Microplusin, a Rhipicephalus (Boophilus) microplus antimicrobial peptide (AMP) is the first fully characterized member of a new family of cysteine-rich AMPs with histidine-rich regions at the N and C termini. In the tick, microplusin belongs to the arsenal of innate defense molecules active against bacteria and fungi. Here we describe the NMR solution structure of microplusin and demonstrate that the protein binds copper II and iron II. Structured as a single ␣-helical globular domain, microplusin consists of five ␣-helices: ␣1 (residues Gly-9 to Arg-21), ␣2 (residues Glu-27 to Asn-40), ␣3 (residues Arg-44 to Thr-54), ␣4 (residues Leu-57 to Tyr-64), and ␣5 (residues Asn-67 to Cys-80). The N and C termini are disordered. This structure is unlike any other AMP structures described to date. We also used NMR spectroscopy to map the copper binding region on microplusin. Finally, using the Gram-positive bacteria Micrococcus luteus as a model, we studied of mode of action of microplusin. Microplusin has a bacteriostatic effect and does not permeabilize the bacterial membrane. Because microplusin binds metals, we tested whether this was related to its antimicrobial activity. We found that the bacteriostatic effect of microplusin was fully reversed by supplementation of culture media with copper II but not iron II. We also demonstrated that microplusin affects M. luteus respiration, a copper-dependent process. Thus, we conclude that the antibacterial effect of microplusin is due to its ability to bind and sequester copper II.

The Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) pandemic represents a global challenge. SARS-CoV-2's ability to replicate in host cells relies on the action of its non-structural proteins, like its main protease (M pro). This cysteine protease acts by processing the viruses' precursor polyproteins. As proteases, together with polymerases, are main targets of antiviral drug design, we here have performed biochemical high throughput screening (HTS) with recombinantly expressed SARS-CoV-2 M pro. A fluorescent assay was used to identify inhibitors in a compound library containing known drugs, bioactive molecules and natural products. These screens led to the identification of 13 inhibitors with IC 50 values ranging from 0.2 μM to 23 μM. The screens confirmed several known SARS-CoV M pro inhibitors as inhibitors of SARS-CoV-2 M pro , such as the organo-mercuric compounds thimerosal and phenylmercuric acetate. Benzophenone derivatives could also be identified among the most potent screening hits. Additionally, Evans blue, a sulfonic acid-containing dye, could be identified as an M pro inhibitor. The obtained compounds could be of interest as lead compounds for the development of future SARS-CoV-2 drugs.

LLLT reduced the lung permeability by a mechanism in which the IL-1beta seems to have an important role.

Background:Cytochrome c release from mitochondria to cytosol is a hallmark of apoptosis and is used to characterize the mitochondria‐dependent pathway of this type of cell death. Techniques currently used to measure cytochrome c release, Western blot and fluorescence microscopy of immunolabeled cells, are time‐consuming and inaccurate, and the latter is still limited by sample size.Methods:We developed a rapid and reliable technique to detect cytochrome c release during drug‐induced apoptosis, using flow cytometry. Plasma membrane of apoptotic HL‐60 cells and thymocytes, treated with staurosporine and dexamethasone, respectively, were selectively permeabilized by digitonin at a low concentration. The released cytochrome c was quickly washed out from cells and that which remained in the mitochondria was immunolabeled after fixing the cells.Results:The fraction of cells that retained their mitochondrial cytochrome c, or the highly fluorescent cells, gradually decreased so that after 4–8 h of drug treatment almost all the cells lost their cytochrome c and emerged as a population of low fluorescent cells. This was confirmed by parallel fluorescence microscopy of cells immunolabeled for cytochrome c.Conclusions:This technique allows the analysis of cytochrome c release from mitochondria of a large number of apoptotic cells in a short period of time and is proposed as an alternative to the methods currently used for this same purpose. © 2006 International Society for Analytical Cytology