Dissolvable microneedles based on Panax notoginseng polysaccharide for transdermal drug delivery and skin dendritic cell activation.

This work explored the feasibility of using biological polysaccharide to fabricate dissolvable microneedles (MNs) for the purpose of transdermal drug delivery and skin dendritic cell (DC) activation. Panax notoginseng polysaccharide (PNPS), a naturally derived immunoactive macromolecule, was used to fabricate dissolvable MNs. The prepared PNPS MNs showed a satisfactory mechanical strength and a skin penetration depth. By Franz diffusion cell assay, the PNPS MNs demonstrated a high transdermal delivery amount of model drugs. Furthermore, with the assistance of MNs, PNPS easily penetrated across the stratum corneum and target ear skin DCs, activating the maturation and migration of immunocytes by increasing the expressions of CD40, CD80, CD86, and MHC II of skin DCs. Consequently, the matured DCs migrated to the auricular draining lymph nodes and increased the proportions of CD4+ T and CD8+ T cells. Thus, PNPS might be a promising biomaterial for transdermal drug delivery, with adjuvant potential.

Dual sgRNA-directed gene deletion in basidiomycete Ganoderma lucidum using the CRISPR/Cas9 system.

Ganoderma lucidum is an important medicinal mushroom in traditional Chinese medicine. However, the lack of adequate genetic tools has hindered molecular genetic research in and the genetic modification of this species. Here, we report that the presence of an intron is necessary for the efficient expression of the heterologous phosphinothricin-resistance and green fluorescent protein genes in G. lucidum. Moreover, we improved the CRISPR/Cas9-mediated gene disruption frequency in G. lucidum by adding an intron upstream of the Cas9 gene. Our results showed that the disruption frequency of the orotidine 5'-monophosphate decarboxylase gene (ura3) in transformants containing the glyceraldehyde-3-phosphate dehydrogenase gene intron in the Cas9 plasmid is 14-18 in 107 protoplasts, which is 10.6 times higher than that in transformants without any intron sequence. Furthermore, genomic fragment deletions in the ura3 and GL17624 genes were achieved via a dual sgRNA-directed CRISPR/Cas9 system in G. lucidum. We achieved a ura3 deletion frequency of 36.7% in G. lucidum. The developed method provides a powerful platform to generate gene deletion mutants and will facilitate functional genomic studies in G. lucidum.

Enhancing Haematococcus pluvialis biomass and γ-aminobutyric acid accumulation by two-step cultivation and salt supplementation.

The primary goal of this study was to assess the roles of chemical factors and bioprocess strategies on a mixotrophic culture of the microalga Haematococcus pluvialis during γ-aminobutyric acid (GABA) production. A two-stage strategy was used to increase the biomass and GABA accumulation of H. pluvialis. In stage I, mixotrophic growth of H. pluvialis in the presence of fulvic acid (FA) produced a high biomass (1.84 g L-1) with a GABA content of 25.45 mg g-1. Furthermore, a maximum GABA content of 38.57 mg g-1 was obtained when cells were cultured with 0.4 g L-1 NaCl under photoautotrophic conditions in stage II, whereas the carbohydrate content of cells sharply decreased from 26.68 to 18.22%. In addition, salt stress upregulated the expression of the gad and cam genes. The results of this study demonstrate an efficient strategy to produce GABA from the microalga H. pluvialis.

Effect of Chinese patent medicine Si-Mo-Tang oral liquid for functional dyspepsia: A systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: Si-Mo-Tang oral liquid (SMT) has been widely used to treat functional dyspepsia (FD), but the effectiveness is still controversial. A systematic review and meta-analysis of randomized controlled trials (RCTs) were performed to assess the efficacy and adverse effects of SMT for FD. METHODS: Investigators searched for articles with publication dates to June 21, 2016, from 9 English and Chinese electronic databases. Comparisons were SMT alone or SMT in combination with western medicine as experimental intervention, and western medicine or placebo as the control. We used the Cochrane collaboration tool for assessing risk of bias to evaluate methodologies. Data were synthesized with RevMan 5.3 software. (PROSPERO Registration #CRD42016042003). RESULTS: Twenty-seven RCTs were included in the review, involving 2,713 participants: 1,383 subjects were in the experimental group and 1,330 in the control group. SMT showed a significant improvement in clinical efficacy (RR 1.14; 95% CI 1.09, 1.20; P<0.00001), but the heterogeneity was also significant (P = 0.0002, I2 = 56%). Because of the different interventions in the 2 groups, we performed subgroup and sensitivity analyses to investigate potential sources of heterogeneity. The heterogeneity was smaller after subgroup analysis and the exclusion of a study by Zhu from 2009. The corresponding pooled RR has no obvious change (RR 1.17; 95% CI 1.13, 1.21; P<0.00001). Subgroup analysis by age and drugs administered in control interventions between SMT and western medicine also showed improvement in the efficacy rate. But a data synthesis that excluded high risk of bias in the blinding of participants and personnel showed no significant difference (RR 1.14; 95% CI 0.97, 1.35; P = 0.12). Three studies measured gastric emptying. Two of these studies reported no significant difference between the experimental and control groups, while 1 study showed that SMT reduced the time of gastric emptying. The relapse rate and adverse effects had no difference between 2 groups. CONCLUSIONS: This meta-analysis suggests that SMT is an effective and safe therapy option for patients with FD. However, because of the high clinical heterogeneity, poor quality, high risk of bias and small sample size of some included studies, further standardized large-scale and strictly designed studies are needed.

[Acupuncture combined with medication for blood-stasis in unstable angina patients with different thrombolysis in myocardial infarction risk stratification].

OBJECTIVE: To observe the intervention effects of acupuncture combined with standardized treatment of western medicine on blood-stasis syndrome in unstable angina (UA) patients with different thrombolysis in myo-cardial infarction (TIMI) risk stratification. METHODS: According to TIMI risk score, a total of 72 UA patients were included, 24 cases in low-risk (0 to 2 points) group, 24 cases in moderate-risk (3 to 4 points) group and 24 cases in high-risk (5 to 7 points) group. Based on routine standardized treatment of western medicine, acupuncture was applied at Xinshu (BL 15), Xuehai (SP 10), Geshu (BL 17), Danzhong (CV 17), Jueyinshu (BL 14), Ximen (PC 4) and Neiguan (PC 6) in three groups. Acupuncture was given once a day, 14 days as a session. After one session, each symptom score and total score of blood stasis syndrome were observed. RESULTS: One patient dropped out respectively, and 23 patients in each group were included in the analysis. After treatment, the score of angina symptom and total score of blood stasis syndrome were significantly improved in three groups (all P<0.01). The improvements of each score and total score of blood stasis syndrome in the high-risk group were superior to those in the low-risk group and moderate-risk group (P<0.05, P<0.01). CONCLUSIONS: With the higher TIMI scores, the blood stasis syndrome is aggravated accordingly. Acupuncture combined with standardized treatment of western medicine could effectively intervene blood stasis syndrome, especially for the score of blood stasis syndrome of patients in high-risk group.

Genetic engineering of Ganoderma lucidum for the efficient production of ganoderic acids.

Ganoderma lucidum is a well-known traditional medicinal mushroom that produces ganoderic acids with numerous interesting bioactivities. Genetic engineering is an efficient approach to improve ganoderic acid biosynthesis. However, reliable genetic transformation methods and appropriate genetic manipulation strategies remain underdeveloped and thus should be enhanced. We previously established a homologous genetic transformation method for G. lucidum; we also applied the established method to perform the deregulated overexpression of a homologous 3-hydroxy-3-methyl-glutaryl coenzyme A reductase gene in G. lucidum. Engineered strains accumulated more ganoderic acids than wild-type strains. In this report, the genetic transformation systems of G. lucidum are described; current trends are also presented to improve ganoderic acid production through the genetic manipulation of G. lucidum.

Enhanced Production of Polysaccharide Through the Overexpression of Homologous Uridine Diphosphate Glucose Pyrophosphorylase Gene in a Submerged Culture of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Higher Basidiomycetes).

This study aimed to improve polysaccharide production by engineering the biosynthetic pathway in Ganoderma lucidum through the overexpression of the homologous UDP glucose pyrophosphorylase (UGP) gene. The effects of UGP gene overexpression on intracellular polysaccharide (IPS) content, extracellular polysaccharide (EPS) production, and transcription levels of 3 genes encoding the enzymes involved in polysaccharide biosynthesis, including phosphoglucomutase (PGM), UGP, and α-1,3-glucan synthase (GLS), were investigated. The maximum IPS content and EPS production in G. lucidum overexpressing the UGP gene were 24.32 mg/100 mg dry weight and 1.66 g/L, respectively, which were higher by 42% and 36% than those of the wild-type strain. The transcription levels of PGM, UGP, and GLS were up-regulated by 1.6, 2.6, and 2.4-fold, respectively, in the engineered strain, suggesting that increased polysaccharide biosynthesis may result from a higher expression of those genes.

Development of an expression plasmid and its use in genetic manipulation of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (higher Basidiomycetes).

We report the construction of a plasmid, pJW-EXP, designed for the expression of homologous and heterologous genes in Ganoderma lucidum. pJW-EXP was generated from the plasmid pMD19-T by inserting the G. lucidum glyceraldehyde-3-phosphate dehydrogenase gene promoter, the G. lucidum iron-sulfur protein subunit of succinate dehydrogenase gene terminator and the homologous carboxin-resistance gene as selection marker. This expression plasmid can be efficiently transformed into Ganoderma through polyethylene glycol-mediated protoplast transformation. Southern blot analysis showed that most of the integrated DNA appeared as multiple copies in the genome. The applicability of the constructed plasmid was tested by expression of the truncated G. lucidum 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene that encodes the catalytic domain of HMGR. Overexpression of the truncated HMGR gene, which is a key gene in the biosynthetic pathway of the antitumor compounds, ganoderic acids, increased the transcription of the HMGR gene and enhanced ganoderic acid accumulation. pJW-EXP can serve as a useful tool in the genetic improvement and metabolic engineering of Ganoderma.

Enhanced biosynthetic gene expressions and production of ganoderic acids in static liquid culture of Ganoderma lucidum under phenobarbital induction.

Static liquid culture of Ganoderma lucidum, a traditional Chinese medicinal mushroom, is a proven technology for producing ganoderic acids, which are secondary metabolites that possess antitumor properties. In this work, the addition of phenobarbital, a P450 inducer, was used to enhance the production of total and individual ganoderic acids in a two-stage cultivation involving a period of initial shake flask culture followed by static liquid culture of G. lucidum. The dosage and time of phenobarbital induction were critical for the enhanced production of ganoderic acids. The addition of 100 muM (final concentration) phenobarbital on day 5 after the shake flask culture was converted to the static liquid culture was found to be optimal, resulting in a maximal amount of total ganoderic acids of 41.4 +/- 0.6 mg/g cell dry weight and increases in the levels of ganoderic acid-Mk, -T, -S, and -Me in the treated cells by 47%, 28%, 36%, and 64%, respectively. Meanwhile, the accumulation of lanosterol, a key intermediate, was found to decrease and transcriptions of three key genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase, squalene synthase, and lanosterol synthase in the triterpene biosynthetic pathway were up-regulated under phenobarbital induction. This work demonstrated a useful strategy for the enhanced production of ganoderic acids by G. lucidum.