cell free protein synthesis

A series of novel chromone derivatives of (N-(4-oxo-2-(trifluoromethyl)-4H-chromen-6-yl) benzamides) were synthesized by treating 7-amino-2-(trifluoromethyl)-4H-chromen-4-one with K2 CO3  and/or NaH, suitable alkyl halides and acetonitrile and/or 1,4-dioxane. The obtained products are in high yields (87 to 96%) with various substituents in short reaction times with no more by-products and confirmed by FT-IR, 1 H, and 13 C-NMR Spectral data. The in vitro cytotoxic activity was examined against two human cancer cell lines, namely the human lung adenocarcinoma (A-549) and the human breast (MCF-7) cancer cell line...

The presence of arsenic (As) and fluoride (F- ) in drinking water is of concern due to the enormous number of individuals exposed to this condition worldwide. Studies in cultured cells and animal models have shown that As- or F-induced hepatotoxicity is primarily associated with redox disturbance and altered mitochondrial homeostasis. To explore the hepatotoxic effects of chronic combined exposure to As and F- in drinking water, pregnant CD-1 mice were exposed to 2 mg/L As (sodium arsenite) and/or 25 mg/L F- (sodium fluoride)...

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...

Ethyl (S)-4-chloro-3-hydroxybutyrate ((S)-CHBE) is an important chiral intermediate in the synthesis of the cholesterol-lowering drug atorvastatin. Studying the use of SpyTag/SpyCatcher and SnoopTag/SnoopCatcher systems for the asymmetric reduction reaction and directed coupling coenzyme regeneration is practical for efficiently synthesizing (S)-CHBE. In this study, Spy and Snoop systems were used to construct a double-enzyme directed fixation system of carbonyl reductase (BsCR) and glucose dehydrogenase (BsGDH) for converting 4-chloroacetoacetate (COBE) to (S)-CHBE and achieving coenzyme regeneration...

Ribosome-inactivating proteins (RIPs) are a group of proteins with rRNA N-glycosylase activity that irreversibly inhibit protein synthesis and consequently cause cell death. Recently, an RIP called ledodin has been found in shiitake; it is cytotoxic, strongly inhibits protein synthesis, and shows rRNA N-glycosylase activity. In this work, we isolated and characterized a 50 kDa cytotoxic protein from shiitake that we named edodin. Edodin inhibits protein synthesis in a mammalian cell-free system, but not in insect-, yeast-, and bacteria-derived systems...

Mono-2-ethylhexyl phthalic acid (MEHP) is the most toxic metabolite of the plasticizer di-2-ethylhexyl phthalic acid (DEHP), and studies have shown that MEHP causes serious reproductive effects. However, its exact mechanisms of action remain elusive. In this study, we aimed to investigate the reproductive effects of MEHP and preliminarily explore its underlying molecular mechanisms. We found that TM3 cells gradually secreted less testosterone and intracellular free cholesterol with increasing MEHP exposure...

Sarcopenia is characterized by loss of muscle strength and muscle mass with aging. The growing number of sarcopenia patients as a result of the aging population has no viable treatment. Exercise maintains muscle strength and mass by increasing peroxisome growth factor activating receptor γ-conjugating factor-1α (PGC-1α) and Akt signaling in skeletal muscle. The present study focused on the carbon monoxide (CO), endogenous activator of PGC-1α and Akt, and investigated the therapeutic potential of CO-loaded red blood cells (CO-RBCs), which is bioinspired from in vivo CO delivery system, as an exercise mimetic for the treatment of sarcopenia...

Bioorthogonal chemistry has gained widespread use in the study of many biological systems of interest, including protein prenylation. Prenylation is a post-translational modification, in which one or two 15- or 20-carbon isoprenoid chains are transferred onto cysteine residues near the C-terminus of a target protein. The three main enzymes─protein farnesyltransferase (FTase), geranylgeranyl transferase I (GGTase I), and geranylgeranyl transferase II (GGTase II)─that catalyze this process have been shown to tolerate numerous structural modifications in the isoprenoid substrate...

The ongoing evolution of SARS-CoV-2 to evade vaccines and therapeutics underlines the need for innovative therapies with high genetic barriers to resistance. Therefore, there is pronounced interest in identifying new pharmacological targets in the SARS-CoV-2 viral life cycle. The small molecule PAV-104, identified through a cell-free protein synthesis and assembly screen, was recently shown to target host protein assembly machinery in a manner specific to viral assembly. In this study, we investigate the capacity of PAV-104 to inhibit SARS-CoV-2 replication in human airway epithelial cells (AECs)...

Cell-free protein synthesis (CFPS) system is an ideal platform for fast and convenient protein research and has been used for macromolecular assembly, unnatural amino acid embedding, glycoprotein production, and more. To realize the construction of an efficient eukaryotic CFPS platform with the advantages of low cost and short time, a CFPS system based on the yeast Pichia pastoris was built in this study. The internal ribosomal entry site (IRES) can independently initiate translation and thus promote protein synthesis...

G-protein coupled receptor-120 (GPR120; FFAR4) is a free fatty acid receptor, widely researched for its glucoregulatory and insulin release activities. This study aimed to investigate the metabolic advantage of FFAR4/GPR120activation using combination therapy. C57BL/6 mice, fed a High Fat Diet (HFD) for 120 days to induce obesity-diabetes, were subsequently treated with a single daily oral dose of FFAR4/GPR120 agonist Compound A (CpdA) (0.1μmol/kg) alone or in combination with sitagliptin (50mg/kg) for 21 days...

Hepatocellular carcinoma (HCC) is the predominant form of liver cancer, characterized by high morbidity and mortality rates, as well as unfavorable treatment outcomes. Tripartite motif-containing protein 47 (TRIM47) has been implicated in various diseases including tumor progression with the activity of E3 ubiquitin ligase. However, the precise regulatory mechanisms underlying the involvement of TRIM47 in HCC remain largely unexplored. Here, we provide evidence that TRIM47 exhibits heightened expression in tumor tissues, and its expression is in intimate association with clinical staging and patient prognosis...

In vitro translation is an important method for studying fundamental aspects of co- and post-translational gene regulation, as well as for protein expression in the laboratory and on an industrial scale. Here, by re-examining and improving a human in vitro translation system (HITS), we were able to develop a minimal system where only four components are needed to supplement human cell lysates. Functional characterization of our improved HITS revealed the synergistic effect of mRNA capping and polyadenylation...

Chinese hamster ovary (CHO) cells are crucial in biopharmaceutical production due to their scalability and capacity for human-like post-translational modifications. However, toxic proteins and membrane proteins are often difficult-to-express in living cells. Alternatively, cell-free protein synthesis can be employed. This study explores innovative strategies for enhancing the production of challenging proteins through the modification of CHO cells by investigating both, cell-based and cell-free approaches. A major result in our study involves the integration of a mutant eIF2 translation initiation factor and T7 RNA polymerase into CHO cell lysates for cell-free protein synthesis...

Iron is essential for all the lives and mitochondria integrate iron into heme and Fe-S clusters for diverse use as cofactors. Here we screened mitochondrial proteins in KU812 human chronic myelogenous leukemia cells by glutathione S- transferase pulldown assay with PCBP2 to identify mitochondrial receptors for PCBP2, a major cytosolic Fe(II) chaperone. LC-MS analyses identified TOM20, sideroflexin-3 (SFXN3), SFXN1 and TOM70 in the affinity-score sequence. Stimulated emission depletion microscopy and proteinase-K digestion of mitochondria in HeLa cells revealed that TOM20 is located in the outer membrane of mitochondria whereas SFXN3 is located in the inner membrane...

Metabolic reprogramming has been defined as a hallmark of malignancies. Prior studies have focused on the single nucleotide polymorphism (SNP) of POLG2 gene, which is reportedly responsible for encoding mitochondrial DNA genes and is implicated in the material and energy metabolism of tumor cells, whereas its function in prostate cancer has been elusive. Gene expression profile matrix and clinical information were downloaded from TCGA (The Cancer Genome Atlas) data portal, and GSE3325 and GSE8511 were retrieved from GEO (Gene Expression Omnibus) database...

The DNA damage inducible SOS response in bacteria serves to increase survival of the species at the cost of mutagenesis. The SOS response first initiates error-free repair followed by error-prone repair. Here, we have employed a multi-omics approach to elucidate the temporal coordination of the SOS response. Escherichia coli was grown in batch cultivation in bioreactors to ensure highly controlled conditions, and a low dose of the antibiotic ciprofloxacin was used to activate the SOS response while avoiding extensive cell death...

Obesity is associated with insulin resistance, hypertension, and coronary artery diseases which are grouped as metabolic syndrome. Rather than being a storage for energy, the adipocytes could synthesis and secret diverse hormones and molecules, named as adipokines. Under obese status, the adipocytes are dysfunctional with excessively producing the inflammatory related cytokines, such as interleukin 1 (IL-1), IL-6, and tumor necrosis factor α (TNF-α). Concerning on the vital role of adipokines, it is proposed that one of the critical pathological factors of obesity is the dysfunctional adipocytic pathways...

Nitric oxide is produced at low micromolar levels following the induction of inducible nitric oxide synthase (iNOS) and is responsible for mediating the inhibitory actions of cytokines on glucose-stimulated insulin secretion by islets of Langerhans. It is through the inhibition of mitochondrial oxidative metabolism, specifically aconitase and complex 4 of the electron transport chain, that nitric oxide inhibits insulin secretion. Nitric oxide also attenuates protein synthesis, induces DNA damage, activates DNA repair pathways, and stimulates stress responses (unfolded protein and heat shock) in β-cells...

Reconstruction and optimization of biosynthetic pathways can help to overproduce target chemicals in microbial cell factories based on genetic engineering. However, the perturbation of biosynthetic pathways on cellular metabolism is not well investigated and profiling the engineered microbes remains challenging. The rapid development of omics tools has the potential to characterize the engineered microbial cell factory. Here, we performed label-free quantitative proteomic analysis and metabolomic analysis of engineered sabinene overproducing Saccharomyces cerevisiae strains...