Recent advances in the metabolic pathways and microbial production of coenzyme Q.

Coenzyme Q (CoQ) serves as an electron carrier in aerobic respiration and has become an interesting target for biotechnological production due to its antioxidative effect and benefits in supplementation to patients with various diseases. Here, we review discovery of the pathway with a particular focus on its superstructuration and regulation, and we summarize the metabolic engineering strategies for overproduction of CoQ by microorganisms. Studies in model microorganisms elucidated the details of CoQ biosynthesis and revealed the existence of multiprotein complexes composed of several enzymes that catalyze consecutive reactions in the CoQ pathways of Saccharomyces cerevisiae and Escherichia coli. Recent findings indicate that the identity and the total number of proteins involved in CoQ biosynthesis vary between species, which raises interesting questions about the evolution of the pathway and could provide opportunities for easier engineering of CoQ production. For the biotechnological production, so far only microorganisms have been used that naturally synthesize CoQ10 or a related CoQ species. CoQ biosynthesis requires the aromatic precursor 4-hydroxybenzoic acid and the prenyl side chain that defines the CoQ species. Up to now, metabolic engineering strategies concentrated on the overproduction of the prenyl side chain as well as fine-tuning the expression of ubi genes from the ubiquinone modification pathway, resulting in high CoQ yields. With expanding knowledge about CoQ biosynthesis and exploration of new strategies for strain engineering, microbial CoQ production is expected to improve.

The process of National Health Insurance coverage for Chuna Manual Therapy in Korea: A qualitative study.

BACKGROUND: Chuna Manual Therapy (CMT) has been widely used in Korea, and coverage in Korean National Health Insurance (NHI) was finally implemented in 2019. The objectives of this study were to analyze the process of NHI coverage for CMT qualitatively, and to summarize important roles, streams, and implications regarding its inclusion in the modern public health insurance system. METHODS: Related literature was collected and 8 key personnel involved in the policy-making process were qualitatively interviewed, and Zahariadis' version of the Multiple Streams Framework (MSF) was applied to analyze the policy agenda setting and the roles of stakeholders. RESULTS: Through the collaborative efforts of various stakeholders, a pilot coverage project for CMT was implemented in 2017, and coverage was expanded nationwide in 2019. MSF showed that it was mainly achieved through three streams: governmental change (political stream), demand from the general public and KM doctors (problem stream), and strengthening/reinforcement of the feasibility and acceptability of the policy (policy steam). Also, the roles of policy entrepreneurs and resulting changes were shown to be significant for the overall process. CONCLUSION: NHI coverage for CMT was realized through collective policy and research efforts from the government and academic sectors. The roles of stakeholders were shown to be significant in the overall process, and documentation of their involvement is hoped to be of use of other countries that utilize traditional and/or manual medicine.

Hepatoprotective Effects of the Cichorium intybus Root Extract against Alcohol-Induced Liver Injury in Experimental Rats.

The effects of the Cichorium intybus root extract (Cii) on alcohol-induced liver disease were investigated using Chang liver cells and male Sprague Dawley rats. Silymarin, a liver-protective agent, was used as a positive control. In cell experiments, after 24 h of treatment with the extract, no cytotoxicity was noted, and death by alcohol was avoided. Migration of Chang liver cells increased after exposure to the extract at a concentration of 400 µg/mL. In animal experiments, alcohol was injected into 6-week-old rats for 1, 3, and 50 days. Oral administration of the drug was performed 30 min before alcohol administration. The control was treated with distilled water, and the drug groups were administered EtOH (40% EtOH + 2.5 mL/kg), EtOH + Cii L (low concentration, 2 mg/kg), EtOH + Cii H (high concentration, 10 mg/kg), or EtOH + silymarin (100 mg/kg). Increased liver weight was observed in the alcohol group, as were increased blood-alcohol concentration and liver damage indicators (glutamic oxalacetic transaminase (GOT), glutamic pyruvate transaminase (GPT), and triglycerides (TG)), decreased alcoholysis enzymes (ADH and ALDH), and increased CYP2E1. In the Cii treatment group, liver weight, blood-alcohol concentration, liver damage indicators (GOT, GPT, and TG), and CYP2E1 were decreased, while alcoholysis enzymes (ADH and ALDH) were increased. The degree of histopathological liver damage was compared visually and by staining with hematoxylin and eosin and oil red O. These results indicated that ingestion of Cii inhibited alcohol-induced liver damage, indicating Cii as a useful treatment for alcohol-induced liver injury.

Long-Term Follow-Up of Inpatients with Failed Back Surgery Syndrome Who Received Integrative Korean Medicine Treatment: A Retrospective Analysis and Questionnaire Survey Study.

INTRODUCTION: this study aimed to investigate the long-term clinical efficacy and satisfaction degree of integrative Korean medicine (KM) treatment for patients with failed back surgery syndrome (FBSS). METHODS: we performed a follow-up questionnaire survey and retrospective analysis of medical records for patients with FBSS who underwent inpatient treatment for ≥ 1 week. The primary evaluation indices were numeric rating scale (NRS) scores for low back pain (LBP) and leg pain at admission and discharge. Sub-evaluation indices included the Oswestry Disability Index (ODI) and EuroQol 5-dimension (EQ-5D) score. The follow-up questionnaire survey obtained information regarding previous surgeries; reasons for satisfaction/dissatisfaction with surgical and KM treatment; and current status. RESULTS: compared with at admission, there was a significant post-treatment decrease in the NRS scores for LBP and leg pain, as well as the ODI score. Further, there was a significant post-treatment increase in the EQ-5D score. Regarding the patients' global impression of change for KM treatment administered during admission and at the follow-up questionnaire survey, 101 (95.3%) patients selected "minimally improved" or better. CONCLUSION: integrative KM treatment could effectively reduce pain, as well as improve function and health-related quality of life, in patients with FBSS.

Coenzyme Q10 Biosynthesis Established in the Non-Ubiquinone Containing Corynebacterium glutamicum by Metabolic Engineering.

Coenzyme Q10 (CoQ10) serves as an electron carrier in aerobic respiration and has become an interesting target for biotechnological production due to its antioxidative effect and benefits in supplementation to patients with various diseases. For the microbial production, so far only bacteria have been used that naturally synthesize CoQ10 or a related CoQ species. Since the whole pathway involves many enzymatic steps and has not been fully elucidated yet, the set of genes required for transfer of CoQ10 synthesis to a bacterium not naturally synthesizing CoQ species remained unknown. Here, we established CoQ10 biosynthesis in the non-ubiquinone-containing Gram-positive Corynebacterium glutamicum by metabolic engineering. CoQ10 biosynthesis involves prenylation and, thus, requires farnesyl diphosphate as precursor. A carotenoid-deficient strain was engineered to synthesize an increased supply of the precursor molecule farnesyl diphosphate. Increased farnesyl diphosphate supply was demonstrated indirectly by increased conversion to amorpha-4,11-diene. To provide the first CoQ10 precursor decaprenyl diphosphate (DPP) from farnesyl diphosphate, DPP synthase gene ddsA from Paracoccus denitrificans was expressed. Improved supply of the second CoQ10 precursor, para-hydroxybenzoate (pHBA), resulted from metabolic engineering of the shikimate pathway. Prenylation of pHBA with DPP and subsequent decarboxylation, hydroxylation, and methylation reactions to yield CoQ10 was achieved by expression of ubi genes from Escherichia coli. CoQ10 biosynthesis was demonstrated in shake-flask cultivation and verified by liquid chromatography mass spectrometry analysis. To the best of our knowledge, this is the first report of CoQ10 production in a non-ubiquinone-containing bacterium.

Long-Term Follow-Up of Spinal Stenosis Inpatients Treated with Integrative Korean Medicine Treatment.

The present prospective observational study aimed to analyze the outcomes of inpatients who received integrative Korean medicine treatment in order to provide evidence on its effects on lumbar spinal stenosis (LSS). Patients with LSS who received inpatient treatment at four Korean medicine hospitals from January 2015 to December 2018 were followed up. Outcomes measured included the numeric rating scale (NRS) scores for back and leg pain, and Oswestry Disability Index (ODI). Changes in outcomes at admission, discharge, and follow-up, as well as associated predictors that could account for the improvement in outcomes were analyzed. The NRS score for back pain, NRS score for leg pain, and ODI decreased by 2.20 points (95% confidence interval (CI), -2.41 to -1.99), 2.28 points (95% CI, -2.59 to -1.96), and 17.31 points (95% CI, -19.6 to -15.02), respectively, at long-term follow-up compared with at admission. Patients with LSS who received inpatient integrative Korean medicine treatment exhibited an improvement in pain and functional disability. Further studies are required to determine the effects of integrative Korean medicine treatment.

Intensive Motion Style Acupuncture Treatment (MSAT) Is Effective for Patients with Acute Whiplash Injury: A Randomized Controlled Trial.

In this single-center, parallel, randomized controlled trial, we aim to examine the effects and safety of motion style acupuncture treatment (MSAT; a combination of acupuncture and Doin therapy) on pain reduction and functional improvement in patients with whiplash-associated disorders (WADs). Ninety-seven patients with cervical pain admitted to the Bucheon Jaseng Hospital of Korean Medicine, South Korea, due to acute whiplash injury were treated with integrative Korean medicine (IKM) with (MSAT group, 48 patients) or without (control group, 49 patients) an additional 3-day MSAT during hospitalization (5-14 days) and followed-up for 90 days. The mean numeric rating scale (NRS) scores of the MSAT and control groups at baseline were 5.67 (95% confidence interval (CI), 5.33, 6.01) and 5.44 (95% CI, 5.06, 5.82), respectively, and on day 5, 3.55 (95% CI, 3.04, 4.06) and 4.59 (95% CI, 4.10-5.07), respectively. The NRS change difference between the groups was -1.07 (95% CI, -1.76, -0.37). The rate of recovery of neck pain (NRS score change ≥ 2 points) was significantly faster in the MSAT than in the control group (log-rank test p = 0.0055). IKM treatment combined with MSAT may be effective in reducing the pain and improving the range of motion in patients with WADs.

Wound Healing Effects of Prunus yedoensis Matsumura Bark in Scalded Rats.

Pruni Cortex has been used to treat asthma, measles, cough, urticaria, pruritus, and dermatitis in traditional Korean medicine. The objective of this study was to investigate the effects of Prunus yedoensis Matsumura bark methanol extract (PYE) on scald-induced dorsal skin wounds in rats. Scalds were produced in Sprague-Dawley rats with 100°C water and treated with 5% and 20% PYE (using Vaseline as a base), silver sulfadiazine (SSD), and Vaseline once a day for 21 days, beginning 24 hours after scald by treatment group allocation. The PYE-treated groups showed accelerated healing from 12 days after scald, demonstrated by rapid eschar exfoliation compared to the control and SSD groups. PYE-treated groups showed higher wound contraction rates and better tissue regeneration in comparison with the control group. Serum analysis showed that transforming growth factor beta 1 and vascular endothelial growth factor levels remained high or gradually increased up to day 14 in both PYE groups and then showed a sharp decline by day 21, implying successful completion of the inflammatory phase and initiation of tissue regeneration. These findings suggested that PYE is effective in promoting scald wound healing in the inflammation and tissue proliferation stages.

Lidocaine/multivalent ion complex as a potential strategy for prolonged local anesthesia.

Local anesthetics are adopted in clinical practice to manage the perioperative and/or postoperative pain. However, their relatively short duration of action limit their ability to meet clinical needs. Herein, we prepared a lidocaine/multivalent ion complex (icLD) using aqueous solutions containing positively charged LD and a multivalent counter-ion as a system for producing prolonged anesthesia. The results of the in vitro and in vivo experiments indicated that the icLD facilitates prolonged LD release even without adjuvants and thus provides nerve blockade for a long duration of action (∼14h) without further increase in neurotoxicity than the LD itself. These findings suggested that the icLD could be a practical strategy for effectively controlling perioperative and/or postoperative pain in clinical practice.

The Role of Neoadjuvant Chemoradiation Therapy in Patients With Borderline Resectable Pancreatic Cancer With Isolated Venous Vascular Involvement.

The rationale for neoadjuvant chemoradiation therapy (Neo-CRT) and the definition of borderline resectable pancreatic cancer (BRPC) are still controversial. In particular, surgical treatment of BRPC with isolated venous vascular involvement (IVVI) is debatable.From January 2000 to December 2013, 84 patients diagnosed with BRPC according to NCCN guidelines were identified, and 70 patients were found to have BRPC with IVVI. We divided all 70 patients into 3 groups: surgery first without Neo-CRT (Group 1); pancreatectomy following Neo-CRT (Group 2); and no operation following Neo-CRT (Group 3). Patient characteristics including oncologic outcomes were analyzed for each of the 3 patients groups.Thirty-seven patients were female and 33 were male, with a mean age of 61.7 ±â€Š9.74 years. Among the 70 BRPC patients with IVVI, 28 patients (40%) belonged to Group 1, 30 patients (42.9%) belonged to Group 2, and 12 patients (17.1%) belonged to Group 3. Pathological tumor size (P < 0.001), pT stage (P = 0.001), pTNM stage (P=0.002), combined vascular resection (P = 0.003), completeness of adjuvant therapy (P = 0.004) were found to be statistically significantly different between Groups 1 and 2. In addition, disease-free survival (P = 0.055) and disease-specific survival (DSS) (P=0.006) were improved in Group 2. Interestingly, when comparing DSS, there was no statistically significant difference between Groups 1 and 3 (P = 0.991).The clinical practice of pancreatectomy following Neo-CRT in BRPC with IVVI provided favorable oncologic outcomes. The effect of Neo-CRT in BRPC with IVVI may be multifactorial, providing proper patient selection, complete adjuvant chemotherapy, and potential therapeutic (downstaging) effect.

Dual growth factor-immobilized microspheres for tissue reinnervation: in vitro and preliminary in vivo studies.

Growth factors (GFs) (basic fibroblast growth factor (bFGF) and/or nerve growth factor (NGF))-immobilized polycaprolactone (PCL)/Pluronic F127 microspheres were prepared using an isolated particulate-melting method and the sequential binding of heparin and GFs onto the microspheres. The GFs immobilized on the microspheres were released in a sustained manner over 28 days, regardless of GF type. From the in vitro culture of muscle-derived stem cells, it was observed that the NGF-immobilized microspheres induced more neurogenic differentiation than the bFGF-immobilized microspheres, as evidenced by a quantitative real-time polymerase chain reaction using specific neurogenic markers (Nestin, GFAP, ß-tubulin, and MAP2) and Western blot (Nestin and ß-tubulin) analyses. The dual bFGF/NGF-immobilized microspheres showed better neurogenic differentiation than the microspheres immobilized with single bFGF or NGF. From the preliminary animal study, the dual bFGF/NGF-immobilized microsphere group also showed effective nerve regeneration, as evaluated by immunocytochemistry using a marker - ß-tubulin. The dual bFGF/NGF-immobilized PCL/Pluronic F127 microspheres may be a promising candidate for nerve regeneration in certain target tissues (i.e. muscles) leading to sufficient reinnervation.

Epigallocatechin-3-O-(3-O-methyl)-gallate-induced differentiation of human keratinocytes involves klotho-mediated regulation of protein kinase-cAMP responsive element-binding protein signaling.

(-)-Epigallocatechin-3-O-gallate (EGCG) has long been known as a potent inducer of keratinocyte differentiation. Although its molecular mechanisms have been extensively studied, its actions on human skin remain to be elucidated. In this study, we demonstrated that methylated EGCG and EGCG increase the expression of klotho, and that klotho functions as a downstream target of EGCG and methylated EGCG in keratinocyte differentiation. We demonstrated that methylated EGCG3 and EGCG induce morphological changes in normal human epidermal keratinocytes (NHEKs) that are related to up-regulation of klotho expression. We also demonstrated that a klotho-induced keratinocyte differentiation marker in NHEKs is inhibited by H-89, a protein kinase (PKA) inhibitor. These results suggest that methylated EGCG and EGCG may function as inducers of keratinocyte differentiation via transcriptional regulation of the klotho protein.

Inhibited apoptosis of C(2)C(1)2 myoblasts by a Eupatorium chinense var. simplicifolium root extract.

We investigated the effects of a Eupatorium chinense var. simplicifolium (EUC) root extract on muscle disorders and explored the underlying mechanism for oxidative stress-induced C(2)C(12) myoblast damage. An EUC pre-treatment reduced the decreased cell viability after an H2O2 treatment. The heat shock protein (HSP) 70 level increased, and the phosphorylation of Jun amino-terminal kinases (JNKs) decreased in the EUC-pre-treated C(2)C(12) myoblasts. The results of the present study demonstrate the potential benefit of a herbal medicine in treating oxidative stress-related muscle disorders.

Comparison of the Amino-Acid Content in Pharmacopuncture Extracts Taken from a Scorpion's Body and from Its Tail.

OBJECTIVE: This study was conducted to investigate the amino-acid compositions of pharmacopuncture extracts taken from the body and from the tail of Buthus martensii Karsch, which are frequently prescribed in Oriental medicine. METHODS: Amino acids in hot water and 70% ethanol extracts taken from the scorpion's whole body and from its tail were screened by using high performance liquid chromatography (HPLC). The experiments were performed with linearity, precision and accuracy. RESULTS: The results of the amino-acid-composition analysis showed that the Buthus martensii Karsch extracts contained various amino acids such as aspartic acid, histidine, alanine, tyrosine, and cystine. The amino-acid analysis showed that the hot water extract was more beneficial than the ethanol extract, except for histidine. The amino acids from the tail and the body of the scorpion were compared, and the concentration of aspartic acid in the extract from the scorpion's tail was two times that found in the extract from its body. The results of validation experiments were all satisfactory. CONCLUSION: Studies on the ingredients in extracts from a scorpion other than buthotoxin may demonstrate that the antiepileptic efficacy, anticancer activity, antithrombotic action and analgesic effect are enhanced. Using only the tail of the scorpion when pharmacopuncture is dispensed may be beneficial because the extracts from the tail of the scorpion have higher potency than those from the whole body.

Effects of Korean red ginseng as an adjuvant to bile acids in medical dissolution therapy for gallstones: a prospective, randomized, controlled, double-blind pilot trial.

Although ginseng, the root of Panax quinquefolium and P. ginseng, was reported to have anti-cholelithogenic effects in animal experiments, there have, to date, been no human studies. We conducted this prospective, controlled, double-blind pilot trial to evaluate the safety and efficiency of Korean red ginseng (KRG), the steamed root of P. ginseng C.A. Meyer. Twenty eight consecutive patients were randomized to receive either KRG (7.5 g divided into three daily doses) or a placebo as an adjuvant to the standard regimen of bile acids for gallstones (500 mg of chenodeoxycholic acid and 500 mg of ursodeoxycholic acid divided into three daily doses) for 24 weeks. No case of serious adverse reaction occurred in both groups. Although the decrease in stone burden was larger in the KRG group (3.4 ± 0.6 ml3) than in the placebo group (2.3 ± 1.1 ml(3)), it did not reach statistical significance (p = 0.09). Also there were no differences in the rate of complete dissolution, subjective improvement in symptoms, and the rate of cholecystectomy due to worsening pain or the development of complications and changes in laboratory tests before and after treatment. In conclusion, the addition of KRG as an adjuvant was safe for patients undergoing bile acid dissolution therapy for gallstones although it did not affect the results. Large-scaled trials to optimize regimens are expectantly needed.

Core/shell nanoparticles for pH-sensitive delivery of doxorubicin.

Core/shell nanoparticles with lipid core were prepared and characterized as pH-sensitive delivery system of anticancer drug. The lipid core is composed of drug-loaded lecithin and the polymeric shell is composed of Pluronics (poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) tri-block copolymer, F-127). Based on the preparation method in the previous report by us, the freeze-drying of drug-loaded lecithin was performed in the F-127 aqueous solution containing trehalose used as a cryoprotectant to form stabilized core/shell nanoparticles. For the application of core/shell nanoparticles as a pH-sensitive drug delivery system for anticancer drug, doxorubicin was loaded into the core/shell nanoparticles and the drug loading amount and drug release behavior in response to pH change were observed.

Tissue anti-adhesion potential of ibuprofen-loaded PLLA-PEG diblock copolymer films.

This study was designed to evaluate the effect of polyethylene glycol (PEG) and nonsteroidal anti-inflammatory drug (ibuprofen) on the prevention of postsurgical tissue adhesion. For this, poly(L-lactic acid) (PLLA)-PEG diblock copolymers were synthesized by ring opening polymerization of L-lactide and methoxy polyethylene glycol (Mw 5000) of different compositions. The synthesized copolymers were characterized by gel permeation chromatography and 1H-nuclear magnetic resonance spectroscopy. PLLA-PEG copolymer films were prepared by solvent casting. The prepared copolymer films were more flexible and hydrophilic than the control PLLA film, as investigated by the measurements of glass transition temperature, water absorption content, and water contact angle. The drug release behavior from the ibuprofen (10 wt%)-loaded copolymer films was examined by high performance liquid chromatography. It was observed that the drug was released gradually up to about 40% of total loading amount after 20 days, depending on PEG composition; more drug release from the films with higher PEG compositions. In vitro cell adhesions on the copolymer films with/without drug were compared by the culture of NIH/3T3 mouse embryo fibroblasts on the surfaces. For in vivo evaluation of tissue anti-adhesion potential, the copolymer films with/without drug were implanted between the cecum and peritoneal wall defects of rats and their tissue adhesion extents were compared. It was observed that the ibuprofen-containing PLLA-PEG films with high PEG composition (particularly PLLA113-PEG113 film with PEG composition, 50 mol%) were very effective in preventing cell or tissue adhesion on the film surfaces, probably owing to the synergistic effects of highly mobile, hydrophilic PEG and anti-inflammatory drug, ibuprofen.