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Isoprene has the potential to replace some petroleum-based chemicals and can be produced through biological systems using renewable carbon sources. Ralstonia eutropha can produce value-added compounds, including intracellular polyhydroxyalkanoate (PHA) through fatty acid and lipid metabolism. In the present study, we engineered strains of R. eutropha H16 and examined the strains for isoprene production. We optimized codons of all the genes involved in isoprene synthesis by the mevalonate pathway and manipulated the promoter regions using pLac and pJ5 elements. Our results showed that isoprene productivity was higher using the J5 promoter (1.9 ± 0.24 µg/l) than when using the lac promoter (1.5 ± 0.2 µg/l). Additionally, the use of three J5 promoters was more efficient (3.8 ± 0.18 µg/l) for isoprene production than a one-promoter system, and could be scaled up to a 5-L batch-cultivation from a T-flask culture. Although the isoprene yield obtained in our study was insufficient to meet industrial demands, our study, for the first time, shows that R. eutropha can be modified for efficient isoprene production and lays the foundation for further optimization of the fermentation process.

Skin cancer should be excised with sufficient margin to reduce recurrence rate. However, the surgeon always has to worry about the reconstruction method of skin defects after excision. In particular, defects in the plantar surface of the foot are difficult to reconstruct due to their position and structure, and various methods are applied by each surgeon. Surgeons think which methods are easier to apply to patients and less morbidity. To alleviate these concerns, we applied artificial dermal substitute to skin defects after skin cancer. Bowen's disease (squamous cell carcinoma in situ) and melanoma in situ on the plantar surface of the foot were subjected to wide excision with sufficient margin. After excision, a skin defect with exposed plantar fascia was applied with a matrix defect and vacuum. A granulation tissue (dermal matrix) was formed and a split-thickness skin graft was performed. Both patients had good functional results and no problems with skin donor sites. Thus, we report a skin graft method that is relatively easy to apply after skin cancer excision on the plantar surface of the foot.

Many volatile compounds, such as isoprene, a precursor used in the synthesis of natural rubber, have been produced through fermentation using genetically engineered microorganisms. Despite this biotechnological success, measuring the concentrations of volatile compounds during fermentation is difficult because of their high volatility. In current systems, off-line analytical methods usually lead to product loss, whereas on-line methods raise the production cost due to the requirement of complex devices. Here, we developed a novel on-line gas chromatography (GC)-based system for analyzing the concentration of isoprene with the aim to minimize the cost and requirement for devices as compared to current strategies. In this system, a programmable logic controller is used to combine conventional GC with a syringe pump module (SPM) directly connected to the exhaust pipe of the fermentor, and isoprene-containing samples are continuously pumped from the SPM into the GC using an air cylinder recycle stream. We showed that this novel system enables isoprene analysis during fermentation with convenient equipment and without the requirement of an expensive desorption tube. Furthermore, this system may be extended to the detection of other volatile organic compounds in fermentation or chemical processes.

When knee osteoarthritis is combined with comorbidity, it is associated with limited physical activity. This study aimed to identify barriers to and facilitators of physical activity among Korean female adults with knee osteoarthritis and comorbidity, such as hypertension, diabetes, and dyslipidemia. A qualitative content analysis study was conducted. Ten female knee osteoarthritis participants with comorbidity were recruited at an orthopedic outpatient center in South Korea. Data were collected using in-depth interviews and were analyzed using a conventional content analysis method. Ten participants with a mean age of 70.7 years participated in this study. Four categories of barriers and three of facilitators were identified. Barriers to physical activity were physical hardships, lack of motivation, environmental restrictions, and lack of knowledge. Categories of facilitators were pain management, self-control in physical activity, and understanding the importance of physical activity. Participants did not express any social or environmental facilitators of physical exercise. Healthcare professionals should include social support and environmental facilities to achieve medical and institutional compliance. Understanding female adults with knee osteoarthritis and comorbidity would support provision of appropriately tailored interventions that account for the characteristics of the comorbidity.

The aim of this study was to test the clinical efficacy of TissueGene-C (TG-C), a cell and gene therapeutic for osteoarthritis consisting of non-transformed and transduced chondrocytes (3:1) retrovirally transduced to overexpress transforming growth factor-β1. A total of 163 Kellgren-Lawrence grade 3 patients with knee osteoarthritis were randomly assigned to receive intra-articular TG-C or placebo. Primary efficacy measures included criteria for subjective assessment by International Knee Documentation Committee (IKDC) and pain severity by Visual Analog Scale (VAS) for 52 weeks. Secondary efficacy measures included IKDC and VAS at 26 and 39 weeks; pain, stiffness, and physical function by the Western Ontario and McMaster Universities Arthritis Index (WOMAC); and pain, symptoms, daily activities, function in sports and recreation, and quality of life by the Knee Injury and Osteoarthritis Outcome Score (KOOS), X-ray, magnetic resonance imaging, and soluble urine and blood biomarkers. TG-C was associated with statistically significant improvement over placebo in the total IKDC score and individual categories, and in the VAS score at 26, 39, and 52 weeks. WOMAC and KOOS scores also improved with TG-C over placebo. Patients treated with TG-C showed trends directed toward thicker cartilage and slower growing rates of subchondral bone surface area in the medial tibia, lateral tibia, lateral patella, and lateral patella femoral regions, although these were not statistically significant (p 0.05). Serum C-terminal telopeptide of type I collagen (CTX-I) and urine CTX-II levels were lower over 1 year in TG-C than placebo-treated patients, with CTX-I level reaching statistical significance. These tendencies supported TG-C as holding great potential as a disease-modifying osteoarthritis drug. The most frequent adverse events in the TG-C group were peripheral edema (9%), arthralgia (8%), joint swelling (6%), and injection site pain (5%). TG-C was associated with statistically significant improvements in function and pain in patients with knee osteoarthritis. The unexpected adverse events were not observed.

The 2C-methyl-D-erythritol 4-phosphate (MEP) pathway is a carbon-efficient route for synthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the building blocks of isoprenoids. However, practical application of a native or recombinant MEP pathway for the mass production of isoprenoids in Escherichia coli has been unsatisfactory. In this study, the entire recombinant MEP pathway was established with plasmids and used for the production of an isoprenoid, protoilludene. E. coli harboring the recombinant MEP pathway plasmid (ME) and a protoilludene synthesis pathway plasmid (AO) produced 10.4mg/L of protoilludene after 48h of culture. To determine the rate-limiting gene on plasmid ME, each constituent gene of the MEP pathway was additionally overexpressed on the plasmid AO. The additional overexpression of IPP isomerase (IDI) enhanced protoilludene production to 67.4mg/L. Overexpression of the Fpr and FldA protein complex, which could mediate electron transfer from NADPH to Fe-S cluster proteins such as IspG and IspH of the MEP pathway, increased protoilludene production to 318.8mg/L. Given that it is required for IspC as well as IspG/H, the MEP pathway has high demand for NADPH. To increase the supply of NADPH, a NADH kinase from Saccharomyces cerevisiae (tPos5p) that converts NADH to NADPH was introduced along with the deletion of a promiscuous NADPH-dependent aldehyde reductase (YjgB) that consumes NADPH. This resulted in a protoilludene production of 512.7mg/L. The results indicate that IDI, Fpr-FldA redox proteins, and NADPH regenerators are key engineering points for boosting the metabolic flux toward a recombinant MEP pathway.

Exenatide (Ex) is a 39-amino acid peptide of glucagon-like peptide-1 (GLP-1) receptor agonist that was approved by the FDA in 2005 as a Type II diabetes treatment. It shows a 53% homology with GLP-1 but has an extended half-life (ca. 2.4 h) relative to GLP-1 (ca. 2–3 min). In this study, to further extend its in vivo half-life, we constructed a fusion protein (Ex-(EBP)10-6xHis) using a biocompatible and inert elastin-based polypeptide (EBP) as a fusion partner. Valine was inserted into the guest position of the pentapeptide (VPGXG), no linker sequence was inserted in between the EBPs, and (EBP)10-6xHis tag was attached to the C-terminus of exenatide. By using a recombinant Saccharomyces cerevisiae expression system, the fusion protein was expressed and secreted to the broth and purified by Ni-NTA affinity chromatography. Compared with the native exenatide, the physical half-life of the fusion protein was ca. 3.7-fold extended while approximately 72% of the in-vitro insulin secreting activity was maintained. However, the biological half-life measured by a glucose tolerance test (GTT) and the hypoglycemic test in mice was not significantly different from that of the native form. The effects of EBPylation on bioactivity and half-life of the fusion protein are similar to those of PEGylation. The result suggests that the bioactivity and half-life should be carefully balanced to obtain optimal fusion proteins. We expect that EBPylation using an optimal repeat number of EBP can be an alternative to chemical modification for therapeutic biobetters with extended half-life.

Skin cancer should be excised with sufficient margin to reduce recurrence rate. However, the surgeon always has to worry about the reconstruction method of skin defects after excision. In particular, defects in the plantar surface of the foot are difficult to reconstruct due to their position and structure, and various methods are applied by each surgeon. Surgeons think which methods are easier to apply to patients and less morbidity. To alleviate these concerns, we applied artificial dermal substitute to skin defects after skin cancer. Bowen’s disease (squamous cell carcinoma in situ) and melanoma in situ on the plantar surface of the foot were subjected to wide excision with sufficient margin. After excision, a skin defect with exposed plantar fascia was applied with a matrix defect and vacuum. A granulation tissue (dermal matrix) was formed and a split-thickness skin graft was performed. Both patients had good functional results and no problems with skin donor sites. Thus, we report a skin graft method that is relatively easy to apply after skin cancer excision on the plantar surface of the foot.

Purpose To investigate the incidence of root tears of the posterior horn of the medial meniscus in total knee replacement arthroplasty for knee osteoarthritis and retrospectively analyze clinical results and factors associated with root tears. Materials and Methods There were 197 knees of 140 enrolled patients who had undergone total knee replacement arthroplasty between September 2010 and May 2014. The presence of a root tear of the posterior horn of the medial meniscus was confirmed in all patients. Statistical analysis was performed to investigate the correlation between root tears and the possible factors of meniscal tears including gender, age, severity of symptoms (visual analogue scale [VAS] score and medial joint line tenderness), grade of osteoarthritis (Kellgren-Lawrence grading scale), body mass index (BMI), varus deformity, and mechanical axis deviation. Results Meniscal tears were observed in 154 knees (78.17%). The root tear had correlation with the severity of osteoarthritis (p

For recombinant production of squalene, which is a triterpenoid compound with increasing industrial applications, in microorganisms generally recognized as safe, we screened Saccharomyces cerevisiae strains to determine their suitability. A strong strain dependence was observed in squalene productivity among Saccharomyces cerevisiae strains upon overexpression of genes important for isoprenoid biosynthesis. In particular, a high level of squalene production (400 ± 45 mg/L) was obtained in shake flasks with the Y2805 strain overexpressing genes encoding a bacterial farnesyl diphosphate synthase (ispA) and a truncated form of hydroxyl-3-methylglutaryl-CoA reductase (tHMG1). Partial inhibition of squalene epoxidase by terbinafine further increased squalene production by up to 1.9-fold (756 ± 36 mg/L). Furthermore, squalene production of 2011 ± 75 or 1026 ± 37 mg/L was obtained from 5-L fed-batch fermentations in the presence or absence of terbinafine supplementation, respectively. These results suggest that the Y2805 strain has potential as a new alternative source of squalene production.

Metabolite production through a multistep metabolic pathway can often be increased by efficient substrate channeling created by spatial sequestration of the metabolic reactions. Here, Tya, a structural component in the Ty1 retrotransposon element that forms virus-like particles (VLPs) in Saccharomyces cerevisiae, was used to spatially organize enzymes involved in a metabolic pathway into a multi-enzyme protein body in yeast. As a proof of principle, Tya fusion to three key enzymes involved in biosynthesis of the isoprenoids farnesene and farnesol was tested to assess its potential to improve productivity. The Tya-fusion protein resulted in three and fourfold increases in farnesene and farnesol production, respectively, as compared with that observed in a non-fused control. Specifically, two-phase partitioning fed-batch fermentations of S. cerevisiae ATCC200589 overexpressing Tya-fused enzymes (tHmg1, IspA, and α-farnesene synthase) yielded 930 ± 40 mg/L of farnesene after 7 days. Additionally, we observed that the Tya-fusion proteins tended to partition into particulate fractions upon 100,000g ultracentrifugation, suggesting the formation of large aggregates of protein bodies, with their particulate structure also observed by transmission electron microscopy. The dramatic increase in the biosynthetic productivity of metabolites via use of a Tya-fusion protein suggested that this approach might be useful for the creation of multi-enzyme complexes to improve metabolic engineering in yeast.

A simplified model in the shape of a wedge box with an opening on the roof was used to generate buffeting conditions at HMC. These measurements performed in controlled conditions are intended to validate the ability of CFD tools to predict buffeting. The results obtained by PowerFLOW are presented in this paper for buffeting and for the boundary layer development on the roof of the model when the roof opening is closed. The flow mechanisms that explain the behavior of the experimental sound pressure level(SPL) curve are described, and an improved setup is used to reproduce the flow structures that lead to the measured SPL.

To report the fracture of proximal humerus in the lateral anchor site after suture bridge repair. A 57-year-old female patient with shoulder pain on the right-side was admitted through the emergency room following a car accident. Seven weeks before the accident, the patient had undergone surgery at a different hospital for the repair of supraspinatus tendon rupture on the right-side via suture bridge technique. Humerus surgical neck fracture was confirmed by X-ray, and proximal humerus fracture at the anchor site was confirmed by magnetic resonance imaging. Following 7 months of conservative treatment resulted in satisfactory bone union and motion of the shoulder joint. We report the need of close observation during and after the arthroscopic repair of the rotator cuff in patients with osteoporosis.

Geraniol, a monoterpene alcohol, has versatile applications in the fragrance industry, pharmacy and agrochemistry. Moreover, geraniol could be an ideal gasoline alternative. In this study, recombinant overexpression of geranyl diphosphate synthase and the bottom portion of a foreign mevalonate pathway in Escherichia coli MG1655 produced 13.3mg/L of geraniol. Introduction of Ocimum basilicum geraniol synthase increased geraniol production to 105.2mg/L. However, geraniol production encountered a loss from its endogenous dehydrogenization and isomerization into other geranoids (nerol, neral and geranial). Three E. coli enzymes (YjgB, YahK and YddN) were identified with high sequence identity to plant geraniol dehydrogenases. YjgB was demonstrated to be the major one responsible for geraniol dehydrogenization. Deletion of yjgB increased geraniol production to 129.7mg/L. Introduction of the whole mevalonate pathway for enhanced building block synthesis from endogenously synthesized mevalonate improved geraniol production up to 182.5mg/L in the yjgB mutant after 48h of culture, which was a double of that obtained in the wild type control (96.5mg/L). Our strategy for improving geraniol production in engineered E. coli should be generalizable for addressing similar problems during metabolic engineering.

Purpose: The purpose of this study was to evaluate the repair integrity and clinical outcomes of delaminated tears following arthroscopic layered suture bridge rotator cuff repair in a prospective fashion. Materials and Methods: A consecutive series of 67 patients with delaminated rotator cuff tears who underwent surgery using the arthroscopic Layered Suture Bridge Technique were followed prospectively. Of 67 shoulders, 26 patients were male and 40 patients were female (one patient had bilateral tears); the mean age was 58.8(40~76) years. The clinical evaluation was performed according to ASES, UCLA, and KSS (Korea Shoulder Scoring System) scores with an average follow up period of 33 months. Repair integrity was estimated using MRI, which was performed six months postoperatively. Results: The average clinical outcome in ASES, UCLA, and KSS scores showed significant improvement at the time of the final follow-up compared to preoperatively, from 50.2 to 92.3, 15 to 31.3, and 54.4 to 90.7, respectively (p

A painful pseudoarthrosis will develop due to a delay in diagnosis and treatment, and surgical care is required. Treatment of pseudoarthrosis is really difficult because the acromion is a thin flat bone that is difficult to be fixed firmly. A 52-year-old woman with multiple trauma had an acromial fracture that was not detected until it had caused pain after ambulation. Open reduction and internal fixation with a variable angle locking compression plate for distal radius and autogenous iliac bone graft were performed. At nine months after the operation, there was partial tear in the supraspinatus tendon, and arthroscopic repair of the supraspinatus tendon was performed. At nine months after the operation, radiographs showed a complete union. At three months after tendon repair, the patient had excellent function of the shoulder. We have reported a case of a successful treatment of nonunion and pseudoarthrosis of acromial fracture that is difficult to be fixed.

Geraniol is an important industrial material and a potential candidate of advanced biofuels. One challenge of microbial geraniol production is the toxicity to hosts. However, the poor understanding on geraniol tolerance mechanism is an obstacle for developing geraniol tolerant host. This study genome-widely screened a shot-gun DNA library of Escherichia coli and found that recA is able to confer geraniol tolerance in E. coli. The recA knockout mutant was found extremely sensitive to geraniol. Based on our data, it was deciphered that recA provided tolerance through SOS response network responding to DNA damage caused by geraniol. RecA-mediated SOS response activates the homologous recombinational repair by RecB and RecN for corrective DNA maintenance. This protection mechanism suggests an effective strategy to combat geraniol toxicity in E. coli.

The XylP gene, which encodes endoxylanase in Bacillus sp. HY-20, was subcloned, and two expression plasmids, pG-xylP and pGMF-xylP were constructed. These plasmids, which contain different signal sequences, XylP s.s and MFα opt s.s, respectively, for the secretory expression of endoxylanase, were transformed into Saccharomyces cerevisiae SEY2102 and FY833, respectively. The recombinant endoxylanases were successfully expressed, with a total activity range of 23.7-70.1 unit/ml according to the expression system and host strain. The endoxylanase activity in SEY2102/pGMF-xylP reached a maximum of 88.1 unit/ml in baffled flask culture. Most of the recombinant endoxylanase was efficiently secreted in the extracellular fraction, and the MFαopt s.s was more efficient for secreting endoxylanase in yeast than the XylP s.s. Therefore, the expression system developed in this study produces large extracellular amounts of endoxylanase using S. cerevisiae as the host strain, and it could be used in bioethanol production and industrial applications.

Improvement of a microorganism's tolerance against organic solvents is required for a microbial factory producing terpenoid based biofuels. The bacterial genes, marA, imp, cls and cti have been found to increase organic solvent tolerance. Thus, the tolerance against the following terpenoids (isopentenol, geraniol, myrcene, and farnesol) was studied with overexpression of marA, imp, cls and cti genes in Escherichia coli. The marA overexpression significantly enhanced the tolerance of E. coli against geraniol, whereas there was no tolerance improvement against the terpenoids by overexpression of cls and cti genes. The imp overexpression even yielded sensitive phenotype to the tested solvents. The colony forming efficiency of the marA overexpressing E. coli was increased by 10(4)-fold in plate overlay of geraniol compared to that of wild type E. coli and a two-fold decrease of intracellular geraniol accumulation was also observed in liquid culture of geraniol. Single knock-out mutations of marA, or one of the following genes (acrA, acrB and tolC) encoding AcrAB-TolC efflux pump made E. coli hypersensitive to geraniol. The geraniol tolerance conferred by marA overexpression was attributed to the AcrAB-TolC efflux pump that is activated by MarA.

To evaluate different codon optimization parameters on the Saccharomyces cerevisiae-derived mating factor α prepro-leader sequence (MFLS) to improve Candida antarctica lipase B (CAL-B) secretory production in Pichia pastoris. Codon optimization based on the individual codon usage (ICU) and codon context (CC) design parameters enhanced secretory production of CAL-B to 7 U/ml and 12 U/ml, respectively. Only 3 U/ml was obtained with the wild type sequence while the sequence optimized using both ICU and CC objectives showed intermediate performance of 10 U/ml. These results clearly show that CC is the most relevant parameter for the codon optimization of MFLS in P. pastoris, and there is no synergistic effect achieved by considering both ICU and CC together. The CC optimized MFLS increased secretory protein production of CAL-B in P. pastoris by fourfold.

Farnesol is a sesquiterpenoid alcohol that has important industrial and medical potential. It is usually synthesized from farnesyl diphosphate (FPP) by farnesol synthase in plants. FPP accumulation can cause up-regulation of phosphatases capable of FPP hydrolysis, resulting in farnesol production in Escherichia coli. We found that PgpB and YbjG, two integral membrane phosphatases, can hydrolyze FPP into farnesol. Overexpression of FPP synthase (IspA) and PgpB, along with a heterologous mevalonate pathway, enabled recombinant E. coli to produce 526.1 mg/L of farnesol. This result indicates that the phosphatases PgpB and YbjG can be used to construct a novel farnesol synthesis pathway for mass production in E. coli.

Additional file 1: Table S1. Primers and strains used for the construction of E. coli AceCo strain. Figure S1. Chromatogram of isoprene standard dissolved in dodecane. Figure S2. Standard curve of isoprene. Figure S3. Effect of IPTG induction on isoprene production and cell growth of MGpsPtM strain (MG1655 harboring pTS-sPtispS-MVA). Figure S4. Effect of low IPTG concentrations on isoprene production rate and culture broth pH in the culture of MGpsPtM strain (MG1655 harboring pTS-sPtispS-MVA). Figure S5. Acetate formation and glycerol consumption of the 0.01 mM IPTG induced culture of MGpsPtM in Fig. 4.

Background: This prospective, randomized study was conducted to compare clinical outcomes of the modified Broström procedure using suture anchor or transosseous suture technique for chronic ankle instability. Methods: Forty patients were followed for more than 2 years after modified Broström procedure. Twenty procedures using a suture anchor and 20 procedures using a transosseous suture were performed by one surgeon. The clinical evaluation consisted of the Karlsson scale and the Sefton grading system. Talar tilt and anterior talar translation were measured on anterior and varus stress radiographs. Results: The Karlsson scale had improved significantly to 90.8 points in the suture anchor group, and to 89.2 points in the transosseous suture group. According to Sefton grading system, 18 patients (90%) in suture anchor group and 17 patients (85%) in transosseous suture group achieved satisfactory results. The talar tilt angle and anterior talar translation improved significantly to 5.9 degrees and 4.2 mm in suture anchor group, and to 5.4 degrees and 4.1 mm in transosseous suture group, respectively. Conclusion: No significant differences existed in clinical and functional outcomes between the two techniques for ligament reattachment. Both modified Broström procedures using the suture anchor and transosseous suture seem to be effective treatment methods for chronic lateral ankle instability. Level of Evidence: I, Prospective Randomized Trial

Sesquiterpenes are important materials in pharmaceuticals and industry. Metabolic engineering has been successfully used to produce these valuable compounds in microbial hosts. However, the microbial potential of sesquiterpene production is limited by the poor heterologous expression of plant sesquiterpene synthases and the deficient FPP precursor supply. In this study, we engineered E. coli to produce α-farnesene using a codon-optimized α-farnesene synthase and an exogenous MVA pathway. Codon optimization of α-farnesene synthase improved both the synthase expression and α-farnesene production. Augmentation of the metabolic flux for FPP synthesis conferred a 1.6- to 48.0-fold increase in α-farnesene production. An additional increase in α-farnesene production was achieved by the protein fusion of FPP synthase and α-farnesene synthase. The engineered E. coli strain was able to produce 380.0 mg/L of α-farnesene, which is an approximately 317-fold increase over the initial production of 1.2 mg/L.

Purpose: This study was performed to assess the usefulness of non-anatomical repair for irreparable large and massive rotator cuff tears by the arthroscopic margin convergence technique. Materials and Methods: Twenty-two patients were followed up more than 1 year after non-anatomical repair for irreparable large and massive rotator cuff tears using the arthroscopic margin convergence technique. The clinical evaluation was performed according to the KSS score, the UCLA score and the Visual analogue scale (VAS). The measurement of the acromio-humeral distance was performed using the shoulder anterior-posterior radiographs. The measurement of fatty degeneration and the healing status was performed using the shoulder MRI after 6 months. Results: Among twenty-two patients, follow up MRI was performed in eleven cases. Three cases were well healed, four cases were partial healed and another four cases were re-torn. The KSS and UCLA scores had significantly improved from a preoperative average of 45.0±8.014 and 10.8±2.302 points to 77.1±10.151 and 30.0±1.521 points, respectively, and the pain VAS had decreased from a preoperative average of 7.7±0.616 points to 3.0±1.021 points at the last follow up. Less favorable results were obtained when the patient had a grade of fatty degeneration higher than grade 3 on the preoperative MRI. Conclusion: Non-anatomical repair for irreparable large and massive rotator cuff tears by the arthroscopic margin convergence technique showed good functional results. It seems to be one of the effective treatment methods for irreparable large and massive rotator cuff tears.

Farnesol (FOH) production has been carried out in metabolically engineered Escherichia coli. FOH is formed through the depyrophosphorylation of farnesyl pyrophosphate (FPP), which is synthesized from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) by FPP synthase. In order to increase FPP synthesis, E. coli was metabolically engineered to overexpress ispA and to utilize the foreign mevalonate (MVA) pathway for the efficient synthesis of IPP and DMAPP. Two-phase culture using a decane overlay of the culture broth was applied to reduce volatile loss of FOH produced during culture and to extract FOH from the culture broth. A FOH production of 135.5 mg/L was obtained from the recombinant E. coli harboring the pTispA and pSNA plasmids for ispA overexpression and MVA pathway utilization, respectively. It is interesting to observe that a large amount of FOH could be produced from E. coli without FOH synthase by the augmentation of FPP synthesis. Introduction of the exogenous MVA pathway enabled the dramatic production of FOH by E. coli while no detectable FOH production was observed in the endogenous MEP pathway-only control.

Purpose: The aim of this study was to evaluate the usefulness of arthroscopic Bony Bankart repair using a One Anchor Double Fixation Technique. Materials and Method: Seventeen patients with a Bony Bankart lesion were treated using the One Anchor Double Fixation Technique (OADF Technique). There were 13 males and 4 females. The average age was 24 years (range 17-42). The average follow-up period was 22.3 months. One 3.0 mm suture anchor with doubly loaded sutures was inserted into the glenoid rim. One suture strand was passed the around the small bony fragment and tied first. Another suture strand was passed through the capsule and tied over the bony fragment. The result was measured using Rowe’s evaluation index & KSS score. The glenoid defect & bony fragment were measured by 3D-CT scan. Results: Rowe’s evaluation index on the final follow-up showed an overall improvement from an average of 54 (range, 23-71) to 83.4 (range 71-90). Of the 17 cases, 13 were excellent, 3 were good, and 1 was fair. KSS scores showed improvement from an average of 71 (range 49-82) to 92.5 (range 82-94). There were no cases where pain continued to the final follow-up, and no cases being re-dislocated during the follow-up period. For six cases, we confirmed the bony healing of the bony Bankart lesion by CT. Conclusion: Bony Bankart lesion repair using this new method achieves excellent clinical results with low recurrence rates and is considered another choice for bony Bankart lesions.

Purpose: Calcium pyrophosphate dihydrate crystal deposition disease(CPPD) is a disease of the elderly and extremely rare in young individuals. If young people develop CPPD crystal deposition disease, it may be associated with metabolic diseases, such as hemochromatosis, hyperparathyroidism, hypophosphatasia, hypomagnesemia, Wilson’s disease, hypothyroidism, and gout. Materials and Methods: Therefore, in young-onset CPPD crystal deposition disease, an investigation of any predisposing metabolic conditions is warranted. Conclusion: We report a case of a young female patient who presented with idiopathic CPPD crystal deposition disease at 25 years of age.

Ethnopharmacology relevanceA previous study indicated non-inferiority of GCSB-5 to celecoxib regarding efficacy and safety in treating OA; however, the gastrointestinal (GI) safety data was limited to 12 weeks. Accordingly, a longer term study with a larger number of patients was necessary to establish the GI safety of GCSB-5.Aim of studyThe primary goal was to determine the safety and efficacy of 24-week use of GCSB-5. The secondary goal was to compare the GI safety data of GCSB-5 with that of the previously reported Celecoxib Long-term Arthritis Safety Study (CLASS).MethodThis was a 24-week, multicenter, single-arm phase IV Study for the safety and efficacy of GCSB-5. A total of 761 patients were enrolled and 756 patients received at least one dose of GCSB-5. Among them, 629 patients (82.7%) completed the 24 week follow up. The primary goal was to determine the safety and efficacy of GCSB-5 for 24 weeks. The secondary goal was to compare the GI safety data of GCSB-5 with that of the previously reported Celecoxib Long-term Arthritis Safety Study (CLASS).ResultsThe incidence of GI disorders of GCSB-5 was 23.7%. The annual rate of perforation, ulcer obstruction, or bleeding (PUB) incidence was 0.0%. The drop-out rate due to GI disorders following GCSB-5 use was 4.8%. Compared to celecoxib data from CLASS, the incidence of GI disorders (23.7% vs. 31.4%, p

Engineering of efflux pumps is a promising way to improve host’s tolerance to biofuels such as medium-chain alcohols (CmOHs); however, this strategy is restricted by poor understanding of the efflux pumps engaged in extrusion of solvents. In this study, several Escherichia coli mutants of multidrug transporters were evaluated for isoprenol tolerance. Susceptible phenotypes were observed in the mutants with individual deletion of six transporters, AcrD, EmrAB, MacAB, MdtBC, MdtJI and YdiM, whereas inactivation of AcrAB transporter resulted in an improved tolerance to isoprenol and other CmOHs. AcrAB is the major transporter forming tripartite transperiplasmic complex with outer membrane channel TolC for direct extrusion of toxic molecules in E. coli. The AcrAB inactivation enables to enhance TolC availability for the multidrug transporters associated with extrusion of CmOHs and increase the tolerance to CmOHs including isoprenol. It is assumed that outer membrane channel TolC plays an important role in extrusion of isoprenol and other CmOHs.

— Shoulder Impingement Caused by Superiorly Displaced GlenoidFracture Fragment at a Boy - A Case Report - Kyoung-Il Jeong, M.D. � , Kyoung-Jin Park, M.D.*, Yong-Min Kim, M.D., Dong-Soo Kim, M.D., Eui-Sung Choi, M.D., Hyun-Chul Shon, M.D., Hun-Sik Choi, M.D. � , Gee-Kang Park, M.D. Department of Orthopedic Surgery, Chungbuk National University Cheongju, KoreaDepartment of Orthopedic Surgery, choi Hospital, Cheong Ju, Korea � We experienced a case of impingement caused by a bone fragment which was impacted between acromion andhumeral head in a child. The bone fragment came from antero-inferior glenoid fracture. Satisfactory clinical resultsand stability were obtained by arthroscopic bone fragment removal. In case that bone fragment is located in the uppershoulder joint and results in impingement, We must consider not only greater tubercle fracture but also glenoid frac-ture. Magnetic resonance imaging can assist in the preoperative diagnosis. 관절면을침범한관절와골절은임상적으로흔하지않은골절로알려져있다 5) . 또한골절된골편이상방전위되어견관절의충돌운동장애를일으키는경우는보고된예가없다. 이에저자들은견관절관절와골절후골편이상방전위되어서충돌운동장애를일으킨예에서관절경적제거술로운동범위회복을얻었고불안정성은관찰되지않았기에문헌고찰과함께보고하고자한다.

Human urokinase-type plasminogen activator (uPA) is poorly secreted and aggregates in the endoplasmic reticulum of yeast cells due to inefficient folding. A screen for Hansenula polymorpha mutants with improved uPA secretion revealed a gene encoding a homologue of the Saccharomyces cerevisiae protein-O-mannosyltransferase Pmt1p. Expression of the H. polymorpha PMT1 gene (HpPMT1) abolished temperature sensitivity of the S. cerevisiae pmt1 pmt2 double mutant. As in S. cerevisiae, inactivation of the HpPMT1 gene affected electrophoretic mobility of the O-glycosylated protein, extracellular chitinase. In contrast to S. cerevisiae, disruption of HpPMT1 alone caused temperature sensitivity. Inactivation of the HpPMT1 gene decreased intracellular aggregation of uPA, suggesting that enhanced secretion of uPA was due to improvement of its folding in the endoplasmic reticulum. Unlike most of the endoplasmic reticulum membrane proteins, HpPmt1p possesses the C-terminal KDEL retention signal. The GenBank Accession No. for the H. polymorpha PMT1 sequence is AY701415. Copyright © 2005 John Wiley & Sons, Ltd.

The Hansenula polymorpha PEP4 gene encoding proteinase A was cloned by Southern blot hybridization using the Saccharomyces cerevisiae PEP4 gene as probe and characterized by gene disruption and overexpression. Nucleotide sequence analysis revealed an open reading frame (ORF) of 1239 nucleotides corresponding to a polypeptide of 413 amino acids, sharing about 67.2% sequence similarity with that of S. cerevisiae proteinase A. That the cloned H. polymorpha PEP4 gene encodes proteinase A was supported by a gene disruption experiment, which showed that the H. polymorpha pep4 mutant strain showed significantly reduced level of carboxypeptidase Y activity when assayed with an artificial substrate. When the PEP4 gene is overproduced in pep4 mutant strain, mature proteinase A could be found in the growth medium. N-terminal amino acid sequencing of extracellular proteinase A revealed the presence of a putative propeptide of 55 amino acids ending with a dibasic peptide (Lys-Arg), probably processed by Kex2p-like endopeptidase of H. polymorpha. The nucleotide sequence of the H. polymorpha PEP4 gene has been submitted to GenBank under Accession No. U67173.

The removal of Gal80 protein by gene disruption turned into efficient GAL promoter-driven heterologous gene expression under anaerobic alcoholic fermentation of Saccharomyces cerevisiae . Using lipase B from Candida antarctica as a reporter, the relative strength of GAL10 promoter ( P GAL10 ) in Δgal80 mutant that does not require galactose as an inducer was compared to those of ADH1 , PDC1 , and PGK promoters, which have been known to work well anaerobically in actively fermenting yeast cells under high glucose concentration. P GAL10 in the Δgal80 mutant showed 0.8-fold ( ADH1 ), fourfold ( PDC1 ), and 50-fold ( PGK ) in promoter strength.

The full-length cDNA of the lumbrokinase fraction 6 (F6) protease gene of Lumbricus rubellus was amplified using an mRNA template, sequenced and expressed in E. coli cells. The F6 protease gene consisted of pro- and mature sequences by gene sequence analysis, and the protease was translated and modified into active mature polypeptide by N-terminal amino acid sequence analysis of the F6 protease. The pro-region of F6 protease consisted of the 44 residues from methionine-1 to lysine-44, and the mature polypeptide sequence (239 amino acid residues and one stop codon; 720 bp) started from isoleucine-45 and continued to the terminal residue. F6 protease gene clones having pro-mature sequence and mature sequence produced inclusion bodies in E. coli cells. When inclusion bodies were orally administrated rats, generated thrombus weight in the rat's venous was reduced by approximately 60 % versus controls. When the inclusion bodies were solubilized in pepsin and/or trypsin solutions, the solubilized enzymes showed hemolytic activity in vitro. It was concluded the F6 protease has hemolytic activity, and that it is composed of pro- and mature regions.

The secretion of Bacillus stearothermophilus L1 lipase in Saccharomyces cerevisiae was investigated by employing a fusion partner, a cellulose-binding domain (CBD) from Trichoderma harzianum endoglucanase II (THEG). The CBD was connected to the N-terminal of L1 lipase through an endogenous linker peptide from THEG. The expression cassette for the fusion protein in S. cerevisiae was constructed using the alpha-amylase signal peptide and the galactose-inducible GAL10 promoter. Secretion of CBD-linker-L1 lipase by this fusion construct was dramatically 7-fold enhanced, compared with that of the mature L1 lipase without CBD-fusion. The fusion protein was secreted into the culture medium, reaching levels of approximately 1.3 g/l in high-cell-density fed-batch cultures. Insertion of a KEX2 cleavage site into the junction between CBD-linker and L1 lipase resulted in the same level of enhanced secretion, indicating that the CBD-linker fusion probably plays a critical role in secretion from endoplasmic reticulum to Golgi apparatus. Therefore, the CBD from THEG can be used both as an affinity tag and as a secretion enhancer for the secretory production of heterologous proteins in S. cerevisiae, since in vivo breakage at the linker was almost negligible.

Saccharomyces cerevisiae sphinganine C4-hydroxylase encoded by the SUR2 gene catalyses the conversion of sphinganine to phytosphingosine. We isolated the SUR2 gene from Pichia ciferrii using nucleotide sequence homology to S. cerevisiae SUR2 to study hydroxylation of sphinganine in the sphingoid base overproducing yeast P. ciferrii. A positive clone was confirmed by nucleotide sequencing. A syringomycin-E resistance phenotype of a S. cerevisiae sur2-null mutant was complemented by expression of the cloned P. ciferrii SUR2 gene. Restoration of phytosphingosine production in the complemented strain was also confirmed, indicating that the cloned gene is a functional homologue of S. cerevisiae SUR2. .

As a novel feeding strategy for optimizing human epidermal growth factor (hEGF) production with a recombinant Hansenula polymorpha DL-1 using the methanol oxidase (MOX) promoter in H. polymorpha DL-1, independent exponential feeding of two substrates was used. A simple kinetic model considering the cell growth on two substrates was established and used to calculate the respective feeding rates of glycerol and methanol. In the fedbatch culture with methanol-only feeding, the optimal set point of specific growth rate on methanol was found to be 0.10 h-1. When the fed-batch cultures were conducted by the independent feeding of glycerol and methanol, the actual specific growth rate on glycerol and methanol was slightly lower than the set point of specific growth rate. By the uncoupled feeding of glycerol and methanol the volumetric productivity of hEGF increased from 6.4 to 8.0 mg/(L.h), compared with methanol-only feeding.

Xanthophyllomyces dendrorhous (Phaffia rhodozyma) is used as a colorant for aquaculture, egg yolks, and crustaceans but its carotenoids can only be absorbed by animals when its cell wall is degraded. Conditions that degraded the cell wall of X. dendrorhous were developed. To measure the degrees of cell wall degradation, the carotenoid extractability (extracted carotenoid by acetone/total carotenoid) unit was used. Treatment with HCl (0.2 M, 9 h, 90 °C) followed by neutralization to pH 3 by NaOH and spray-drying increased carotenoid extractability to 100% with minimal decomposition.

A novel approach to isolation and functional characterization of the Hansenula polymorpha genes basing on the use of two strains of different origin is described. One of these strains is better suited for the isolation of genomic DNA fragments, while the other is preferable for their functional analysis. Thirty three genomic sequences governing expression of a reporter protein have been isolated. Analysis of the sequence encoding a homolog of the Saccharomyces cerevisiae cofilin revealed two introns. Another isolated DNA fragment encoded a homolog of the S. cerevisiae Vps10p. Disruption of the corresponding gene resulted in secretion of a vacuolar protein, carboxypeptidase Y, into the culture medium.

Lactic acid fermentation of Jerusalem artichoke tuber was performed with strains of Lactobacillus paracasei without acidic or enzymatic inulin hydrolysis prior to fermentation. Some strains of L. paracasei, notably KCTC13090 and KCTC13169, could ferment hot-water extract of Jerusalem artichoke tuber more efficiently compared with other Lactobacillus spp. such as L. casei type strain KCTC3109. The L. paracasei strains could utilize almost completely the fructo-oligosaccharides present in Jerusalem artichoke. Inulin-fermenting L. paracasei strains produced c.a. six times more lactic acid compared with L. casei KCTC3109. Direct lactic fermentation of Jerusalem artichoke tuber extract at 111.6g/L of sugar content with a supplement of 5 g/L of yeast extract by L. paracasei KCTC13169 in a 5L jar fermentor produced 92.5 ce:hsp sp="0.25"/>g/L of lactic acid with 16.8 g/L fructose equivalent remained unutilized in 72 h. The conversion efficiency of inulin-type sugars to lactic acid was 98% of the theoretical yield.

The pH dependency of cell growth and human epidermal growth factor (hEGF) production was studied with a recombinant Hansenula polymorpha strain. Maximum specific growth rate and apparent specific production rate of hEGF was highly dependent on culture pH over the range of 4.0–7.0. The optimal pH for the fastest growth was 5.0 and for the apparent production of hEGF 6.0. A simple kinetic model considering the degradation of hEGF during culture was established. Then it was used to estimate the intrinsic specific production rate of hEGF and simulate expression behavior according to culture pH. The intrinsic specific production rate of hEGF was relatively unaffected by culture pH over the range of 5.0–7.0. An optimal pH switching time exists for a given methanol concentration in the fed-batch culture maintaining constant methanol concentration.