Assessment of Escherichia coli-derived Recombinant Human Bone Morphogenic Protein-2 on Fertilization and Early Embryonic Development in Rats.

OBJECTIVE: Bone morphogenetic protein-2 (BMP-2) impacts fertility in women by affecting the menstrual cycle and embryonic development. We aimed to determine the reproductive toxicity of Escherichia coli (E. coli)-derived recombinant human BMP-2 (rhBMP-2) by measuring changes in the reproductive performance and organs in rhBMP-2-treated rats. METHODS: Overall, 88 male and female rats each were categorized into one control and three experimental groups. rhBMP-2 was intravenously administered to the experimental groups at 0.05, 0.15, and 0.50 mg/kg/day, respectively. The male rats were administered rhBMP-2 daily, starting from 28 days before mating until the day of necropsy (48 days), after which they were euthanized and necropsied. The female rats were administered rhBMP-2 daily, starting from 14 days before mating until 7 days after fertilization (22-36 days), after which they were necropsied 13 days after fertilization. RESULTS: No rhBMP-2-related death occurred throughout the study period. All rhBMP-2-treated groups showed swelling in the tail at the site of rhBMP-2 administration. In the high-dose rhBMP-2 group, the male rats showed a slight reduction in body weight and food consumption, whereas the female rats showed a reduction in the weights of the ovary and oviduct. Examining the fertilization status and necropsy showed no effect of rhBMP-2 on fertility and early embryonic development. The no-observed-adverse-effect level of rhBMP-2 was 0.50 mg/kg/day in all rats. CONCLUSION: rhBMP-2 had no reproductive toxicity on the reproductive performance and organs in female and male rats. Therefore, these results provide new toxicology information on E. coli-derived rhBMP-2 as a therapeutic protein.

Production of recombinant human acid ß-glucosidase with high mannose-type N-glycans in rice gnt1 mutant for potential treatment of Gaucher disease.

Gaucher disease is an inherited metabolic disease caused by genetic acid ß -glucosidase (GBA) deficiency and is currently treated by enzyme replacement therapy. For uptake into macrophages, GBA needs to carry terminal mannose residues on their N-glycans. Knockout mutant rice of N-acetylglucosaminyltransferase-I (gnt1) have a disrupted N-glycan processing pathway and produce only glycoproteins with high mannose residues. In this study, we introduced a gene encoding recombinant human GBA into both wild-type rice (WT) and rice gnt1 calli. Target gene integration and mRNA expression were confirmed by genomic DNA PCR and Northern blotting, respectively. Secreted rhGBAs in culture media from cell lines originating from both WT (WT-GBA) and rice gnt1 (gnt1-GBA) were detected by Western blotting. Each rhGBA was purified by affinity and ion exchange chromatography. In vitro catalytic activity of purified rhGBA was comparable to commercial Chinese hamster ovary cell-derived rhGBA. N-glycans were isolated from WT-GBA and gnt1-GBA and analyzed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The amounts of high mannose-type N-glycans were highly elevated in gnt1-GBA (100%) compared to WT-GBA (1%).

The clinical and radiological results of individualized surgical treatment depending on pathologic abnormalities in recurrent patellar dislocation: low recurrence rate, but unintended patella baja.

PURPOSE: To evaluate the clinical and radiological outcomes and chondral lesion change using individualized surgery for recurrent patellar dislocation. METHODS: A total of 31 knees with recurrent patellar dislocation underwent surgery depending on individual pathologic abnormalities. Pathologic abnormalities including medial laxity, lateral tightness, increased tibial tuberosity (TT)-to-trochlear groove distance (>20 mm), and patella alta (Caton-Deschamps ratio >1.2) were evaluated in each patient. The abnormalities were corrected through medial patellofemoral ligament reconstruction, TT distalization, TT anteromedialization, and lateral retinacula release. The mean follow-up period was 33 months. RESULTS: There was one recurrent case (3.2%), requiring additional surgery. The mean Kujala scores were significantly (P = 0.002) improved from 75.8 (SD 12.4) to 84.6 (SD 13.1). Tegner scores were significantly improved from 3.7 (range 1-9) to 5.4 (range 2-9) (P < 0.001), as were and visual analogue scale pain scores from 4.7 (SD 2.5) to 2.6 (SD 2.2) (P = 0.001). Caton-Deschamps ratio was significantly decreased from 1.1 (SD 0.2) to 0.9 (SD 0.1) (P < 0.001), regardless of TT distalization. Chondral lesions of the patella and trochlear groove were improved or maintained in 57.1 and 71.4% of patients, respectively. CONCLUSION: Individualized surgery in recurrent patellar dislocation was effective and safe with a low recurrence rate. However, the possibility of unintended patella baja, which might be related to post-operative anterior knee pain, should be considered. LEVEL OF EVIDENCE: IV.

Heterologous expression of pikromycin biosynthetic gene cluster using Streptomyces artificial chromosome system.

BACKGROUND: Heterologous expression of biosynthetic gene clusters of natural microbial products has become an essential strategy for titer improvement and pathway engineering of various potentially-valuable natural products. A Streptomyces artificial chromosomal conjugation vector, pSBAC, was previously successfully applied for precise cloning and tandem integration of a large polyketide tautomycetin (TMC) biosynthetic gene cluster (Nah et al. in Microb Cell Fact 14(1):1, 2015), implying that this strategy could be employed to develop a custom overexpression scheme of natural product pathway clusters present in actinomycetes. RESULTS: To validate the pSBAC system as a generally-applicable heterologous overexpression system for a large-sized polyketide biosynthetic gene cluster in Streptomyces, another model polyketide compound, the pikromycin biosynthetic gene cluster, was preciously cloned and heterologously expressed using the pSBAC system. A unique HindIII restriction site was precisely inserted at one of the border regions of the pikromycin biosynthetic gene cluster within the chromosome of Streptomyces venezuelae, followed by site-specific recombination of pSBAC into the flanking region of the pikromycin gene cluster. Unlike the previous cloning process, one HindIII site integration step was skipped through pSBAC modification. pPik001, a pSBAC containing the pikromycin biosynthetic gene cluster, was directly introduced into two heterologous hosts, Streptomyces lividans and Streptomyces coelicolor, resulting in the production of 10-deoxymethynolide, a major pikromycin derivative. When two entire pikromycin biosynthetic gene clusters were tandemly introduced into the S. lividans chromosome, overproduction of 10-deoxymethynolide and the presence of pikromycin, which was previously not detected, were both confirmed. Moreover, comparative qRT-PCR results confirmed that the transcription of pikromycin biosynthetic genes was significantly upregulated in S. lividans containing tandem clusters of pikromycin biosynthetic gene clusters. CONCLUSIONS: The 60 kb pikromycin biosynthetic gene cluster was isolated in a single integration pSBAC vector. Introduction of the pikromycin biosynthetic gene cluster into the pikromycin non-producing strains resulted in higher pikromycin production. The utility of the pSBAC system as a precise cloning tool for large-sized biosynthetic gene clusters was verified through heterologous expression of the pikromycin biosynthetic gene cluster. Moreover, this pSBAC-driven heterologous expression strategy was confirmed to be an ideal approach for production of low and inconsistent natural products such as pikromycin in S. venezuelae, implying that this strategy could be employed for development of a custom overexpression scheme of natural product biosynthetic gene clusters in actinomycetes.

Redesign of antifungal polyene glycosylation: engineered biosynthesis of disaccharide-modified NPP.

Polyene macrolides such as nystatin A1 and amphotericin B have been known to be potent antifungal antibiotics for several decades. Because the therapeutic application of polyenes is restricted by severe side effects such as nephrotoxicity, various chemical and biological studies to modify the polyene structure have been conducted to develop less-toxic polyene antifungals. A newly discovered nystatin-like polyene compound NPP was shown to contain an aglycone that was identical to nystatin but harbored a unique di-sugar moiety, mycosaminyl-N-acetyl-glucosamine, which led to higher solubility and reduced hemolytic toxicity. Additionally, a NPP-specific second sugar extending gene, nppY, was recently identified to be responsible for the transfer of a second sugar, N-acetyl-glucosamine, in NPP biosynthesis. In this study, we investigated biosynthesis of the glycoengineered NPP analog through genetic manipulation of the NPP A1 producer, Pseudonocardia autotrophica KCTC9441. NypY is another second sugar glycosyltransferase produced by Pseudonocardia sp. P1 that is responsible for the transfer of a mannose to the mycosaminyl sugar residue of nystatin. We blocked the transfer of a second sugar through nppY disruption, then expressed nypY in P. autotrophica △nppY mutant strain. When compared with nystain A1 and NPP A1, the newly engineered mannosylated NPP analog showed reduced in vitro antifungal activity, while exhibiting higher nephrotoxical activities against human hepatocytes. These results suggest for the first time that not only the number of sugar residues but also the type of extended second sugar moiety could affect biological activities of polyene macrolides.

Vertical Lifting: A New Optimal Thread Lifting Technique for Asians.

BACKGROUND: With the evolution of facial rejuvenation methods, thread lifts have gained popularity among patients who seek cosmetic improvement. Absorbable wedge-shaped polydioxanone (PDO) sutures are currently available, and they are extremely popular in the aesthetic clinics in Korea. In case of midface and mandibular jowl lift, threads are most often inserted in an oblique manner with vectors of rejuvenation directed toward the temple. However, specific characteristics of skeletal anatomy should be considered when deciding which technique to use in Asians. Herein, the authors introduce a vertical lifting technique most suitable for Asians, in which short (6 cm in length), wedge-shaped PDO sutures are inserted vertically downward in the anterior malar and submalar areas. OBJECTIVE: To describe a novel technique adopted to counteract the descent and laxity of the Asian face. The authors also aimed to assess the overall safety and efficacy of wedge-shaped PDO thread lift in facial rejuvenation. METHODS: A retrospective chart review was performed on cases of facial laxity treated with vertical thread lifting. A total of 39 Korean patients were included. All participants underwent a single treatment session. The results were assessed objectively using serial photography and subjectively based on the patients' satisfaction scores. Complications were also recorded. RESULTS: Most patients (89.7%) considered the results satisfactory. Consensus ratings by 2 independent dermatologists showed that the objective outcomes at the 6-month follow-up were largely categorized as very much improved (10.3%), much improved (43.6%), and improved (33.3%). The incidence of complications was low, and the complications were minor. CONCLUSION: Thread lifting with short, wedge-shaped PDO sutures is safe and effective for facial rejuvenation. The authors' vertical lifting technique has several advantages over the current approaches. First, the vectors directly oppose the vertical sagging of the face, which makes the technique highly efficient. The technique also carries a lower risk of accentuating the cheekbones, a feature which most Asians do not prefer.

Improved recovery and biological activities of an engineered polyene NPP analogue in Pseudonocardia autotrophica.

NPP A1 produced by Pseudonocardia autotrophica is a unique disaccharide-containing polyene macrolide. NPP A1 was reported to have higher water solubility and lower hemolytic toxicity than nystatin A1 while retaining its antifungal activity. An engineered NPP A1 analogue, NPP A2, was generated by inactivation of the nppL gene, encoding a P450 monooxygenase in P. autotrophica. The resulting compound exhibited the corresponding chemical structure of NPP A1 but lacked a C10 hydroxyl group. In this study, newly developed crystallization recovery methods for NPP A2 purification, followed by an evaluation of in vitro antifungal activity and hemolytic activity, were performed. The crystallization methods were designed to eliminate the undesired viscous impurities encountered during the NPP A2 purification process, resulting in improved purity from 5.3 to 83.5% w/w. NPP A2 isolated from the improved purification process also exhibited two times higher antifungal activity and 1.8 times higher hemolytic toxicity than those of NPP A1. These results suggest that the minor structural modification of disaccharide-containing polyene macrolides, such as removing a C10 hydroxyl group, might require an alternative recovery process, such as crystallization, to confirm its improved biological activity.

Arthroscopic modified Mason-Allen technique for large U- or L-shaped rotator cuff tears.

PURPOSE: While a conventional single- or double-row repair technique could be applied for repair of C-shaped tears, a different surgical strategy should be considered for repair of U- or L-shaped tears because they typically have complex patterns with anterior, posterior, or both mobile leaves. This study was performed to examine the outcomes of the modified Mason-Allen technique for footprint restoration in the treatment of large U- or L-shaped rotator cuff tears. METHODS: Thirty-two patients who underwent an arthroscopic modified Mason-Allen technique for large U- or L-shaped rotator cuff tears between January 2012 and December 2013 were included in this study. Margin convergence was first performed to reduce the tear gap and tension, and then, an arthroscopic Mason-Allen technique was performed to restore the rotator cuff footprint in a side-to-end repair fashion. All patients were evaluated preoperatively and for a minimum of 2 years of follow-up with a visual analog scale (VAS) for pain, Constant score, and ultrasonography. RESULTS: There was significant improvement in all VAS and Constant scores compared with the preoperative values (P < 0.001). Functional results by Constant scores included 9 cases that were classified as excellent, 11 cases as good, 8 cases as fair, and 2 cases as poor. Binary logistic regression analysis revealed that heavy work, pseudoparalysis, joint space narrowing, fatty degeneration of the SST and IST, and a positive tangent sign were found to significantly correlate with functional outcomes. Multivariable logistic regression analysis revealed that only fatty degeneration of the SST was a risk factor for fair/poor clinical outcomes. Complications occurred in 5 of the 32 patients (15.6 %), and the reoperation rate due to complications was 6.3 % (2 of 32 patients). CONCLUSIONS: An arthroscopic modified Mason-Allen technique was sufficient to restore the footprint of the rotator cuff in our data. Overall satisfactory results were achieved in most patients, with the exception of those with severe fatty degeneration. An arthroscopic modified Mason-Allen technique could be an effective and reliable alternative for patients with large U- or L-shaped rotator cuff tears. LEVEL OF EVIDENCE: Case Series, Therapeutic Level IV.

Discrepancy of alignment in different weight bearing conditions before and after high tibial osteotomy.

PURPOSE: To evaluate the differences in the amount of varus malalignment and valgus (over) correction in relation to three different weight bearing conditions from whole leg AP radiographs (single-limb (SL) stance, double-limb (DL) stance, supine position (S)) before and after high tibial osteotomy (HTO), and to evaluate which alignment parameters affect the changes for patients in three different weight bearing conditions. METHODS: A total of 40 consecutive patients (43 knees) with varus osteoarthritis underwent navigation assisted open wedge HTO. Mechanical axis angle (MA) was measured before and after surgery from hip-to-ankle radiographs taken with patients in three different weight bearing conditions. To find significant factors that affect the alignment differences, several variables including patient demographics, soft tissue laxity, pelvic obliquity, and ground mechanical axis deviation of tibia (calculated by the angle between two lines, tibial anatomical axis and weight-bearing line) were evaluated. RESULTS: Pre-operatively, mean MA measured on SL stance radiographs was significantly more varus than on DL stance (10.1° ± 2.4° and 8.0° ± 2.6°, respectively, p < 0.001), which was significantly more varus than on supine position (6.6° ± 2.6°, p < 0.001). Meanwhile, in patients with post-operatively valgus corrected knee, MA did not show the same pattern of change as with pre-operative varus knee. Mean MA measured on DL stance radiographs was more valgus than in supine position (-3.0o ± 2.4o and -2.6o ± 3.1o, p = 0.455), while mean MA on SL stance radiographs (-2.0o ± 2.1o) was significantly less valgus than on DL stance (p = 0.002). The ground mechanical axis deviation of tibia showed a significant correlation with MA difference between SL and DL stance radiographs before (ß = -0.341, p = 0.045) and after surgery (ß = -0.536, p = 0.001). CONCLUSION: In pre-operative varus knee, the mean MA on SL stance was changed to more varus than on DL stance, which changed to less valgus in post-operative valgus knee. The understanding in discrepancy of alignment in different weight bearing conditions before and after HTO should be considered for the appropriate realignment of the limb. STUDY DESIGN: Level II Prospective comparative study.

Glycan structure and serum half-life of recombinant CTLA4Ig, an immunosuppressive agent, expressed in suspension-cultured rice cells with coexpression of human ß1,4-galactosyltransferase and human CTLA4Ig.

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) is an immunosuppressive therapeutic, and recently produced rice cell-derived hCTLA4Ig (hCTLA4Ig(P)) reportedly exhibits in vitro immunosuppressive activities equivalent to those of Chinese hamster ovary cell-derived hCTLA4Ig (hCTLA4Ig(M)). However, limitations of hCTLA4Ig(P) include shortened in vivo half-life as well as the presence of nonhuman N-glycans containing (ß1-2)-xylose and α1,3-fucose, which cause immunogenic reactions in humans. In the present study, human ß1,4-galactose-extended hCTLA4Ig(P) (hCTLA4Ig(P)-Gal) was expressed through the coexpression of human ß1,4-galactosyltransferase (hGalT) and hCTLA4Ig in an attempt to overcome these unfavorable effects. The results indicated that both encoding hGalT and hCTLA4Ig were successfully coexpressed, and the analysis of N-glycan and its relative abundance in purified hCTLA4Ig(P)-Gal indicated that not only were the two glycans containing (ß1-4)-galactose newly extended, but also glycans containing both ß1,2-xylose and α1,3-fucose were markedly reduced and high-mannose-type glycans were increased compared to those of hCTLA4Ig(P), respectively. Unlike hCTLA4Ig(P), hCTLA4Ig(P)-Gal was effective as an acceptor via (ß1-4)-galactose for in vitro sialylation. Additionally, the serum half-life of intravenously injected hCTLA4Ig(P)-Gal in Sprague-Dawley rats was 1.9 times longer than that of hCTLA4Ig(P), and the clearance pattern of hCTLA4Ig(P)-Gal was close to that for hCTLA4Ig(M). These results indicate that the coexpression with hGalT and hCTLA4Ig(P) is useful for both reducing glycan immunogens and increasing in vivo stability. This is the first report of hCTLA4Ig as an effective therapeutics candidate in glycoengineered rice cells.

Isolation of Streptomyces inhibiting multiple-phytopathogenic fungi and characterization of lucensomycin biosynthetic gene cluster.

Soil microorganisms with diverse bioactive compounds such as Streptomyces are appreciated as valuable resources for the discovery of eco-friendly fungicides. This study isolated a novel Streptomyces from soil samples collected in the organic green tea fields in South Korea. The isolation process involved antifungal activity screening around 2400 culture extracts, revealing a strain designated as S. collinus Inha504 with remarkable antifungal activity against diverse phytopathogenic fungi. S. collinus Inha504 not only inhibited seven phytopathogenic fungi including Fusarium oxysporum and Aspergillus niger in bioassays and but also showed a control effect against F. oxysporum infected red pepper, strawberry, and tomato in the in vivo pot test. Genome mining of S. collinus Inha504 revealed the presence of the biosynthetic gene cluster (BGC) in the chromosome encoding a polyene macrolide which is highly homologous to the lucensomycin (LCM), a compound known for effective in crop disease control. Through genetic confirmation and bioassays, the antifungal activity of S. collinus Inha504 was attributed to the presence of LCM BGC in the chromosome. These results could serve as an effective strategy to select novel Streptomyces strains with valuable biological activity through bioassay-based screening and identify biosynthetic gene clusters responsible for the metabolites using genome mining approach.

Air-toxin technique: Introduction and analysis of effect using a photoimaging system.

BACKGROUND: As an alternative to other approaches to facial rejuvenation, the air-toxin technique (dermal injection of air with micro-doses of botulinum toxin) is a promising method. We aimed to describe this novel technique and assess the overall safety and efficacy of the air-toxin technique in facial rejuvenation. MATERIALS AND METHODS: A retrospective chart review was performed on cases treated with the air-toxin technique. A total of 47 Korean patients underwent a single treatment session. Treatment efficacy was assessed objectively with serial VISIA® (Canfield Scientific, NJ, USA) skin analysis data and subjectively based on the patients' assessment scores at Week 12. RESULTS: A significant improvement in wrinkle (p < 0.001), UV spot (p < 0.001), and porphyrin (p = 0.005) was objectively seen at 12 weeks follow-up. Patients noted improvement in the categories wrinkle (p < 0.01), redness (p = 0.029), texture (p < 0.001), dilated pore (p < 0.001), and lifting (p < 0.001) at Week 12 compared to baseline. No major adverse event was reported. CONCLUSIONS: Our data show that the air-toxin technique is efficacious in facial rejuvenation as shown by improved skin analysis parameters and patient assessment scores. Notably, there was some discrepancy between the objective and subjective parameters of improvement.

Modulation of osteogenic differentiation by Escherichia coli-derived recombinant bone morphogenetic protein-2.

Recombinant human bone morphogenetic protein-2 (rhBMP-2), a key regulator of osteogenesis, induces the differentiation of mesenchymal cells into cartilage or bone tissues. Early orthopedic and dental studies often used mammalian cell-derived rhBMP-2, especially Chinese hamster ovary (CHO) cells. However, CHO cell-derived rhBMP-2 (C-rhBMP-2) presents disadvantages such as high cost and low production yield. To overcome these problems, Escherichia coli-derived BMP-2 (E-rhBMP-2) was developed; however, the E-rhBMP-2-induced signaling pathways and gene expression profiles during osteogenesis remain unclear. Here, we investigated the E-rhBMP-2-induced osteogenic differentiation pattern in C2C12 cells and elucidated the difference in biological characteristics between E-rhBMP-2 and C-rhBMP-2 via surface plasmon resonance, western blotting, qRT-PCR, RNA-seq, and alkaline phosphatase assays. The binding affinities of E-rhBMP-2 and C-rhBMP-2 towards BMP receptors were similar, both being confirmed at the nanomolecular level. However, the phosphorylation of Smad1/5/9 at 3 h after treatment with E-rhBMP-2 was significantly lower than that on treatment with C-rhBMP-2. The expression profiles of osteogenic marker genes were similar in both the E-rhBMP-2 and C-rhBMP-2 groups, but the gene expression level in the E-rhBMP-2 group was lower than that in the C-rhBMP-2 group at each time point. Taken together, our results suggest that the osteogenic signaling pathways induced by E-rhBMP-2 and C-rhBMP-2 both follow the general Smad-signaling pathway, but the difference in intracellular phosphorylation intensity results in distinguishable transcription profiles on osteogenic marker genes and biological activities of each rhBMP-2. These findings provide an extensive understanding of the biological properties of E-rhBMP-2 and the signaling pathways during osteogenic differentiation.

Streptomyces BAC Cloning of a Large-Sized Biosynthetic Gene Cluster of NPP B1, a Potential SARS-CoV-2 RdRp Inhibitor.

As valuable antibiotics, microbial natural products have been in use for decades in various fields. Among them are polyene compounds including nystatin, amphotericin, and nystatin-like Pseudonocardia polyenes (NPPs). Polyene macrolides are known to possess various biological effects, such as antifungal and antiviral activities. NPP A1, which is produced by Pseudonocardia autotrophica, contains a unique disaccharide moiety in the tetraene macrolide backbone. NPP B1, with a heptane structure and improved antifungal activity, was then developed via genetic manipulation of the NPP A1 biosynthetic gene cluster (BGC). Here, we generated a Streptomyces artificial chromosomal DNA library to isolate a large-sized NPP B1 BGC. The NPP B1 BGC was successfully isolated from P. autotrophica chromosome through the construction and screening of a bacterial artificial chromosome (BAC) library, even though the isolated 140-kb BAC clone (named pNPPB1s) lacked approximately 8 kb of the right-end portion of the NPP B1 BGC. The additional introduction of the pNPPB1s as well as co-expression of the 32-kb portion including the missing 8 kb led to a 7.3-fold increase in the production level of NPP B1 in P. autotrophica. The qRT-PCR confirmed that the transcription level of NPP B1 BGC was significantly increased in the P. autotrophica strain containing two copies of the NPP B1 BGCs. Interestingly, the NPP B1 exhibited a previously unidentified SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibition activity in vitro. These results suggest that the Streptomyces BAC cloning of a large-sized, natural product BGC is a valuable approach for titer improvement and biological activity screening of natural products in actinomycetes.

BAC cloning and heterologous expression of a giant biosynthetic gene cluster encoding antifungal neotetrafibricin in streptomyces rubrisoli.

Polyene natural products including nystatin A1, amphotericin B, ECO-02301, and mediomycin belong to a large family of valuable antifungal polyketide compounds typically produced by soil actinomycetes. A previous study (Park et al., Front. Bioeng. Biotechnol., 2021, 9, 692340) isolated Streptomyces rubrisoli Inha501 with strong antifungal activity and analyzed a large-sized biosynthetic gene cluster (BGC) of a linear polyene compound named Inha-neotetrafibricin (I-NTF) using whole genome sequencing and bioinformatics. In the present study, an entire I-NTF BGC (∼167 kb) was isolated through construction and screening of Streptomyces BAC library. Overexpression of the cloned I-NTF BGC in the wild-type S. rubrisoli Inha501 and its heterologous expression in S. lividans led to 2.6-fold and 2.8-fold increase in I-NTF yields, respectively. The qRT-PCR confirmed that the transcription levels of I-NTF BGC were significantly increased in both homologous and heterologous hosts containing the BAC integration of I-NTF BGC. In addition, the I-NTF aglycone-producing strains were constructed by a target-specific deletion of glycosyltransferase gene present in I-NTF BGC. A comparison of the in vitro biological activities of I-NTF and I-NTF aglycone confirmed that the rhamnose sugar motif of I-NTF plays a critical role in both antifungal and antibacterial activities. These results suggest that the Streptomyces BAC cloning of a large-sized natural product BGC is a valuable approach for natural product titer improvement and biological activity screening of natural product in actinomycetes.

Establishment of a glycoengineered CHO cell line for enhancing antennary structure and sialylation of CTLA4-Ig.

Cytotoxic T-lymphocyte-associated protein 4-Ig (CTLA4-Ig) produced using Chinese hamster ovary (CHO) cell lines is a fusion protein of CTLA4 and the Fc region of antibody. In the present study, we identified and overexpressed genes capable of increasing sialic acid levels in CTLA4-Ig to develop cell lines using glycoengineering technology. CTLA4-Ig was produced using CHO cells overexpressing N-acetylglucosaminyltransferase (GnT) and α2,6-sialyltransferase (α2,6-ST). The conditions were wild type (WT), overexpression (GnT-IV, GnT-V, and α2,6-ST), and co-overexpression (GnT-IV and α2,6-ST, and GnT-V and α2,6-ST). GnT-IV and GnT-V were transfected into CHO cells to determine tri-antennary structure formation in CTLA4-Ig. CHOGnT-IV (cells overexpressing GnT-IV) showed the highest tri-antennary structures of glycans. Compared to CHOWT, neutral and mono-sialylated glycans decreased (-10.9% and -18.6%, respectively), while bi- and tri-sialylated N-glycans increased (4.1% and 85.7%, respectively) in CHOGnT-IV∙ST (cells co-overexpressing GnT-IV and α2,6-ST). The sum of the relative quantities of neutral N-glycans decreased from 32.0% to 28.5%, while that of sialylated N-glycans increased from 68.0% to 71.5% in CHOGnT-IV∙ST. These results are the first to demonstrate the co-overexpression of especially GnT-IV and α2,6-ST, which is an effective strategy to increase sialic acid levels and the tri-antennary structure of CTLA4-Ig produced using CHO cell lines.

Screening and Isolation of a Novel Polyene-Producing Streptomyces Strain Inhibiting Phytopathogenic Fungi in the Soil Environment.

Microbial-based eco-friendly biological substances are needed to protect crops from phytopathogenic fungi and replace toxic chemical fungicides that cause serious environmental issues. This study screened for soil antifungal Streptomyces strains, which produce rich, diverse, and valuable bioactive metabolites in the soil environment. Bioassay-based antifungal screening of approximately 2,400 Streptomyces strains led to the isolation of 149 strains as tentative antifungal producers. One Streptomyces strain showing the most potent antifungal activities against Candida albicans and Fusarium oxysporum was identified as a putative anti-phytopathogenic soil isolate that is highly homologous to Streptomyces rubrisoli (named S. rubrisoli Inha 501). An in vitro antifungal assay, pot-test, and field-test against various phytopathogenic fungi confirmed that S. rubrisoli Inha 501 is a potential novel phytopathogenic fungicide producer to protect various crops in the soil environment. Whole-genome sequencing of S. rubrisoli Inha 501 and an anti-SMASH genome mining approach revealed an approximately 150-kb polyene biosynthetic gene cluster (BGC) in the chromosome. The target compound isolation and its BGC analysis confirmed that the giant linear polyene compound exhibiting the anti-phytopathogenic activity in S. rubrisoli Inha 501 was highly homologous to the previously reported compound, neotetrafibricin A. These results suggest that a bioassay-based screening of a novel antifungal Streptomyces strain followed by its genome mining for target compound BGC characterization would be an efficient approach to isolating a novel candidate phytopathogenic fungicide that can protect crops in the soil environment.

Arthroscopic Posterior Cruciate Ligament Reconstruction: The Achilles Tendon Allograft versus the Quadriceps Tendon Allograft.

We aimed to compare and analyze the outcomes of arthroscopic posterior cruciate ligament (PCL) reconstruction with the Achilles tendon allograft and the quadriceps tendon allograft. Twenty-nine patients who received the same procedure of arthroscopic PCL reconstruction within our inclusion criteria were reviewed retrospectively. There were 13 patients in the Achilles tendon allograft group and 16 patients in the quadriceps tendon allograft group. At least in 2 years of follow-up period, we evaluated the patients using the posterior drawer test, KT 2000 test, Lysholm knee scoring scale, Tegner activity scale score, International Knee Documentation Committee (IKDC) subjective knee form score, and Telos stress radiography. Between the two groups, no differences were found in preoperative patient demographic factors (age, gender, mean time of surgery, average follow-up period, cause of injury, and combined injury) (p > 0.05). Results of the posterior drawer test, KT 2000 test, Telos stress radiography, Lysholm score, Tegner activity score, and IKDC subjective score were not significantly different between the two groups at preoperative evaluation and after surgery (p > 0.05). On comparing preoperative evaluation and follow-up after surgery, the Achilles tendon allograft group showed significant improvement in the results of the KT 2000 test, Telos stress radiology, and Lysholm score, whereas the quadriceps tendon allograft group showed significant improvement in the results of the KT 2000 test, Telos stress radiology, Lysholm score, Tegner activity score, and IKDC subjective score (p < 0.05). The quadriceps tendon for arthroscopic PCL reconstruction is good alternative allograft for the Achilles tendon for arthroscopic PCL reconstruction. This is a retrospective comparative study.

Wedge-shaped polydioxanone threads in a folded configuration ("Solid fillers"): A treatment option for deep static wrinkles on the upper face.

BACKGROUND: Facial wrinkles are key signs of aging which affect one's quality of life and psychological well-being. Absorbable wedge-shaped polydioxanone (PDO) sutures are popular in the esthetic clinics of Korea and may be a treatment option for static wrinkles on the upper face. OBJECTIVE: To describe a novel technique applicable to deep static wrinkles on the upper face and to assess its safety and efficacy. PATIENTS AND METHODS: A retrospective chart review was performed on the static wrinkle cases which were treated with folded, wedge-shaped PDO threads. A total of 33 Korean patients with glabellar and forehead wrinkles were included. All participants underwent a single treatment session. The results were assessed objectively using serial photography and subjectively based on the patients' satisfaction scores. Complications were also recorded. RESULTS: Our patients displayed moderate (15.2%), severe (39.4%), and very severe (45.4%) static wrinkles at baseline. The majority of patients (81.8%) considered the results satisfactory. Consensus ratings by two independent dermatologists showed that the objective outcomes at 2-month follow-up were very much improved (30.3%), much improved (30.3%), and improved (24.2%). The incidence of complications was low, and the complications were minor. CONCLUSION: Wedge-shaped PDO sutures in a folded configuration ("solid fillers") are a promising treatment option for deep static wrinkles on the upper face. They may be applied as an adjunct, or as an alternative to conventional therapies such as botulinum toxin and soft tissue filler injection.

Risk Factors That Influence Subsequent Recurrent Instability in Terrible Triad Injury of the Elbow.

OBJECTIVE: To identify risk factors associated with subsequent recurrent instability and to identify predictors of poor outcomes in terrible triad injury of the elbow. DESIGN: Retrospective cohort study. SETTING: University trauma center. PATIENTS/PARTICIPANTS: Seventy-six patients who were surgically treated for terrible triad injury of the elbow. INTERVENTION: Review of charts and standardized x-ray images before surgery and 2 years after surgery. Patients were categorized into 2 groups: recurrent instability (group A) or concentric stability (group B). MAIN OUTCOME MEASUREMENT: Primary outcome measures were injury mechanism, time between injury and operation, fracture type, ligament injury, radial head fixation, coronoid fixation, ligament repair, period of postoperative immobilization, joint space restoration, healing progress, secondary operation, functional outcomes, and complications. Secondary outcome measures were age, sex, height, body mass index, bone mineral density, and comorbidities (hypertension and diabetes). Outcomes were measured before surgery and 2 years after surgery and were compared between groups. RESULTS: Recurrent instability occurred in 19.7% of cases; revision surgeries were performed in 12 cases (80%). High-energy trauma (P = 0.012), time between injury and operation (P = 0.001), radial head comminution (P = 0.001), medial collateral injury (P = 0.041), and coronoid nonrepair (P = 0.030) were associated with recurrent instability. Posttraumatic arthritis developed more often in group A (P = 0.001). CONCLUSIONS: Recurrent instability was associated with high-energy trauma, time between injury and operation, Mason type III radial head fracture, medial collateral injury, and coronoid nonrepair. Patients with recurrent instability were more likely to require secondary surgery and develop posttraumatic arthritis than those with concentric stability. LEVEL OF EVIDENCE: Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.