Screening of COVID-19 polymerase chain reaction tests using saliva for pregnant women and their partners in Himeji city.

A screening of coronavirus disease 2019 (COVID-19) polymerase chain reaction (PCR) tests using saliva for pregnant women and their partners was performed at all 12 maternity facilities located in Himeji city between May 29 and September 5, 2020. Pregnant women at 37 or more weeks of gestation or who experienced threatened labor and their partners who cared for an infant underwent a saliva PCR test with informed consent. As a result, all of 1475 pregnant women and 1343 partners tested negative for COVID-19 PCR. There were no cases of false positive or false negative PCR tests. This cohort study revealed for the first time that a screening of COVID-19 PCR tests using saliva may be useful to sustain perinatal medical care during the pandemic period in Japan.

Design and synthesis of binding growth factors.

Growth factors play important roles in tissue regeneration. However, because of their instability and diffusible nature, improvements in their performance would be desirable for therapeutic applications. Conferring binding affinities would be one way to improve their applicability. Here we review techniques for conjugating growth factors to polypeptides with particular affinities. Conjugation has been designed at the level of gene fusion and of polypeptide ligation. We summarize and discuss the designs and applications of binding growth factors prepared by such conjugation approaches.

Recombinant human gelatin substitute with photoreactive properties for cell culture and tissue engineering.

The human recombinant collagen I α1 chain monomer (rh-gelatin) was modified by the incorporation of an azidophenyl group to prepare photoreactive human gelatin (Az-rh-gelatin), with approximately 90% of the lysine residues conjugated with azidobenzoic acid. Slight changes in conformation (circular dichroism spectra) and thermal properties (gelation and melting points) were noticed after modification. Ultraviolet (UV) irradiation could immobilize the Az-rh-gelatin on polymer surfaces, such as polystyrene and polytetrafluoroethylene. Az-rh-gelatin was stably retained on the polymer surfaces, while unmodified gelatin was mostly lost by brief washing. Human mesenchymal cells grew more efficiently on the immobilized surface than on the coated surface. The immobilized Az-rh-gelatin on the polymer surfaces was able to capture engineered growth factors with collagen affinity, and the bound growth factors stimulated the growth of cells dose-dependently. It was also possible to immobilize Az-rh-gelatin in micropatterns (stripe, grid, and so on) using photomasks, and the cells grew according to the patterns. These results suggest that the photoreactive human gelatin, in combination with collagen-binding growth factors, will be clinically useful for surface modification of synthetic materials for cell culture systems and tissue engineering.

Recombinant hBMP4 incorporated with non-canonical amino acid for binding to hydroxyapatite.

A novel growth factor containing non-canonical amino acids was designed and synthesized to enhance the binding to hydroxyapatite (HA). The designed protein was human bone morphogenetic protein 4 (hBMP4) incorporating diphosporylated serines (pSpS) that was found in salivary protein statherin and was reported to be responsible for binding to HA. Recombinant hBMP4 and a short peptide sequences containing pSpS were ligated by enzymatice reaction of sortase A, which exchanges the terminal amino acids of two polypeptides. Resulting hBMP4 containing pSpS (hBMP4-pSpS) bound HA more efficiently than hBMP-4 tagged with canonical serines (hBMP4-SS). The HA-bound hBMP-4-pSpS exhibited osteogenesis inducing activity to multipotential mesenchyme cells (C3H10T1/2) as evidenced by increased expression of osteogenic markers, which was not seen by hBMP4-SS. This novel protein with non-canonical serines will be applicable to bone regeneration materials in combination with HA.

Pharmacological and Genetic Inhibition of Translocator Protein 18 kDa Ameliorated Neuroinflammation in Murine Endotoxemia Model.

ABSTRACT: Sepsis-associated encephalopathy (SAE) is a diffuse brain dysfunction associated with sepsis. The development of an effective strategy for early diagnosis and therapeutic intervention is essential for the prevention of poor prognosis of SAE. Translocator protein 18 kDa (TSPO) is a mitochondrial protein implicated in steroidogenesis and inflammatory responses. Despite accumulating evidence that implicates TSPO in the neuroinflammatory response of the central nervous system, the possible role of TSPO in SAE remains unclear. The aim of this study is to address a role of TSPO in neuroinflammation using mice 24 h after systemic injection of LPS, which consistently demonstrated microglial activation and behavioral inhibition. Quantitative polymerase chain reaction analysis revealed that hippocampal TSPO expression was induced following the systemic LPS injection, associated with an increase in pro-inflammatory cytokines such as tumor necrosis factor-α and interleukin-1ß. Interestingly, pretreatment with the TSPO antagonist, ONO-2952, or germ-line deletion of the TSPO gene exhibited an anti-inflammatory effect with significant suppression of LPS-induced production of those cytokines. These effects demonstrated by the ONO-2952 or TSPO knockout were associated with significant recovery from behavioral inhibition, as shown by improved locomotor activity in the open field analysis. Histological analysis revealed that ONO-2952 pretreatment suppressed the LPS-induced activation of TSPO-expressing microglia in the hippocampus of mice. Collectively, these results suggest that TSPO plays a critical role in the SAE mouse model. Based on this finding, monitoring TSPO activity, as well as the progress of endotoxemia and its sequelae in the animal model, would deepen our understanding of the underlying molecular mechanism of SAE.

Antidepressant effect of the translocator protein antagonist ONO-2952 on mouse behaviors under chronic social defeat stress.

In preclinical models, it has been reported that social defeat stress activates microglial cells in the CNS. Translocator protein 18 kDa (TSPO) is a mitochondrial protein expressed on microglia in the CNS that has been proposed to be a useful biomarker for brain injury and inflammation. We hypothesized that a TSPO antagonist, ONO-2952, would inhibit the neuroinflammation induced by microglial hyperactivation and associated depressive-like behaviors. An in vitro analysis showed that ONO-2952 suppressed the release of pro-inflammatory cytokines and mitochondrial reactive oxygen species in cultured microglia stimulated by lipopolysaccharide. In mice submitted to chronic social defeat stress, microglia predominantly expressed TSPO in limbic areas implicated in depressive-like behaviors, including the amygdala, ventral hippocampus and nucleus accumbens, in which an increase in the production of pro-inflammatory cytokines in vivo were associated. Treating animals with ONO-2952 during chronic social defeat stress ameliorated impairments in social avoidance and anxiety-like behaviors and suppressed pro-inflammatory cytokine production, suggesting that ONO-2952 exerted an anti-stress effect in this animal model of depression. Thus, targeting TSPO as a candidate for the development of antidepressants that reduce susceptibility to chronic stress could pave the way toward therapeutic interventions for relapse prophylaxis in depression.

Inhibition of catechol-O-methyltransferase in the cynomolgus monkey by opicapone after acute and repeated administration.

INTRODUCTION: The aim of the study was to clarify the dose response for inhibition of catechol-O-methyltransferase (COMT) by opicapone, a third generation COMT inhibitor, after acute and repeated administration to the cynomolgus monkey with pharmacokinetic evaluation at the higher dose. METHODS: Three cynomolgus monkeys were used in the study. In the first experiment, COMT inhibition was evaluated over 24 h after the first and at 24 h after the last of 14 daily oral administrations of vehicle, 1, 10 and 100 mg/kg opicapone using a crossover design. In the second experiment, the effect of the maximally effective dose, 100 mg/kg, was retested under the same conditions with additional monitoring of plasma opicapone levels to explore the relationship between pharmacokinetics and pharmacodynamics. RESULTS: Opicapone dose-dependently inhibited COMT activity, significantly so at 10 and 100 mg/kg. Maximal inhibition was 13.1%, 76.4% and 93.2% at 1, 10 and 100 mg/kg respectively, and COMT remained significantly inhibited at 24 h after 10 and 100 mg/kg (42.6% and 60.2% respectively). Following repeated administration of opicapone residual COMT inhibition at 24 h was 15-25% greater at all doses. In contrast to its pharmacodynamic effect, opicapone was rapidly absorbed and eliminated, with no accumulation in plasma following repeated administration. CONCLUSION: Opicapone showed sustained and dose-dependent COMT inhibition despite being rapidly eliminated from plasma and with no evidence for accumulation in plasma after 14 days administration. Opicapone fills the unmet need for a compound with sustained COMT inhibition which will improve levodopa bioavailability in patients with Parkinson's disease.

Collagen-Binding Hepatocyte Growth Factor (HGF) alone or with a Gelatin- furfurylamine Hydrogel Enhances Functional Recovery in Mice after Spinal Cord Injury.

The treatment of spinal cord injury (SCI) is currently a significant challenge. Hepatocyte growth factor (HGF) is a multipotent neurotrophic and neuroregenerative factor that can be beneficial for the treatment of SCI. However, immobilized HGF targeted to extracellular matrix may be more effective than diffusible, unmodified HGF. In this study, we evaluated the neurorestorative effects of an engineered HGF with a collagen biding domain (CBD-HGF). CBD-HGF remained in the spinal cord for 7 days after a single administration, while unmodified HGF was barely seen at 1 day. When a gelatin-furfurylamine (FA) hydrogel was applied on damaged spinal cord as a scaffold, CBD-HGF was retained in gelatin-FA hydrogel for 7 days, whereas HGF had faded by 1 day. A single administration of CBD-HGF enhanced recovery from spinal cord compression injury compared with HGF, as determined by motor recovery, and electrophysiological and immunohistochemical analyses. CBD-HGF alone failed to improve recovery from a complete transection injury, however CBD-HGF combined with gelatin-FA hydrogel promoted endogenous repair and recovery more effectively than HGF with hydrogel. These results suggest that engineered CBD-HGF has superior therapeutic effects than naïve HGF. CBD-HGF combined with hydrogel scaffold may be promising for the treatment of serious SCI.

Incorporating Si3 N4 into PEEK to Produce Antibacterial, Osteocondutive, and Radiolucent Spinal Implants.

Polyetheretherketone (PEEK) is a popular polymeric biomaterial which is primarily used as an intervertebral spacer in spinal fusion surgery; but it is developed for trauma, prosthodontics, maxillofacial, and cranial implants. It has the purported advantages of an elastic modulus which is similar to native bone and it can be easily formed into custom 3D shapes. Nevertheless, PEEK's disadvantages include its poor antibacterial resistance, lack of bioactivity, and radiographic transparency. This study presents a simple approach to correcting these three shortcomings while preserving the base polymer's biocompatibility, chemical stability, and elastic modulus. The proposed strategy consists of preparing a PEEK composite by dispersing a minor fraction (i.e., 15 vol%) of a silicon nitride (Si3 N4 ) powder within its matrix. In vitro tests of PEEK composites with three Si3 N4 variants-ß-Si3 N4 , α-Si3 N4 , and ß-SiYAlON-demonstrate significant improvements in the polymer's osteoconductive versus SaOS-2 cells and bacteriostatic properties versus gram-positive Staphylococcus epidermidis bacteria. These properties are clearly a consequence of adding the bioceramic dispersoids, according to chemistry similar to that previously demonstrated for bulk Si3 N4 ceramics in terms of osteogenic behavior (vs both osteosarcoma and mesenchymal progenitor cells) and antibacterial properties (vs both gram-positive and gram-negative bacteria).

A fusion protein of hepatocyte growth factor for immobilization to collagen.

We describe here a fusion protein consisting of hepatocyte growth factor (HGF; an angiogenic factor) and a collagen-binding domain (CBD) polypeptide of fibronectin (FN). This fusion protein (CBD-HGF), produced by a baculovirus expression system, exhibited much stronger collagen binding activity than native HGF in the range of 0.4-6.4microg/ml. Its binding at the lowest concentration exceeded that of HGF at the highest concentration. In addition, the collagen-bound CBD-HGF promoted growth of endothelial cells (ECs) to a greater degree at least 4 days longer than HGF added to the culture medium; about 5-fold greater increase in cell number after 10 days. These findings suggest that the fused CBD moiety not only helped immobilize HGF on collagen but also helped stabilize the fusion molecule, resulting in prolonged activity. The angiogenic activity of CBD-HGF in animal tissues was examined by subcutaneously implanting collagen sponges containing bound CBD-HGF. Blood vessel formation in the sponges after 7 days was 4-6-fold extensive as compared to the control sponges without sample. Implanted sponges with native HGF did not show significant difference from control. These results indicate that CBD-HGF is suitable for in vitro culture of ECs, and that this fusion protein can be used to confer HGF activity on biomaterials for use in tissue engineering.

Hepatocyte growth factor fusion protein having collagen-binding activity (CBD-HGF) accelerates re-endothelialization and intimal hyperplasia in balloon-injured rat carotid artery.

AIM: Hepatocyte growth factor (HGF) is known to stimulate endothelial cell proliferation. However, re-endothelialization is not enhanced when the native protein is administered to the injured artery, probably due to the short half-life of HGF at the site of injury. Therefore, the effects of an HGF fusion protein having collagen-binding activity (CBD-HGF) on re-endothelialization and neointimal formation was studied in the balloon-injured rat carotid artery. METHODS: The left common carotid artery of male Sprague-Dawley rats was injured with an inflated balloon catheter, and then treated with CBD-HGF 10 microg/mL), HGF (10 micro g/mL) or saline (control) for 15 min. After 14 days, the rats were injected with Evans blue and sacrificed. RESULTS: The re-endothelialized area was significantly greater in the CBD-HGF- treated rats than in the control or HGF -treated rats. Neointimal formation was significantly more pronounced in the CBD-HGF treated rats than in other rat groups. Both HGF and CBD-HGF stimulated proliferation of vascular smooth muscle cells as well as endothelial cells in vitro. Consistent with this, cultured smooth muscle cells were shown to express the HGF receptor (c-Met). CONCLUSION: CBD-HGF accelerates re-endothelialization and neointimal formation in vivo. CBD fusion protein is a useful vehicle to deliver vascular growth factors to injured arteries.

Ex vivo expansion of human cord blood hematopoietic progenitor cells using glutaraldehyde-fixed human bone marrow stromal cells.

Human stromal cells were immortalized and fixed with glutaraldehyde to support an ex vivo expansion of human cord blood hematopoietic progenitor cells. In addition, this enabled glutaraldehyde-fixed stromal cells to be stored at 4 degrees C. Although freeze-dried glutaraldehyde-fixed stromal cells did not increase the number of the progenitor cells, the percent decrease in the number of CD34(+) cells in the presence of freeze-dried glutaraldehyde-fixed stromal cells was less than that in the absence of the stromal cells. Thus, glutaraldehyde-fixed stromal cells can serve as a stabilizing device for hematopoietic cell expansion.

Emulsifier content and side effects of oil-based adjuvant vaccine in swine.

Side effects caused by the excessive emulsifier in oil-based adjuvant vaccine were examined practically in swine using one oil-in-water type adjuvant vaccine against swine pleuropneumonia. The vaccine was prepared from cell-free-antigen of Actinobacillus pleuropneumoniae, liquid paraffin, and several polyoxyethylenesorbitan and sorbitan oleates. Based on findings about safety in mice and emulsion stability, 2 vaccines containing either 11.25% or 6.25% emulsifier content were injected intramuscularly twice in swine, as the highest and lowest limits, respectively, within the practical range. All pigs showed temporary fever and malaise with anorexia for several days after each injection. The fever of the higher emulsifier content group took significantly longer to recover than the lower. Malaise also showed a similar tendency. On the other hand, antibody response was sufficiently induced with no significant difference between the 2 groups. Lowering the emulsifier content is a very simple but effective solution for mitigation of side effects without the reduction of adjuvanticity. For safe and high-quality oil-based adjuvant vaccines, not only antigen and base-oil, but emulsifier content must be optimized.

Culture of human umbilical vein endothelial cells on immobilized vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) was immobilized on substrata in photoreactive gelatin to control the adhesion and growth of vascular endothelial cells. The gelatin and VEGF were mixed in water and cast on a polystyrene dish or a silane-coated glass plate. The surface was then photoirradiated in the presence or absence of a photomask and washed. Toughness of the immobilized material was confirmed by ethanol treatment. Human umbilical vein endothelial cells (HUVECs) grew on the immobilized VEGF but not on a nontreated surface. Growth of HUVEC increased significantly with an increase in the amount of immobilized VEGF, and the effects were inhibited by treatment with anti-VEGF antibody. Thus, immobilized VEGF specifically interacted with HUVECs to permit growth in culture. Micropatterning of HUVEC cultures was also achieved using micropattern-immobilized VEGF. This patterning technique may be useful for the formation of blood vessel networks in vitro.

Experimental infection of calves with Pasteurella multocida Serovar A: 3 isolated in Japan.

Pasteurella multocida, serovar A: 3, selected by pathogenicity in mice from among 10 strains isolated from pneumonic lesions of calves, was adjusted to 10(7), 10(8) and 10(10) colony-forming units (CFU), and inoculated intratracheally into four calves. All calves showed pyrexia and had lungs with congestion and hepatization. Inoculation with 10(10) CFU of bacteria produced respiratory symptoms and abscesses in lungs. This information will aid elucidation of the pathogenicity of P. multocida and the development of vaccines.

Differentiation of Sea Urchin Micromeres: Correlation between Specific Protein Synthesis and Spicule Formation: (micromere/differentiation/protein synthesis/sea urchin).

Sea urchin micromeres were isolated from the 16-cell stage embryos and cultured until they differentiated into spicule-forming cells. Electrophoretic analysis of proteins labeled with [35 S]-methionine showed that the differentiation accompanied the synthesis of five cell-specific proteins. These proteins appeared prior to spicule formation and were synthesized continuously or maintained stably while the cultured micromeres formed spicules. In contrast, these proteins were hardly detectable during development of the meso- and macromeres. Correlation between synthesis of the specific proteins and spicule formation was further examined in culture conditions which inhibit spicule formation. In Zn2+ -containing or serum-free medium, the micromere descendants failed to form spicules and exhibited markedly reduced synthesis of one of the specific proteins (32 K daltons). After removal of Zn2+ , or addition of serum, however, spicules were formed with delay but concomitantly with an increase in the synthesis of this protein. This clear correlation suggests the participation of the 32 K protein in the process of spicule formation.

SPICULE FORMATION IN VITRO BY THE DESCENDANTS OF PRECOCIOUS MICROMERE FORMED AT THE 8-CELL STAGE OF SEA URCHIN EMBRYO.

The micromeres at the 16-cell stage of sea urchin embryo have already been endowed with a faculty to self-differentiate into spicule-forming cells (11). The present experiment was designed to test whether the factor(s) necessary for such self-differentiation had already been localized at the 8-cell stage in an area corresponding to the presumptive micromere region in Hemicentrotus pulcherrimus. Since the blastomeres at the 8-cell stage are all equal in size in normal embryo, unequal 3rd cleavage, by which small blastomeres are pinched off toward the vegetal pole (precocious micromeres), was experimentally induced either by treatment with 4NQO (4-nitroquinoline-1-oxide) at the 2-cell stage or by continuous culture in Ca-free sea water. The precocious micromeres were cultured in vitro in natural sea water containing horse serum. Descendants of the precocious micromeres formed spicules. In comparison their spicule formation with that by the descendants of the micromere of normal embryo, no differences were found regarding 1) time of initiation of spicule formation, 2) rate of growth of spicule, 3) size and shape of resultant spicule and 4) percentage of clones which formed spicule. The fact indicates that factor(s) indispensable for self-differentiation into spicule-forming cells have already been localized near the vegetal pole as early as the 8-cell stage.

Micromere Differentiation in the Sea Urchin Embryo: Two-Dimensional Gel Electrophoretic Analysis of Newly Synthesized Proteins: (sea urchin/micromere/protein synthesis/differentiation).

A method for large-scale culture of isolated blastomeres of sea urchin embryos in spinner flasks was developed. Micromeres and meso-, macromeres isolated from sea urchin embryos at the 16-cell stage were cultured by this method and the patterns of protein synthesis by their descendants were examined by two-dimensional gel electrophoresis of [35 S] methionine-labeled proteins. Six distinct proteins with molecular weights of 140-kDa, 105-kDa, 43-kDa, 32-kDa, and 28-kDa (two components) were specifically synthesized by differentiating micromeres. Quantitative analysis of the two-dimensional gel patterns demonstrated that all these proteins, except the 32-kDa protein, appeared at the time of ingression of primary mesenchyme cells (PMC's) in vivo, several hours earlier than the onset of spicule formation. The synthesis of 32-kDa protein was paralleled to active spicule formation and the uptake of Ca2+ . Cell-free translation products directed by poly (A)+ RNAs isolated from descendant cells of micromeres and meso-, macromeres were compared by two-dimensional gel electrophoresis. Several spots specific to the micromere lineage were detected. However, none of them comigrated with the proteins synthesized specifically by the cultured micromeres. The results suggest that the expression of these proteins specific to differentiating micromeres may involve post-translational modification.

Neuroprotective effects of ONO-1924H, an inhibitor of poly ADP-ribose polymerase (PARP), on cytotoxicity of PC12 cells and ischemic cerebral damage.

N-[3-(4-Oxo-3,4-dihydro-phthalazin-1-yl)phenyl]-4-(morpholin-4-yl) butanamide methanesulfonate monohydrate (ONO-1924H) is a novel inhibitor of poly ADP-ribose polymerase (PARP). In this study, we examined the effects of ONO-1924H on cytotoxicity induced by hydrogen peroxide in PC12 cells in vitro and cerebral damage and neurological deficits induced by middle cerebral artery (MCA) thrombus occlusion in vivo in rat. In the in vitro cytotoxicity assay, exposure to 0.5 mmol/L hydrogen peroxide induced cell death in differentiated PC12 cells. ONO-1924H, a PARP inhibitor (Ki=0.21 micromol/L), reduced cell death in a concentration-dependent manner that was correlated with inhibition of PARP activation. A 50% reduction in cell death (EC50) was achieved with 2.4 micromol/L ONO-1924H. In the MCA occlusion model, ONO-1924H was injected intravenously at doses of 3, 10 and 30 mg/kg/h for 3 h, and cerebral damage and neurological deficits were estimated 24 h after MCA occlusion. ONO-1924H treatment led to a significant decrease in cerebral damage in the 10 mg/kg/h-treated group (P < 0.05) and the 30 mg/kg/h-treated group (P < 0.01). Further, ONO-1924H at doses of 30 mg/kg/h significantly (P < 0.05) improved neurological deficits. These findings suggest that the novel PARP inhibitor, ONO-1924H, affords effective neuroprotection and is a useful therapeutic candidate for the treatment of ischemic stroke.

Direct in vitro selection of titanium-binding epidermal growth factor.

Epidermal growth factor (EGF) with affinity to TiO2 surfaces was obtained by direct in vitro selection. A random peptide library was generated for fusion to the N-terminal of EGF, and polypeptides exhibiting affinity were selected in vitro by ribosome display. The best-performing polypeptide sequence was selected for synthesis using a solid-phase method and showed high affinity to TiO2 after refolding. Molecular dynamic simulations indicated that the interaction of the selected peptide segment with the TiO2 surface was comparable to that of a previously reported titanium-binding peptide, TBP-1. The hydroxyl groups in the selected peptide segment were found to be critical for the binding interaction. NIH3T3 cell culture for two days in the presence of the TiO2-binding EGF showed that it was able to enhance cell proliferation as much as unmodified EGF in solution. As a result, the selected EGF construct was able to induce cell proliferation on titanium surfaces. This direct in vitro selection technique should extend the possibilities for the design of other surface-binding growth factors.