Publications by authors named "Silvia Cardoso"

62 Publications

Juvenile Polyposis of Infancy Presenting as Protein-Losing Enteropathy.

ACG Case Rep J 2021 Mar 3;8(3):e00542. Epub 2021 Mar 3.

Department of Pediatrics, Faculty of Medical Sciences, State University of Campinas, Campinas, São Paulo, Brazil.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.14309/crj.0000000000000542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7932797PMC
March 2021

Donor-Derived Myeloid Heme Oxygenase-1 Controls the Development of Graft-Versus-Host Disease.

Front Immunol 2020 18;11:579151. Epub 2021 Jan 18.

Institute for Medical Immunology, Université Libre de Bruxelles, Gosselies, Belgium.

Graft-versus-host disease (GVHD) remains a major clinical drawback of allogeneic hematopoietic stem cell transplantation (HSCT). Here, we investigated how the stress responsive heme catabolizing enzyme heme oxygenase-1 (HO-1, encoded by ) regulates GVHD in response to allogeneic hematopoietic stem cell transplantation in mice and humans. We found that deletion of the allele, specifically in the myeloid compartment of mouse donor bone marrow, promotes the development of aggressive GVHD after allogeneic transplantation. The mechanism driving GVHD in mice transplanted with allogeneic bone marrow lacking HO-1 expression in the myeloid compartment involves enhanced T cell alloreactivity. The clinical relevance of these observations was validated in two independent cohorts of HSCT patients. Individuals transplanted with hematopoietic stem cells from donors carrying a long homozygous (GT) repeat polymorphism (L/L) in the promoter, which is associated with lower HO-1 expression, were at higher risk of developing severe acute GVHD as compared to donors carrying a short (GT) repeat (S/L or S/S) polymorphism associated with higher HO-1 expression. In this study, we showed the unique importance of donor-derived myeloid HO-1 in the prevention of lethal experimental GVHD and we corroborated this observation by demonstrating the association between human (GT) microsatellite polymorphisms and the incidence of severe acute GVHD in two independent HSCT patient cohorts. Donor-derived myeloid HO-1 constitutes a potential therapeutic target for HSCT patients and large-scale prospective studies in HSCT patients are necessary to validate the HO-1 L/L genotype as an independent risk factor for developing severe acute GVHD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2020.579151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7849683PMC
January 2021

Loss of α-gal during primate evolution enhanced antibody-effector function and resistance to bacterial sepsis.

Cell Host Microbe 2021 03 25;29(3):347-361.e12. Epub 2021 Jan 25.

Instituto Gulbenkian de Ciência, 2780-156 Oeiras, Portugal. Electronic address:

Most mammals express a functional GGTA1 gene encoding the N-acetyllactosaminide α-1,3-galactosyltransferase enzyme, which synthesizes Gal-α1-3Gal-β1-4GlcNAc (α-gal) and are thus tolerant to this self-expressed glycan. Old World primates including humans, however, carry loss-of-function mutations in GGTA1 and lack α-gal. Presumably, fixation of such mutations was propelled by natural selection, favoring the emergence of α-gal-specific immunity, conferring resistance to α-gal-expressing pathogens. Here, we show that loss of Ggta1 function in mice enhances resistance to bacterial sepsis, irrespectively of α-Gal-specific immunity. Rather, the absence of α-gal from IgG-associated glycans increases IgG effector function via a mechanism associated with enhanced IgG-Fc gamma receptor (FcγR) binding. The ensuing survival advantage against sepsis comes alongside a cost of accelerated reproductive senescence in Ggta1-deleted mice. Mathematical modeling of this trade-off suggests that high exposure to virulent pathogens exerts sufficient selective pressure to fix GGTA1 loss-of-function mutations, as likely occurred during the evolution of primates toward humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chom.2020.12.017DOI Listing
March 2021

Subclinical atherosclerosis in children and adolescents with congenital heart disease.

Cardiol Young 2020 Dec 11:1-8. Epub 2020 Dec 11.

Postgraduate Program in Public Health, Federal University of Santa Catarina, Florianopolis, Brazil.

Background: Subclinical atherosclerosis in childhood can be evaluated by carotid intima-media thickness, which is considered a surrogate marker for atherosclerotic disease in adulthood. The aims of this study were to evaluate carotid intima-media thickness and, to investigate associated factors.

Methods: Cross-sectional study with children and adolescents with congenital heart disease (CHD). Socio-demographic and clinical characteristics were assessed. Subclinical atherosclerosis was evaluated by carotid intima-media thickness. Cardiovascular risk factors, such as physical activity, screen time, passive smoke, systolic and diastolic blood pressure, waist circumference, dietary intake, lipid parameters, glycaemia, and C-reactive protein, were also assessed. Factors associated with carotid intima-media thickness were analysed using multiple logistic regression.

Results: The mean carotid intima-media thickness was 0.518 mm and 46.7% had subclinical atherosclerosis (carotid intima-media thickness ≥ 97th percentile). After adjusting for confounding factors, cyanotic CHD (odds ratio: 0.40; 95% confidence interval: 0.20; 0.78), cardiac surgery (odds ratio: 3.17; 95% confidence interval: 1.35; 7.48), and be hospitalised to treat infections (odds ratio: 1.92; 95% confidence interval: 1.04; 3.54) were associated with subclinical atherosclerosis.

Conclusion: Clinical characteristics related to CHD were associated with subclinical atherosclerosis. This finding suggests that the presence of CHD itself is a risk factor for subclinical atherosclerosis. Therefore, the screen and control of modifiable cardiovascular risk factors should be made early and intensively to prevent atherosclerosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1017/S1047951120004448DOI Listing
December 2020

M. tuberculosis Reprograms Hematopoietic Stem Cells to Limit Myelopoiesis and Impair Trained Immunity.

Cell 2020 Oct;183(3):752-770.e22

Meakins-Christie Laboratories, Department of Medicine, Department of Microbiology and Immunology, Department of Pathology, McGill University, Montreal, QC, Canada; McGill International TB Centre, McGill University Health Centre, Montreal, QC, Canada. Electronic address:

A greater understanding of hematopoietic stem cell (HSC) regulation is required for dissecting protective versus detrimental immunity to pathogens that cause chronic infections such as Mycobacterium tuberculosis (Mtb). We have shown that systemic administration of Bacille Calmette-Guérin (BCG) or β-glucan reprograms HSCs in the bone marrow (BM) via a type II interferon (IFN-II) or interleukin-1 (IL1) response, respectively, which confers protective trained immunity against Mtb. Here, we demonstrate that, unlike BCG or β-glucan, Mtb reprograms HSCs via an IFN-I response that suppresses myelopoiesis and impairs development of protective trained immunity to Mtb. Mechanistically, IFN-I signaling dysregulates iron metabolism, depolarizes mitochondrial membrane potential, and induces cell death specifically in myeloid progenitors. Additionally, activation of the IFN-I/iron axis in HSCs impairs trained immunity to Mtb infection. These results identify an unanticipated immune evasion strategy of Mtb in the BM that controls the magnitude and intrinsic anti-microbial capacity of innate immunity to infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2020.09.062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7599081PMC
October 2020

Heme Oxygenase-1 Induction by Blood-Feeding Arthropods Controls Skin Inflammation and Promotes Disease Tolerance.

Cell Rep 2020 10;33(4):108317

Vector Molecular Biology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA. Electronic address:

Hematophagous vectors lacerate host skin and capillaries to acquire a blood meal, resulting in leakage of red blood cells (RBCs) and inflammation. Here, we show that heme oxygenase-1 (HO-1), a pleiotropic cytoprotective isoenzyme that mitigates heme-mediated tissue damage, is induced after bites of sand flies, mosquitoes, and ticks. Further, we demonstrate that erythrophagocytosis by macrophages, including a skin-residing CD163CD91 professional iron-recycling subpopulation, produces HO-1 after bites. Importantly, we establish that global deletion or transient inhibition of HO-1 in mice increases inflammation and pathology following Leishmania-infected sand fly bites without affecting parasite number, whereas CO, an end product of the HO-1 enzymatic reaction, suppresses skin inflammation. This indicates that HO-1 induction by blood-feeding sand flies promotes tolerance to Leishmania infection. Collectively, our data demonstrate that HO-1 induction through erythrophagocytosis is a universal mechanism that regulates skin inflammation following blood feeding by arthropods, thus promoting early-stage disease tolerance to vector-borne pathogens.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2020.108317DOI Listing
October 2020

Druggable targets from coronaviruses for designing new antiviral drugs.

Bioorg Med Chem 2020 11 8;28(22):115745. Epub 2020 Sep 8.

Chemistry and Biotechnology Institute, Federal University of Alagoas, Campus A.C. Simões, Lourival Melo Mota Avenue, Maceió 57072-970, Brazil; Laboratory of Medicinal Chemistry, Pharmaceutical Sciences Institute, Federal University of Alagoas, Campus A.C. Simões, Lourival Melo Mota Avenue, Maceió 57072-970, Brazil. Electronic address:

Severe respiratory infections were highlighted in the SARS-CoV outbreak in 2002, as well as MERS-CoV, in 2012. Recently, the novel CoV (COVID-19) has led to severe respiratory damage to humans and deaths in Asia, Europe, and Americas, which allowed the WHO to declare the pandemic state. Notwithstanding all impacts caused by Coronaviruses, it is evident that the development of new antiviral agents is an unmet need. In this review, we provide a complete compilation of all potential antiviral agents targeting macromolecular structures from these Coronaviruses (Coronaviridae), providing a medicinal chemistry viewpoint that could be useful for designing new therapeutic agents.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmc.2020.115745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7836322PMC
November 2020

Neuromuscular activation analysis of the trunk muscles during hippotherapy sessions.

J Bodyw Mov Ther 2020 Jul 7;24(3):235-241. Epub 2020 Mar 7.

Federal University of Viçosa, Brazil.

Introduction: Hippotherapy allows the development of affective, sensory, motor, and cognitive areas, besides providing the practitioner with several movements and stimuli necessary for therapeutic progress. However, there is a limited amount of scientific evidence regarding the suitability of the mount material, mount type, and hippotherapy session duration, as well as regarding the activation of specific muscle groups during the practice and its applicability to activities of daily living.

Objective: This study aimed to study the neuromuscular activation behavior of the iliocostalis, longissimus, multifidus, and upper trapezius muscles of children during four hippotherapy session time points using a functional task. It also compared two different mount materials for riding.

Methodology: A total of 30 children were randomly assigned to one of three groups: Saddle Hippotherapy Group, Blanket Hippotherapy Group, and Control Group. Data were collected with an electromyograph in a functional task that comprised trunk movements to pick up an object. Assessments took place at four times during the session.

Results: There was a significant increase in the neuromuscular activation of the iliocostalis, longissimus, and multifidus muscles after a 30-min session. The trapezius muscle showed increased neuromuscular activation after only 10 min. It continued to increase (but without a statistical difference) after and 20 and 30 min.

Conclusion: Hippotherapy promoted neuromuscular activation of the trunk muscles in children, assessed through a functional task, and was influenced by both session time and mount material. Specifically, the greater neuromuscular performance occurred when an exercise was performed using saddle and stirrup and lasted 30 min.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jbmt.2020.02.029DOI Listing
July 2020

Incidence and Prediction of Unstable Blood Glucose Level among Critically Ill Patients: A Cohort Study.

Int J Nurs Knowl 2021 Apr 24;32(2):96-102. Epub 2020 Jul 24.

Patrícia de Oliveira Salgado, PhD, RN, is an Assistant Professor in Department of Medicine and Nursing, Federal University of Viçosa (UFV), Viçosa, MG, Brazil.

Purpose: To evaluate the incidence and the prediction of unstable blood glucose level among critically ill patients hospitalized in an intensive care unit.

Methods: A cohort study was conducted with 62 adult patients hospitalized at an intensive care unit of a hospital located in Minas Gerais, Brazil, between March and July of 2017. Patient's demographic information, along with scores for Simplified Acute Physiology Score III, primary medical diagnosis, discharge status, diagnosis of diabetes and/or sepsis, length of stay, glycemic variability, type of nutrition, types of medications and treatments, and oxygen therapy were collected daily. A daily venous blood sample was collected to measure blood glucose levels during the patient's hospitalization period. Bivariate analysis was used to explore the association among the potential diagnostic indicators and the outcome of unstable blood glucose levels. Multivariate Cox regression was used to identify the potential predictors for the outcome.

Findings: Of the total of 62 participants, 45.1% (n=28) had unstable blood glucose level. Among the 28 patients with unstable blood glucose levels, half of them (n=14, 50%) had hypoglycemia and the other half had hyperglycemia (n=14, 50%). Decreased number of days hospitalized and the use of intensive glucose control with regular insulin were associated with decreased odds of developing hyperglycemia. The presence of mechanical ventilation was associated with a higher risk for the development of hypoglycemia.

Conclusions: This study provides knowledge and evidence of diagnostic indicators for unstable blood glucose levels that are not currently included in the NANDA-International terminology for the nursing diagnosis Risk for unstable blood glucose level (00179).

Implications For Nursing Practice: This study identified important diagnostic indicators that nurses can observe during the assessment to identify patients that are at risk for developing unstable blood glucose level and provide the appropriate care.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/2047-3095.12299DOI Listing
April 2021

Interleukin-1 promotes autoimmune neuroinflammation by suppressing endothelial heme oxygenase-1 at the blood-brain barrier.

Acta Neuropathol 2020 10 11;140(4):549-567. Epub 2020 Jul 11.

Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany.

The proinflammatory cytokine interleukin 1 (IL-1) is crucially involved in the pathogenesis of multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Herein, we studied the role of IL-1 signaling in blood-brain barrier (BBB) endothelial cells (ECs), astrocytes and microglia for EAE development, using mice with the conditional deletion of its signaling receptor IL-1R1. We found that IL-1 signaling in microglia and astrocytes is redundant for the development of EAE, whereas the IL-1R1 deletion in BBB-ECs markedly ameliorated disease severity. IL-1 signaling in BBB-ECs upregulated the expression of the adhesion molecules Vcam-1, Icam-1 and the chemokine receptor Darc, all of which have been previously shown to promote CNS-specific inflammation. In contrast, IL-1R1 signaling suppressed the expression of the stress-responsive heme catabolizing enzyme heme oxygenase-1 (HO-1) in BBB-ECs, promoting disease progression via a mechanism associated with deregulated expression of the IL-1-responsive genes Vcam1, Icam1 and Ackr1 (Darc). Mechanistically, our data emphasize a functional crosstalk of BBB-EC IL-1 signaling and HO-1, controlling the transcription of downstream proinflammatory genes promoting the pathogenesis of autoimmune neuroinflammation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00401-020-02187-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7498485PMC
October 2020

Oxidative stress markers in cognitively intact patients with diabetic neuropathy.

Brain Res Bull 2019 08 5;150:196-200. Epub 2019 Jun 5.

Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil. Electronic address:

Various forms of vascular injury are frequently associated with type-2 diabetes mellitus (DM2). Macro-angiopathy has alarming signs and symptoms such as those seen with stroke or heart attack, however the presentation of small vessel disease is generally more subtle and therefore usually unnoticed for a long period of time. While it may affect any organ, complications involving the nervous system such as diabetic poly-neuropathy (DPN) are especially debilitating, and it may also be a risk factor for other brain disorders such as dementia. The underlying mechanisms are likely to be multi-faceted, but piling evidence indicates oxidative stress as one of the crucial factors. Here we evaluate the oxidative profile of patients with DM2. The total anti-oxidant capacity appears to be reduced in DM2 with or without complications. Of the specific bio-markers studied, the levels of tissue-damage indicator malon-dialdehyde (MDA) were significantly lower in the DM2 + DPN population only. These results suggest that diabetic patients present with wavering oxidative status, and the low MDA concentrations in patients with complications such as DPN may represent either an exhausted anti-oxidative defense system or a response to anti-inflammatory medications. The findings may also support the use of anti-oxidants such as vitamins A and E.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.brainresbull.2019.06.001DOI Listing
August 2019

Ferritin regulates organismal energy balance and thermogenesis.

Mol Metab 2019 06 21;24:64-79. Epub 2019 Mar 21.

Instituto Gulbenkian de Ciência, Oeiras, Portugal. Electronic address:

Objective: The ferritin heavy/heart chain (FTH) gene encodes the ferroxidase component of the iron (Fe) sequestering ferritin complex, which plays a central role in the regulation of cellular Fe metabolism. Here we tested the hypothesis that ferritin regulates organismal Fe metabolism in a manner that impacts energy balance and thermal homeostasis.

Methods: We developed a mouse strain, referred herein as Fth, expressing a tamoxifen-inducible Cre recombinase under the control of the Rosa26 (R26) promoter and carrying two LoxP (fl) sites: one at the 5'end of the Fth promoter and another the 3' end of the first Fth exon. Tamoxifen administration induces global deletion of Fth in adult Fth mice, testing whether FTH is required for maintenance of organismal homeostasis.

Results: Under standard nutritional Fe supply, Fth deletion in adult Fth mice led to a profound deregulation of organismal Fe metabolism, oxidative stress, inflammation, and multi-organ damage, culminating in death. Unexpectedly, Fth deletion was also associated with a profound atrophy of white and brown adipose tissue as well as with collapse of energy expenditure and thermogenesis. This was attributed mechanistically to mitochondrial dysfunction, as assessed in the liver and in adipose tissue.

Conclusion: The FTH component of ferritin acts as a master regulator of organismal Fe homeostasis, coupling nutritional Fe supply to organismal redox homeostasis, energy expenditure and thermoregulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molmet.2019.03.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6531837PMC
June 2019

Renal control of disease tolerance to malaria.

Proc Natl Acad Sci U S A 2019 03 4;116(12):5681-5686. Epub 2019 Mar 4.

Instituto Gulbenkian de Ciência, 2780-156 Oeiras, Portugal;

Malaria, the disease caused by spp. infection, remains a major global cause of morbidity and mortality. Host protection from malaria relies on immune-driven resistance mechanisms that kill However, these mechanisms are not sufficient per se to avoid the development of severe forms of disease. This is accomplished instead via the establishment of disease tolerance to malaria, a defense strategy that does not target directly. Here we demonstrate that the establishment of disease tolerance to malaria relies on a tissue damage-control mechanism that operates specifically in renal proximal tubule epithelial cells (RPTEC). This protective response relies on the induction of heme oxygenase-1 (; HO-1) and ferritin H chain () via a mechanism that involves the transcription-factor nuclear-factor E2-related factor-2 (). As it accumulates in plasma and urine during the blood stage of infection, labile heme is detoxified in RPTEC by HO-1 and FTH, preventing the development of acute kidney injury, a clinical hallmark of severe malaria.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1822024116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6431151PMC
March 2019

Prevalence of vitamin D deficiency and its relationship with factors associated with recurrent wheezing.

J Bras Pneumol 2019 Feb 11;45(1):e20170431. Epub 2019 Feb 11.

. Departamento de Medicina e Enfermagem, Universidade Federal de Viçosa, Viçosa (MG) Brasil.

Objective: To determine the prevalence of vitamin D deficiency/insufficiency in children 0-18 years of age with recurrent wheezing and/or asthma residing in the microregion of Viçosa, Minas Gerais, Brazil, and treated at a referral center, and to determine its association with major risk factors for wheezing.

Methods: A cross-sectional study was performed using a semi-structured questionnaire, which was administered by trained interviewers to the legal guardians of the study participants. Data were obtained regarding general characteristics of recurrent wheezing; general sociodemographic, environmental, and biologic factors; and atopy-related factors. The magnitude of the statistical association was assessed by calculating ORs and their corresponding 95% CIs by using multiple logistic regression.

Results: We included 124 children in the study. The prevalence of vitamin D deficiency/insufficiency in the sample was 57.3%. Vitamin D deficiency/insufficiency was found to be associated with wheezing in the first year of life, personal history of atopic dermatitis, environmental pollution, and vitamin D supplementation until 2 years of age.

Conclusions: The prevalence of vitamin D deficiency/insufficiency was high in our sample. Vitamin D concentrations were directly associated with vitamin D supplementation until 2 years of age and were inversely associated with wheezing events in the first year of life, personal history of atopic dermatitis, and environmental pollution.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1590/1806-3713/e20170431DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6534403PMC
February 2019

Purinergic Antagonist Suramin Aggravates Myocarditis and Increases Mortality by Enhancing Parasitism, Inflammation, and Reactive Tissue Damage in -Infected Mice.

Oxid Med Cell Longev 2018 30;2018:7385639. Epub 2018 Sep 30.

Departament of General Biology, Federal University of Viçosa, MG, Brazil.

Suramin (Sur) acts as an -NTPDase inhibitor in and a P2-purinoceptor antagonist in mammalian cells. Although the potent antitrypanosomal effect of Sur has been shown , limited evidence suggests that this drug can be dangerous to -infected hosts. Therefore, we investigated the dose-dependent effect of Sur-based chemotherapy in a murine model of Chagas disease. Seventy uninfected and -infected male C57BL/6 mice were randomized into five groups: SAL = uninfected; INF = infected; SR5, SR10, and SR20 = infected treated with 5, 10, or 20 mg/kg Sur. In addition to its effect on blood and heart parasitism, the impact of Sur-based chemotherapy on leucocytes myocardial infiltration, cytokine levels, antioxidant defenses, reactive tissue damage, and mortality was analyzed. Our results indicated that animals treated with 10 and 20 mg/kg Sur were disproportionally susceptible to , exhibiting increased parasitemia and cardiac parasitism (amastigote nests and parasite load ( DNA)), intense protein, lipid and DNA oxidation, marked myocarditis, and mortality. Animals treated with Sur also exhibited reduced levels of nonprotein antioxidants. However, the upregulation of catalase, superoxide dismutase, and glutathione-S-transferase was insufficient to counteract reactive tissue damage and pathological myocardial remodeling. It is still poorly understood whether Sur exerts a negative impact on the purinergic signaling of -infected host cells. However, our findings clearly demonstrated that through enhanced parasitism, inflammation, and reactive tissue damage, Sur-based chemotherapy contributes to aggravating myocarditis and increasing mortality rates in -infected mice, contradicting the supposed relevance attributed to this drug for the treatment of Chagas disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1155/2018/7385639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6186315PMC
November 2018

Synergism of Plant Compound With Traditional Antimicrobials Against spp. Isolated From Bovine Mastitis.

Front Microbiol 2018 6;9:1203. Epub 2018 Jun 6.

Bacterial Disease Laboratory, Department of Veterinary, Universidade Federal de Viçosa, Viçosa, Brazil.

Mastitis is an inflammation of the mammary gland that causes major losses in the dairy industry. spp. are among the main agents of this disease. Increased resistance to antibiotics is one of the causes of therapeutic failure. Plants, due to their broad chemodiversity, are an interesting source of new molecules with antibacterial activity. Using these compounds along with traditional antibiotics is a possible method for reversing resistance. The objective of this work was to determine the interactions between the activities of guttiferone-A and 7-epiclusianone, two active substances isolated from the fruits of , and traditional antibiotics against spp. isolated from bovine mastitis and known to be resistant to them. First, the MIC for the antibiotics and bioactive compounds was determined, followed by their activities, alone and in combination. Then, their cytotoxicity was measured in bovine mammary epithelial cells. Finally, molecular docking simulations were performed to elucidate molecular details of the interactions between β-lactamase and the compounds binding to it (clavulanic acid, ampicillin, 7-epiclusianone, and guttiferone-A). The bacterial isolates were resistant to ampicillin and gentamicin. Both antibiotics showed predominantly synergistic antibacterial activities in combination with guttiferone-A or 7-epiclusianone. These two active substances were not cytotoxic at synergistic concentrations and both showed strong binding to β-lactamase, which may explain the reversal of ampicillin resistance. These substances are promising for the treatment of bovine mastitis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2018.01203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5998740PMC
June 2018

Synthesis of newly functionalized 1,4-naphthoquinone derivatives and their effects on wound healing in alloxan-induced diabetic mice.

Chem Biol Interact 2018 Aug 11;291:55-64. Epub 2018 Jun 11.

Laboratory of Cell Biology, Campus A.C. Simões, Federal University of Alagoas, CEP 57.072-970, Maceio, Alagoas, Brazil. Electronic address:

Naphthoquinone derivatives have various pharmacological properties. Here, we describe the synthesis of new 1,4-naphthoquinone derivatives inspired by lawsone and β-lapachone and their effects on both migration of fibroblasts in vitro and dermal wound healing in diabetic mice. NMR and FTIR spectroscopy aided characterization of chemical composition and demonstrated the molecular variations after the synthesis of four different derivatives, namely 2-bromo-1,4-naphthoquinone (termed derivative S3), 2-N-phenylamino-1,4-naphthoquinone (derivative S5), 2-N-isonicotinoyl-hydrazide-1,4-naphthoquinone (derivative S6), and 1-N-isonicotinoyl-hydrazone-[2-hydroxy-3-(3-methyl-2-butenyl)]-1,4-naphthoquinone (derivative S7). Our results indicate that derivatives S3, S5, S6 and S7 were non-toxic to the 3T3 fibroblast cell line. In scratch assays, derivatives S3 and S6, but not S5 or S7, stimulated the migration of fibroblasts. Compared with untreated diabetic mice, S3, S6 and S7 treatments accelerated wound closure. However, derivative S3 was optimal for the stimulation of epithelization, thereby increasing the number of keratinocyte layers and blood vessels, and reducing diffuse cellular infiltration, compared to derivatives S6 and S7. Our results suggest that novel 1,4-naphthoquinone derivatives promote fibroblast migration and accelerate wound closure under diabetic conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cbi.2018.06.007DOI Listing
August 2018

Bacteriophage Isolated from Sewage Eliminates and Prevents the Establishment of Biofilm.

Adv Pharm Bull 2018 Mar 18;8(1):85-95. Epub 2018 Mar 18.

Departamento de Biologia Geral, Universidade Federal de Viçosa, 36570-900, Viçosa, Minas Gerais, Brasil.

Biofilm growth exerts a negative impact on industry and health, necessitating the development of strategies to control. The objective of this work was study the lytic activity of the phage isolated from the sewage network in the formation and degradation of Escherichia coli biofilms. E. coli cultures were incubated in 96-well polystyrene microplates under controlled conditions to evaluate the biofilm formation. The E. coli cultures and established biofilms were treated with the suspensions of the vB_EcoM-UFV017 (EcoM017) bacteriophage obtained from sewage for 24 hours. The E. coli bacterial density was measured using absorbance at 600 nm and the biofilms were measured by crystal violet staining. Polystyrene coupons were used as support for Scanning Electron Microscopy and Confocal Microscopy to evaluate biofilm formation. The E. coli strains formed biofilms in polystyrene microplates after 48 hours' incubation. The highest EcoM017 phage titer, in the prevention and degradation experiments, reduced the bacterial growth and the quantity of biofilm formed by E. coli in 90.0% and 87.5%, respectively. The minimum dose capable of reducing the biofilms of this bacterium was 10 PFU/mL after 24 hours. The preformed E. coli biofilm mass was reduced 79% post exposure to the phage in the degradation assay. Microscopic analysis confirmed the results obtained in the plates assays. The EcoM017 phage prevented biofilm formation and degraded the E. coli-established ones. The EcoM017 phage isolated from sewage can reduce bacterial attachment and lyse the E. coli associated biofilm cells, offering biotechnological potential applicability for this phage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15171/apb.2018.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5896399PMC
March 2018

Exposure to deltamethrin induces oxidative stress and decreases of energy reserve in tissues of the Neotropical fruit-eating bat Artibeus lituratus.

Ecotoxicol Environ Saf 2018 Feb 21;148:684-692. Epub 2017 Nov 21.

Departamento de Biologia Geral, Universidade Federal de Viçosa, 36570-900 Viçosa, MG, Brazil. Electronic address:

Deltamethrin (DTM) is a synthetic pyrethroid insecticide highly used by farmers and home users. This pesticide has lipophilic properties that facilitate a high absorption and can cause toxicity in non-target organisms. During foraging, the fruit-eating bats Artibeus lituratus are exposed to pesticides. However, the knowledge of the toxicity of pesticides on the physiology of bats is relatively scarce. This study aimed to check the toxicity of short-term exposure to low concentration of DTM on fruit-eating bat A. lituratus. After seven days of exposure to two doses of DTM (0.02 and 0.04mg/kg of papaya), the fruit bats showed an increase in the enzyme aspartate aminotransferase, alanine aminotransferase, and hyperglycemia. The liver and pectoral muscle presented oxidative stress. In the liver, the hydrogen peroxide (HO) and nitric oxide (NO) were increased as well as the antioxidant glutathione (GSH), the activity of glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT) but in a lesser extent. Yet, total lipids were increased while hepatic glycogen content is reduced. The pectoral muscle showed NO, SOD, CAT, malondialdehyde (MDA), and carbonyl increased protein levels in both concentrations of DTM. All these results show that low doses of DTM can cause hepatic and muscular toxicity and induce changes in carbohydrate metabolism. Physiological changes caused by exposure to DTM in bats may have direct consequences in flight capacity, reproduction, and metabolism of these animals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ecoenv.2017.11.024DOI Listing
February 2018

Metabolic Adaptation Establishes Disease Tolerance to Sepsis.

Cell 2017 Jun;169(7):1263-1275.e14

Instituto Gulbenkian de Ciência, 2780-156 Oeiras, Portugal. Electronic address:

Sepsis is an often lethal syndrome resulting from maladaptive immune and metabolic responses to infection, compromising host homeostasis. Disease tolerance is a defense strategy against infection that preserves host homeostasis without exerting a direct negative impact on pathogens. Here, we demonstrate that induction of the iron-sequestering ferritin H chain (FTH) in response to polymicrobial infections is critical to establish disease tolerance to sepsis. The protective effect of FTH is exerted via a mechanism that counters iron-driven oxidative inhibition of the liver glucose-6-phosphatase (G6Pase), and in doing so, sustains endogenous glucose production via liver gluconeogenesis. This is required to prevent the development of hypoglycemia that otherwise compromises disease tolerance to sepsis. FTH overexpression or ferritin administration establish disease tolerance therapeutically. In conclusion, disease tolerance to sepsis relies on a crosstalk between adaptive responses controlling iron and glucose metabolism, required to maintain blood glucose within a physiologic range compatible with host survival.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2017.05.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5480394PMC
June 2017

Eosinophilic Esophagitis: Latent Disease in Patients with Anaphylactic Reaction to Cow's Milk.

J Allergy Clin Immunol Pract 2018 Mar - Apr;6(2):451-456.e1. Epub 2017 May 26.

Division of Clinical Immunology and Allergy, School of Medicine, University of São Paulo, São Paulo, Brazil.

Background: Food allergy and eosinophilic esophagitis are a substantial and evolving public health issue. Clinicians should know the relationship between these diseases and how one may predispose to the other. This can help minimize misdiagnosis.

Objective: The objective of this study was to assess esophageal eosinophilia and eosinophilic esophagitis frequency in patients with persistent cow's milk allergy and anaphylaxis manifestations.

Methods: Patients with persistent cow's milk allergy with anaphylaxis manifestations were enrolled from 2012 through 2016 at the São Paulo University Hospital, Brazil. All of them were submitted to endoscopy despite the presence or absence of gastrointestinal symptoms. Demographics data, atopic comorbidities, medication use, endoscopic findings, and esophageal eosinophilia frequency were evaluated.

Results: Eighty-nine patients were selected. The median age was 8 years. It was observed that 34 of 89 patients (38.2%; 95% confidence interval [CI]: 28.14%-49.16%) presented with esophageal eosinophilia. Five patients (7.1%) presented proton pump inhibitor-responsive esophageal eosinophilia, and 10 patients (14.2%) presented eosinophilic esophagitis. We found that 29.4% were asymptomatic patients, 23.5% had nonspecific symptoms, 23.5% had persistent typical symptoms, and 23.5% had intermittent typical symptoms. There was an association with inflammatory endoscopy findings in 21 patients (61.7%).

Conclusions: This description demands scientific attention because it is the highest frequency of esophageal eosinophilia yet described in a group of patients with cow's milk allergy presenting with anaphylaxis. Eosinophilic esophagitis is a condition that can coexist "silently" with an IgE-mediated food allergy and is most often underestimated and underdiagnosed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jaip.2017.04.037DOI Listing
November 2019

Vaccination of Mice with Virulence-Associated Protein G (VapG) Antigen Confers Partial Protection against Infection through Induced Humoral Immunity.

Front Microbiol 2017 11;8:857. Epub 2017 May 11.

Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, Universidade de São PauloSão Paulo, Brazil.

is a facultative intracellular bacterium causing severe pyogranulomatous pneumonia, ulcerative enterocolitis, and mesenteric lymphadenopathy in foals aged less than 6 months. Less frequently, this pathogen affects various other species, such as pigs, cattle, cats, and even humans. Although rhodococcosis is treated with a combination of antimicrobial agents, resistance is developed in some cases, and thus, antimicrobial susceptibility must be monitored and managed. Considering these limitations of the current therapy and unavailability of a vaccine to prevent the disease, research is particularly focused on the development of an effective vaccine against rhodococcosis. Most vaccines undergoing development utilize the virulence-associated protein (Vap) A antigen, which was identified previously as a key virulence factor of . Nevertheless, other proteins, such as VapG, present in most virulent strains, are also encoded by vap genes located on the bacterial virulence plasmid. In the present study, we evaluated the effect of VapG immunization on the survival of -challenged mice. We used attenuated as a carrier for VapG (-+), a procedure previously adopted to develop a VapA-based vaccine. We observed that vaccination with -+ induced both an increased IFN-γ, IL-12, and TNF-α production, and a decreased bacterial burden in organs of the -challenged mice. Nevertheless, -+ vaccination protected only 50% of the mice challenged with a lethal dose of . Interestingly, we observed an increased frequency of B cells in the spleen of -+-vaccinated mice and showed that -+-vaccinated -challenged B-cell-knockout mice did not reduce the bacterial burden. Given these results, we discussed the potential role of the humoral immune response induced by -+ vaccination in conferring protection against infection, as well as the employment of VapG antigen for obtaining hyperimmune plasma to prevent rhodoccocosis in young foals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2017.00857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5425581PMC
May 2017

MAST CELLS DISTINGUISH EOSINOPHILIC ESOPHAGITIS IN PEDIATRIC PATIENTS.

Arq Gastroenterol 2017 Jul-Sept;54(3):192-196. Epub 2017 May 8.

Departamento de Patologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, SP, Brasil.

Background: : Mast cells exert a substantial role in gastrointestinal allergic diseases. Therefore, it is reasonable to presume that mast cell may aid diagnosis in eosinophilic gastroenteropathy.

Objective: : To evaluate whether mast cell count in the esophageal epithelium can discriminate eosinophilic esophagitis, proton-pump inhibitor (PPI)-responsive eosinophilic esophagitis and gastroesophageal reflux esophagitis.

Methods:: Retrospectively we reviewed the files of 53 consecutive patients (age: 7.8 years; range: 8-14 years) with definitive diagnose established during clinical follow up in a universitary outpatient clinic as follow: eosinophilic esophagitis (N=23), PPI-responsive eosinophilic esophagitis (N=15) and gastroesophageal reflux esophagitis (N=15). Eosinophil count in the esophageal epithelium in slides stained with H-E was reviewed and immunohistochemistry for mast cell tryptase was performed.

Results:: Count of eosinophils/high-power field (HPF) higher than 15 were found in 14 out of 15 reflux esophagitis patients. The mean count of eosinophils/HPF was similar in eosinophilic esophagitis patients and in those with PPI-responsive eosinophilic esophagitis (42 and 39 eosinophils/HPF, respectively, P=0.47). Values of mast cell tryptase (+) were higher in eosinophilic esophagitis [median: 25 mast cells/HPF; range (17-43) ] and in PPI-responsive eosinophilic esophagitis patients [25 (16-32) ], compared to reflux esophagitis [4 (2-14) ], P<0.001. There was no difference between the mean count of mast cells/HPF in the esophageal epithelium of eosinophilic esophagitis patients and PPI-responsive eosinophilic esophagitis patients, respectively, 26 and 24 mast cells/HPF, P=0.391.

Conclusion: : Tryptase staining of mast cells differentiates eosinophilic esophagitis from reflux esophagitis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1590/S0004-2803.201700000-23DOI Listing
November 2017

A new TRAF-like protein from B. oleracea ssp. botrytis with lectin activity and its effect on macrophages.

Int J Biol Macromol 2017 Jan 19;94(Pt A):508-514. Epub 2016 Oct 19.

Departamento de Biologia Geral, Universidade Federal de Viçosa, 36570-900 Viçosa, MG, Brazil. Electronic address:

Lectins are involved in a wide range of biological mechanisms, like immunomodulatory agent able to activate the innate immunity. In this study, we purified and characterized a new lectin from cauliflower (Brassica oleracea ssp. botrytis - BOL) by three sequential chromatographic steps and confirmed the purity by SDS-PAGE. Additionally, we evaluated the role of the lectin in innate immunity by a phagocytosis assay, production of HO and NO. BOL was characterized like a non-glycosylated protein that showed a molecular mass of ∼34kDa in SDS-PAGE. Its N-terminal sequence (ETRAFREERPSSKIVTIAG) did not reveal any similarity to the other lectins; nevertheless, it showed 100% homology to a putative TRAF-like protein from Brassica rapa and Brassica napus. This is a first report of the TRAF-protein with lectinic activity. The BOL retained its complete hemagglutination activity from 4°C up to 60°C, with stability being more apparent between pH 7.0 and 8.0. Moreover, the lectin was able to stimulate phagocytosis and induce the production of HO and NO. Therefore, BOL can be explored as an immunomodulatory agent by being able to activate the innate immunity and favor antigen removal.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijbiomac.2016.10.061DOI Listing
January 2017

Erratum to: Antigen production using heterologous expression of dengue virus-2 non structural protein 1 (NS1) in Nicotiana tabacum (Havana) for immunodiagnostic purposes.

Plant Cell Rep 2016 Oct;35(10):2205

Laboratory of Molecular Immunovirology, Department of General Biology, Federal University of Viçosa, Av. PH Rolfs, s/n Campus Universitário, Viçosa, MG, CEP 36570-000, Brazil.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00299-016-2043-6DOI Listing
October 2016

Leishmania chagasi heparin-binding protein: Cell localization and participation in L. chagasi infection.

Mol Biochem Parasitol 2015 Nov 21;204(1):34-43. Epub 2015 Dec 21.

Departamento de Biologia Geral, Universidade Federal de Viçosa, Viçosa, Minas Gerais 36570-900, Brazil.

Visceral leishmaniasis is a fatal human disease caused by the intracellular protozoan parasite Leishmania chagasi that is captured by host cells in a process involving classics receptors mediated phagocytosis. The search for molecules involved in this process is important to design strategies to disease control. In this work, we verified the presence of heparin-binding protein (HBP) in L. chagasi promastigotes forms. HBP is a lectin of the group of ubiquitous proteins, whose main characteristic is to bind to carbohydrates present in glycoproteins or glycolipids, which is poorly studied in Leishmania species. L. chagasi HBP (HBPLc) was purified by affinity chromatography using heparin-agarose column in FPLC automated system. Its localization in the parasite was assessed by immunolabeling and electronic transmission microscopy tests using anti-HBPLc polyclonal antibodies, which showed HBP spread over the parasite outer surface and internally next to the kynetoplast. In addition, we verified that HBPLc participates in the process of parasite infection, since its blocking with heparin generated a partial reduction in the internalization of Leishmania by RAW macrophages "in vitro". According to these results, it is believed that, in further "in vivo" studies, interference on this parasitic protein may provide us prophylactic and therapeutic alternatives against visceral leishmaniasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molbiopara.2015.12.005DOI Listing
November 2015

Modulation of oxidative and inflammatory cardiac response by nonselective 1- and 2-cyclooxygenase inhibitor and benznidazole in mice.

J Pharm Pharmacol 2015 Nov 23;67(11):1556-66. Epub 2015 Jun 23.

Department of General Biology, Federal University of Viçosa, Viçosa, MG, Brazil.

Objectives: This study investigated the combined effects of benznidazole (BZ) and ibuprofen (IB) on the oxidative and inflammatory status of the cardiac tissue in vivo.

Methods: Swiss mice were randomized in groups receiving BZ (100 mg/kg) and IB (400 mg/kg) alone or combined (BZ + IB 200 or 400 mg/kg). Control animals were concurrently treated with 1% carboxymethyl cellulose. All treatments were administered orally for 7 days.

Key Findings: BZ treatment increased cardiac production of nitrogen/oxygen-reactive species, malondialdeyde, carbonyl proteins, prostaglandins as well as the activities of catalase, superoxide dismutase and glutathione peroxidase. These parameters were attenuated by IB, with the best results at higher dose. Individually, BZ and IB significantly reduced the tissue levels of chemokine ligand 2, tumour necrosis factor-α and IL-10, but no reduction was observed when the treatments were combined.

Conclusions: BZ triggers an oxidative and nitrosative route, which is associated with increased prostaglandin synthesis and marked damages to the lipids and proteins of the cardiac tissue. IB treatment attenuated reactive stresses triggered by BZ, which was an independent effects of this drug on the endogenous antioxidant enzymes. Individually, but not together, BZ and IB reduced the cardiac inflammatory status, indicating a beneficial and complex drug interaction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jphp.12451DOI Listing
November 2015

Identification and characterization of an antimicrobial peptide of Hypsiboas semilineatus (Spix, 1824) (Amphibia, Hylidae).

Toxicon 2015 Jun 12;99:16-22. Epub 2015 Mar 12.

Federal University of Viçosa, Departament of General Biology, Av. P.H. Rolfs s/n, 36570-000 Viçosa, MG, Brazil. Electronic address:

The multidrug-resistant bacteria have become a serious problem to public health. In this scenery the antimicrobial peptides (AMPs) derived from animals and plants emerge as a novel therapeutic modality, substituting or in addition to the conventional antimicrobial. The anurans are one of the richest natural sources of AMPs. In this work several cycles of cDNA cloning of the skin of the Brazilian treefrog Hypsiboas semilineatus led to isolation of a precursor sequence that encodes a new AMP. The sequence comprises a 27 residue signal peptide, followed by an acidic intervening sequence that ends in the mature peptide at the carboxy terminal. The AMP, named Hs-1, has 20 amino acids residues, mostly arranged in an alpha helix and with a molecular weight of 2144.6 Da. The chemically synthesized Hs-1 showed an antimicrobial activity against all Gram-positive bacteria tested, with a range of 11-46 μM, but it did not show any effect against Gram-negative bacteria, which suggest that Hs-1 may have a selective action for Gram-positive bacteria. The effects of Hs-1 on bacterial cells were also demonstrated by transmission electron microscopy. Hs-1 is the first AMP to be described from H. semilineatus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.toxicon.2015.03.006DOI Listing
June 2015

Antigen production using heterologous expression of dengue virus-2 non-structural protein 1 (NS1) in Nicotiana tabacum (Havana) for immunodiagnostic purposes.

Plant Cell Rep 2015 Jun 18;34(6):919-28. Epub 2015 Feb 18.

Laboratory of Molecular Immunovirology, Department of General Biology, Federal University of Viçosa, Av. PH Rolfs, s/n Campus Universitário, Viçosa, MG, CEP 36570-000, Brazil.

Key Message: Expression of dengue-2 virus NS1 protein in Nicotiana tabacum plants for development of dengue immunodiagnostic kits. Dengue is one of the most important diseases caused by arboviruses in the world. A significant increase in its geographical distribution has been noticed over the last 20 years, with continuous transmission of several serotypes and emergence of the hemorrhagic fever in areas where the disease was previously not prevalent. Although the methodological processes for dengue diagnosis are in deep development and improvement, a limitation for the realization of dengue diagnostic tests is the difficulty of large-scale production of the antigen to be used in diagnostic tests. Due to this demand, the purpose of this study was to obtain the non-structural protein 1 (NS1) from dengue-2 serotype by heterologous expression in Nicotiana tabacum (Havana). After confirmation of the NS1 protein gene integration in the plant genome, the heterologous protein was characterized using SDS-PAGE and immunoblotting. In an immunoenzymatic test, the recombinant NS1 protein presents an antigen potential for development of dengue immunodiagnostic kits.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00299-015-1753-5DOI Listing
June 2015

Construction of recombinant Kluyveromyces marxianus UFV-3 to express dengue virus type 1 nonstructural protein 1 (NS1).

Appl Microbiol Biotechnol 2015 Feb 2;99(3):1191-203. Epub 2014 Aug 2.

Laboratório de Fisiologia de Micro-organismos, Departamento de Microbiologia, BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, Brazil.

The yeast Kluyveromyces marxianus is a convenient host for industrial synthesis of biomolecules. However, despite its potential, there are few studies reporting the expression of heterologous proteins using this yeast. Here, we report expression of a dengue virus protein in K. marxianus for the first time. The dengue virus type 1 nonstructural protein 1 (NS1) was integrated into the K. marxianus UFV-3 genome at the LAC4 locus using an adapted integrative vector designed for high-level expression of recombinant protein in Kluyveromyces lactis. The NS1 gene sequence was codon-optimized to increase the level of protein expression in yeast. The synthetic gene was cloned in frame with K. lactis α-mating factor signal peptide, and the recombinant plasmid obtained was used to transform K. marxianus UFV-3 by electroporation. The transformed cells, selected in yeast extract peptone dextrose containing 200 μg mL(-1) Geneticin, were mitotically stable. Analysis of recombinant strains by RT-PCR and protein detection using blot analysis confirmed both transcription and expression of extracellular NS1 polypeptide. After induction with galactose, the NS1 protein was analyzed by sodium dodecyl sulfate-PAGE and immunogenic detection. Protein production was investigated under two conditions: with galactose and biotin pulses at 24-h intervals during 96 h of induction and without galactose and biotin supplementation. Protease activity was not detected in post-growth medium. Our results indicate that recombinant K. marxianus is a good host for the production of dengue virus NS1 protein, which has potential for diagnostic applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00253-014-5963-5DOI Listing
February 2015