A Major Downregulation of Circulating microRNAs in Zika Acutely Infected Patients: Potential Implications in Innate and Adaptive Immune Response Signaling Pathways.

Zika virus (ZIKV) is an arbovirus mainly transmitted by mosquitos of the genus Aedes. The first cases of ZIKV infection in South America occurred in Brazil in 2015. The infection in humans causes diverse symptoms from asymptomatic to a syndrome-like dengue infection with fever, arthralgia, and myalgia. Furthermore, ZIKV infection during pregnancy is associated with fetal microcephaly and neurological disorders. The identification of host molecular mechanisms responsible for the modulation of different signaling pathways in response to ZIKV is the first step to finding potential biomarkers and therapeutic targets and understanding disease outcomes. In the last decade, it has been shown that microRNAs (miRNAs) are important post-transcriptional regulators involved in virtually all cellular processes. miRNAs present in body fluids can not only serve as key biomarkers for diagnostics and prognosis of human disorders but also contribute to cellular signaling offering new insights into pathological mechanisms. Here, we describe for the first time ZIKV-induced changes in miRNA plasma levels in patients during the acute and recovery phases of infection. We observed that during ZIKV acute infection, among the dysregulated miRNAs (DMs), the majority is with decreased levels when compared to convalescent and control patients. We used systems biology tools to build and highlight biological interactions between miRNAs and their multiple direct and indirect target molecules. Among the 24 DMs identified in ZIKV + patients, miR-146, miR-125a-5p, miR-30-5p, and miR-142-3p were related to signaling pathways modulated during infection and immune response. The results presented here are an effort to open new vistas for the key roles of miRNAs during ZIKV infection.

Autophagy-targeted therapy to modulate age-related diseases: Success, pitfalls, and new directions.

Autophagy is a critical metabolic process that supports homeostasis at a basal level and is dynamically regulated in response to various physiological and pathological processes. Autophagy has some etiologic implications that support certain pathological processes due to alterations in the lysosomal-degradative pathway. Some of the conditions related to autophagy play key roles in highly relevant human diseases, e.g., cardiovascular diseases (15.5%), malignant and other neoplasms (9.4%), and neurodegenerative conditions (3.7%). Despite advances in the discovery of new strategies to treat these age-related diseases, autophagy has emerged as a therapeutic option after preclinical and clinical studies. Here, we discuss the pitfalls and success in regulating autophagy initiation and its lysosome-dependent pathway to restore its homeostatic role and mediate therapeutic effects for cancer, neurodegenerative, and cardiac diseases. The main challenge for the development of autophagy regulators for clinical application is the lack of specificity of the repurposed drugs, due to the low pharmacological uniqueness of their target, including those that target the PI3K/AKT/mTOR and AMPK pathway. Then, future efforts must be conducted to deal with this scenery, including the disclosure of key components in the autophagy machinery that may intervene in its therapeutic regulation. Among all efforts, those focusing on the development of novel allosteric inhibitors against autophagy inducers, as well as those targeting autolysosomal function, and their integration into therapeutic regimens should remain a priority for the field.

Microbiota da pele: novos desafios / Skin microbiota: new challenges

Comunidades complexas de bactérias, fungos e vírus prosperam em nossa pele. A composição dessas comunidades depende das características da pele, como concentração das glândulas sebáceas, teor de umidade e temperatura, bem como da genética do hospedeiro e fatores ambientais exógenos. Estudos metagenômicos recentes descobriram uma diversidade surpreendente dentro desses ecossistemas e promoveram uma nova visão dos organismos comensais durantes as diferentes fases da vida humana. Portanto nesta revisão de literatura buscamos compreender as interações micróbio-hospedeiro e descobrir os fatores que impulsionam a colonização microbiana nos ajudará a entender a patogênese das doenças de pele e a desenvolver novas terapêuticas pro-microbianas e antimicrobianas.

Complex communities of bacteria, fungi and viruses thrive on our skin. The composition of these communities depends on the characteristics of the skin, such as concentration of sebaceous glands, moisture and temperature content, as well as the host's genetics and exogenous environmental factors. Recent metagenomic studies have discovered a surprising diversity within these ecosystems and have promoted a new view of commensal organisms during the different stages of human life. Thus, this review explores microbe-host interactions and discovering the factors that drive microbial colonization will help us understand the pathogenesis of skin diseases and develop new promicrobial and antimicrobial therapies

Cellular compartments challenged by membrane photo-oxidation.

The lipid composition impacts directly on the structure and function of the cytoplasmic as well as organelle membranes. Depending on the type of membrane, specific lipids are required to accommodate, intercalate, or pack membrane proteins to the proper functioning of the cells/organelles. Rather than being only a physical barrier that separates the inner from the outer spaces, membranes are responsible for many biochemical events such as cell-to-cell communication, protein-lipid interaction, intracellular signaling, and energy storage. Photochemical reactions occur naturally in many biological membranes and are responsible for diverse processes such as photosynthesis and vision/phototaxis. However, excessive exposure to light in the presence of absorbing molecules produces excited states and other oxidant species that may cause cell aging/death, mutations and innumerable diseases including cancer. At the same time, targeting key compartments of diseased cells with light can be a promising strategy to treat many diseases in a clinical procedure called Photodynamic Therapy. Here we analyze the relationships between membrane alterations induced by photo-oxidation and the biochemical responses in mammalian cells. We specifically address the impact of photosensitization reactions in membranes of different organelles such as mitochondria, lysosome, endoplasmic reticulum, and plasma membrane, and the subsequent responses of eukaryotic cells.

Modulação da autofagia como estratégia terapêutica contra o câncer / Autophagy modulation as a therapeutic strategy against cancer

A autofagia é uma via metabólica essencial para a manutenção da homeostase celular, e pode desempenhar diferentes papéis dentro do contexto fisiológico e patológico. Por este motivo tem sido foco de muitos estudos por ser um alvo terapêutico promissor, principalmente contra o câncer, onde atua de maneira ambígua, podendo suprimir ou promover o tumor de acordo com o contexto. Compreender a base molecular desse mecanismo é de interesse emergente para se alcançar terapias eficazes utilizando a modulação da autofagia. Neste trabalho, realizou-se uma revisão da literatura para abordar o papel da autofagia na biologia do câncer e como ela pode ser usada como estratégia terapêutica antitumoral através de sua ativação ou inibição no tratamento de vários tipos e estágios do câncer.

Autophagy is an essential metabolic pathway for the maintenance of cellular homeostasis and may play different roles within the physiological and pathological context. For this reason, it has been the focus of many studies because it is a promising therapeutic target, especially against cancer, in which plays a duo role, and it may suppress or promote the tumor according to the context. Understanding the molecular basis of this mechanism is an emerging interest to provide effective therapies using autophagy modulation. In this paper, we performed a literature review to address the role of autophagy in cancer biology and how it can be used as an antitumoral therapeutic strategy through its activation or inhibition to treat various types and stages of cancer.

Porphyrin-Loaded TyroSpheres for the Intracellular Delivery of Drugs and Photoinduced Oxidant Species.

In order to understand the intracellular delivery of drugs and to improve the cell killing efficiency of photosensitizers (PSs) used in photodynamic therapy (PDT), we prepared TyroSphere nanoparticles, which are triblock polymer [poly(ethylene glycol)-block-oligo(desaminotyrosyltyrosine octyl ester suberate)-block-poly(ethylene glycol)] aggregates, loaded with amphiphilic porphyrins with either positive (CisDiMPyP) or negative (TPPS2a) charges. Their physicochemical and photochemical properties were investigated, as well as the efficiency and mechanism of PDT death in a cervical cancer cell line (HeLa). The photophysical properties of both PSs were improved when loaded in the nanocarrier, with a decrease in aggregation as well as an increase in the yield of singlet oxygen generation. The physical and chemical stability of TyroSphere nanoparticles allows them to enter cells and to promote the slow intracellular delivery of part of the PSs. Confocal steady-state and lifetime-resolved fluorescence imaging microscopy data showed that the released PSs are free to target their natural intracellular targets, which are mitochondria and lysosomes for CisDiMPyP and TPPS2a, respectively. The photodynamic efficiency of cell killing was increased considerably compared with the free PSs (∼3×), but the mechanism of cell death was the same as that of the free PSs, which are acute necro-apoptosis for CisDiMPyP and autophagy malfunction for TPPS2a, reflecting the specific damage in mitochondria and lysosomes, respectively. We are confident that TyroSpheres provide a novel and efficient platform to administrate PDT photosensitizers, as well as other drugs with intracellular targets.

Identifying Specific Subcellular Organelle Damage by Photosensitized Oxidations.

The search for conditions that maximize the outcome of Photodynamic Therapy (PDT) continues. Recent data indicate that PDT-induced cell death depends more on the specific intracellular location of the photosensitizer (PS) than on any other parameter. Indeed, knowledge of the PS intracellular location allows the establishment of clear relationships between the mechanism of cell death and the PDT efficacy. In order to determine the intracellular localization sites of a given PS, classical co-localization protocols, which are based in the comparison of the emissive profiles of organelle-specific probes to those of the PS, are usually performed. Since PSs are usually not efficient fluorophores, co-localization protocols require relatively high PS concentrations (micromolar range), distorting the whole proposal of the experiment, as high PS concentration means accumulation in many low-affinity sites. To overcome this difficulty, herein we describe a method that identifies PS intracellular localization by recognizing and quantifying the photodamage at intracellular organelles. We propose that irradiation protocols and characterization of major sites of photodamage results from many cycles of photosensitized oxidations, furnishing an integrated picture of the PS location. By comparing the results of protocols based in either method, we showed that the analysis of the damaged organelles can be conducted at optimal conditions (low PS concentrations), providing clear correlations with cell death mechanisms, which is not the case for the results obtained with co-localization protocols. Experiments using PSs that target either mitochondria or lysosomes were described and investigated in detail, showing that evaluating organelle damage is as simple as performing co-localization protocols.

Antioxidant role on the protection of melanocytes against visible light-induced photodamage.

Visible light can induce the generation of singlet oxygen and can cause oxidative stress, especially in melanocytes due to melanin photosensitization. Currently, there is no organic UV-filter that provide visible light protection. Previous studies showed that some antioxidants, such as apigenin (API), chrysin (CRI) and beta-carotene (BTC) besides neutralizing radical chain reactions can also quench singlet oxygen via physical or chemical quenching and exhibit potential for use in photoprotection. Therefore, the aim of this study is to evaluate the efficacy of API, CRI and BTC on the protection against cell death induced by melanin photosensitization and understand the underlying mechanisms that are involved in the protection. Precise protocols of melanogenesis and quantification of singlet oxygen generation were developed. Viability of B16-F10 cells with melanin basal levels and after melanogenesis induction was evaluated after visible light exposure in the presence and absence of API, CRI and BTC. Results showed that API and BTC protected cells from photoinduced cell death API exhibiting superior photoprotective effect. We noticed that the efficiency of cell protection and the rate of singlet oxygen suppression are not well correlated, at least for the studied series of antioxidants, indicating that the anti-radical capacity should be playing a major role in protecting cells against the damage induced by melanin photosensitization. In terms of sun care strategies, both API and BTC offer protection against visible light-induced damages and may be effective topical antioxidants to be added to sunscreens.

Enhanced efficiency of cell death by lysosome-specific photodamage.

Mobilization of specific mechanisms of regulated cell death is a promising alternative to treat challenging illness such as neurodegenerative disease and cancer. The use of light to activate these mechanisms may provide a route for target-specific therapies. Two asymmetric porphyrins with opposite charges, the negatively charged TPPS2a and the positively charged CisDiMPyP were compared in terms of their properties in membrane mimics and in cells. CisDiMPyP interacts to a larger extent with model membranes and with cells than TPPS2a, due to a favorable electrostatic interaction. CisDiMPyP is also more effective than TPPS2a in damaging membranes. Surprisingly, TPPS2a is more efficient in causing photoinduced cell death. The lethal concentration on cell viability of 50% (LC50) found for TPPS2a was ~3.5 (raw data) and ~5 (considering photosensitizer incorporation) times smaller than for CisDiMPyP. CisDiMPyP damaged mainly mitochondria and triggered short-term phototoxicity by necro-apoptotic cell death. Photoexcitation of TPPS2a promotes mainly lysosomal damage leading to autophagy-associated cell death. Our data shows that an exact damage in lysosome is more effective to diminish proliferation of HeLa cells than a similar damage in mitochondria. Precisely targeting organelles and specifically triggering regulated cell death mechanisms shall help in the development of new organelle-target therapies.

Mechanism of Aloe Vera extract protection against UVA: shelter of lysosomal membrane avoids photodamage.

The premature aging (photoaging) of skin characterized by wrinkles, a leathery texture and mottled pigmentation is a well-documented consequence of exposure to sunlight. UVA is an important risk factor for human cancer also associated with induction of inflammation, immunosuppression, photoaging and melanogenesis. Although herbal compounds are commonly used as photoprotectants against the harmful effects of UVA, the mechanisms involved in the photodamage are not precisely known. In this study, we investigated the effects of Aloe Vera (Aloe barbadensis mil) on the protection against UVA-modulated cell killing of HaCaT keratinocytes. Aloe Vera exhibited the remarkable ability of reducing both in vitro and in vivo photodamage, even though it does not have anti-radical properties. Interestingly, the protection conferred by Aloe Vera was associated with the maintenance of membrane integrity in both mimetic membranes and intracellular organelles. The increased lysosomal stability led to a decrease in lipofuscinogenesis and cell death. This study explains why Aloe Vera extracts offer protection against photodamage at a cellular level in both the UV and visible spectra, leading to its beneficial use as a supplement in protective dermatological formulations.

Melanin photosensitization and the effect of visible light on epithelial cells.

Protecting human skin from sun exposure is a complex issue that involves unclear aspects of the interaction between light and tissue. A persistent misconception is that visible light is safe for the skin, although several lines of evidence suggest otherwise. Here, we show that visible light can damage melanocytes through melanin photosensitization and singlet oxygen (1O2) generation, thus decreasing cell viability, increasing membrane permeability, and causing both DNA photo-oxidation and necro-apoptotic cell death. UVA (355 nm) and visible (532 nm) light photosensitize 1O2 with similar yields, and pheomelanin is more efficient than eumelanin at generating 1O2 and resisting photobleaching. Although melanin can protect against the cellular damage induced by UVB, exposure to visible light leads to pre-mutagenic DNA lesions (i.e., Fpg- and Endo III-sensitive modifications); these DNA lesions may be mutagenic and may cause photoaging, as well as other health problems, such as skin cancer.

Phage display identification of CD100 in human atherosclerotic plaque macrophages and foam cells.

Atherosclerosis is a complex disease in which vessels develop plaques comprising dysfunctional endothelium, monocyte derived lipid laden foam cells and activated lymphocytes. Considering that humans and animal models of the disease develop quite distinct plaques, we used human plaques to search for proteins that could be used as markers of human atheromas. Phage display peptide libraries were probed to fresh human carotid plaques, and a bound phage homologous to plexin B1, a high affinity receptor for CD100, was identified. CD100 is a member of the semaphorin family expressed by most hematopoietic cells and particularly by activated T cells. CD100 expression was analyzed in human plaques and normal samples. CD100 mRNA and protein were analyzed in cultured monocytes, macrophages and foam cells. The effects of CD100 in oxLDL-induced foam cell formation and in CD36 mRNA abundance were evaluated. Human atherosclerotic plaques showed strong labeling of CD100/SEMA4D. CD100 expression was further demonstrated in peripheral blood monocytes and in in vitro differentiated macrophages and foam cells, with diminished CD100 transcript along the differentiation of these cells. Incubation of macrophages with CD100 led to a reduction in oxLDL-induced foam cell formation probably through a decrease of CD36 expression, suggesting for the first time an atheroprotective role for CD100 in the human disease. Given its differential expression in the numerous foam cells and macrophages of the plaques and its capacity to decrease oxLDL engulfment by macrophages we propose that CD100 may have a role in atherosclerotic plaque development, and may possibly be employed in targeted treatments of these atheromas.

The role of GADD45A in resistance to oxidative stress-mediated cell death in human colon tumor cell lines

Objective: GADD45A is a growth arrest-associated gene, directly involved in the maintenance of genomic stability. In fact, in the absence of this protein cells became sensitized to death by ultraviolet irradiation or cisplatin. However, this role with respect to cell damage triggered by Reactive Oxygen Species (ROS) is not well understood. Thus, in an attempt to evaluate its role in oxidative stress-induced apoptosis, we analyzed its expression after induction of ROS in human colon cell lines. Material and methods: Cell lines derived from human colon tumors (RKO-AS45-1 and HuTu-80) were treated with Menadione (MEN) and Hydrogen Peroxide (HP). Next, the expression of GADD45A was evaluated by semi-quantitative RT-PCR analysis. Results: The results indicated that expression of this gene was associated with resistance to apoptosis. Additionally, cells with high expression of GADD45A were resistant to treatment with oxidative stress-inducing compounds. Conclusion: In conclusion, we propose that the expression of the GADD45A gene can be used as a functional tool to predict cellular responses to antitumor treatments.

Análise do perfil de expressão gênica em melanomas humanos / Analysis of gene expression profiling in human melanoma

O melanoma cutâneo é a principal causa de morte por câncer de pele no Brasil, apresentando como principais fatores etiológicos e de predisposição a exposição à radiação UV e, as alterações genéticas e epigenéticas. Um modelo aceito para o processo tumorigênico do melanoma cutâneo é a transformação contínua dos melanócitos normais à displasia melanocítica, e por último ao melanoma maligno. ... Ao empregar a metodologia de busca de módulos funcionais descrita por SEGAL et al. (2003; 2004) identificamos alguns módulos funcionais que apresentaram mudança de estado de ativação com significância estatística (p0,05), segundo o grupo de melanoma: desenvolvimento da epiderme; atividade de peptidase; comunicação celular; adesão celular; receptor neuroativo ligante e assinatura metastática. Além disso, através da abordagem hipóteses baseadas em dados, identificamos milhares de pares de genes funcionalmente relacionados que apresentaram significativa quebra de correlação segundo o grupo de melanoma. Dentre estes destacamos aqueles presentes nos módulos funcionais desenvolvimento da epiderme (KLK7 x LAMA3; KLK7 x EVPL; KLK7 x BNC e KLK7 x SCEL); atividade de peptidase (KLK6 x KLK13); comunicação celular (COL3A1 x COL6A3); adesão celular (GPR56 x POSTN) e assinatura metastática (LUM x COL6A3 e IGFBP4 x IGF2). E finalmente, a partir das redes de relevância baseadas no processo de “descamação da epiderme” e no “sistema IGF” propusemos: 1) um mecanismo de transposição da camada basal ou de migração intra-epidérmica modulado, principalmente, por kalicreínas e por suas proteases-inibidoras; e 2) um mecanismo de sobrevivência e proliferação modulado por IGFBP-4 e por suas serina-proteases reguladoras (KLKs e MMPs).

Cutaneous melanoma is the leading cause of skin cancer death in occidental countries, having as major etiological and predisposing factors the exposion to UV radiation, and genetic and epigenetic susceptibility. The tumorigenesis of melanoma is explained by a continuous malignant transformation, from normal melanocytes to melanocytic displasia and melanoma. The underlying molecular events that explain malignant melanoma genesis have been only partially characterized, and only a small number of genes have been identified as playing key roles in melanoma initiation and progression. Among these, some cell cycle regulators, apoptotic, signal transduction, cell adhesion, and matrix digestion genes have been demonstrated to be deregulated in this neoplasm. In current project, we determined the gene expression profiling in primary and metastatic human melanoma disease. We improved the methodology developing by SEGAL et al. (2003; 2004) to stablish some functional maps which demonstrated significant alteration (p≤0,05) when primary and metastatic disease were compared: epidermis development; peptidase activity; cell communication; cell adhesion; neuroactive ligand receptor and metastatic signature. Furthermore, we determined according to relevance networks concept pair of genes functionally related that showed significant alteration in your correlations coefficients when primary and metastatic disease were compared. Among these we report those related to epidermis development - KLK7 (Kallikrein-7 precursor) x LAMA3 (Laminin, alpha 3); KLK7 x EVPL (Envoplakin); KLK7 x BNC (Basonuclin 1) and KLK7 x SCEL (Sciellin); peptidase activity - KLK6 (Kallikrein-6 precursor) x KLK13 (Kallikrein-13 precursor); cell communication - COL3A1 (Collagen, type III, alpha 1) x COL6A3 (Collagen, type VI, alpha 3cell adhesion - GPR56 (G protein-coupled receptor 56) x POSTN (Periostin precursor) and metastatic signature - LUM (Lumican precursor) x COL6A3 and IGFBP4 (Insulin-like growth factor-binding protein 4) x IGF2 (Insulin-like growth factor-2). And finally, we proposed two biological processes for melanoma progression that were based on epidermis desquamation and IGF system relevance networks (AU)