Quantum Sensing of Free Radical Generation in Mitochondria of Single Heart Muscle Cells during Hypoxia and Reoxygenation.

Cells are damaged during hypoxia (blood supply deprivation) and reoxygenation (oxygen return). This damage occurs in conditions such as cardiovascular diseases, cancer, and organ transplantation, potentially harming the tissue and organs. The role of free radicals in cellular metabolic reprogramming under hypoxia is under debate, but their measurement is challenging due to their short lifespan and limited diffusion range. In this study, we employed a quantum sensing technique to measure the real-time production of free radicals at the subcellular level. We utilize fluorescent nanodiamonds (FNDs) that exhibit changes in their optical properties based on the surrounding magnetic noise. This way, we were able to detect the presence of free radicals. To specifically monitor radical generation near mitochondria, we coated the FNDs with an antibody targeting voltage-dependent anion channel 2 (anti-VDAC2), which is located in the outer membrane of mitochondria. We observed a significant increase in the radical load on the mitochondrial membrane when cells were exposed to hypoxia. Subsequently, during reoxygenation, the levels of radicals gradually decreased back to the normoxia state. Overall, by applying a quantum sensing technique, the connections among hypoxia, free radicals, and the cellular redox status has been revealed.

Diamond Relaxometry as a Tool to Investigate the Free Radical Dialogue between Macrophages and Bacteria.

Although free radicals, which are generated by macrophages play a key role in antimicrobial activities, macrophages sometimes fail to kill Staphylococcus aureus (S. aureus) as bacteria have evolved mechanisms to withstand oxidative stress. In the past decades, several ROS-related staphylococcal proteins and enzymes were characterized to explain the microorganism's antioxidative defense system. Yet, time-resolved and site-specific free radical/ROS detection in bacterial infection were full of challenges. In this work, we utilize diamond-based quantum sensing for studying alterations of the free radical response near S. aureus in macrophages. To achieve this goal we used S. aureus-fluorescent nanodiamond conjugates and measured the spin-lattice relaxation (T1) of NV defects embedded in nanodiamonds. We observed an increase of intracellular free radical generation when macrophages were challenged with S. aureus. However, under a high intracellular oxidative stress environment elicited by lipopolysaccharides, a lower radical load was recorded on the bacteria surfaces. Moreover, by performing T1 measurements on the same particles at different times postinfection, we found that radicals were dominantly scavenged by S. aureus from 80 min postinfection under a high intracellular oxidative stress environment.

Recent Applications of Contact Lenses for Bacterial Corneal Keratitis Therapeutics: A Review.

Corneal keratitis is a common but severe infectious disease; without immediate and efficient treatment, it can lead to vision loss within a few days. With the development of antibiotic resistance, novel approaches have been developed to combat corneal keratitis. Contact lenses were initially developed to correct vision. Although silicon hydrogel-based contact lenses protect the cornea from hypoxic stress from overnight wear, wearing contact lenses was reported as an essential cause of corneal keratitis. With the development of technology, contact lenses are integrated with advanced functions, and functionalized contact lenses are used for killing bacteria and preventing infectious corneal keratitis. In this review, we aim to examine the current applications of contact lenses for anti-corneal keratitis.

Intracellular Quantum Sensing of Free-Radical Generation Induced by Acetaminophen (APAP) in the Cytosol, in Mitochondria and the Nucleus of Macrophages.

Acetaminophen overdoses cause cell injury in the liver. It is widely accepted that liver toxicity is initiated by the reactive N-acetyl-para-aminophenol (APAP) metabolite N-acetyl-p-benzoquinone imine (NAPQI), which first depletes glutathione and then irreversibly binds to mitochondrial proteins and nuclear DNA. As a consequence, mitochondrial respiration is inhibited, and DNA strands break. NAPQI also promotes the oxidative stress since glutathione is one of the main free-radical scavengers in the cell. However, so far it is unknown where exactly free radicals are generated. In this study, we used relaxometry, a novel technique that allows nanoscale magnetic resonance imaging detection of free radicals. The method is based on fluorescent nanodiamonds, which change their optical properties based on their magnetic surrounding. To achieve subcellular resolution, these nanodiamonds were targeted to cellular locations, that is, the cytoplasm, mitochondria, and the nucleus. Since relaxometry is sensitive to spin noise from radicals, we were able to measure the radical load in these different organelles. For the first time, we measured APAP-induced free-radical production in an organelle-specific manner, which helps predict and better understand cellular toxicity.

Quantum Sensing of Free Radicals in Primary Human Dendritic Cells.

Free radicals are crucial indicators for stress and appear in all kinds of pathogenic conditions, including cancer, cardiovascular diseases, and infection. However, they are difficult to detect due to their reactivity and low abundance. We use relaxometry for the detection of radicals with subcellular resolution. This method is based on a fluorescent defect in a diamond, which changes its optical properties on the basis of the magnetic surroundings. This technique allows nanoscale MRI with unprecedented sensitivity and spatial resolution. Recently, this technique was used inside living cells from a cell line. Cell lines differ in terms of endocytic capability and radical production from primary cells derived from patients. Here we provide the first measurements of phagocytic radical production by the NADPH oxidase (NOX2) in primary dendritic cells from healthy donors. The radical production of these cells differs greatly between donors. We investigated the cell response to stimulation or inhibition.

Pharmacodynamic Studies of Fluorescent Diamond Carriers of Doxorubicin in Liver Cancer Cells and Colorectal Cancer Organoids.

BACKGROUND: We recently reported on preferential deposition of bare fluorescent diamond particles FDP-NV-700/800nm (FDP-NV) in the liver following intravenous administration to rats. The pharmacokinetics of FDP-NV in that species indicated short residency in the circulation by rapid clearance by the liver. Retention of FDP-NV in the liver was not associated with any pathology. These observations suggested that cancer therapeutics, such as doxorubicin, linked to FDP-NV, could potentially serve for anti-cancer treatment while sparing toxicities of peripheral organs. PURPOSE: To generate proof-of-concept (POC) and detail mechanisms of action of doxorubicin-coated FDP-NV-700/800nm (FDP-DOX) as a prospective chemotherapeutic for metastatic liver cancer. METHODS: FDP-DOX was generated by adsorption chemistry. Experimental design included concentration and time-dependent efficacy studies as compared with naïve (baren) FDP-NV in in vitro liver cancer cells models. Uptake of FDP-NV and FDP-DOX by HepG-2, Hep-3B and hCRC organoids were demonstrated by flow-cytometry and fluorescent microscopy. FDP-DOX pharmacodynamic effects included metabolic as well as cell death biomarkers Annexin V, TUNEL and LDH leakage. DOX desorpted from FDP-DOX was assessed by confocal microscopy and chemical assay of cells fractions. RESULTS: FDP-DOX efficacy was dose- and time-dependent and manifested in both liver cancer cell lines and human CRC organoids. FDP-DOX was rapidly internalized into cancer cells/organoids leading to cancer growth inhibition and apoptosis. FDP-DOX disrupted cell membrane integrity as evident by LDH release and suppressing mitochondrial metabolic pathways (AlamarBlue assay). Access of free DOX to the nuclei was confirmed by direct UV-Visible fluorescent assay and confocal microscopy of DOX fluorescence. CONCLUSION: The rapid uptake and profound cancer inhibition observed using FDP-DOX in clinically relevant cancer models, highlight FDP-DOX promise for cancer chemotherapeutics. We also conclude that the in vitro data justify further investment in in vivo POC studies.

pH Sensitive Dextran Coated Fluorescent Nanodiamonds as a Biomarker for HeLa Cells Endocytic Pathway and Increased Cellular Uptake.

Fluorescent nanodiamonds are a useful for biosensing of intracellular signaling networks or environmental changes (such as temperature, pH or free radical generation). HeLa cells are interesting to study with these nanodiamonds since they are a model cell system that is widely used to study cancer-related diseases. However, they only internalize low numbers of nanodiamond particles very slowly via the endocytosis pathway. In this work, we show that pH-sensitive, dextran-coated fluorescent nanodiamonds can be used to visualise this pathway. Additionally, this coating improved diamond uptake in HeLa cells by 5.3 times (*** p < 0.0001) and decreased the required time for uptake to only 30 min. We demonstrated further that nanodiamonds enter HeLa cells via endolysosomes and are eventually expelled by cells.

Micro Versus Macro - The Effect of Environmental Confinement on Cellular Nanoparticle Uptake.

While the microenvironment is known to alter the cellular behavior in terms of metabolism, growth and the degree of endoplasmic reticulum stress, its influence on the nanoparticle uptake is not yet investigated. Specifically, it is not clear if the cells cultured in a microenvironment ingest different amounts of nanoparticles than cells cultured in a macroenvironment (for example a petri dish). To answer this question, here we used J774 murine macrophages and fluorescent nanodiamonds (FND) as a model system to systematically compare the uptake efficiency of cells cultured in a petri dish and in a microfluidic channel. Specifically, equal numbers of cells were cultured in two devices followed by the FND incubation. Then cells were fixed, stained and imaged to quantify the FND uptake. We show that the FND uptake in the cells cultured in petri dishes is significantly higher than the uptake in a microfluidic chip where the alteration in CO2 environment, the cell culture medium pH and the surface area to volume ratio seem to be the underlying causes leading to this observed difference.

A novel inhibitor of the new antibiotic resistance protein OptrA.

The antibiotic resistance (ARE) subfamily of ABC (ATP-binding cassette) proteins confers resistance to a variety of clinically important ribosome-targeting antibiotics and plays an important role in infections caused by pathogenic bacteria. However, inhibitors of ARE proteins have rarely been reported. Here, OptrA, a new member of the ARE proteins, was used to study inhibitors of these types of proteins. We first confirmed that destroying the catalytic activity of OptrA could restore the sensitivity of host cells to antibiotics. Then, fragment-based screening, a drug screening method, was used to screen for inhibitors of OptrA. The competitive saturation transfer difference experiments, docking, and molecular dynamics were used to determine the binding sites and mode of interactions between OptrA and fragment screening hits. In this study, we first find a novel and specific inhibitor of OptrA (CP1), which suppressed the ATPase activity of OptrA in vitro by 30%. A hydrogen bond formed between the 8-position phenylcyclic cyano group in CP1 and the amino acid residue Lys-271 allows CP1 to form a stable complex with OptrA protein. These findings provide a theoretical basis for the further optimization of the inhibitor structure to obtain inhibitors with higher efficiencies.

Novel short antimicrobial peptide isolated from Xenopus laevis skin.

A rich source of bioactive peptides, including a large number of antimicrobial peptides, has been found in amphibian skin. In this study, a novel short antimicrobial peptide was purified from Xenopus laevis skin and characterised through reversed-phase high-performance liquid chromatography, Edman degradation and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. The peptide was composed of six amino acids with a sequence of DEDLDE and thus named X. laevis antibacterial peptide-P2 (XLAsp-P2). Transmission electron microscopy revealed that this peptide showed potential antimicrobial abilities against bacteria by damaging the bacterial cell membrane. XLAsp-P2 maybe inhibit bacterial growth by binding to the microbial genomic DNA. The peptide also exhibited a weak haemolytic activity against rabbit red blood cells. Therefore, XLAsp-P2 is a novel short anionic antibacterial peptide with broad activities. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Polypeptides from the Skin of Rana chensinensis Exert the Antioxidant and Antiapoptotic Activities on HaCaT Cells.

Studies have shown that frog skin secretes many types of peptides that are good for human skin. In this study, acid and enzymatic extracts of Rana skin peptides (acid/enzymatic Rana skin peptides, ARPs/ERPs) were obtained. The chemical and physical properties of the ARPs and ERPs were identified through UV scanning, HGLC, FRIT, and MS. MTS and flow cytometry were used to test the proproliferative and antiapoptotic effects of the ARPs and ERPs on human immortalized keratinocytes (HaCaT cells). To elucidate the antiapoptotic mechanisms, the mRNA and protein levels of EGF (epidermal growth factor, which enhances stimulation of cellular proliferation in both cells and epithelial tissues) and caspase-3 were evaluated using quantitative RT-PCR. The results indicated that the ARPs and ERPs were extracted from the Rana skin with yields of 0.65% and 0.52%, respectively. Treatment with ARPs (1.6 g/L) and ERPs (0.8 g/L) showed a 1.66-fold (p < 0.001) and 2.1-fold (p < 0.001) enhancement in the proliferation rates of HaCaT cells. The rate of apoptosis decreased by 2.6 fold (p < 0.01) and 3.4 fold (p < 0.01) under the UVB stimulation, respectively, at the same time, the up-regulation of EGF and down-regulation of caspase-3 were found. These results suggested that we can dig into the potential value of ARPs/ERPs in a new field.

Highly Efficient and Rapid Detection of the Cleavage Activity of Cas9/gRNA via a Fluorescent Reporter.

The RNA-guided endonuclease clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) derived from CRISPR systems is a simple and efficient genome-editing technology applied to various cell types and organisms. So far, the extensive approach to detect the cleavage activity of customized Cas9/guide RNA (gRNA) is T7 endonuclease I (T7EI) assay, which is time and labor consuming. In this study, we developed a visualized fluorescent reporter system to detect the specificity and cleavage activity of gRNA. Two gRNAs were designed to target porcine immunoglobulin M and nephrosis 1 genes. The cleavage activity was measured by using the traditional homology-directed repair (HDR)-based fluorescent reporter and the single-strand annealing (SSA)-based fluorescent reporter we established in this study. Compared with the HDR assay, the SSA-based fluorescent reporter approach was a more efficient and dependable strategy for testing the cleavage activity of Cas9/gRNA, thereby providing a universal and efficient approach for the application of CRISPR/Cas9 in generating gene-modified cells and organisms.

Missense mutations in the signal peptide of the porcine GH gene affect cellular synthesis and secretion.

CONTEXT: In previous investigations, we have demonstrated the mutations in the signal peptide of porcine GH gene were associated with the body size. METHODS: In this study, the fusion gene expression vectors which consisted of eight signal peptide mutants of GH gene and EGFP gene were constructed according to three missense mutations (p.Val9Ala, p.Gln22Arg and p.Asp25Gly), and they were transfected into the GH3 cell line. RESULTS: The inhibition levels of EGFP gene transcriptions with different signal peptide mutants were significantly different. Typically, the allelic variants carrying Val in codon nine showed higher protein synthesis (P < 0.05), and the allelic variants carrying neutral Gln in codon 22 and Gly in codon 25 showed higher secretion proportion (P < 0.05) compared with the other groups as assessed by western blotting. In silico RNA folding prediction indicated that the mutations gave rise to different RNA secondary structures, suggesting that they might affect translation and protein synthesis. CONCLUSION: We conclude that the missense mutations within the signal sequence influence the expression and the secretion of the protein. To the best of our knowledge, this is the first report addressing the functional consequences of the mutations in the signal peptide of porcine GH gene.

A novel short anionic antibacterial peptide isolated from the skin of Xenopus laevis with broad antibacterial activity and inhibitory activity against breast cancer cell.

A vastarray of bioactive peptides from amphibian skin secretions is attracting increasing attention due to the growing problem of bacteria resistant to conventional antibiotics. In this report, a small molecular antibacterial peptide, named Xenopus laevis antibacterial peptide-P1 (XLAsp-P1), was isolated from the skin of Xenopus laevis using reversed-phase high-performance liquid chromatography. The primary structure of XLAsp-P1, which has been proved to be a novel peptide by BLAST search in AMP database, was DEDDD with a molecular weight of 607.7 Da analysed by Edman degradation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). The highlight of XLAsp-P1 is the strong in vitro potency against a variety of Gram-positive and Gram-negative bacteria with minimum inhibitory concentrations (MICs) starting at 10 µg/mL and potent inhibitory activity against breast cancer cell at tested concentrations from 5 to 50 µg/mL. In addition, only 6.2 % of red blood cells was haemolytic when incubated with 64 µg/mL (higher than MICs of all bacterial strain) of XLAsp-P1. The antimicrobial mechanism for this novel peptide was the destruction of the cell membrane investigated by transmission electron microscopy. All these showed that XLAsp-P1 is a novel short anionic antibacterial peptide with broad antibacterial activity and inhibitory activity against breast cancer cell.