Severe acute respiratory syndrome coronavirus 2 may exploit human transcription factors involved in retinoic acid and interferon-mediated response: a hypothesis supported by an in silico analysis.

The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19), resulting in acute respiratory disease, is a worldwide emergency. Because recently it has been found that SARS-CoV is dependent on host transcription factors (TF) to express the viral genes, efforts are required to understand the molecular interplay between virus and host response. By bioinformatic analysis, we investigated human TF that can bind the SARS-CoV-2 sequence and can be involved in viral transcription. In particular, we analysed the key role of TF involved in interferon (IFN) response. We found that several TF could be induced by the IFN antiviral response, specifically some induced by IFN-stimulated gene factor 3 (ISGF3) and by unphosphorylated ISGF3, which were found to promote the transcription of several viral open reading frame. Moreover, we found 22 TF binding sites present only in the sequence of virus infecting humans but not bat coronavirus RaTG13. The 22 TF are involved in IFN, retinoic acid signalling and regulation of transcription by RNA polymerase II, thus facilitating its own replication cycle. This mechanism, by competition, may steal the human TF involved in these processes, explaining SARS-CoV-2's disruption of IFN-I signalling in host cells and the mechanism of the SARS retinoic acid depletion syndrome leading to the cytokine storm. We identified three TF binding sites present exclusively in the Brazilian SARS-CoV-2 P.1 variant that may explain the higher severity of the respiratory syndrome. These data shed light on SARS-CoV-2 dependence from the host transcription machinery associated with IFN response and strengthen our knowledge of the virus's transcription and replicative activity, thus paving the way for new targets for drug design and therapeutic approaches.

Morphology of synthetic DOPA-eumelanin deposited on glass and mica substrates: an atomic force microscopy investigation.

Bright field microscopy and atomic force microscopy techniques are used to investigate morphological properties of synthetic eumelanin, obtained by oxidation of L-DOPA solution, deposited on glass and mica substrates. Deposits of eumelanin are characterized by aggregates with different shape and size. On a micrometric scale, filamentous as well as granular structures are present on glass and mica substrates, with a larger density on the former than on the latter. On a nanometric scale, filamentous aggregates, several microns long and about 100 nm wide and high, and granular aggregates, ∼50 nm high and 100 nm wide, are found on both substrates, whereas point-like deposits less than 10nm high and less than 50 nm wide are found on mica substrate. Dynamic light scattering measurements and atomic force microscopy images support the evidence that eumelanin presents only nanometric point-like aggregates in aqueous solution, whereas such nanoaggregates organize themselves according to granular and filamentous structures when deposition occurs, as a consequence of interactions with the substrate surface.

Lactobacillus plantarum passage through an oro-gastro-intestinal tract simulator: carrier matrix effect and transcriptional analysis of genes associated to stress and probiosis.

Dietary probiotics should reach the intestine viable and in high numbers; therefore, they should tolerate the stress associated to the gastro-intestinal (GI) environment. Indeed, all along the different GI sections, probiotics are challenged by several sources of stress, including low pH, bile and digestive enzymes. Bacterial cells are equipped with various defense mechanisms to allow survival in hostile environments. The food matrix used to deliver beneficial bacteria may contribute to their probiotic action, e.g. by enhancing survival to stress and gut colonization. In this study, the survival of the lactic acid bacterium Lactobacillus plantarum WCFS1, a model probiotic strain, was examined in a human oro-gastric-intestinal (OGI) in vitro system, using different carrier matrices to compare protective and buffering properties. Higher survival was observed in complex and/or nutrient-rich matrices, and when potential prebiotics were added. The molecular response of L. plantarum to the OGI transit was analyzed by studying the transcriptional levels of genes involved in stress response and probiosis. The OGI steps of higher mortality corresponded to greater induction of stress genes, thus implying their involvement in adaptation to the gut environment. Plantaricins were significantly upregulated all along the different OGI sections; adhesion genes were mainly induced by gastric environment.

Factors affecting quality and safety of fresh-cut produce.

The quality of fresh-cut fruit and vegetable products includes a combination of attributes, such as appearance, texture, and flavor, as well as nutritional and safety aspects that determine their value to the consumer. Nutritionally, fruit and vegetables represent a good source of vitamins, minerals, and dietary fiber, and fresh-cut produce satisfies consumer demand for freshly prepared, convenient, healthy food. However, fresh-cut produce deteriorates faster than corresponding intact produce, as a result of damage caused by minimal processing, which accelerates many physiological changes that lead to a reduction in produce quality and shelf-life. The symptoms of produce deterioration include discoloration, increased oxidative browning at cut surfaces, flaccidity as a result of loss of water, and decreased nutritional value. Damaged plant tissues also represent a better substrate for growth of microorganisms, including spoilage microorganisms and foodborne pathogens. The risk of pathogen contamination and growth is one of the main safety concerns associated with fresh-cut produce, as highlighted by the increasing number of produce-linked foodborne outbreaks in recent years. The pathogens of major concern in fresh-cut produce are Listeria monocytogenes, pathogenic Escherichia coli mainly O157:H7, and Salmonella spp. This article describes the quality of fresh-cut produce, factors affecting quality, and various techniques for evaluating quality. In addition, the microbiological safety of fresh-cut produce and factors affecting pathogen survival and growth on fresh-cut produce are discussed in detail.

The melanogenic system of the liver pigmented macrophages of Rana esculenta L.--tyrosinase activity.

The enzyme system responsible for Amphibian Kupffer Cell (KC) melanogenesis has not been entirely elucidated. This research demonstrates that the KC melanosomes of Rana esculenta L. possess a tyrosine-hydroxylase (TH) activity, showing that a tyrosinase is the enzyme involved in the melanogenesis. The TH reaction depends on catalytic Dopa as a cofactor and is not affected by catalase or H2O2, showing that it is catalysed by the tyrosinase and not by the peroxidase present in the melanosomes. The TH reaction is activated by Cu2+ ions but not by other tyrosinase activators such as limited proteolysis, protein ageing, and Sodium Dodecyl Sulphate (SDS). SDS inhibited the KC TH activity even below the critical micelle concentration. All these results suggest that the KC-tyrosinase differs in structure from other known tyrosinases. Using anti-KC-tyrosinase antobodies, we observed that the sites of the tyrosinase location within the cell are the same as those described in the melanocytes. In the immunoblots, the anti-KC-tyrosinase antibodies also recognised two protein bands, at the higher molecular weight ranges, in the protein electrophoretic pattern. Moreover, the tyrosinase activity was limited to the highest molecular weight band of about 260 kDa, suggesting that the enzyme activity could depend on a molecular aggregate. The melanin produced in the liver was found to be a 5,6-dihydroxyindole-rich eumelanin similar to the Sepia melanin.

Tyrosinase gene expression in the Kupffer cells of Rana esculenta L.

The liver of Amphibia and Reptilia shows a dark-brown pigmentation due to the presence of particular melanin-containing cells that are different from melanocytes and derive from cells of macrophage lineage (Kupffer Cells), which have been shown to have an autonomous capacity to synthesize melanins. To date, as far as we know, there are no reports in the literature about the genetic system of tyrosinase as regards these melanin-synthesizing cells; we carried out the present study to analyze how the tyrosinase gene may function. We showed that the Kupffer cells of Rana esculenta L. do indeed have a transcriptionally active tyrosinase gene. Evidence of this was obtained by reverse transcription polymerase chain reaction analysis carried out on both the liver tissue and the Kupffer cells in culture. Moreover, analysis of the cells in culture enabled us to observe that, by increasing the culture time from 0 to 72 hr, an appreciable increase occurred in the amplification products of the tyrosinase gene, as well as in the level of dopa oxidase activity and in the quantity of melanin in the cells. The results of the present study demonstrate that frog Kupffer cells possess an active tyrosinase gene and that the increase of the tyrosinase mRNA accumulation closely correlates with phenotypic differentiation, in terms of increased dopa oxidase activity and melanosome content. This provides further strong support of the hypothesis that amphibian Kupffer cells possess an endogenous ability to synthesize melanin and suggests the involvement of the transcriptional level of control in the modulation of their melanogenic activity.

Ultrastructural and functional study of the liver pigment cells from Rana esculenta L.

A study of the liver pigment cells of Rana esculenta L. has been performed on both liver in toto and cells in culture. Ultrastructural and cytochemical analyses showed a close relationship between this visceral pigment cell system and the cells of hepatic macrophage lineage. Like the latter, the liver pigment cells present phagocytic activity, in the sinusoids and in vitro, and give a positive response to tests for peroxidase and lipase. The liver pigment cells are isolated, together with the Kupffer cells, from the sinusoidal cell fraction of the liver. In culture, they maintain their melanogenetic ability, demonstrated by the presence of dopaoxidase activity in the soluble, membranous, and melanosome fractions. Analysis of the cultures showed that as culture time increased, so did melanosome dopaoxidase activity, the number of pigmented fields, and the level of pigmentation of the cells. The values of dopaoxidase activity of the pigment cells in culture show the same seasonal oscillations as the system in toto, indicating that the cells maintain an internal clock, at least in the first 72 h of culture. There is evidence that the pigment cells are macrophages which can express a melanogenetic function. Our results and other experimental data provide a basis for hypothesizing that the pigment cells in Rana esculenta L. liver may derive from, or have a common origin with, the Kupffer cells.

Morpho-functional characterization of cultured pigment cells from Rana esculenta L. liver.

A simple method to isolate and culture liver pigment cells from Rana esculenta L. is described which utilizes a pronase digestion of perfused liver, followed by sedimentation on a Ficoll gradient. A first characterization of isolated and cultured cells is also reported. They show both positivity for nonspecific esterases, and phagocytosis ability, like the cells of phagocytic lineage. Furthermore, after stimulation with a phorbol ester, these cells generate superoxide anions. At phase contrast microscope, liver pigment cells present variability in size, morphology, and in their content of dark-brown granules. Inasmuch as a cell extract obtained from cultured cells exhibits a specific protein band with dopa-oxidase activity, when run on nondenaturing polyacrylamide gel electrophoresis, liver pigment cells from Rana esculenta L. should not be considered as melanophages, but as cells that can actively synthesize melanin. The method presented here seems to be useful to more directly investigate this extra-cutaneous melanin-containing cell system and to clarify its physiologic relevance.

Effects of copper on the tyrosinase of liver pigment cells from Rana esculenta L.

1. The liver pigment cells of R. esculenta L. constitute a peculiar pigment cell system of histiocytic nature and contain a tyrosinase-like activity localized in the protein component of melanosomes. 2. The effects of addition and/or removal of Cu on the DOPA-oxidase activity of the system were studied. 3. It was concluded that: (a) this tyrosinase behaves as a Cu-enzyme; (b) Cu could be involved in the regulation of the enzyme activity; and (c) mixtures of apoenzyme and active enzyme coexist in the melanosomes.

Melanogenesis in the pigment cells of Rana esculenta L. liver: evidence for tyrosinase-like activity in the melanosome protein fraction.

This work demonstrates the presence of a tyrosinase-like activity in the pigment cells of frog Rana esculenta L. liver. The activity was evidenced in the protein melanosome fraction extracted with differential centrifugation methods. The study of this activity, carried out with spectrophotometric methods, indicates 1) the system presents the characteristics of an allosteric enzyme; 2) the grade of cooperativity shows oscillations going from negative cooperativity toward the substrate, evident in the warmest months of the year, to an absence of cooperativity in the coldest months of the year; and 3) the levels of activity of the system also vary according to season, with the highest levels appearing in the coldest months of the year. Given that this extracutaneous system of pigment cells, different from melanocytes, is able to carry out melanogenesis, we suggest its inclusion in the classification of pigment cells.