Rational design approach to improve the solubility of the ß-sandwich domain 1 of a thermophilic protein.

The ß-sandwich domain 1 (SD1) of islandisin is a stable thermophilic protein with surface loops that can be redesigned for specific target binding, architecturally comparable to the variable domain of immunoglobulin (IgG). SD1's propensity to aggregate due to incorrect folding and subsequent accumulation in Escherichia coli inclusion bodies limits its use in biotechnological applications. We rationally designed SD1 for improved variants that were expressed in soluble forms in E. coli while maintaining the intrinsic thermal stability of the protein (melting temperature (Tm) = 73). We used FoldX's ΔΔG predictions to find beneficial mutations and aggregation-prone regions (APRs) using Tango. The S26K substitution within protein core residues did not affect protein stability. Among the soluble mutants studied, the S26K/Q91P combination significantly improved the expression and solubility of SD1. We also examined the effects of the surface residue, pH, and concentration on the solubility of SD1. We showed that the surface polarity of proteins had little or no effect on solubility, whereas surface charges played a substantial role. The storage stability of several SD1 variants was impaired at pH values near their isoelectric point, and pH levels resulting in highly charged groups. We observed that mutations that create an uneven distribution of charged groups on the SD1 surface could enhance protein solubility by eliminating favorable protein-protein surface charge interactions. Our findings suggest that SD1 is mutationally tolerant to new functionalities, thus providing a novel perspective for the application of rational design to improve the solubility of targeted proteins.

Sar1A overexpression in Chinese hamster ovary cells and its effects on antibody productivity and secretion.

Chinese hamster ovary (CHO) cells are the most widely used for therapeutic antibody production. In cell line development, engineering secretion processes such as folding-related protein upregulation is an effective way of constructing cell lines with high recombinant protein productivity. However, there have been few studies on the transport of recombinant proteins between the endoplasmic reticulum (ER) and the Golgi apparatus. In this study, Sar1A, a protein involved in COPII vesicle formation, was focused on to improve antibody productivity by enhancing COPII vesicle-mediated antibody transport from the ER to the Golgi apparatus, and to clarify its effect on the secretion process. The constructed Sar1A-overexpressing CHO cell lines were batch-cultured, in which they showed an increased specific antibody production rate. The intracellular antibody accumulation and the specific localization of the intracellular antibodies were investigated by chase assay using a translation inhibitor and observed by immunofluorescence-based imaging analysis. The results showed that Sar1A overexpression reduced intracellular antibody accumulation, especially in the ER. The effects of the engineered antibody transport on the antibody's glycosylation profile and the unfolded protein response (UPR) pathway were analyzed by liquid chromatography-mass spectrometry and UPR-related gene expression evaluation, respectively. Sar1A overexpression lowered glycan galactosylation and induced a stronger UPR at the end of the batch culture. Sar1A overexpression enhanced the antibody productivity of CHO cells by modifying their secretion process. This approach could also contribute to the production of not only monoclonal antibodies but also other therapeutic proteins that require transport by COPII vesicles.

[Stanford Type B Acute Aortic Dissection Complicated by Lower Limb Ischemia Late after Abdominal Aortic Graft Replacement].

Leg ischemia is a serious complication of acute aortic dissection. Few cases of lower extremity ischemia due to dissection late after abdominal aortic graft replacement have been reported. Critical limb ischemia occurs when true lumen blood flow is obstructed by the false lumen at the proximal anastomosis of the abdominal aortic graft. Usually, the inferior mesenteric artery (IMA) is reimplanted to the aortic graft to avoid intestinal ischemia. We therein report a case of Stanford type B acute aortic dissection, in which previously reimplanted IMA prevented bilateral lower extremity ischemia. A 58-years-old male with a history of abdominal aortic replacement experienced sudden onset of epigastralgia and subsequent pain in the back and the right lower limb and was admitted to the authors' hospital. Computed tomography (CT) revealed Stanford type B acute aortic dissection, and occlusion of the abdominal aortic graft, and the right common iliac artery. However, the left common iliac artery was perfused through the reconstructed IMA during previous abdominal aortic replacement. The patient underwent thoracic endovascular aortic repair and thrombectomy, and had an uneventful recovery. For residual arterial thrombi in the abdominal aortic graft, oral warfarin potassium was administered for 16 days until the day of discharge. Since then, the thrombus has dissolved and the patient has been doing well without any lower extremity disorders.

Cloning, recombinant expression, purification, and functional characterization of AGAAN antibacterial peptide.

A recombinant version of the AGAAN antimicrobial peptide (rAGAAN) was cloned, expressed, and purified in this study. Its antibacterial potency and stability in harsh environments were thoroughly investigated. A 15 kDa soluble rAGAAN was effectively expressed in E. coli. The purified rAGAAN exhibited a broad antibacterial spectrum and was efficacious against seven Gram-positive and Gram-negative bacteria. The minimal inhibitory concentration (MIC) of rAGAAN against the growth of M. luteus (TISTR 745) was as low as 60 µg/ml. Membrane permeation assay reveals that the integrity of the bacterial envelope is compromised. In addition, rAGAAN was resistant to temperature shock and maintained a high degree of stability throughout a reasonably extensive pH range. The bactericidal activity of rAGAAN ranged from 36.26 to 79.22% in the presence of pepsin and Bacillus proteases. Lower bile salt concentrations had no significant effect on the function of the peptide, whereas higher concentrations induced E. coli resistance. Additionally, rAGAAN exhibited minimal hemolytic activity against red blood cells. This study indicated that rAGAAN may be produced on a large scale in E. coli and that it had an excellent antibacterial activity and sufficient stability. This first work to express biologically active rAGAAN in E. coli yielded 8.01 mg/ml at 16 °C/150 rpm for 18 h in Luria Bertani (LB) medium supplemented with 1% glucose and induced with 0.5 mM IPTG. It also assesses the interfering factors that influence the activity of the peptide, demonstrating its potential for research and therapy of multidrug-resistant bacterial infections.

Effect of co-overexpression of the cargo receptor ERGIC-53/MCFD2 on antibody production and intracellular IgG secretion in recombinant Chinese hamster ovary cells.

Therapeutic antibodies are attractive biopharmaceuticals because of their high therapeutic effects, fewer side effects, and prolonged half-life in the blood. Chinese hamster ovary (CHO) cells are the most widely used host cell lines to produce therapeutic antibodies in industries. High-producing recombinant CHO cells can be established via overexpression of endogenous proteins. In this study, we focused on the intracellular traffic of an antibody-producing CHO cell line, CHO-HcD6. Assembled antibodies were accumulated in the endoplasmic reticulum (ER) in the cell. We hypothesized that the accumulation was due to the insufficient number of cargo receptors in the cell and focused on a cargo receptor, the ERGIC-53-MCFD2 complex, which transports expressed proteins from the ER to the Golgi apparatus. Overexpression of the cargo receptor transport was expected to improve antibody production. Exogenous ERGIC-53 and MCFD2 were transfected into CHO-HcD6 cells, and overexpressing CHO-HcD6 cells were constructed. As a result of overexpression, antibody productivity increased in batch cultivation. However, the chase assay results and immunofluorescence microscopic observations revealed intracellular IgG accumulation in the overexpressing cells. These results suggest that overexpression of cargo receptors not only promoted extracellular secretion but also enhanced the retention of intracellular antibodies.

Secretion of a low-molecular-weight species of endogenous GRP94 devoid of the KDEL motif during endoplasmic reticulum stress in Chinese hamster ovary cells.

GRP94 (glucose-regulated protein 94) is a well-studied chaperone with a lysine, aspartic acid, glutamic acid and leucine (KDEL) motif at its C-terminal, which is responsible for GRP94 localization in the endoplasmic reticulum (ER). GRP94 is upregulated during ER stress to help fold unfolded proteins or direct proteins to ER-associated degradation. In a previous study, engineered GRP94 without the KDEL motif stimulated a powerful immune response in vaccine cells. In this report, we show that endogenous GRP94 is naturally secreted into the medium in a truncated form that lacks the KDEL motif in Chinese hamster ovary cells. The secretion of the truncated form of GRP94 was stimulated by the induction of ER stress. These truncations prevent GRP94 recognition by KDEL receptors and retention inside the cell. This study sheds light on a potential trafficking phenomenon during the unfolded protein response that may help understand the functional role of GRP94 as a trafficking molecule.

[Repeated Heart Failure after Transcatheter Aortic Valve Implantation Using Self-expandable Bioprosthetic Valve:Report of a Case].

The patient was an 81-year-old man. Transcatheter aortic valve implantation( TAVI) was performed for severe aortic stenosis using Evolut R. The patient moved to intensive care unit without an adverse event after the operation. But repeated acute heart failure occurred several times during hospital stay. Mitral regurgitation (MR) was worsened from mild at baseline to moderate or more by transthoracic echocardiography. Various factors that worsened MR after TAVI have been reported, and treatment strategy for severe aortic stenosis patients with MR should be carefully developed.

Giant aneurysmal coronary artery fistula originating from the left circumflex artery: Two case reports.

Coronary artery fistula (CAF) is one of the most common coronary artery anomalies. The most common fistulas originate from the right coronary artery and drain into the right heart structures. Due to the variety of coronary fistulas, the surgical treatment strategy is individualized for each case. We report two cases of giant aneurysmal CAF originating from the left circumflex artery. One case required coronary artery bypass grafting, while the other did not.

Production of monoclonal shark-derived immunoglobulin new antigen receptor antibodies using Chinese hamster ovary cell expression system.

Cartilaginous fishes such as sharks have adaptive immune systems based on immunoglobulins similar to those in mammals. During their evolution, cartilaginous fishes individually have acquired their adaptive immune system called immunoglobulin new antigen receptor (IgNARs). IgNARs maintain their functions in the harsh environment of shark serum, which contains a high concentration of urea to prevent water loss in seawater. Therefore, IgNARs have high structural stability, and are expected to be used as next-generation antibodies in applications different from those of conventional IgG antibodies. However, no recombinant expression system for IgNAR, which has a molecular weight of approximately 147 kDa as a dimer and multiple N-glycosylation sites, has yet been constructed. This has stalled research into IgNAR development. Here, we constructed a recombinant expression system for IgNAR using Chinese hamster ovary (CHO) cells, widely used as hosts for IgG antibody production. Using this system, IgNAR was successfully expressed and purified as a human IgG Fc fusion protein and showed antigen-binding ability. After Protein A affinity purification, followed by specific cleavage and removal of the human Fc-region, the final yield of IgNAR was 1.07 mg/L-medium. Moreover, this CHO cell expression system modified IgNAR with various N-glycans, including high-mannose and complex types. This expression system will allow us to analyze the structure, physicochemical properties, and biological functions of IgNAR. This fundamental information will advance the development of IgNARs for industrial and biotechnological applications.

Acute type A aortic dissection with a tear in the posterior wall of the ascending aorta early after off-pump coronary artery bypass grafting.

The patient was a 68-year-old man who underwent triple-vessel OPCAB uneventfully with aortic proximal anastomosis at one site using the saphenous vein with a mechanical device, not a side clamp, with mild traction of the ascending aorta by aortic taping. On postoperative day 7, computed tomography revealed extremely localized AAD with a tear on the posterior wall of the ascending aorta. Emergent ascending aortic replacement was successfully performed. Surprisingly, the tear extended laterally along the traction site of the tape. To our knowledge, this is the first report of AAD early after OPCAB originating at a location other than the sites of proximal anastomosis or side clamping. Proximal anastomosis with a mechanical device to the towed aorta may indirectly or directly injure the intima of the posterior wall, causing this complication. Manipulating the aorta under abnormal pressure should be avoided.

Insertion loop-mediated folding propagation governs efficient maturation of hyperthermophilic Tk-subtilisin at high temperatures.

The serine protease Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakarensis possesses three insertion loops (IS1-IS3) on its surface, as compared to its mesophilic counterparts. Although IS1 and IS2 are required for maturation of Tk-subtilisin at high temperatures, the role of IS3 remains unknown. Here, CD spectroscopy revealed that IS3 deletion arrested Tk-subtilisin folding at an intermediate state, in which the central nucleus was formed, but the subsequent folding propagation into terminal subdomains did not occur. Alanine substitution of the aspartate residue in IS3 disturbed the intraloop hydrogen-bonding network, as evidenced by crystallographic analysis, resulting in compromised folding at high temperatures. Taking into account the high conservation of IS3 across hyperthermophilic homologues, we propose that the presence of IS3 is important for folding of hyperthermophilic subtilisins in high-temperature environments.

Early results of expanding the anatomical indications for using a Gore Iliac branch endoprosthesis to treat aortoiliac and iliac aneurysms.

PURPOSE: To assess the safety and anatomical suitability of using a Gore Iliac Branch Endoprosthesis (IBE) in aortoiliac and iliac aneurysm repair. METHODS: Between 2017 and 2020, 20 patients underwent endovascular aneurysm repair (EVAR) with a Gore IBE device (bilateral IBE, n = 1) after expanding the instructions for use (IFU) criteria. We evaluated the early clinical outcomes and suitability of the IFU criteria, retrospectively. RESULTS: Six patients (30%) met all the IFU criteria. Anatomical suitability according to the IFU criteria for the collective total of 21 IBE limbs was confirmed for 10 (47.6%) proximal common iliac arteries, 21 (100%) external iliac arteries, 18 (85.7%) internal iliac arteries, and in the length from the lowest renal artery to the iliac bifurcation in 15 (71.8%) patients. Assisted primary technical success was achieved in all patients with various bail-out techniques. One patient (5%) required a bare-stent insertion 7 days after EVAR for severe stenosis in the ipsilateral limb caused by a small terminal aorta. There was no case of occlusion of an iliac branch component device. CONCLUSIONS: Gore IBEs were implanted safely and effectively with various bail-out techniques to repair aortoiliac and iliac aneurysms in our Japanese patients with a low rate of inclusion IFU criteria.

Effect of Nitrogen Gas Plasma Generated by a Fast-Pulsed Power Supply Using a Static Induction Thyristor on Scrapie Prion.

Previous studies show that nitrogen gas plasma generated by a fast-pulsed power supply using a static induction thyristor has both virucidal and bactericidal effects. In this study, nitrogen gas plasma was further evaluated for its potential effects on prions, which are well known to be the most resistant pathogen to both chemical and physical inactivation. Aliquots (10 µL) of mouse brain homogenate infected with Chandler scrapie prion were spotted onto cover glasses and subjected to nitrogen gas plasma. Treated samples were recovered and subjected to further analyses. Control prion samples were prepared in exactly the same way but without plasma treatment. Protein misfolding cyclic amplification (PMCA) showed that nitrogen gas plasma treatment at 1.5 kilo pulse per second for 15 or 30 min caused a reduction in the in vitro propagation level of PrPres (proteinase K-resistant prion protein), which was used as an index of abnormal prion protein (PrPSc). Moreover, mice injected with prion treated with plasma for 30 min showed longer survival than mice injected with control prion, indicating that nitrogen gas plasma treatment decreased prion infectivity. Altogether, these results suggest that nitrogen gas plasma treatment can inactivate scrapie prions by decreasing the propagation activity and infectivity of PrPSc.

[Rupture of Tuberculous Infective Abdominal Aortic Aneurysm after Intravesical Bacillus Calmette-Guérin Instillation Therapy].

The patient was a 74-year-old male who had undergone intravesical Bacillus Calmette-Guérin(BCG) instillation therapy for bladder cancer. He visited our hospital with chief complaints of fever and abdominal pain. Abdominal aortic aneurysmal rupture and iliopsoas muscle abscess were confirmed by computed tomography( CT). We performed semi-emergency surgery, including replacement of the abdominal aorta with a synthetic graft, iliopsoas abscess debridement, and omentopexy. A rifampicin-bonded synthetic graft was used because of the possibility of tuberculous involvement after BCG instillation therapy. Examination of the tissues collected during surgery were positive for tuberculosis deoxyribonucleic acid (DNA) in a polymerase chain reaction (PCR), and showed multiple giant cell granulomas with caseous necrosis, which both strongly suggested involvement of tuberculosis. Therefore, 4 types of antituberculous drugs were administered for 40 days. This case shows that an infective aneurysm should be suspected when fever and abdominal pain develop after intravesical BCG instillation therapy.

Effect of Noncompacted Myocardial Resection on Isolated Left Ventricular Noncompaction.

Isolated left ventricular noncompaction, where broad trabeculae and deep intertrabecular recesses are observed in the left ventricular myocardium resulting from an arrest in normal embryogenesis, is a rare cardiomyopathy. We present a report on isolated trabeculectomy and postoperative echocardiographic follow-up showing recovery of cardiac function for isolated left ventricular noncompaction.

[Thrombus in the Right Atrial Appendage Detected Incidentally during Coronary Artery Bypass Grafting;Report of a Case].

A 71-year-old male was admitted to our hospital for treatment of complete atrioventricular block. By cardiac catheterization, chronic occlusion was confirmed in the right coronary artery and circumflex branch, and coronary artery bypass grafting was planned. Although atrioventricular block disappeared by discontinuance of ß-blocker, paroxysmal atrial flutter had been confirmed before surgery. After an incision into the pericardium, changes in the color of the right atrial appendage were observed, and a thrombus was detected at the site by epicardial echography. The right atrial appendage was removed together with the thrombus, and coronary artery bypass grafting was performed as scheduled. Pathological findings suggested myocardial tissues with ischemic changes and an organized thrombus exhibiting granulomatous changes. This case suggested the need to observe the right atrial appendage carefully during cardiac surgery in patients with risk factors for atrial thrombus.

Affinity shift of ATP upon glycerol binding to a glycerol kinase from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1.

Glycerol kinase (GK) is a key enzyme of glycerol metabolism. It participates in glycolysis and lipid membrane biosynthesis. A hexamer of GK from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1(Tk-GK) was identified as a substrate-binding form of the enzyme. Here, the X-ray crystal structure analysis and the biochemical analysis was done and the relationships between its unique oligomer structure and substrate binding affinity were investigated. Wild type GK and mutant K271E GK, which disrupts the hexamer formation interface, were crystallized with and without their substrates and analyzed at 2.19-3.05 Å resolution. In the absence of glycerol, Tk-GK was a dimer in solution. In the presence of its glycerol substrate, however, it became a hexamer consisting of three symmetrical dimers about the threefold axis. Through glycerol binding, all Tk-GK molecules in the hexamer were in closed form as a result of domain-motion. The closed form of Tk-GK had tenfold higher ATP affinity than the open form of Tk-GK. The hexamer structure stabilized the closed conformation and enhanced ATP binding affinity when the GK was bound to glycerol. This molecular mechanism is quite simple activity regulation mechanism among known GKs.

Placement of a Zenith® Dissection Endovascular System in the Descending Thoracic Aorta Can Hamper Further Surgical Aortic Operations.

INTRODUCTION: The efficacy of endovascular treatment for complicated Stanford type B acute aortic dissection is being established. However, aortic events sometimes occur, and some cases require surgical intervention. REPORT: A 52 year old man underwent ascending aorta replacement for Stanford type A acute aortic dissection in August 2016. Post-operative computed tomography (CT) showed residual dissection from the aortic arch to the right common iliac artery and a large re-entry in the right common iliac artery (RCIA). Two months after the operation, CT revealed enlargement of the false lumen of the thoracic aorta and the thoracic aortic diameter. Aiming to reduce the false lumen and remodel the aorta, a three stage operation was performed, as described below. Four months after the dissection, total aortic arch replacement and a frozen elephant trunk insertion were performed as the first stage. Subsequently, as a second stage operation, thoracic endovascular repair (TEVAR) was performed using a Zenith® Dissection Endovascular System (Cook Japan Co., Ltd, Tokyo, Japan), with the aim of expanding the true aortic lumen. The implanted devices were a stent graft for the proximal part and two bare stents for the middle and distal part. As a third stage operation, abdominal aortic endovascular treatment was performed with the purpose of closing the re-entry from the RCIA. However, two years after the three stage operation, CT showed that the thoracic aorta was over 60 mm in diameter. Graft replacement of the thoraco-abdominal aorta was performed. The bare stents were expected to be easily removable from the aorta, but unexpectedly, they were strongly attached to the intima, which made it extremely difficult to perform surgical and aortic operations. DISCUSSION: Surgical operations for the aorta can become more difficult after bare stent placement in the aorta.

Analysis of the immunoglobulin G (IgG) secretion efficiency in recombinant Chinese hamster ovary (CHO) cells by using Citrine-fusion IgG.

Biopharmaceuticals represented by immunoglobulin G (IgG) are produced by the cultivation of recombinant animal cells, especially Chinese hamster ovary (CHO) cells. It is thought that the intracellular secretion process of IgG is a bottleneck in the production of biopharmaceuticals. Many studies on the regulation of endogenous secretory protein expression levels have shown improved productivity. However, these strategies have not universally improved the productivity of various proteins. A more rational and efficient establishment of high producer cells is required based on an understanding of the secretory processes in IgG producing CHO cells. In this study, a CHO cell line producing humanized IgG1, which was genetically fused with fluorescent proteins, was established to directly analyze intracellular secretion. The relationship between the amount of intracellular and secreted IgG was analyzed at the single cell level by an automated single-cell analysis and isolation system equipped with dual color fluorescent filters. The amounts of intracellular and secreted IgG showed a weak positive correlation. The amount of secreted IgG analyzed by the system showed a weak negative linear correlation with the specific growth of isolated clones. An immunofluorescent microscopy study showed that the established clones could be used to analyze the intracellular secretion bottleneck. This is the first study to report the use of fluorescent protein fusion IgG as a tool to analyze the secretion of recombinant CHO cells.

Secretion analysis of intracellular "difficult-to-express" immunoglobulin G (IgG) in Chinese hamster ovary (CHO) cells.

The Chinese hamster ovary (CHO) cell line is the most widely used host cell for therapeutic antibody production. Although its productivity has been improved by various strategies to satisfy the growing global demand, some difficult-to-express (DTE) antibodies remain at low secretion levels. To improve the production of various therapeutic antibodies, it is necessary to determine possible rate-limiting steps in DTE antibody secretion in comparison with other high IgG producers. Here, we analyzed the protein secretion process in CHO cells producing the DTE immunoglobulin G (IgG) infliximab. The results from chase assays using a translation inhibitor revealed that infliximab secretion could be nearly completed within 2 h, at which time the cells still retained about 40% of heavy chains and 65% of light chains. Using fluorescent microscopy, we observed that these IgG chains remained in the endoplasmic reticulum and Golgi apparatus. The cells inefficiently form fully assembled heterodimer IgG by making LC aggregates, which may be the most serious bottleneck in the production of DTE infliximab compared with other IgG high producers. Our study could contribute to establish the common strategy for constructing DTE high-producer cells on the basis of rate-limiting step analysis.