Production of a correctly assembled fibrinogen using transgenic silkworms.

Fibrinogen from human blood is used as a main component of coagulants, including surgical tissue sealants. The development of a recombinant human fibrinogen (rFib) is anticipated to eliminate the risks of blood-borne infections. Here, we report the efficient production of rFib in a transgenic silkworm system. A silkworm line carrying cDNAs of the fibrinogen Aα and γ chains (Aα/γ-silkworm) produced Aα and γ chains in its cocoons, however, the Bß chains were not detected from cocoons of another silkworm line carrying the cDNA of fibrinogen Bß chains (Bß-silkworm). A silkworm line for all three fibrinogen chains was generated by crossing Aα/γ-silkworms with Bß-silkworms, which secreted Aα2Bß2γ2 fibrinogen (rFib) into cocoons at high contents. The N-terminal amino acid sequences of the three rFib chains were identical to those of the corresponding chains of native fibrinogen (nFib). The N-glycan profile of the rFib comprised oligomannose-type (53%), complex-type (34%), and paucimannose-type (13%); neither high-mannose-type (six or more mannose residues) nor core-fucosylated glycans were observed. The coagulation activity of the rFib was evaluated for the amount of thrombin-released fibrinopeptide A (FpA) and the kinetics for turbidity increase (non-covalent network formation) in the solution. FpA release rates were equivalent between rFib and nFib; by contrast, the kinetics of the turbidity increase for rFib were accelerated nearly two-fold, for both the rate and maximum value, compared to those of nFib. These results demonstrate that the rFib produced in the transgenic silkworm system is comparable to nFib in both physical and coagulative properties. This rFib is a promising candidate component for safe hemostatic pharmaceuticals.

Membrane fusion and infection abilities of baculovirus virions are preserved during freezing and thawing in the presence of trehalose.

Budded viruses (BVs) of baculovirus such as Autographa californica nucleopolyhedrovirus (AcNPV) have recently been studied as biological nanomaterials, and methods for their longer-term storage without deterioration would be desirable. The cryopreservation of virions with a naturally occurring saccharide like trehalose as a cryoprotectant is known to be useful for maintaining the viral structure and function. In this study, we examined how useful trehalose is as protectant for BV cryopreservation during repeated freeze-thaw cycles: 1) membrane fusion between liposomes (multilamellar vesicles, MLVs) and BVs, 2) infection of insect culture cells (Sf9 cells) by RFP-expressing BVs, and 3) morphologies of these BVs were investigated by fluorescent dequenching assay, fluorescence microscopy, and transmission electron microscopy (TEM), respectively. The results suggest that the BVs deteriorate in quality with each freeze-thaw cycle, and this deterioration can be diminished with the use of trehalose to an extent similar to that seen with storage on ice.

Tumor-dependent secretion of close homolog of L1 results in elevation of its circulating level in mouse model for human lung tumor.

Close homolog of L1 (CHL1) and its truncated form mainly play crucial roles in mouse brain development and neural functions. Herein, we newly identified that truncated form of CHL1 is produced and released from lung tumor tissue in a mouse model expressing human EML4-ALK fusion gene. Both western blot and direct ELISA analysis revealed that mouse CHL1 level in serum (including serum extracellular vesicles) was significantly elevated in EML4-ALK transgenic mice. The correlation between the tumor size and the amount of CHL1 secretion could be examined in this study, and showed a significant positive correlation in a tumor size-dependent manner. Considering these results, the measurement of circulating CHL1 level may contribute to assess a tumor progression in human lung tumor patients.

Novel recombinant feline interferon carrying N-glycans with reduced allergy risk produced by a transgenic silkworm system.

BACKGROUND: The generation of recombinant proteins for commercialisation must be cost-effective. Despite the cost-effective production of recombinant feline interferon (rFeIFN) by a baculovirus expression system, this rFeIFN carries insect-type N-glycans, with core α 1,3 fucosyl residues that act as potential allergens. An alternative method of production may yield recombinant glycoproteins with reduced antigenicity. RESULTS: A cDNA clone encoding the fifteenth subtype of FeIFN-α (FeIFN-α15) was isolated from a Japanese domestic cat. This clone encoded a protein of 189 amino acids with a molecular mass of 21.1 kDa. The rFeIFN-α15 was expressed using a transgenic silkworm system, which was expected to yield an N-glycan structure with reduced antigenicity compared with the protein produced by the baculovirus system. The resulting rFeIFN-α15 accumulated in the sericin layer of silk fibres and was easily extracted and purified by column chromatography. The N-terminal amino acid sequence of purified rFeIFN-α15 was identical to the mature form of natural sequence. Moreover, its N-glycans did not include detectable core α 1,3 fucosyl residues. Its anti-vesicular stomatitis virus activity (2.6 × 108 units/mg protein) was comparable to that of the baculovirus-expressed rFeIFN. CONCLUSIONS: The lower allergy risk of rFeIFN produced by the transgenic silkworm system than by the baculovirus expression system is due to the former lacking core α 1,3 fucosyl residues in its N-glycans. The rFeIFN-α15 produced by the transgenic silkworm system may be a prospective candidate for the next generation of rFeIFN in veterinary medicine.

Electron crystallography of ultrathin 3D protein crystals: atomic model with charges.

Membrane proteins and macromolecular complexes often yield crystals too small or too thin for even the modern synchrotron X-ray beam. Electron crystallography could provide a powerful means for structure determination with such undersized crystals, as protein atoms diffract electrons four to five orders of magnitude more strongly than they do X-rays. Furthermore, as electron crystallography yields Coulomb potential maps rather than electron density maps, it could provide a unique method to visualize the charged states of amino acid residues and metals. Here we describe an attempt to develop a methodology for electron crystallography of ultrathin (only a few layers thick) 3D protein crystals and present the Coulomb potential maps at 3.4-Å and 3.2-Å resolution, respectively, obtained from Ca(2+)-ATPase and catalase crystals. These maps demonstrate that it is indeed possible to build atomic models from such crystals and even to determine the charged states of amino acid residues in the Ca(2+)-binding sites of Ca(2+)-ATPase and that of the iron atom in the heme in catalase.

The method used to culture host cells (Sf9 cells) can affect the qualities of baculovirus budding particles expressing recombinant proteins.

Budded virus (BV) particles of baculovirus (Autographa californica nucleopolyhedrovirus, AcNPV) are harvested from the supernatant of liquid culture of Sf9 host cells by ultracentrifugation. Using polyacrylamide gel electrophoresis, Western blot and transmission electron microscopy (TEM) of BV samples fractionated closely by sucrose density gradient centrifugation, we observed that BVs exhibited different qualities depending on whether they had been harvested from the supernatant from a standing (static), shaking (suspension), or standing/shaking (pre-/post-infection) culture of Sf9 cells. The amount of BV protein apparently increased in the order of standing, standing/shaking, and shaking procedure, and the yield of intact particles showed an opposite trend. TEM observation clearly showed that appropriate fractions of the standing and standing/shaking cultures contained more intact BV particles than those from the shaking culture. These results suggest that the qualities of recombinant BV particles may be related to the culture conditions of the host cells.

Expression of 3-Mercaptopyruvate Sulfurtransferase in the Mouse.

3-Mercaptopyruvate sulfurtransferase (MST) is one of the principal enzymes for the production of hydrogen sulfide and polysulfides in mammalians, and emerging evidence supports the physiological significance of MST. As a fundamental study of the physiology and pathobiology of MST, it is necessary to establish the tissue distribution of MST in mice. In the present study, the expression of MST in various organs of adult and fetal mice was analyzed by Western blotting and enzyme-immunohistochemistry. Moreover, the histology of MST gene-deficient mice was examined. Western blotting revealed that all organs examined had MST. The brain, liver, kidneys testes, and endocrine organs contained large amounts of MST, but the lungs, spleen, thymus, and small intestine did not. Immunohistochemically, the MST expression pattern varies in a cell-specific manner. In the brain, neural and glial cells are positively stained; in the lung, bronchiolar cells are preferentially stained; in the liver, hepatocytes around central veins are more strongly stained; renal convoluted cells are strongly stained; and pancreatic islets are strongly stained. Fetal tissues were studied, and MST expression was found to be similar before and after birth. Histological observation revealed no remarkable findings in MST gene-deficient mice. The present study revealed fundamental information regarding the MST expression of various organs in adult and fetal mice, and the morphological phenotype of MST gene-deficient mice.

Identification of Core Alpha 1,3-Fucosyltransferase Gene From Silkworm: An Insect Popularly Used to Express Mammalian Proteins.

Silkworm has great potential as production system of recombinant mammalian proteins. When the protein products are used for medical purpose, it is required to reduce the risk of an allergy, the content of core alpha 1,3-fucosyl residue attached to the N-glycan of proteins, for example. We isolated the gene of an enzyme responsible for the transfer of core alpha 1,3-fucosyl residue, core alpha 1,3-fucosyltransferase (Fuc-T C3), from silkworm. A candidate cDNA for silkworm Fuc-T C3 was isolated as a homolog of the fruit fly enzyme gene fucTA. The gene was located on chromosome 7 of the silkworm genome and was composed of seven exons, which spanned approximately 10 kb on the genome. The coding region of the gene was 1,350 bp and encoded a 450-amino acid protein with a molecular mass of 52.2 kDa. Deduced amino acid sequence of the coding region showed one transmembrane domain in its N-terminal and typical motifs common to fucosyltransferases including Fuc-T C3s of other organisms in its C-terminal. The extract of CHO cells transfected with the cDNA showed Fuc-T C3 activity using GDP-fucose and DABS-GnGn peptide as substrates. These results showed this cDNA clone actually encodes silkworm Fuc-T C3.

Cell pairing using microwell array electrodes based on dielectrophoresis.

We report a simple device with an array of 10,000 (100 × 100) microwells for producing vertical pairs of cells in individual microwells with a rapid manipulation based on positive dielectrophoresis (p-DEP). The areas encircled with micropoles which fabricated from an electrical insulating photosensitive polymer were used as microwells. The width (14 µm) and depth (25 µm) of the individual microwells restricted the size to two vertically aligned cells. The DEP device for the manipulation of cells consisted of a microfluidic channel with an upper indium tin oxide (ITO) electrode and a lower microwell array electrode fabricated on an ITO substrate. Mouse myeloma cells stained in green were trapped within 1 s in the microwells by p-DEP by applying an alternating current voltage between the upper ITO and the lower microwell array electrode. The cells were retained inside the wells even after switching off the voltage and washing with a fluidic flow. Other myeloma cells stained in blue were then trapped in the microwells occupied by the cells stained in green to form the vertical cell pairing in the microwells. Cells stained in different colors were paired within only 1 min and a pairing efficiency of over 50% was achieved.

pH switching that crosses over the isoelectric point (pI) can improve the entrapment of proteins within giant liposomes by enhancing protein-membrane interaction.

The ability to encapsulate various molecules including proteins within giant liposomes is needed for studies on model cell membranes and artificial cells. In this report, we demonstrate how to improve the efficiency of protein entrapment with the gentle hydration (natural swelling) method, which is a well-known protocol for the preparation of giant liposomes. We found that when the initial pH of a solution was kept below the pI of a target protein during hydration and then changed to above the pI, the protein could be entrapped more efficiently compared to the sample that was kept at above the pI during the hydration. An examination of the ratio of the mass of entrapped protein to the moles of phospholipid in liposomes (dioleoylphosphatidylcholine (DOPC)/dioleoylphosphatidylglycerol (DOPG)) indicated that entrapment of target proteins like bovine serum albumin, myoglobin, and lysozyme could be improved using this procedure, and this trend was consistent with microscopic observations at the level of a single giant liposome. The conditions that resulted in good efficiencies were affected by the NaCl concentration and the temperature of the hydration solution, implying that protein entrapment in giant liposomes may be enhanced by associative interaction between lipid lamellar membranes and target proteins.

Incorporation of adenylate cyclase into membranes of giant liposomes using membrane fusion with recombinant baculovirus-budded virus particles.

Recombinant transmembrane adenylate cyclase (AC) was incorporated into membranes of giant liposomes using membrane fusion between liposomes and baculovirus-budded virus (BV). AC genes were constructed into transfer vectors in a form fused with fluorescent protein or polyhistidine at the C-terminus. The recombinant BVs were collected by ultracentrifugation and AC expression was verified using western blotting. The BVs and giant liposomes generated using gentle hydration were fused under acidic conditions; the incorporation of AC into giant liposomes was demonstrated by confocal laser scanning microscopy through the emission of fluorescence from their membranes. The AC-expressing BVs were also fused with liposomes containing the substrate (ATP) with/without a specific inhibitor (SQ 22536). An enzyme immunoassay on extracts of the sample demonstrated that cAMP was produced inside the liposomes. This procedure facilitates direct introduction of large transmembrane proteins into artificial membranes without solubilization.

Combined Neutrophil-to-Lymphocyte Ratio Score Is Associated With Chemotherapeutic Response and Predicts Prognosis in Patients With Advanced Pancreatic Cancer.

BACKGROUND/AIM: Neutrophil-to-lymphocyte ratio (NLR) is a prognostic indicator for several malignancies, including pancreatic cancer. We developed a novel combined NLR score (cNLRS) based on baseline NLR and change in NLR after chemotherapy (ΔNLR), and examined its prognostic value and role in chemotherapeutic response in patients with advanced pancreatic cancer. PATIENTS AND METHODS: This study retrospectively assessed 210 advanced pancreatic cancer patients receiving chemotherapy between 2010 and 2021. The cNLRS was developed and its association with chemotherapeutic response and prognosis was investigated. RESULTS: The cNLRS consisted of baseline NLR ≥2.5 and ΔNLR ≥0, both of which were remained as independent poor predictors of prognosis adjusting for other traditional clinicopathological features. A high cNLRS served as an independent prognostic factor of reduced overall survival. Of note, the cNLRS was significantly associated with disease control rate and treatment duration not only in 1st line treatment but also in 2nd line treatment. CONCLUSION: The cNLRS established as a useful prognostic biomarker might be associated with chemotherapeutic response and could predict survival in advanced patients with pancreatic ductal adenocarcinoma treated with chemotherapy.

Diagnostic accuracy of preoperative adhesion mapping by ultrasonography for laparoscopic surgery in patients with past abdominal surgery with special reference to loose adhesion.

PURPOSE: Endoscopic surgery is widely accepted for both elective and emergent abdominal surgery. This study was performed to assess the accuracy of preoperative adhesion mapping by abdominal ultrasonography (US). METHODS: Intra-abdominal intestinal adhesions on the abdominal wall in 50 patients with a history of abdominal surgery were prospectively assessed by the visceral slide test with US before laparoscopic surgery from 2019 to 2022. Adhesion was assessed in six separate abdominal zones during US. Actual adhesion on the abdominal wall was confirmed during laparoscopic surgery. RESULTS: The sliding distances in upper right, upper central, upper left, lower right, lower central, and lower left zones in patients with versus without intestinal adhesion were 4.4 versus 1.4 cm (P = .004), 3.4 versus 2.5 cm, 4.3 versus 1.3 cm (P = .011), 3.1 versus 1.5 cm (P = .0014), 3.3 versus 1.1 cm (P = .013), and 3.4 versus 0.8 cm (P = .0061), respectively. Receiver operating characteristic analysis revealed the optimal value of sliding distance as 2.5 cm and the area under the curve as 0.86. The specificity of US assessment of adhesion was lower in the central zone than in lateral zones. Loose adhesion mostly seen around the scar was attributed to either filmy tissue or omental adhesion, leading to visceral sliding during US. CONCLUSION: This study revealed the reason for insufficient accuracy of preoperative US assessment of intestinal adhesion around the scar area because of loose adhesion. The upper lateral area might be optimal for first port insertion.

Advanced Cholangiocarcinoma With High Tumor Mutation Burden Achieving Complete Response to Immune Check Point Inhibitor.

BACKGROUND/AIM: Genomic examination of tumor tissue has been clinically accepted, and the identification of actionable mutations for molecular-targeted therapy may provide substantial survival benefit for patients with advanced malignancies. CASE REPORT: A female patient in her 60s showed a stenosis of the afferent loop of the small intestine because of circumferential metastatic tumor 14 months after curative surgery for hilar cholangiocarcinoma. Chemotherapy with gemcitabine plus cisplatin was administered for 18 months. An oncopanel examination was performed during chemotherapy, and a high tumor mutation burden was revealed. At 38 months after surgery, a new recurrent tumor, 2.7 cm in size, was observed in the abdominal wall, which was histologically proven to be metastatic adenocarcinoma. Atezolizumab was administered. After three cycles of treatment, treatment was switched to pembrolizumab because of its acceptance by healthcare insurance. The recurrent tumors in the abdominal wall and small intestine disappeared 6 months after the administration of immune checkpoint inhibitor, and the patient has continued pembrolizumab, surviving for 76 months after surgery without any clinical evidence of tumor. CONCLUSION: Immune checkpoint blockade successfully prolonged the survival of a patient with advanced hilar cholangiocarcinoma with high tumor mutation burden, although the optimal number of mutations for such a successful response needs to be clarified.

The First Successful Living Donor Liver Transplantation for Acute-on-Chronic Liver Failure Caused by Severe Acute Necrotizing Pancreatitis: A Case Report.

Liver transplantation (LT) is the only life-saving option when acute-on-chronic liver failure (ACLF) does not improve with conservative therapy. Acute pancreatitis (AP) can cause chronic liver disease progression to ACLF. However, deceased donor LT for patients with AP has had mixed results, and no consensus has been established regarding the indication for LT. We report the first successful living donor LT (LDLT) for ACLF caused by severe AP. The 38-year-old patient with alcoholic liver disease was transferred to our institute with worsening refractory ascites. During the pretransplant workup, she developed severe acute necrotizing pancreatitis, resulting in grade 3 ACLF. The patient's clinical course was further complicated by high levels of donor-specific antibodies and immune thrombocytopenia. The AP gradually improved after intensive care combined with artificial liver support. The patient successfully underwent urgent LDLT with upfront splenectomy and desensitization therapy, including plasm exchange, high-dose intravenous immunoglobulin, and anti-thymocyte globulin. No infection or recurrence of AP was observed postoperatively. We conclude that LDLT is a feasible option for ACLF patients caused by severe AP if a deceased donor is not readily available.

Absorption of proteoglycan via clathrin-mediated endocytosis in the small intestine of rats.

The mechanism underlying proteoglycan (PG) absorption in the intestine is not clear. Hence we analyzed the transport of salmon PG in the rat jejunum, ileum, and colon by the everted-sac method. The jejunum showed the largest capacity for PG transport. Jejunal transport of PG was also greater than that of chondroitin A and C. An inhibitor of clathrin-mediated endocytosis reduced jejunal PG transport. We conclude that intestinal PG transport is highest in the jejunum, and is partially dependent on clathrin-mediated endocytosis.

A case of accessory hepatic duct entering cystic duct successfully treated by laparoscopic cholecystectomy for cholecystolithiasis.

Although laparoscopic cholecystectomy is a well-established surgical procedure, an accessory hepatic duct (AcHD) entering the cystic duct is poorly understood. A 77-year-old woman with symptomatic cholecystlithiasis was referred to our hospital. Abdominal ultrasonography indicated several small stones in the gall bladder. Magnetic resonance cholangiopancreatography (MRCP) did not reveal an anomalous cystic duct. Dissecting the gall bladder bed at operation, AcHD entering the cystic duct was suspected. Intraoperative cholangiography revealed that B5 branch entered the cystic duct. We ligated the AcHD, and divided it. Laparoscopic cholecystectomy was completed, and the patient was discharged without any complication. A week after the operation, MRCP showed that ventral branch of B5 was dilated. The patient showed no symptom for more than a year. The present case exhibited extremely rare AcHD entering the cystic duct, which was hardly recognized before surgery. It is possible to recognize such anomalous variants with standard laparoscopic approach based on 2018 Tokyo Guidelines and with attention to the possibilities of AcHD entering the cystic duct.

Reappraisal of anatomical liver resection for hepatoblastoma in children.

Background: The principle of hepatoblastoma (HB) treatment is complete resection. The removal of tumor-bearing section(s) or hemiliver is widely accepted. However, neither the standardized anterior approach for right hepatectomy nor parenchymal sparing anatomical liver resection has been described for HB. Methods: We retrospectively reviewed the clinical course of two pediatric HB patients who underwent extended right hepatectomy using the anterior approach with the liver hanging maneuver and one who underwent parenchymal sparing anatomical liver resection of S4 apical+S8 ventral/dorsal+S7. The critical aspects of surgical techniques are described in detail. Results: In all three patients, R0 resection was achieved without complications and are currently alive without recurrence after an average follow-up of 23 months. Intraoperative cardiac hemodynamics were stable, even in a trisomy 18 patient with cardiac disease. Conclusions: Our findings suggest that these innovative techniques established in adults are safe and feasible for HB in children. These techniques also allow optimal anatomical liver resection to accomplish curative surgery while maintaining the functional reserve of the remnant liver.

Binding activity of avidin to the biotin within mesoporous silica materials for bioanalytical applications.

It has been reported that the activity of protein improved when it was adsorbed inside the pores of mesoporous silica (MPS). The current study investigated the activity of immobilized avidin to the biotin on MPS with various pore sizes (diameter=2.4-45.0 nm). The binding amount of immobilized avidin to biotin is 123 to 160 ng biotin/10 µg avidin on 2.7- to 5.4-nm pore MPS, but that on 12- to 45-nm pore MPS was markedly decreased (33-42 ng biotin/10 µg). Moreover, the binding amount was approximately 2- and 3-fold higher on the glycidoxypropyl (Gly)-functionalized 5.4- and 45-nm pore MPS in comparison with methyl (Me)-functionalized 5.4- and 45-nm pore MPS, respectively. Furthermore, avidin immobilized in native and Gly-grafted 45-nm pore MPS retained more than 70% and 50% binding activity to biotin, respectively, after incubating at 90°C for 3 h. In contrast, the activity was greatly reduced in the native and Gly-grafted 5.4-nm pore MPS under the same conditions (<36.9%). The immobilization also protected against effects of 0.01 M HCl and 50% MeOH; all of immobilized avidin proteins showed high activity (>50%) with biotin compared with that observed with free avidin (MeOH [<18.2%] and HCl [<32.7%]).

Efficient expression of recombinant soluble human FcγRI in mammalian cells and its characterization.

The extracellular domain of human FcγRI which interacts with a human IgG was expressed as recombinant soluble human FcγRI (rshFcγRI) by Chinese hamster ovary (CHO) cell. Stable CHO cell clones with efficient expression of rshFcγRI were established based on a dihydrofolate reductase (DHFR)/methotrexate (MTX) gene-amplification system. The CHO clones efficiently produced rshFcγRI under high-density continuous culture in a bioreactor. After 53 days of culture, the number of cells had reached approximately 4 × 106 cells/mL in the bioreactor and the average production of rshFcγRI had reached 7.4 mg L-medium⁻¹ day⁻¹. Secreted rshFcγRI was purified to a homogeneous state using cation exchange and affinity chromatographies. The binding affinities of rshFcγRI to human IgG subclasses were determined using surface plasmon resonance analysis. The binding affinities of rshFcγRI to human IgG1/κ and IgG3/κ were high (1.59 × 10⁻¹° and 2.81 × 10⁻¹° M, respectively), whereas that of rshFcγRI to human IgG4/κ was lower binding affinity (1.41 × 10⁻8 M). Binding to IgG2/κ was not detectable. Examination of circular dichroism spectra indicated that rshFcγRI was rich in ß-structures and loop or turn structures, but there were few α-helices. These results may be valuable for further studies of the structure and function of human FcγRI.