Search Results (60)

Microbial contamination is the leading cause of foodborne diseases and spoilage in food and personal care products. Previous studies by our group have demonstrated that vine tea extract (VTE) and dihydromyricetin (DMY) inhibit the growth of Escherichia coli. In this study, we further explored the inhibitory mechanisms of VTE and DMY against E. coli through a label-free proteomics approach. The proteomic analysis detected 130 and 81 differentially expressed proteins (DEPs) in E.coli following VTE and DMY treatment, respectively. The analysis indicated that VTE and DMY inhibit bacterial growth through multiple-target mechanisms. Specifically, they inhibit E. coli growth by disrupting the cationic antimicrobial peptide resistance pathway, amino acid biosynthesis and metabolism, and nucleotide metabolism. Additionally, VTE disrupts various secondary metabolic pathways, while DMY interferes with E. coli ribosome assembly and function, and disrupts cell membrane lipid homeostasis by interfering with fatty acid metabolism. RT-qPCR validation confirmed transcriptional alterations in genes encoding key target proteins. Molecular docking results indicated that DMY may affect bacterial protein synthesis, cationic antimicrobial peptide resistance, and transcriptional regulation by binding to target proteins such as RplB, RplV, LpxA, and YafC. In conclusion, this study systematically deciphered the multi-target inhibitory mechanisms of VTE and DMY against E. coli, providing a theoretical basis for developing plant-derived antimicrobial agents. Full article

The first demonstration of wheat germ extract (WGE)-based in vitro translation synthesising a protein from exogenously introduced messenger ribonucleic acid (mRNA) was published approximately fifty years ago. Since then, there have been numerous crucial improvements to the WGE-based in vitro translation, resulting in a significant increase in yield and the development of high-throughput protein-producing platforms. These developments have transformed the original setup into a versatile eukaryotic protein production method with broad applications. The present review explores the theoretical background of the implemented modifications and brings a panel of examples for WGE applications in high-throughput protein studies and synthesis of challenging-to-produce proteins such as protein complexes, extracellular proteins, and membrane proteins. It also highlights the unique advantages of in vitro translation as an open system for synthesising radioactively labelled proteins, as illustrated by numerous publications using WGE to meet the protein demands of these studies. This review aims to orientate readers in finding the most appropriate WGE arrangement for their specific needs and demonstrate that a deeper understanding of the system modifications will help them make further adjustments to the reaction conditions for synthesising difficult-to-express proteins. Full article

Background: Vaccine development against Chlamydia, a prevalent sexually transmitted infection (STI), is imperative due to its global public health impact. However, significant challenges arise in the production of effective subunit vaccines based on recombinant protein antigens, particularly with membrane proteins like the Major Outer Membrane Protein (MOMP). Methods: Cell-free protein synthesis (CFPS) technology is an attractive approach to address these challenges as a method of high-throughput membrane protein and protein complex production coupled with nanolipoprotein particles (NLPs). NLPs provide a supporting scaffold while allowing easy adjuvant addition during formulation. Over the last decade, we have been working toward the production and characterization of MOMP-NLP complexes for vaccine testing. Results: The work presented here highlights the expression and biophysical analyses, including transmission electron microscopy (TEM) and dynamic light scattering (DLS), which confirm the formation and functionality of MOMP-NLP complexes for use in animal studies. Moreover, immunization studies in preclinical models compare the past and present protective efficacy of MOMP-NLP formulations, particularly when co-adjuvanted with CpG and FSL1. Conclusion: Ex vivo assessments further highlight the immunomodulatory effects of MOMP-NLP vaccinations, emphasizing their potential to elicit robust immune responses. However, further research is warranted to optimize vaccine formulations further, validate efficacy against Chlamydia trachomatis, and better understand the underlying mechanisms of immune response. Full article

Exosomes are small membrane vesicles in a cell culture. They are secreted by most cells and originate from the endosomal pathway. A variety of proteins, lipids, and genetic materials have been shown to be carried by exosomes. Once taken up by neighboring or distant cells, the bioactive compounds in exosomes can regulate the condition of recipient cells. Typically, producing exosomes in large quantities requires cell culture, resulting in high production costs. However, exosomes are abundant in milk and can be isolated on a large scale at a low cost. In our study, we found that milk exosomes can promote the synthesis and reconstruction of stratum corneum lipids, enhance skin barrier function, and provide greater protection for the skin. Furthermore, milk exosomes have anti-inflammatory properties that can reduce skin irritation, redness, and other symptoms, giving immediate relief. They also exhibit antioxidant activity, which helps neutralize free radicals and slows down the skin aging process. Additionally, milk exosomes inhibit melanin production, aiding in skin whitening. Ongoing research has uncovered the benefits of milk exosomes for skin improvement and their application in cosmetics, skin healthcare, and other fields, and these applications are continuing to expand. Full article

Brain lipid homeostasis is an absolute requirement for proper functionality of nerve cells and neurological performance. Current evidence demonstrates that lipid alterations are linked to neurodegenerative diseases, especially Alzheimer’s disease (AD). The complexity of the brain lipidome and its metabolic regulation has hampered the identification of critical processes associated with the onset and progression of AD. While most experimental studies have focused on the effects of known factors on the development of pathological hallmarks in AD, e.g., amyloid deposition, tau protein and neurofibrillary tangles, neuroinflammation, etc., studies addressing the causative effects of lipid alterations remain largely unexplored. In the present study, we have used a multifactor approach combining diets containing different amounts of polyunsaturated fatty acids (PUFAs), estrogen availabilities, and genetic backgrounds, i.e., wild type (WT) and APP/PS1 (FAD), to analyze the lipid phenotype of the frontal cortex in middle-aged female mice. First, we observed that severe n-3 PUFA deficiency impacts the brain n-3 long-chain PUFA (LCPUFA) composition, yet it was notably mitigated by hepatic de novo synthesis. n-6 LCPUFAs, ether-linked fatty acids, and saturates were also changed by the dietary condition, but the extent of changes was dependent on the genetic background and hormonal condition. Likewise, brain cortex phospholipids were mostly modified by the genotype (FAD>WT) with nuanced effects from dietary treatment. Cholesterol (but not sterol esters) was modified by the genotype (WT>FAD) and dietary condition (higher in DHA-free conditions, especially in WT mice). However, the effects of estrogen treatment were mostly observed in relation to phospholipid remodeling in a genotype-dependent manner. Analyses of lipid-derived variables indicate that nerve cell membrane biophysics were significantly affected by the three factors, with lower membrane microviscosity (higher fluidity) values obtained for FAD animals. In conclusion, our multifactor analyses revealed that the genotype, diet, and estrogen status modulate the lipid phenotype of the frontal cortex, both as independent factors and through their interactions. Altogether, the outcomes point to potential strategies based on dietary and hormonal interventions aimed at stabilizing the brain cortex lipid composition in Alzheimer’s disease neuropathology. Full article

The rising incidence of extensively drug-resistant (XDR) Klebsiella pneumoniae, including carbapenem- and colistin-resistant strains, leads to the limitation of available effective antibiotics. Miang, known as chewing tea, is produced from Camellia sinensis var. assamica or Assam tea leaves fermentation. Previous studies revealed that the extract of Miang contains various phenolic and flavonoid compounds with numerous biological activities including antibacterial activity. However, the antibacterial activity of Miang against XDR bacteria especially colistin-resistant strains had not been investigated. In this study, the compositions of phenolic and flavonoid compounds in fresh, steamed, and fermented Assam tea leaves were examined by HPLC, and their antibacterial activities were evaluated by the determination of the MIC and MBC. Pyrogallol was detected only in the extract from Miang and showed the highest activities with an MIC of 0.25 mg/mL and an MBC of 0.25–0.5 mg/mL against methicillin-susceptible Staphylococcus aureus, methicillin-resistant S. aureus, Escherichia coli ATCC 25922, colistin-resistant E. coli, and colistin-resistant K. pneumoniae. The effects on morphology and proteomic changes in K. pneumoniae NH54 treated with Miang extract were characterized by SEM and label-free quantitative shotgun proteomics analysis. The results revealed that Miang extract caused the decrease in bacterial cell wall integrity and cell lysis. The up- and downregulated expression with approximately a 2 to >5-fold change in proteins involved in peptidoglycan synthesis and outer membrane, carbohydrate, and amino acid metabolism were identified. These findings suggested that Miang containing pyrogallol and other secondary metabolites from fermentation has potential as an alternative candidate with an antibacterial agent or natural active pharmaceutical ingredient against XDR bacteria including colistin-resistant bacteria. Full article

The cell-free aqueous extract from the coelomic fluid of Holothuria tubulosa was prepared and examined for its glucose-lowering effect on HepG2 cells in vitro. In particular, employing a combination of cytochemical, flow cytometric, PCR, and protein blot techniques, we evaluated its role on glucose internalization and storage and on the upregulation and surface translocation of the two glucose transporters GLUT-2 and -4. The changes in expression, synthesis, and/or activation of the GLUT2-related transcription factor hepatocyte nuclear factor-1 alpha (HNF1α) and the GLUT-4-translocation regulatory factors insulin receptor substrate-1 (IRS-1) and AKT were also studied. Our results showed the improved glucose response by HepG2 cells, leading to an evident increase in glucose consumption/uptake and glycogen storage upon exposure. Moreover, the extract induced molecular reprogramming involving the upregulation of (i) IRS1 gene expression, (ii) the transcription and translation levels of HNF1α, AKT, and GLUT-4, (iii) the phosphorylation level of AKT, (iv) the synthesis of GLUT-2 protein, and (v) the translocation of GLUT-2 and -4 transporters onto the plasma membrane. Cumulatively, our results suggest that the coelomic fluid extract from H. tubulosa can be taken into consideration for the development of novel treatment agents against diabetes mellitus. Full article

Altered metabolism of lipids is a key factor in many diseases including cancer. Therefore, investigations into the impact of unsaturated and saturated fatty acids (FAs) on human body homeostasis are crucial for understanding the development of lifestyle diseases. In this paper, we focus on the impact of palmitic (PA), linoleic (LA), and eicosapentaenoic (EPA) acids on human colon normal (CCD-18 Co) and cancer (Caco-2) single cells using Raman imaging and spectroscopy. The label-free nature of Raman imaging allowed us to evaluate FAs dynamics without modifying endogenous cellular metabolism. Thanks to the ability of Raman imaging to visualize single-cell substructures, we have analyzed the changes in chemical composition of endoplasmic reticulum (ER), mitochondria, lipid droplets (LDs), and nucleus upon FA supplementation. Analysis of Raman band intensity ratios typical for lipids, proteins, and nucleic acids (I₁₆₅₆/I₁₄₄₄, I₁₄₄₄/I₁₂₅₆, I₁₄₄₄/I₇₅₀, I₁₃₀₄/I₁₂₅₆) proved that, using Raman mapping, we can observe the metabolic pathways of FAs in ER, which is responsible for the uptake of exogenous FAs, de novo synthesis, elongation, and desaturation of FAs, in mitochondria responsible for energy production via FA oxidation, in LDs specialized in cellular fat storage, and in the nucleus, where FAs are transported via fatty-acid-binding proteins, biomarkers of human colon cancerogenesis. Analysis for membranes showed that the uptake of FAs effectively changed the chemical composition of this organelle, and the strongest effect was noticed for LA. The spectroscopy studies have been completed using XTT tests, which showed that the addition of LA or EPA for Caco-2 cells decreases their viability with a stronger effect observed for LA and the opposite effect observed for PA. For normal cells, CCD-18 Co supplementation using LA or EPA stimulated cells for growing, while PA had the opposite impact. Full article

Thelephora ganbajun Zang, a rare wild macrofungus, has significant culinary and medicinal value. However, it also has a high cost attributed to its inability to achieve artificial cultivation and its strict environmental requirements. To reveal the intricacies of its development, we conducted a comprehensive analysis of the proteome and metabolome in three pivotal developmental stages: the mycelium, the primordium, and the fruiting body. In our investigation, genes exhibiting various expression levels across multi-omics analyses were identified as potential candidates implicated in growth, development, or metabolic regulation. The aim of this study was to provide a clearer direction for understanding the fundamental metabolic activities and growth stages of this species. Label-free proteomic sequencing revealed a critical juncture in ectomycorrhiza formation, particularly during the transition from the mycelium to the primordium. Secreted proteins, signaling proteins, membrane proteins, and proteins with unidentified functions were rapidly synthesized, with certain amino acids contributing to the synthesis of proteins involved in signaling pathways or hormone precursor substances. In the metabolomics analysis, the classification of secondary metabolites revealed a noteworthy increase in lipid substances and organic acids, contributing to cell activity. The early mycelial development stage exhibited vigorous cell metabolism, contrasting with a decline in cell division activity during fruiting body formation. In our findings, the integration of metabolomic and transcriptomic data highlighted the potential key role of folate biosynthesis in regulating early ectomycorrhiza development. Notably, the expression of alkaline phosphatase and dihydrofolate synthase genes within this pathway was significantly up-regulated in the mycelium and fruiting body stages but down-regulated in the primordium stage. This regulation primarily influences dihydrofolate reductase activity and B vitamin synthesis. Full article

Poly (β-amino ester) (PBAE) is an exceptional non-viral vector that is widely used in gene delivery, owing to its exceptional biocompatibility, easy synthesis, and cost-effectiveness. However, it carries a high surface positive charge that may cause cytotoxicity. Therefore, hydrophilic d-α-tocopherol polyethylene glycol succinate (TPGS) was copolymerised with PBAE to increase the biocompatibility and to decrease the potential cytotoxicity of the cationic polymer-DNA plasmid polyplex nanoparticles (NPs) formed through electrostatic forces between the polymer and DNA. TPGS-b-PBAE (TBP) copolymers with varying feeding molar ratios were synthesised to obtain products of different molecular weights. Their gene transfection efficiency was subsequently evaluated in HEK 293T cells using green fluorescent protein plasmid (GFP) as the model because free GFP is unable to easily pass through the cell membrane and then express as a protein. The particle size, ζ-potential, and morphology of the TBP2-GFP polyplex NPs were characterised, and plasmid incorporation was confirmed through gel retardation assays. The TBP2-GFP polyplex NPs effectively transfected multiple cells with low cytotoxicity, including HEK 293T, HeLa, Me180, SiHa, SCC-7 and C666-1 cells. We constructed a MUC2 (Mucin2)-targeting CRISPR/cas9 gene editing system in HEK 293T cells, with gene disruption supported by oligodeoxynucleotide (ODN) insertion in vitro. Additionally, we developed an LMP1 (latent membrane protein 1)-targeting CRISPR/cas9 gene editing system in LMP1-overexpressing SCC7 cells, which was designed to cleave fragments expressing the LMP1 protein (related to Epstein–Barr virus infection) and thus to inhibit the growth of the cells in vivo. As evidenced by in vitro and in vivo experiments, this system has great potential for gene therapy applications. Full article

Vaccinia virus (Orthopoxvirus) F17 protein is a major virion structural phosphoprotein having a molecular weight of 11 kDa. Recently, it was shown that F17 synthesised in infected cells interacts with mTOR subunits to evade cell immunity and stimulate late viral protein synthesis. Several years back, we purified an 11 kDa protein that inhibited protein synthesis in reticulocyte lysate from virions, and that possesses all physico-chemical properties of F17 protein. To investigate this discrepancy, we used defective vaccinia virus particles devoid of the F17 protein (designated iF17⁻ particles) to assess their ability to inhibit protein synthesis. To this aim, we purified iF17⁻ particles from cells infected with a vaccinia virus mutant which expresses F17 only in the presence of IPTG. The SDS-PAGE protein profiles of iF17⁻ particles or derived particles, obtained by solubilisation of the viral membrane, were similar to that of infectious iF17 particles. As expected, the profiles of full iF17⁻ particles and those lacking the viral membrane were missing the 11 kDa F17 band. The iF17⁻ particles did attach to cells and injected their viral DNA into the cytoplasm. Co-infection of the non-permissive BSC40 cells with a modified vaccinia Ankara (MVA) virus, expressing an mCherry protein, and iF17⁻ particles, induced a strong mCherry fluorescence. Altogether, these experiments confirmed that the iF17⁻ particles can inject their content into cells. We measured the rate of protein synthesis as a function of the multiplicity of infection (MOI), in the presence of puromycin as a label. We showed that iF17⁻ particles did not inhibit protein synthesis at high MOI, by contrast to the infectious iF17 mutant. Furthermore, the measured efficiency to inhibit protein synthesis by the iF17 mutant virus generated in the presence of IPTG, was threefold to eightfold lower than that of the wild-type WR virus. The iF17 mutant contained about threefold less F17 protein than wild-type WR. Altogether these results strongly suggest that virion-associated F17 protein is essential to mediate a stoichiometric inhibition of protein synthesis, in contrast to the late synthesised F17. It is possible that this discrepancy is due to different phosphorylation states of the free and virion-associated F17 protein. Full article

The aim of this study was to investigate NAD⁺/NADH redox regulation in astrocytes by Ginsenoside Rb1 subjected to oxygen–glucose deprivation/reoxygenation (OGD/R) and to reveal the neuroprotective mechanism of ginseng. Neonatal mouse brain was used to culture primary astrocytes. The third generation of the primary astrocytes was used for the experiments. OGD/R was introduced by culturing the cells in a glucose-free media under nitrogen for 6 h followed by a regular culture for 24 h. Ginsenoside Rb1 attenuated OGD/R-induced astrocyte injury in a dose-dependent manner. It improved the mitochondrial function of OGD/R astrocytes indicated by improving mitochondrial distribution, increasing mitochondrial membrane potential, and enhancing mitochondrial DNA copies and ATP production. Ginsenoside Rb1 significantly lifted intracellular NAD⁺/NADH, NADPH/NADP⁺, and GSH/GSSG in OGD/R astrocytes. It inhibited the protein expression of both PARP1 and CD38, while attenuating the SIRT1 drop in OGD/R cells. In line with its effects on PARP1, Ginsenoside Rb1 significantly reduced the expression of poly-ADP-ribosylation (PARylation) proteins in OGD/R cells. Ginsenoside Rb1 also significantly increased the expression of NAMPT and NMNAT2, both of which are key players in NAD/NADH synthesis. The results suggest that the regulation of NAD⁺/NADH redox involves the protective effects of ginsenoside Rb1 against OGD/R-induced astrocyte injury. Full article

Integral membrane proteins are important components of a cell. Their structural and functional studies require production of milligram amounts of proteins, which nowadays is not a routine process. Cell-free protein synthesis is a prospective approach to resolve this task. However, there are few known membrane mimetics that can be used to synthesize active membrane proteins in high amounts. Here, we present the application of commercially available “Facade” detergents for the production of active rhodopsin. We show that the yield of active protein in lipid bicelles containing Facade-EM, Facade-TEM, and Facade-EPC is several times higher than in the case of conventional bicelles with CHAPS and DHPC and is comparable to the yield in the presence of lipid-protein nanodiscs. Moreover, the effects of the lipid-to-detergent ratio, concentration of detergent in the feeding mixture, and lipid composition of the bicelles on the total, soluble, and active protein yields are discussed. We show that Facade-based bicelles represent a prospective membrane mimetic, available for the production of membrane proteins in a cell-free system. Full article

The emergence of antibiotic resistance poses a serious threat to humankind, emphasizing the need for alternative antimicrobial agents. This study focuses on investigating the antibacterial, antibiofilm, and anti-quorum-sensing (anti-QS) activities of saponin-derived silver nanoparticles (AgNPs-S) obtained from Ajwa dates (Phoenix dactylifera L.). The design and synthesis of these novel nanoparticles were explored in the context of developing alternative strategies to combat bacterial infections. The Ajwa date saponin extract was used as a reducing and stabilizing agent to synthesize AgNPs-S, which was characterized using various analytical techniques, including UV–Vis spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and transmission electron microscopy (TEM). The biosynthesized AgNPs-S exhibited potent antibacterial activity against both Gram-positive and Gram-negative bacteria due to their capability to disrupt bacterial cell membranes and the leakage of nucleic acid and protein contents. The AgNPs-S effectively inhibited biofilm formation and quorum-sensing (QS) activity by interfering with QS signaling molecules, which play a pivotal role in bacterial virulence and pathogenicity. Furthermore, the AgNPs-S demonstrated significant antioxidant activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals and cytotoxicity against small lung cancer cells (A549 cells). Overall, the findings of the present study provide valuable insights into the potential use of these nanoparticles as alternative therapeutic agents for the design and development of novel antibiotics. Further investigations are warranted to elucidate the possible mechanism involved and safety concerns when it is used in vivo, paving the way for future therapeutic applications in combating bacterial infections and overcoming antibiotic resistance. Full article

Impaired iron metabolism has been increasingly observed in many diseases, but a deeper, mechanistic understanding of the cellular impact of altered iron metabolism is still lacking. In addition, deficits in neuronal energy metabolism due to reduced glucose import were described for Alzheimer’s disease (AD) and its comorbidities like obesity, depression, cardiovascular disease, and type 2 diabetes mellitus. The aim of this review is to present the molecular link between both observations. Insufficient cellular glucose uptake triggers increased ferritin expression, leading to depletion of the cellular free iron pool and stabilization of the hypoxia-induced factor (HIF) 1α. This transcription factor induces the expression of the glucose transporters (Glut) 1 and 3 and shifts the cellular metabolism towards glycolysis. If this first line of defense is not adequate for sufficient glucose supply, further reduction of the intracellular iron pool affects the enzymes of the mitochondrial electron transport chain and activates the AMP-activated kinase (AMPK). This enzyme triggers the translocation of Glut4 to the plasma membrane as well as the autophagic recycling of cell components in order to mobilize energy resources. Moreover, AMPK activates the autophagic process of ferritinophagy, which provides free iron urgently needed as a cofactor for the synthesis of heme- and iron–sulfur proteins. Excessive activation of this pathway ends in ferroptosis, a special iron-dependent form of cell death, while hampered AMPK activation steadily reduces the iron pools, leading to hypoferremia with iron sequestration in the spleen and liver. Long-lasting iron depletion affects erythropoiesis and results in anemia of chronic disease, a common condition in patients with AD and its comorbidities. Instead of iron supplementation, drugs, diet, or phytochemicals that improve energy supply and cellular glucose uptake should be administered to counteract hypoferremia and anemia of chronic disease. Full article