Cell-free protein synthesis with technical additives - expanding the parameter space of in vitro gene expression.

Biocatalysis has established itself as a successful tool in organic synthesis. A particularly fast technique for screening enzymes is the in vitro expression or cell-free protein synthesis (CFPS). The system is based on the transcription and translation machinery of an extract-donating organism to which substrates such as nucleotides and amino acids, as well as energy molecules, salts, buffer, etc., are added. After successful protein synthesis, further substrates can be added for an enzyme activity assay. Although mimicking of cell-like conditions is an approach for optimization, the physical and chemical properties of CFPS are not well described yet. To date, standard conditions have mainly been used for CFPS, with little systematic testing of whether conditions closer to intracellular conditions in terms of viscosity, macromolecules, inorganic ions, osmolarity, or water content are advantageous. Also, very few non-physiological conditions have been tested to date that would expand the parameter space in which CFPS can be performed. In this study, the properties of an Escherichia coli extract-based CFPS system are evaluated, and the parameter space is extended to high viscosities, concentrations of inorganic ion and osmolarity using ten different technical additives including organic solvents, polymers, and salts. It is shown that the synthesis of two model proteins, namely superfolder GFP (sfGFP) and the enzyme truncated human cyclic GMP-AMP synthase fused to sfGFP (thscGAS-sfGFP), is very robust against most of the tested additives.

Radioprotective effect of radiation-induced Lactococcus lactis cell-free extract against 60Coγ injury in mice.

Ionizing radiation (IR) is widely used in the diagnosis and treatment of various cancers. However, IR can cause damage to human health by producing reactive oxygen species. Lactococcus lactis is a type of microorganism that is beneficial to human health and has a strong antioxidant capacity. In this study, the protective effect of normal and IR-induced L. lactis IL1403 cell-free extracts (CFE and IR-CFE, respectively) against oxidative damage in vitro and the radioprotective effect of IR-CFE in vivo was evaluated using 60Coγ-induced oxidative damage model in mice. Results showed that IR-CFE exhibited a stronger oxidative damage-protective effect than CFE for L. lactis IL1403 under H2O2 in vitro. Moreover, IR-CFE also showed strong radioprotective effect on hepatocyte cells (AML-12) under radiation condition, and the effect was better than that of CFE. Animal experiment indicated that IR-CFE could reduce the IR-induced damage to the hematopoietic system by increasing the number of white blood cells and red blood cells in peripheral blood of irradiated mice. It was also observed that IR-CFE could markedly alleviate the 60Coγ-induced oxidative stress via increasing the activities of superoxide dismutase and glutathione peroxidase, enhancing the levels of glutathione, and decreasing the contents of malondialdehyde in serum, liver, and spleen. In addition, IR-CFE also could reduce the activities of alanine transaminase and aspartate aminotransferase in serum, thereby reducing radiation damage to the liver. These results suggested that IR-CFE could be considered as potential candidates for natural radioprotective agents. This study provides a theoretical basis for improving the application of lactic acid bacteria.

Lipolytic Postbiotic from Lactobacillus paracasei Manages Metabolic Syndrome in Albino Wistar Rats.

The current study investigates the capacity of a lipolytic Lactobacillus paracasei postbiotic as a possible regulator for lipid metabolism by targeting metabolic syndrome as a possibly safer anti-obesity and Anti-dyslipidemia agent replacing atorvastatin (ATOR) and other drugs with proven or suspected health hazards. The high DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS [2,2'-azino-bis (3-ethyl benzothiazoline-6-sulphonic acid)] scavenging activity and high activities of antioxidant enzyme such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-px) of the Lactobacillus paracasei postbiotic (cell-free extract), coupled with considerable lipolytic activity, may support its action against metabolic syndrome. Lactobacillus paracasei isolate was obtained from an Egyptian cheese sample, identified and used for preparing the postbiotic. The postbiotic was characterized and administered to high-fat diet (HFD) albino rats (100 and 200 mg kg-1) for nine weeks, as compared to atorvastatin (ATOR; 10 mg kg-1). The postbiotic could correct the disruption in lipid metabolism and antioxidant enzymes in HFD rats more effectively than ATOR. The two levels of the postbiotic (100 and 200 mg kg-1) reduced total serum lipids by 29% and 34% and serum triglyceride by 32-45% of the positive control level, compared to only 25% and 35% in ATOR's case, respectively. Both ATOR and the postbiotic (200 mg kg-1) equally decreased total serum cholesterol by about 40% and 39%, while equally raising HDL levels by 28% and 30% of the positive control. The postbiotic counteracted HFD-induced body weight increases more effectively than ATOR without affecting liver and kidney functions or liver histopathology, at the optimal dose of each. The postbiotic is a safer substitute for ATOR in treating metabolic syndrome.

Lactobacillus rhamnosus cell-free extract targets virulence and antifungal drug resistance in Candida albicans.

Candidiasis caused by multidrug-resistant Candida species continues to be difficult to eradicate. The use of live probiotic bacteria has gained a lot of interest in the treatment of candidiasis; however, whole-cell probiotic use can often be associated with a high risk of sepsis. Strategies manipulating cell-free methods using probiotic strains could lead to the development of novel antifungal solutions. Therefore, we evaluated the effect of three probiotic cell-free extracts (CFEs) on the growth, virulence traits, and drug efflux pumps in C. albicans. On the basis of its minimum inhibitory concentration, Lactobacillus rhamnosus was selected and assessed against various virulence traits and drug resistance mechanisms. The results showed that L. rhamnosus CFE significantly inhibited hyphae formation and reduced secretion of proteinases and phospholipases. Moreover, L. rhamnosus inhibited the drug efflux proteins in resistant C. albicans strains thus reversing drug resistance. Gene expression data confirmed downregulation of genes associated with microbial virulence and drug resistance following treatment of C. albicans with L. rhamnosus CFE. Through gas chromatography - mass spectrometry chemical characterization, high contents of oleic acid (24.82%) and myristic acid (13.11%) were observed in this CFE. Collectively, our findings indicate that L. rhamnosus may potentially be used for therapeutic purposes to inhibit C. albicans infections.

Biosynthesis of gold nanoparticles using cell-free extracts of Magnusiomyces ingens LH-F1 for nitrophenols reduction.

A green and eco-friendly method for the synthesis of gold nanoparticles (AuNPs) was developed using the cell-free extracts of a yeast strain Magnusiomyces ingens LH-F1. UV-vis spectra showed a distinct absorption band at ~ 540 nm, corresponding to the surface plasmon resonance of AuNPs. Transmission electron microscopy images revealed that the shapes of AuNPs were almost spherical and pseudo-spherical. Fourier transform infrared spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses suggested that some proteins containing amino- and carboxyl-groups in the cell-free extracts were absorbed on the surface of nanoparticles, which could act as reducing and capping agents for AuNPs synthesis. Furthermore, with the concentration of cell-free extracts increasing from 25 to 200 mg L-1, the average size of AuNPs decreased from 28.3 to 20.3 nm. Meanwhile, the morphology became more uniform with less irregular shapes. In addition, the as-synthesized AuNPs showed an excellent catalytic activity for nitrophenols reduction (i.e., 4-nitrophenol, 3-nitrophenol and 2-nitrophenol) in the presence of excess NaBH4. The catalytic rate constant of nitrophenols reduction was also dependent on cell-free extract concentration. The larger AuNPs synthesized by less cell-free extracts were covered with a thinner corona and showed better capacity for reducing nitrophenols. This study suggested that the as-synthesized AuNPs could be employed as efficient catalysts in reduction of organic contaminants.

The effects of interspecific interactions between bloom forming cyanobacteria and Scenedesmus quadricauda (chlorophyta) on their photophysiology.

Eutrophication and enhanced external nutrient loading of lakes and seas are most clearly reflected by increased cyanobacterial blooms, which are often toxic. Freshwater cyanobacteria produce a number of bioactive secondary metabolites, some of which have allelopathic properties, significantly influencing the biological processes of other algae, thereby affecting species composition and succession of the phytoplankton. The goal of this work was to investigate the influence of bloom-forming cyanobacterial exudates on the photophysiology of the green alga Scenedesmus quadricauda by chlorophyll fluorescence analysis. We were able to prove the effect of algal cell-free filtrates on the performance of S. quadricauda and demonstrate for the first time that the freshwater picocyanobacterium Cyanobium gracile has strong negative impact on the coexisting green alga. Neither the cyanotoxin (MYC, CYN and ATX) producing, nor the non-toxic strains showed any systematic effect on the production of S. quadricauda. Various strains of the cyanobacterium Cylindrospermopsis raciborskii inhibited the performance of the green alga independently of their origin. Our results urge further studies for a better understanding of the factors affecting the release of allelopathic compounds and the mechanisms of their effects on target organisms.

CDC6 controls dynamics of the first embryonic M-phase entry and progression via CDK1 inhibition.

CDC6 is essential for S-phase to initiate DNA replication. It also regulates M-phase exit by inhibiting the activity of the major M-phase protein kinase CDK1. Here we show that addition of recombinant CDC6 to Xenopus embryo cycling extract delays the M-phase entry and inhibits CDK1 during the whole M-phase. Down regulation of endogenous CDC6 accelerates the M-phase entry, abolishes the initial slow and progressive phase of histone H1 kinase activation and increases the level of CDK1 activity during the M-phase. All these effects are fully rescued by the addition of recombinant CDC6 to the extracts. Diminution of CDC6 level in mouse zygotes by two different methods results in accelerated entry into the first cell division showing physiological relevance of CDC6 in intact cells. Thus, CDC6 behaves as CDK1 inhibitor regulating not only the M-phase exit, but also the M-phase entry and progression via limiting the level of CDK1 activity. We propose a novel mechanism of M-phase entry controlled by CDC6 and counterbalancing cyclin B-mediated CDK1 activation. Thus, CDK1 activation proceeds with concomitant inhibition by CDC6, which tunes the timing of the M-phase entry during the embryonic cell cycle.

Microbial cell-free extracts as sources of enzyme activities to be used for enhancement flavor development of ewe milk cheese.

Freeze-dried cell-free extracts (CFE) from Lactobacillus casei LC01, Weissella cibaria 1XF5, Hafnia alvei Moller ATCC 51815, and Debaryomyces hansenii LCF-558 were used as sources of enzyme activities for conditioning the ripening of ewe milk cheese. Compared with control cheese (CC), CFE did not affect the gross composition and the growth of the main microbial groups of the cheeses. As shown through urea-PAGE electrophoresis of the pH 4.6-soluble nitrogen fraction and the analysis of free AA, the secondary proteolysis of the cheeses with CFE added was markedly differed from that of the CC. Compared with CC, several enzyme activities were higher in the water-soluble extracts from cheeses made with CFE. In agreement, the levels of 49 volatile compounds significantly differentiated CC from the cheeses made with CFE. The level of some alcohols, ketones, sulfur compounds, and furans were the lowest in the CC, whereas most aldehydes were the highest. Each CFE seemed to affect a specific class of chemical compounds (e.g., the CFE from H. alvei ATCC 51815 mainly influenced the synthesis of sulfur compounds). Apart from the microbial source used, the cheeses with the addition of CFE showed higher score for acceptability than the control cheese. Cheese ripening was accelerated or conditioned using CFE as sources of tailored enzyme activities.

Enzymatic cyanide degradation by cell-free extract of Rhodococcus UKMP-5M.

The cell-free extract of locally isolated Rhodococcus UKMP-5M strain was used as an alternative to develop greener and cost effective cyanide removal technology. The present study aims to assess the viability of the cell-free extract to detoxify high concentrations of cyanide which is measured through the monitoring of protein concentration and specific cyanide-degrading activity. When cyanide-grown cells were subjected to grinding in liquid nitrogen which is relatively an inexpressive and fast cell disruption method, highest cyanide-degrading activity of 0.63 mM min(-1) mg(-1) protein was obtained in comparison to enzymatic lysis and agitation with fine glass beads. The cell-free extracts managed to degrade 80% of 20 mM KCN within 80 min and the rate of cyanide consumption increased linearly as the concentration of protein was raised. In both cases, the addition of co-factor was not required which proved to be advantageous economically. The successful formation of ammonia and formate as endproducts indicated that the degradation of cyanide by Rhodococcus UKMP-5M proceeded via the activity of cyanidase and the resulting non-toxic products are safe for disposal into the environment. Further verification with SDS-PAGE revealed that the molecular weight of the active enzyme was estimated to be 38 kDa, which is consistent with previously reported cyanidases. Thus, the utilization of cell-free extracts as an alternative to live microbial in cyanide degradation offers numerous advantageous such as the potential to tolerate and degrade higher concentration of cyanide and total reduction in the overall cost of operation since the requirement for nutrient support is irrelevant.

Studying Translesion DNA Synthesis Using Xenopus In Vitro Systems.

Cell-free extracts derived from Xenopus eggs have been widely used to decipher molecular pathways involved in several cellular processes including DNA synthesis, the DNA damage response, and genome integrity maintenance. We set out assays using Xenopus cell-free extracts to study translesion DNA synthesis (TLS), a branch of the DNA damage tolerance pathway that allows replication of damaged DNA. Using this system, we were able to recapitulate TLS activities that occur naturally in vivo during early embryogenesis. This chapter describes protocols to detect chromatin-bound TLS factors by western blotting and immunofluorescence microscopy upon induction of DNA damage by UV irradiation, monitor TLS-dependent mutagenesis, and perform proteomic screening.

Cell-Free Supernatant of Bacillus thuringiensis Displays Anti-Biofilm Activity Against Staphylococcus aureus.

Staphylococcus aureus is an important bacterial pathogen responsible for biofilm formation in medical devices. Due to the increasing antibiotic resistance of S. aureus, it is necessary to search for new anti-biofilm agents. In this study, the cell-free supernatant of Bacillus thuringiensis inhibited biofilm formation up to 93% and dispersed biofilms up to 83% without affecting the growth of S. aureus. The ethyl acetate extract of B. thuringiensis cell-free supernatant exhibited a dose-dependent anti-biofilm activity against S. aureus with the biofilm inhibition concentration ranging from 8 to 64 µg/mL. Scanning electron microscopy revealed that the cell-free supernatant extract of B. thuringiensis resulted in a significant reduction in S. aureus biofilms. The ethyl acetate extract of cell-free supernatant of B. thuringiensis was found to contain various compounds with structural similarity to known anti-biofilm compounds. In particular, squalene, cinnamic acid derivatives, and eicosapentaene seem to act synergistically against S. aureus biofilms. Hence, B. thuringiensis cell-free supernatant proved to be effective against S. aureus biofilms. The results clearly show the potential of natural molecules produced by B. thuringiensis as alternative therapies with anti-biofilm activity instead of bactericidal properties.

Effect of Yogurt Addition on the Stability of Anthocyanin during Cold Storage of Strawberry, Raspberry, and Blueberry Smoothies.

The addition of yogurt to fruit smoothies enhances their nutritional value by introducing components not naturally found in fruit products. However, the addition of fermented products can affect the stability of fruit bioactive components in fruits, such as anthocyanins. This study aimed to evaluate the effect of varying yogurt additions (0, 10, 20, and 30%) on the stability of anthocyanins during a 4-week refrigerated storage period. The smoothies were obtained from purees of strawberry, raspberry, and blueberry, combined with apple juice and apple puree. In addition, to elucidate the causes of the observed changes in the smoothies, model studies were conducted using purified anthocyanin extracts obtained from the analyzed fruits. We assessed the effects of pH, hydrogen peroxide concentration, and the addition of cell-free extracts from Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus on changes in anthocyanin content during storage. We found that adding yogurt led to a decrease in anthocyanin stability during the 4-week cold storage period. Specifically, a 30% yogurt addition decreased anthocyanin stability in all tested beverages, while a 20% yogurt addition impacted the strawberry and raspberry smoothies. The degree to which yogurt affected anthocyanin stability was dependent on the source of the raw material. The most notable impact was observed in strawberry smoothies and the least in blueberry smoothies. The variability could be attributed to differences in anthocyanin profiles among the fruits, the chemical composition of the beverages, and the observed difference in the survival rates of lactic acid bacteria. Model studies showed that during the storage of anthocyanin extracts, the addition of hydrogen peroxide and cell-free extract had a significant effect, whereas pH within the examined range (3.0-4.5) did not affect anthocyanin stability.

Xenopus cell-free extracts and their applications in cell biology study.

Xenopus has proven to be a remarkably versatile model organism in the realm of biological research for numerous years, owing to its straightforward maintenance in laboratory settings and its abundant provision of ample-sized oocytes, eggs, and embryos. The cell cycle of these oocytes, eggs, and early embryos exhibits synchrony, and extracts derived from these cells serve various research purposes. Many fundamental concepts in biochemistry, cell biology, and development have been elucidated through the use of cell-free extracts derived from Xenopus cells. Over the past few decades, a wide array of cell-free extracts has been prepared from oocytes, eggs, and early embryos of different Xenopus species at varying cell cycle stages. Each of these extracts possesses distinct characteristics. This review provides a concise overview of the Xenopus species employed in laboratory research, the diverse types of cell-free extracts available, and their respective properties. Furthermore, this review delves into the extensive investigation of spindle assembly in Xenopus egg extracts, underscoring the versatility and potency of these cell-free systems in the realm of cell biology.

Enterococcus spp. Cell-Free Extract: An Abiotic Route for Synthesis of Selenium Nanoparticles (SeNPs), Their Characterisation and Inhibition of Escherichia coli.

Selenite (SeO32-), the most toxic and most reactive selenium (Se) oxyanion, can be reduced to elemental selenium (Se0) nanoparticles by a variety of bacteria, including Enterococcus spp. Previously, the orthodox view held that the reduction of SeO32- to Se0 by a wide range of bacteria was solely accomplished by biological processes; however, recent studies have shown that various bacterial strains secrete metal-reducing metabolites, thereby indirectly catalysing the reduction of these metal species. In the current study, selenium nanoparticles were synthesised from the abiotic reduction of selenite with the use of Enterococcus spp. cell-free extract. Once separated from the cell-free extract, the particles were analysed using Fourier-transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), Transmission electron microscopy (TEM) and a Zetasizer. The results revealed that the SeNPs were spherical in shape, containing both amorphous and crystalline properties, and the sizes with the highest frequency ranged close to 200 nm. Additionally, the obtained nanoparticles exhibited antimicrobial properties by directly inhibiting the viability of an E. coli bacterial strain. The results demonstrate not only the potential of abiotic production of SeNPs, but also the potential for these particles as microbial inhibitors in medical or similar fields.

A Simple Recombinant E. coli Cell Lysate-Based Biocatalyst for ATP-Dependent Multi-step Reactions.

The utility of ATP-dependent multi-enzymatic reactions is limited by their requirement for stoichiometric amounts of this expensive cofactor or additional purified enzymes for its recycling. Here we describe a simple method for the production of recombinant cell-free extracts (or lysates) of E. coli that support ATP-dependent biotransformations. The inexpensive preparation described is obtained with modest processing from a single recombinant bacterial culture of E. coli. In addition to recombinantly overexpressed enzymes that catalyze the primary ATP-dependent reactions of interest, endogenous kinases that are naturally present in the extract catalyze recycling of the requisite ATP. This means that only catalytic amounts of cofactor are necessary to drive the biotransformation, and without the requirement for additional purified enzymes. This approach has been applied successfully to an array of in vitro enzymatic cascades with multiple ATP-dependent steps.

Antifungal Activity of Biosynthesized Silver Nanoparticles (AgNPs) against Aspergilli Causing Aspergillosis: Ultrastructure Study.

Currently, nanoparticles and nanomaterials are widely used for biomedical applications. In the present study, silver nanoparticles (AgNPs) were successfully biosynthesized using a cell-free extract (CFE) of Bacillus thuringiensis MAE 6 through a green and ecofriendly method. The size of the biosynthesized AgNPs was 32.7 nm, and their crystalline nature was confirmed by XRD, according to characterization results. A surface plasmon resonance spectrum of AgNPs was obtained at 420 nm. Nanoparticles were further characterized using DLS and FTIR analyses, which provided information on their size, stability, and functional groups. AgNPs revealed less cytotoxicity against normal Vero cell line [IC50 = 155 µg/mL]. Moreover, the biosynthesized AgNPs exhibited promising antifungal activity against four most common Aspergillus, including Aspergillus niger, A. terreus, A. flavus, and A. fumigatus at concentrations of 500 µg/mL where inhibition zones were 16, 20, 26, and 19 mm, respectively. In addition, MICs of AgNPs against A. niger, A. terreus, A. flavus, and A. fumigatus were 125, 62.5, 15.62, and 62.5 µg/mL, respectively. Furthermore, the ultrastructural study confirmed the antifungal effect of AgNPs, where the cell wall's integrity and homogeneity were lost; the cell membrane had separated from the cell wall and had intruded into the cytoplasm. In conclusion, the biosynthesized AgNPs using a CFE of B. thuringiensis can be used as a promising antifungal agent against Aspergillus species causing Aspergillosis.

Practical Assessment of an Interdisciplinary Bacteriophage Delivery Pipeline for Personalized Therapy of Gram-Negative Bacterial Infections.

Despite numerous advances in personalized phage therapy, smooth logistics are challenging, particularly for multidrug-resistant Gram-negative bacterial infections requiring high numbers of specific lytic phages. We conducted this study to pave the way for efficient logistics for critically ill patients by (1) closely examining and improving a current pipeline under realistic conditions, (2) offering guidelines for each step, leading to safe and high-quality phage supplies, and (3) providing a tool to evaluate the pipeline's efficiency. Due to varying stipulations for quality and safety in different countries, we focused the pipeline on all steps up to a required phage product by a cell-free extract system. The first of three study runs included patients with respiratory bacterial infections from four intensive care units, and it revealed a cumulative time of up to 23 days. Ultimately, adjustment of specific set points of the vulnerable components of the pipeline, phage isolation, and titration increased the pipeline's efficiency by 15% and decreased the maximum required time to 13 days. We present a site-independent practical approach to establish and optimize pipelines for personalized phage delivery, the co-organization of pipeline components between different institutions, non-binding guidelines for every step, and an efficiency check for phage laboratories.

Key reaction components affect the kinetics and performance robustness of cell-free protein synthesis reactions.

Cell-free protein synthesis (CFPS) reactions have grown in popularity with particular interest in applications such as gene construct prototyping, biosensor technologies and the production of proteins with novel chemistry. Work has frequently focussed on optimising CFPS protocols for improving protein yield, reducing cost, or developing streamlined production protocols. Here we describe a statistical Design of Experiments analysis of 20 components of a popular CFPS reaction buffer. We simultaneously identify factors and factor interactions that impact on protein yield, rate of reaction, lag time and reaction longevity. This systematic experimental approach enables the creation of a statistical model capturing multiple behaviours of CFPS reactions in response to components and their interactions. We show that a novel reaction buffer outperforms the reference reaction by 400% and importantly reduces failures in CFPS across batches of cell lysates, strains of E. coli, and in the synthesis of different proteins. Detailed and quantitative understanding of how reaction components affect kinetic responses and robustness is imperative for future deployment of cell-free technologies.

Cell-free extract assisted synthesis of ZnO nanoparticles using aquatic bacterial strains: Biological activities and toxicological evaluation.

The introduction of novel bacterial strains and the development of microbial approaches for nanoparticles biosynthesis could minimize the negative environmental impact and eliminate the concern and challenges of the available approaches. In this study, a biological method based on microbial cell-free extract was used for biosynthesis of ZnO NPs using two new aquatic bacteria, Marinobacter sp. 2C8 and Vibrio sp. VLA. The synthesized ZnO NPs were characterized by UV-Visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), atomic force microscope (AFM), dynamic light scattering (DLS) and zeta potential. The UV-Visible absorption peak was found to be at 266 and 250 nm for ZnO-2C8 NPs and ZnO-VLA NPs, respectively. FTIR study suggested that the hydroxyl, amine, and carboxyl groups of bacterial proteins are mainly responsible for stabilizing the biosynthesized ZnO NPs. The formation of hexagonal wurtzite structure of ZnO NPs was confirmed by the XRD pattern. The morphology of the nanoparticles was found to be spherical with the average particle size of about 10.23 ± 2.48 nm and 20.26 ± 4.44 nm for ZnO-2C8 NPs and ZnO-VLA NPs, respectively. The values of zeta potential indicate the high stability of the biosynthesized ZnO NP. Zeta potential values indicated the high stability of the biosynthesized ZnO NP and were obtained -20.54 ± 7.15 and -23.87 ± 2.29 mV for ZnO-2C8 NPs and ZnO-VLA NPs, respectively. The biosynthesized ZnO NPs had antibacterial activity against Gram-negative and Gram-positive strains and possessed excellent antibiofilm activity with the maximum inhibition of about 96.55% at 250 µg/mL. The DPPH activity of ZnO-2C8 NPs and ZnO-VLA NPs were found 88.9% and 85.7% for 2500 µg/mL concentration, respectively. The toxicity test revealed the biocompatibility of the biosynthesized ZnO NPs. The results suggested that this approach is a very good route for synthesizing ZnO NPs with potential applications in biotechnology.

Regulatory mechanisms of energy metabolism and inflammation in oleic acid-treated HepG2 cells from Lactobacillus acidophilus NX2-6 extract.

In this study, the cell-free extracts (CFE) of Lactobacillus acidophilus NX2-6 were utilized to treat oleic acid (OA)-induced hepatic steatosis. It was found that CFE treatment improved lipid metabolism in OA-induced hepatic steatosis model by downregulating several lipogenic genes but increasing expression levels of lipolysis-related genes. In addition, gene expression analysis revealed that CFE treatment promoted mitochondrial biogenesis and fission by upregulating the mRNA levels of PGC-1α, PGC-1ß, Sirt1, NRF1, and Fis1. CFE treatment also increased protein expression of p-AMPKα, PGC-1α, ACOX1, and Sirt1 in OA-treated cells, suggesting that CFE possessed ability to improve energy metabolism. Furthermore, CFE treatment also reversed OA-induced oxidative stress by increasing CAT activity and protein level of Nrf-2 as well as reducing protein expression of ATF6, XBP1, GRP78, p50, and p-ERK, indicating that CFE could inhibit endoplasmic reticulum stress and sterile inflammation. Thus, L. acidophilus NX2-6 had potential to fight against NAFLD. PRACTICAL APPLICATIONS: Diet-induced hepatic steatosis is one of major public health concerns all over the world. Hepatic steatosis is accompanied by disregulation of lipid metabolism and energy metabolism, endoplasmic reticulum stress, oxidative stress as well as chronic inflammation. It is reported that probiotics are considered as emerging therapeutic strategy to alleviate hepatic steatosis. This study indicated potential applications of dead probiotics in the prevention of hepatic steatosis and development of functional foods.