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The oxidation of proteins and, in particular, of tryptophan (Trp) residues leads to chemical modifications that can affect the structure and function. The oxidative damage to proteins in photochemical processes is relevant in the skin and eyes and is related to a series of pathologies triggered by exposure to electromagnetic radiation. In this work, we studied the photosensitized formation of N-formylkynurenine (NFKyn) from Trp in different reaction systems. We used two substrates: free Trp and a peptide of nine amino acid residues, with Trp being the only oxidizable residue. Two different photosensitizers were employed: Rose Bengal (RB) and pterin (Ptr). The former is a typical type II photosensitizer [acts by producing singlet oxygen (1O2)]. Ptr is the parent compound of oxidized or aromatic pterins, natural photosensitizers that accumulate in human skin under certain pathological conditions and act mainly through type I mechanisms (generation of radicals). Experimental data were collected in steady photolysis, and the irradiated solutions were analyzed by chromatography (HPLC). Results indicate that the reaction of Trp with 1O2 initiates the process leading to NFKyn, but different competitive pathways take place depending on the photosensitizer and the substrate. In Ptr-photosensitization, a type I mechanism is involved in secondary reactions accelerating the formation of NFKyn when free Trp is the substrate.

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Bioluminescence resonance energy transfer photodynamic therapy, which uses light generated by bioluminescent proteins to activate photosensitizers and produce reactive oxygen species without the need for external irradiation, has shown promising results in cancer models. However, the characterization of delivery systems that can incorporate the components of this therapy for preferential delivery to the tumor remains necessary. In this work, we have characterized parvovirus B19-like particles (B19V-VLPs) as a platform for a photosensitizer and a bioluminescent protein. By chemical and biorthogonal conjugation, we conjugated rose Bengal photosensitizer and firefly luciferase to B19V-VLPs and a protein for added specificity. The results showed that B19V-VLPs can withstand decoration with all three components without affecting its structure or stability. The conjugated luciferase showed activity and was able to activate rose Bengal to produce singlet oxygen without the need for external light. The photodynamic reaction generated by the functionalized VLPs-B19 can decrease the viability of tumor cells in vitro and affect tumor growth and metastasis in the 4 T1 model. Treatment with functionalized VLPs-B19 also increased the percentage of CD4 and CD8 cell populations in the spleen and in inguinal lymph nodes compared to vehicle-treated mice. Our results support B19V-VLPs as a delivery platform for bioluminescent photodynamic therapy components to solid tumors.

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Purpose: In vitro evaluation of rose bengal (RB) photoactivated by our custom-built green light-emitting diode (LED) source for the growth inhibition of bacterial strains and rapidly growing mycobacterial (RGM) isolates in infectious keratitis. Methods: Six corneal clinical bacteria isolates were included in this study: two Gram-positive bacteria (methicillin-resistant Staphylococcus aureus [MRSA] and Staphylococcus epidermidis), two Gram-negative bacteria (Pseudomonas aeruginosa and Serratia marcescens), and two RGM (Mycobacterium chelonae and Mycobacterium abscessus). Microorganisms were cultured and incubated at specific conditions and prepared in suspensions to adjust their concentration to 104 cells/mL. Different treatments were conducted in triplicates: Group I, no treatment; Group II, treated with 0.1% rose bengal alone (exposed to dark for 30 minutes); Group III, exposed to custom green LED for 30 minutes (12.87 J/cm2); and Group IV, treated with 0.1% rose bengal and exposed to custom green LED for 30 minutes. Agar plates were incubated at specific conditions and photographed after growth for pixel analyses. Results: Complete growth inhibition of all bacteria and RGM was observed in Group IV. MRSA and S. epidermidis in Group II also showed complete growth inhibition. Conclusions: The custom-built green LED presented good activity by photoactivating RB and inhibiting micro-organism growth. For the first time, we demonstrated the expressive growth inhibition effect of RB against S. epidermidis, RGM, and S. marcescens. Clinical treatment with RB may offer an alternate adjunct therapy for corneal surface infections. Translational Relevance: Validating in vitro the custom-built green LED encourages the clinical application for the treatment of infectious keratitis.

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In order, understanding the antimicrobial action of photodynamic therapy and how this technique can contribute to its application in the control of pathogens. The objective of the study was to employ a proteomic approach to investigate the protein profile of Staphylococcus aureus after antimicrobial photodynamic therapy mediated by rose bengal (RB-aPDT). S. aureus was treated with RB (10 nmoL L-1 ) and illuminated with green LED (0.17 J cm-2 ) for cell viability evaluation. Afterward, proteomic analysis was employed for protein identification and bioinformatic tools to classify the differentially expressed proteins. The reduction in S. aureus after photoinactivation was ~2.5 log CFU mL-1 . A total of 12 proteins (four up-regulated and eight down-regulated) correspond exclusively to alteration by RB-aPDT. Functionally, these proteins are distributed in protein binding, structural constituent of ribosome, proton transmembrane transporter activity and ATPase activity. The effects of photodamage include alterations of levels of several proteins resulting in an activated stress response, altered membrane potential and effects on energy metabolism. These 12 proteins required the presence of both light and RB suggesting a unique response to photodynamic effects. The information about this technique contributes valuable insights into bacterial mechanisms and the mode of action of photodynamic therapy.

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Photosensitized crosslinking of proteins in tissues has many medical applications including sealing wounds, strengthening tissues, and beneficially altering tissue properties. Rose Bengal (RB) is used most frequently as the photosensitizer but is not as efficient as would be desired for broad utilization in medicine. Aggregation of RB, at the high concentrations used for medical treatments, decreases the yield of singlet oxygen, which mediates protein crosslinking. We hypothesized that nanocages that sequester RB would inhibit self-association, increasing photosensitization efficiency. We tested cucurbituril and cyclodextrin nanocages, demonstrating that hydroxypropyl-functionalized cyclodextrins are most effective in inhibiting RB aggregation. For these RB/cyclodextrin solutions, we investigated the effect of nanocaging on the photobleaching and oxygen consumption kinetics under 530 nm LED light in aqueous phosphate-buffered solutions. At 100 µm RB, the initial oxygen consumption rates increased by 58% and 80% compared with uncaged RB for the ß and γ (2-hydroxypropyl) cyclodextrins, respectively. For 1 mm RB, the enhancement in these rates was much greater, about 200% and 300%, respectively. In addition, at 1 mm RB these two cyclodextrins increased the RB photobleaching rate by ~20% and ~75%. These results suggest that nanocages can minimize RB aggregation and may lead to higher-efficiency photo-medical therapies.

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The present study investigates the insecticidal and biochemical effects of two essential oils (EOs) and two photosensitizers against cotton aphids in a laboratory setting. The EOs evaluated were clove (Syzygium aromaticum L.) and basil (Ocimum basilicum), while the photosensitizers were rose bengal and rhodamine B. The individual median lethal concentrations (LC50) revealed that clove was ~4.44 times more potent than basil, and rhodamine B was ~1.34 times more potent than rose bengal. The mortality rates increased using higher concentrations of the photosensitizers and prolonging exposure time to sunlight. The most effective combination against adult aphids was found to be a mixture of sub-lethal concentrations of clove and rhodamine B, resulting in a mortality rate of 92.31%. Conversely, the combination of basil and rose bengal exhibited the lowest efficacy with a mortality rate of 33.33%. Biochemical analyses indicate that Rhodamine B, basil, and the basil-rhodamine B mixture (mixture C) significantly reduced trehalase activity. However, the protease activity significantly increased in aphids treated with rose bengal, clove, and the clove-rose bengal mixtures (mixtures A and B). The lipase activity is notably decreased upon treatment with rhodamine B and clove. Glutathione S-transferase (GST) activity decreased in aphids treated with rose bengal and the basil-rhodamine B mixtures (mixtures C and D), suggesting that GST did not play a role in detoxifying these compounds, thereby explaining the susceptibility of A. gossypii. Overall, the combination of essential oils and photosensitizers has demonstrated a synergistic effect in controlling Aphis gossypii, offering great potential as an effective strategy for aphid management.

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Melanoma is a type of tumor that originates from melanocytes. Irradiation of melanin with UVA and visible light can produce reactive oxygen species (ROS) such as singlet molecular oxygen (1 O2 ). The objective of this study was to examine DNA damage in melanoma cells (B16-F10) with different melanin contents, subjected to 1 O2 generation. To this end, we used the photosensitizer Rose Bengal acetate (RBAc) and irradiation with visible light (526 nm) (RBAc-PDT). We used the modified comet assay with the repair enzymes hOGG1 and T4 endonuclease V to detect the DNA damage associated with 8-oxo-7,8-dihydro-2'-deoxyguanosine and cyclobutane pyrimidine dimers lesions, respectively. We observed increased formation of hOGG1- and T4endoV-sensitive DNA lesions after light exposure (with or without RBAc). Furthermore, 18 h after irradiation, hOGG1-sensitive DNA lesions increased compared to that at the initial time point (0 h), which shows that a high melanin content contributes to post-irradiation formation of them, mainly via sustained oxidative stress, as confirmed by the measurement of ROS levels and activity of antioxidant enzymes. Contrastingly, the number of T4endoV-sensitive DNA lesions decreased over time (18 h). Our data indicate that in melanoma cells, a higher amount of melanin may affect DNA damage levels when subjected to RBAc-PDT.

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Glanders and brucellosis are zoonotic infectious diseases that affect equids in several countries worldwide. On Marajó Island (Amazon region of Brazil), Marajoara and Puruca horses, which are well adapted to the climatic and territorial adversities of the region, play a fundamental role in the local economy and in the sociocultural lives of the population. However, these animals have undergone a drastic reduction in number, markedly due to precarious veterinary care, unknown causes of morbidity and mortality, and disordered crossing with other breeds introduced to the island. Thus, this study aimed to investigate the occurrence of glanders and brucellosis in equids on a property located in the municipality of Soure, Marajó Island (Brazil). Serum samples were collected from 388 animals (357 horses and 31 mules), maintained in an extensive breeding system, in a property that was also extensively breeding buffaloes, goats, and sheep, with contact among species. The sera were tested for glanders using an indirect ELISA (ELISAi), and the results were confirmed by immunoblotting. The diagnosis of brucellosis was made using the Rose Bengal test (RBT) and confirmed through the Serum Agglutination test (SAT) and 2-mercaptoethanol test. In the case of glanders, 2.31% (9/388) of animals were positive in ELISAi test, of which eight had results confirmed by immunoblotting, representing 2.06% seropositivity in the entire herd. For brucellosis, serum samples from 6.7% (26/388) horses were reactive in the RBT, of which 4.12% (18/388) had a titer ≥50 and 2.06% (8/388) had a titer ≥100 in the SAT. This is the first study to report the occurrence of glanders and equine brucellosis in the municipality of Soure/Marajó Island. Monitoring the occurrence of such diseases is extremely important since they affect the herds economically and zootechnically, in addition to their high zoonotic potential. The number of animals sampled in this study, as well as the way they are raised and managed, is representative of the total equid population of the island. These results, combined with previous studies on buffaloes, indicate that these diseases are endemic in the Marajo Island.

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The antimicrobial photodynamic activity (aPDA) in fungal and bacterial strains of supramolecular adducts formed between the anionic photosensitizer (PS) Rose Bengal (RB2-) and aromatic polycations derived from (p-vinylbenzyl)triethylammonium chloride was evaluated. Stable supramolecular adducts with dissociation constants Kd ≈ 5 µM showed photosensitizing properties suitable for generating singlet oxygen (ΦΔ = 0.5 ± 0.1) with the added advantage of improving the photostability of the xanthenic dye. However, the aPDA of both free and supramolecular RB2- was highly dependent on the type of microorganism treated, indicating the importance of specific interactions between the different cell wall structures of the microbe and the PSs. Indeed, in the case of Gram-positive Staphylococcus aureus, the aPDA of molecular and supramolecular PSs was highly effective. Instead, in the case of Gram-negative Escherichia coli, only the RB2-:polycation adducts showed aPDA, while RB2- alone was inefficient, but in the case of Candida tropicalis, the opposite behavior was observed. Therefore, the present results indicate the potential of supramolecular chemistry to obtain aPDA à la carte depending on the target microbe and the PS properties.

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Considering the lack of information about livestock diseases on Brazilian oceanic islands, the occurrence of bovine brucellosis was investigated on the island of Fernando de Noronha, state of Pernambuco, Brazil. Serum samples were collected in October 2009, from all the 105 cows raised on the island at that time. These were examined concurrently using the Rose Bengal test and the Complement Fixation Test. All the samples were negative in both tests, indicating that the cows on the island were likely free from infection by smooth forms of Brucella. These results can partly be explained by the prohibition of introduction and importation of both small and large-sized animals that had been implemented through District Decree 19 of February 28, 2004.(AU)

Tendo em vista a inexistência de informações sobre a ocorrência da brucelose bovina em ilhas oceânicas brasileiras, investigou-se a presença da infecção na ilha de Fernando de Noronha, Estado de Pernambuco, Brasil. Soros de todas as 105 fêmeas bovinas existentes, colhidos em outubro de 2009, foram examinados concomitantemente pelo teste do Antígeno Acidificado Tamponado e pela Reação de Fixação de Complemento. Todas as amostras foram negativas em ambos os testes, indicando que provavelmente os animais presentes na ilha encontravam-se livres da infecção por Brucella. Estes resultados podem ser explicados, em partes, pela proibição da introdução e importação de grandes e pequenos animais, implementada pelo Decreto Distrital 19, de 28 de fevereiro de 2004.(AU)

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