Highly sensitive strigolactone perception by a divergent clade KAI2 receptor in a facultative root parasitic plant, Phtheirospermum japonicum.

Phtheirospermum japonicum, a member of the Orobanchaceae family, is a facultative root parasitic plant that can survive without parasitizing the host. In contrast, obligate root parasitic plants such as Striga and Orobanche, which are also members of the Orobanchaceae family, cannot survive in the absence of the host. The germination of obligate root parasitic plants is typically induced by host root-derived strigolactones (SLs) at very low concentrations. The KAI2/HTL family proteins have been found to be involved in the perception of karrikin (KAR), a smoke-derived germination inducer and unidentified endogenous ligand, in non-parasitic plants. Obligate root parasitic plants possess uniquely diverged KAI2 clade genes, which are collectively referred to as KAI2d. Many of those have been shown to function as SL receptors. Intriguingly, the KAI2d clade genes are also conserved in P. japonicum, even though this plant does not require SLs for germination. The biochemical and physiological functions of the KAI2d proteins in P. japonicum remain unclear. Here, we report that some of these proteins can function as SL receptors in P. japonicum. Moreover, we found that one of them, PjKAI2d4, is highly sensitive to SLs when expressed in Arabidopsis, and it is similar to the sensitive SL receptors found in Striga and Orobanche. These results suggest that the KAI2d clade SL receptors play a crucial role not only in obligate parasites but also in facultative parasitic plants.

Radicle growth regulation of root parasitic plants by auxin-related compounds.

Root parasitic plants in the Orobancheceae, such as Striga and Orobanche, cause significant damage to crop production. The germination step of these root parasitic plants is induced by host-root-derived strigolactones (SLs). After germination, the radicles elongate toward the host and invade the host root. We have previously discovered that a simple amino acid, tryptophan (Trp), as well as its metabolite, the plant hormone indole-3-acetic acid (IAA), can inhibit radicle elongation of Orobanche minor. These results suggest that auxin plays a crucial role in the radicle elongation step in root parasitic plants. In this report, we used various auxin chemical probes to dissect the auxin function in the radicle growth of O. minor and Striga hermonthica. We found that synthetic auxins inhibited radicle elongation. In addition, auxin receptor antagonist, auxinole, rescued the inhibition of radicle growth by exogenous IAA. Moreover, a polar transport inhibitor of auxin, N-1-naphthylphthalamic acid (NPA), affected radicle bending. We also proved that exogenously applied Trp is converted into IAA in O. minor seeds, and auxinole partly rescued this radicle elongation. Our data demonstrate a pivotal role of auxin in radicle growth. Thus, manipulation of auxin function in root parasitic plants should offer a useful approach to combat these parasites.

A Divergent Clade KAI2 Protein in the Root Parasitic Plant Orobanche minor Is a Highly Sensitive Strigolactone Receptor and Is Involved in the Perception of Sesquiterpene Lactones.

Strigolactones (SLs) were initially discovered as germination inducers for root parasitic plants. In 2015, three groups independently reported the characterization of the SL receptor in the root parasitic plant Striga hermonthica, which causes significant damage to crop production, particularly in sub-Saharan Africa. The characterized receptors belong to HYPOSENSITIVE TO LIGHT/KARRIKIN INSENSITIVE2 (HTL/KAI2), which is a member of the α/ß-hydrolase protein superfamily. In non-parasitic plants, HTL/KAI2 perceives the smoke-derived germination inducer karrikin and a yet-unidentified endogenous ligand. However, root parasitic plants evolved a specific clade of HTL/KAI2 that has diverged from the KAI2 clade of non-parasitic plants. The S. hermonthica SL receptors are included in this specific clade, which is called KAI2 divergent (KAI2d). Orobanche minor is an obligate root holoparasitic plant that grows completely dependent on the host for water and nutrients because of a lack of photosynthetic ability. Previous phylogenetic analysis of KAI2 proteins in O. minor has demonstrated the presence of at least five KAI2d clade genes. Here, we report that KAI2d3 and KAI2d4 in O. minor have the ability to act as the SL receptors. They directly interact with SLs in vitro, and when expressed in Arabidopsis, they rescue thermo-inhibited germination in response to the synthetic SL analog GR24. In particular, KAI2d3 showed high sensitivity to GR24 when expressed in Arabidopsis, suggesting that this receptor enables highly sensitive SL recognition in O. minor. Furthermore, we provide evidence that these KAI2d receptors are involved in the perception of sesquiterpene lactones, non-strigolactone-type germination inducers.

Desmethyl type germinone, a specific agonist for the HTL/KAI2 receptor, induces the Arabidopsis seed germination in a gibberellin-independent manner.

DWARF14 (D14) and HTL/KAI2 (KAI2) are paralogous receptors in the α/ß-hydrolase superfamily. D14 is the receptor for a class of plant hormones, strigolactones (SLs), and KAI2 is the receptor for the smoke-derived seed germination inducer, Karrikin (KAR), in Arabidopsis. Germinone (Ger) was previously reported as a KAI2 agonist with germination-inducing activity for thermo-inhibited Arabidopsis seed. However, Ger was not specific to KAI2, and could also bind to D14. It was reported that SL analogs with a desmethyl-type D-ring structure are specifically recognized by KAI2. On the basis of this observation, we synthesized a desmethyl-type germinone (dMGer). We found that dMGer is highly specific to KAI2. Moreover, dMGer induced Arabidopsis seed germination more effectively than did Ger. In addition, dMGer induced the seed germination of Arabidopsis in a manner independently of GA, a well-known germination inducer in plants.

Rac1 activation in oral squamous cell carcinoma as a predictive factor associated with lymph node metastasis.

OBJECTIVES: Secondary lymph node metastasis (SLNM) indicates a poor prognosis, and limiting it can improve the survival rate in early-stage tongue squamous cell carcinoma (TSCC). Many factors have been identified as predictors of SLNM; however, there is no unified view. Ras-related C3 botulinum toxin substrate 1 (Rac1) was found to be a promoter of the epithelial-mesenchymal transition (EMT) and is also attracting attention as a new therapeutic target. This study aims to investigate the role of Rac1 in metastasis and its relationship with pathological findings in early-stage TSCC. MATERIALS AND METHODS: Rac1 expression levels of 69 cases of stage I/II TSCC specimens and their association with clinicopathological characteristics were evaluated by immunohistochemical staining. The role of Rac1 in oral squamous cell carcinoma (OSCC) was examined after Rac1 in OSCC cell lines was silenced in vitro. RESULTS: High Rac1 expression was significantly associated with the depth of invasion (DOI), tumor budding (TB), vascular invasion, and SLNM (p < 0.05). Univariate analyses revealed that Rac1 expression, DOI, and TB were factors significantly associated with SLNM (p < 0.05). Moreover, our multivariate analysis suggested that Rac1 expression was the only independent determinant of SLNM. An in vitro study revealed that Rac1 downregulation tended to decrease cell migration and proliferation. CONCLUSION: Rac1 was suggested to be an important factor in the metastasis of OSCC, and it could be useful as a predictor of SLNM.

Tryptophan derivatives regulate the seed germination and radicle growth of a root parasitic plant, Orobanche minor.

Root parasitic plant germination is induced by the host-derived chemical, strigolactone (SL). We found that a major microbial culture broth component, tryptone, inhibits the SL-inducible germination of a root parasitic plant, Orobanche minor. l-tryptophan (l-Trp) was isolated as the active compound from tryptone. We further found that l-Trp related compounds (1b-11), such as a major plant hormone auxin (8, indole-3-acetic acid; IAA), also inhibit the germination and post-radicle growth of O. minor. We designed a hybrid chemical (13), in which IAA is attached to a part of SL, and found that this synthetic analog induced the germination of O. minor, and also inhibited post-radicle growth. Moreover, contrary to our expectations, we found that N-acetyl Trp (9) showed germination stimulating activity, and introduction of a substitution at C-5 position increased its activity (12a-12f). Our data, in particular, the discovery of a structurally hybrid compound that has two activities that induce spontaneous germination and inhibit subsequent radical growth, would provide new types of germination regulators for root parasitic plants.

Mechanical Low-Pass Filtering of Cells for Detection of Circulating Tumor Cells in Whole Blood.

The detection of circulating tumor cells (CTCs) from liquid biopsies using microfluidic devices is attracting a considerable amount of attention as a new, less-invasive cancer diagnostic and prognostic method. One of the drawbacks of the existing antibody-based detection systems is the false negatives for epithelial cell adhesion molecule detection of CTCs. Here we report a mechanical low-pass filtering technique based on a microfluidic constriction and electrical current sensing system for the novel CTC detection in whole blood without any specific antigen-antibody interaction or biochemical modification of the cell surface. The mechanical response of model cells of CTCs, such as HeLa, A549, and MDA-MB-231 cells, clearly demonstrated different behaviors from that of Jurkat cells, a human T-lymphocyte cell line, when they passed through the 6-µm wide constriction channel. A 6-µm wide constriction channel was determined as the optimum size to identify CTCs in whole blood with an accuracy greater than 95% in tens of milliseconds. The mechanical filtering of cells at a single cell level was achieved from whole blood without any pretreatment (e.g., dilution of lysing) and prelabeling (e.g., fluorophores or antibodies).

Usefulness of fluorescence visualization-guided surgery for early-stage tongue squamous cell carcinoma compared to iodine vital staining.

BACKGROUND: In the most cases of oral squamous cell carcinoma (OSCC), oral epithelial dysplasia (OED) is found adjacent to the primary tumor. The delineation of surgical margins for OSCC is critical to minimize the risk for local recurrence. The aim of this study is to demonstrate that the fluorescence visualization (FV)- device can delineated the lesion visualizes OED of adjacent primary tumors by histopathologically comparison to conventional iodine vital staining. MATERIAL AND METHODS: The study involved 40 patients with superficial tongue squamous cell carcinoma treated from July 2016 to July 2018 at the Oral Cancer Center, Tokyo Dental College. RESULTS: Cytokeratin 13 (CK13) expression rate in the area of fluorescence visualization loss (FVL) was significantly lower than that in the area of fluorescence visualization retention (FVR). In addition, CK17, Ki-67, and p53 expression rates were significantly higher in FVL than FVR. There was no significant difference in the delineation rate or area between FVL and iodine-unstained area. High-grade dysplasia was observed most frequently at the FV and iodine-unstained boundary, but no significant pathological differences were found. CONCLUSION: We strongly suggest the FV-guided surgery is a useful method for accurate resection in early-stage tongue squamous cell carcinoma.

Temporary Closure of Inpatient Ward due to Mumps Virus Reinfection in Elderly Patient.

We report a case of suspected reinfection with the mumps virus in an elderly patient which resulted in temporary closure of an inpatient ward. A 65-year-old man with colorectal cancer was admitted to the digestive surgery ward at our hospital to undergo a stoma closure operation. He was subsequently referred to our department with right swelling in the preauricular region on postoperative day 4. The swelling subsided within a few days, and the patient was discharged. A serum titer test revealed a high level of antibodies to the mumps virus, however. Therefore, staff who had come into close contact with the patient were examined and the decision taken to stop admitting new patients to the ward. When symptoms are detected in a patient has already had mumps, it is important to consider the possibility of reinfection. Furthermore, it is necessary for medical workers to undergo a serum antibody test to the mumps virus and receive a further vaccination if antibody levels are too low to confer immunity.

Fatal Bleeding in Conjunction with Mandibular Medication-related Osteonecrosis of the Jaw (MRONJ).

Here, we report a case of fatal bleeding in conjunction with mandibular medicationrelated osteonecrosis of the jaw (MRONJ). A 75-year-old Japanese man was referred to our department with osteonecrosis of the jaw due to bisphosphonate (BP) for multiple bone metastases from prostate cancer. Aggressive surgical intervention was ruled out due to a poor prognosis in terms of life expectancy. Death occurred due to hemorrhagic shock resulting from massive oral bleeding caused by necrosis of the mandible. Numerous reports have suggested that jaw necrosis is induced not only by BP, but also RANKL antibody, steroids, and molecularly-targeted agents. This suggests that the number of cases of MRONJ is likely to increase among elderly patients in whom general health is already poor. The American Association of Oral and Maxillofacial Surgery recommends aggressive treatment only in cases of stage 3 disease. Therefore, such a therapeutic strategy may only be available for cases of jaw necrosis in which the general health status of the patient is otherwise good. To prevent a life-threatening outcome in cases of MRONJ, physicians, who are responsible for determining the drug strategy, should cooperate with oral surgeons in determining the best therapeutic strategy.

Squamous Cell Carcinoma of Maxillary Gingiva Progressing to Disseminated Carcinomatosis of Bone Marrow.

Disseminated carcinomatosis of the bone marrow (DCBM) is characterized by diffuse metastasis to bone marrow and sudden mortality. To the best of our knowledge, no studies to date have reported progression of oral squamous cell carcinoma to DCBM. Herein, we report a case of squamous cell carcinoma in the maxillary gingiva suspected of progressing to DCBM. A 64-year-old woman presented with white lesions on the left maxillary gingiva. The lesions were diagnosed as squamous cell carcinoma (T2, N0, M0), and partial maxillectomy performed. Two years and 5 months after surgery, metastasis was noted in the left cervical lymph node and left radical neck dissection carried out. The subsequent diagnosis was right cervical lymph node metastasis and multiple bone metastases. The patient also presented with thrombocytopenia, anemia, and elevated levels of alkaline phosphatase, probably due to metastatic bone disease. Although various antitumor therapies were administered, the patient died 6 months after diagnosis of multiple bone metastases.

Preliminary Study of Temporal Change in Free Flap Volume after Tongue Reconstruction.

The purpose of this study was to investigate three-dimensionally temporal change in flap volume after free flap reconstructive surgery in tongue cancer patients. The results revealed an average change in flap volume of 82.3% at 1 year postoperatively. Change in tongue volume at approximately 6 months postoperatively showed a correlation with triglyceride levels. A correlation was also found between albumin levels and tongue volume at 1 year onwards postoperatively. The goal of such surgery in patients with tongue cancer is to reconstruct a functional tongue. Taking postoperative change in tongue volume into consideration is therefore of importance in minimizing postoperative dysphagia.

Production of wild-type and mutant-type human DAP12 proteins by Bombyx mori nucleopolyhedrovirus vector.

A Japanese patient with Nasu-Hakola disease was found to have a serine-to-asparagine (S39N) substitution in human DNAX-activation protein 12 (DAP12). To elucidate the functional abnormalities of mutant-type DAP12, we expressed wild-type and mutant-type recombinant DAP12 protein with Bombyx mori nucleopolyhedrovirus (BmNPV) vector, and successfully purified the respective proteins from the hemolymph of recombinant BmNPV infected B. mori larvae.

Impact of skeletal muscle mass on postoperative complications in oral cancer surgery.

BACKGROUND: Sarcopenia is characterized by a progressive and generalized loss of skeletal muscle mass and strength. The aim of this retrospective study was to investigate the impact of skeletal muscle mass on adverse events in free-flap reconstruction for defects after oral cancer resection. RESULTS: Of 120 patients, recipient-site adverse events occurred in 56 patients (46.7%), and recipient-site surgical site infections occurred in 45 patients (37.5%). Skeletal muscle index was significantly associated with recipient-site adverse events in univariate analysis (P < 0.05). Lower body mass index and skeletal muscle index were significantly associated with recipient-site surgical site infection in univariate analysis (P < 0.05). In the multiple logistic regression model, a lower skeletal muscle index was a significant risk factor for recipient-site adverse events and surgical site infections (adverse events odds ratio; 3.17/P = 0.04; surgical site infection odds ratio; 3.76/P = 0.02). CONCLUSIONS: The SMI at level Th12 was an independent factor for postoperative AEs, especially SSI, in OSCC patients with free-flap reconstruction.

A 5-year activity report from the Oral Cancer Center, Tokyo Dental College.

The Tokyo Dental College Oral Cancer Center was established on April 1st, 2006 at our Ichikawa General Hospital for the purpose of providing multimodal treatment for oral cancer. This report summarizes the Center's activities over the last 5 years. The total number of oral cancer patients treated was 360 (April 2006 to March 2011), with 205 primary cases. We investigated the following treatment-related items: 1) site, 2) age, 3) sex, 4) pathological examination, 5) staging, 6) systemic disorder, 7) double cancer, 8) treatment, and 9) prognosis. Out of 205 patients, 60% were men and 40% were women. Men in their 60s and women in their 80s were seen the most. The most common site was the tongue, at 42%, followed by the mandibular gingiva, maxillary gingiva, oral floor, and buccal mucosa. Squamous cell carcinomas were seen most frequently, at 94% (15% were stage I, 33% stage II, 15% stage III, and 34% stage IV). The most common treatment method was surgical treatment, at 83%. The 5-year survival rate at all stages was 85.4%. At the Oral Cancer Center, oral surgeons take the initiative in establishing treatment in cooperation with other departments and branches. Since the establishment of the Ambulatory Center for Maxillary Prosthetics in October 2011, 26 patients have undergone treatment. Related departments and branches work in teams, enabling comprehensive treatment, from the preoperative state to postoperative functional recovery. We wish to use these strengths to improve oral cancer treatment in Japan and will continue to work toward providing the best possible care for our patients.

Ameloblastic carcinoma of the mandible: a case report.

BACKGROUND: Ameloblastic carcinoma is a malignant form of ameloblastoma and a very rare odontogenic tumor. We report a case of ameloblastic carcinoma that occurred after removal of a right-sided mandibular dental implant. CASE PRESENTATION: A 72-year-old female patient visited her family dentist with a complaint of pain around a lower right implant placed 37 years previously. Although the dental implant was removed with the diagnosis of peri-implantitis, the patient experienced dullness of sensation in the lower lip and was followed up by her dentist, but after no improvement. She was referred to a highly specialized institution where she was diagnosed with osteomyelitis and treated the patient with medication; however, there was no improvement. In addition, granulation was observed in the same area leading to a suspicion of malignancy, and the patient was referred to our oral cancer center. The diagnosis of squamous cell carcinoma was made after a biopsy at our hospital. Under general anesthesia, the patient underwent mandibulectomy, right-sided neck dissection, free flap reconstruction with an anterolateral thigh flap, immediate reconstruction with a metal plate, and tracheostomy. Histological analysis of the resected specimen on hematoxylin and eosin staining showed structures reminiscent of enamel pulp and squamous epithelium in the center of the tumor. The tumor cells were highly atypical, with nuclear staining, hypertrophy, irregular nuclear size, and irregular nuclear shape, all of which were suggestive of cancer. Immunohistochemical analysis showed that Ki-67 was expressed in more than 80% of the targeted area, and the final diagnosis was primary ameloblastic carcinoma. CONCLUSION: After reconstructive flap transplantation, occlusion was re-established using a maxillofacial prosthesis. The patient remained disease-free at the 1-year 3-month follow-up.

A New Series of Strigolactone Analogs Derived From Cinnamic Acids as Germination Inducers for Root Parasitic Plants.

Root parasitic plants such as Striga and Orobanche cause significant damage on crop production, particularly in sub-Saharan Africa. Their seeds germinate by sensing host root-derived signaling molecules called strigolactones (SLs). SL mimics can be used as suicidal germination inducers for root parasitic plants. Previous attempts to develop such chemicals have revealed that the methylbutenolide ring (D-ring), a common substructure in all the naturally occurring SLs, is critical for SL agonistic activity, suggesting that it should be possible to generate new SL mimics simply by coupling a D-ring with another molecule. Because structural information regarding SLs and their receptor interaction is still limited, such an approach might be an effective strategy to develop new potent SL agonists. Here, we report development of a series of new SL analogs derived from cinnamic acid (CA), the basis of a class of phenylpropanoid natural products that occur widely in plants. CA has an aromatic ring and a double-bond side-chain structure, which are advantageous for preparing structurally diverse derivatives. We prepared SL analogs from cis and trans configuration CA, and found that all the cis-CA-derived SL analogs had stronger activities as seed germination inducers for the root parasitic plants, Orobanche minor and Striga hermonthica, compared with the corresponding trans-CA-derived analogs. Moreover, introduction of a substitution at the C-4 position increased the germination-stimulating activity. We also found that the SL analogs derived from cis-CA were able to interact directly with SL receptor proteins more effectively than the analogs derived from trans-CA. The cis isomer of CA was previously reported to have a growth promoting effect on non-parasitic plants such as Arabidopsis. We found that SL analogs derived from cis-CA also showed growth promoting activity toward Arabidopsis, suggesting that these new SL agonists might be useful not only as suicidal germination inducers for root parasitic weeds, but also as plant growth promoters for the host plants.

Features of mandibular cortical bone morphology in osteoporotic fracture patients.

PURPOSE: The diagnostic criteria for osteoporosis are based on the bone mineral density (BMD) level in the lumbar spine and femur bone. Patients with osteoporotic fractures were diagnosed with osteoporosis. While systemic BMD and mandibular cortical bone morphology are correlated, this has not been studied in patients with a history of osteoporotic fractures. Therefore, purpose of this study was researching the mandibular cortical bone morphology in patients with osteoporotic fractures. METHODS: The subjects were 55 female and 20 male patients with osteoporotic fractures. Patients were divided into 30 primary osteoporosis patients and 45 secondary osteoporosis patients according to the medical history. Patients underwent BMD and panoramic radiography examinations during orthopedic treatment for fractures. A dual-energy X-ray absorptiometry system was used to measure BMD. Mandibular cortex index (MCI) and mandibular cortex width (MCW) were evaluated using machine-learning measurement software. RESULTS: In the analysis of MCI, the ratio of class 2 and 3 was 73% of both primary osteoporosis and secondary osteoporosis. The average MCW was 2.19 mm for primary osteoporosis and 2.30 mm for secondary osteoporosis. The sensitivity values by MCI and MCW were 73% and 76% for both primary and secondary osteoporosis, which were similar detection powers. In addition, the false-negative rates by MCI and MCW were 27% and 24%. CONCLUSION: We suggested that MCI and MCW are indicators of osteoporotic conditions in patients with primary and secondary osteoporosis. Our results show that MCI and MCW are non-inferior to the sensitivity values for lumbar BMD in patients with osteoporotic fractures.

A rare case of positional changes of carotid artery depicted within a single MR study and a wandering carotid artery depicted on a serial MR studies.

The retropharyngeal carotid artery (RCA) is a relatively rare anatomical malposition, and positional changes in the RCA is also extremely rare. In addition, there are some reports of "wandering carotid artery" which means that the carotid artery reciprocate positional changes (wandering) between its normal position and retropharyngeal regions, during follow-up evaluations. A male patient in his 50s with a chief complaint of globus pharyngeus. A pulsatile swelling of the posterior pharyngeal wall of the right side was found on clinical examination. In this case, the right carotid artery showed the clinical course of a wandering carotid artery, which reciprocated between its normal position and the retropharyngeal space during three serial MR investigations. Interestingly, both the most recent MR study and the MR study performed 4 years ago showed that the carotid artery at the level of the hyoid bone moved laterally (positional normalization) during the single MR investigation. This is a first case which showed a rare clinical course of a wandering carotid artery on serial follow-up MR studies and positional changes of carotid artery within a single MR study. It is important for clinicians to be aware of these phenomena, in order to avoid fatal and unexpected complications during clinical procedures.

Effect of postoperative radiotherapy for free flap volume changing after tongue reconstruction.

OBJECTIVE: The purpose of this study was to evaluate the effect of postoperative radiotherapy (RT) on temporal volume changes with a cutaneous free flap (CF) and a myocutaneous free flap (MCF). METHODS: The subjects were 24 men and 11 women (mean age, 50.5 ± 15.5 years) with tongue or floor of mouth cancer. Twenty-seven cases of CF and eight cases of MCF were selected. The flap volume change of the reconstructed tongue was calculated using computed tomography (CT) images taken immediately and at one year postoperatively using the DICOM image processing software OsiriX®. RESULTS: The reduction rate in flap volume at one year postoperatively was 82.0 ± 15% in CF without RT, 70.3 ± 26.1% in CF with RT, 88.5 ± 14.7% in MCF without RT, and 99.5 ± 16% in MCF with RT. The MCF volume was significantly higher compared to the CF volume. Although postoperative RT reduced the CF volume by 30%, there was only a slight reduction in the MCF volume. CONCLUSIONS: We evaluated the effect of postoperative RT on volume reduction in 35 cases of the reconstructed tongue with CF and MCF using a computer-assisted volume rendering technique. In this study, the effect of RT on volume reduction was different between the CF and MCF.