Antibody-like proteins that capture and neutralize SARS-CoV-2.

To combat severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and any unknown emerging pathogens in the future, the development of a rapid and effective method to generate high-affinity antibodies or antibody-like proteins is of critical importance. We here report high-speed in vitro selection of multiple high-affinity antibody-like proteins against various targets including the SARS-CoV-2 spike protein. The sequences of monobodies against the SARS-CoV-2 spike protein were successfully procured within only 4 days. Furthermore, the obtained monobody efficiently captured SARS-CoV-2 particles from the nasal swab samples of patients and exhibited a high neutralizing activity against SARS-CoV-2 infection (half-maximal inhibitory concentration, 0.5 nanomolar). High-speed in vitro selection of antibody-like proteins is a promising method for rapid development of a detection method for, and of a neutralizing protein against, a virus responsible for an ongoing, and possibly a future, pandemic.

Effects of high calcium levels on the disturbed extrusion of the second polar body during in vitro fertilization in C3H/He mouse substrains.

We previously reported that high concentrations (≥3.42 mM) of calcium during in vitro fertilization (IVF) disturbed the extrusion of the second polar body (PBII) in C3H/He inbred mice. In this study, the substrain specificity of this phenomenon was examined under 1.71-6.84 mM calcium concentration in ova from six C3H/He mouse commercially available substrains in Japan. PBII extrusion in ova from J substrains was not affected by calcium concentrations (<10% at any calcium level), but was grossly disturbed at high calcium levels in the ova of other substrains. This result has practical applications for the efficient production of normal zygotes by IVF, therefore contributing to the reduction in the numbers of donor animals for further zygote or embryo manipulation. Care must be taken in choosing IVF medium for particular strains and substrains.

Predicting the therapeutic effect of carbamazepine in trigeminal neuralgia by analysis of neurovascular compression utilizing magnetic resonance cisternography.

The aim of this study was to determine whether the evaluation of neurovascular compression (NVC) using new criteria on magnetic resonance (MR) cisternography improves the prediction of the curative effects of carbamazepine (CBZ) in trigeminal neuralgia (TN). In this study, a new analysis of NVC using MR cisternography was performed retrospectively for 280 patients with clinical signs and symptoms suggesting TN. The new analysis examined whether the site of the NVC was less than 3mm from the edge of the exit point and within the first third of the root entry zone on MR cisternography. Prediction of the curative effects of CBZ using the new evaluation of distance to the NVC improved on the predictive ability of the previous method of calculating the NVC volume (Mantel-Haenszel coefficient, P<0.01). In particular, initial treatment with CBZ 100mg/day for 2 weeks appeared more effective for patients with NVC volumes of ≤5mm3 plus a distance to the NVC of ≤3mm, than for those with NVC volumes >5mm3. The evaluation of NVC on MR cisternography using this new approach appears to be more useful than the previous method for predicting the initial treatment response in patients with TN.

Importance of absorbable surgical sutures for the prevention of stitch abscess after surgery in patients with oral squamous cell carcinoma.

BACKGROUND: To elucidate the significance of absorbable surgical sutures in the occurrence of stitch abscess after surgery in patients with oral squamous cell carcinoma (SCC). MATERIAL AND METHODS: The subjects were 251 patients who underwent excision and/or reconstruction and/or neck dissection for oral SCC using absorbable surgical sutures. Detection rates and characteristics of patients with stitch abscess were retrospectively evaluated by comparing between our present and previous data. RESULTS: There was only one stitch abscess among the 251 patients. A significant difference in the incidence of stitch abscess was found between the present data and our previous data. Of course, no significant correlations were found between the occurrence of stitch abscess using absorbable surgical sutures and the various factors seen in our previous analysis. CONCLUSIONS: A complete switch of surgical sutures from silk to absorbable surgical sutures is needed for surgery in patients with oral SCC.

Alterations in 18F-FDG accumulation into neck-related muscles after neck dissection for patients with oral cancers.

BACKGROUND: 18F-fluoro-2-deoxy-D-glucose (18F-FDG) accumulations are commonly seen in the neck-related muscles of the surgical and non-surgical sides after surgery with neck dissection (ND) for oral cancers, which leads to radiologists having difficulty in diagnosing the lesions. To examine the alterations in 18F-FDG accumulation in neck-related muscles of patients after ND for oral cancer. MATERIAL AND METHODS: 18F-FDG accumulations on positron emission tomography (PET)-computed tomography (CT) in neck-related muscles were retrospectively analyzed after surgical dissection of cervical lymph nodes in oral cancers. RESULTS: According to the extent of ND of cervical lymph nodes, the rate of patients with 18F-FDG-PET-positive areas increased in the trapezius, sternocleidomastoid, and posterior neck muscles of the surgical and/or non-surgical sides. In addition, SUVmax of 18F-FDG-PET-positive areas in the trapezius and sternocleidomastoid muscles were increased according to the extent of the ND. CONCLUSIONS: In evaluating 18F-FDG accumulations after ND for oral cancers, we should pay attention to the 18F-FDG distributions in neck-related muscles including the non-surgical side as false-positive findings.

Variety and complexity of fluorine-18-labelled fluoro-2-deoxy-D-glucose accumulations in the oral cavity of patients with oral cancers.

OBJECTIVES: To elucidate the points that require attention when interpreting fluorine-18-labelled fluoro-2-deoxy-d-glucose ((18)F-FDG)/positron emission tomography (PET) images by demonstration of (18)F-FDG accumulation in various areas of the oral cavity other than primary lesions in patients with oral cancers. METHODS: (18)F-FDG accumulations with a maximal standardized uptake value of over 2.5 in various areas of the oral cavity other than primary lesions were identified in 82 patients with oral cancers. RESULTS: (18)F-FDG/PET-positive areas, excluding primary tumours, included the front intrinsic muscles of the tongue (89.0%), upper and lower marginal parts of the orbicularis oris muscle (64.6%), sublingual glands, palatine tonsil, pharyngeal tonsil, and lingual tonsil. In addition, some areas in the jaws also showed accumulation. CONCLUSIONS: In patients with oral cancers, areas of (18)F-FDG accumulation in the oral cavity should be precisely identified and appropriately diagnosed, because accumulations can be seen in areas other than the primary tumour.

Functional evaluations of the parotid and submandibular glands using dynamic magnetic resonance sialography.

PURPOSE: To evaluate the functional differences between the parotid and submandibular glands using dynamic MR sialography. METHODS: In 30 volunteers, the time-dependent changes (dynamic changes) in the maximum area of the detectable parotid and submandibular gland ducts on dynamic MR sialographic images were analysed. RESULTS: Dynamic changes in the parotid gland ducts were detectable on MR sialographic images in all volunteers, but images of the submandibular gland ducts were detectable in only 23 volunteers. In addition, the dynamic changes in the submandibular gland ducts in these 23 subjects were less than those seen in the parotid gland ducts. A relationship was found between the changing ratio of parotid (Pearson r=0.448, P=0.013) or submandibular gland ducts (Pearson r=0.418, P=0.047) and the salivary flow rate during the stimulation period. CONCLUSIONS: Dynamic MR sialography allows evaluation of rest and stimulated functioning and morphological evaluation of the parotid and submandibular glands. This technique appears to have many possible applications in the dental, medical and biological fields.

Dynamic magnetic resonance sialography as a new diagnostic technique for patients with Sjögren's syndrome.

OBJECTIVE: To evaluate the clinical utility of dynamic magnetic resonance (MR) sialographic images as a diagnostic tool for patients with Sjögren's syndrome. METHODS: The morphological findings and various kinds of functional parameters in volunteers on dynamic MR sialographic images were compared with those in five patients with definite Sjögren's syndrome. RESULTS: On the MR sialographs of all five patients with Sjögren's syndrome, the so-called 'apple-tree appearance' was seen. The difference in two functional parameters using the dynamic MR sialographic data was elucidated between the two groups. The maximum area of the detectable ducts in the group of patients was significantly smaller (P < 0.001) than that in the group of volunteers. The ratio of change in the detectable ducts in the group of patients was significantly lower (P = 0.011) than that in the group of volunteers. CONCLUSIONS: Our study suggests that dynamic MR sialographic data in addition to MR sialographic images might be useful for the diagnosis of Sjögren's syndrome.

Clinical significance and variation of the advanced calcified stylohyoid complex detected by panoramic radiographs among 80-year-old subjects.

OBJECTIVES: To evaluate the clinical significance and variation of a calcified stylohyoid complex associated with advanced calcification as detected on panoramic radiographs among 80-year-old subjects. METHODS: Panoramic radiographs of 659 patients (262 men and 397 women) and data of their general and dental health conditions were used for the evaluation of the relationship between a calcified stylohyoid complex and general health among 80-year-olds. This study was part of the 8020 Data Bank Survey, which was designed to collect the baseline data of general and dental health conditions in 80-year-old subjects. The morphology of the calcified stylohyoid complexes was allocated to one of 12 patterns. The lengths of the calcified stylohyoid complex were measured directly on the radiographs from the caudal margin of the tympanic plate to the tip of the styloid process. RESULTS: All of the stylohyoid complexes from the 659 panoramic radiographs could be allocated to one of 12 morphological patterns, and some of their radiographic findings suggested calcification of the carotid artery and lymph nodes. In 80-year-old subjects, the average length of the calcified stylohyoid complexes was 36.7 mm, and ranged from 0.0 mm to 153.0 mm. There appeared to be correlations between the length of the calcified stylohyoid complex and serum calcium concentration and heel bone density, but not with the Community Periodontal Index (CPI). CONCLUSIONS: Dentists should recognize the existence of morphological variation in calcified stylohyoid complexes, especially in length, apparent on panoramic radiographs of 80-year-old patients. In addition, the longer length of the calcified stylohyoid complexes in 80-year-old patients may be a predictor of bone density and high serum calcium concentration level. The findings from the present study may provide potentially life-saving information about elderly people.

Correlation between the incidence of central nodal necrosis in cervical lymph node metastasis and the extent of differentiation in oral squamous cell carcinoma.

OBJECTIVES: To investigate the incidence of central nodal necrosis (CNN) in the cervical lymph nodes of patients with oral squamous cell carcinoma (SCC) and the factors that influence the formation of CNN. METHODS: Lymph nodes shown as CNN on computed tomography (CT) films in 107 lymph nodes from 27 patients with oral SCC were selected. Lymph nodes with CNN on CT films were compared with the pathological findings of lymph nodes on specimens. We compared many kinds of factors influencing the formation of CNN, including the differentiated type, with the incidence of CNN. RESULTS: Significant relationships were found between the incidence of CNN in metastatic lymph nodes and the presence of well-differentiated SCC and the presence of keratinization in tumour cells. CONCLUSIONS: The results indicated that if a patient had SCC with low-grade differentiation, CNN in small lymph nodes would be difficult to detect on CT scan. Therefore, noting changes in lymph node density in the absence of CNN on CT scans is necessary in case the primary tumour is low-grade SCC.

Estradiol induces PKA activation through the putative membrane receptor in the living hippocampal neuron.

For the past decade, estrogen actions rapid in onset, short in duration through the putative membrane estrogen receptor have attracted considerable attention. Nevertheless, there is so far limited evidence for estrogenic nongenomic regulation of the adenyl cyclase-cAMP-PKA system, especially in the brain. The present study reconfirms that 17beta-estradiol induces membrane-mediated PKA activation with a short latency in a living hippocampal neural cell with use of BSA-conjugated beta-estradiol which does not reach the nuclear estrogen receptor following my previous publication.

Effects of nicotine on neurogenesis and plasticity of hippocampal neurons.

To study nicotine's effects on neurogenesis in the dentate gyrus of the hippocampus, nicotine was injected intraperitoneally into adult rats. After sacrificing, the hippocampal formation was processed for immunohistochemical staining of PSA-NCAM, NeuN and GFAP. Nicotine decreased numbers of PSA-NCAM(+) and NeuN(+) cells dose-dependently.

Cleavage of nucleolin and AgNOR proteins during apoptosis induced by anticancer drugs in human salivary gland cells.

BACKGROUND: To investigate the behavior of nuclear proteins in apoptosis induced by anticancer drugs in cultured human salivary gland (HSG) cells. METHODS: Dynamic alternations of nucleolin and argyrophilic nucleolar organizer region (AgNOR) proteins in anticancer drug-induced apoptosis of HSG cells and in a cell-free apoptotic system were examined using Western blot analysis and immunocytochemical method. RESULTS: The 110-kDa form of nucleolin and AgNOR protein decreased and the 80- and 95-kDa forms appeared during apoptosis in HSG cells and in a cell-free apoptotic system. In addition, the induction of DNA ladder formation coincided with the appearance of alternation of nucleolin and AgNOR proteins in a cell-free apoptosis. Nucleolin diffusely spread out into the nuclear material in the apoptotic body of HSG cells. CONCLUSIONS: The present results indicate that alternations of nucleolin and AgNOR proteins are associated with the induction of DNA fragmentation and the final active phase of apoptosis induced by anticancer drugs in malignant salivary gland cells.

Utility of three dimension fast asymmetric spin-echo (3D-FASE) sequences in MR sialographic sequences: model and volunteer studies.

OBJECTIVES: To evaluate the utility of 3D-FASE for the visualization of salivary gland ducts for use in MR sialographic sequences. METHODS: We compared MR sialographic images and virtual endoscopic views from 3D-FASE with those from three kinds of sequences described by previous reports in a 3D parotid gland duct model and volunteer. The four sequences were two-dimension fast spin-echo (2D-FSE), three-dimension fast spin-echo (3D-FSE), two-dimension fast asymmetric spin-echo (2D-FASE), and three-dimension fast asymmetric spin-echo (3D-FASE). RESULTS: In the 3D parotid gland duct model, image visibility on visual score was clearest with 3D-FSE, followed by 3D-FASE (P = 0.028). In the volunteers, the visualization of images improved in the following order: 3D-FASE > 3D-FSE > 2D-FSE > 2D-FASE. CONCLUSIONS: The technique of 3D-FASE sequencing is more suitable and useful for MR sialography with an appropriate acquisition time.

Radiographic examination of the mandibular (glenoid) fossa in ancient and modern man.

OBJECTIVE: To compare the morphology of the mandibular (glenoid) fossa between ancient and modern Japanese. METHODS: There were 221 specimens from the ancient period and 206 specimens from the modern period. Radiographs of the mandibular fossa were obtained using a modified Schuller technique and were studied. The length, depth, and area of the mandibular fossa and angulation of the frontal slope of the mandibular fossa on the radiographs were automatically computed. In addition, morphological changes of the mandibular fossa in the ancient or modern periods on radiographs were evaluated in order to assess tendency of temporomandibular joint disorder (TMD). RESULT: The ratio of mandibular fossa having morphological alternation in the modern period was greater than that in the Yayoi. Differences in the length and area between the right and left sides became progressively larger from the Yayoi to modern periods. The angulation of the anterior slope of the mandibular fossa in the modern period was smaller than that in other periods. CONCLUSION: The morphology of the mandibular fossa was shown to have changed and asymmetry has increased from the Yayoi to modern periods. It can be speculated that this change in morphology of the mandibular fossa in the modern period is the result of an increase of TMD.

Instability of background fat intensity suppression using fat-saturated (FS) MR imaging techniques according to region and reconstruction procedure in patients with oral cancer.

The purpose of the present study is to evaluate the background fat intensity suppression instability of each area in the head and neck region, and in the post-reconstruction with metal plate and myocutaneous flap, of patients with oral cancer using fat-saturated (FS) images. STIR and FS T2-weighted images at pre- and post-surgery in 59 patients with oral cancer were scored for uniformity of fat suppression and tissue conspicuity in each region of the head and neck. The scores of FS on uniformity of fat suppression pre-operatively were worse than those of STIR in the mandibular level, but not lesion and tissue conspicuity. However, the deterioration both of scores between pre- and post-surgery using FS was worse than that using STIR using metal plate and/or myocutaneous flap. At diagnosis, we should recognize on MR images using FS that instability of the status of fat suppression might be brought about by respective area and reconstruction with metal plate and myocutaneous flap of patients with oral cancer.

Okadaic acid stimulates expression of Fas receptor and Fas ligand by activation of nuclear factor kappa-B in human oral squamous carcinoma cells.

In the present study, we used western blot and RT-PCR analysis to examine the expression of proteins and mRNAs of Fas receptor and Fas ligand in human oral squamous carcinoma SCC-25 cells treated with okadaic acid. Treatment with okadaic acid enhanced the expression of proteins and mRNAs of both Fas receptor and Fas ligand in SCC-25 cells. The amount of IkappaB-alpha in whole cell lysates decreased, while the level of NF-kappaB in nucleus increased, in the okadaic acid-treated cells. Okadaic acid-treatment also alters the cellular localization of NF-kappaB, from cytoplasm to nuclei. To investigate the activation of NF-kappaB in okadaic acid-treated SCC-25 cells, we performed electrophoretic mobility gel shift assay using nuclear extracts and the consensus oligonucleotide for NF-kappaB DNA binding site. The binding of nuclear proteins to the oligonucleotide of NF-kappaB increased when the cells had been treated with 20 nM okadaic acid for 4 h. We transfected the cells with pFLF1, which has the promoter region of Fas receptor gene containing NF-kappaB binding site. A luciferase reporter gene assay demonstrated that the activity in the cells transfected with pFLF1 and treated with 20 nM okadaic acid increased in a time-dependent manner and that the activity was more than three-fold over that in the control cells. Our results suggest that NF-kappaB activated at early stages in the okadaic acid-treated SCC-25 cells stimulated the promoter activity of Fas receptor in the cells leading to the apoptotic death of these cells.

Evaluation of coronal CT findings of rare cases of isolated medial orbital wall blow-out fractures.

OBJECTIVES: To report rare cases of isolated medial orbital wall blow-out fractures and to evaluate their coronal CT findings. METHODS: Nine cases of medial orbital wall blow-out fractures, diagnosed by coronal CT in a general medical hospital between 1995 and 1999, were studied. CT was performed using direct coronal acquisition with a slice thickness of 5 mm. Images were obtained at bone window settings. Clinical examination findings from the medical records were used to give information on the cause of injury, ocular symptoms and the days after injury that the CT examination was performed. RESULTS: Most of the patients (67%) were young male adults. The cause of injury was most frequently the result of fist fighting or athletics (78%). Diplopia and/or limitation of the gaze were the main ocular symptoms (78%). The average time from injury to CT examination was 4 days (range 0-16 days). Seven of the nine patients with medial orbital wall fractures had isolated medial orbital wall blow-out fractures. Herniation of orbital contents toward the ethmoid sinus was observed among all nine fractures. Only three patients (33%) had a swelling and/or medial deviation of the medial rectus muscle, all three having isolated medial orbital wall blow-out fractures. CONCLUSIONS: Nine cases of medial orbital wall blow-out fractures were examined, seven of which were isolated medial wall blow-out fractures. CT findings of this type of fracture included herniation of the orbital contents toward the ethmoid sinus. However, swelling and/or medial deviation of the medial rectus muscle was also seen.

Estrogen induces elevation of cAMP-dependent protein kinase activity in immortalized hippocampal neurons: imaging in living cells.

17beta-estradiol-stimulated PKA activation in living differentiated H19-7 cells was visualized using DR2, a cell-permeable fluorescent PKA substrate. Estradiol induced marked changes of fluorescence in cytosolic areas and these were inhibited by a PKA inhibitor.

Alteration of argyrophilic nucleolar organizer region associated (Ag-NOR) proteins in apoptosis-induced human salivary gland cells and human oral squamous carcinoma cells.

The level of argyrophilic nucleolar organizer regions (AgNORs) and AgNOR-associated proteins (Ag-NOR proteins) varies with cell activity, including ribosomal biogenesis occurring in proliferating cells. Proteins associated with some AgNORs are detected by a specific silver staining. To investigate a possible relationship between apoptosis and the AgNORs or Ag-NOR proteins, we examined the changes of AgNORs and Ag-NOR proteins during apoptosis in a human salivary gland cell line, HSG cells, and a human oral squamous carcinoma cell line, SCC-25 cells. Apoptosis was induced by treatment of HSG and SCC-25 cells with okadaic acid. Proteins prepared from HSG and SCC-25 cells treated with varying concentrations of okadaic acid (OA) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by transferring to transfer membranes and staining for Ag-NOR proteins by modified Western blot analysis. Four major bands (110 kDa, 43 kDa, 39kDa, and 37 kDa) were detected in the proteins obtained from the control cells. The level of the 110-kDa protein decreased in the proteins prepared from OA-induced apoptotic cells; however, the reaction intensity of the other three bands was changed in apoptotic cells. An additional band of an 80-kDa Ag-NOR protein appeared and increased in the apoptotic cells. Cellular fractionation of HSG cells and SCC-25 cells was done with or without apoptotic induction. An 80-kDa Ag-NOR protein was detected in the nuclear fraction prepared from the apoptotic cells, while the 110-kDa protein decreased in the nuclear fraction of these cells. The 110-kDa Ag-NOR protein may be nucleolin (C23) as deduced from its AgNOR staining features, including molecular weight. The 80-kDa protein may be the cleavage product of the 110-kDa protein. In the cell-free apoptotic system, in which intact nuclei of HSG cells were incubated with the cytosol fraction of apoptotic HSG and SCC-25 cells, the 80-kDa Ag-NOR protein was detected in nuclei incubated with the cytosol fraction of apoptotic cells, while the level of the 110-kDa protein decreased. The changes of Ag-NOR proteins in nuclei prepared from SCC-25 cells incubated with cytosol fractions prepared from HSG and SCC-25 cells were identical to those of the HSG cells. The alternation of AgNORs in apoptosis-induced HSG cells was also examined using double staining with Hoechst 33342 and silver nitrate. Hoechst staining revealed typical apoptotic nuclei, which exhibited highly fluorescent condensed chromatin in OA-treated HSG cells. Silver grains representing AgNORs were not detected in the cells undergoing apoptosis. The dual-imposition view confirmed that AgNORs, which are visible as dots in nucleoli in the control cells, disappeared from the apoptotic nuclei of HSG cells. Our results indicate that the 110-kDa nucleolar Ag-NOR protein is associated with apoptosis and is cleaved during apoptosis.