Effects UV-A and visible light on skin cells and hair

A luz solar apresenta ondas eletromagnéticas em ampla faixa espectral, incluindo as regiões do ultravioleta (UV-C, UV-B, UV-A), visível e infravermelho. Cada região interage com a pele de forma dependente da fotofísica e da fotoquímica dos seus respectivos compostos absorvedores. A luz UV-A causa a geração de espécies reativas de oxigênio e de nitrogênio (EROs e ERNs) através da fotossensibilização de moléculas endógenas (co-enzimas de flavina, porfirinas, melaninas). Quando fotossensibilizadores produzem quantidades de EROs e ERNs maiores do que a capacidade celular de supressão destas espécies, caracteriza-se um quadro de desbalanço redox, que causa lesão em biomoléculas como os ácidos nucleicos, lipídeos e as proteínas. Essas lesões podem levar à morte celular ou a outras transformações fenotípicas e genotípicas e também estimulam a liberação de citocinas pró-inflamatórias. Com a finalidade de melhor compreender a dinâmica dos mecanismos de resposta celular após exposição ao UV-A e ao visível, nós caracterizamos inicialmente as propriedades fotofísicas da melanina e detectamos a produção de oxigênio singlete (1O2) pela fotossensibilização no visível e a supressão desta espécie excitada pela reação do oxigênio singlete com a dupla ligação reativa dos grupos indóis presentes na melanina. Estes processos também foram observados no cabelo e levaram-nos a propor um modelo que explica o efeito da luz visível na estrutura e cor dos cabelos. Demonstramos também que a feomelanina produz mais (30%) 1O2 do que a eumelanina, que sofre maior modificação na sua estrutura por fotodegradação. O efeito destes processos na pele foi estudado a nível celular. Demonstramos que células epiteliais com maior teor de melanina apresentaram maior geração de 1O2 que causa lesão no DNA e morte necro-apoptótica após irradiação com luz visível. A foto-oxidação da melanina pela luz visível nos motivou a estudar um pigmento que fosse foto-protetor não somente contra luz UV-B mas também contra luz visível. A pigmentação com Acetil-Tirosina se mostrou atóxica e protetora contra luz UV-B e visível ao contrário do pigmento com tirosina, que se mostrou protetor do UV-B mas tóxico no visível. Este efeito foi relacionado com a localização celular do polímero e não com a estrutura do mesmo. A luz UV-A, por sua vez, promove o acúmulo de lipofuscina dentro dos vacúolos autofágicos de queratinócitos da pele e que também ativa a fototoxicidade pela luz visível. A lipofuscina dentro dos vacúolos autofágicos é foto-oxidada pela luz visível, causando lesão no DNA e morte celular programada tipo II. Doses UV-A que desencadeiam a liberação de citocinas também foram caracterizados.Sunlight presents electromagnetic radiation over a wide spectral range, including the regions of ultraviolet (UV-C, UV-B, UV-A), visible and infrared. Each region interacts with skin dependending on the photophysics and photochemistry of the respective absorbing compounds. UV-A light causes the generation of reactive oxygen and nitrogen species (ROS and RNS) by photosensitization of endogenous molecules (flavin coenzymes, porphyrins, melanins). When photosensitizers produce amounts of ROS and RNS larger than the cell capacity to suppress these species, a set of redox imbalance, which damages biomolecules such as nucleic acids, lipids and proteins. This damage cause cell death and to other phenotypic and genotypic changes and also stimulates the release of proinflammatory cytokines. In order to better understand the dynamics of the mechanisms of cellular responses after exposure to UV-A and visible light, we initially characterized the photophysical properties of melanin and detected the production of singlet oxygen (1O2) by photosensitization in the visible, as well as the suppression of these excited species by reaction of singlet oxygen with the double bonds of the reactive groups presented in the melanin indols. These processes were also observed in hair and led us to propose a model that explains the effects of visible light on the structure and color of hair. We also demonstrated that pheomelanin produces more (30%) 1O2 than eumelanin, which undergoes a quick change on its structure by photodegradation. The effect of these processes in the skin was studied at the cellular level. We demonstrated that epithelial cells with larger melanin content have stronger generation of 1O2, which causes DNA damage and necro-apoptotic death after irradiation with visible light. The photo-oxidation of melanin by visible light has motivated us to study a pigment that was not only able to protect against UV-B but also against visible. Pigmentation with Acetyl-Tyrosine proved nontoxic and protective against UV-B and visible light instead of pigmentation with Tyrosine, which shielded against UV-B but showed toxicity in the visible. This effect was associated with the polymer, cell location and not with its structure. UV-A light, in turn, promotes the accumulation of lipofuscin, within autophagic vacuoles of keratinocytes also enabling phototoxicity in the visible light. The lipofuscin within the autophagic vacuoles is fotooxidized by visible light, causing DNA damage and programmed cell death type II. Linear dose of UV-A that trigger the release of cytokines were also characterized

Filogeny and filogeografy of porcine circovirus 2 (PCV2), construction and characterization of a recombinant adenovirus that express capsid protein of PCV2

O circovirus suíno 2 (PCV2) é o principal agente causador da síndrome da postweaning multisystemic wasting syndrome (PMWS) e está associado a diferentes síndromes em suínos. O genoma do PCV2 possui três ORFs principais, sendo a ORF2 a codificadora da proteína do capsídeo. No primeiro estudo, sequências de nucleotídeos da ORF2 de 30 isolados brasileiros foram analizadas e comparadas com outras sequências depositadas no GenBank usando uma abordagem filogenética e filogeográfica. No segundo estudo, a fim de controlar a circovirose suína, nós construímos um sistema de expressão gênica da ORF2 do PCV2 em vetor adenovírus. Primeiramente, um total de onze amostras de seis estados brasileiros foram coletadas e as ORF2 foram clonadas em vetor pGEM T-easy e sequenciadas. Nossos resultados, portanto, mostraram uma alta variabilidade de sequências no Brasil além de os isolados brasileiros serem classificados nos subgrupos 1AB, 2D e 2, e que o vírus foi introduzido no Brasil mais que uma vez. Em seguida, a ORF2 de um desses isolados foi amplificada pela reação da polimerase em cadeia (PCR) e clonada em vetor pGEM T-easy. Células de Escherichia coli DH5α foram transformadas com o plasmídeo recombinante (pGEM-ORF2) e o material genético amplificado. A clonagem foi confirmada por sequenciamento e ensaio de restrição com a enzima EcoRI. Confirmada a clonagem, o plasmídeo foi clivado pelas enzimas AclI e NheI, liberando a ORF2. Este segmento foi clonado no vetor pAD5-Blue previamente clivado pelas enzimas ClaI e XbaI. Células de E. coli TOP10 foram transformadas com a construção (pAd5-Blue ORF2); as colônias recombinantes foram selecionadas e o material genético multiplicado. Este plasmídeo foi linearizado pela enzima PacI e trasnfectado em células da linhagem HEK 293. A expressão viral in vitro foi confirmada por imunoperoxidase em monocamada, Western blotting e imunofluorescência. Os vírus purificados foram titulados em 2 x108 TCID50/50μL. Duas doses de vírus purificado foram inoculadas em camundongos BALB/c para verificação da resposta imune. Soro e baço dos animais foram coletados para ensaios de resposta humoral e celular, respectivamente. A resposta imune celular se caracterizou por uma eficiente expressão de mRNA de INF-γ bem como a proliferação de linfócitos T CD8+. Em relação à resposta humoral, o candidato vacinal apresentou produção de anticorpos principalmente após a segunda dose evidenciado na coleta de soro do dia 45. De acordo com os resultados encontrados neste trabalho, acredita-se que este modelo de vacina possa ser utilizado em suíno SPF e convercional uma vez que em modelo murino, o adenovírus recombinante foi capaz de estimular resposta imune celular, em especial linfócitos T CD8+, o que destaca eficiênica na eliminação do PCV2.The porcine circovirus 2 (PCV2) is the main agent causing the postweaning multisystemic wasting syndrome (PMWS) and is associated with different syndromes in pigs. The genome of PCV2 has three major ORFs, the ORF2 encodes a protein of capsid. First in this study, the ORF2 nucleotide sequences of 30 Brazilian isolates were analyzed and compared to other GenBank sequences using phylogenic and phylogeographic approaches. Secound, in order for the control of swine circovirose, our constructed a system of gene expression of the RF2 of PCV2 in an adenovirus vector. The first work, a total of eleven samples of six Brazilian states were collected and the ORF2 were cloned into vector pGEM T-easy and sequencing. Our results show high sequence variability in Brazil, since, in this work, the Brazilian isolates were classified into subgroup 1AB, 2D and 2, which reveals that the virus was introduced in Brazil more than once. The second work, one this isolate of ORF2 was amplified by the polymerase chain reaction (PCR). Cells of Escherichia coli DH5α were transformed with the recombinant plasmid (pGEM-ORF2) and amplified genetic product. The cloning was confirmed by and a test of the restriction enzyme EcoRI. Confirmed the cloning, the plasmid was cleaved by restriction enzyme NheI and AclI, releasing the ORF2. This segment was cloned in the vector-Blue pAD5 previously cleaved with the enzymes XbaI and ClaI. Cells of E. coli TOP10 were transformed with the construction (pAd5 Blue-ORF2), the recombinant colonies were selected and the genetic product multiplied. This plasmid was cleaved by the enzyme PacI, linearized and trasnfected in HEK 293 cells. The confirmation of viral expression in vitro was detected by Immunoperoxidase Monolayer Assay (IPMA), Western Blotting and Immunofluorescence Assay (IFA) tecnics. The purified viruses were titled in 2 x108 TCID50/50μL. Two doses of purified virus were inoculated into BALB/c animals to check the immune response. Serum and spleen of animals were collected for testing the humoral and cellular response respectively. The cellular immune response marked a high expression of mRNA for INF-γ and high proliferation of CD8+ T cells. Regarding the humoral response, the candidate vaccine showed antibody production after the second dose mainly evidenced in the collected of serum of day 45. According to the results found in this work, this type of vaccine can be used in SPF and convercional pigs since in mouse model, the recombinant adenovirus was able to stimulate cellular immune response, particularly cells T CD8+, that emphasizes the elimination of PCV2.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Melanin, lipofuscin and the effects of visible light in the skin

Photoexcitation of endogenous photosensitizers (ePS) in the human skin by sunlight culminates with the formation of electronic excited states, such as triplet excited states, singlet oxygen, and a variety of reactive oxygen and nitrogen species, either free radicals or two-electron oxidants. Considering the endogenous skin photosensitizers, melanin is the most abundant and is involved in photoprotection mechanisms, while lipofuscin is an unintentionally pigment accumulated during cell aging or oxidative stress. Both pigments severely increase the phototoxicity of visible light (VL) to skin cells. In the presence of these pigments, VL induces significant oxidative damage in nucleic acids, lipids and proteins, triggering regulated and unregulated cell death mechanisms. Besides, there is accumulation of premutagenic DNA lesions, indicated by the formamidopyrimidine glycosylase (Fpg)- and endonuclease III (Endo III)-sensitive sites in the nuclear DNA. As consequence of the photoinduced oxidative damage, skin cells, under the stimulus of VL release pro-inflammatory cytokines and metalloproteinases, prompting further cell death and skin aging. In this review we describe the photochemical properties of both melanin and lipofuscin pigments and discuss the consequences of their accumulation in terms of the phototoxicity of VL to the human skin

DNA lesions triggered by visible light in skin cells: In the search for comprehensive sun protection

Skin cells present many endogenous photosensitizers (ePS) that interact with light, generating oxidizing species, causing molecular damage in proteins, lipids, and nucleic acids, and consequently triggering cellular and organelle malfunction. Several cell lines with terminal differentiation are susceptible to accumulating non-digestible pigments, such as lipofuscin or melanin-lipofuscin. Besides being hallmarks of aging, both pigments can work as photosensitizers, increasing and expanding the toxicity of sunlight to the range of visible light (VL, 400–700 nm). In here we review the literature to describe the mechanisms by which the photosensitized oxidation reactions induced by VL cause DNA damage. We aim to provide the mechanistic background needed to improve the current strategies of photoprotection

Infecção do trato urinário: estudo de sensibilidade e resistência bacteriana em pacientes internados

Infecção do Trato Urinário (ITU) é uma doença bacteriana que acomete principalmente o sexo feminino com maior prevalência em crianças e idosos. Os antibióticos possuem uma capacidade de inibir o crescimento bem como destruir as bactérias que causam a ITU. O uso de antibióticos sem condução do antibiograma tem aumentado o número de isolados resistentes. O foco deste estudo foi investigar a incidência e a resistência aos antibióticos das bactérias causadoras da ITU em hospitais de Colatina. Laudos da urocultura dos pacientes hospitalizados no período de 2015 a 2019 foram investigados. Faixa etária, sexo, agentes etiológicos mais freqüentes e resistência aos antibióticos foram analisados. A avaliação estatística foi realizada pelo programa Origin 8,0 a p<0,05. Os resultados mostraram maior incidência de ITU em crianças e idosos com acometimento maior em pacientes do sexo feminino (56%). Klebsiella pneumoniae (31%), Escherichia coli (29%), Enterococcus sp (20%), Pseudomonas aeruginosa (7%), Proteus (7%) e Stapylococcus coagulase negativa (6%) foram as bactérias mais prevalentes. Klebsiella pneumoniae mostrou-se resistente em 41% dos antibióticos administrados, Stapylococcus coagulase negativa 38%, Pseudomonas aeruginosa 30%, Proteus 29%, Escherichia coli 21% e Enterococcus sp 19%. Amicacina, gentamicina, imipenem e vancomicina foram os antibióticos mais eficazes para o tratamento da ITU. Pacientes internados em hospitais de Colatina com ITU apresentaram resistência bacteriana em torno de 40% para a maioria dos antibióticos administrados. Portanto faz-se necessário o uso do antibiograma para evitar o aumento da resistência bacteriana nos ambientes hospitalares de Colatina – ES.Urinary tract infection (UTI) is a bacterial disease that mainly affects females, with a higher prevalence in children and the elderly. Antibiotics have the ability to inhibit growth and destroy bacteria that cause UTI. The use of antibiotics without conducting antibiograms increased the number of resistant isolates. The focus of this study was to investigate the incidence and antibiotic resistance of bacteria that cause UTIs in Colatina hospitals. Uroculture reports of patients hospitalized between 2015 and 2019 were investigated. Statistical evaluation was performed using Origin 8.0 at p <0.05. The results showed a higher incidence of UTI in children and the elderly, with a greater number of female patients (56%). Klebsiella pneumoniae (31%), Escherichia coli (29%), Enterococcus sp. (20%), Pseudomonas aeruginosa (7%), Proteus (7%), and Staphylococcus sp. (6%) were the most prevalent. Klebsiella pneumoniae was resistant to 41% of the antibiotics administered. The other bacteria showed 38% resistance to Staphylococcus sp., 30% to Pseudomonas aeruginosa, 29% to Proteus, 21% to Escherichia coli, and 19% to Enterococcus sp. Amikacin, gentamicin, imipenem, and vancomycin were the most effective antibiotics for the treatment of UTIs. Patients admitted to Colatina hospitals with UTI showed a bacterial resistance of approximately 40% for most antibiotics administered. Therefore, it is necessary to use antibiograms to avoid increasing bacterial resistance in hospital environments in Colatina, ES

Quercetin as a shuttle for labile iron

The antioxidant activity of flavonoids may involve their ability to complex body iron in non-redox-active forms. In this study, it was found that the catechol flavonoids rutin and quercetin are able to suppress redox-active labile plasma iron (LPI) in both buffered solution and in iron-overloaded sera. Both flavonoids are effective in loading the metal into the iron-transport protein transferrin. Iron derivatives of quercetin and rutin are able to permeate cell membranes, however, only free quercetin is able to gain access to the cytosol and decrease intracellular labile iron pools. These results suggest that the antioxidant activity of quercetin may be dependent on its ability to shuttle labile iron from cell compartments followed by its transfer to transferrin. (C) 2011 Elsevier Inc. All rights reserved.Brazilian government agency CNPqCNPq (Brazilian government agency)FAPESP (Brazilian government agency)Brazilian government agency FAPES

Generation and suppression of singlet oxygen in hair by photosensitization of melanin

We have studied the spectroscopic properties of hair (white, blond, red, brown, and black) under illumination with visible light, giving special emphasis to the photoinduced generation of singlet oxygen ((1)O(2)). Irradiation of hair shafts (lambda(ex)>400 nm) changed their properties by degrading the melanin. Formation of C3 hydroperoxides in the melanin indol groups was proven by (1)H NMR. After 532-nm excitation, all hair shafts presented the characteristic (1)O(2) emission (lambda(em) = 1270 nm), whose intensity varied inversely with the melanin content. (1)O(2) lifetime was also shown to vary with hair type, being five times shorter in black hair than in blond hair, indicating the role of melanin as a (1)O(2) suppressor. Lifetime ranged from tenths of a nanosecond to a few microseconds, which is much shorter than the lifetime expected for (1)O(2) in the solvents in which the hair shafts were suspended, indicating that (1)O(2) is generated and suppressed inside the hair structure. Both eumelanin and pheomelanin were shown to produce and to suppress (1)O(2), with similar efficiencies. The higher amount of (1)O(2) generated in blond hair and its longer lifetime is compatible with the stronger damage that light exposure causes in blond hair. We propose a model to explain the formation and suppression of (1)O(2) in hair by photosensitization of melanin with visible light and the deleterious effects that an excess of visible light may cause in hair and skin. 2011 Published by Elsevier Inc.FAPESPFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Farma Service BioextractFarma Service Bioextrac

Development and evaluation of a recombinant DNA vaccine candidate expressing porcine circovirus 2 structural protein Desenvolvimento e avaliação de um candidato à vacina de DNA recombinante expressando a proteína estrutural do circovírus suíno

Porcine circovirus 2 (PCV2) is generally associated with the porcine circovirosis syndrome, which is considered an important disease of swine and has potentially serious economic impact on the swine industry worldwide. This article describes the construction of a recombinant plasmid expressing the PCV2 structural protein and the evaluation of cellular and humoral immune responses produced by this recombinant vaccine in BALB/c mice. The vaccine candidate was obtained and analyzed in vivo, in an effort to determine the ability to induce a specific immune response in mice. DNA was extracted from a Brazilian PCV2 isolate and the gene coding for Cap protein was amplified by PCR and inserted into an expression plasmid. Groups of BALB/c mice were inoculated intra-muscularly and intradermally in a 15-day interval, with 100 µg and 50 µg of the vaccine construct, respectively. Another group was inoculated intramuscularly with 100 µg of empty plasmid, corresponding to the control group. Seroconversion and cellular response in BALB/c mice were compared and used for vaccine evaluation. Seroconversion was analyzed by ELISA. After a series of 3 immunizations the spleen cells of the immunized animals were used to perform lymphocyte proliferation assays. Seroconversion to PCV2 was detected by ELISA in the animals inoculated with the vaccine construct when compared with control groups. Lymphocyte proliferation assays showed a stronger cell proliferation in the inoculated animals compared with the control group. Thus, the vaccine candidate construct demonstrated to be able to induce both humoral and cellular responses in inoculated mice.O circovírus suíno 2 (PCV2) é geralmente associado à síndrome da circovirose suína, que é considerada uma importante doença de suínos e possui um sério impacto econômico na suinocultura mundial. Este trabalho descreve a construção de um plasmídeo recombinante que expressa a proteína estrutural do PCV2 e a avaliação das respostas imune humoral e celular por meio de vacinação em camundongos BALB/c. O candidato vacinal foi submetido a análises in vivo, determinando a capacidade de induzir resposta imune específica em camundongos. O DNA de um isolado brasileiro de PCV2 foi extraído e o gene que codifica para a proteína do capsídeo foi amplificado por PCR e inserido num plasmídeo de expressão. Grupos de camundongos BALB/c foram inoculados por via intramuscular e intradérmica a cada 15 dias, com 100µg e 50µg da construção vacinal, respectivamente. Outro grupo foi inoculado com 100µg do plasmídeo original, correspondente ao grupo controle. A soroconversão e a resposta celular dos grupos de camundongos BALB/c vacinados foram comparados como parâmetros de avaliação vacinal. A soroconversão foi avaliada por um teste de ELISA. Após 3 imunizações, as células esplênicas dos animais imunizados foram utilizadas nos ensaios de linfoproliferação. A soroconversão para o PCV2 foi detectada por ELISA nos animais inoculados com a construção vacinal quando comparados com o grupo controle. Nos ensaios de linfoproliferação foi observada uma grande proliferação celular nos animais inoculados comparados ao grupo controle. Portanto, o candidato vacinal demonstrou ser capaz de induzir tanto uma resposta humoral e celular nos camundongos inoculados

Mixed-Cell Type Choroidal Melanoma in a Middle-Aged Woman

Introduction: Melanomas are malignant neoplasms that occur in various anatomical sites, including the eye. Ocular melanomas account for 5% of all melanomas and are mainly described in Caucasian and older individuals. This study describes the clinical and pathological characteristics of uveal (choroid) melanoma in a Caucasian patient. Case report: A 41-year-old Caucasian female patient, brown eyes, without history of ophthalmological diseases or family history of cancer experienced pain and loss of visual acuity in the left eye. On clinical examination, an increase of ocular pressure was detected. Ultrasound showed a mushroom-like neoformation. Moreover, magnetic resonance imaging showed a mass with spontaneous hypersignal on T1-weighted images, intense gadolinium enhancement, and marked hyposignal on T2-weighted images. The patient was referred to the Oncology Ophthalmology department for enucleation due to suspected uveal melanoma. Anatomopathological analysis revealed a blackened mass in the eyeball. Histologically, the mass comprised spindle cells (50%) and epithelioid cells (50%). A diagnosis of choroidal melanoma was established based on the identification of ophthalmoscopic, imaging, and histological characteristics of the tumor. Conclusion: Choroidal melanomas usually occur in males, clear-eyed, and older individuals. A wide variety of ocular lesions may mimic choroidal melanoma, which should be included in the differential diagnosis of choroidal nevus and peripheral hemorrhages