KasQ an Epimerase Primes the Biosynthesis of Aminoglycoside Antibiotic Kasugamycin and KasF/H Acetyltransferases Inactivate Its Activity.

Kasugamycin (KSM), an aminoglycoside antibiotic, is composed of three chemical moieties: D-chiro-inositol, kasugamine and glycine imine. Despite being discovered more than 50 years ago, the biosynthetic pathway of KSM remains an unresolved puzzle. Here we report a structural and functional analysis for an epimerase, KasQ, that primes KSM biosynthesis rather than the previously proposed KasF/H, which instead acts as an acetyltransferase, inactivating KSM. Our biochemical and biophysical analysis determined that KasQ converts UDP-GlcNAc to UDP-ManNAc as the initial step in the biosynthetic pathway. The isotope-feeding study further confirmed that 13C, 15N-glucosamine/UDP-GlcNH2 rather than glucose/UDP-Glc serves as the direct precursor for the formation of KSM. Both KasF and KasH were proposed, respectively, converting UDP-GlcNH2 and KSM to UDP-GlcNAc and 2-N'-acetyl KSM. Experimentally, KasF is unable to do so; both KasF and KasH are instead KSM-modifying enzymes, while the latter is more specific and reactive than the former in terms of the extent of resistance. The information gained here lays the foundation for mapping out the complete KSM biosynthetic pathway.

Switchable generation of rectangular noise-like pulse and dissipative soliton resonance in a fiber laser.

We report on the switchable generation of a rectangular noise-like pulse (NLP) and a dissipative soliton resonance (DSR) in a fiber laser with highly nonlinear effect at very low pump power. The NLP centered at 1530.5 nm demonstrates a new characteristic that its profile evolves gradually from rectangular shape to Gaussian-like shape with the increasing pump power. By appropriately manipulating the polarization controller (PC), the laser switches emit a DSR pulse centered at 1551.3 nm. The duration of the DSR could broaden from 17.4 ns to the cavity round trip time with increasing the pump power, while keeping the pulse profile and the intensity unaltered. This type of fiber laser may not only facilitate further investigations of the characteristics of NLP and DSR but also serve as a multi-functional optical source for potential applications.

Enhancement of ethyl (S)-4-chloro-3-hydroxybutanoate production at high substrate concentration by in situ resin adsorption.

Asymmetric reduction of ethyl 4-chloro-3-oxobutyrate (COBE) by carbonyl reductases presents an efficient way to produce Ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), an important chiral intermediate for the synthesis of hydroxymethylglutaryl-CoA reductase inhibitors such as Lipitor®. In this study, an NADPH-dependent carbonyl reductase (SrCR) from Synechocystis sp. was characterized to demonstrate a broad substrate spectrum, and the highest activity (53.1U/mg protein) with COBE. To regenerate the cofactor NADPH, Bacillus subtilis glucose dehydrogenase was successfully coexpressed with SrCR. Owing to the product inhibition, no more than 400mM of COBE could be completely reduced to (S)-CHBE using the recombinant Escherichia coli/pET-SrCR-GDH. The macroporous adsorption resin HZ 814 was applied to adsorb (S)-CHBE in situ to alleviate the product inhibitio. Consequently, 3000mM (494g/L) of COBE was bioconverted within 8h, resulting in a (S)-CHBE yield of 98.2%, with 99.4% ee and total turnover number of 15,000, revealed great industrial potential of (S)-CHBE production.

The status of diagnosis and treatment to intracranial hypotension, including SIH.

Intracranial hypotension, especially spontaneous intracranial hypotension (SIH), is a well-recognized entity associated with cerebrospinal fluid (CSF) leaks, and has being recognized better in resent years, while still woefully inadequate. An increasing number of factors including iatrogenic factors are realized to involve in development and progression of intracranial hypotension. The diagnosis remains difficult due to the various clinical manifestations, some of which are nonspecific and easily to be neglected. Multiple imaging tests are optional in CSF leakage identification while clinicians are still confronted with difficulties when making selection resulting from superiorities and disadvantages of different imaging tests. Treatments for intracranial hypotension are multifarious but evidence is anecdotal. Values of autologous epidural blood patching (EBP), the mainstay of first-line interventional treatment currently, is getting more and more regards while there are no systematic review of its efficacy and risks. Hereby, the purpose of this review was to reveal the present strategy of intracranial hypotension diagnosis and treatment by reviewing literatures, coupled with our experience in clinical work.

Synthesis and antitumor activity of novel substituted uracil-1'(N)-acetic acid ester derivatives of 20(S)-camptothecins.

A series of novel substituted uracil-1'(N)-acetic acid esters (6-20) of camptothecins (CPTs) were synthesized by the acylation method. These new compounds were evaluated for in vitro antitumor activity against tumor cell lines, A549, Bel7402, BGC-823, HCT-8 and A2780. In vitro results showed that most of the derivatives exhibited comparable or superior cytotoxicity compare to CPT (1) and topotecan (TPT, 2), with 12 and 13 possessing the best efficacy. Four compounds, 9, 12, 13 and 16, were selected to be evaluated for in vivo antitumor activity against H22, BGC-823 and Bel-7402 in mice. In vivo testing results indicated that 12 and 13 had antitumor activity against mouse liver carcinoma H22 close to Paclitaxel and cyclophosphamide. 12 had similar antitumor activity against human gastric carcinoma BGC-823 in nude mice compared to irinotecan (3) and possessed better antitumor activity against human hepatocarcinoma Bel-7402 in nude mice than 2. It is also discovered that 12 showed a similar mechanism but better inhibitory activity on topoisomerase I (Topo I) compared to 2. These findings indicate that 20(S)-O-fluorouracil-1'(N)-acetic acid ester derivative of CPTs, 12, could be developed as an antitumor drug candidate for clinical trial.

How reliable is the Ki-67 cytological index in grading pancreatic neuroendocrine tumors? A meta-analysis.

OBJECTIVE: To investigate the accuracy of the cytological Ki-67 index in distinguishing intermediate and high-grade (G2 + G3) from low-grade (G1) pancreatic neuroendocrine tumors (PNETs). METHODS: Two investigators independently searched databases to identify eligible studies using the following term: ('Ki-67') AND ('pancreatic endocrine tumor' OR 'pancreatic neuroendocrine tumor' OR 'pancreatic endocrine tumour' OR 'pancreatic neuroendocrine tumour' OR 'pancreatic endocrine tumors' OR 'pancreatic neuroendocrine tumors' OR 'pancreatic endocrine tumours' OR 'pancreatic neuroendocrine tumours'), and meta-analysis was performed to calculate the pooled sensitivity, specificity, positive (PLR) and negative likelihood ratio (NLR), and diagnostic odds ratio (DOR). RESULTS: A total of 263 lesions from 13 studies were included in the study. The pooled sensitivity and specificity of Ki-67 (cut-off value: 2%) in the differential diagnosis of G2 + G3 from G1 PNETs were 64% and 87%, respectively. The pooled PLR, NLR and DOR were 3.96, 0.42 and 11.21, respectively. The area under the summary receiver operating characteristic curve (AUROC) was 0.8397. While the cut-off value of Ki-67 index was set as 5%, the sensitivity and specificity were increased up to 69% and 93%, respectively, and the AUROC was increased to 0.955. CONCLUSION: The cytological Ki-67 index is very useful in distinguishing intermediate and high-grade from low-grade PNETs, and a cut-off value of 5% had a better predictive value compared with that of 2%.

Endoscopic submucosal dissection for early gastric cancer in elderly patients: a meta-analysis.

BACKGROUND: The effectiveness of endoscopic submucosal dissection (ESD) has been increasingly reported. However, studies addressing the safety and application value of ESD in elderly patients with early gastric cancer (EGC) were still lacking. This meta-analysis was intended to evaluate the feasibility and safety of ESD in elderly patients with EGC. METHODS: A systematic search was conducted in PubMed, EBSCO, Cochrane Library, EMBASE, and Web of Science. Studies were screened out if data of elderly and non-elderly gastric cancer patients were reported separately. The qualities of included studies were assessed using Newcastle-Ottawa Quality Assessment Scale. The pooled odd ratios (ORs) with 95 % confidence intervals (CIs) were calculated. Statistical analysis was conducted using the Review Manager 5.2 (Cochrane Collaboration, Oxford, UK). RESULTS: Nine studies (eight in Japan, one in China), including a total of 30,100 lesions, met the inclusion criteria. The "en bloc" and histological complete resection rates of the elderly and non-elderly groups were similar [OR, 0.98, 95 % CI, 0.56 to 1.71; P = 0.93 and OR, 0.79, 95 % CI, 0.58 to 1.07; P = 0.13, respectively]. As for procedure-related complications, similar perforation rates [OR, 1.19, 95 % CI, 0.94 to 1.51; P = 0.15], and bleeding rates [OR, 1.13, 95 % CI, 0.83 to 1.56); P = 0.43] between the elderly and non-elderly groups were observed. Whereas, the elderly patients had a higher procedure-related pneumonia rate compared with non-elderly ones [OR, 2.18, 95 % CI, 1.55 to 3.08; P < 0.01]. CONCLUSIONS: The ESD procedure appears to be a safe technique in elderly patients with EGC while appropriate approach should be taken to avoid procedure-related pneumonia.

Engineered Expression Vectors Significantly Enhanced the Production of 2-Keto-D-gluconic Acid by Gluconobacter oxidans.

2-Keto-D-gluconic acid (2KGA), a precursor of the important food antioxidant erythorbic acid, can be produced by Gluconobacter oxidans. To genetically engineer G. oxidans for improved 2KGA production, six new expression vectors with increased copy numbers based on pBBR1MCS-5 were constructed via rational mutagenesis. The utility of the mutant vectors was demonstrated by the increased ga2dh mRNA abundance, enzyme activity, and 2KGA production when the ga2dh gene was overexpressed using these vectors. Among the obtained constructs, G. oxidans/pBBR-3510-ga2dh displayed the highest oxidative activity toward gluconic acid (GA). In a batch biotransformation process, the G. oxidans/pBBR-3510-ga2dh strain exhibited 2KGA productivity (0.63 g/g CWW/h) higher than that obtained using strain G. oxidans/pBBR-ga2dh (0.40 g/g CWW/h). When sufficient oxygen was supplied during the biotransformation, up to 480 g/L GA was exhausted in 45 h by the G. oxidans/pBBR-3510-ga2dh strain and approximately 486 g/L 2KGA was produced, generating the productivity of 0.54 g/g CWW/h.

Functional expression of a novel alkaline-adapted lipase of Bacillus amyloliquefaciens from stinky tofu brine and development of immobilized enzyme for biodiesel production.

Using enrichment procedures, a lipolytic strain was isolated from a stinky tofu brine and was identified as Bacillus amyloliquefaciens (named B. amyloliquefaciens Nsic-8) by morphological, physiological, biochemical tests and 16S rDNA sequence analysis. Meanwhile, the key enzyme gene (named lip BA) involved in ester metabolism was obtained from Nsic-8 with the assistance of homology analysis. The novel gene has an open reading frame of 645 bp, and encodes a 214-amino-acid lipase (LipBA). The deduced amino acid sequence shows the highest identity with the lipase from B. amyloliquefaciens IT-45 (NCBI database) and belongs to the family of triacylglycerol lipase (EC 3.1.1.3). The lipase gene was expressed in Escherichia coli BL21(DE3) using plasmid pET-28a. The enzyme activity and specific activity were 250 ± 16 U/ml and 1750 ± 153 U/mg, respectively. The optimum pH and temperature of the recombinant enzyme were 9.0 and 40 °C respectively. LipBA showed much higher stability under alkaline conditions and was stable at pH 7.0-11.0. The Km and Vmax values of purified LipBA using 4-nitrophenyl palmitate as the substrate were 1.04 ± 0.06 mM and 119.05 ± 7.16 µmol/(ml min), respectively. After purification, recombinant lipase was immobilized with the optimal conditions (immobilization time 3 h at 30 °C, with 92 % enzyme recovery) and the immobilized enzyme was applied in biodiesel production. This is the first report of the lipase activity and lipase gene obtained from B. amyloliquefaciens (including wild strain and recombinant strain) and the recombinant LipBA with the detailed enzymatic properties. Also the preliminary study of the transesterification shows the potential value in biodiesel production applications.

Improving Gluconobacter oxydans performance in the in situ removal of the inhibitor for asymmetric resolution of racemic 1-phenyl-1,2-ethanediol.

Gluconobacter oxydans DSM2003 was used to catalyze the oxidation of racemic 1-phenyl-1,2-ethanediol (PED) for the production of (S)-enantiomer. The oxidative product mandelic acid produced strong inhibition to this reaction and largely reduced the activity of biocatalyst, which was the key problem in the reaction. In order to overcome this bottleneck, an anion exchange resin was selected and introduced as adsorbent for the in situ removal of the inhibitor from the reaction system. This method increased the substrate concentration from 12 to 60 g/L and the yield of (S)-PED by approximately five times from 4.9 g/L, on the premise that the enantiomeric excess (ee) value of (S)-PED remained above 96% and the reaction time was no more than 20 h. Moreover, the final space-time yield was over 1.2g/L/h, which was higher than that reported from previous studies.

Determination of dihydroxyacetone and glycerol in fermentation process by GC after n-methylimidazole catalyzed acetylation.

A gas chromatographic method that accurately measures glycerol and dihydroxyacetone from a fermentation broth is described in this paper. The method incorporates a sample derivatization reaction using n-methylimidazole as catalyst in the presence of acetic anhydride. Resulting derivatives are separated on a DB-5 capillary column and flame ionization detector. Results show that 10 µL n-methylimidazole and 75 µL acetic anhydride are sufficient to complete the acetylation for glycerol and dihydroxyacetone at room temperature for 5 min. The present method exhibits good linearity at a concentration range of 1-100 g/L with excellent regression (R(2) > 0.9997). The limits of detection are 0.025 and 0.013 g/L for dihydroxyacetone and glycerol, respectively. The method has been successfully applied to the monitoring and control of the fermentation process, and recoveries are in the range of 95.5-98.8% with relative standard deviations below 1%.

Highly selective oxidation of benzyl alcohol using engineered Gluconobacter oxydans in biphasic system.

The Gluconobacter oxydans M5 with disruption of the pyrroloquinoline quinine-dependent membrane-bound aldehyde dehydrogenase (ALDH) was used for the oxidation of benzyl alcohol. The selectivity toward benzaldehyde showed an obvious increase for the engineered strain, which reached the 67.3%, while the wild strain had only 2.8%. Meantime, the aqueous/isooctane (1:1) biphasic system was used for the further improvement of selectivity. By these methods, nearly 100% selectivity and conversion rate could be obtained within 1 h at the optimum initial benzyl alcohol concentration of 5.0 g/l.

Optimization of immobilization for selective oxidation of benzyl alcohol by Gluconobacter oxydans using response surface methodology.

This study used the Box-Behnken design and response surface methodology to optimize immobilization of Gluconobacter oxydans in Ca-alginate gel for the production of benzaldehyde in a biphasic system. Immobilization parameters, such as Na-alginate concentration, cell load, and bead diameter, were optimized. The mathematical model developed was validated and proven to be statistically adequate and accurate in predicting the response. For both activity and stability responses, the best results were achieved at alginate concentration of 2.55% (w/v), cell load of 49.26 mg/ml, and 2.2 mm bead diameter. Under these conditions, retention activity of 87.6% could be attained for the immobilized cell. In addition, the oxidative activity of immobilized cells was retained at 53.2% compared with that of free cells after 10 repeated batch reactions, while only 15.7% of activity remained in free cells.

Enhanced production of dihydroxyacetone from glycerol by overexpression of glycerol dehydrogenase in an alcohol dehydrogenase-deficient mutant of Gluconobacter oxydans.

Gluconobacter oxydans can rapidly and incompletely oxidize glycerol to dihydroxyacetone (DHA), a versatile product extensively used in cosmetic, chemical and pharmaceutical industries. To improve DHA production, the glycerol dehydrogenase (GDH) responsible for DHA formation was overexpressed in G. oxydans M5AM, in which the gene coding for the membrane-bound alcohol dehydrogenase (ADH) was interrupted. Real-time PCR and enzyme activity assay revealed that the absence of ADH together with the overexpression of GDH gene resulted in an increased GDH activity in the resulting strain M5AM/GDH, which led to a substantially enhanced production of DHA in a resting cell system. In a batch biotransformation process, M5AM/GDH exhibited a 2.4-fold increased DHA productivity of 2.4g/g CDW/h from 1.0g/g CDW/h, yielding 96g/L DHA from 100g/L glycerol. When 140g/L glycerol was supplied, a final DHA concentration of 134g/L was accumulated within 14h. In four repeated batch runs, 385g DHA over a time period of 34h was achieved from 400g glycerol with an average productivity of 2.2g/g CDW/h. These results indicated that this newly developed strain G. oxydans M5AM/GDH with high productivity and increased tolerance against product inhibition has potential for DHA production in an industrial bioconversion process.

Pyrroloquinoline quinone biosynthesis in Escherichia coli through expression of the Gluconobacter oxydans pqqABCDE gene cluster.

We have expressed the pqqABCDE gene cluster from Gluconobacter oxydans, which is involved in pyrroloquinoline quinone (PQQ) biosynthesis, in Escherichia coli, resulting in PQQ accumulation in the medium. Since the gene cluster does not include the tldD gene needed for PQQ production, this result suggests that the E. coli tldD gene, which shows high homology to the G. oxydans tldD gene, carries out that function. The synthesis of PQQ activated d-glucose dehydrogenase in E. coli and the growth of the recombinant was improved. In an attempt to increase the production of PQQ, which acts as a vitamin or growth factor, we transformed E. coli with various recombinant plasmids, resulting in the overproduction of the PQQ synthesis enzymes and, consequently, PQQ accumulation--up to 6 mM--in the medium. This yield is 21.5-fold higher than that obtained in previous studies.

Membrane-bound pyrroloquinoline quinone-dependent dehydrogenase in Gluconobacter oxydans M5, responsible for production of 6-(2-hydroxyethyl) amino-6-deoxy-L-sorbose.

A membrane-bound protein purified from Gluconobacter oxydans M5 was confirmed to be a pyrroloquinoline quinone-dependent D-sorbitol dehydrogenase. Gene disruption and complementation experiments demonstrated that this enzyme is responsible for the oxidation of 1-(2-hydroxyethyl) amino-1-deoxy-D-sorbitol (1NSL) to 6-(2-hydroxyethyl) amino-6-deoxy-L-sorbose (6NSE), which is the precursor of an antidiabetic drug, miglitol.