Mitochondrial dysfunction induced by callyspongiolide promotes autophagy-dependent cell death.

Callyspongiolide is a marine macrolide known to induce caspaseindependent cancer cell death. While its toxic effects have been known, the mechanism leading to cell death is yet to be identified. We report that Callyspongiolide R form at C-21 (cally2R) causes mitochondrial dysfunction by inhibiting mitochondrial complex I or II, leading to a disruption of mitochondrial membrane potential and a deprivation of cellular energy. Subsequently, we observed, using electron microscopy, a drastic formation of autophagosome and mitophagy. Supporting these data, LC3, an autophagosome marker, was shown to co-localize with LAMP2, a lysosomal protein, showing autolysosome formation. RNA sequencing results indicated the induction of hypoxia and blocking of EGF-dependent pathways, which could be caused by induction of autophagy. Furthermore, mTOR and AKT pathways preventing autophagy were repressed while AMPK was upregulated, supporting autophagosome progress. Finally, the combination of cally2R with known anti-cancer drugs, such as gefitinib, sorafenib, and rapamycin, led to synergistic cell death, implicating potential therapeutic applications of callyspongiolide for future treatments. [BMB Reports 2021; 54(4): 227-232].

Anti­photoaging and anti­oxidative activities of natural killer cell conditioned medium following UV­B irradiation of human dermal fibroblasts and a reconstructed skin model.

Conditioned media from various sources comprise numerous growth factors and cytokines and are known to promote the regeneration of damaged tissues. Among these, natural killer cell conditioned medium (NK­CdM) has been shown to stimulate collagen synthesis and the migration of fibroblasts during the wound healing process. With a long­term aim of developing a treatment for skin photoaging, the ability of NK­CdM to prevent ultraviolet­B (UV­B) damage was assessed in neonatal human dermal fibroblasts (NHDFs) and an in vitro reconstructed skin model. The factors present in NK­CdM were profiled using an antibody array analysis. Protein and mRNA levels in UV­B exposed NHDFs treated with NK­CdM were measured by western blotting and quantitative reverse transcription­PCR, respectively. The total antioxidant capacity of NK­CdM was determined to assess its ability to suppress reactive oxygen species. The anti­photoaging effect of NK­CdM was also assessed in a 3D reconstituted human full skin model. NK­CdM induced proliferation of UV­B­treated NHDFs, increased procollagen expression, and decreased matrix metalloproteinase (MMP)­1 expression. NK­CdM also exhibited a potent antioxidant activity as measured by the total antioxidant capacity. NK­CdM inhibited UV­B­induced collagen degradation by inactivating MAPK signaling. NK­CdM also elicited potential anti­wrinkle effects by inhibiting the UV­B­induced increase in MMP­1 expression levels in a 3D reconstituted human full skin model. Taken together, the suppression of both UV­B­induced MMP­1 expression and JNK activation by NK­CdM suggests NK­CdM as a possible candidate anti­skin aging agent.

Quality by Design characterization of the perfusion culture process for recombinant FVIII.

A Quality by Design (QbD) concept was applied to characterize a cell culture process for production of the recombinant Factor VIII (rFVIII). We characterized the production bioreactor process and defined the design space by applying risk assessment to determine potential critical process parameters (CPPs) impacting critical quality attributes (CQAs). Characterization studies were subsequently performed using a qualified scaled-down model (SDM) and a multi-factorial design of experiment (DOE) approach to determine both the individual and combined impacts of the potential CPPs on CQAs. Among the operating parameters characterized, production temperature, production pH and a shift in the timing of production affected rFVIII activity and tyrosine sulfation level. Finally, we identified CPPs and established a design space for the cell culture process to identify appropriate conditions for routine manufacturing.

The impact of coronal alignment of device on radiographic degeneration in the case of total disc replacement.

BACKGROUND CONTEXT: Numerous studies have been conducted on the importance of radiographic parameters after a total disc replacement (TDR). Most of them have focused on sagittal alignment. There has been no research on what influence the coronal alignment or tilting of device has on radiographic parameters. PURPOSE: The aim was to investigate the influences of coronal tilting of device on radiographic parameters and degeneration. STUDY DESIGN/SETTING: This was a prospective comparative study. PATIENT SAMPLE: A total of 180 patients with single-level cervical disc disease who underwent TDR were included. OUTCOME MEASURES: Overall and functional spinal unit (FSU) sagittal range of motion (ROM), coronal alignment (or tilting) of device, and postoperative radiographic degeneration (RD) were analyzed. METHODS: Static anteroposterior, lateral X-rays, and dynamic lateral radiographs were assessed preoperatively, postoperatively, at 1.5, 3, 6, 9, 12, 18, 24 months, and every 6 months thereafter until final follow-up. A correlation with various parameters that could result in RD was investigated, For this, the patients were divided into two groups (Group I, RD; Group II, no RD) and subdivided into Group I-A (<5°; low coronal tilt) and Group I-B (≥5°; high coronal tilt) to analyze whether coronal tilting of device was correlated with RD. RESULTS: No statistical differences were found in preoperative overall and FSU ROM, postoperative overall and FSU ROM between Groups I and II. However, there was significant difference in coronal tilting of device between Groups I (4.50±2.83°) and II (2.04±1.15°; p=.001). There were no significant differences in preoperative overall and FSU ROM, postoperative overall and FSU ROM between Group I-A and I-B. But, RD incidence rate at surgical segment in Group I-A was 23.1%, whereas that in Group I-B was 75.0% (p=.001). The influence level of a difference in the incidence rate was found to be 10.0 of the odds ratio. Radiographic degeneration incidence rate at adjacent levels in Group I-A was 8.33%, whereas that in Group I-B was 25.0% (p=.013). The influence level of a difference in the incidence rate was found to be 3.67 of the odds ratio. CONCLUSIONS: It is considered that maintaining appropriate coronal alignment of device is important in long-term success after a cervical TDR.

Efficacy, Safety, and Pharmacokinetics of Beroctocog Alfa in Patients Previously Treated for Hemophilia A.

PURPOSE: Beroctocog alfa is a second generation recombinant factor VIII manufactured by removing the B-domain from factor VIII. This prospective clinical trial was conducted to evaluate the efficacy, safety, and pharmacokinetics of beroctocog alfa in patients of ages ≥12 years previously treated for severe hemophilia A. MATERIALS AND METHODS: Seventy subjects received beroctocog alfa as an on-demand treatment for acute hemorrhage. RESULTS: The final hemostatic effect was excellent in 35 subjects (50%) and good in 26 subjects (37.1%). The drug showed an overall efficacy rate of 87.1%. The majority of acute hemorrhages was treated by administering the study drug once (86.2%) or twice (10.0%), and the mean dose administered per single infusion was 28.55±6.53 IU/kg. Ten subjects underwent 12 surgical procedures, and hemostatic efficacy was excellent in seven cases (58.3%) and good in five cases (41.7%), showing a 100% efficacy rate. A total of 52 of 88 subjects (59.0%) experienced 168 adverse events. There were 18 serious adverse events (10.7%) in 11 subjects, and two (mild dyspnea and facial edema) in one subject were related to the study drug. Only one subject formed a de novo factor VIII inhibitor, for an occurrence rate of 1.4% (one-sided 95% upper confidence limit: 3.85%). The final elimination half-life was 13.3 h and 12.6 h at baseline and 6 months after administration, respectively. CONCLUSION: Our results suggest that beroctocog alfa is safe and efficacious as either an on-demand treatment for acute hemorrhage or a surgical prophylaxis in patients with hemophilia A.

Acute spinal subdural hematoma after vigorous back massage: a case report and review of literature.

STUDY DESIGN: A case report and review of literature. OBJECTIVE: We report on a patient with traumatic spinal subdural hematoma after vigorous back massage while on vacation. SUMMARY OF BACKGROUND DATA: Traumatic spinal subdural hematoma is extremely rare, and to our knowledge, this is the first case reported after violent back massage. We emphasize a high index of suspicion for early recognition and treatment for a good neurological recovery. METHODS: A 41-year-old male was brought to our hospital with severe back pain, motor and sensory impairments of the bilateral lower extremities, and urinary dysfunction after vigorous back massage. Magnetic resonance images revealed an acute spinal subdural hematoma in the thoracolumbar region. After careful monitoring of his neurological status, the patient was successfully managed with conservative treatment. RESULTS: After 2 weeks of hospitalization, complete motor power recovery was achieved with only minor sensory deficit. At a follow-up of more than 12 months, the patient has no residual neurological deficits. CONCLUSION: Spinal subdural hematoma secondary to physical trauma is quite rare. This case brings new information that traumatic spinal subdural hematoma can be caused by violent massage. LEVEL OF EVIDENCE: N/A.

Is cervical lordosis relevant in laminoplasty?

BACKGROUND CONTEXT: Laminoplasty aims to decompress the spinal cord and stabilize the cervical spine in patients with multilevel cervical lesions. Not every patient with cervical compressive myelopathy is a good candidate for laminoplasty. Most studies recommend that neutral or kyphotic alignments are contraindications for laminoplasty. However, cervical sagittal alignment does not have a strong and consistent effect on the clinical outcomes of laminoplasty. Moreover, many reports on the effect of cervical sagittal alignment did not designate the ideal definition of alignment and used different definitions of lordosis. PURPOSE: To identify the effect of preoperative cervical alignment according to two different definitions after midline splitting double-door laminoplasty. STUDY DESIGN: Retrospective cohort study. PATIENT SAMPLE: From August 2008 to September 2010, 58 patients were diagnosed with cervical myelopathy and treated with midline splitting double-door laminoplasty. OUTCOME MEASURES: The clinical results were assessed with the modified Japanese Orthopedic Association (JOA) score, neck disability index (NDI), and visual analog scale (VAS) and were compared to analyze the rate of change between preoperative and postoperative values. Postoperative radiological results at the final follow-up examinations were compared between groups to obtain the change in range of motion and sagittal alignment. METHOD: The effect of cervical alignment on JOA, NDI, and VAS scales and also on change of alignment and change of range of motion (ROM) at the final follow-up examinations was analyzed statistically between two groups according to two different definitions such as Toyama classification and Cobb angle. RESULTS: No difference was found between the two groups according to Toyama classification in terms of the postoperative improvement rate of the modified JOA score (p=.086), decreasing rate of the VAS (p=.940) or NDI (p=.211), postoperatively. Additionally, no difference was found for the decreasing rate of ROM (p=.427) or sagittal alignment (p=.864) based on the radiological evaluation results. Also, there was no difference between two groups according to Cobb angle in terms of the modified JOA score (p=.743), VAS (p=.548), or NDI (p=.32), postoperatively. Additionally, no difference was found for the ROM (p=1.000) or sagittal alignment (p=.440) based on the radiological evaluation results. CONCLUSIONS: Despite nonlordosis cervical sagittal alignment, double-door laminoplasty would be effective for patients with cervical myelopathy because of cervical spondylotic myelopathy or ossification of the posterior longitudinal ligament. Furthermore, sagittal alignment is not the absolute and sole factor that surgeons should consider when determining the optimal treatment strategy.

Comparability studies of new 3rd generation recombinant human factor VIII GreenGene F after improvement of formulation and viral inactivation/removal process.

A new 3rd generation recombinant factor VIII (rFVIII), GreenGene F (WHO INN: beroctocog alfa), which is a highly homogenous B-domain deleted FVIII protein comprising of two peptides as heavy chain (A1 and A2 domain) and light chain (A3, C1, and C2 domain) at 80 and 90 kDa, was developed from its predecessor product GreenGene (2nd generation product previously approved by Korea FDA after clinical studies in South Korea) by process improvements of i) addition of Solvent/Detergent treatment for virus inactivation, ii) nanofiltration (20 nm pore size) for viral removal and iii) alterations to an albumin-free formulation to minimize the risk of viral contamination. An assessment of comparability between the two products was made to see if process improvements for safer product manufacturing affected the rFVIII structural and functional characteristics. Physicochemical and physiological characteristics were observed, in vivo efficacy following a single intravenous administration to FVIII knock-out mice and toxicity by various GLP in vivo tests were evaluated. All results showed equivalence, proving that no changes in protein characteristics of rFVIII occurred from process changes in formulation, viral inactivation, and viral removal which minimize the risk of pathogen transmission to enhance safety.

Mixture of three amino acids as stabilizers replacing albumin in lyophilization of new third generation recombinant factor VIII GreenGene F.

A formulation with stabilizers replacing albumin was developed for lyophilization of recombinant factor VIII (FVIII), GreenGene F (WHO INN: beroctocog alfa), to achieve stability and eliminate safety issues of blood-derived albumin. L-Arginine (hydrophilic amino acid, positively charged side chain), L-glutamic acid (hydrophilic amino acid, negatively charged side chain), and L-isoleucine (hydrophobic amino acid, nonpolar) were selected as stabilizers, and the mixture of the three amino acids were optimized. The mixture had results comparative with albumin and other commonly used stabilizers showing good preservation of recombinant FVIII during lyophilization, robust stability with consistently high recovery of FVIIII, very low aggregate formation, and good storage stability without alterations in protein characteristics. In vivo test results showed that the efficacy was maintained and had no signs of toxicity. The study demonstrated that the three amino acid mixture acts as a good stabilizer for lyophilization of recombinant FVIII and as a safe excipient.

Natural form of noncytolytic flexible human Fc as a long-acting carrier of agonistic ligand, erythropoietin.

Human IgG1 Fc has been widely used as a bioconjugate, but exhibits shortcomings, such as antibody- and complement-mediated cytotoxicity as well as decreased bioactivity, when applied to agonistic proteins. Here, we constructed a nonimmunogenic, noncytolytic and flexible hybrid Fc (hyFc) consisting of IgD and IgG4, and tested its function using erythropoietin (EPO) conjugate, EPO-hyFc. Despite low amino acid homology (20.5%) between IgD Fc and IgG4 Fc, EPO-hyFc retained "Y-shaped" structure and repeated intravenous administrations of EPO-hyFc into monkeys did not generate EPO-hyFc-specific antibody responses. Furthermore, EPO-hyFc could not bind to FcγR I and C1q in contrast to EPO-IgG1 Fc. In addition, EPO-hyFc exhibited better in vitro bioactivity and in vivo bioactivity in rats than EPO-IgG1 Fc, presumably due to the high flexibility of IgD. Moreover, the mean serum half-life of EPO-hyFc(H), a high sialic acid content form of EPO-hyFc, was approximately 2-fold longer than that of the heavily glycosylated EPO, darbepoetin alfa, in rats. More importantly, subcutaneous injection of EPO-hyFc(H) not only induced a significantly greater elevation of serum hemoglobin levels than darbepoetin alfa in both normal rats and cisplatin-induced anemic rats, but also displayed a delayed time to maximal serum level and twice final area-under-the-curve (AUC(last)). Taken together, hyFc might be a more attractive Fc conjugate for agonistic proteins/peptides than IgG1 Fc due to its capability to elongate their half-lives without inducing host effector functions and hindering bioactivity of fused molecules. Additionally, a head-to-head comparison demonstrated that hyFc-fusion strategy more effectively improved the in vivo bioactivity of EPO than the hyperglycosylation approach.

Delayed intra-articular migration of the IntraFix outer sheath after anterior cruciate ligament reconstruction: a case report.

We describe a case of foreign body synovitis caused by delayed intra-articular migration of the outer sheath 5 months after anterior cruciate ligament (ACL) reconstruction with a quadrupled tibialis allograft tendon using the IntraFix device for tibial fixation. The postoperative course was unremarkable. At 5 months after surgery, the patient experienced a sudden catching sensation and a slight pain without any obvious twisting or trauma. At 6 months after surgery, extension deficit was 20°. At arthroscopy, intra-articular migration of the outer sheath from the tibial tunnel and reactive synovitis were observed. The outer sheath in the joint and the inner screw in the tibial tunnel were removed successfully. The ACL graft was well incorporated under good tension. Patient was able to return to her previous level of all daily activities with no further episodes of swelling. To our knowledge, described here is the only case of foreign body synovitis due to intra-articular migration of the unbroken sheath.

Analysis of factors that may influence range of motion after cervical disc arthroplasty.

BACKGROUND CONTEXT: Cervical artificial disc replacement is increasingly becoming popular among spine surgeons. Cervical disc arthroplasty aims to afford spinal stability and then balance this with flexibility. One of the fundamental benefits from performing cervical arthroplasty instead of fusion is preservation of motion in both the functional spinal unit (FSU) and the overall cervical spine. Eventually, preservation of segmental motion is believed to prevent the development of adjacent segment degeneration. But to justify its use, disc replacement prosthesis must demonstrate actual motion in vivo and preserve range of motion (ROM) after surgery as long as it allows. Without preservation of motion, disc prosthesis becomes just a functional arthrodesis equivalent. PURPOSE: The purpose of this study was to analyze the possible factors affecting cervical spine ROM after single-level cervical disc arthroplasty. STUDY DESIGN/SETTING: This is a retrospective radiological study of patients with symptomatic single-level cervical disc disorder who received the cervical disc prosthesis (Bryan Cervical Disc Prosthesis; Medtronic Sofamor Danek, Memphis, TN, USA). PATIENT SAMPLE: Procedure was performed in 39 patients. OUTCOME MEASURES: The outcome measures were statistical correlation of possible factors and ROM. METHODS: We investigated possible factors that could affect cervical ROM after surgical intervention using cervical disc replacement. For this, we focused on two main components, namely, patient factors and technical factors. First, we examined patient factors, such as age, sex, preoperative FSU ROM, and preoperative overall cervical spine (whole cervical spine) ROM. Second, we then investigated technical factors, such as the amount of bone resection, disc insertion angle, and disc insertion depth. Then, our study searched if there was any statistical correlation between these factors and the postoperative cervical ROM. RESULTS: Significant correlation was found between the postoperative overall cervical spine ROM and preoperative overall cervical spine ROM (p<.0001, R(2)=0.9062). Postoperative FSU ROM is closely correlated to both the preoperative FSU ROM (p<.0001) and the disc insertion angle (p=.0097). However, no significant correlation was noted between age, sex, disc insertion angle, and disc insertion depth. CONCLUSION: Significant correlation was found between the postoperative overall cervical spine ROM and preoperative overall cervical spine ROM. Postoperative FSU ROM is closely correlated to both the preoperative FSU ROM and the disc insertion angle. Careful preoperative evaluation of the patient's radiographs and meticulous surgical technique during the surgical procedure could aid in achieving the goals and benefits of cervical disc arthroplasty.

Pseudoaneurysm of the medial superior genicular artery after arthroscopic partial meniscectomy.

We describe a case of 43-year-old man who had a pseudoaneurysm of the medial superior genicular artery after arthroscopic partial meniscectomy with standard anterolateral and anteromedial portals. Pseudoaneurysm of the medial superior genicular artery has been reported at the previous superomedial portal site after arthroscopy. Described herein is a unique case that involved the medial superior genicular artery at the previous anteromedial portal site after arthroscopy. The pseudoaneurysm was successfully treated with transcatheter embolization.

Neutralization of hepatitis B virus (HBV) by human monoclonal antibody against HBV surface antigen (HBsAg) in chimpanzees.

The virus neutralizing efficacy of HB-C7A, a human monoclonal antibody raised against the surface antigen of hepatitis B virus (HBsAg), was proved using hepatitis B virus (HBV)-naïve chimpanzees. One control chimpanzee which received 100CID(50) of HBV, subtype adw, without HB-C7A antibody became infected by HBV as evidenced by the appearance of HBV DNA on week 10 and subsequent appearance of HBsAg, anti-HBc and anti-HBs in the serum. Two experimental chimpanzees were inoculated intravenously with same dose of HBV as the control chimpanzee, which was previously incubated with 0.1mg and 10mg of HB-C7A antibody prior to inoculation. HBV infection was not observed in the antibody-treated chimpanzees during 12 months of follow-up, exhibiting neither detectable HBsAg nor anti-HBc antibody. This work demonstrates the neutralization of HBV by HB-C7A monoclonal antibody and shows the possibility of prevention of HBV infection using this antibody in liver transplantation and exposure to HBV.