Suction Drain Tip Cultures in Predicting a Surgical Site Infection.

STUDY DESIGN: Retrospective study. PURPOSE: This study aimed to evaluate the prognostic value of drain tip culture after spinal surgery with a large number of participants. OVERVIEW OF LITERATURE: The routine culture of suction drain tips that are placed in the surgical site of spinal surgeries has been performed in many institutions to detect surgical site infection (SSI). However, few reports have evaluated drain tip culture as a prognostic for SSI after spinal surgery. Materials and. METHODS: This study retrospectively included 1,415 consecutive patients who underwent spinal surgery between January 2016 and December 2021. Patients diagnosed with infectious diseases were excluded. Prophylactic antibiotics were administered intraoperatively and 24 hours postoperatively. Drains were removed when the volume of postoperative fluid drainage was <50 mL and <100 mL in patients who underwent cervical and thoracic surgery and lumbar surgery in the preceding 24 hours, respectively, and cultures were made. We evaluated the correlation between the results of positive drain tip culture and SSI. RESULTS: Positive drain tip cultures were found in 51 cases (3.6%). SSI was identified in 34 cases (2.4%). The most frequently isolated microorganism was methicillin-resistant Staphylococcus epidermidis (61.8%). The sensitivity, specificity, and positive, and negative predictive values of drain tip culture were 50.0%, 97.4%, 32.1%, and 98.8%, respectively. The same bacteria were isolated from the surgical lesion in 16 of 17 SSI cases with a positive drain tip culture, thereby giving a bacteria matching rate between tissue culture and drain tip culture of 94.1%. The number of surgery levels, drain remaining period, and drain tip culture positivity were significantly increased in the SSI group. CONCLUSIONS: Drain tip cultures might be useful for predicting SSI. Drain tip culture had a high positivity rate in the SSI group, and the coincidence rate for the causative pathogen was high.

Modification of ginsenoside saponin composition via the CRISPR/Cas9-mediated knockout of protopanaxadiol 6-hydroxylase gene in Panax ginseng.

Background: The roots of Panax ginseng contain two types of tetracyclic triterpenoid saponins, namely, protopanaxadiol (PPD)-type saponins and protopanaxatiol (PPT)-type saponins. In P. ginseng, the protopanaxadiol 6-hydroxylase (PPT synthase) enzyme catalyses protopanaxatriol (PPT) production from protopanaxadiol (PPD). In this study, we constructed homozygous mutant lines of ginseng by CRISPR/Cas9-mediated mutagenesis of the PPT synthase gene and obtained the mutant ginseng root lines having complete depletion of the PPT-type ginsenosides. Methods: Two sgRNAs (single guide RNAs) were designed for target mutations in the exon sequences of the two PPT synthase genes (both PPTa and PPTg sequences) with the CRISPR/Cas9 system. Transgenic ginseng roots were generated through Agrobacterium-mediated transformation. The mutant lines were screened by ginsenoside analysis and DNA sequencing. Result: Ginsenoside analysis revealed the complete depletion of PPT-type ginsenosides in three putative mutant lines (Cr4, Cr7, and Cr14). The reduction of PPT-type ginsenosides in mutant lines led to increased accumulation of PPD-type ginsenosides. The gene editing in the selected mutant lines was confirmed by targeted deep sequencing. Conclusion: We have established the genome editing protocol by CRISPR/Cas9 system in P. ginseng and demonstrated the mutated roots producing only PPD-type ginsenosides by depleting PPT-type ginsenosides. Because the pharmacological activity of PPD-group ginsenosides is significantly different from that of PPT-group ginsenosides, the new type of ginseng mutant producing only PPD-group ginsenosides may have new pharmacological characteristics compared to wild-type ginseng. This is the first report to generate target-induced mutations for the modification of saponin biosynthesis in Panax species using CRISPR-Cas9 system.

Characterization of a Pentacyclic Triterpene Acetyltransferase Involved in the Biosynthesis of Taraxasterol and ψ-Taraxasterol Acetates in Lettuce.

Triterpenoids exist in a free state and/or in conjugated states, such as triterpene glycosides (saponins) or triterpene esters. There is no information on the enzyme participating in the production of triterpene esters from free triterpenes. Lettuce (Lactuca sativa) contains various pentacyclic triterpene acetates (taraxasterol acetates, ψ-taraxasterol acetates, taraxerol acetates, lupeol acetates, α-amyrin acetates, ß-amyrin acetates, and germanicol acetate). In this study, we report a novel triterpene acetyltransferase (LsTAT1) in lettuce involved in the biosynthesis of pentacyclic triterpene acetates from free triterpenes. The deduced amino acid sequences of LsTAT1 showed a phylogenetic relationship (43% identity) with those of sterol O-acyltransferase (AtSAT1) of Arabidopsis thaliana and had catalytic amino acid residues (Asn and His) that are typically conserved in membrane-bound O-acyltransferase (MBOAT) family proteins. An analysis of LsTAT1 enzyme activity in a cell-free system revealed that the enzyme exhibited activity for the acetylation of taraxasterol, ψ-taraxasterol, ß-amyrin, α-amyrin, lupeol, and taraxerol using acetyl-CoA as an acyl donor but no activity for triterpene acylation using a fatty acyl donor. Lettuce oxidosqualene cyclase (LsOSC1) is a triterpene synthase that produces ψ-taraxasterol, taraxasterol, ß-amyrin and α-amyrin. The ectopic expression of both the LsOSC1 and LsTAT1 genes in yeast and tobacco could produce taraxasterol acetate, ψ-taraxasterol acetate, ß-amyrin acetate, and α-amyrin acetate. However, expression of the LsTAT1 gene in tobacco was unable to induce the conversion of intrinsic sterols (campesterol, stigmasterol, and ß-sitosterol) to sterol acetates. The results demonstrate that the LsTAT1 enzyme is a new class of acetyltransferase belong to the MBOAT family that have a particular role in the acetylation of pentacyclic triterpenes and are thus functionally different from sterol acyltransferase conjugating fatty acyl esters.

Identification of triterpenes and functional characterization of oxidosqualene cyclases involved in triterpene biosynthesis in lettuce (Lactuca sativa).

Lettuce (Lactuca sativa) is a member of the family Asteraceae and is most often used for green salads. Triterpenes are the largest class of natural compounds in plants and have beneficial health effects. Here, we identified various triterpene esters (taraxasterol acetates, ψ-taraxasterol acetates, taraxerol acetates, lupeol acetates, α-amyrin acetates, ß-amyrin acetates, and germanicol acetate) and free triterpenes (α-amyrin, ß-amyrin, taraxerol, and taraxasterol) in both the leaves and roots of lettuce. Triterpene biosynthesis occurs through the action of oxidosqualene cyclase (OSC), which generates various types of triterpenes from 2,3-oxidosqualene. None of the OSC genes involved in triterpene biosynthesis in lettuce have been characterized. Five putative lettuce OSC genes (LsOSC1, LsOSC2, LsOSC3, LsOSC4, and LsOSC5) were selected from a transcriptome database. These five genes were functionally characterized via heterologous expression in yeast. The first two enzymes were multifunctional triterpene synthase and the last three genes were monofunctional. Transgenic yeast expressing LsOSC1 produced five triterpenes, namely, taraxasterol, Ψ-taraxasterol, α-amyrin, ß-amyrin, and dammarenediol-II. Yeast expressing LsOSC2 produced baurenol and Ψ-taraxasterol. LsOSC3, LsOSC4, and LsOSC5 expression led to ß-amyrin, taraxerol, and lupeol production, respectively. Transcriptional activity assessment of the five genes revealed that all the OSC genes were more actively transcribed in roots than in leaves, and LsOSC5 among the five OSC genes showed the highest expression in both the leaves and the roots. In conclusion, we identified structurally diverse free triterpenes and triterpene esters in lettuce plants and characterized five OSC genes, which are key enzymes involved in the biosynthesis of diverse triterpenes in lettuce.

Biorefinery of instant noodle waste to biofuels.

Instant noodle waste, one of the main residues of the modern food industry, was employed as feedstock to convert to valuable biofuels. After isolation of used oil from the instant noodle waste surface, the starch residue was converted to bioethanol by Saccharomyces cerevisiae K35 with simultaneous saccharification and fermentation (SSF). The maximum ethanol concentration and productivity was 61.1g/l and 1.7 g/lh, respectively. After the optimization of fermentation, ethanol conversion rate of 96.8% was achieved within 36 h. The extracted oil was utilized as feedstock for high quality biodiesel conversion with typical chemical catalysts (KOH and H2SO4). The optimum conversion conditions for these two catalysts were estimated; and the highest biodiesel conversion rates were achieved 98.5% and 97.8%, within 2 and 3h, respectively. The high conversion rates of both bioethanol and biodiesel demonstrate that novel substrate instant noodle waste can be an attractive biorefinery feedstock in the biofuels industry.

Development of glycerol-utilizing Escherichia coli strain for the production of bioethanol.

The production of bioethanol was studied using recombinant Escherichia coli with glycerol as a carbon source. Glycerol is an attractive feedstock for biofuels production since it is generated as a major byproduct in biodiesel industry; therefore, we investigated the conversion of glycerol to bioethanol using E. coli BL21 (DE3) which harbors several genes in ethanol production pathway of Enterobacter aerogenes KCTC 2190. Fermentation was carried out at 34°C for 42h, pH 7.6, using defined production medium. Under optimal conditions, bioethanol production by the recombinant E. coli BL21 (DE3), strain pEB, was two-fold (3.01g/L) greater than that (1.45g/L) by the wild-type counterpart. The results obtained in this study will provide valuable guidelines for engineering bioethanol producers.

Production of cephalosporin C using crude glycerol in fed-batch culture of Acremonium chrysogenum M35.

In this study, cephalosporin C production by Acremonium chrysogenum M35 cultured with crude glycerol instead of rice oil and methionine was investigated. The addition of crude glycerol increased cephalosporin C production by 6-fold in shake-flask culture, and also the amount of cysteine. In fed-batch culture without methionine, crude glycerol resulted only in overall improvement in cephalosporin C production (about 700%). In addition, A. chrysogenum M35 became highly differentiated in fed-batch culture with crude glycerol, compared with the differentiation in batch culture. The results presented here suggest that crude glycerol can replace methionine and plant oil as cysteine and carbon sources during cephalosporin C production by A. chrysogenum M35.

Antioxidant and antimelanogenic properties of chestnut flower extract.

In this study, we analyzed the antioxidant and antimelanogenic properties of a variety of solvent extracts of pre-bloom and full-bloom chestnut flowers. Among the solvent extracts, a pre-bloom methanol extract (preM) and an ethanol extract (preE) showed the highest amounts of phenolics (467.92+/-0.45 and 456.24+/-5.88 mg of gallic acid equivalent/g of extract) and flavonoids (60.96+/-1.86 and 41.59+/-8.57 mg of quercetin equivalent/g of extract). These extracts exhibited the highest DPPH radical and reducing activities, as well as the greatest mushroom tyrosinase inhibition activity. In addition, preE effectively protected the skin against ultraviolet (UV) rays. Further, extracts were tested for cytotoxicity on human melanoma cells (SK-MEL-2), and we observed that all the extracts were non-cytotoxic for the cells. Their effects on tyrosinase and melanin inhibitory action were further assessed, and we found that all the extracts reduced the tyrosinase activity and melanin formation of SK-MEL-2 cells as effectively as arbutin. Moreover, the protein level expression of tyrosinase decreased dramatically. However, the protein levels of the other melanogenic enzymes, tyrosinase-related protein 1 (TRP1) and dopachrome tautomerase (DCT), were not altered significantly. Therefore, the antimelanogenic effects of chestnut flower extracts were attributable to their inhibitory effects on tyrosinase via their anti-oxidative action, making them a strong candidate for use in food, cosmetics, and pharmaceutical applications.

PESTAS: a web server for EST analysis and sequence mining.

SUMMARY: We have developed a web server for the high-throughput annotation of expressed sequence tags (ESTs) called pipeline for EST analysis service (PESTAS). PESTAS processes entire datasets with an automated pipeline of 13 analytic services, then deposits the data into the MySQL database and transforms it into three kinds of reports: preprocessing, assembling and annotation. All annotated information is provided to the scientist and can be downloaded through a web browser. To get more relevant functional annotation results, a curation function was introduced with which biologists can easily change the best-hit annotation information. We included a gene chip module that detects gene expression differences between libraries by comparing accession number counts from BLAST search results. PESTAS also provides access to the pathway information of KEGG, which is useful for mapping the relationships among networks of annotated enzymes, and is especially valuable for those researchers interested in biological pathways. AVAILABILITY: PESTAS is available at http://pestas.kribb.re.kr/.

Purification and characterization of fibrinolytic enzyme from cultured mycelia of Armillaria mellea.

A fibrinolytic enzyme was purified from the cultured mycelia of Armillaria mellea by ion-exchange chromatography followed by gel filtration, and was designated A. mellea metalloprotease (AMMP). The purification protocol resulted in a 627-fold purification of the enzyme, with a final yield of 6.05%. The apparent molecular mass of the purified enzyme was estimated to be 21kDa by SDS-PAGE, fibrin-zymography and gel filtration chromatography, which revealed a monomeric form of the enzyme. The optimal reaction pH value and temperature were, pH 6.0, and 33 degrees C, respectively. This protease effectively hydrolyzed fibrinogen, preferentially digesting the Aalpha-chain over the Bbeta- and r-chains. Enzyme activity was inhibited by Cu(2+) and Co(2+), but enhanced by the addition of Ca(2+) and Mg(2+) ions. Furthermore, AMMP activity was potently inhibited by EDTA, and was found to exhibit a higher specificity for the substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The first 24 amino acid residues of the N-terminal sequence were MFSLSSRFFLYTLCL SAVAVSAAP, which is extremely similar to the 24 amino acid residues of the N-terminal sequence of the fruiting body of A. mellea. These data suggest that the fibrinolytic enzyme AMMP, obtained from the A. mellea exhibits a profound fibrinolytic activity. The mycelia of A. mellea may thus represent a potential source of new therapeutic agents to treat thrombosis.