Identification of Potential Mechanisms of Rk1 Combination with Rg5 in the Treatment of Type II Diabetes Mellitus by Integrating Network Pharmacology and Experimental Validation.

In this study, we aimed to explore the potential targets and functional mechanisms of Rk1 combined with Rg5 (Rk1+Rg5) against type II diabetes mellitus (T2DM). Network pharmacology and molecular docking were used to predict and verify the targets and signaling pathways of Rk1+Rg5 against T2DM. The results were further confirmed by a db/db mouse model and a model using PA-induced L6 cells. According to network pharmacology, a total of 250 core targets of Rk1+Rg5 towards T2DM were identified; the insulin resistance signaling pathways were enriched by KEGG. Results of molecular docking indicated good binding affinity of Rk1 and Rg5 to Akt1. In vivo and in vitro studies further showed that Rk1+Rg5 is an inhibitor of skeletal muscle insulin resistance. The results showed that Rk1+Rg5 significantly improved the hyperglycemic state of db/db mice, alleviated dyslipidemia, and promoted skeletal muscle glucose uptake. This phenomenon was closely related to the alleviation of the insulin resistance in skeletal muscles. Finally, the combination activated the Akt signaling pathway and promoted GLUT4 translocation to the cell membrane for glucose uptake. Altogether, our findings, for the first time, demonstrate that the combination of Rk1 and Rg5 could be beneficial for anti-T2DM, possibly involving ameliorated insulin resistance.

Diversity and Antimicrobial Activity of Endophytic Fungi Isolated from Chloranthus japonicus Sieb in Qinling Mountains, China.

The plant Chloranthus japonicus Sieb is known for its anticancer properties and mainly distributed in China, Japan, and Korea. In this study, we firstly investigated the diversity and antimicrobial activity of the culturable endophytic fungi from C. japonicus. A total of 332 fungal colonies were successfully isolated from 555 tissue segments of the medicinal plant C. japonicus collected from Qinling Mountains, China. One hundred and thirty representative morphotype strains were identified according to ITS rDNA sequence analyses and were grouped into three phyla (Ascomycota, Basidiomycota, Mucoromycota), five classes (Dothideomycetes, Sordariomycetes, Eurotiomycetes, Agaricomycetes, Mucoromycetes), and at least 30 genera. Colletotrichum (RA, 60.54%) was the most abundant genus, followed by Aspergillus (RA, 11.75%) and Diaporthe (RA, 9.34%). The Species Richness Index (S, 56) and the Shannon-Wiener Index (H', 2.7076) indicated that C. japonicus harbored abundant fungal resources. Thirteen out of 130 endophytic fungal ethyl acetate extracts exhibited inhibitory activities against at least one pathogenic bacterium or fungus. Among of these, F8158, which was identified as Trichoderma cf. harzianum, exhibited good antagonistic capacities (the percent inhibition of mycelial growth ranged from 47.72~88.18) for different pathogens and has a potential application in biological control. In addition, it is noteworthy that the strain F8157 (Thanatephorus cucumeris, an opportunistic pathogen) showed antibacterial and antifungal activity, which is reported firstly in this study, and should be investigated further. Taken together, these results indicated that the endophytic fungi from C. japonicus may be of potential interest in screening bio-control agents and discovering of new bioactive compounds.

New strategy for expression of recombinant hydroxylated human collagen α1(III) chains in Pichia pastoris GS115.

Type III collagen is one of the most abundant proteins in the human body, which forms collagen fibrils and provides the stiff, resilient characteristics of many tissues. In this paper, a new method for secretory expression of recombinant hydroxylated human collagen α1(III) chain in Pichia pastoris GS115 was applied. The gene encoding for full-length human collagen α1(III) chain (COL3A1) without N-terminal propeptide and C-terminal propeptide was cloned in the pPIC9K expression vector. The prolyl 4-hydroxylase (P4H, EC 1.14.11.2) α-subunit (P4Hα) and ß-subunit (P4Hß) genes were cloned in the same expression vector, pPICZB. Fluorogenic quantitative PCR indicates that COL3A1 and P4H genes have been expressed in mRNA level. SDS-PAGE shows that secretory expression of recombinant human collagen α1(III) chain was successfully achieved in P. pastoris GS115. In addition, the result of amino acids composition analysis shows that the recombinant human collagen α1(III) chain contains hydroxyproline by coexpression with the P4H. Furthermore, liquid chromatography coupled with tandem mass spectrometry analysis demonstrates that proline residues of the recombinant human collagen α1(III) chain were hydroxylated in the X or Y positions of Gly-X-Y triplets.

Enhancing human-like collagen accumulation by deleting the major glucose transporter ptsG in recombinant Escherichia coli BL21.

Collagen has been proven to be a valuable biomedical material for many medical applications. Human-like collagen (HLC) is a novel important biomedical material with diverse medical applications. In this work, recombinant Escherichia coli BL21 3.7 ∆ptsG was constructed, the characters of ptsG mutant strain were analyzed, and real-time quantitative polymerase chain reaction (PCR) was applied to investigate the effect of ptsG gene deletion on the transcriptional level of the phosphotransferase system (PTS) genes responsible for glucose transport. The HLC production and cell growth ability were 1.33- and 1.24-fold higher than those of its parent strain in the fermentation medium, respectively, and 1.16- and 1.17-fold in the modified minimal medium individually. The acetate accumulation decreased by 42%-56% compared to its parent strain in the fermentation medium, and 70%-87% in the modified minimal medium. The results of RT-qPCR showed that the transcriptional level of crr, ptsH, ptsI, and blgF in ptsG mutant all decreased dramatically, which inferred a decrease in the glucose uptake rate, but the transcriptional level of FruB and manX increased slightly, which demonstrated the activation of fructose- and mannose-specific transport pathways in the ptsG mutant. This study demonstrates that ptsG deletion is an effective strategy to reduce acetate accumulation and increase biomass and HLC production.

Protective Effect of Ginsenoside CK against Autoimmune Hepatitis Induced by Concanavalin A.

Ginsenoside CK, a kind of rare ginsenoside transformed from protopanaxadiol saponins extracted from the genus Panax, has been proven to possess favorable bioactivities such as anti-inflammatory, anti-cancer, anti-diabetes, and hepatoprotective effects. The current study is targeted to determine the effect of ginsenoside CK on hepatitis induced by concanavalin A (Con A). Mice were treated with different dosages of ginsenoside CK for 7 days, and Con A (15 mg/kg) was intravenously injected to induce autoimmune hepatitis (AIH) after the last administration. The results demonstrated that pretreatment with ginsenoside CK (40 mg/kg) could obviously ameliorate the increase in serum indicators related to liver function such as AST, ALT, and ALP, and hepatic lesions induced by Con A. Meanwhile, ginsenoside CK suppressed hepatocyte apoptosis, which was observed in pathological data, and immunoblotting results showed that the expression of Bax, Bcl-2, and other proteins was regulated by CK. Furthermore, the release of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and IL-6 in mice with AIH were lowered by the administration of 40 mg/kg of ginsenoside CK. Furthermore, ginsenoside CK elevated the gene expression of Nrf2 and Sirt1 and augmented downstream target genes such as HO-1. In addition, a significant inhibition effect of the TLR4/NF-κB signal was observed in 40 mg/kg CK-pretreated mice compared with the model group. To sum up, the results indicated that ginsenoside CK has a notable hepatoprotective effect against AIH by activating Sirt1/Nrf2 and suppressing the TLR4/NF-κB signaling pathway.

Burkholderia ambifaria XN08: A plant growth-promoting endophytic bacterium with biocontrol potential against sharp eyespot in wheat.

Plant growth-promoting bacteria (PGPB) have been considered promising biological agents to increase crop yields for years. However, the successful application of PGPB for biocontrol of sharp eyespot in wheat has been limited, partly by the lack of knowledge of the ecological/environmental factors affecting the colonization, prevalence, and activity of beneficial bacteria on the crop. In this study, an endophytic bacterium XN08 with antagonistic activity against Rhizoctonia cerealis (wheat sharp eyespot pathogenic fungus), isolated from healthy wheat plants, was identified as Burkholderia ambifaria according to the sequence analysis of 16S rRNA. The antibiotic synthesis gene amplification and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) analyses were used to characterize the secondary metabolites. The results showed that the known powerful antifungal compound named pyrrolnitrin was produced by the strain XN08. In addition, B. ambifaria XN08 also showed the capacity for phosphate solubilization, indole-3-acetic acid (IAA), protease, and siderophore production in vitro. In the pot experiments, a derivate strain carrying the green fluorescent protein (GFP) gene was used to observe its colonization in wheat plants. The results showed that GFP-tagged B. ambifaria could colonize wheat tissues effectively. This significant colonization was accompanied by an enhancement of wheat plants' growth and an induction of immune resistance for wheat seedlings, which was revealed by the higher activities of polyphenol oxidase (PPO), peroxidase (POD), and phenylalanine ammonia-lyase (PAL). As far as we know, this is the first report describing the colonization traits of B. ambifaria in wheat plants. In addition, our results indicated that B. ambifaria XN08 might serve as a new effective biocontrol agent against wheat sharp eyespot disease caused by R. cerealis.

Diversity and Ginsenoside Biotransformation Potential of Cultivable Endophytic Fungi Associated With Panax bipinnatifidus var. bipinnatifidus in Qinling Mountains, China.

To obtain novel fungi with potent ß-glucosidase for minor ginsenoside production, Panax bipinnatifidus var. bipinnatifidus, which is a traditional medicinal plant containing various ginsenosides, was first employed to isolate endophytic fungi in this study. A total of 93 representative morphotype strains were isolated and identified according to ITS rDNA sequence analyses, and they were grouped into three phyla (Ascomycota, Basidiomycota, and Mucoromycota), five classes (Dothideomycetes, Sordariomycetes, Eurotiomycetes, Agaricomycetes, and Mucoromycetes), and 24 genera. Plectosphaerella (RA, 19.35%) was the most abundant genus, followed by Paraphoma (RA, 11.83%) and Fusarium (RA, 9.70%). The species richness index (S, 34) and the Shannon-Wiener index (H', 3.004) indicated that P. bipinnatifidus harbored abundant fungal resources. A total of 26 endophytic fungal ethyl acetate extracts exhibited inhibitory activities against at least one pathogenic bacterium or fungus. In total, 11 strains showed strong ß-glucosidase activities and also presented with the ability of ginsenoside biotransformation with varied glycoside-hydrolyzing pathways. Excitingly, three genera, namely, Ilyonectria, Sarocladium, and Lecanicillium, and all 11 taxa were first found to have the ability to transform ginsenosides in our study. The results indicated that P. bipinnatifidus could be a new fungi resource with potential novel natural compounds with antimicrobial activity and potent ß-glucosidase for varied minor ginsenoside production.

Knockdown of NRSF Alleviates Ischemic Brain Injury and Microvasculature Defects in Diabetic MCAO Mice.

Diabetes is one of the well-established risk factors of stroke and is associated with a poor outcome in patients with stroke. Previous studies have shown that the expression of neuron restrictive silencer factor (NRSF) is elevated in diabetes as well as ischemic stroke. However, the role of NRSF in regulating an outcome of diabetic ischemic stroke has not been completely understood. Here, we hypothesized that diabetes-induced NRSF elevation can aggravate brain injury and cognition impairment in ischemic stroke. The diabetic ischemic stroke mice model was established by 8 weeks of high-fat-diet feeding and 5 days of streptozotocin injection followed by 30 min of middle cerebral artery occlusion (MCAO). We found that diabetes enhanced the MCAO-induced elevation of NRSF in the hippocampus in accompany with an elevation of its corepressors, HDAC1, and mSin3A, and decrease of ß-TrCP. By using histological/immunofluorescence staining and neurobehavioral testing, our results showed that the brain damage and learning/memory impairment were aggravated in diabetic ischemic mice but significantly attenuated after stereotaxic injection of NRSF-shRNA. Meanwhile, by performing whole-brain clearing with PEGASOS, microvascular reconstruction, western blotting, and ELISA, we found that NRSF-shRNA markedly alleviated the vasculature disorders and rescued the suppression of NRP-1, VEGF, and VEGFR2 in the hippocampus of diabetic ischemic mice. Therefore, our results demonstrated for the first time that the elevation of hippocampal NRSF plays an important role in alleviating brain injury and cognitive disabilities in diabetic ischemic mice, potentially via the reduction of NRP-1/VEGF signaling.

High glucose and palmitic acid induces neuronal senescence by NRSF/REST elevation and the subsequent mTOR-related autophagy suppression.

Cell senescence is a basic aging mechanism. Previous studies have found that the cellular senescence in adipose tissue and other tissues, such as the pancreas, muscle and liver, is associated with the pathogenesis and progression of type 2 diabetes; however, strong evidence of whether diabetes directly causes neuronal senescence in the brain is still lacking. In this study, we constructed a high glucose and palmitic acid (HGP) environment on PC12 neuronal cells and primary mouse cortical neurons to simulate diabetes. Our results showed that after HGP exposure, neurons exhibited obvious senescence-like phenotypes, including increased NRSF/REST level, mTOR activation and cell autophagy suppression. Downregulation of NRSF/REST could remarkably alleviate p16, p21 and γH2A.X upregulations induced by HGP treatment, and enhance mTOR-autophagy of neurons. Our results suggested that the diabetic condition could directly induce neuronal senescence, which is mediated by the upregulation of NRSF/REST and subsequent reduction of mTOR-autophagy.

A genetic algorithm for the optimization of the thermoinduction protocol for high-level production of recombinant human-like collagen from Escherichia coli.

Production of recombinant human-like collagen (RHLC) by thermoinduction of recombinant Escherichia coli BL 21 during high cell density cultivation was investigated in a 30 L bioreactor. The effects of induction temperature (T), pH, and carbon-to-nitrogen molar ratio of the nutrient medium (C/N) were examined. The optimal thermoinduction protocol for RHLC production was determined by using a model coupling genetic algorithm and artificial neural networks. The optimal operating conditions were as follows: maintenance of induction temperature at 42°C for 3 H and then at 39.4°C until the end, induction pH at 7.03, and C/N at 4.8 (mol/mol). The theoretical maximum concentration of RHLC was 12.5 g/L, whereas the experimental value was 12.1 g/L under the optimal induction conditions.

Probiotic Potential of Lactobacillus Strains Isolated From Fermented Vegetables in Shaanxi, China.

The objective of this study was to assess in vitro probiotic potential of Lactobacillus strains derived from artisanal fermented vegetables in Shaanxi, China. In total, 74 acid-producing Gram-positive strains with rod-shaped under the microscope were isolated from 16 samples of spontaneously fermented vegetables. Out of 74 strains, 26 showed high survival rate under low pH and high bile salts conditions and were subjected to molecular identification by 16S rRNA gene sequencing analysis. The results showed that 15 isolates belonged to Lactobacillus plantarum, 9 isolates belonged to Lactobacillus brevis, and the 2 remaining strains belonged to Weissella viridescens. The 24 Lactobacillus strains were investigated for their survival rate to transit simulated gastrointestinal tract, cell surface hydrophobicity, auto-aggregation, co-aggregation with pathogen, adhesion to Caco-2, antimicrobial activity, antibiotics susceptibility, radical scavenging ability, α-glucosidase inhibition, and the cholesterol assimilation. The results showed that the probiotic characteristics were strain-dependent, and several strains exhibited great probiotic potential with specific health benefits, which indicated that they might be excellent candidates for production of functional foods. Interestingly, it was first found that L. plantarum generally had higher antibacterial activities, α-glucosidase inhibition ability, and antibiotics susceptibility compared to L. brevis in this study. The results indicated that Lactobacillus strains isolated from fermented vegetables in Shaanxi, China, could be exploited as a promising novel probiotic source.

Identification of Two Novel Linear Neutralizing Epitopes within the Hexon Protein of Canine Adenovirus Using Monoclonal Antibodies.

Canine adenovirus (CAdV) has a high prevalence in canine populations. High affinity neutralizing antibodies against conserved epitopes can provide protective immunity against CAdV and protect against future outbreaks. In this study, we identified two CAdV-2-specific neutralizing monoclonal antibodies (mAbs), 2C1 and 7D7, which recognized two linear-dependent epitopes. MAb 2C1 potently neutralized CAdV-2 with a 50% neutralization titer (NT50) of 4096, and mAb 7D7 partially neutralized CAdV-2 with a 50% NT50 of 64. Immunoprecipitation, Western blot and protein spectral analysis indicated that both neutralizing mAbs recognized the hexon protein (Hex) of CAdV-2. Through a 12-mer random peptide phage display and synthetic peptides analysis, we finely mapped the neutralizing epitopes to two 10-amino acid (aa) peptides within the CAdV Hex: 634RIKQRETPAL643 located on the surface region; and 736PESYKDRMYS745 located in the inner region of the expected 3D structure of trimeric Hex. Importantly, the two epitopes are highly conserved among all CAdV isolates by sequence alignment analysis. Thus, these results provide insights into the interaction between virus and mAbs at the aa level and may have potential applications in the development of novel therapeutic or epitope-based vaccines, antibody therapeutics and a diagnostic method suitable for the rapid detection of all CAdVs.

A novel palmitic acid hydroxy stearic acid (5-PAHSA) plays a neuroprotective role by inhibiting phosphorylation of the m-TOR-ULK1 pathway and regulating autophagy.

AIMS: Type 2 diabetes mellitus (T2DM) can lead to brain dysfunction and a series of neurological complications. Previous research demonstrated that a novel palmitic acid (5-PAHSA) exerts effect on glucose tolerance and chronic inflammation. Autophagy was important in diabetic-related neurodegeneration. The aim of the present study was to investigate whether 5-PAHSA has specific therapeutic effects on neurological dysfunction in diabetics, particularly with regard to autophagy. METHODS: 5-PAHSA was successfully synthesized according to a previously described protocol. We then carried out a series of in vitro and in vivo experiments using PC12 cells under diabetic conditions, and DB/DB mice, respectively. PC12 cells were treated with 5-PAHSA for 24 h, while mice were administered with 5-PAHSA for 30 days. At the end of each experiment, we analyzed glucolipid metabolism, autophagy, apoptosis, oxidative stress, cognition, and a range of inflammatory factors. RESULTS: Although there was no significant improvement in glucose metabolism in mice administered with 5-PAHSA, ox-LDL decreased significantly following the administration of 5-PAHSA in serum of DB/DB mice (p < 0.0001). We also found that the phosphorylation of m-TOR and ULK-1 was suppressed in both PC12 cells and DB/DB mice following the administration of 5-PAHSA (p < 0.05 and p < 0.01), although increased levels of autophagy were only observed in vitro (p < 0.05). Following the administration of 5-PAHSA, the concentration of ROS decreased in PC12 cells and the levels of CRP increased in high-dose group of 5-PAHSA (p < 0.01). There were no significant changes in terms of apoptosis, other inflammatory factors, or cognition in DB/DB mice following the administration of 5-PAHSA. CONCLUSION: We found that 5-PAHSA can enhance autophagy in PC12 cells under diabetic conditions. Our data demonstrated that 5-PAHSA inhibits phosphorylation of the m-TOR-ULK1 pathway and suppressed oxidative stress in PC12 cells, and exerted influence on lipid metabolism in DB/DB mice.

Isolation, diversity, and antimicrobial activity of fungal endophytes from Rohdea chinensis (Baker) N.Tanaka (synonym Tupistra chinensis Baker) of Qinling Mountains, China.

Endophytic fungi have been emerged as fruitful resources for producing structurally fascinating and biologically active secondary metabolites. However, endophytic fungi from medicinal plants of Qinling Mountains-the most important natural climatic boundary between the subtropical and warm temperate zones of China with an astonishingly high level of biodiversity-have rarely been explored as potential sources of novel fungal species and active secondary metabolites. In this study, a total of 371 fungal colonies were successfully isolated from 510 tissue segments of the medicinal Tupistra chinensis Baker collected from Qinling Mountains, China. Roots of T. chinensis Baker are used as a folk medicine to ameliorate pharyngitis and treat rheumatic diseases. A total of 100 representative morphotype strains were identified according to ITS rDNA sequence analyses and were grouped into three phyla (Ascomycota, Basidiomycota, Mucoromycota), seven classes (Dothideomycetes, Sordariomycetes, Eurotiomycetes, Microbotryomycetes, Agaricomycetes, Leotiomycetes, Mortierellomycetes), and at least 35 genera. The genera of Collectotrichum (IF, 29.92%), Fusarium (IF, 8.36%), Aspergillus (IF, 8.09%), and Dactylonectria (IF, 5.39%) were most frequently isolated from the tissues of T. chinensis Baker. The Species Richness Index (S, 65) and the Shannon-Wiener Index (H', 3.7914) indicated that T. chinensis Baker harbored abundant fungal resources. Moreover, five isolates were potential new taxa because of low similarity of ITS sequences ranged from 95.09%∼96.61%. Fifteen out of 100 endophytic fungal ethyl acetate extracts exhibited inhibitory activities against at least one pathogenic bacterium or fungus. Two important lead compounds produced by two stains (F8047 and F8075) with high antimicrobial activities were identified using high performance liquid chromatography (HPLC) and ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-QTOF MS) analyses. In addition, it was noteworthy that the strain F8001, which may be a potential new species, showed antimicrobial activity and should be investigated further. Overall, these results indicated that the endophytic fungi from T. chinensis Baker could be exploited as a novel source of bioactive compounds.

Comparative study of different molecular weight pullulan productions by Aureobasidium pullulans CGMCC No.11602.

Pullulan productions by Aureobasidium pullulans CGMCC No.11602 were conducted using the initial culture (IC) medium and the optimized culture (OC) medium, respectively, in which pullulan with significantly different molecular weights was obtained. Under the IC medium condition, the pullulan molecular weight (M w) reached 288,403 Da with a yield of 64.12 g/L after 96 h fermentation period. However, the pullulan molecular weight was much higher (M w, 3,715,352 Da), while the yield of pullulan was lower (40.12 g/L) using the OC medium. The FTIR spectra indicated that pullulan produced using the IC and OC medium were similar. Transcriptome analysis showed that a total of 871 differentially expressed genes (DEGs) were screened and "N-glycan biosynthesis" and "other types of O-glycan biosynthesis" were the most highly represented differential metabolic pathways (DMPs). Specifically, the genes involved in the two DMPs consistently pointed to glucosyltransferase genes (GTF), all of which were up-regulated in the OC medium when compared with those in the IC medium. Further studies showed that the activity and the relative quantity (RQ) of GTF transcription were remarkable higher, which were coincident with the slower decrease in the molecular weight of pullulan in the OC medium than those in the IC medium during the late stage of fermentation. The results indicated that GTF may be involved in the regulation of pullulan molecular weight.

Efficient production of pullulan by Aureobasidium pullulans grown on mixtures of potato starch hydrolysate and sucrose.

Pullulan is a natural exopolysaccharide with many useful characteristics. However, pullulan is more costly than other exopolysaccharides, which limits its effective application. The purpose of this study was to adopt a novel mixed-sugar strategy for maximizing pullulan production, mainly using potato starch hydrolysate as a low-cost substrate for liquid-state fermentation by Aureobasidium pullulans. Based on fermentation kinetics evaluation of pullulan production by A. pullulans 201253, the pullulan production rate of A. pullulans with mixtures of potato starch hydrolysate and sucrose (potato starch hydrolysate:sucrose=80:20) was 0.212h-1, which was significantly higher than those of potato starch hydrolysate alone (0.146h-1) and mixtures of potato starch hydrolysate, glucose, and fructose (potato starch hydrolysate:glucose:fructose=80:10:10, 0.166h-1) with 100gL-1 total carbon source. The results suggest that mixtures of potato starch hydrolysate and sucrose could promote pullulan synthesis and possibly that a small amount of sucrose stimulated the enzyme responsible for pullulan synthesis and promoted effective potato starch hydrolysate conversion effectively. Thus, mixed sugars in potato starch hydrolysate and sucrose fermentation might be a promising alternative for the economical production of pullulan.

Hydroxylation of Human Type III Collagen Alpha Chain by Recombinant Coexpression with a Viral Prolyl 4-Hydroxylase in Escherichia coli.

High-level expression of recombinant collagen by genetic engineering is urgently required. Recombinant collagen is different from natural collagen in its hydroxyproline (Hyp) content and thermal stability. To obtain hydroxylated collagen for applications in biomedicine and biomaterials, the human collagen α1(III) chain was co-expressed with the viral prolyl 4-hydroxylase A085R in Escherichia coli. Unlike previous reports using human prolyl 4-hydroxylase, this study examined the hydroxylation of full-length human collagen α1(III) chain (COL3A1) by viral prolyl 4-hydroxylase. The genes encoding these two proteins were controlled by different promoters, Ptac and PRPL, on a recombinant pKK223-3 plasmid. The sequencing results verified that the target genes were successfully inserted into the recombinant vector. Based on quantitative PCR, SDS-PAGE, and western blotting, successful expression by E. coli BL21(DE3) was detected at the mRNA and protein levels for both loci. Liquid chromatography-mass spectrometry (LC-MS/MS) results suggested that the highest Hyp yield was obtained when the two proteins were induced with 0.5 mM IPTG and heat-shock treatment at 50 °C, corresponding to high enzyme expression and low human collagen α1(III) chain expression levels. A biological activity analysis indicated that the recombinant collagen with the highest hydroxylation level supported the growth of baby hamster kidney cells, similar to observations for native collagen. The production of hydroxylated collagen in this study establishes a new method for collagen hydroxylation and provides a basis for the application of recombinant collagen expressed in E. coli.

HLC/pullulan and pullulan hydrogels: their microstructure, engineering process and biocompatibility.

New locally injectable biomaterials that are suitable for use as soft tissue fillers are needed to address a significant unmet medical need. In this study, we used pullulan and human-like collagen (HLC) based hydrogels with various molecular weights (MWs) in combination therapy against tissue defects. Briefly, pullulan was crosslinked with NaIO4 to form a pullulan hydrogel and then may coupled with HLC using the reaction between the -NH2 end-group of HLC and the -CHO group present on the aldehyde pullulan to form the HLC/pullulan hydrogel, wherein the NaIO4 acted as the crosslinking and oxidizing agent. The good miscibility of pullulan and HLC in the hydrogels was confirmed via Fourier transform infrared spectroscopy, scanning electron microscopy, compression testing, enzyme degradation testing, cell adhesions, live/dead staining and subcutaneous filling assays. Here, pullulan hydrogels with various MWs were fabricated and physicochemically characterized. Limitations of the pullulan hydrogels included inflammation, poor mechanical strength, and degradation. By contrast, the properties of the HLC/pullulan hydrogels strongly enhanced. The efficacy of these hydrogels was evaluated both in vitro and in vivo. Our results indicate that HLC/pullulan hydrogels may have therapeutic value as efficient soft tissue fillers, with reduced inflammation, improved cell adhesion and delayed hydrogel degradation.

Novel hydrogels based on carboxyl pullulan and collagen crosslinking with 1, 4-butanediol diglycidylether for use as a dermal filler: initial in vitro and in vivo investigations.

Novel hydrogels based on carboxyl pullulan (PC) and human-like collagen (HLC) crosslinking with 1,4-butanediol diglycidyl ether (BDDE) are promising soft fillers for tissue engineering due to their highly tunable properties. Recent studies, however, have shown that incorporating hyaluronic acid and BDDE results in hydrogels with a microporous structure, a large pore size and high porosity, which reduce cell adhesion and enhance degradation in vivo. To improve biocompatibility and prevent biodegradation, the use of PC to replace hyaluronic acid in the fabrication of PC/BDDE (PCB) and PC/BDDE/HLC (PCBH) hydrogels was investigated. Preparation of gels with PC is a promising strategy due to the high reactivity, superb selectivity, and mild reaction conditions of PC. In particular, the Schiff base reaction of HLC and PC produces the novel functional group -RCONHR' in PCBH hydrogels. Twenty-four weeks after subcutaneous injection of either PCB or PCBH hydrogel in mice, the surrounding tissue inflammation, enzymatic response and cell attachment were better compared to hyaluronic acid-based hydrogels. However, the biocompatibility, cytocompatibility and non-biodegradability of PCBH were milder than those of the PCB hydrogels both in vivo and in vitro. These results show that the proposed use of PC and HLC for the fabrication of hydrogels is a promising strategy for generating soft filler for tissue engineering.

Novel multifunctional PB and PBH hydrogels as soft filler for tissue engineering.

In this study, we designed multifunctionalized hydrogel scaffolds and injectable particles based on high-molecular-weight (MW) pullulan and human-like collagen (HLC) crosslinked with 1,4-butanediol diglycidyl ether (BDDE) for combination therapy tissue restoration. The properties of the pullulan/BDDE (PB) and pullulan/BDDE/human-like collagen (PBH) hydrogels were characterized via swelling ratio measurements, mechanical tests, and enzymatic degradation in vitro and via subcutaneous injections in vivo. The results demonstrate that the dry hydrogels completely returned to their original state in deionized water. The elastic modulus of the PBH53 dry hydrogels is higher than that of the other hydrogels after exposure to bending stress and compression stress with a maximum value of 7858.93 MPa. In addition, the in vitro live/dead staining and cell adhesion of the PBH hydrogels exhibited a superior fibroblast morphology without high levels of cell death, which were considerably better than those of PB hydrogels. In vivo, PB and PBH particles with good biocompatibility and anti-biodegradation were successfully prepared via the granulation of wet PB and PBH hydrogels for efficient subcutaneous injection in Kunming mice and New Zealand rabbits. Therefore, the PB and PBH hydrogels were found to be acceptable, safe, soft materials for use in skin restoration, cartilage treatment, and lacrimal dryness therapy.