In-vivo half-life and hypoglycemic bioactivity of a fusion protein of exenatide and elastin-based polypeptide from recombinant Saccharomyces cerevisiae.

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 graft using MatriDerm® for plantar defects after excision of skin cancer.

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.

Development of novel on-line capillary gas chromatography-based analysis method for volatile organic compounds produced by aerobic fermentation.

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.

Clinical Evaluation of the Root Tear of the Posterior Horn of the Medial Meniscus in Total Knee Arthroplasty for Osteoarthritis.

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<0.05), varus deformity (p<0.05), mechanical axis deviation (p<0.05), and BMI (p<0.05). CONCLUSIONS: Factors considered to represent the severity of osteoarthritis were found to be associated with root tears of the medial meniscus posterior horn. Increased BMI seemed to be associated with the increased incidence of root tears of the medial meniscus posterior horn.

Avulsion fracture of the acromial physis in a 14-year-old boy: a case report.

Scapular fractures are uncommon and among them acromial fractures are even more uncommon. Because the vast majority of acromial fractures are either non-displaced or minimally displaced, symptomatic and nonoperative management was performed. We describe a case of avulsion fracture of the acromial physis displaced by acromioclavicular ligament treated with open reduction and internal fixation, and include a review of the literature.

Dynamic positional deformity of the hallux.

The present study reports a case with concomitant tethering of the flexor tendon and extensor tendon of the hallux after closed tibiofibular shaft fractures. We have obtained good clinical results using tenotomy of the flexor hallucis longus tendon and Z-plasty lengthening of the extensor hallucis longus tendon. Because few studies have described the clinical results and operative methods for this type of combined deformity, we report a case with dynamic positional deformity of the hallux.

Outcomes of the modified Brostrom procedure using suture anchors for chronic lateral ankle instability--a prospective, randomized comparison between single and double suture anchors.

The present prospective, randomized study was conducted to compare the clinical outcomes of the modified Brostrom procedure using single and double suture anchors for chronic lateral ankle instability. A total of 50 patients were followed up for more than 2 years after undergoing the modified Brostrom procedure. Of the 50 procedures, 25 each were performed using single and double suture anchors by 1 surgeon. The Karlsson scale had improved significantly to 89.8 points and 90.6 points in the single and double anchor groups, respectively. Using the Sefton grading system, 23 cases (92%) in the single anchor group and 22 (88%) in the double anchor group achieved satisfactory results. The talar tilt angle and anterior talar translation on stress radiographs using the Telos device had improved significantly to an average of 5.7° and 4.6 mm in the single anchor group and 4.5° and 4.3 mm in the double anchor group, respectively. The double anchor technique was superior with respect to the postoperative talar tilt. The single and double suture anchor techniques produced similar clinical and functional outcomes, with the exception of talar tilt as a reference of mechanical stability. The modified Brostrom procedure using both single and double suture anchors appears to be an effective treatment method for chronic lateral ankle instability.

Nonfusion method in thoracolumbar and lumbar spinal fractures.

STUDY DESIGN: a retrospective study of surgically managed patients. OBJECTIVE: to evaluate the results of posterior stabilization of thoracolumbar fracture using nonfusion method followed by the removal of metal implants within an appropriate period. Changes in the sagittal alignment and the restoration of segmental motion were also investigated. SUMMARY OF BACKGROUND DATA: posterior fusion using a transpedicular screw system remains the treatment of choice for the management of thoracolumbar and lumbar fractures. However, fusion methods result in the permanent loss of segmental motion. If both stability and motion could be achieved, functional results would improve considerably. METHODS: twenty-three patients under 40 years of age (mean, 28.0 years) with thoracolumbar or lumbar spine fractures were managed by this nonfusion method. Implants were removed at a mean 9.7 months after initial fracture fixation, and patients were observed for more than 18 months. Sagittal alignments of metal fixed segments, heights of vertebral bodies, recovered motion ranges in flexion/extension, right-left bending view were measured radiologically. Clinical aspects, such as gross deformities and functional abilities, were also investigated. RESULTS: heights of fractured bodies were well maintained at final follow-up. Initial mean sagittal angle was 17.2° kyphosis, which became 2.8° lordosis after fixation of fractures. This angle was 1.7° kyphosis just before implant removal, 2.4° kyphosis just after implant removal, and showed 5.9° kyphosis at final follow-up. Mean segmental motion was 14.2° in the sagittal plane and 13.1° in the coronal plane at final follow-up. Most patients were satisfied with final gross appearance and functional outcome. CONCLUSION: the described nonfusion method appears to be effective in achieving favorable sagittal alignment and regaining motions of fixed segments. The present study suggests that the nonfusion method is one of the most effective methods for managing thoracolumbar fractures, especially in young active people.

Mini-open muscle resection procedure under local anesthesia for lateral and medial epicondylitis.

BACKGROUND: This study examined the clinical results of surgical treatment using a mini-open muscle resection procedure under local anesthesia for intractable lateral or medial epicondylitis. METHODS: Forty two elbows (41 patients) were treated surgically for lateral or medial epicondylitis. The indication for surgery was refractory pain after six months of conservative treatment, or a history of more than three local injections of steroid, or severe functional impairment in the occupational activities. The treatment results were assessed in terms of the pain using the visual analogue scale (VAS), Roles & Maudsley score, and Nirschl & Pettrone grade. RESULTS: The preoperative VAS scores of pain were an average of 5.36 at rest, 6.44 at daily activities, and 8.2 at sports or occupational activities. After surgery, the VAS scores improved significantly (p < 0.01): 0.3 at rest, 1.46 at daily activities, and 2.21 at sports or occupational activities. The preoperative Roles & Maudsley score was acceptable in 6 cases, and poor in 36 cases, which was changed to excellent in 23 cases, good in 16 cases, acceptable in 3 cases after surgery. According to the grading system by Nirschl & Pettrone, 23 cases were excellent, 18 cases were good, and the remaining 1 case was fair. Overall, 41 cases (97.6%) achieved satisfactory results. Postoperative complications were encountered in three cases. Subcutaneous seroma due to the leakage of joint fluid in two patients was managed by additional surgery and suction drainage, and resulted in a satisfactory outcome. One patient complained of continuous pain on occupational activity, but her pain at rest was improved greatly. CONCLUSIONS: The mini-open muscle resection procedure under local anesthesia appears to be one of effective methods for intractable lateral or medial epicondylitis.

Phosphate-responsive promoter of a Pichia pastoris sodium phosphate symporter.

To develop a functional phosphate-regulated promoter in Pichia pastoris, a phosphate-responsive gene, PHO89, which encodes a putative sodium (Na(+))-coupled phosphate symporter, was isolated. Sequencing analyses revealed a 1,731-bp open reading frame encoding a 576-amino-acid polypeptide with 12 putative transmembrane domains. The properties of the PHO89 promoter (P(PHO89)) were investigated using a bacterial lipase gene as a reporter in 5-liter jar fermentation experiments. P(PHO89) was tightly regulated by phosphate and was highly activated when the cells were grown in a phosphate-limited external environment. Compared to translation elongation factor 1alpha and the glyceraldehyde-3-phosphate dehydrogenase promoter, P(PHO89) exhibited strong transcriptional activity with higher specific productivity (amount of lipase produced/cell/h). Furthermore, a cost-effective and simple P(PHO89)-based fermentation process was developed for industrial application. These results demonstrate the potential for efficient use of P(PHO89) for controlled production of recombinant proteins in P. pastoris.

Cloning and characterization of the Hansenula polymorpha PEP4 gene encoding proteinase A.

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.

Molecular cloning, sequencing, and expression of a fibrinolytic serine-protease gene from the earthworm Lumbricus rubellus.

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.