In situ quantification of poly(3-hydroxybutyrate) and biomass in Cupriavidus necator by a fluorescence spectroscopic assay

Fluorescence spectroscopy offers a cheap, simple, and fast approach to monitor poly(3-hydroxybutyrate) (PHB) formation, a biodegradable polymer belonging to the biodegradable polyester class polyhydroxyalkanoates. In the present study, a fluorescence and side scatter-based spectroscopic setup was developed to monitor in situ biomass, and PHB formation of biotechnological applied Cupriavidus necator strain. To establish PHB quantification of C. necator, the dyes 2,2-difluoro-4,6,8,10,12-pentamethyl-3-aza-1-azonia-2-boranuidatricyclo[7.3.0.03,7]dodeca-1(12),4,6,8,10-pentaene (BODIPY493/503), ethyl 5-methoxy-1,2-bis(3-methylbut-2-enyl)-3-oxoindole-2-carboxylate (LipidGreen2), and 9-(diethylamino)benzo[a]phenoxazin-5-one (Nile red) were compared with each other. Fluorescence staining efficacy was obtained through 3D-excitation-emission matrix and design of experiments. The coefficients of determination were ≥ 0.98 for all three dyes and linear to the high-pressure liquid chromatography obtained PHB content, and the side scatter to the biomass concentration. The fluorescence correlation models were further improved by the incorporation of the biomass-related side scatter. Afterward, the resulting regression fluorescence models were successfully applied to nitrogen-deficit, phosphor-deficit, and NaCl-stressed C. necator cultures. The highest transferability of the regression models was shown by using LipidGreen2. The novel approach opens a tailor-made way for a fast and simultaneous detection of the crucial biotechnological parameters biomass and PHB content during fermentation. Key points • Intracellular quantification of PHB and biomass using fluorescence spectroscopy. • Optimizing fluorescence staining conditions and 3D-excitation-emission matrix. • PHB was best obtained by LipidGreen2, followed by BODIPDY493/503 and Nile red. Graphical abstract

Identification of the Glucose Metabolism Biomarkers of NASH: Weighted Gene Co-Expression Network Analysis

Background The genetic mechanism of glucose metabolism has not been elucidated in nonalcoholic steatohepatitis (NASH), and many genes are took part in glucose metabolism of NASH. In this study, we used the weighted gene co-expression network analysis (WGCNA) to find the key genes associated with glucose metabolism; Methods Data sets GSE96971 and GSE89632 from Gene Expression Omnibus (GEO) were analyzed by WGCNA. We screened the Hub gene from the GSE96971 dataset, and the selected Hub genes were verified by GSE89632 dataset. We then analyzed the dataset using the Gene Ontology (GO) term enrichment and the Kyoto Encyclopedia of Genome (KEGG) path analysis. Expression levels of the hub genes are assessed by qPCR analysis. The function of hub genes was verified by Nile Red staining and relative glucose consumption detection; Results The hub genes are mannosidase beta like (MANBAL), myc proto-oncogene protein (MYC), caspase 4 (CASP4), CDK5 regulatory subunit associated protein 3 (CDK5RAP3) and ZFP36 ring finger protein (ZFP36) in the datasets GSE96971 and the GSE89632. Further, these genes are mainly involved in the integral component of membrane and plasma membrane, the PI3K-AKT signaling pathway and the olfactory transduction according to the GO and KEGG results. These hub genes were significantly up-regulated in the palmitic acid (PA) cell model and methionine-choline-deficient medium (MCD) cell model. After knocking out the hub genes in PA model and the MCD model of NASH, relative glucose consumption was increased and lipid deposition was reduced compared with the control group; Conclusions MANBAL, MYC, CASP4, CDK5RAP3 and ZFP36 are elevated and involved in the pathogenesis of NASH. Further research on these genes are warranted.

Biocompatibility and Physiological Thiolytic Degradability of Radically-made Thioester-functional Copolymers

Being non-degradable, vinyl polymers have limited biomedical applicability. Unfortunately, backbone esters incorporated through conventional radical ring-opening methods do not undergo appreciable abiotic hydrolysis under physiologically relevant conditions. Here, PEG acrylate and di(ethylene glycol) acrylamide-based copolymers containing backbone thioesters were prepared through the radical ring-opening copolymerization of the thionolactone dibenzo[c,e]oxepin-5(7H)-thione. The thioesters degraded fully in the presence of 10 mM cysteine at pH 7.4, with the mechanism presumed to involve an irreversible S–N switch. Degradations with N-acetylcysteine and glutathione were reversible through the thiol–thioester exchange polycondensation of R–SC(=O)–polymer–SH fragments with full degradation relying on an increased thiolate:thioester ratio. Treatment with 10 mM glutathione at pH 7.2 (mimicking intracellular conditions) triggered an insoluble–soluble switch of a temperature-responsive copolymer at 37 °C and the release of encapsulated Nile Red (as a drug model) from core-degradable diblock copolymer micelles. Copolymers and their cysteinolytic degradation products were found to be non-cytotoxic, making thioester backbone-functional polymers promising for drug delivery applications.

Enzyme-Responsive Amphiphilic Peptide Nanoparticles for Biocompatible and Efficient Drug Delivery

Self-assembled peptide nanostructures recently have gained much attention as drug delivery systems. As biomolecules, peptides have enhanced biocompatibility and biodegradability compared to polymer-based carriers. We introduce a peptide nanoparticle system containing arginine, histidine, and an enzyme-responsive core of repeating GLFG oligopeptides. GLFG oligopeptides exhibit specific sensitivity towards the enzyme cathepsin B that helps effective controlled release of cargo molecules in the cytoplasm. Arginine can induce cell penetration, and histidine facilitates lysosomal escape by its buffering capacity. Herein, we propose an enzyme-responsive amphiphilic peptide delivery system (Arg-His-(Gly-Phe-Lue-Gly)3, RH-(GFLG)3). The self-assembled RH-(GFLG)3 globular nanoparticle structure exhibited a positive charge and formulation stability for 35 days. Nile Red-tagged RH-(GFLG)3 nanoparticles showed good cellular uptake compared to the non-enzyme-responsive control groups with d-form peptides (LD (LRH-D(GFLG)3), DL (DRH-L(GFLG)3), and DD (DRH-D(GFLG)3). The RH-(GFLG)3 nanoparticles showed negligible cytotoxicity in HeLa cells and human RBCs. To determine the drug delivery efficacy, we introduced the anticancer drug doxorubicin (Dox) in the RH-(GFLG)3 nanoparticle system. LL-Dox exhibited formulation stability, maintaining the physical properties of the nanostructure, as well as a robust anticancer effect in HeLa cells compared to DD-Dox. These results indicate that the enzyme-sensitive RH-(GFLG)3 peptide nanoparticles are promising candidates as drug delivery carriers for biomedical applications.

Feasibility Study on Biomonitoring of Microplastics in Fish Gastrointestinal Tracts

Monitoring the occurrence and trends of microplastic contamination in the marine environment is key to establish microplastic (MP) data baselines, to work out policy mitigation measures, and to assess the effectiveness of waste regulations. To establish MP contamination baselines in the marine environment, marine biota species can be selected as monitoring matrices to track plastic pollution in the environment. The aim of this work was to evaluate the feasibility of biomonitoring MPs in fish gastrointestinal tract (GIT). A selection of suitable fish species was performed, based on species distribution, sampling effort, commercial value of species, sustainable development of fish populations, migration behaviour, and scientific evidence for occurrence of MPs in the fish GIT. Sampling and MP extraction protocols were developed and validated on fish GIT samples acquired in the Southern North Sea. The fish species selection protocol enabled the selection of ubiquitous distributed and non-endangered fish species relevant for MP monitoring in the North Sea. The fish GIT sampling protocol considered background contamination measures and sampling fillet as procedural blanks. Advantages and disadvantages of onboard dissection were discussed. The MPs extraction protocol was based on matrix digestion, density separation, and Nile red staining of particles followed by fluorescent microscopy observation. The confirmation of MPs identification and the analysis of the polymer composition was done using micro-Fourier transform infrared (μFTIR) spectroscopy. The MP analysis indicated a low number of MPs in the fish GIT. The mean number of particles per single fish GIT was 0.48 ± 0.81 (Nile red staining observations) to 0.26 ± 0.64 (corrected for background contamination). A power analysis (sampling effort) indicated that to detect significant differences, in a balanced-ANOVA type of analysis, between species and/or sampling areas, the sample size would require a minimum of 109 up to 370 individual fish. The feasibility of MP biomonitoring in fish GIT was assessed by a SWOT-analysis, which indicated that fish GIT is a suitable matrix for biomonitoring of MPs, but that the large number of samples needed to identify significant differences can be a major drawback. A potential implementation strategy for MP biomonitoring within Europe was suggested.

Following the light: 3D-printed COF@poly(2-hydroxyethyl methacrylate) dual emissive composite with response to polarity and acidity

A new imine-based covalent organic framework (COF) functionalized in its cavities with moieties of the chromophore Nile Red has been synthesized and characterized. Using mechanical (ultrasonics) and chemical (acid-self-exfoliation) methods,...

pixOL: pixel-wise dipole-spread function engineering for simultaneously measuring the 3D orientation and 3D localization of dipole-like emitters

Interactions between biomolecules are characterized by both where they occur and how they are organized, e.g., the alignment of lipid molecules to form a membrane. However, spatial and angular information are mixed within the image of a fluorescent molecule-the microscopy's dipole spread function (DSF). We demonstrate the pixOL algorithm for simultaneously optimizing all pixels within a phase mask to produce an engineered Green's tensor-the dipole extension of point-spread function engineering. The pixOL DSF achieves optimal precision for measuring simultaneously the 3D orientation and 3D location of a single molecule, i.e., 1.14 degree orientation, 0.24 sr wobble angle, 8.17 nm lateral localization, and 12.21 nm axial localization precisions over an 800-nm depth range using 2500 detected photons. The pixOL microscope accurately and precisely resolves the 3D positions and 3D orientations of Nile red within a spherical supported lipid bilayer, resolving both membrane defects and differences in cholesterol concentration, in 6 dimensions.

Microemulsions and nanoemulsions modified with cationic surfactants for improving the solubility and therapeutic efficacy of loaded drug indomethacin

In this work, a noncovalent strategy was successfully used to modify colloidal stability and in vitro and in vivo efficacy of two amphiphilic formulations of the anti-inflammatory drug indomethacin. Namely, nanoemulsions and microemulsions based on oleic acid and nonionic surfactants have been produced and compared. The influence of cationic surfactants cetyltrimethylammonium bromide and its carbamate bearing analogue on the size characteristics, stability and ability to provide prolonged action of loaded drug indomethacin has been evaluated. Adding the positively charged molecules in the surface layer of nanoemulsions and microemulsions has shown the stability increase along with maintaining the size characteristics and homogeneity in time. Moreover, the carbamate modified analogue demonstrated beneficial behavior. Indomethacin loaded in microemulsions and nanoemulsions showed prolonged-release (10 to 15% release for 5 h) compared to a free drug (complete release for 5 h). The rate of release of indomethacin from nanoemulsions was slightly higher than from microemulsions and insignificantly decreased with an increase in the concentration of the cationic surfactant. For carbamate surfactant nanocarrier loaded with fluorescence probe Nile red, the ability to penetrate into the cell was supported by flow cytometry study and visualized by fluorescence microscopy. In vitro tests on anti-inflammatory activity of the systems demonstrated that the blood cell membrane stabilization increased in the case of modified microemulsion. The anti-inflammatory activity of the encapsulated drug was tested in rats using a carrageenan-induced edema model. Nanoemulsions without cationic surfactants appeared more efficient compared to microemulsions. Indomethacin emulsion formulations with carbamate surfactant added showed slower carrageenan-induced edema progression compared to unmodified compositions. Meanwhile, the edema completely disappeared upon treatment with emulsion loaded indomethacin after 4 h in the case of microemulsions versus 5 h in the case of nanoemulsions.

Jet Colliding and Mixing Efficiency

Two experimental methods, the Nile Red dye extraction and the Williamson ether synthesis in biphasic conditions, were used to characterize the mixing performance of a new cheap impinging jet colliding mixer from Gjosa and to compare it to other commercial micromixers (Caterpillar CPMM-R300, T-mixer, LTF MR-MX and LTF MR-MS). The Nile Red method shows that the Caterpillar mixer is the best one. Excellent results are also achieved with two Gjosa mixers in series. These results are not reflected in the Williamson ether synthesis, where the best mixer is the Gjosa one.