograft mouse model, administration of sulforaphane showed remarkable inhibition of tumor growth without apparent toxicity noticed. We found that sulforaphane induced the degradation of heat shock protein 90 (Hsp90) client proteins and blocked the interaction of Hsp90 with its cochaperone p50Cdc37 in pancreatic cancer cells. Using Nuclear Magnetic Resonance Spectroscopy (NMR) with an isoleucine-specific labeling strategy, we overcame the protein size limit of conventional NMR and studied the interaction of sulforaphane with full-length Hsp90 dimer (170 kDa) in solution. NMR revealed multiple chemical shifts in sheet 2 and the adjacent loop in Hsp90 N-terminal domain after incubation of Hsp90 with sulforaphane. Liquid Chromatography coupled to Mass Spectrometry (LC-MS) further mapped a short peptide in this region that was tagged with sulforaphane. These data suggest a new mechanism of sulforaphane that disrupts protein-protein interaction in Hsp90 complex for its chemopreventive activity.
PMCID: PMC3386376  PMID: 22444872
sulforaphane; Hsp90; p50Cdc37; NMR; pancreatic cancer
21.  Haemoglobin levels for population from Gambo, a rural area of Ethiopia, and their association with anaemia and malaria 
Malaria Journal  2013;12:435.
Knowledge of appropriate reference intervals is critical not only to provide optimal clinical care, but also to enrol populations in medical research. The aim of this study was to generate normal ranges of laboratory values for haemoglobin among healthy Ethiopian adults and children and to determine if anaemia is a possible indicator of malaria in women and children in this area of Ethiopia.
This study was carried out from January 2008 to May 2010. The reference sample population with malaria-negative consisted of 454 individuals, divided women, men and children. The malaria-infected sample population consisted of 117 individuals. The reference ranges were based on the guidelines from the Clinical and Laboratory Standards Institute. Haemoglobin concentration was determined by Hemo-Control EKF Diagnostic Analyser on whole blood. Testing for malaria-positive and negative infection was done by microscopy and by PCR.
The lower limits for adult haemoglobin range obtained from this population were slightly higher than those derived from other African populations, but were equal to those established by other studies in Ethiopia and the World Health Organization (WHO). Regarding children, the minimum values were lower than those obtained from different African populations and those established by WHO. The malaria-negative group had anaemia in 35.6% of cases and in the malaria-positive group in 70.9%. There was a stronger, statistically significant association between anaemia and malaria-positive samples than between anaemia and malaria-negative samples in women and both groups of children.
The results from this study are a contribution in the definition of the haemoglobin parameters in African populations, which could be taken as standards for interpretation of laboratory results. The haemoglobin indices in adults from Gambo tended to be higher than other African populations and in children were lower than other studies in Africa. The results also suggest that anaemia is not useful as a supportive diagnostic criterion to monitor and evaluate malaria in women and children from Ethiopia, because a 29.1% of malaria cases will be not detected, because of not having anaemia.
PMCID: PMC3866573  PMID: 24289142
Haemoglobin; Anaemia; Malaria; Ethiopia
22.  Extended-spectrum β-lactamase-producing enterobacteriaceae in community-acquired urinary tract infections in São Luís, Brazil 
Brazilian Journal of Microbiology  2013;44(2):469-471.
The number of ESBL-producing Enterobacteriaceae in community-acquired urinary tract infections worldwide is probably underestimated because of the technical difficulties encountered with their detection. In this study, out of 5,672 urine samples analyzed, 916 were positive for uropathogens, 472 of them being enterobacteria of which 7.6% produced β-lactamases. Analysis of the isolated from 36 patients showed a high level of antibiotic resistance, with 52.7% and 80.5% of isolates expressing blaTEM and blaCTX-M, respectively.
PMCID: PMC3833145  PMID: 24294239
enterobacteriaceae; antimicrobial resistance; ESBL; community-acquired infections
23.  miR-363-5p regulates endothelial cell properties and their communication with hematopoietic precursor cells 
Recent findings have shown that the blood vessels of different organs exert an active role in regulating organ function. In detail, the endothelium that aligns the vasculature of most organs is fundamental in maintaining organ homeostasis and in promoting organ recovery following injury. Mechanistically, endothelial cells (EC) of tissues such as the liver, lungs or the bone marrow (BM) have been shown to produce “angiocrine” factors that promote organ recovery and restore normal organ function. Controlled production of angiocrine factors following organ injury is therefore essential to promote organ regeneration and to restore organ function. However, the molecular mechanisms underlying the coordinated production and function of such “angiocrine” factors are largely undisclosed and were the subject of the present study. In detail, we identified for the first time a microRNA (miRNA) expressed by BM EC that regulates the expression of angiocrine genes involved in BM recovery following irradiation. Using a microarray-based approach, we identified several miRNA expressed by irradiated BMEC. After validating the variations in miRNA expression by semi-quantitative PCR, we chose to study further the ones showing consistent variations between experiments, and those predicted to regulate (directly or indirectly) angiogenic and angiocrine factors. Of the mi-RNA that were chosen, miR-363-5p (previously termed miR-363*) was subsequently shown to modulate the expression of numerous EC-specific genes including some angiocrine factors. By luciferase reporter assays, miR-363-5p is shown to regulate the expression of angiocrine factors tissue inhibitor of metalloproteinases-1 (Timp-1) and thrombospondin 3 (THBS3) at post-transcriptional level. Moreover, miR-363-5p reduction using anti-miR is shown to affect EC angiogenic properties (such as the response to angiogenic factors stimulation) and the interaction between EC and hematopoietic precursors (particularly relevant in a BM setting). miR-363-5p reduction resulted in a significant decrease in EC tube formation on matrigel, but increased hematopoietic precursor cells adhesion onto EC, a mechanism that is shown to involve kit ligand-mediated cell adhesion. Taken together, we have identified a miRNA induced by irradiation that regulates angiocrine factors expression on EC and as such modulates EC properties. Further studies on the importance of miR-363-5p on normal BM function and in disease are warranted.
PMCID: PMC3874849  PMID: 24257019
Bone marrow; Endothelial cell; Hematopoietic progenitors; miRNA; Cell interactions
24.  Correction: Proteomic Analysis of Liver in Rats Chronically Exposed to Fluoride 
PLoS ONE  2013;8(11):10.1371/annotation/54b8fb45-bb22-469e-96b3-77e99ba62b77.
PMCID: PMC3826724  PMID: 24250769
25.  Relative Risk of Visceral Leishmaniasis in Brazil: A Spatial Analysis in Urban Area 
Visceral leishmaniasis (VL) is a vector-borne disease whose factors involved in transmission are poorly understood, especially in more urban and densely populated counties. In Brazil, the VL urbanization is a challenge for the control program. The goals were to identify the greater risk areas for human VL and the risk factors involved in transmission.
This is an ecological study on the relative risk of human VL. Spatial units of analysis were the coverage areas of the Basic Health Units (146 small-areas) of Belo Horizonte, Minas Gerais State, Brazil. Human VL cases, from 2007 to 2009 (n = 412), were obtained in the Brazilian Reportable Disease Information System. Bayesian approach was used to model the relative risk of VL including potential risk factors involved in transmission (canine infection, socioeconomic and environmental features) and to identify the small-areas of greater risk to human VL.
Principal Findings
The relative risk of VL was shown to be correlated with income, education, and the number of infected dogs per inhabitants. The estimates of relative risk of VL were higher than 1.0 in 54% of the areas (79/146). The spatial modeling highlighted 14 areas with the highest relative risk of VL and 12 of them are concentrated in the northern region of the city.
The spatial analysis used in this study is useful for the identification of small-areas according to risk of human VL and presents operational applicability in control and surveillance program in an urban environment with an unequal spatial distribution of the disease. Thus the frequent monitoring of relative risk of human VL in small-areas is important to direct and prioritize the actions of the control program in urban environment, especially in big cities.
Author Summary
Visceral leishmaniasis (VL) is a vector-borne disease whose factors involved in transmission are poorly understood, especially in more urban and densely populated counties. In Brazil, the increasing occurrence of human VL cases in urban centers is a challenge for the control program. We aimed to identify the risk areas for VL and the risk factors involved in transmission in Belo Horizonte, a large urban area of the Brazil. At the same geographical space, we analyzed human VL cases (n = 412), canine infection and socioeconomic and environmental features. We identified a concentration of high-risk small-areas of human VL cases in the northern part of the city, marked by worse levels of education and income, and higher number of infected dogs per inhabitants. The spatial analysis used is useful for the identification of small-areas with a greater risk of VL and displays operational applicability in the control program in an urban environment with an unequal spatial distribution of the disease. Thus, the frequent monitoring of risk of human VL according to small-areas is important to direct and prioritize the actions of the control program in urban environment, especially in big cities.
PMCID: PMC3820760  PMID: 24244776

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