Expression and characterization of 1,4-α-glucan branching enzyme from Microvirga sp. MC18 and its application in the preparation of slowly digestible starch.

Nutraceuticals containing modified starch with increased content of slowly-digestible starch (SDS) may reduce the prevalence of obesity, diabetes and cardiovascular diseases due to its slow digestion rate. Enzymatic methods for the preparation of modified starch have attracted increasing attention because of their low environmental impact, safety and specificity. In this study, the efficient glucan branching enzyme McGBE from Microvirga sp. MC18 was identified, and its relevant properties as well as its potential for industrial starch modification were evaluated. The purified McGBE exhibited the highest specificity for potato starch, with a maximal specific activity of 791.21 U/mg. A time-dependent increase in the content of α-1,6 linkages from 3.0 to 6.0% was observed in McGBE-modified potato starch. The proportion of shorter chains (degree of polymerization, DP < 13) increased from 29.2 to 63.29% after McGBE treatment, accompanied by a reduction of the medium length chains (DP 13-24) from 52.30 to 35.99% and longer chains (DP > 25) from 18.51 to 0.72%. The reduction of the storage modulus (G') and retrogradation enthalpy (ΔHr) of potato starch with increasing treatment time demonstrated that McGBE could inhibit the short- and long-term retrogradation of starch. Under the optimal conditions, the SDS content of McGBE-modified potato starch increased by 65.8% compared to native potato starch. These results suggest that McGBE has great application potential for the preparation of modified starch with higher SDS content that is resistant to retrogradation.

Biocidal effects of volatile organic compounds produced by the myxobacterium Corrallococcus sp. EGB against fungal phytopathogens.

Myxobacteria have excellent biocontrol activity against various phytopathogens due to their rich spectrum of secondary metabolites and active predatory characteristics. In this study, the mycelial growth of Fusarium oxysporum f. sp. cucumerinum (FOC) was found to be significantly inhibited by volatile compounds (VOCs) produced by Corallococcus sp. EGB. A total of 32 compounds were identified among the VOCs produced by strain EGB, of which isooctanol exhibited the highest antifungal activity, with dosages of 3.75 and 4.0 µL/plate being sufficient to suppress FOC and Penicillum digitatum, respectively. Isooctanol was found to damage the cell wall and cell membranes of FOC and P. digitatum. Apoptosis-like cell death of FOC and P. digitatum induced by isooctanol was observed subsequently due to the accumulation of reactive oxygen species (ROS). The transcription level of genes related to cell wall integrity (CWI) pathway and redox reactions were significantly upregulated by 15- to 40-fold, indicating the stress caused by isooctanol. Postharvest storage experiments showed that the disease severity of post-harvest oranges infected with P. digitatum could be significantly reduced by isooctanol at 114.2 µL/L.

A chemical proteomics approach for global mapping of functional lysines on cell surface of living cell.

Cell surface proteins are responsible for many crucial physiological roles, and they are also the major category of drug targets as the majority of therapeutics target membrane proteins on the surface of cells to alter cellular signaling. Despite its great significance, ligand discovery against membrane proteins has posed a great challenge mainly due to the special property of their natural habitat. Here, we design a new chemical proteomic probe OPA-S-S-alkyne that can efficiently and selectively target the lysines exposed on the cell surface and develop a chemical proteomics strategy for global analysis of surface functionality (GASF) in living cells. In total, we quantified 2639 cell surface lysines in Hela cell and several hundred residues with high reactivity were discovered, which represents the largest dataset of surface functional lysine sites to date. We discovered and validated that hyper-reactive lysine residues K382 on tyrosine kinase-like orphan receptor 2 (ROR2) and K285 on Endoglin (ENG/CD105) are at the protein interaction interface in co-crystal structures of protein complexes, emphasizing the broad potential functional consequences of cell surface lysines and GASF strategy is highly desirable for discovering new active and ligandable sites that can be functionally interrogated for drug discovery.

Isolation and genome sequencing of a novel lytic Pseudoalteromonas phage SL20.

Phage SL20, a novel lytic Pseudoalteromonas phage, was isolated from the coastal waters of the Yellow Sea, China. The microbiological characterization demonstrated that phage SL20 was relatively stable from 35 to 55 °C and the optimal pH was approximately 6.0. A latent period of approximately 24 min was indicated by a one-step growth curve. The burst size was approximately 12 ± 3 PFU/cell. The genome had a length of 120,295 bp with a G + C content of 35.84%, and predicted 95 ORFs. The phylogenetic tree based on DNA helicase showed that Pseudoalteromonas phage SL20 was related to the Pseudoalteromonas phage H101 and was a member of the family Shandongvirus. The isolation and genomic analysis of SL20 has improved our understanding of host-phage interactions and the ecology of the marine bacteria Pseudoalteromonas.

[An experimental study on the basic fibroblast growth factor slow-releasing microsphere for knee osteoarthritis in the rabbit].

OBJECTIVE: To study the therapeutic effect of chitosan-coated basic fibroblast growth factor (bFGF) slow-releasing microspheres on the knee osteoarthritis in the rabbit. METHODS: From November 2008 to July 2009, 54 New Zealand rabbits were divided into 6 groups at random, which were the control group, the model group, the PBS-M group, the bFGF-S group, the 10-bFGF-M group and the 100-bFGF-M group, respectively. The model of knee osteoarthritis was induced by the injection of papain in the rabbit. Except the control and model groups, all the experimental groups were implanted 1 ml intervention solution at the third and sixth weeks, including the PBS microspheres, bFGF solution, 10 µg bFGF microspheres and 100 µg bFGF microspheres, respectively. The rabbits were sacrificed at the ninth week after operation, and then articular cartilage was conducted the morphological and histopathological evaluation. RESULTS: The damage of articular cartilage in the model group was more serious than that in the control group, with statistical differences according to the Ink score (t = 8.22, P = 0.00) and Mankin score (t = 17.20, P = 0.00). The damage of articular cartilage in the PBS-M and bFGF-S groups were similar with that in the model group, according to the Ink score (t = 0.26, P = 0.79; t = 0.80, P = 0.45) and Mankin score (t = 1.51, P = 0.17; t = 0.56, P = 0.60). The Ink and Mankin scores in the 10-bFGF-M and 100-bFGF-M groups were better than that in the model group (Ink score: t = 3.58, P = 0.01; t = 6.82, P = 0.00; Mankin score: t = 3.41, P = 0.01; t = 5.00, P = 0.00), with the 100-bFGF-M group much better (t = 5.29, P = 0.00; t = 2.80, P = 0.02). CONCLUSIONS: The bFGF slow-releasing microsphere can keep its effective intra-articular concentration, which may accelerate the synthesis of proteoglycan and inhibit its decomposition to reverse the damage of articular cartilage.

Structural and digestion properties of potato starch modified using an efficient starch branching enzyme AqGBE.

Modified potato starch with slower digestion may aid the development of new starch derivatives with improved nutritional values, and strategies to increase nutritional fractions such as resistant starch (RS) are desired. In this study, a correspondence between starch structure and enzymatic resistance was provided based on the efficient branching enzyme AqGBE, and modified starches with different amylose content (Control, 100%; PS1, 90%; PS2, 72%; PS3, 32%; PS4, 18%) were prepared. Through SEM observation, NMR and X-ray diffraction analyses, we identified that an increased proportion of α-1,6-linked branches in potato starch changes its state of granule into large pieces with crystallinity. Molecular weight and chain-length distribution analysis showed a decrease of molecular weight (from 1.1 × 106 to 1.1 × 105 g/mol) without an obvious change of chain-length distribution in PS1, while PS2-4 exhibited an increased proportion of DP 6-12 with a stable molecular weight distribution, indicating a distinct model of structural modification by AqGBE. The enhancement of peak viscosity was related to increased hydrophobic interactions and pieces state of PS1, while the contents of SDS and RS in PS1 increased by 37.7 and 49.4%, respectively. Our result provides an alternative way to increase the RS content of potato starch by branching modification.

Thiazide Diuretics and the Incidence of Osteoporotic Fracture: A Systematic Review and Meta-Analysis of Cohort Studies.

Background: Thiazide diuretics may improve bone mineral density. However, results are inconsistent for studies evaluating the association between thiazides and risk of osteoporotic fracture. We performed an updated meta-analysis of cohort studies to determine the association between thiazides use and fracture risk. Methods: Relevant studies were identified via systematic search of PubMed and Embase. A random-effect model was used for meta-analysis. Subgroup analyses were performed to explore the potential influences of study characteristics on the outcome. Results: Seventeen cohort studies with 3,537,504 participants were included. The pooled results showed that use of thiazide diuretics at baseline did not significantly affect the risk of overall osteoporotic fracture incidence as compared with controls (risk ratio [RR]: 0.96, 95% confidence interval [CI]: 0.83 to 1.09, p = 0.51) with significant heterogeneity (p for Cochrane's Q test < 0.001, I2 = 90%). Results of subgroup analyses indicated that general status of the participants may be an important determinant for the association between thiazide diuretics and subsequent risk of osteoporotic fracture. Use of thiazide diuretics was associated with significantly reduced risk of fracture in patients with acute status including new-onset stroke or spinal cord injury (RR: 0.70, 95% CI: 0.57 to 0.86, p < 0.001), but not in those with good conditions such as community-dwelling population or hypertensive patients (p for subgroup difference = 0.02). Conclusions: Use of thiazide diuretics is not associated with significantly affected risk of overall osteoporotic fracture. However, the association may be different according to the general status of the participants.

Expression of microRNA­27a in a rat model of osteonecrosis of the femoral head and its association with TGF­ß/Smad7 signalling in osteoblasts.

The present study assessed whether microRNA (miR)­27a is an influential factor in steroid­induced osteonecrosis of the femoral head (ONFH) and investigated the underlying mechanism of action. The results indicated that serum miR­27a was decreased in a rat model of ONFH compared with that in control rats. It was also observed that increased miR­27a expression promoted osteogenic differentiation and cell proliferation, inhibited caspase­3/9 and B­cell lymphoma­2­associated X protein expression and induced alkaline phosphatase (ALP) activity and bone morphogenetic protein (BMP)­2, runt­related transcription factor (Runx)2 and osteonectin mRNA expression in osteoblastic MC3T3­E1 cells. miR­27a mimics also induced transforming growth factor (TGF)­ß and Smad7 protein expression in MC3T3­E1 cells. Furthermore, transfection with TGF­ß expression plasmid was able to enhance the effects of miR­27a mimics on osteoblastic differentiation, cell proliferation, ALP activity, BMP­2, Runx2 and osteonectin mRNA expression, and Smad7 protein expression in the MC3T3­E1 cells. Transfection with a TGF­ß or Smad7 expression plasmid also enhanced the effects of miR­27a mimics on osteoblastic differentiation, cell proliferation, ALP activity and osteonectin mRNA expression in the MC3T3­E1 cells. Taken together, the results of the present study suggested that the induction of TGF­ß/Smad7 signaling in osteoblasts may be a potential mechanism by which miR­27a regulates steroid­induced ONFH.

Isoliquiritigenin Inhibits IL-1ß-Induced Production of Matrix Metalloproteinase in Articular Chondrocytes.

Osteoarthritis (OA) is a major joint disease in which inflammatory cytokine interleukin-1ß (IL-1ß) and matrix metalloproteinases (MMPs) play a pivotal role. Isoliquiritigenin has been reported to have anti-inflammation activity. In this study, the effect of isoliquiritigenin on IL-1ß-induced production of matrix metalloproteinase and nuclear factor κB (NF-κB) activation was analyzed. We treated primary cultured articular chondrocytes with isoliquiritigenin and the expressions of MMPs were analyzed on mRNA and protein level. The phosphorylation of IκBa and p65 was analyzed to detect NF-κB activation. We also used in vivo model by treating mice with isoliquiritigenin and detecting the level of MMPs. IL-1ß induced NF-κB activation and MMP-1, MMP-3, MMP-9, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 production on chondrocytes. A 10-µM isoliquiritigenin treatment significantly inhibited IL-1ß-induced NF-κB activation and these MMPs production on chondrocytes. Injecting isoliquiritigenin into rat knee joint also inhibited IL-1ß-induced NF-κB activation and MMPs production in articular cartilage. Isoliquiritigenin treatment inhibited IL-1ß-induced MMPs production and NF-κB activation both in vitro and in vivo, suggesting a potential therapeutic role of isoliquiritigenin to treat osteoarthritis.

Comparison of hyaluronic acid and PRP intra-articular injection with combined intra-articular and intraosseous PRP injections to treat patients with knee osteoarthritis.

The aim of this study was to evaluate the benefit provided by intraosseous infiltration combined with intra-articular injection of platelet-rich plasma to treat mild and moderate stages of knee joint degeneration (Kellgren-Lawrence score II-III) compared with other treatments, specifically intra-articular injection of PRP and of HA. Eighty-six patients with grade II to grade III knee OA according to the Kellgren-Lawrence classification were randomly assigned to intra-articular combined with intraosseous injection of PRP (group A), intra-articular PRP (group B), or intra-articular HA (group C). Patients in group A received intra-articular combined with intraosseous injection of PRP (administered twice, 2 weeks apart). Patients in group B received intra-articular injection of PRP every 14 days. Patients in group C received a series of five intra-articular injections of HA every 7 days. All patients were evaluated using the Visual Analogue Scale (VAS) and Western Ontario and McMaster Universities (WOMAC) score before the treatment and at 1, 3, 6, 12, and 18 months after treatment. There were significant improvements at the end of the 1st month. Notably, group A patients had significantly superior VAS and WOMAC scores than were observed in groups B and C. The VAS scores were similar in groups B and group C after the 6th month. Regarding the WOMAC scores, groups B and C differed at the 1st, 3rd, 6th, and 12th months; however, no significant difference was observed at the 18th month. The combination of intraosseous with intra-articular injections of PRP resulted in a significantly superior clinical outcome, with sustained lower VAS and WOMAC scores and improvement in quality of life within 18 months.

Identification of pathogenic genes related to rheumatoid arthritis through integrated analysis of DNA methylation and gene expression profiling.

The purpose of our study was to identify new pathogenic genes used for exploring the pathogenesis of rheumatoid arthritis (RA). To screen pathogenic genes of RA, an integrated analysis was performed by using the microarray datasets in RA derived from the Gene Expression Omnibus (GEO) database. The functional annotation and potential pathways of differentially expressed genes (DEGs) were further discovered by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Afterwards, the integrated analysis of DNA methylation and gene expression profiling was used to screen crucial genes. In addition, we used RT-PCR and MSP to verify the expression levels and methylation status of these crucial genes in 20 synovial biopsy samples obtained from 10 RA model mice and 10 normal mice. BCL11B, CCDC88C, FCRLA and APOL6 were both up-regulated and hypomethylated in RA according to integrated analysis, RT-PCR and MSP verification. Four crucial genes (BCL11B, CCDC88C, FCRLA and APOL6) identified and analyzed in this study might be closely connected with the pathogenesis of RA.

[Short-term effectiveness of arthroscopic single-bundle reconstruction of anterior cruciate ligament being centered within native ligament's tibial and femoral insertions].

OBJECTIVE: To evaluate the short-term effectiveness of arthroscopic single-bundle reconstruction of anterior cruciate ligament (ACL) being centered within the native ligament's tibial and femoral insertions with independent drilling of tibial and femoral tunnels. METHODS: Between September 2008 and September 2010, 33 patients with chronic ACL ruptures underwent arthroscopic reconstruction with four-stranded hamstring tendons in single-bundle. There were 19 males and 14 females, aged 22-33 years (mean, 26.4 years). Injuries were caused by traffic accident in 15 cases, by falling in 13 cases, and by sports in 5 cases. The location was the left knee in 20 cases and the right knee in 13 cases. The average time from injury to surgery was 6 months (range, 2-20 months). ACL reconstruction could be optimized when single-bundle grafts were centered within the native ligament's tibial and femoral insertions with independent drilling of tibial and femoral tunnels. KT-1000 test, Lachman test, and pivot-shift test were used to evaluate the knee stability, and the International Knee Documentation Committee (IKDC) and Lysholm scores to assess the knee function. RESULTS: Primary healing of incision was obtained in all patients, who had no complications of intra-articular infection, deep venous thrombosis of the lower extremity, and injury of blood vessels and nerves. All the patients were followed up 18.6 months on average (range, 13-24 months). At 1 year after operation, the results of Lachman test were negative in 31 cases and I degree positive in 2 cases, showing significant difference when compared with preoperative results (I degree positive in 4, II degree positive in 26, and III degree positive in 3) (Z = -5.42, P = 0.00). The results of pivot-shift test were negative in 31 cases, I degree positive in 2 cases, showing significant difference when compared with preoperative results (I degree positive in 15 and II degree positive in 18) (Z = -5.17, P = 0.00). The KT-1000 results of examination (134 N) showed that the side difference of anterior laxity was (1.2 +/- 0.7) mm at 25 degrees flexion and (0.8 +/- 0.6) mm at 70 degrees flexion, showing significant differences when compared with preoperative ones [(7.8 +/- 2.1) mm and (5.0 +/- 1.8) mm] (t = 16.19, P = 0.00; t = 13.28, P = 0.00). The IKDC score was significantly increased from 39.6 +/- 4.5 at preoperation to 95.1 +/- 1.6 at postoperation (t = - 78.88, P = 0.00), and Lysholm score was significantly increased from 48.3 +/- 3.6 at preoperation to 92.0 +/- 2.5 at postoperation (t = -42.00, P = 0.00). CONCLUSION: It is a reliable procedure to restore the stability of the knee that arthroscopic single-bundle reconstruction of ACL is centered within the native ligament's tibial and femoral insertions with independent drilling of tibial and femoral tunnels.