Cellular In Vitro Responses Induced by Human Mesenchymal Stem/Stromal Cell-Derived Extracellular Vesicles Obtained from Suspension Culture.

Mesenchymal stem/stromal cells (MSCs) and their extracellular vesicles (MSC-EVs) have been described to have important roles in tissue regeneration, including tissue repair, control of inflammation, enhancing angiogenesis, and regulating extracellular matrix remodeling. MSC-EVs have many advantages for use in regeneration therapies such as facility for dosage, histocompatibility, and low immunogenicity, thus possessing a lower possibility of rejection. In this work, we address the potential activity of MSC-EVs isolated from adipose-derived MSCs (ADMSC-EVs) cultured on cross-linked dextran microcarriers, applied to test the scalability and reproducibility of EV production. Isolated ADMSC-EVs were added into cultured human dermal fibroblasts (NHDF-1), keratinocytes (HaCat), endothelial cells (HUVEC), and THP-1 cell-derived macrophages to evaluate cellular responses (i.e., cell proliferation, cell migration, angiogenesis induction, and macrophage phenotype-switching). ADMSC viability and phenotype were assessed during cell culture and isolated ADMSC-EVs were monitored by nanotracking particle analysis, electron microscopy, and immunophenotyping. We observed an enhancement of HaCat proliferation; NHDF-1 and HaCat migration; endothelial tube formation on HUVEC; and the expression of inflammatory cytokines in THP-1-derived macrophages. The increased expression of TGF-ß and IL-1ß was observed in M1 macrophages treated with higher doses of ADMSC-EVs. Hence, EVs from microcarrier-cultivated ADMSCs are shown to modulate cell behavior, being able to induce skin tissue related cells to migrate and proliferate as well as stimulate angiogenesis and cause balance between pro- and anti-inflammatory responses in macrophages. Based on these findings, we suggest that the isolation of EVs from ADMSC suspension cultures makes it possible to induce in vitro cellular responses of interest and obtain sufficient particle numbers for the development of in vivo concept tests for tissue regeneration studies.

A Target-Site Mutation Confers Cross-Resistance to ALS-Inhibiting Herbicides in Erigeron sumatrensis from Brazil.

Cases of weed resistant to herbicides have changed the dynamics of agricultural areas in Brazil, and in recent years, Erigeron species have caused major problems to farmers in the country, mainly in relation to the ineffectiveness of herbicide treatments used. The objective of this study was to confirm the cross-resistance to ALS inhibitors in populations of Erigeron sumatrensis as well as to investigate the existence of mutations in the site of action of ALS-inhibiting herbicides. To do this, 30 populations collected in the 2016/2017 crop season were grown in a greenhouse. Dose-response (chlorimuron-ethyl and cloransulam-methyl), inhibition of cytochrome P-450 with malathion, and ALS gene sequencing experiments were carried out in the F1 generations of two fleabane populations. The results proved the cross-resistance to chlorimuron-ethyl and cloransulam-methyl herbicides applied in the post-emergence of the resistant population of E. sumatrensis. The higher activity of P450 enzymes is unlikely responsible for the resistance of the population studied. The resistance mechanism found in R was the target site mutation Pro197Ser at the ALS gene. This is the first study in Brazil to identify a target-site change as a survival mechanism in E. sumatrensis for the resistance to ALS-inhibiting herbicides.

Alternative Methods as Tools for Obesity Research: In Vitro and In Silico Approaches.

The study of adipogenesis is essential for understanding and treating obesity, a multifactorial problem related to body fat accumulation that leads to several life-threatening diseases, becoming one of the most critical public health problems worldwide. In this review, we propose to provide the highlights of the adipogenesis study based on in vitro differentiation of human mesenchymal stem cells (hMSCs). We list in silico methods, such as molecular docking for identification of molecular targets, and in vitro approaches, from 2D, more straightforward and applied for screening large libraries of substances, to more representative physiological models, such as 3D and bioprinting models. We also describe the development of physiological models based on microfluidic systems applied to investigate adipogenesis in vitro. We intend to identify the main alternative models for adipogenesis evaluation, contributing to the direction of preclinical research in obesity. Future directions indicate the association of in silico and in vitro techniques to bring a clear picture of alternative methods based on adipogenesis as a tool for obesity research.

Re-emergence of Gamma-like-II and emergence of Gamma-S:E661D SARS-CoV-2 lineages in the south of Brazil after the 2021 outbreak.

BACKGROUND: We report a genomic surveillance of SARS-CoV-2 lineages circulating in Paraná, southern Brazil, from March 2020 to April 2021. Our analysis, based on 333 genomes, revealed that the first variants detected in the state of Paraná in March 2020 were the B.1.1.33 and B.1.1.28 variants. The variants B.1.1.28 and B.1.1.33 were predominant throughout 2020 until the introduction of the variant P.2 in August 2020 and a variant of concern (VOC), Gamma (P.1), in January 2021. The VOC Gamma, a ramification of the B.1.1.28 lineage first detected in Manaus (northern Brazil), has grown rapidly since December 2020 and was thought to be responsible for the deadly second wave of COVID-19 throughout Brazil. METHODS: The 333 genomic sequences of SARS-CoV-2 from March 2020 to April 2021 were generated as part of the genomic surveillance carried out by Fiocruz in Brazil Genomahcov Fiocruz. SARS-CoV-2 sequencing was performed using representative samples from all geographic areas of Paraná. Phylogenetic analyses were performed using the 333 genomes also included other SARS-CoV-2 genomes from the state of Paraná and other states in Brazil that were deposited in the GISAID. In addition, the time-scaled phylogenetic tree was constructed with up to 3 random sequences of the Gamma variant from each state in Brazil in each month of 2021. In this analysis we also added the sequences identified as the B.1.1.28 lineage of the Amazonas state and and the Gamma-like-II (P.1-like-II) lineage identified in different regions of Brazil. RESULTS: Phylogenetic analyses of the SARS-CoV-2 genomes that were previously classified as the VOC Gamma lineage by WHO/PANGO showed that some genomes from February to April 2021 branched in a monophyletic clade and that these samples grouped together with genomes recently described with the lineage Gamma-like-II. Additionally, a new mutation (E661D) in the spike (S) protein has been identified in nearly 10% of the genomes classified as the VOC Gamma from Paraná in March and April 2021.Finally, we analyzed the correlation between the lineage and the Gamma variant frequency, age group (patients younger or older than 60 years old) and the clinical data of 86 cases from the state of Paraná. CONCLUSIONS: Our results provided a reliable picture of the evolution of the SARS-CoV-2 pandemic in the state of Paraná characterized by the dominance of the Gamma strain, as well as a high frequencies of the Gamma-like-II lineage and the S:E661D mutation. Epidemiological and genomic surveillance efforts should be continued to unveil the biological relevance of the novel mutations detected in the VOC Gamma in Paraná.

Pirahy virus: Identification of a new and potential emerging arbovirus in South Brazil.

Genomic and epidemiological surveillance are paramount for the discovery of new viruses with the potential to cross species barriers. Here, we present a new member of the genus Alphavirus found in Trichoprosopon and Wyeomia mosquitoes, tentatively named Pirahy virus (PIRAV). PIRAV was isolated from mosquito pools collected in a rural area of Piraí do Sul, South Brazil. In vitro assays revealed that PIRAV replicates and causes cytopathic effects in vertebrate cell lines such as Vero E6, SH-SY5Y, BHK-21 and UMNSAH/DF-1. Genomic signature analysis supports these results showing a dinucleotide and codon usage balance compatible with several hosts. Phylogenetic analyses placed PIRAV basal to the Venezuelan equine encephalitis complex. Genome analyses, electron microscopy, and biological characterization show findings that may alert for the emergence of a new arbovirus in South America.

Downregulation of IGF2 expression in third trimester placental tissues from Zika virus infected women in Brazil.

OBJECTIVES: Screening for genes differentially expressed in placental tissues, aiming to identify transcriptional signatures that may be involved in ZIKV congenital pathogenesis. METHODS: Transcriptome data from placental tissues of pregnant women naturally infected with Zika virus during the third trimester were compared to those from women who tested negative for Zika infection. The findings were validated using both a cell culture model and an immunohistochemistry/morphological analysis of naturally infected placental tissues. RESULTS: Transcriptome analysis revealed that Zika virus infection induces downregulation of insulin-like growth factor II (IGF2) gene, an essential factor for fetal development. The Caco-2 cell culture model that constitutively expresses IGF2 was used for the transcriptome validation. Asiatic and African Zika virus strains infection caused downregulated IGF2 gene expression in Caco-2 cells, whereas other flaviviruses, such as dengue serotype 1, West Nile and wild-type yellow fever viruses, had no effect on this gene expression. Immunohistochemical assays on decidual tissues corroborated our transcriptome analysis, showing that IGF2 is reduced in the decidua of Zika virus-infected women. CONCLUSIONS: Our results draw attention to IGF2 modulation in uterine tissues, and this finding is expected to support future studies on strategies to ameliorate the harmful effects of Zika virus infection during pregnancy.

Non-target-Site Resistance in Lolium spp. Globally: A Review.

The Lolium genus encompasses many species that colonize a variety of disturbed and non-disturbed environments. Lolium perenne L. spp. perenne, L. perenne L. spp. multiflorum, and L. rigidum are of particular interest to weed scientists because of their ability to thrive in agricultural and non-agricultural areas. Herbicides are the main tool to control these weeds; however, Lolium spp. populations have evolved multiple- and cross-resistance to at least 14 herbicide mechanisms of action in more than 21 countries, with reports of multiple herbicide resistance to at least seven mechanisms of action in a single population. In this review, we summarize what is currently known about non-target-site resistance in Lolium spp. to acetyl CoA carboxylase, acetohydroxyacid synthase, microtubule assembly, photosystem II, 5-enolpyruvylshikimate-3-phosphate synthase, glutamine synthetase, very-long chain fatty acids, and photosystem I inhibitors. We suggest research topics that need to be addressed, as well as strategies to further our knowledge and uncover the mechanisms of non-target-site resistance in Lolium spp.

The citrus flavonoid naringenin impairs the in vitro infection of human cells by Zika virus.

The Zika virus (ZIKV) is an arthropod-borne virus that belongs to the Flaviviridae family. The ZIKV infection is usually asymptomatic or is associated with mild clinical manifestations; however, increased numbers of cases of microcephaly and birth defects have been recently reported. To date, neither a vaccine nor an antiviral treatment has become available to control ZIKV replication. Among the natural compounds recognized for their medical properties, flavonoids, which can be found in fruits and vegetables, have been found to possess biological activity against a variety of viruses. Here, we demonstrate that the citrus flavanone naringenin (NAR) prevented ZIKV infection in human A549 cells in a concentration-dependent and ZIKV-lineage independent manner. NAR antiviral activity was also observed when primary human monocyte-derived dendritic cells were infected by ZIKV. NAR displayed its antiviral activity when the cells were treated after infection, suggesting that NAR acts on the viral replication or assembly of viral particles. Moreover, a molecular docking analysis suggests a potential interaction between NAR and the protease domain of the NS2B-NS3 protein of ZIKV which could explain the anti-ZIKV activity of NAR. Finally, the results support the potential of NAR as a suitable candidate molecule for developing anti-ZIKV treatments.

Identification of a novel alphavirus related to the encephalitis complexes circulating in southern Brazil.

In early 2017, an outbreak caused by an unknown and supposedly viral agent in the Marilena region of southern Brazil was investigated. Since the etiological agent causing the outbreak was not identified from human samples, mosquitoes from this region were collected. Three out of 121 mosquito pools collected from the region tested positive for alphavirus in molecular tests. Next generation sequencing results revealed the presence of a novel alphavirus, tentatively named here as Caainguá virus (CAAV). DNA barcoding analyses indicated that different species of Culex are hosts for CAAV. This new virus was basal to the New World encephalitic alphaviruses in a comprehensive and robust phylogenetic approach using complete genomes. Viral particles were observed in the cytosol and inside of intracellular compartments of cells in mosquito-derived cell cultures. Despite being noninfectious in vertebrate derived cell cultures, primary culturing of CAAV in human mononuclear cells suggests monocytes and lymphocytes as CAAV targets. However, the epidemiological link of CAAV on the human outbreak should be further explored.

Identification of insect-specific flaviviruses in areas of Brazil and Paraguay experiencing endemic arbovirus transmission and the description of a novel flavivirus infecting Sabethes belisarioi.

Viral infection was examined with pan-flavivirus and pan-alphavirus sets of primers in mosquitoes collected in four South American regions with confirmed pathogenic arbovirus circulation. Positive pools for flavivirus infection were sequenced and screened for specific arboviruses, which were not detected. However, NS5 gene sequencing showed that most sequences corresponded to the insect-specific Culex flavivirus. One sequence retrieved from an Aedes albopictus pool grouped with the insect-specific Aedes flavivirus and two Sabethes belisarioi pools were infected by a previously unknown flavivirus, tentatively named Sabethes flavivirus (SbFV). Phylogenetic inference placed SbFV as ancestral to a clade formed by Culiseta flavivirus, Mercadeo, and Calbertado. SbFV polyprotein showed an average aminoacidic identity of 51% in comparison to these flaviviruses. In vitro studies suggest that SbFV infects insect cells, but not vertebrate cells, therefore, we propose it as a new insect-specific flavivirus. These results highlight the wide distribution of insect-specific flaviviruses concomitant with the circulation of emergent arboviruses.

Zika Virus Infection at Different Pregnancy Stages: Anatomopathological Findings, Target Cells and Viral Persistence in Placental Tissues.

Zika virus (ZIKV) infection in humans has been associated with congenital malformations and other neurological disorders, such as Guillain-Barré syndrome. The mechanism(s) of ZIKV intrauterine transmission, the cell types involved, the most vulnerable period of pregnancy for severe outcomes from infection and other physiopathological aspects are not completely elucidated. In this study, we analyzed placental samples obtained at the time of delivery from a group of 24 women diagnosed with ZIKV infection during the first, second or third trimesters of pregnancy. Villous immaturity was the main histological finding in the placental tissues, although placentas without alterations were also frequently observed. Significant enhancement of the number of syncytial sprouts was observed in the placentas of women infected during the third trimester, indicating the development of placental abnormalities after ZIKV infection. Hyperplasia of Hofbauer cells (HCs) was also observed in these third-trimester placental tissues, and remarkably, HCs were the only ZIKV-positive fetal cells found in the placentas studied that persisted until birth, as revealed by immunohistochemical (IHC) analysis. Thirty-three percent of women infected during pregnancy delivered infants with congenital abnormalities, although no pattern correlating the gestational stage at infection, the IHC positivity of HCs in placental tissues and the presence of congenital malformations at birth was observed. Placental tissue analysis enabled us to confirm maternal ZIKV infection in cases where serum from the acute infection phase was not available, which reinforces the importance of this technique in identifying possible causal factors of birth defects. The results we observed in the samples from naturally infected pregnant women may contribute to the understanding of some aspects of the pathophysiology of ZIKV.

Development and evaluation of a novel high-throughput image-based fluorescent neutralization test for detection of Zika virus infection.

Zika virus (ZIKV) is an emerging arbovirus belonging to the genus flavivirus that comprises other important public health viruses, such as dengue (DENV) and yellow fever (YFV). In general, ZIKV infection is a self-limiting disease, however cases of Guillain-Barré syndrome and congenital brain abnormalities in newborn infants have been reported. Diagnosing ZIKV infection remains a challenge, as viral RNA detection is only applicable until a few days after the onset of symptoms. After that, serological tests must be applied, and, as expected, high cross-reactivity between ZIKV and other flavivirus serology is observed. Plaque reduction neutralization test (PRNT) is indicated to confirm positive samples for being more specific, however it is laborious intensive and time consuming, representing a major bottleneck for patient diagnosis. To overcome this limitation, we developed a high-throughput image-based fluorescent neutralization test for ZIKV infection by serological detection. Using 226 human specimens, we showed that the new test presented higher throughput than traditional PRNT, maintaining the correlation between results. Furthermore, when tested with dengue virus samples, it showed 50.53% less cross reactivity than MAC-ELISA. This fluorescent neutralization test could be used for clinical diagnosis confirmation of ZIKV infection, as well as for vaccine clinical trials and seroprevalence studies.

Popcorn genotypes resistance to fall armyworm / Genótipos de milho pipoca resistentes à lagarta-do-cartucho

ABSTRACT: The aim of this study was to evaluate popcorn genotypes for resistance to the fall armyworm, Spodoptera frugiperda. The experiment used a completely randomized design with 30 replicates. The popcorn genotypes Aelton, Arzm 05 083, Beija-Flor, Colombiana, Composto Chico, Composto Gaúcha, Márcia, Mateus, Ufvm Barão Viçosa, Vanin, and Viviane were evaluated,along with the common maize variety Zapalote Chico. Newly hatched fall armyworm larvae were individually assessed with regard to biological development and consumption of food. The data were subjected to multivariate analyses of variance and genetic divergence among genotypes was evaluated through the clustering methods of Tocher based on generalized Mahalanobis distances and canonical variable analyses. Seven popcorn genotypes, namely, Aelton, Arzm 05 083, Composto Chico, Composto Gaúcha, Márcia, Mateus, and Viviane,were shown to form a cluster (cluster I) that had antibiosis as the mechanism of resistance to the pest. Cluster I genotypes and the Zapalote Chico genotype could be used for stacking genes for antibiosis and non-preference resistance.

RESUMO: O objetivo do estudo foi avaliar genótipos de milho pipoca para resistência à lagarta-do-cartucho, Spodoptera frugiperda. O experimento foi conduzido em delineamento inteiramente casualizado com 30 repetições. Foram avaliados os genótipos de milho pipoca Aelton, Arzm 05 083, Beija-Flor, Colombiana, Composto Chico, Composto Gaúcha, Márcia, Mateus, Ufvm Barão Viçosa, Vanin, Viviane e Zapalote Chico. Larvas recém-eclodidas foram individualizadas para avaliação do desenvolvimento biológico e consumo alimentar. Os dados foram submetidos à análise multivariada de variância e a divergência genética entre genótipos foi avaliada através dos métodos de agrupamento de Tocher, com base na distância generalizada de Mahalanobis e análise de variáveis canônicas. Sete genótipos,Aelton, Arzm 05 083, Composto Chico, CompostoGaúcha, Márcia, Mateus e Viviane formaram um cluster (cluster I) que apresentaram antibiose com o mecanismo de resistência à praga. Os genótipos do cluster I e Zapalote Chico podem ser usados para empilhar genes para resistência por antibiose e não-preferência.

Development and validation of a point-of-care test for detecting hantavirus antibodies in human and rodent samples.

Hantaviruses are etiologic agents of a zoonotic disease transmitted mainly from wild rodents to humans, causing Hemorrhagic Fever with Renal Syndrome in Eurasia and the Hantavirus Cardiopulmonary Syndrome in the Americas (HCPS), reaching a lethality rate of 40% in Brazil. Hantavirus diagnostic and seroprevalence are often based on the presence of IgM and IgG antibodies against the virus. Here we propose a rapid test assay able to identify hantavirus antibodies with sensibility and specificity similar to ELISA assays. We analyzed five groups of samples, including healthy human population and small mammals of endemic areas, suspected cases of HCPS, patients with non-related infections and a serum panel from a different geographical region. The test presented good rates of sensibility (87-100%) and specificity (97-100%) for all groups, being a promising tool suitable for both rodent and human hantavirus epidemiological surveys.

Effect of seed coat on the seed germination and seedling development of Calophyllum brasiliense Cambess. (Clusiaceae) / Efeito do envoltório na germinação da semente e no desenvolvimento de plântulas de Calophyllum brasiliense Cambess. (Clusiaceae)

This work aimed to study the effect of the Calophyllum brasiliense seed coat on the seed germination process. To this end, three experiments were conducted in laboratory, greenhouse and screenhouse. From a total of six treatments, five are related to the seed coat (mechanical scarification; mechanical scarification followed by 2 hours in water, chemical scarification, hot water immersion and complete seed coat removal) and one control. Laboratory and greenhouse experiments were conducted in a completely randomized design (CRD). Screenhouse experiment was conducted in a completely randomized block design (RBD). We evaluated the total percentage, the speed index and the average time of germination or emergence. Data were subjected to analysis of variance and means compared by LSD test, at 5%. Under the conditions of this work, it was possible to infer that, in laboratory, mechanical scarification followed by 2 hours in water increases the proportion and germination speed index (GSI), in the greenhouse, the complete seed coat removal increases the percentage and emergence speed index (ESI), and in the screenhouse, mechanical scarification followed by 2 hours in water and chemical scarification presented the best results. The average germination time was not significantly different in the three experiments evaluated.

Objetivou-se com este trabalho estudar o efeito do envoltório da semente de Calophyllum brasiliense no processo de germinação. Foram montados três experimentos, sendo estes conduzidos em laboratório, casa de vegetação e telado. Avaliaram-se seis tratamentos, sendo cinco relacionados ao envoltório (escarificação mecânica; escarificação mecânica seguido por 2h em água; escarificação química; imersão em água quente; e retirada total do envoltório) e a testemunha. Os experimentos em laboratório e casa de vegetação foram conduzidos em delineamento inteiramente casualizado. No telado, o experimento foi conduzido em delineamento em blocos casualizados. Avaliaram-se a percentagem total; o índice de velocidade e o tempo médio de germinação ou emergência. Os dados foram submetidos à análise de variância e as médias comparadas pelo teste t (LSD) a 5% de probabilidade. Foi possível inferir que no laboratório, a escarificação mecânica, seguido por 2h em água, aumenta a percentagem e o índice de velocidade de germinação (IVG); na casa de vegetação, a retirada total do envoltório aumenta a percentagem e o índice de velocidade de emergência (IVE); e no telado, a escarificação mecânica, seguida por 2h em água, e a escarificação química apresentam os melhores resultados. O tempo de germinação não apresentou diferença significativa nos três experimentos avaliados.

Effects of the melanin precursor 5,6-dihydroxy-indole-2-carboxylic acid (DHICA) on DNA damage and repair in the presence of reactive oxygen species.

Eumelanin is a heterogeneous polymer composed of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6-dihydroxyindole (DHI). Studies have shown that DHICA promotes single strand breaks in plasmid DNA exposed to ultraviolet B radiation (UVB, 313 nm) and in DNA from human keratinocytes exposed to ultraviolet A radiation (UVA, 340-400 nm). Singlet molecular oxygen ((1)O2) is the main reactive species formed by UVA radiation on the skin. In this context, we now report that DHICA can cause single strand breaks in plasmid DNA even in the absence of light radiation. Interestingly, when DHICA was pre-oxidized by (1)O2, it lost this harmful capacity. It was also found that DHICA could interact with DNA, disturbing Fpg activity and decreasing its recognition of lesions by ∼50%. Additionally, the free nucleoside deoxyguanosine (dGuo) was used to evaluate whether DHICA would interfere with the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and spiroiminodihydantoin (dSp) by (1)O2 or with the formation of 8-oxodGuo by hydroxyl radical (OH). We observed that when dGuo was oxidized by (1)O2 in the presence of DHICA, 8-oxodGuo formation was increased. However, when dGuo was oxidized by OH in the presence of DHICA, 8-oxodGuo levels were lower than in the absence of the precursor. Overall, our data reveal an important role for this eumelanin precursor in both the promotion and the protection of DNA damage and imply that it can impair DNA repair.

Effect of bio-regulator and foliar fertilizers on chemical composition and yield of soybean.

Current study evaluates the effects of bio-regulator associated with foliar fertilizers on the yield components, productivity and chemical composition of soybean. The experimental design was entirely randomized blocks, with four replications. The treatments consisted of: T1-absolute control, T2-application of 0.25 L h(-1) Stimulate in R1 stage of development, T3-application of 0.25 L h(-1) Stimulate and 3 L h(-1) Sett in R1, T4-application of 0.25 L h(-1) Stimulate and 3 L h(-1) Sett in R1 and 0.25 L h(-1) Stimulate and 2 L h(-1) Mover in R5.1; T5-application of 0.25 L h(-1) Stimulate and 3 L h(-1) Sett in R1 and 2 L h(-1) Mover in R5.1, T6-application of 3 L h(-1) Sett in R1 and 0.25 L h(-1) Stimulate and 2 L h(-1) Mover in R5.1 and T7-application of 0.25 L h(-1) Stimulate and 2 L h(-1) Mover in R1. Application of Sett and Mover is a potentially efficient handling as it favors the soybean agronomic performance in R1 stage. Chemical composition of processed grains has influence with applying bio-regulator and foliar fertilizers.

Novel properties of melanins include promotion of DNA strand breaks, impairment of repair, and reduced ability to damage DNA after quenching of singlet oxygen.

Melanins have been associated with the development of melanoma and its resistance to photodynamic therapy (PDT). Singlet molecular oxygen ((1)O(2)), which is produced by ultraviolet A solar radiation and the PDT system, is also involved. Here, we investigated the effects that these factors have on DNA damage and repair. Our results show that both types of melanin (eumelanin and pheomelanin) lead to DNA breakage in the absence of light irradiation and that eumelanin is more harmful than pheomelanin. Interestingly, melanins were found to bind to the minor grooves of DNA, guaranteeing close proximity to DNA and potentially causing the observed high levels of strand breaks. We also show that the interaction of melanins with DNA can impair the access of repair enzymes to lesions, contributing to the perpetuation of DNA damage. Moreover, we found that after melanins interact with (1)O(2), they exhibit a lower ability to induce DNA breakage; we propose that these effects are due to modifications of their structure. Together, our data highlight the different modes of action of the two types of melanin. Our results may have profound implications for cellular redox homeostasis, under conditions of induced melanin synthesis and irradiation with solar light. These results may also be applied to the development of protocols to sensitize melanoma cells to PDT.

Time course of skeletal muscle loss and oxidative stress in rats with Walker 256 solid tumor.

Reactive oxygen species oxidize proteins and modulate the proteasomal system in muscle-wasting cancer cachexia. On day 5 (D5), day 10 (D10), and day 14 (D14) after tumor implantation, skeletal muscle was evaluated. Carbonylated proteins and thiobarbituric acid reactive substances were measured. Chemiluminescence was employed for lipid hydroperoxide estimation. Glutathione, superoxide dismutase, and total radical antioxidant capacity were evaluated. The proteasomal system was assessed by mRNA atrogin-1 expression. Increased muscle wasting, lipid hydroperoxide, and superoxide dismutase, and decreased glutathione levels and total radical antioxidant capacity, were found on D5 in accordance with increased mRNA atrogin-1 expression. All parameters were significantly modified in animals treated with α-tocopherol. The elevation in aldehylde levels and carbonylated proteins observed on D10 were reversed by α-tocopherol treatment. Oxidative stress may trigger signal transduction of the proteasomal system and cause protein oxidation. These pathways may be associated with the mechanism of muscle wasting that occurs in cancer cachexia.

Genistein abrogates pre-hemolytic and oxidative stress damage induced by 2,2'-Azobis (Amidinopropane).

The pre-hemolytic mechanism induced by free radicals initiated from water-soluble 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH) and its reversal by genistein was investigated in human erythrocytes. The time course of K+ efflux compared to the occurrence of hemolysis suggests that AAPH-induced hemolysis occurs indirectly via pore formation and band 3 oxidation as expected. However, genistein inhibited hemolysis, LDH release and membrane protein oxidation but not K+ efflux. This indicated that erythrocyte protein oxidation possibly in the hydrophobic core plays a significant role in the membrane pre-hemolytic damage. Chemiluminescence (CL) analysis carried out in non-lysed erythrocytes treated with AAPH showed a dramatic increase in CL indicating both reduced levels of antioxidants and increased membrane lipid peroxide. The V0 value was also increased up to 6 times, denoting a high degree of membrane peroxidation very early in erythrocyte membrane damage. The whole process was inhibited by genistein in a dose-dependent manner. These results indicate that the genistein inhibited both hemolysis and pre-hemolytic damage and also hindered membrane lipid peroxide formation and protein oxidation. In addition, it is suggested that pre-hemolytic damage is mediated mainly by the oxidation of both phospholipid and protein located in the deeper hydrophobic region of the membrane.