Progression of liver fibrosis and associated factors among chronic hepatitis B patients at a general hospital in Northern Vietnam.

Evaluation of liver fibrosis is necessary to make the therapeutic decision and assess the prognosis of CHB patients. The current study aimed to describe the progression and identify some influencing factors in patients with chronic hepatitis B at a General Hospital in Northern Vietnam. The longitudinal study included 55 eligible subjects diagnosed Hepatitis-B-virus. Dependent variable was the aspartate aminotransferase/platelet ratio index and we collected some demographic variables and disease related and behaviour variables. Bayesian Model Averaging was used to select variables into model. Mixed-effect linear models were used to evaluate the change of the aspartate aminotransferase/platelet ratio index over time and identify related factors. the aspartate aminotransferase/platelet ratio index differences between examinations, age of participants, working status were statistically significant. This pattern indicated that the average the aspartate aminotransferase/platelet ratio index of the population decreased by 0.005 (95% CI=-0.009; -0.001) after each patient's visit, and increased by 0.013 if the patient's age increased by 1 year (95% CI=0.005; 0.0219). For non-working patients, the aspartate aminotransferase/platelet ratio index was lower, coefficient was -0.054 (95% CI=-0.108; 0.001). Other variables such as gender, education level, time for disease detection, drinking tea, alcohol consumption, forgetting to take medicine and the aspartate aminotransferase/platelet ratio index were not significantly different. The study showed that the majority of study subjects had average the aspartate aminotransferase/platelet ratio index, and were relatively well controlled and treated during the study. Age and working status are factors that influence the the aspartate aminotransferase/platelet ratio index.

Research and Development Prospects for Sugarcane Industry in Vietnam.

Sugarcane is one of the most important industrial crops in Vietnam and covers a total of 127,000 hectares of plantation area. In the season 2020-2021, Vietnam has produced 0.763 million tons of sugar (accounting for 0.34% total world sugar production). A current sugarcane production of 7.498 million tons is being used mainly for sugar production for direct consumption, ethanol production, bio-electricity and fertilization. To ensure crop sustainability, various policies and plans have been implemented. Crop breeding and zoning improvement programme significantly influence sugarcane production and sugar yield. Over 25 years since the programme "one million ton of sugar" was promoted, Vietnam currently possesses 25 sugar mills with a total capacity of 110,000 tons of sugarcane per day. Major problems of sugarcane industry as well as research and development have been discussed in this review. Recent research and development work focused on the added values of co-products to ensure sustainability of the sugarcane industry. Molasses will be used for ethanol production, and bagasse is used as the biomass for the alternative energy. Sugarcane and sugar would be the main feedstocks for those bio-economy growths in Vietnam. To keep the sustainable development of the sugar industry, and to meet the demand of the food and non-food requirements, it is necessary to upgrade the sugar value chain through the adoption and the development of co-products of the sugar industry.

Endophytic actinomycetes associated with Cinnamomum cassia Presl in Hoa Binh province, Vietnam: Distribution, antimicrobial activity and, genetic features.

Endophytic microbes associated with medicinal plants are considered to be potential producers of various bioactive secondary metabolites. The present study investigated the distribution, antimicrobial activity and genetic features of endophytic actinomycetes isolated from the medicinal plant Cinnamomum cassia Presl collected in Hoa Binh province of northern Vietnam. Based on phenotypic characteristics, 111 actinomycetes were isolated from roots, stems and leaves of the host plants by using nine selective media. The isolated actinomycetes were mainly recovered from stems (n = 67; 60.4%), followed by roots (n = 29; 26.1%) and leaves (n = 15; 13.5%). The isolates were accordingly assigned into 5 color categories of aerial mycelium, of which gray is the most dominant (n = 42; 37.8%), followed by white (n = 33; 29.7%), yellow (n = 25; 22,5%), red (n = 8; 7.2%) and green (n = 3; 2.7%). Of the total endophytic actinomycetes tested, 38 strains (occupying 34.2%) showed antimicrobial activity against at least one of nine tested microbes and, among them, 26 actinomycetes (68.4%) revealed anthracycline-like antibiotics production. Analysis of 16S rRNA gene sequences deposited on GenBank (NCBI) of the antibiotic-producing actinomycetes identified 3 distinct genera, including Streptomyces, Microbacterium, and Nocardia, among which Streptomyces genus was the most dominant and represented 25 different species. Further genetic investigation of the antibiotic-producing actinomycetes found that 28 (73.7%) and 11 (28.9%) strains possessed genes encoding polyketide synthase (pks) and nonribosomal peptide synthetase (nrps), respectively. The findings in the present study highlighted endophytic actinomycetes from C. cassia Presl which possessed broad-spectrum bioactivities with the potential for applications in the agricultural and pharmaceutical sectors.

Characterization of Endophytic Streptomyces griseorubens MPT42 and Assessment of Antimicrobial Synergistic Interactions of its Extract and Essential Oil from Host Plant Litsea cubeba.

The present study aimed to evaluate the synergistic effects of the crude ethyl acetate extract (CEAE) from endophytic actinomycete MPT42 and essential oil (EO) of the same host plant Litsea cubeba. The isolate MPT42, exhibiting broad-spectrum antimicrobial activities and harboring all three antibiotic-related biosynthetic genes pks-I, pks-II, and nrps, was identified as Streptomycete griseorubens based on an analysis of the morphology, physiology, and 16S rDNA sequence. Minimum inhibitory concentrations (MICs) and the fractional inhibitory concentration index were used to estimate the synergistic effects of various combined ratios between CEAE or antibiotics (erythromycin, vancomycin) and EO toward 13 microbial strains including pathogens. L. cubeba fruit EO, showing the main chemical constituents of 36.0% citral, 29.6% carveol, and 20.5% limonene, revealed an active-low against tested microbes (MICs ≥ 600 µg/mL). The CEAE of S. griseorubens culture exhibited moderate-strong antimicrobial activities against microbes (MICs = 80-600 µg/mL). Analysis of the mechanism of action of EO on Escherichia coli ATCC 25922 found that bacterial cells were dead after 7 h of the EO treatment at 1 MIC (5.5 mg/mL), where 62% cells were permeabilized after 2 h and 3% of them were filament (length ≥ 6 µm). Combinations of CEAE, erythromycin, or vancomycin with EO led to significant synergistic antimicrobial effects against microbes with 4-16 fold reduction in MIC values when compared to their single use. Interestingly, the vancomycin-EO combinations exhibited a strong synergistic effect against five Gram-negative bacterial species. This could assume that the synergy was possibly due to increasing the cell membrane permeability by the EO acting on the bacterial cells, which allows the uptake and diffusion of antimicrobial substances inside the cell easily. These findings in the present study therefore propose a possible alternative to combat the emergence of multidrug-resistant microbes in veterinary and clinics.

Prokaryotic soluble expression and purification of bioactive human fibroblast growth factor 21 using maltose-binding protein.

Human fibroblast growth factor 21 (hFGF21) has been characterized as an important regulator of glucose and lipid metabolism homeostasis. Here, to produce hFGF21 efficiently in Escherichia coli, the expression and solubility of hFGF21 were tested and optimised by fusing the protein with one of eight tags: hexahistidine (His6), thioredoxin (Trx), small ubiquitin-related modifier (Sumo), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilisation substance protein A (NusA), human protein disulphide isomerase (PDI), and the b'a' domain of PDI (PDIb'a'). Each tag increased solubility of the protein when the expression temperature was 18°C. Unlike many other tags that were tested, MBP significantly enhanced the solubility of the protein also in the culture condition at 37°C. Thus, the MBP-hFGF21 construct was further pursued for optimisation of affinity chromatography purification. After tag removal, 8.1 mg of pure hFGF21 was obtained as a final product from 500 mL of starting culture. The protein was then characterised by mass spectroscopy and an in vitro functional assay using NIH-3T3 cells transfected with a ß-klotho reporter gene. These characteristics are similar to those of commercial hFGF21. Thus, the MBP tag is useful for efficient prokaryotic production and purification of bioactive hFGF21.

Endophytic Actinobacteria Associated with Dracaena cochinchinensis Lour.: Isolation, Diversity, and Their Cytotoxic Activities.

Dracaena cochinchinensis Lour. is an ethnomedicinally important plant used in traditional Chinese medicine known as dragon's blood. Excessive utilization of the plant for extraction of dragon's blood had resulted in the destruction of the important niche. During a study to provide a sustainable way of utilizing the resources, the endophytic Actinobacteria associated with the plant were explored for potential utilization of their medicinal properties. Three hundred and four endophytic Actinobacteria belonging to the genera Streptomyces, Nocardiopsis, Brevibacterium, Microbacterium, Tsukamurella, Arthrobacter, Brachybacterium, Nocardia, Rhodococcus, Kocuria, Nocardioides, and Pseudonocardia were isolated from different tissues of D. cochinchinensis Lour. Of these, 17 strains having antimicrobial and anthracyclines-producing activities were further selected for screening of antifungal and cytotoxic activities against two human cancer cell lines, MCF-7 and Hep G2. Ten of these selected endophytic Actinobacteria showed antifungal activities against at least one of the fungal pathogens, of which three strains exhibited cytotoxic activities with IC50-values ranging between 3 and 33 µg·mL-1. Frequencies for the presence of biosynthetic genes, polyketide synthase- (PKS-) I, PKS-II, and nonribosomal peptide synthetase (NRPS) among these 17 selected bioactive Actinobacteria were 29.4%, 70.6%, and 23.5%, respectively. The results indicated that the medicinal plant D. cochinchinensis Lour. is a good niche of biologically important metabolites-producing Actinobacteria.

Correction: Prokaryotic soluble overexpression and purification of human VEGF165 by fusion to a maltose binding protein tag.

[This corrects the article DOI: 10.1371/journal.pone.0156296.].

Soluble Prokaryotic Expression and Purification of Human Interferon Alpha-2b Using a Maltose-Binding Protein Tag.

Human interferon alpha-2b (IFNα-2b) has therapeutic applications as an antiviral and antiproliferative drug and has been used for a wide range of indications. Efficient production of IFNα-2b in Escherichia coli has been difficult because the protein tends to form inclusion bodies. This obstacle has garnered interest in efficiently expressing IFNα-2b and overcoming its poor solubility. In this study, seven N-terminal fusion partners - hexahistidine (His6), thioredoxin, glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A, protein disulfide bond isomerase (PDI), and b'a' domain of PDI - were tested for soluble overexpression of codon-optimized IFNα-2b in E. coli. Low temperature increased the expression level of all of the tagged proteins except for the GST fusion. All the tags, except for His6 and GST, improved solubility. We purified IFNα-2b from the MBP-tagged fusion using immobilized metal affinity chromatography and anion exchange chromatography, and obtained a final yield of 7.2 mg from an initial 500-ml culture. The endotoxin level was 0.46 EU/µg. Biological activity was demonstrated using a luciferase assay, which showed a dose-dependent response with a calculated EC50 of 10.3 ± 5.9 pM. Our results demonstrate that using an MBP-tagged fusion is an efficient way to produce pure IFNα-2b.

Prokaryotic Soluble Overexpression and Purification of Human VEGF165 by Fusion to a Maltose Binding Protein Tag.

Human vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and plays a central role in the process of tumor growth and metastatic dissemination. Escherichia coli is one of the most common expression systems used for the production of recombinant proteins; however, expression of human VEGF in E. coli has proven difficult because the E. coli-expressed VEGF tends to be misfolded and forms inclusion bodies, resulting in poor solubility. In this study, we successfully produced semi-preparative amounts of soluble bioactive human VEGF165 (hVEGF). We created seven N-terminal fusion tag constructs with hexahistidine (His6), thioredoxin (Trx), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A (NusA), human protein disulfide isomerase (PDI), and the b'a' domain of PDI (PDIb'a'), and tested each construct for soluble overexpression in E. coli. We found that at 18°C, 92.8% of the MBP-tagged hVEGF to be soluble and that this tag significantly increased the protein's solubility. We successfully purified 0.8 mg of pure hVEGF per 500 mL cell culture. The purified hVEGF is stable after tag cleavage, contains very low levels of endotoxin, and is 97.6% pure. Using an Flk1+ mesodermal precursor cell (MPC) differentiation assay, we show that the purified hVEGF is not only bioactive but has similar bioactivity to hVEGF produced in mammalian cells. Previous reports on producing hVEGF in E. coli have all been based on refolding of the protein from inclusion bodies. To our knowledge, this is the first report on successfully expressing and purifying soluble hVEGF in E. coli.

Characterization and evaluation of antimicrobial and cytotoxic effects of Streptomyces sp. HUST012 isolated from medicinal plant Dracaena cochinchinensis Lour.

A highly potent secondary metabolite producing endophytic strain, Streptomyces sp. HUST012 was isolated from the stems of the medicinal plant Dracaena cochinchinensis Lour. Strain HUST012 showed antimicrobial and antitumor activities which were significantly much higher than those of dragon's blood extracted from D. cochinchinensis Lour. On further analysis, the strain was found to produce two metabolites, SPE-B11.8 (elucidated to be a novel metabolite (Z)-tridec-7-ene-1,2,13-tricarboxylic acid) and SPE-B5.4 (elucidated as Actinomycin-D). The Minimum Inhibitory Concentration values of SPE-B11.8 against a set of test bacterial organisms (Methicillin-resistant Staphylococcus epidermis ATCC 35984, Methicillin-resistant Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Klebsiella pneumoniae ATCC 13883) ranged between 15.63 and 62.5 µg/ml while that for SPE-B5.4 ranged between 0.04 and 2.24 µg/ml. The compound SPE-B11.8 showed cytotoxic effect at 41.63 and 29.54 µg/ml IC 50-values against Hep G2 and MCF-7, respectively, while the compound SPE-B5.4 exhibited stronger activities against them at 0.23 and 0.18 µg/ml IC 50-values.

Soluble overexpression and purification of bioactive human CCL2 in E. coli by maltose-binding protein.

Human chemokine (C-C motif) ligand 2 (hCCL2) is a small cytokine in the CC chemokine family that attracts monocytes, memory T lymphocytes, and natural killer cells to the site of tissue injury- or infection-induced inflammation. hCCL2 has been implicated in the pathogeneses of diseases characterized by monocytic infiltrates, including psoriasis, rheumatoid arthritis, atherosclerosis, multiple sclerosis, and insulin-resistant diabetes. The prokaryotic overexpression of hCCL2 has been investigated previously in an attempt to develop biomedical applications for this factor, but this has been hampered by protein misfolding and aggregation into inclusion bodies. In our present study, we screened 7 protein tags-Trx, GST, MBP, NusA, His8, PDI, and PDIb'a'-for their ability to allow the soluble overexpression of hCCL2. Three tags-MBP, His8, and PDI-solubilized more than half of the expressed hCCL2 fusion proteins. Lowering the expression temperature to 18 °C significantly further improved the solubility of all fusion proteins. MBP was chosen for further study based on its solubility, expression level, ease of purification, and tag size. MBP-CCL2 was purified using conventional chromatography and cleaved using TEV or Factor Xa proteases. Biological activity was assessed using luciferase and cell migration assays. Factor Xa-cleaved hCCL2 was found to be active and TEV-cleaved hCCL2 showed relatively less activity. This is probably because the additional glycine residues present at the N-terminus of hCCL2 following TEV digestion interfere with the binding of hCCL2 to its receptor.

Soluble prokaryotic expression and purification of crotamine using an N-terminal maltose-binding protein tag.

Crotamine is a peptide toxin found in the venom of the rattlesnake Crotalus durissus terrificus. Interestingly, crotamine demonstrates promising anticancer, antimicrobial, and antifungal activities. The crotamine peptide can also deliver plasmids into rapidly dividing cells, such as cancer and stem cells, and demonstrates potent analgesic effects. Efficiently producing crotamine in mammalian cells is difficult because it is both cell-permeable and cytotoxic. Prokaryotic expression of this peptide is also difficult to maintain because it does not fold properly in the cytoplasm, resulting in aggregation and in the formation of inclusion bodies. In our current study, we show for the first time that N-terminal fusion with three protein tags-N-utilization substance protein A (NusA), protein disulfide isomerase b'a' domain (PDIb'a'), and maltose-binding protein (MBP)-enables the soluble overexpression of crotamine in the cytoplasm of Escherichia coli. MBP-tagged crotamine was purified using Ni affinity, anion exchange, and MBP chromatography. The tag was cleaved using TEV protease, and the final product was pure on a silver-stained gels. In total, 0.9 mg pure crotamine was obtained from each liter of bacterial culture with endotoxin level approximately 0.15 EU/µg, which is low enough to use in biomedical applications. The identity and intramolecular disulfide bonds were confirmed using MALDI-TOF MS analysis. Purified crotamine inhibited the hKv1.3 channel (but not hKv1.5) in a dose-dependent manner with IC50 value of 67.2 ± 44.7 nM (n = 10), indicating the correct protein folding. The crotamine product fused with MBP at its N-terminus also inhibited the hKv1.3 channel, suggesting that the N-terminus is not involved in the channel binding of the toxin.

Prokaryotic soluble overexpression and purification of bioactive human growth hormone by fusion to thioredoxin, maltose binding protein, and protein disulfide isomerase.

Human growth hormone (hGH) is synthesized by somatotroph cells of the anterior pituitary gland and induces cell proliferation and growth. This protein has been approved for the treatment of various conditions, including hGH deficiency, chronic renal failure, and Turner syndrome. Efficient production of hGH in Escherichia coli (E. coli) has proven difficult because the E. coli-expressed hormone tends to aggregate and form inclusion bodies, resulting in poor solubility. In this study, seven N-terminal fusion partners, hexahistidine (His6), thioredoxin (Trx), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A (NusA), protein disulfide bond isomerase (PDI), and the b'a' domain of PDI (PDIb'a'), were tested for soluble overexpression of codon-optimized hGH in E. coli. We found that MBP and hPDI tags significantly increased the solubility of the hormone. In addition, lowering the expression temperature to 18°C also dramatically increased the solubility of all the fusion proteins. We purified hGH from MBP-, PDIb'a'-, or Trx-tagged hGH expressed at 18°C in E. coli using simple chromatographic techniques and compared the final purity, yield, and activity of hGH to assess the impact of each partner protein. Purified hGH was highly pure on silver-stained gel and contained very low levels of endotoxin. On average, ∼37 mg, ∼12 mg, and ∼7 mg of hGH were obtained from 500 mL-cell cultures of Trx-hGH, MBP-hGH, and PDIb'a'-hGH, respectively. Subsequently, hGH was analyzed using mass spectroscopy to confirm the presence of two intra-molecular disulfide bonds. The bioactivity of purified hGHs was demonstrated using Nb2-11 cell.

Soluble expression of human leukemia inhibitory factor with protein disulfide isomerase in Escherichia coli and its simple purification.

Human leukemia inhibitory factor (hLIF) is a multifunctional cytokine that is essential for maintaining the pluripotency of embryonic stem cells. hLIF may be also be useful in aiding fertility through its effects on increasing the implantation rate of fertilized eggs. Thus these applications in biomedical research and clinical medicine create a high demand for bioactive hLIF. However, production of active hLIF is problematic since eukaryotic cells demonstrate limited expression and prokaryotic cells produce insoluble protein. Here, we have adopted a hybrid protein disulfide isomerase design to increase the solubility of hLIF in Escherichia coli. Low temperature expression of hLIF fused to the b'a' domain of protein disulfide isomerase (PDIb'a') increased the soluble expression in comparison to controls. A simple purification protocol for bioactive hLIF was established that includes removal of the PDIb'a' domain by cleavage by TEV protease. The resulting hLIF, which contains one extra glycine residue at the N-terminus, was highly pure and demonstrated endotoxin levels below 0.05 EU/µg. The presence of an intramolecular disulfide bond was identified using mass spectroscopy. This purified hLIF effectively maintained the pluripotency of a murine embryonic stem cell line. Thus we have developed an effective method to produce a pure bioactive version of hLIF in E. coli for use in biomedical research.

Expression and purification of biologically active human FGF2 containing the b'a' domains of human PDI in Escherichia coli.

Among the members of the fibroblast growth factor (FGF) family that affect the growth, differentiation, migration, and survival of many cell types, FGF2 is the most abundant in the central nervous system. Because of its wound healing effects, FGF2 has potential as a therapeutic agent. The protein is also added to the culture media to maintain stem cells. Expression and purification procedures for FGF2 that are highly efficient and low cost have been intensively investigated for the past two decades. Our current study focuses on the purification of FGF2 fused with b'a' domains of human protein disulfide isomerase to elevate overexpression, solubility, and stability with a simplified experimental procedure using only ion exchange chromatography, as well as on the confirmation of the biological activity of FGF2 on fibroblast Balb/c 3T3 cells and hippocampal neural cells.

Cu/Zn incorporation during purification of soluble human EC-SOD from E. coli stabilizes proper disulfide bond formation.

Extracellular superoxide dismutase (EC-SOD) is the only enzyme that removes superoxide radical in the extracellular space. The reduction of EC-SOD is linked to many diseases, suggesting that the protein may have therapeutic value. EC-SOD is reported to be insoluble and to make inclusion bodies when overexpressed in the cytoplasm of Escherichia coli. The refolding process has the advantage of high yield, but has the disadvantage of frequent aggregation or misfolding during purification. For the first time, this study shows that fusion with maltose-binding protein (MBP), N-utilization substance protein A, and protein disulfide isomerase enabled the soluble overexpression of EC-SOD in the cytoplasm of E. coli. MBP-tagged human EC-SOD (hEC-SOD) was purified by MBP affinity and anion exchange chromatography, and its identity was confirmed by MALDI-TOF MS analysis. The purified protein showed good enzyme activity in vitro; however, there was a difference in metal binding. When copper and zinc were incorporated into hEC-SOD before MBP tag cleavage, the enzymatic activity was higher than when the metal ions were bound to the purified protein after MBP tag cleavage. Therefore, the enzymatic activity of hEC-SOD is associated with metal incorporation and protein folding via disulfide bond.