Differential N-terminal processing of beta and gamma actin in vivo

Actin is one of the most essential and abundant intracellular proteins, playing an essential physiological role as the major constituent of the actin cytoskeleton. Two cytoplasmic actins, beta- and gamma-actin, are encoded by different genes, but their amino acid sequences differ only by four conservative substitutions at the N-terminus, making it very difficult to dissect their individual regulation in vivo. The majority of actins are N-terminally acetylated, following the removal of N-terminal Met. Here, we analyzed beta and gamma cytoplasmic actin N-termini in vivo and found that beta actin, unlike gamma actin, specifically undergoes sequential removal of N-terminal amino acid Asp residues. This processing affects ∼1-3% of beta actin in different cell types. We identified candidate enzymes capable of mediating this type of processing, and used CRISPR/Cas-9 to delete them, individually or together, in mammalian cell lines. This deletion abolishes most of the beta actin N-terminal processing and results in changes in F-actin levels, cell spreading, filopodia formation, and cell migration, suggesting that the beta actin processing mediated by these enzymes is physiologically important to beta actin function. We propose that selective N-terminal processing of beta actin by sequential removal of Asp contributes to differentiating the functions of non-muscle actin isoforms in vivo.

Development of Droplet Microfluidics Capable of Quantitative Estimation of Single-Cell Multiplex Proteins

This study presented constriction microchannel based droplet microfluidics realizing quantitative measurements of multiplex types of single-cell proteins with high throughput. Cell encapsulation with evenly distributed fluorescence labelled antibodies stripped from targeted proteins by proteinase K was injected into the constriction microchannel with the generated fluorescence signals captured and translated into protein numbers leveraging the equivalent detection volume formed by constriction microchannels in both droplet measurements and fluorescence calibration. In order to form the even distribution of fluorescence molecules within each droplet, the stripping effect of proteinase K to decouple binding forces between targeted proteins and fluorescence labelled antibodies was investigated and optimized. Using this microfluidic system, binding sites for beta-actin, alpha-tubulin, and beta-tubulin were measured as 1.15±0.59×106, 2.49±1.44×105, and 2.16±1.01×105 per cell of CAL 27 (N cell=15486), 0.98±0.58×106, 1.76±1.34×105 and 0.74±0.74×105 per cell of Hep G2 (N cell=18266). Neural net pattern recognition was used to differentiate CAL 27 and Hep G2 cells, producing successful rates of 59.4% (beta-actin), 64.9% (alpha-tubulin), 88.8% (beta-tubulin), and 93.0% in combination, validating the importance of quantifying multiple types of proteins. As a quantitative tool, this approach could provide a new perspective for single-cell proteomic analysis.

Macrophage-Derived Small Exosomes: Efficient Transmission and Cytotoxicity to Cancer Cells

Tumor-associated macrophages are a key component in the tumor microenvironment, secreting extracellular vesicles (EVs) such as exosomes and other various factors for intercellular communication. However, macrophage-derived EVs heterogeneity and their cytotoxicity to cancer cells has not been well understood. Here, we aimed to separately isolate various types of macro-phage-EVs by size exclusion chromatography (SEC) method and investigate EV transmission and cytotoxicity to oral cancer cells. For fluorescence-labeling of cellular and EV membranes, palmitoylation signal-fused GFP and tdTomato were expressed in THP-1 monocytic cells and HSC-3 oral cancer cells, respectively. We found that fluorescence-labeled EVs secreted by macrophages were highly transmissive to oral cancer cells than those from parental monocytic cells. In a co-culture system and conditioned medium (CM), a macrophage-secreted unidentified factor was cytotoxic to oral cancer cells. We fractionated macrophage-derived EVs by the SEC method and performed western blotting to characterize various EV types. Three fractions were characterized: small exosomes (EXO-S: < 50 nm) fraction containing HSP90α, HSP90β, CD63 (EV marker) and β-actin; large exosomes (EXO-L: 50-200 nm) fraction containing CD9 (EV marker) and HSP90β; large EVs (100-500 nm) fraction. Notably, the macrophage-derived small exosomes fraction was cytotoxic to oral cancer cells, while large exosomes and large EVs were not. There-fore, it was implicated that macrophage-derived small exosomes are cytotoxic with high trans-mission potential to cancer cells.

Anti-cancer activity and in vitro to in vivo mechanistic recapitulation of novel ruthenium-based metallodrugs in the zebrafish model

Ruthenium is popular as a metal-core for chemotherapeutics, due to versatile molecular coordination. Because new metallodrugs are synthesized at high rates, our studies included assays in zebrafish to expedite the initial evaluation as anti-cancer agents. Here we evaluated novel metallodrugs PMC79 and LCR134), and cisplatin, a widely-used platinum-based chemotherapeutic. We hypothesized that this model could characterize anti-cancer properties and recapitulate previous in vitro results in vivo. Our findings suggest anti-cancer properties of PMC79 and LCR134 were similar with less toxicity than cisplatin. Exposures from 24-72 hrs at or below the LOAELs of PMC79 and LCR134 (3.9 µM and 13.5 µm, respectively), impaired blood vessel development and tailfin regeneration. Blood vessel examination through live-imaging of larvae revealed distinct regional anti-angiogenic impacts. The significant decrease in gene expression of the VEGF-HIF pathway and beta-actin could explain the morphological effects observed in the whole organism following exposure. Tailfin amputation in larvae exposed to PMC79 or LCR134 inhibited tissue regrowth and cell division, but did not impact normal cell proliferation unlike cisplatin. This suggests Ru-drugs may be more selective in targeting cancerous cells than cisplatin. Additionally, in vitro mechanisms were confirmed. PMC79 disrupted cytoskeleton formation in larvae and P-glycoprotein transporters in vivo was inhibited at low doses which could limit off-target effects of chemotherapeutics. Our results demonstrate the value for using the zebrafish in metallodrug research to evaluate mechanisms and off-target effects. In light of the findings reported in this paper, future investigation of PMC79 and LCR134 are warranted in higher vertebrate models.

Betaine Induced Autophagy to Protect Neurons Against Amyloid Beta in Alzheimer’s Disease

Background: Alzheimer disease is a neurodegenerative disease, which is accompanied by the accumulation of amyloid beta and tau hyper phosphorylation. The objective of this study is to investigate amyloid-beta clearance through autophagy in HMC3 cells by betaine.Methods: In this study HMC3 cells were used. LC3, P62, p-AKT, p-MTOR, Beclin1, ATG 5, ATG 7, ATG 12 and beta-actin were studied by western blot. LC3 was studied by confocal microscopy. Neuroprotection and cell viability were assessed by MTT assay. Confocal microscopy and ELISA studied amyloid-beta clearance. Student's T-test was used to compare the treated vs untreated groups.Results: We have identified betaine an autophagy inducer in microglia HMC3 cells. We have further tried to establish the mechanism of betaine for autophagy induction. Betaine was able to induce autophagy through the PI3K/AKT pathway. Further, we investigated the clearance of amyloid-beta by betaine through autophagy induction. Betaine was also able to reduce the toxicity of amyloid-beta in human SHSY-5Y cells. A known down of LC3 reverses the protective effect of betaine against amyloid-beta in differentiated SHSY5Y cells.Conclusion: Betaine induced autophagy, clears amyloid-beta and protects SHSY-5Y cells against amyloid-beta. Based on these data, betaine can be further studied for anti-Alzheimer therapy.

Detection of Non-Tuberculosis Mycobacteria by Real Time PCR

Background: Identification of non-tuberculosis mycobacteria by culture and phenotypic description is commonly used; however, it takes 4 to 6 weeks or even a longer time for slow growing species as well as for identification of some species that may be missed by biochemical characteristics methods. This study aimed to evaluate Real Time PCR for Detection of NTM by Amplification of Internal Transcribed Spacer (ITS) and 16S rRNA. Methods: In our investigation, using Real Time PCR and two pairs of unique primers targeted to ITS and 16S rRNA genes as well as Beta- actin as an internal control, Non tuberculosis mycobacteria species were detected. Results: Real time PCR was performed on the prepared dilutions. In addition, the threshold of sensitivity in this study was 10pg. To test the specificity, the genome of several bacteria responsible for respiratory infections was used, in which only the test response related to the non-tuberculosis mycobacterium genome and internal control was positive. Conclusion: In this research, an effective and up-to-date Real Time PCR method was used to design a diagnostic kit from all aspects. To avoid any error or mistake and to minimize the false results, internal control was used. The ability to design diagnostic kits allows us to increase efficiency, minimize mistakes, and save a considerable amount of time and cost.

ACTIN CYTOSKELETON ORGANIZATION IN OOCYTES AT VARIOUS STAGES OF DROSOPHILA MELANOGASTER OOGENESIS AFTER EXPOSURE IN A MICROGRAVITY SIMULATOR OVER THE COMPLETE GAMETOGENESIS CYCLE

The paper deals with the effects of modeled microgravity on actin cytoskeleton in oocytes at various stages of Drosophila melanogaster oogenesis over the complete gametogenesis cycle. Total actin content and F-actin singly was determined using immunohistochemical analysis. The results point to the growth of both total beta-actin and its polymer recognized by phalloidin. This finding can have key implications for evaluation of risks for the reproductive potential from the spaceflight factors.