Direct production of 4-hydroxybenzoic acid from cellulose using cellulase-displaying Pichia pastoris.

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. ß-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.

LONG-HOSP Score: A Novel Predictive Score for Length of Hospital Stay in Acute Lower Gastrointestinal Bleeding - A Multicenter Nationwide Study.

INTRODUCTION: Length of stay (LOS) in hospital affects cost, patient quality of life, and hospital management; however, existing gastrointestinal bleeding models applicable at hospital admission have not focused on LOS. We aimed to construct a predictive model for LOS in acute lower gastrointestinal bleeding (ALGIB). METHODS: We retrospectively analyzed the records of 8,547 patients emergently hospitalized for ALGIB at 49 hospitals (the CODE BLUE-J Study). A predictive model for prolonged hospital stay was developed using the baseline characteristics of 7,107 patients and externally validated in 1,440 patients. Furthermore, a multivariate analysis assessed the impact of additional variables during hospitalization on LOS. RESULTS: Focusing on baseline characteristics, a predictive model for prolonged hospital stay was developed, the LONG-HOSP score, which consisted of low body mass index, laboratory data, old age, nondrinker status, nonsteroidal anti-inflammatory drug use, facility with ≥800 beds, heart rate, oral antithrombotic agent use, symptoms, systolic blood pressure, performance status, and past medical history. The score showed relatively high performance in predicting prolonged hospital stay and high hospitalization costs (area under the curve: 0.70 and 0.73 for derivation, respectively, and 0.66 and 0.71 for external validation, respectively). Next, we focused on in-hospital management. Diagnosis of colitis or colorectal cancer, rebleeding, and the need for blood transfusion, interventional radiology, and surgery prolonged LOS, regardless of the LONG-HOSP score. By contrast, early colonoscopy and endoscopic treatment shortened LOS. CONCLUSIONS: At hospital admission for ALGIB, our novel predictive model stratified patients by their risk of prolonged hospital stay. During hospitalization, early colonoscopy and endoscopic treatment shortened LOS.

Countercurrent chromatography as a frontier tool to discover new mechanical roles in liquid-liquid phase separation of cellular biomolecules.

The development of countercurrent chromatography (CCC) technology enabled us to achieve higher peak resolutions and more shortened separation times even for protein separation using aqueous two-phase solvent systems composed of polyethylene glycol and inorganic salts (or dextrans). By eliminating the solid support matrix, all analytes can be recovered from the coiled column after the separation is completed. 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. 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 in the CCC apparatus, the coiled separation column, the choice of a 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.

Exchange of endogenous and heterogeneous yeast terminators in Pichia pastoris to tune mRNA stability and gene expression.

In the yeast Saccharomyces cerevisiae, terminator sequences not only terminate transcription but also affect expression levels of the protein-encoded upstream of the terminator. The non-conventional yeast Pichia pastoris (syn. Komagataella phaffii) has frequently been used as a platform for metabolic engineering but knowledge regarding P. pastoris terminators is limited. To explore terminator sequences available to tune protein expression levels in P. pastoris, we created a 'terminator catalog' by testing 72 sequences, including terminators from S. cerevisiae or P. pastoris and synthetic terminators. Altogether, we found that the terminators have a tunable range of 17-fold. We also found that S. cerevisiae terminator sequences maintain function when transferred to P. pastoris. Successful tuning of protein expression levels was shown not only for the reporter gene used to define the catalog but also using betaxanthin production as an example application in pathway flux regulation. Moreover, we found experimental evidence that protein expression levels result from mRNA abundance and in silico evidence that levels reflect the stability of mRNA 3'-UTR secondary structure. In combination with promoter selection, the novel terminator catalog constitutes a basic toolbox for tuning protein expression levels in metabolic engineering and synthetic biology in P. pastoris.

Fecal microbiota transplantation as therapy for recurrent Clostridioides difficile infection is associated with amelioration of delirium and accompanied by changes in fecal microbiota and the metabolome.

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.

Alterations of Brain Quantitative Proteomics Profiling Revealed the Molecular Mechanisms of Diosgenin against Cerebral Ischemia Reperfusion Effects.

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.

Risk Factors for Post-Endoscopic Retrograde Pancreatography Pancreatitis: A Retrospective Chart Review in a Regional Hospital in Japan.

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 (<75 years; 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 (<75 years), female sex, a history of pancreatitis, and performance of pancreatography were potential risk factors for PEP, whereas the risk of PEP was not different between ERCP performed during the off hours versus regular hours.

An Investigation of Nine Patients with Gastrointestinal Immune-Related Adverse Events Caused by Immune Checkpoint Inhibitors.

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.

A novel foam based separation strategy for extracting minute target impurities.

The purpose of this study was to extract impurities from compounds using a simple separatory bottle to purify target compounds with a foam column and allow for the further characterization of impurities. Charged dyes were used as target compounds due to the ease of detection of dyes and isolated impurities. Foaming agents were used in a glass bottle with a modified cap to separate a target impurity using an appropriately charged ligand. By passing N2 gas through the solution, the surfactants sodium dodecyl sulfate and cetylpyridinium chloride generated foams that separated the dyes, Methylene blue and Orange G, respectively, from a solution containing both dyes. Sodium dodecyl sulfate condensed Methylene blue from the solution with high purity while cetylpyridinium chloride condensed Orange G with less purity. A range of concentrations (0.01-0.5 mmol/L) of dyes were used for separation. The condensability (volume and/or concentration) of the target compound increased as its concentration decreased. This novel separation method is a simple, rapid, inexpensive, and effective way to prepare samples and allows for the characterization of these impurities using sensitive analytical detection techniques.

A Stable, Autonomously Replicating Plasmid Vector Containing Pichia pastoris Centromeric DNA.

The methylotrophic yeast Pichia pastoris is widely used to produce recombinant proteins, taking advantage of this species' high-density cell growth and strong ability to secrete proteins. Circular plasmids containing the P. pastoris-specific autonomously replicating sequence (PARS1) permit transformation of P. pastoris with higher efficiency than obtained following chromosomal integration by linearized DNA. Unfortunately, however, existing autonomously replicating plasmids are known to be inherently unstable. In this study, we used transcriptome sequencing (RNA-seq) data and genome sequence information to independently identify, on each of the four chromosomes, centromeric DNA sequences consisting of long inverted repeat sequences. By examining the chromosome 2 centromeric DNA sequence (Cen2) in detail, we demonstrate that an ∼111-bp region located at one end of the putative centromeric sequence had autonomous replication activity. In addition, the full-length Cen2 sequence, which contains two long inverted repeat sequences and a nonrepetitive central core region, is needed for the accurate replication and distribution of plasmids in P. pastoris Thus, we constructed a new, stable, autonomously replicating plasmid vector that harbors the entire Cen2 sequence; this episome facilitates genetic manipulation in P. pastoris, providing high transformation efficiency and plasmid stability.IMPORTANCE Secretory production of recombinant proteins is the most important application of the methylotrophic yeast Pichia pastoris, a species that permits mass production of heterologous proteins. To date, the genetic engineering of P. pastoris has relied largely on integrative vectors due to the lack of user-friendly tools. Autonomously replicating Pichia plasmids are expected to facilitate genetic manipulation; however, the existing systems, which use autonomously replicating sequences (ARSs) such as the P. pastoris-specific ARS (PARS1), are known to be inherently unstable for plasmid replication and distribution. Recently, the centromeric DNA sequences of P. pastoris were identified in back-to-back studies published by several groups; therefore, a new episomal plasmid vector with centromere DNA as a tool for genetic manipulation of P. pastoris is ready to be developed.

Deletion of DNA ligase IV homolog confers higher gene targeting efficiency on homologous recombination in Komagataella phaffii.

The non-conventional yeast Komagataella phaffii, formerly Pichia pastoris, is a popular host for recombinant protein production. The relatively lower gene targeting efficiency observed in this species occurs due to high levels of non-homologous recombination activity. In the current study, we explored the function of the K. phaffii homolog of DNA ligase IV (Dnl4p) by creating a DNL4-disrupted strain. To assess the roles of non-homologous end joining (NHEJ)-related proteins in this species, strains deleted for either or both genes encoding Dnl4p or the telomeric Ku complex subunit (Ku70p) were generated. These deletions were constructed by either of two distinct marker-recycling methods (yielding either a seamless gene deletion or a Cre-loxP-mediated gene deletion). The resulting dnl4- and/or ku70-deleted K. phaffii strains were used to evaluate gene targeting efficiency in gene knock-out and gene knock-in experiments. The Dnl4p-defective strain showed improved gene targeting efficiency for homologous recombination compared to the wild-type and Ku70p-deffective strains. The dnl4 ku70 double knock-out strain exhibited a further improvement in gene targeting efficiency. Thus, the K. phaffii dnl4 and dnl4 ku70 deletion strains are expected to serve as useful platforms for functional analysis and strain development in this species.

Outcomes of Patients Undergoing Endoscopic Hemostasis for the Upper Gastrointestinal Bleeding Were Not Influenced by the Timing of Hospital Emergency Visits: A Situation Prevailing in Japan.

BACKGROUND: The aim of the present study was to determine differences in the prognosis of patients in Japan who underwent emergency endoscopic hemostasis (i) during regular hours versus off hours and (ii) as outpatients versus hospitalized patients. METHODS: The present retrospective study included 443 patients who underwent emergency endoscopic hemostasis for non-variceal upper gastrointestinal bleeding from January 2008 to December 2014. These patients were classified into 2 groups: hospitalized patients and outpatients. The outpatients were further subclassified into those who visited the hospital during regular hours and those who visited during off hours. RESULTS: The outcomes of outpatients who underwent emergency hemostasis during off hours did not differ from patients treated during regular hours. Multivariate analysis revealed that outcomes of hospitalized patients, including mortality, need for blood transfusion and length of hospitalization, were worse than those of outpatients; it also revealed that patient age, malnutrition rate and prevalence of diabetes and neoplasms were higher among hospitalized patients than those in outpatients. CONCLUSIONS: The clinical outcomes of patients who underwent emergency endoscopic hemostasis for upper gastrointestinal bleeding during off hours did not differ from those of patients treated during regular hours. Outcomes were worse among hospitalized patients, mainly because of their bad general condition.

High Cost of Hospitalization for Colonic Diverticular Bleeding Depended on Repeated Bleeding and Blood Transfusion: Analysis with Diagnosis Procedure Combination Data in Japan.

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) and the high-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.

Safety investigation on total steroid saponins extracts from Dioscorea zingiberensis C.H. Wright: Sub-acute and chronic toxicity studies on dogs.

Sub-acute and chronic toxic effects of total steroidal saponins (TSSN) extracts from Dioscorea zingiberensis C.H. Wright on various internal organs and biochemical indicators have never been studied before and this study is the first of its kind to demonstrate sub-acute and chronic toxicities of TSSN on dogs. Administration of TSSN extracts at doses up to 3000 mg/Kg daily for 14 days, no biochemical and organ changes were observed on the experimental groups of dogs. Further, chronic toxicity study through oral administration of TSSN extracts at the gradual doses of 50, 250 and 500 mg/Kg for 90 days followed by a 2-week recovery assay revealed absence of significant architectural and morphological changes in internal organs which were confirmed through histopathological examination and merely no significant alteration in the biochemical indicators including hematologic and urine analysis and electrocardiogram compared to the control dogs. This toxicological evaluation came across with the finding that the herbal preparation can be considered as nontoxic and animals could tolerate the extracts at doses up to 500 mg/Kg with LD50 greater than 3000 mg/Kg. It may serve as a preliminary scientific evidence for further therapeutic investigations.

Enhanced cell-surface display and secretory production of cellulolytic enzymes with Saccharomyces cerevisiae Sed1 signal peptide.

Recombinant yeast strains displaying aheterologous cellulolytic enzymes on their cell surfaces using a glycosylphosphatidylinositol (GPI) anchoring system are a promising strategy for bioethanol production from lignocellulosic materials. A crucial step for cell wall localization of the enzymes is the intracellular transport of proteins in yeast cells. Therefore, the addition of a highly efficient secretion signal sequence is important to increase the amount of the enzymes on the yeast cell surface. In this study, we demonstrated the effectiveness of a novel signal peptide (SP) sequence derived from the Saccharomyces cerevisiae SED1 gene for cell-surface display and secretory production of cellulolytic enzymes. Gene cassettes with SP sequences derived from S. cerevisiae SED1 (SED1SP), Rhizopus oryzae glucoamylase (GLUASP), and S. cerevisiae α-mating pheromone (MFα1SP) were constructed for cell-surface display of Aspergillus aculeatus ß-glucosidase (BGL1) and Trichoderma reesei endoglucanase II (EGII). These gene cassettes were integrated into the S. cerevisiae genome. The recombinant strains with the SED1SP showed higher cell-surface BGL and EG activities than those with the conventional SP sequences (GLUASP and MFα1SP). The novel SP sequence also improved the secretory production of BGL and EG in S. cerevisiae. The extracellular BGL activity of the recombinant strains with the SED1SP was 1.3- and 1.9-fold higher than the GLUASP and MFα1SP strains, respectively. Moreover, the utilization of SED1SP successfully enhanced the secretory production of BGL in Pichia pastoris. The utilization of the novel SP sequence is a promising option for highly efficient cell-surface display and secretory production of heterologous proteins in various yeast species. Biotechnol. Bioeng. 2016;113: 2358-2366. © 2016 Wiley Periodicals, Inc.

Higher Frequency of Reflux Symptoms and Acid-Related Dyspepsia in Women than Men Regardless of Endoscopic Esophagitis: Analysis of 3,505 Japanese Subjects Undergoing Medical Health Checkups.

BACKGROUND/AIMS: This study aimed at (i) clarifying the factors associated with high scores on the modified frequency scale for the symptoms of gastroesophageal reflux disease (FSSG) among 3,505 relatively healthy subjects undergoing routine medical health checkups with gastrointestinal endoscopy and (ii) comparing risk factors for high FSSG scores between subjects with and without reflux esophagitis. METHODS: In total, 3,505 subjects (male/female: 1,922/1,583) who underwent upper gastrointestinal endoscopy during health medical checkups at 5 hospitals in Saga, Japan from January 2013 to December 2013 were enrolled. All subjects completed a modified FSSG questionnaire, which comprised 7 questions regarding reflux symptoms and 7 questions regarding acid-related dyspepsia. Each question was assigned a score based on the frequency of symptoms. RESULTS: Younger age, female gender, hiatal herniation, and endoscopic reflux esophagitis were risk factors for a FSSG score with a high total. Subjects with high scores but without esophagitis were women, and hiatal herniation and Barrett's esophagus were frequently seen in patients with reflux esophagitis. CONCLUSION: Younger age, female gender, hiatal hernia, and endoscopic esophagitis were risk factors for a high FSSG score, and women tended to complain of upper gastrointestinal symptoms more frequently than did men among subjects without endoscopic esophagitis.

Single-step enrichment of basophils from human peripheral blood by a novel method using a Percoll density gradient.

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.

Purification of active myrosinase from plants by aqueous two-phase counter-current chromatography.

INTRODUCTION: Myrosinase (thioglucoside glucohydrolase; E.C. 3.2.1.147), is a plant enzyme of increasing interest and importance to the biomedical community. Myrosinase catalyses the formation of isothiocyanates such as sulforaphane (from broccoli) and 4-(α-l-rhamnopyranosyloxy)benzyl isothiocyanate (from moringa), which are potent inducers of the cytoprotective phase-2 response in humans, by hydrolysis of their abundant glucosinolate (ß-thioglucoside N-hydroxysulphate) precursors. OBJECTIVE: To develop an aqueous two-phase counter-current chromatography (CCC) system for the rapid, three-step purification of catalytically active myrosinase. METHODS: A high-concentration potassium phosphate and polyethylene glycol biphasic aqueous two-phase system (ATPS) is used with a newly developed CCC configuration that utilises spiral-wound, flat-twisted tubing (with an ovoid cross-section). RESULTS: Making the initial crude plant extract directly in the ATPS and injecting only the lower phase permitted highly selective partitioning of the myrosinase complex before a short chromatography on a spiral disk CCC. Optimum phase retention and separation of myrosinase from other plant proteins afforded a 60-fold purification. CONCLUSION: Catalytically active myrosinase is purified from 3-day broccoli sprouts, 7-day daikon sprouts, mustard seeds and the leaves of field-grown moringa trees, in a CCC system that is predictably scalable.

Development of a method to extract and purify target compounds from medicinal plants in a single step: online hyphenation of expanded bed adsorption chromatography and countercurrent chromatography.

Pure compounds extracted and purified from natural sources are crucial to lead discovery and drug screening. This study presents a novel two-dimensional hyphenation of expanded bed adsorption chromatography (EBAC) and high-speed countercurrent chromatography (HSCCC) for extraction and purification of target compounds from medicinal plants in a single step. The EBAC and HSCCC were hyphenated via a six-port injection valve as an interface. Fractionation of ingredients of Salvia miltiorrhiza and Rhizoma coptidis was performed on the hyphenated system to verify its efficacy. Two compounds were harvested from Salvia miltiorrhiza, one was 52.9 mg of salvianolic acid B with an over 95% purity and the other was 2.1 mg of rosmarinic acid with a 74% purity. Another two components were purified from Rhizoma coptidis, one was 4.6 mg of coptisine with a 98% purity and one was 4.1 mg of berberine with a 82% purity. The processing time was nearly 50% that of the multistep method. The results indicate that the present method is a rapid and green way to harvest targets from medicinal plants in a single step.

Single-step total fractionation of single-wall carbon nanotubes by countercurrent chromatography.

Development of simple processes to fractionate synthetic mixtures of single-wall carbon nanotubes (SWCNTs) into individual species is crucial to many applications. Existing methods for single-chirality SWCNT purification are cumbersome, often requiring multiple steps and different conditions for different species. Here, we report a method to achieve total fractionation of a synthetic SWCNT mixture by countercurrent chromatography, resulting in purification of many single-chirality SWCNT species in a single run. This method is based on a tunable partition of sodium deoxycholate dispersed SWCNTs in a polyethylene glycol/dextran aqueous two-phase system. By running the mobile phase with 0.02% of sodium deoxycholate and a gradient of sodium dodecyl sulfate from 0.1% to 0.7% (w/w), we observe clear diameter-dependent elution, with ∼ 90% total recovery. Among all the fractions collected, a number of them are enriched in single-chirality (9,4), (7,5), (7,6), (8,3), (6,5) species, while most of the remaining ones contain no more than 2-3 major species. We also observe strong (n,m)-dependent elution peak width due to the enantiomer-resolved partition. These results demonstrate countercurrent chromatography (CCC) as an effective way to obtain high purity (n, m) species, and suggest the potential of CCC as an analytical tool for chirality distribution mapping of synthetic SWCNT mixtures.