Cell alterations induced by a biotherapic for influenza

Introduction: Influenza viruses have been responsible for highly contagious acute respiratory illnesses with high mortality, mainly in the elderly, which encourages the development of new drugs for the treatment of human flu. The biotherapics are medicines prepared from biological products, which are not chemically defined. They are compounded following the homeopathic procedures indicated for infectious diseases with known etiology [1]. Aim: The purpose of the present study is to verify cellular alterations induced by a biotherapic prepared from the infectious influenza A virus. Methodology: This biotherapic was prepared for this study in the homeopathic potency of 30X according to the Brazilian Homeopathic Pharmacopeia [2]. The concentration of 10% was not cytotoxic to cells, as verified by neutral red assay. The cellular alterations observed in MDCK cells were analyzed by optical microscopy for the quantification of mitosis, nucleoli and lipid bodies. The mitochondrial activity was assessed by MTT assay and the phosphosfructokinase-1 (PFK-1) enzyme activity was analyzed on the MDCK cells treated for 5, 10 and 30 days. Macrophages J778.G8 were treated with this biotherapic to evaluate the immunostimulatory cytokine release. Results: The cellular alterations observed in MDCK cells were verified by optical microscopy. The number of lipid bodies present in MDCK cells stimulated for 10 days was significantly lower (p

β-carbolines RCC and C5 induce death of Leishmania amazonensis intracellular amastigotes

Background: Cutaneous leishmaniasis is caused by Leishmania spp., and its treatment is limited. The β-carbolines have shown activity against kinetoplastids. Aim: To evaluate the activity and effects of the β-carbolines, N-{2-[(4,6-bis(isopropylamino)-1,3,5-triazin-2-yl)amino]ethyl}-1-(4-methoxyphenyl)-β-carboline-3-carboxamide (RCC) and N-benzyl-1-(4-methoxy)phenyl-9H-beta-carboline-3-carboxamide (C5), against L. amazonensis intracellular amastigotes and to suggest their mechanism of action. Methods: We analyzed the activity and cytotoxicity of β-carbolines and the morphological alterations by electron microscopy. Mitochondrial membrane potential, production nitric oxide, reactive oxygen species, lipidic bodies, autophagic vacuoles and ATP were also evaluated. Results & conclusion: The results showed that RCC and C5 are active against intracellular amastigotes and were able to induce oxidative stress and ultrastructural alterations such as accumulation of lipid bodies and autophagic vacuoles, leading to parasite death.

Lipid and Fatty Acids Accumulation Features of Entomoneis cf. paludosa during Exponential and Stationary Growth Phases in Laboratory Culture

Diatoms are capable of accumulating substantial amounts of triacylglycerides in their cells, which differ in the composition of fatty acids depending on the conditions of cultivation, making them attractive subjects in biotechnology. In the present study, we characterized the structural features of lipid bodies in the diatom Entomoneis cf. paludosa (W. Smith) Reimer strain 8.0727-B and revealed the peculiarities of fatty acid composition in cultures during the stationary and exponential growth phases. Laser scanning confocal microscopy revealed an increased number of lipid bodies in the cytoplasm during the stationary phase of culture growth. Electron microscopy of ultrathin sections showed that an extreme increase in the number and size of plastoglobules in the cells occurs in the stationary phase of culture growth. The gas chromatography with mass spectrometric detection method revealed differences in the fatty acid composition depending on the growth phase. The studied strain can be recommended as a source of hexadecanoic and octadecanoic fatty acids from the culture during the stationary growth phase, as well as eicosapentaenoic fatty acid from the culture during the exponential growth phase.

Compartmentalized Reconstitution of Post-squalene Pathway for 7-Dehydrocholesterol Overproduction in Saccharomyces cerevisiae

7-Dehydrocholesterol (7-DHC) is the direct precursor to manufacture vitamin D3. Our previous study has achieved 7-DHC synthesis in Saccharomyces cerevisiae based on the endogenous post-squalene pathway. However, the distribution of post-squalene enzymes between the endoplasmic reticulum (ER) and lipid bodies (LD) might raise difficulties for ERG proteins to catalyze and deliver sterol intermediates, resulting in unbalanced metabolic flow and low product yield. Herein, we intended to rearrange the subcellular location of post-squalene enzymes to alleviate metabolic bottleneck and boost 7-DHC production. After identifying the location of DHCR24 (C-24 reductase, the only heterologous protein for 7-DHC biosynthesis) on ER, all the ER-located enzymes were grouped into four modules: ERG1/11/24, ERG25/26/27, ERG2/3, and DHCR24. These modules attempted to be overexpressed either on ER or on LDs. As a result, expression of LD-targeted DHCR24 and ER-located ERG1/11/24 could promote the conversion efficiency among the sterol intermediates to 7-DHC, while locating module ERG2/3 into LDs improved the whole metabolic flux of the post-squalene pathway. Coexpressing LD-targeted ERG2/3 and DHCR24 (generating strain SyBE_Sc01250035) improved 7-DHC production from 187.7 to 308.2 mg/L at shake-flask level. Further expressing ER-targeted module ERG1/11/24 in strain SyBE_Sc01250035 dramatically reduced squalene accumulation from 620.2 mg/L to the lowest level (by 93.8%) as well as improved 7-DHC production to the highest level (to 342.2 mg/L). Then targeting module ERG25/26/27 to LDs further increased 7-DHC titer to 360.6 mg/L, which is the highest shake-flask level production for 7-DHC ever reported. Our study not only proposes and further proves the concept of pathway compartmentalized reconstitution to regulate metabolic flux but also provides a promising chassis to produce other steroidal compounds through the post-squalene pathway.

A Lipid Bodies-Associated Galactosyl Hydrolase Is Involved in Triacylglycerol Biosynthesis and Galactolipid Turnover in the Unicellular Green Alga Chlamydomonas reinhardtii

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the main constituent lipids of thylakoid and chloroplast envelop membranes. Many microalgae can accumulate large amounts of triacylglycerols (TAGs) under adverse environmental conditions, which is accompanied by degradation of the photosynthetic membrane lipids. However, the process mediating the conversion from galactolipids to TAG remains largely unknown. In this study, we performed genetic and biochemical analyses of galactosyl hydrolases (CrGH) identified in the proteome of lipid bodies of the green microalga Chlamydomonas reinhardtii. The recombinant CrGH was confirmed to possess galactosyl hydrolase activity by using o-nitrophenyl-β-D-galactoside as the substrate, and the Michaelis constant (Km) and Kcat of CrGH were 13.98 μM and 3.62 s−1, respectively. Comparative lipidomic analyses showed that the content of MGDG and DGDG increased by 14.42% and 24.88%, respectively, in the CrGH-deficient mutant as compared with that of the wild type cc4533 grown under high light stress conditions, and meanwhile, the TAG content decreased by 32.20%. Up-regulation of CrGH at both a gene expression and protein level was observed under high light stress (HL) conditions. In addition, CrGH was detected in multiple subcellular localizations, including the chloroplast envelope, mitochondria, and endoplasmic reticulum membranes. This study uncovered a new paradigm mediated by the multi-localized CrGH for the conversion of the photosynthetic membranes to TAGs.

Ultrastructural characterization of microlipophagy induced by the interaction of vacuoles and lipid bodies around generative and sperm cells in Arabidopsis pollen

During pollen maturation, various organelles change their distribution and function during development as male gametophytes. We analyzed the behavior of lipid bodies and vacuoles involved in lipophagy in Arabidopsis pollen using serial section SEM and conventional TEM. At the bicellular pollen stage, lipid bodies in the vegetative cells lined up at the surface of the generative cell. Vacuoles then tightly attached, drew in, and degraded the lipid bodies and eventually occupied the space of the lipid bodies. Degradation of lipid began before transfer of the entire contents of the lipid body. At the tricellular stage, vacuoles instead of lipid bodies surrounded the sperm cells. The degradation of lipid bodies is morphologically considered microautophagy. The atg2-1 Arabidopsis mutant is deficient in one autophagy-related gene (ATG). In this mutant, the assembly of vacuoles around sperm cells was sparser than that in wild-type pollen. The deficiency of ATG2 likely prevents or slows lipid degradation, although it does not prevent contact between organelles. These results demonstrate the involvement of microlipophagy in the pollen development of Arabidopsis.

Eosinophils and Neutrophils—Molecular Differences Revealed by Spontaneous Raman, CARS and Fluorescence Microscopy

Leukocytes are a part of the immune system that plays an important role in the host’s defense against viral, bacterial, and fungal infections. Among the human leukocytes, two granulocytes, neutrophils (Ne) and eosinophils (EOS) play an important role in the innate immune system. For that purpose, eosinophils and neutrophils contain specific granules containing protoporphyrin-type proteins such as eosinophil peroxidase (EPO) and myeloperoxidase (MPO), respectively, which contribute directly to their anti-infection activity. Since both proteins are structurally and functionally different, they could potentially be a marker of both cells’ types. To prove this hypothesis, UV−Vis absorption spectroscopy and Raman imaging were applied to analyze EPO and MPO and their content in leukocytes isolated from the whole blood. Moreover, leukocytes can contain lipidic structures, called lipid bodies (LBs), which are linked to the regulation of immune responses and are considered to be a marker of cell inflammation. In this work, we showed how to determine the number of LBs in two types of granulocytes, EOS and Ne, using fluorescence and coherent anti-Stokes Raman scattering (CARS) microscopy. Spectroscopic differences of EPO and MPO can be used to identify these cells in blood samples, while the detection of LBs can indicate the cell inflammation process.