Discovery of Gambogic acid as an antibacterial adjuvant against vancomycin-resistant enterococci in vitro and in vivo.

BACKGROUND: The emergence and spread of vancomycin-resistant enterococci (VRE) have posed a significant challenge to clinical treatment, underscoring the need to develop novel strategies. As therapeutic options for VRE are limited, discovering vancomycin enhancer is a feasible way of combating VRE. Gambogic acid (GA) is a natural product derived from the resin of Garcinia hanburyi Hook.f. (Clusiaceae), which possesses antibacterial activity. PURPOSE: This study aimed to investigate the potential of GA as an adjuvant to restore the susceptibility of VRE to vancomycin. METHODS: In vitro antibacterial and synergistic activities were evaluated against vancomycin-susceptible and resistant strains by the broth microdilution method for the Minimal Inhibitory Concentrations (MICs) determination, and checkerboard assay and time-kill curve analysis for synergy evaluation. In vivo study was conducted on a mouse multi-organ infection model. The underlying antibacterial mechanism of GA was also explored. RESULTS: GA showed a potent in vitro activity against all tested strains, with MICs ranging from 2 to 4 µg/ml. The combination of GA and vancomycin exhibited a synergistic effect against 18 out of 23 tested VRE strains, with a median fractional inhibitory concentration index (FICI) of 0.254, and demonstrated a synergistic effect in the time-kill assay. The combination therapy exhibited a significant reduction in tissue bacterial load compared with either compound used alone. GA strongly binds to the ParE subunit of topoisomerase IV, a bacterial type II DNA topoisomerase, and suppresses its activity. CONCLUSIONS: The study suggests that GA has a significant antibacterial activity against enterococci, and sub-MIC concentrations of GA can restore the activity of vancomycin against VRE in vitro and in vivo. These findings indicate that GA has the potential to be a new antibacterial adjuvant to vancomycin in the treatment of infections caused by VRE.

Genetic engineering of complex feed enzymes into barley seed for direct utilization in animal feedstuff.

Currently, feed enzymes are primarily obtained through fermentation of fungi, bacteria, and other microorganisms. Although the manufacturing technology for feed enzymes has evolved rapidly, the activities of these enzymes decline during the granulating process and the cost of application has increased over time. An alternative approach is the use of genetically modified plants containing complex feed enzymes for direct utilization in animal feedstuff. We co-expressed three commonly used feed enzymes (phytase, ß-glucanase, and xylanase) in barley seeds using the Agrobacterium-mediated transformation method and generated a new barley germplasm. The results showed that these enzymes were stable and had no effect on the development of the seeds. Supplementation of the basal diet of laying hens with only 8% of enzyme-containing seeds decreased the quantities of indigestible carbohydrates, improved the availability of phosphorus, and reduced the impact of animal production on the environment to an extent similar to directly adding exogenous enzymes to the feed. Feeding enzyme-containing seeds to layers significantly increased the strength of the eggshell and the weight of the eggs by 10.0%-11.3% and 5.6%-7.7% respectively. The intestinal microbiota obtained from layers fed with enzyme-containing seeds was altered compared to controls and was dominated by Alispes and Rikenella. Therefore, the transgenic barley seeds produced in this study can be used as an ideal feedstuff for use in animal feed.

Enhanced phytoremediation of selenium using genetically engineered rice plants.

Selenium (Se) is a micronutrient essential for human and animal health. However, Se is toxic at high levels because the nonspecific substitution of cysteine by selenocysteine could lead to protein malfunction. In an attempt to prevent nonspecific selenocysteine incorporation into proteins, we simultaneously overexpressed the gene encoding selenocysteine lyase from Homo sapiens (HsSL), which specifically catalyzes the decomposition of selenocysteine into elemental Se0 and alanine, and the gene encoding selenocysteine methyltransferase from Astragalus bisulcatus (AbSMT), which methylates selenocysteine into methylselenocysteine in rice. The transgenic plants showed normal growth under standard conditions. Se treatment resulted in higher levels of alanine and methylselenocysteine in transgenic plants than in wild-type plants, which indicated that this approach might have successfully redirected Se flow in the plant. Overexpression of HsSL and AbSMT in rice also endows transgenic plants with hyposensitivity to Se stress at the seed germination stage. The transgenic plants showed enhanced selenate and selenite tolerance, which was simultaneously supported by fresh weight values. Moreover, our phytoremediation assay revealed that the transgenic plants exhibited greatly improved Se elimination capabilities and accumulated about 38.5% and 128.6% more Se than wild-type plants when treated with selenate and selenite, respectively. This study offers hope that genetically modified plants could play a role in the restoration of Se-contaminated environment.

Metabolic engineering of rice endosperm for betanin biosynthesis.

Betanin has been widely used as an additive for many centuries, and its use has increased because of its market application as an additive, high free radical scavenging activity, and safety, health-promoting properties. The main source of betanin is red beet, but many factors notably affect the yield of betanin from red beets. Betanin is not produced in cereal grains. Thus, developing biofortified crops with betanin is another alternative to health-promoting food additives. Here, rice endosperm was bioengineered for betanin biosynthesis by introducing three synthetic genes (meloS, BvDODA1S, and BvCYP76AD1S). The overexpression of these genes driven by rice endosperm-specific promoter established the betanin biosynthetic pathways in the endosperm, resulting in new types of germplasm - 'Betanin Rice' (BR). The BR grains were enriched with betanin and had relatively high antioxidant activity. Our results proved that betanin can be biosynthesized de novo in rice endosperm by introducing three genes in the committed betanin biosynthetic pathway. The betanin-fortified rice in this study can be used as a functional grain to promote health and as a raw material to process dietary supplements.

Study on efficacy and safety of Tong-luo Qu-tong plaster treatment for knee osteoarthritis: study protocol for a randomized, double-blind, parallel positive controlled, multi-center clinical trial.

BACKGROUND: Knee osteoarthritis (KOA) is a common chronic musculoskeletal disorder that seriously affects quality of life. Patients with KOA frequently develop one or more of the following typical symptoms: joint pain, stiffness, joint friction noise and impaired functionality. Traditional Chinese medicine (TCM) has been shown to have a superior effect and a particular advantage in the treatment of KOA; among TCM, the Tong-luo Qu-tong plaster is the convenient and most commonly used method in China to improve symptoms including pain, stiffness and limited mobility in patients with KOA, as it causes few adverse effects. But there is a lack of high-quality clinical evidences to support the therapeutic effect that Chinese adhesive plaster can have in relieving pain and stiffness. The purpose of this study will be to evaluate the efficacy and safety of Tong-luo Qu-tong plaster in patients with KOA. METHODS/DESIGN: This study will be a randomized, double-blind, parallel positive controlled, multi-center clinical trial, a non-inferiority trial design was adopted. A total of 2000 participants older than 40 years, with KOA, will be randomly allocated into an experimental group (n = 1500) and a control group (n = 500). All participants will receive a conventional conservative treatment lasting for 14 days as two courses, once daily. Tong-luo Qu-tong plaster will be administered externally to participants in the experimental group, while the control group will receive a Qi-zheng Xiao-tong plaster. The outcome of the total Western Ontario and McMaster Universities Arthritis Index scores, TCM syndrome quantitative score and visual analog scale scores will be measured during the assessment visits (baseline and 1-week and 2-week follow up). In addition, adverse events related to clinical symptoms and signs and results of laboratory tests will be documented during the clinical trials. DISCUSSION: This study will provide reliable evidence of the effectiveness and safety of Tong-luo Qutong plaster in patients with KOA. If the results are favorable, it is expected that the patients with KOA will benefit from this study, many patients may have a good alternative treatment for KOA. TRIAL REGISTRATION: ClinicalTrials.gov, ID: NCT03309501 . Registered on 8 November 2017.

Ectopic overexpression of SsCBF1, a CRT/DRE-binding factor from the nightshade plant Solanum lycopersicoides, confers freezing and salt tolerance in transgenic Arabidopsis.

The C-repeat (CRT)/dehydration-responsive element (DRE) binding factor (CBF/DREB1) transcription factors play a key role in cold response. However, the detailed roles of many plant CBFs are far from fully understood. A CBF gene (SsCBF1) was isolated from the cold-hardy plant Solanum lycopersicoides. A subcellular localization study using GFP fusion protein indicated that SsCBF1 is localized in the nucleus. We delimited the SsCBF1 transcriptional activation domain to the C-terminal segment comprising amino acid residues 193-228 (SsCBF1(193-228)). The expression of SsCBF1 could be dramatically induced by cold, drought and high salinity. Transactivation assays in tobacco leaves revealed that SsCBF1 could specifically bind to the CRT cis-elements in vivo to activate the expression of downstream reporter genes. The ectopic overexpression of SsCBF1 conferred increased freezing and high-salinity tolerance and late flowering phenotype to transgenic Arabidopsis. RNA-sequencing data exhibited that a set of cold and salt stress responsive genes were up-regulated in transgenic Arabidopsis. Our results suggest that SsCBF1 behaves as a typical CBF to contribute to plant freezing tolerance. Increased resistance to high-salinity and late flowering phenotype derived from SsCBF1 OE lines lend more credence to the hypothesis that plant CBFs participate in diverse physiological and biochemical processes related to adverse conditions.

Isolation and functional characterization of the ShCBF1 gene encoding a CRT/DRE-binding factor from the wild tomato species Solanum habrochaites.

Plant growth and productivity are greatly affected by low ambient temperature. Complex cascades of gene expression in cold stress response are regulated by transcription factors. In this study, a cDNA clone, named ShCBF1, was isolated from Solanum habrochaites seedlings (a wild relative of cultivated tomato). It was classified as one of CBF family members based on multiple sequence alignment. The expression analysis confirmed that ShCBF1 was induced by low temperature, high salinity and drought stress. Experiments of subcellular localization in tobacco leaf cells indicated that it was localized in nucleus. Transient expression assay using onion epidermal cells revealed that the ShCBF1 protein could function similarly to AtCBF1 in activating the expression of reporter genes with a CRT/DRE element in their promoter. Moreover, ectopic overexpression of ShCBF1 in Arabidopsis enhanced freezing and high salinity tolerance of transgenic plants by improving the expression levels of some stress-responsive marker genes. Taken together, our results suggest that ShCBF1 behaves as a typical plant CBF transcription factor and might be involved in plant response to various environmental stresses.

An outbreak of Mycoplasma pneumoniae caused by a macrolide-resistant isolate in a nursery school in China.

Eighteen out of 45 children were reported to have a respiratory illness during an outbreak at a temporary dormitory in a nursery school in China in 2011. To study the outbreak and to determine the risk factors for infection, an epidemiological investigation was performed. A standardized questionnaire was completed for a total of 45 children with the help of their guardians and parents. In addition, acute- and convalescent-phase serum samples and throat swabs from the children were taken for laboratory diagnosis. The diagnosis of a Mycoplasma-like illness was based on the following clinical criteria. The criteria were onset of illness after 31 May 2011, characterized by a cough, fever(>37.5 °C), or at least 3 of the following symptoms: fever, sore throat, cough or expectoration, and runny or stuffy nose. PCR-restriction fragment length polymorphism (PCR-RFLP), determination of MICs, and sequencing were performed to determine the genotype, antibiotic resistance, and sequence polymorphisms of the isolated strains, respectively. The paired sera revealed that 15 patients were infected with Mycoplasma pneumoniae. Epidemiology confirmed that this was a point source outbreak, characterized by a short incubation period, a high secondary attack rate, and a long period of hospitalization. PCR-RFLP analysis revealed that the 12 isolated strains of M. pneumoniae shared the same subtype P1 gene, and 23S rRNA sequence analysis showed that these strains harbored two macrolide-resistant gene-related point mutations at position 2063 and 2617. In this outbreak, the major risk factor was the distance between the bed of the first patient and the beds of close contacts (beds less than three meters apart). The strains isolated in this study were found to harbor two point mutations conferring macrolide resistance, indicating the importance of pathogen and drug resistance surveillance systems.