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The methylotrophic yeast species Komagataella phaffii (synonym: Pichia pastoris) is widely used as a host for recombinant protein production. Although several genetic engineering techniques are being employed on K. phaffii, advanced methods such as in vivo DNA assembly in this yeast species are required for synthetic biology applications. In this study, we established a technique for accomplishing one-step in vivo assembly of multiple DNA fragments and genomic integration in K. phaffii. To concurrently achieve an accurate multiple DNA assembly and a high-efficient integration into the target genomic locus in vivo, a K. phaffii strain, lacking a non-homologous end joining-related protein, DNA ligase IV (Dnl4p), that has been reported to improve gene targeting efficiency by homologous recombination, was used. Using green fluorescent protein along with the lycopene biosynthesis, we showed that our method that included a Dnl₄ₚ-defective strain permits direct and easy engineering of K. phaffii strains.

4-hydroxybenzoic acid (4-HBA) is an industrially important aromatic compound, and there is an urgent need to establish a bioprocess to produce this compound in a sustainable and environmentally friendly manner from renewable feedstocks such as cellulosic biomass. Here, we developed a bioprocess to directly produce 4-HBA from cellulose using a recombinant Pichia pastoris strain that displays heterologous cellulolytic enzymes on its cell surface via the glycosylphosphatidylinositol (GPI)-anchoring system. β-glucosidase (BGL) from Aspergillus aculeatus, endoglucanase (EG) from Trichoderma reesei, and cellobiohydrolase (CBH) from Talaromyces emersonii were co-displayed on the cell surface of P. pastoris using an appropriate GPI-anchoring domain for each enzyme. The cell-surface cellulase activity was further enhanced using P. pastoris SPI1 promoter- and secretion signal sequences. The resulting strains efficiently hydrolyzed phosphoric acid swollen cellulose (PASC) to glucose. Then, we expressed a highly 4-HBA-resistant chorismate pyruvate-lyase (UbiC) from Providencia rustigianii in the cellulase-displaying strain. This strain produced 975 mg/L of 4-HBA from PASC, which corresponding to 36.8% of the theoretical maximum yield, after 96 h of batch fermentation without the addition of commercial cellulase. This 4-HBA yield was over two times higher than that obtained from glucose (12.3% of the theoretical maximum yield). To our knowledge, this is the first report on the direct production of an aromatic compound from cellulose using cellulase-displaying yeast.

The development of countercurrent chromatography (CCC) technology enabled to perform higher peak resolutions and more shortened separation times even for protein separation using aqueous two-phase solvent systems composed of polyethylene glycol (PEG) and inorganic salts (or dextrans). By eliminating solid support matrix, all analytes can be recovered from the coiled column after completing the separation. Recently, it has been found that droplets of biomolecules formed by liquid-liquid phase separation in cells closely relate to the transcription, regulation of signal transduction and formation of amyloids, etc. Meanwhile, although CCC is a separation technique based on liquid-liquid partitioning of analytes between two immiscible phases, the mechanism of separation could suggest some idea concerning the formation of biomolecule droplets in cells. This article describes the recent advances of the CCC apparatus, the coiled separation column, the choice of suitable two-phase solvent system and the application to separation and purification of bioactive macromolecules such as proteins and enzymes and also discusses the possibility of CCC as a tool to reveal new mechanical roles of biomolecule droplets in the cellular environments.

Bioactive plant-based secondary metabolites such as stilbenoids, flavonoids, and benzylisoquinoline alkaloids (BIAs) are produced from l-tyrosine (l-Tyr) and have a wide variety of commercial applications. Therefore, building a microorganism with high l-Tyr productivity (l-Tyr chassis) is of immense value for large-scale production of various aromatic compounds. The aim of this study was to develop an l-Tyr chassis in the nonconventional yeast

Steroid saponins are the medicinal compounds and nutrition ingredients of medicine food homology (MFH) Dioscorea zingiberensis C. H. Wright (D. zingiberensis) yam. Our phytochemical investigation of the edible rhizomes resulted in 9 new furostanol steroid saponins named dioscins A–I (1–9), together with 11 known steroid saponins. Their chemical structures were elucidated based on spectroscopic and chemical analyses. The new dioscins were evaluated for their anti-inflammatory and beneficial effects against cerebral ischemia reperfusion (I/R) injury on RAW264.7 and PC12 cells in vitro, respectively. Dioscins A, B, and G revealed considerable anti-I/R effect through an anti-inflammatory mechanism based on the decreasing concentration of pro-inflammatory (TNF-α and IL-6) and down-regulating the NF-κB expression. The present research demonstrated that daily consumption of this yam plant probably prevented the I/R occurrence via the anti-inflammatory property of steroid saponins, and it also enriched the steroid saponin library, providing the possibility to develop MFH-containing steroid saponins into functional foods for maintenance of human health or drugs for the treatment of I/R disease.

The methylotrophic yeast species

Background: Motion of the two mutually immiscible liquids in hydrodynamic countercurrent chromatographic systems is speculated based on the observation of their behavior in a closed coiled tube rotating in unit gravity. Materials and Methods: The experiment revealed an up and down pattern of four stages of twophase volume ratio occupied at the head end of the coil according to the rotation speed. These two-phase behaviors are comprehensively explained on the bases of interplay between the unit gravity and centrifugal force generated by rotation of the coil. This theory is successfully extended to explain the two-phase behavior in a coil undergoing the type-I and type-J planetary motions. Results and Discussion: The type-I planetary motion produces the centrifugal force distribution similar to that of slowly rotating coil in unit gravity (Stage I), where both phases competitively move toward the head of the coil. In contrast, the type-J planetary motion displays complex distribution patterns of centrifugal force according to the location of the coil on the holder hence the two-phase motion varies with the ß values. When ß is 0.5 – 0.75, the force pattern simulates that of the rotating coil in unit gravity at 120 rpm (Stage III) where the lighter phase moves toward the head leaving the heavier phase behind. Conclusion: This clearly demonstrates the importance of the proper choice of ß values in highspeed countercurrent chromatography utilizing the type-J planetary motion.

It is a research hotspot to use natural compounds in treatment of cerebral ischemia reperfusion (I/R) for a refractory disease throughout the worldwide without available drugs or treatments at present. Our previous study has demonstrated that diosgenin (DIO), a starting material to synthesize various steroid anti-inflammatory drugs in medical industry, showed medicinal effect against I/R via inhibiting aberrant inflammatory reaction induced by I/R. However, the detailed anti-inflammatory network of DIO in treatment of I/R still remains to be further explored.HIKESHI was firstly identified as a novel target of DIO used for I/R by rat brain proteomic analysis, and mechanistic efforts were focused based on this gene. Hopefully, extensive detailed molecular mechanisms of DIO against I/R was established and confirmed.The effect of DIO against I/R was examined in vitro and in vivo, which cells (SH-SY5Y and PC12) and rats were experienced to ORD/RP and MCAO exposures, respectively, to establish I/R modes. Staining was used to evaluate the pathological procedure of DIO used for I/R. Protein changes including expression, interaction, and activity during DIO's anti-I/R effect were assessed with real time PCR, western blot, Co-IP, luciferase reporter assay.In the current study, HIKESHI and HSP70 were both upregulated, when I/R cells and rats were treated with DIO in vitro and in vivo. Mechanistically, DIO stimulated the binding of HIKESHI to HSP70 and facilitated the translocation of HSP70 into nucleus. Subsequently, HSP70 blunted the transcription activity of NF-κB after physical interaction with this transcription factor, and therefore led to the suppression of its downstream pro-inflammatory cytokine (TNF-α, IL-1β, and IL-6) release into surrounding I/R lesion area. Conversely, HIKESHI or HSP70 knockdowns attenuated the nuclear translocation and restraint on NF-κB-mediated inflammation, finally resulting in the abolishment of DIO-induced anti-I/R effect. NF-κB activation also relieved the inhibitory inflammation and reversed DIO's effect against I/R, suggesting that NF-κB was the downstream target of HIKESHI and HSP70 in I/R treatment with DIO.These findings established a novel HIKESHI/HSP70/NF-κB signaling pathway associated with DIO-treated I/R, which might be as therapeutic targets or drugs with potential implications for the therapeutic use of I/R in clinic.

Steroid saponins are the medicinal compounds and nutrition ingredients of medicine food homology (MFH)

The preparative capability of the multilayer coil has been demonstrated in the separation of 1 g of the DNP amino acid mixture under the optimal operational conditions determined by the preliminary studies on short coils as described above. These centrifuge schemes are equipped with a novel flow-through mechanism which entirely eliminates the need for the conventional rotary-seal device, thus providing the following advantages: Because of these advantages, these schemes can become a reliable elution system, comparable to other chromatographic schemes. This simple synchronous planetary motion of the holder eliminates the need for the rotating seal. Recently, the phase distribution produced by the above scheme has been more systematically studied in the author's laboratory on a horizontal flow-through CPC with a 10-cm radius of revolution.

Background: Endoscopic retrograde pancreatography (ERCP) is sometimes complicated by post-ERCP pancreatitis (PEP), which is a severe adverse effect. Objective: The present study was performed to (i) evaluate the risk factors for PEP and (ii) compare the risk of PEP after ERCP performed in the off hours versus regular hours. Methods: This retrospective study included 374 patients who underwent ERCP from January 2013 to December 2017. Among these patients, 38 (10.2%) developed PEP. The potential risk factors for PEP were evaluated by multivariate regression analysis, and the risk of PEP was compared between ERCP performed during regular hours and off hours. Results: The independent risk factors for PEP were a relatively younger age (p = 0.024), female sex (p = 0.002), a history of pancreatitis (p = 0.044), and performance of pancreatography (p = 0.010). Use of a diclofenac suppository and performance of pancreatic stenting were not preventive for PEP after ERCP. The complication rate of PEP did not differ between ERCP performed during the off hours versus regular hours. Conclusions: A relatively younger age (

The purpose of this study is to separate impurities from dyes using a simple separatory bottle in order to detect to allow for the characterization of these impurities using analytical separation techniques. Foaming agents were used in a glass bottle with a modified cap, to separate a target impurity using the appropriately charged ligand. By running N(2) gas through the solution, the cationic surfactant cetylpyridinium chloride (CPC) and anionic surfactant sodium dodecyl sulfate (SDS) generated foams that separated the anionic dye Orange G and cationic dye Methylene blue, respectively, from a solution containing both dyes. It was found that SDS was able to condense minute quantities of methylene blue from the solution, with high purity. CPC was also able to condense minute quantities of Orange G from the solution, however with less purity. A range of 100nmol to 50μmol of each target dye was tested. For both surfactants, the condensability was shown to increase exponentially as concentration of the target dye decreased. This novel separation method could potentially prove to be a simple, cheap, and effective way to prepare samples and identify components of impurities using HPLC, NMR, and mass spec as well as be used to purify difficult to separate charged compounds.

Objective This historical control study was performed to evaluate i) the rebleeding rate of bleeding colon diverticula treated with endoscopic band ligation (EBL) versus endoscopic clipping (EC) and ii) risk factors for rebleeding of diverticula initially treated by endoscopic hemostasis. Methods From January 2010 to December 2012, 68 patients were treated with EC, and from January 2013 to August 2016, 67 patients were treated with EBL. All patients in each group were followed up for one year to check for rebleeding. Results The rebleeding rate was lower in the EBL group (7 of 67, 10%) than in the EC group (21 of 68, 31%; p

Recently, the multilayer coil was introduced in type-I CCC system, which showed the excellent peak resolution. Its performance was further improved by intermittently pressed tubing. In the ...

In our recent study, the design and performance of type-I counter-current chromatography (CCC) were changed and improved by multilayer coil. In the present study, the performance of protein separation using the multilayer coil configuration in type-I CCC was investigated under various flow rates. In order to overcome the noise signal interrupting the target peaks, a hollow fiber membrane dialyzer was inserted between CCC column outlet and inlet of the monitor. The separation was performed with a two-phase solvent system composed of polyethylene glycol 1000/dibasic potassium phosphate each at 12.5% (w/w) in deionized water and lysozyme and myoglobin were used as the test samples. The retention of stationary phase in the head to tail elution mode was lower than that in the tail to head elution mode, but Rs (peak resolution) was opposite. The intermittently pressed tubing can efficiently improve the performance of protein separation by type-I CCC with the multilayer coil. The best Rs was 1.54 and obtained at the flow rate of 0.10 ml/min under a revolution speed of 200 rpm. The present result indicated that type-I CCC can be as a potential powerful tool of protein separation for the first time.

Recurrent Clostridioides difficile infection (rCDI) is a frustrating condition that may affect a person's quality of life for months. Microbiome-based therapy such as fecal microbiota transplantation (FMT) has been effective for the treatment of rCDI by correcting the imbalance of the gut microbiota. Appropriate antibiotic treatment is recommended for at least two recurrences before offering FMT. Here, we report the case of a 92-year-old woman who experienced five recurrences of Clostridioides difficile infection (CDI) (six episodes in total) complicated by dementia and delirium, both of which were dramatically improved by FMT, which was associated with alterations in fecal microbiota and the metabolome. Analyses of whole microbial communities and metabolomic analyses were performed on stool specimens collected from the patient on the first episode, the third episode, the day of FMT (before FMT), and 2, 8, and 23 weeks after the FMT and from the donor. The patient had various fecal dysbioses on the first and third episodes and on the day of FMT. Two weeks after FMT, diversity of the gut bacteriome as well as the virome increased dramatically and was reflected in a positive clinical outcome for this patient. Metabolomic analysis revealed that short-chain fatty acids, which have been reported to be associated with improved memory function, were increased after FMT.

The partition efficiency of the double-spaced coil for eccentric and toroidal coils on countercurrent chromatographic separation of proteins was evaluated using the small-scale cross-axis coil planet centrifuge (CPC) equipped with circular and elliptic cylindrical columns. Standard cytochrome c, myoglobin and lysozyme samples were used for separation with the 12.5% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate system. In the circular column, the double-spaced eccentric coil yielded better peak resolution than the double-spaced toroidal coil, and the double-spaced eccentric coil yielded better peak resolution than the single-spaced eccentric coil. In the elliptic column, the double-spaced eccentric coil also produced better peak resolution than the double-spaced toroidal coil, but the single-spaced eccentric coil yielded better peak resolution than the double-spaced eccentric coil. The overall results indicated that the double-spaced eccentric coil for the circular column and the single-spaced eccentric coil for the elliptic column yielded better protein separation using the small-scale cross-axis CPC with aqueous two-phase solvent systems.

An impurity in the color additives D&C Red No. 30 (R30) and D&C Red No. 30 lakes (R30L) was newly identified and characterized as 7-chloro-5-methyl-2H-1,4-benzothiazin-3(4H)-one (BTZ), and its extent and level in certified batches of these color additives was determined. BTZ was extracted from the dye with ethanol, resulting in a crude extract enriched to a concentration of over 60%. BTZ was then separated from a portion of the enriched extract by high-speed counter-current chromatography using a spiral-tube assembly column with intermittently pressed tubing of 60 ml capacity. It was the first reported use of such a column to separate a small, moderately hydrophobic compound. The two-phase solvent system was also moderately hydrophobic, consisting of hexane-ethyl acetate-methanol-water (5:2:5:2), and the retention of the organic stationary phase measured after the separation was 83.3%. The separation yielded BTZ of two purity grades, the higher of which (~95.5%) was used as a standard to quantify the impurity in 37 batches of R30 and R30L using an HPLC method developed and validated for that purpose. Analyses revealed a wide range of BTZ levels across batches

Diosgenin (DIO), the starting material for the synthesis of steroidal anti-inflammatory drugs in the pharmaceutical industry, has been previously demonstrated to display pharmaceutical effects against cerebral ischemic reperfusion (I/R). However, the alterations of brain proteome profiles underlying this treatment remain elusive. In the present study, the proteomics analysis of the brain tissues from I/R rats after DIO treatment was performed using an integrated TMT-based quantitative proteomic approach coupled with the liquid chromatography with tandem mass spectrometry technology. A total of 5043 proteins (ProteomeXchange identifier: PXD016303) were identified, of which 58 common differentially expressed proteins were significantly dysregulated in comparison between sham versus I/R and I/R versus DIO. The eight validated proteins including EPG5, STAT2, CPT1A, EIF2AK2, GGCT, HIKESHI, TNFAIP8, and EMC6 by quantitative polymerase chain reaction and western blotting consistently supported the TMT-based proteomic results, which were mainly associated with autophagy and inflammation response. Considering the anti-inflammatory characters of DIO, the biological functions of STAT2 and HIKESHI that are the probable direct anti-inflammatory targets were further investigated during the course of I/R treated with DIO. In addition, the combination of verified STAT2 and HIKESHI in peripheral blood samples from stroke patients resulted in the area under the curve value of 0.765 with P < 0.004 to distinguish stroke patients from healthy controls. Taken together, the current findings first mapped comprehensive proteomic changes after I/R was treated with DIO to better decipher the molecular mechanisms mainly based on the anti-inflammatory aspect underlying this therapeutic effect, providing a foundation for developing potentially therapeutic targets of anti-I/R of DIO and clinically prognostic biomarkers of stroke.

Objective The aim of this study was to clarify the safety of colorectal endoscopic submucosal dissection (ESD) during the era of health insurance coverage starting from April 2012 in Japan. Methods Between April 2012 and May 2016, ESD was applied to 398 lesions in 373 patients. Risk factors for serious complications of colorectal ESD, perforation and post-ESD bleeding, were evaluated focusing on the resected specimen size, location, growth pattern, invasion depth, histopathology, postoperative clipping, and procedure time. In addition, the relationship between serious complications and patients' background characteristics was analyzed. Results Among 373 patients, perforation occurred in 12 patients and post-ESD bleeding in 19 patients. A univariate analysis showed that the risk factors for perforation were the lesion size, the resected specimen size, and a long operation time. A multivariate analysis showed that a long operation time was a risk factor for perforation during colorectal ESD. A univariate analysis indicated that significant risk factors for postoperative bleeding were a long operation time, rectal lesion, and cancer. All patients with serious complications were treated by an endoscopic procedure without blood transfusion or the need to convert to open surgery. Conclusion The present study suggests that colorectal ESD may be accepted with relative safety in Japan as a common therapeutic approach for early colorectal cancer.

Ethnopharmacological relevance Dioscorea zingiberensis C. H. Wright (D. zingiberensis), Dioscoreaceae, is used extensively in traditional Chinese medicines. The aim of the current review paper is to give a comprehensive overview of the traditional usage and phytochemistry of the plant. Clinical studies performed and products prepared from the plant and active principles will be mentioned. In addition a review of the taxonomy of the genus Dioscorea is given. Materials and methods A systematic search on literature has been performed in databases like Google Scholar, Science Direct, Scifinder, Web of Science, and in Chinese databases (China Knowledge Resource Integrated, i.e., CNKI and Wanfang) including Ph.D. and M.Sc. dissertations. These from the period 1983–2016 have been searched. Results D. zingiberensis is an endemic plant in China widely distributed in some provinces, especially Hubei and Shaanxi. The rhizomes are the medicinal part in Dun-Ye-Guan-Xin-Ning tablets and are used for isolation of diosgenin. Steroidal saponins are believed to be the active principles. More than 70 compounds have been identified. Several of these have been tested in preclinical assays and clinical trials. A wide spectrum of biological effects including cardiovascular, anti-thrombosis, hyperlipidemia, neuroprotection, anti-inflammatory, and anthelmintic effect has been verified. Conclusions Because of the promising results from the investigations on the plant material of D. zingiberensis, further in depth analyses ought to be performed to evaluate its potential as either a traditional drug or a source of bioactive principle. The presently performed studies do not explain mechanism of action, pharmacokinetics (ADME properties), or toxicity. All of these topics need more elaborate investigations.

Diosgenin wastewater produced from Dioscorea zingiberensis C. H. Wright in diosgenin enterprise is the by-product of numerous operations chiefly including washing raw materials, acid hydrolysis, and cleaning the apparatus with water. It requires the removal of high concentrations of various organic and inorganic contaminants from this wastewater in order to avoid pollution to the environment and fresh water system. Currently, several different methods such as chemical, physicochemical, biological recovery resource, constructed wetland, and integrated methods of these processes are being applied for treating diosgenin wastewater. Aiming to get a comprehensive overview, available reports from experimental scale to industry scale about the treatment of this wastewater were collected and summarized. Therefore, this review paper will provide appropriate choice for the efficient treatment of diosgenin wastewater and supportive information for the other scientists who want to continue this valuable research in the future.

Centrifugal precipitation chromatography (CpC) is a powerful chromatographic technique invented in the year 2000 but so far very little applied. The method combines dialysis, counter-current and salting out processes. The separation rotor consists of two identical spiral channels separated by a dialysis membrane (6–8 K MW cut-off) in which the upper channel is eluted with an ammonium sulfate gradient and the lower channel with water, and the mixtures are separated according to their solubility in ammonium sulfate as a chromatographic technique. In the present study, the method was successfully applied for separation and purification of R-phycoerythrin (R-PE), a protein widely used as a fluorescent probe, from the red alga Gracilaria lemaneiformis. The separation was performed with the elution of ammonium sulfate from 50% to 0% in 21.5 h at a flow rate of 0.5 ml/min, while the lower channel was eluted with water at a flow rate of 0.05 ml/min after sample charge, and the column was rotated at 200 rpm. After a single run, the absorbance ratio A565/A280 (a criterion for the purity of R-PE) was increased from 0.5 of the crude to 6.5. The purified R-PE exhibited a typical “three peaks” spectrum with absorbance maximum at 497, 538 and 565 nm. The Native-PAGE showed one single protein band and 20 kDa (subunits α and β) and 30 kDa (subunit γ) can be observed in SDS-PAGE analysis which were consistent with the (αβ)6γ subunit composition of R-PE. The results indicated that CpC is an efficient method to obtain protein with the high purity from a complex source.

I/R (cerebral ischemia reperfusion injury) is the secondary complication of ischemic stroke patients that are immediately treated with drug thrombolysis or vascular recanalization in clinic. Diosgenin (DIO) purified from medicine food homologous (MFH) Dioscorea yam source is served as a fatal starting material to synthesize multifarious steroidal anti-inflammatory drugs in medicinal field, and has previously been demonstrated the potential prevention of I/R. However, the detailed mechanisms of neuroprotective effects against I/R remain elusively understood. Here, a global proteomic dynamics of rat right hemisphere brains was executed to investigate the protein expression patterns with a quantitative LC-MSn. In total, 5043 proteins were identified and 418 ones were determined to be significantly dysregulated DEPs (differentially expressed proteins) in comparison of Sham verse I/R and I/R verse DIO after onset stage of I/R, among which 5 DEPs namely BICD2, HNRNPK, CEP41, PPM1K, and ARL2BP, whose biological functions were mainly clustered into the mediation of nervous system, were selected for further validation in vitro and in vivo, and the change tendency expectedly supported the proteomic findings. Additionally, the AUC value of the combined ROC of these 5 DEPs was 0.988 with P

Four flavonoids including apigenin-7,4'-dimethylether, genkwanin, quercetin, and kaempferol were isolated in a preparative or semi-preparative scale from the leaves of wild Aquilaria sinensis using an improved preparative high-speed counter-current chromatography apparatus. The separations were performed with a two-phase solvent system composed of hexane-ethyl acetate, methanol-water at suitable volume ratios. The obtained fractions were analyzed by HPLC, and the identification of each target compound was carried out by ESI-MS and NMR. The yields of the above four target flavonoids were 4.7, 10.0, 11.0 and 4.4%, respectively. All these four flavonoids exhibited nitrite scavenging activities with the clearance rate of 12.40 ± 0.20%, 5.84 ± 0.03%, 28.10 ± 0.17% and 5.19 ± 0.11%, respectively. Quercetin was originally isolated from the Thymelaeaceae family, while kaempferol was isolated from the Aquilaria genus for the first time. In cytotoxicity test these two flavonoids exhibited moderate inhibitory activities against HepG2 cells with the IC50 values of 12.54 ± 1.37 and 38.63 ± 4.05 μM, respectively.

Specifications in the Code of Federal Regulations for the color additive DC Red No. 17 (Colour Index 26100) limit the levels of two subsidiary colors, 1-(phenylazo)-2-naphthol (Sudan I) and 1-[[2-(phenylazo)phenyl]azo]-2-naphthalenol (Sudan III o-isomer), to 3% and 2%, respectively. The present work reports the development of a high-performance liquid chromatography (HPLC) method for the quantitative determination of these subsidiary colors. Since Sudan III o-isomer needed to be synthesized for use as a reference material, a two-step procedure was devised: (i) preparative-scale synthesis of the intermediate 2-aminoazobenzene (2AAB) and its purification by counter-current chromatography and (ii) diazotization of 2AAB and coupling with 2-naphthol. Characterization of the newly synthesized Sudan III o-isomer is also reported. Sudan I and Sudan III o-isomer were quantified by using five-point calibration curves with data points ranging from 0.108 to 3.240% and 0.077 to 2.227% by weight, respectively. The HPLC method is rapid (14 min for the total analysis cycle) and simple to implement. It was applied to the analysis of test portions from 25 batches of DC Red No. 17 submitted to the U.S. Food and Drug Administration (USFDA) for certification, and it has recently been implemented by USFDA for routine batch certification of that color additive.

This study aimed to evaluate the influence of Helicobacter pylori infection and its eradication on the upper gastrointestinal symptoms of relatively healthy Japanese subjects. A total of 3,005 subjects (male/female: 1,549/1,456) undergoing medical health checkups were enrolled in the present study, at five hospitals in Saga, Japan, from January to December 2013. They had no significant findings following upper gastrointestinal endoscopy. All subjects completed a questionnaire that addressed a frequency scale for symptoms of gastroesophageal reflux disease. The questionnaire comprised seven questions regarding reflux symptoms and seven regarding acid-related dyspepsia, which were answered with a score based on the frequency of symptoms. Helicobacter pylori infection was identified by a rapid urease test and/or H. pylori antibody titer, and an eradication history was confirmed by the subjects’ medical records. Helicobacter pylori infection was positive in 894 subjects out of 3,005 (29.8%). Eradication of Helicobacter pylori was successfully achieved in 440 subjects of 458 treated. Helicobacter pylori infection had no influence on the acid-related dyspepsia evaluated by the questionnaire, whereas the mean reflux score was relatively high in the Helicobacter pylori native negative subjects compared to Helicobacter pylori native positive. Eradication of Helicobacter pylori and time span after the eradication had no effect on the upper gastrointestinal symptoms evaluated by the questionnaire. Helicobacter pylori infection and history of eradication did not affect acid-related dyspepsia symptoms in Japanese healthy subjects.

Coil satellite centrifuge (CSC) produces the complex satellite motion consisting of the triplicate rotation of the coiled column around three axes including the sun axis (the angular velocity, ω1), the planet axis (ω2) and the satellite axis (the central axis of the column) (ω3) according to the following formula: ω1=ω2+ω3. Improved peak resolution in the separation of 4-methylumbelliferyl sugar derivatives was achieved using the conventional multilayer coiled columns with ethyl acetate/1-butanol/water (3: 2: 5, v/v) for the lower mobile phase at the combination of the rotation speeds (ω1, ω2, ω3)=(300, 150, 150rpm), and (1:4:5, v/v) for the upper mobile phase at (300:100:200rpm). The effect of the satellite motion on the peak resolution and the stationary phase retention was evaluated by each CSC separation with the different rotation speeds of ω2 and ω3 under the constant revolution speed at ω1=300rpm. With the lower mobile phase, almost constant peak resolution and stationary phase retention were yielded regardless of the change of ω2 and ω3, while with the upper mobile phase these two values were sensitively varied according to the different combination of ω2 and ω3. For example, when ω2=147 or 200rpm is used, no stationary phase was retained in the coiled column while ω2=150rpm could retain enough volume of stationary phase for separation. On the other hand, the combined rotation speeds at (ω1, ω2, ω3)=(300, 300, 0rpm) or (300, 0, 300rpm) produced insufficient peak resolution regardless of the choice of the mobile phase apparently due to the lack of rotation speed except at (300, 0, 300rpm) with the upper mobile phase. At lower rotation speed of ω1=300rpm, better peak resolution and stationary phase retention were obtained by the satellite motion (ω3) than by the planetary motion (ω2), or ω3>ω2. The effect of the hydrophobicity of the two-phase solvent systems on the stationary phase retention was further examined using the n-hexane/ethyl acetate/1-butanol/methanol/water system at different volume ratios. In the satellite motion at (ω1, ω2, ω3)=(300, 150, 150rpm), almost constant stationary phase retention was obtained with the lower mobile phase regardless of the hydrophobicity of the solvent system whereas the stationary phase retention varied according to the volume ratio of the two-phase solvent system for the upper mobile phase. However, stable stationary phase retention was observed with either phase used as the mobile phase. In order to analyze the acceleration acting on the coiled column, an acceleration sensor was set on the column holder by displacing the multilayer column. The combination of the rotation speeds at (300, 100, 200rpm) showed double loops in the acceleration track, whereas (300, 150, 150rpm) showed a single loop, and all other combinations showed, complex tracks. The overall results indicate that the satellite motion is seriously affected by the combination of rotation speeds and the hydrophobicity of the two-phase solvent system when the upper phase was used as the mobile phase for separation.

Background: Bleeding from a colonic diverticulum is serious in aged patients. The aim of this study was to determine the risk factors for high-cost hospitalization of colonic diverticular bleeding using the diagnosis procedure combination (DPC) data. Methods: From January 2009 to December 2015, 78 patients with colonic diverticular bleeding were identified by DPC data in Saga Medical School Hospital. All patients underwent colonic endoscopy within 3 days. The patients were divided into 2 groups: the low-cost group (DPC cost of 500,000 yen). Results: Univariate analysis revealed that aging, hypertension, rebleeding, a low hemoglobin concentration at admission, and blood transfusion were risk factors for high hospitalization cost. Multivariate analysis revealed that rebleeding (OR 5.3; 95% CI 1.3-21.3; p = 0.017) and blood transfusion (OR 3.8; 95% CI 1.01-14.2; p = 0.048) were definite risk factors for high hospitalization cost. Conclusion: Rebleeding and blood transfusion were related to high hospitalization cost for colonic diverticular bleeding.

Introduction/Aims: Immune checkpoint inhibitors (ICIs) sometimes cause immune-related adverse events (irAEs), of which there is little information in the literatures. The objective of this study was to characterize the clinical features of gastrointestinal irAEs (GI irAEs). Materials and Methods: From a total of 250 patients who were administered anti-PD-1 antibodies (nivolumab and pembrolizumab), we retrospectively identified 9 patients with grade 2 or higher GI irAE based on medical records. Patient characteristics, clinical features, imaging and pathological findings, and treatment course were evaluated. Results: Grade 2 or higher GI irAEs were observed in 9 (3.6%) patients. Of the 9 patients who experienced GI irAE, 8 were male, and mean age was 63.2 years. Five patients received nivolumab and 4 received pembrolizumab. The GI irAEs observed were diarrhea in 7 patients and bloody stool in 2 patients. Grade 2 GI irAEs were identified in 3 patients and grade 3 GI irAEs in 6 patients. The average time from ICI administration to the onset of GI irAEs was 22.2 weeks (range 7–56 weeks) for nivolumab and 19.7 weeks (range 11–28 weeks) for pembrolizumab. Endoscopic findings showed ulcerative colitis-like findings in 3 of 7 patients, and pathological examination revealed crypt epithelial cell apoptosis in 6 of 7 patients. Eight of the 9 patients received steroids, and 2 patients received infliximab additionally. All GI irAEs were manageable. Conclusions: Because of the lack of specific clinical, imaging, and pathological findings, information of ICI use was indispensable for diagnosis. Although GI irAEs are controllable by steroid and infliximab, further studies regarding management strategy will be needed.

The partition efficiencies of three different coiled columns, conventional multilayer coiled column, eccentric coiled column and toroidal coiled column, were evaluated by the separation of 4-methylumbelliferyl sugar derivatives using the coil satellite centrifuge (CSC) with an organic-aqueous two-phase solvent system composed of ethyl acetate/1-butanol/water. The CSC apparatus was reinforced the planet axis to maintain the stable satellite motion, which was completed by combining the rotation of three axes including the sun axis (the angular velocity, ω(1)), the planet axis (ω(2)) and the satellite axis (ω(3)) under the relation at ω(1) = ω(2) + ω(3). In the present study, four different rotation speed combination types were used for the separation at the ratio (ω(1), ω(2), ω(3)) = I. (300, 150, 150), II. (300, 100, 200), III. (300, 147, 153) and IV. (300, 200, 100 rpm) under different revolution speeds of ω(1) = 300, 400 and 500 rpm. In the conventional multilayer coiled column with the upper mobile phase, the rotation speed combination type II yielded the best peak resolution while the rotation speed combination types III and IV had extremely low stationary phase retention even at higher revolution speeds. This inconvenience was eliminated by using the eccentric and the toroidal coiled column. The rotation speed combination type II for the eccentric coiled column and the type IV for the toroidal coiled column produced the best separation in both the upper and the lower mobile phases among four different rotation speed combination types. The overall results indicated that better peak resolution was obtained by the eccentric coiled column than by the toroidal coiled column except for the separation with the upper mobile phase at the low revolution speed.

The present work describes the application of high-speed counter-current chromatography to the preparative separation of a previously unreported impurity in the color additive D&C Red No. 17 (R17, Colour Index No. 26100, Sudan III). Due to the hydrophobic nature of the impurity, a hydrophobic two-phase solvent system (hexane-ethanol-water, 5:4:1) was used for its separation. The separated impurity was chemically characterized by spectroscopic methods as a disazo triazene, 1,3-bis(4-phenylazophenyl)triazene (PAPT). This impurity was synthesized and used as a reference material to quantify it in 15 batches of the color additive produced by various domestic and foreign manufacturers and certified by the U.S. Food and Drug Administration (FDA). Analysis of test portions by high-performance liquid chromatography showed a range of PAPT levels, from "not detected" (

As a classic foaming agent, bovine serum albumin (BSA), can be adsorbed on gas–liquid interface to generate stable foam. In the present study, the foam disappeared when Evans Blue (EB) interacted with BSA. Therefore, this phenomenon was used to separate the non-defoaming Rhodamine 6G (R6G) from the defoaming EB. After optimizing the parameters such as BSA concentration, pH and gas flow rate, R6G was enriched with spilled foam. The enrichment ratio and recovery of R6G were 5.32 and 88.29%, respectively, in pH 7 solution at a gas flow rate of 0.25 mL/min using 0.20% BSA as foaming agent. The present strategy provides an effective method for foam separation and dye recycling.

The aim of the present study is to explore the potential cerebroprotection of diosgenin against the transient focal cerebral ischemia-reperfusion (I/R) injury and its possible underlying mechanisms. The diosgenin at two dose levels, namely 100 and 200mgkg(-1), was intragastrically administrated once daily for 7-day period prior to the surgery. Then, the rats were subjected to middle cerebral artery occlusion (MCAO) using the intraluminal thread for 90min. After 24h reperfusion, several diagnostic indicators were evaluated and all animals were sacrificed to harvest their brains and blood for subsequent biochemical analyses. The results indicated that diosgenin treatment significantly inhibited the death rate and improved the impaired neurological functions along with neurological deficit scores and cerebral infarct size as compared with the rats exposed to I/R insult without agents administration. The increase in the number of apoptotic cells determined by TUNEL in the hippocampus CA1 and cortex was also apparently attenuated in the diosgenin treatment group, which was closely correlated with suppression of Caspase-3 activity and Bax/Bcl-2 ratio. In addition the elevated concentrations of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in blood serum of the I/R treated rats were reduced almost to their normal level. Further results obtained from the Western blotting analysis revealed that the protein expression of IκBα in the injured brain was up-regulated, while the p65 subunit of NF-κB was down-regulated in nucleus after the treatment. Collectively, this neuroprotection of diosgenin against I/R injury may be attained through its anti-apoptosis, anti-inflammation and intervening the NF-κB signal pathway properties. Due to the satisfactory findings, diosgenin might be a powerful therapeutic agent to combat the similar disease in future clinic.

A simple tool is introduced which can modify the shape of tubing to enhance the partition efficiency in high-speed countercurrent chromatography. It consists of a pair of interlocking identical gears, each coaxially holding a pressing wheel to intermittently compress plastic tubing in 0 - 10 mm length at every 1 cm interval. The performance of the processed tubing is examined in protein separation with 1.6 mm ID PTFE tubing intermittently pressed in 3 mm and 10 mm width both at 10 mm intervals at various flow rates and revolution speeds. A series of experiments was performed with a polymer phase system composed of polyethylene glycol and dibasic potassium phosphate each at 12.5% (w/w) in deionized water using three protein samples. Overall results clearly demonstrate that the compressed tubing can yield substantially higher peak resolution than the non-processed tubing. The simple tubing modifier is very useful for separation of proteins with high-speed countercurrent chromatography.

We have developed a novel continuous flow-through cell separation method using a Percoll density gradient. This method can continuously separate a large number of cells into five fractions according to their densities. To apply this method to the separation of basophils, Percoll density gradients were modified to improve basophil enrichment. When a set of Percoll density gradients was prepared (1.071, 1.075, 1.080, 1.084, and 1.090 g/mL) the basophils in a healthy volunteer were enriched by an average of 23.1 and 63.5% at Percoll densities of 1.075 (fraction 3) and 1.080 g/mL (fraction 4), respectively. On average, the yield of basophils was 1.66 × 10(5) cells in fraction 3 and 1.61 × 10(5) cells in fraction 4 from 9 mL of peripheral blood. The expression of CD203c (cluster of differentiation 203c) on separated basophils was upregulated by anti-immunoglobulin E stimulation similar to basophils in whole blood. Histamine release induced by calcium ionophore was also observed in the separated basophils. The present method will be useful for basophil enrichment since it preserves their function without using counterflow elutriation and immunological reagents, and this method will be effective as a preparative separation for cell purification by flow cytometry.