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.Fil: Martins, Waleska Kerllen. Anhanguera University; BrasilFil: Silva, Maryana do Nascimento da. Anhanguera University; BrasilFil: Pandey, Kiran. University of New York; Estados UnidosFil: Maejima, Ikuko. Gunma University; JapónFil: Ramalho, Ercília. Anhanguera University; BrasilFil: Olivon, Vania Claudia. Anhanguera University; BrasilFil: Diniz, Susana Nogueira. Anhanguera University; BrasilFil: Grasso, Daniel Hector. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentin

Photobiomodulation: Cellular, molecular, and clinical aspects

Photobiomodulation (PBM) is a noninvasive photonic-based therapy, capable of dealing with immune-inflammatory, neurological, and musculoskeletal disorders, as well as healing oral and chronic skin wounds. During PBM light is applied at a specific wavelength, either in the visible or near-infrared (NIR) ranges. Photophysical and photochemical processes might stimulate or inhibit various biological processes, depending on the target tissue, the wavelength of light, irradiance, fluence, repetition rate (pulse frequency), spot size, optical data of the tissue to be irradiated and treatment regimen. There are several randomized clinical studies demonstrating the PBM benefits as main or adjuvant therapies. Of importance to this review, there is a large piece of evidence in the management of skin or venous ulcers, and diabetic foot. In this review, the PBM´s efficacy as adjuvant therapy to deal with chronic human ulcers were discussed concerning the photophysical parameters and clinical aspects. Beside, we overview the state-of-the-art regarding the cellular and molecular modulatory mechanisms photoactivated by red and NIR light

Rapid screening of potential autophagic inductor agents using mammalian cell lines

Recent progress in understanding the molecular basis of autophagy has demonstrated its importance in several areas of human health. Affordable screening techniques with higher sensitivity and specificity to identify autophagy are, however, needed to move the field forward. In fact, only laborious and/or expensive methodologies such as electron microscopy, dye-staining of autophagic vesicles, and LC3-II immunoblotting or immunoassaying are available for autophagy identification. Aiming to fulfill this technical gap, we describe here the association of three widely used assays to determine cell viability - Crystal Violet staining (CVS), 3-[4, 5-dimethylthiaolyl]-2, 5-diphenyl-tetrazolium bromide (MTT) reduction, and neutral red uptake (NRU) - to predict autophagic cell death in vitro. The conceptual framework of the method is the superior uptake of NR in cells engaging in autophagy. NRU was then weighted by the average of MTT reduction and CVS allowing the calculation of autophagic arbitrary units (AAU), a numeric variable that correlated specifically with the autophagic cell death. The proposed strategy is very useful for drug discovery, allowing the investigation of potential autophagic inductor agents through a rapid screening using mammalian cell lines B16-F10, HaCaT, HeLa, MES-SA, and MES-SA/Dx5 in a unique single microplate.CAPES PNPD/FINEP 02533/09-

Further contributions to the flora of lichens and lichenicolous fungi of the Azores.

Several lichens are reported new to the flora of the Azores. Mycoporum sparsellum, and the lichenicolous coelomycete Laeviomyces fallaciosus are reported for the first time in Europe. Arthothelium crozalsianum, Bacidia friesiana, Belonia incarnata, Julella sericea, Micarea assimilata, Mycomicrothelia confusa and Roselliniopsis ventosa are recorded for the first time in Laurimacaronesia. New for the Azores are Acarospora umbilicata, Buellia aethalea, B. subdisciformis, Byssoloma marginatum, Canoparmelia texana, Catillaria atomarioides, Chaenotheca furfuracea, Chromatochlamys muscorum, Cladonia cyathomorpha, C. pocillum, C. rangiformis var. gracillima, Cliostomom griffithii, Endocarpon pusillum, Hypotrachyna taylorensis, Opegrapha ochrocheila, O. vermicellifera, O. vulgata, Parmotrema mellissii, Peltula euploca, Pertusaria hymenea, Phaeophyscia hispidula, Porina aenea, P. borreri, Pyrenula acutispora, Ramalina lacera, R. subpusilla, Rinodina anomala, R. intermedia, Scoliciosporum umbrinum, Strigula taylorii, Toninia mesoidea, Xanthoria candelaria and X. fallax. Fourteen additional lichenicolous fungi double the list of species from the Azores

Infiltrating cells of the intima in coronary and carotid plaques are CD100 positive.

<p>CD100 labeling (E-H) and negative controls (A-D) in artery tissue sections of carotid plaques (A, B, E, F) and coronary plaques (C, D, G, H) by immunohistochemistry. Positive CD100 staining was observed only in preserved endothelium (arrowhead), and in infiltrating cells in the intima (asterisks). Scale bars: 10µm (<i>A</i>, <i>E</i>, <i>C</i> and <i>G</i>), and 50µm (<i>B</i>, <i>F</i>, <i>D</i> and <i>H</i>).</p

CD100 is expressed only in endothelia of normal coronary and carotid arteries.

<p>CD100 labeling (E-H) and negative controls (A-D) in normal carotid (A, B, E, F) and coronary (C, D, G, H) artery tissue sections by immunohistochemistry. Positive CD100 staining was observed only in endothelia of normal arteries (arrowheads). Scale bars: 10µm (<i>A</i>, <i>E</i>, <i>C</i> and <i>G</i>) and 50µm (<i>B</i>, <i>F</i>, <i>D</i> and <i>H</i>).</p

Resting and IFN-γ stimulated monocytes show increased levels of CD100 mRNA compared to macrophages and foam cells (A, B).

<p>Quantitative RT-PCR (qRT-PCR) for CD100 (A) or STAT-1 (B) in PB monocytes, macrophages and foam cells stimulated (+) or not (-) with IFN-γ. Folds relative to non stimulated monocytes (mono-), normalized with GAPDH. (a) * = significant <i>p</i> values (p≤0.05) in the comparisons in unstimulated and stimulated conditions.</p

Cultured monocytes express higher amounts of CD100 protein than <i>in vitro</i> differentiated macrophages and foam cells.

<p>CD100 and β-actin protein expression was evaluated in activated T lymphocytes (TL – positive control) and PB monocytes (Mono), macrophages (Mac) and foam cells (Foam) A. Western blot showing CD100 and β-actin bands B. Densitometry of CD100 (sum of 120 and 150kDa bands) and β-actin protein bands, showing CD100/β-actin. Mean ± SD of 3 independent experiments.</p

(A, B): CD36 mRNA is reduced in cultured macrophages treated with oxLDL in the presence of CD100 and IL-10.

<p>Relative quantitative RT-PCR (qRT-PCR) for CD36 in macrophages (<b>A</b>) and foam cells (<b>B</b>) incubated with oxLDL in the presence of CD100 or IL-10. Data normalized with GAPDH and expressed as a ratio (fold) relative to cells incubated only with oxLDL. Mean ± SD of 3 independent experiments (** p≤0.01).</p

<i>In vitro</i> cultured monocytes, macrophages and foam cells express CD100.

<p>Immunofluorescence of PB monocytes (A-D), macrophages (E-H) and foam cells (I-L) showing blue nuclei in DAPI (A, E, I), CD68 (<i>green</i>; B, F, J), CD100 (<i>red</i>; C, G, K) and co-localization of all markers in D, H and L. Scale bars: 5µm.</p