Protein Sensing Device with Multi-Recognition Ability Composed of Self-Organized Glycopeptide Bundle.

We designed and synthesized amphiphilic glycopeptides with glucose or galactose at the C-terminals. We observed the protein-induced structural changes of the amphiphilic glycopeptide assembly in the lipid bilayer membrane using transmission electron microscopy (TEM) and Fourier transform infrared reflection-absorption spectra (FTIR-RAS) measurements. The glycopeptides re-arranged to form a bundle that acted as an ion channel due to the interaction among the target protein and the terminal sugar groups of the glycopeptides. The bundle in the lipid bilayer membrane was fixed on a gold-deposited quartz crystal microbalance (QCM) electrode by the membrane fusion method. The protein-induced re-arrangement of the terminal sugar groups formed a binding site that acted as a receptor, and the re-binding of the target protein to the binding site induced the closing of the channel. We monitored the detection of target proteins by the changes of the electrochemical properties of the membrane. The response current of the membrane induced by the target protein recognition was expressed by an equivalent circuit consisting of resistors and capacitors when a triangular voltage was applied. We used peanut lectin (PNA) and concanavalin A (ConA) as target proteins. The sensing membrane induced by PNA shows the specific response to PNA, and the ConA-induced membrane responded selectively to ConA. Furthermore, PNA-induced sensing membranes showed relatively low recognition ability for lectin from Ricinus Agglutinin (RCA120) and mushroom lectin (ABA), which have galactose binding sites. The protein-induced self-organization formed the spatial arrangement of the sugar chains specific to the binding site of the target protein. These findings demonstrate the possibility of fabricating a sensing device with multi-recognition ability that can recognize proteins even if the structure is unknown, by the protein-induced self-organization process.

Ligand-inducible dimeric antibody for selecting antibodies against a membrane protein based on mammalian cell proliferation.

A method for selecting antibodies against a membrane protein is important for attaining a variety of antibody-based diagnostics and therapies. In this study, we propose a novel system to select specific antibodies against a membrane protein based on mammalian cell proliferation as a readout. The system employs a chimeric membrane protein in which a target membrane protein of interest is fused to the intracellular signaling domain of a cytokine receptor. The chimeric membrane protein transduces a cell proliferation signal through dimerization when co-expressed with a specific single-chain Fv fused with a mutant of FK-binding protein 12 (scFv-Fk) that can be conditionally dimerized by a synthetic ligand AP20187. To demonstrate this system, ErbB2 and gp130 were chosen as the target membrane protein and cytokine receptor, respectively. Consequently, co-expression of the ErbB2/gp130 chimera and ErbB2-specific scFv-Fk rendered the cells proliferative in response to AP20187. The system also allowed selection of high-affinity binders from a mixture composed of dominant low-affinity binders. This system may be extended to affinity maturation of scFvs by modulating AP20187 concentration in the selection process.

Intraoperative molecular assessment for lymph node metastasis in head and neck squamous cell carcinoma using one-step nucleic acid amplification (OSNA) assay.

BACKGROUND: Conventional intraoperative pathological examination for Sentinel node navigation surgery (SNNS) has been controversial. We evaluated the efficacy of one-step nucleic acid amplification (OSNA) assay for intraoperative diagnosis of cervical lymph node (CLN) metastasis compared with histopathological examination in patients with head and neck squamous cell carcinoma (HNSCC). METHODS: A total of 175 CLNs dissected from 56 patients with HNSCC who underwent surgery at Aichi Cancer Center, Kyorin University, Gunma University or Fukushima Medical University, between April 2008 and December 2011 were enrolled. CLN samples were sectioned into four equal pieces, with two of each used for OSNA assay and other histopathological examinations. The diagnostic value of OSNA assay in HNSCC patients in predicting the results of histopathological diagnosis was evaluated using the area under the receiver operating characteristic (AUROC) curve. RESULTS: OSNA assay showed acceptable efficacy in the detection of pathological CLN metastasis (AUROC 0.918, 95 % confidence interval [CI] 0.852-0.984). Regarding the CK19mRNA cutoff value, the optimum cutoff point in HNSCC patients was 131 copies/µl (sensitivity: 82.4, 95 % CI 65.5-93.2; specificity: 99.3, 95 % CI 96.1-100.0; positive likelihood ratio 116.1; negative likelihood ratio 0.2]. CONCLUSIONS: We demonstrated that OSNA assay is useful in intraoperative diagnosis for CLN metastasis in patients with HNSCC. OSNA assay could be applied for SNNS in HNSCC patients.

Calcium-mediated rapid movements defend against herbivorous insects in Mimosa pudica.

Animals possess specialized systems, e.g., neuromuscular systems, to sense the environment and then move their bodies quickly in response. Mimosa pudica, the sensitive plant, moves its leaves within seconds in response to external stimuli; e.g., touch or wounding. However, neither the plant-wide signaling network that triggers these rapid movements nor the physiological roles of the movements themselves have been determined. Here by simultaneous recording of cytosolic Ca2+ and electrical signals, we show that rapid changes in Ca2+ coupled with action and variation potentials trigger rapid movements in wounded M. pudica. Furthermore, pharmacological manipulation of cytosolic Ca2+ dynamics and CRISPR-Cas9 genome editing technology revealed that an immotile M. pudica is more vulnerable to attacks by herbivorous insects. Our findings provide evidence that rapid movements based on propagating Ca2+ and electrical signals protect this plant from insect attacks.

Comparison of lung cancer cell lines representing four histopathological subtypes with gene expression profiling using quantitative real-time PCR.

BACKGROUND: Lung cancers are the most common type of human malignancy and are intractable. Lung cancers are generally classified into four histopathological subtypes: adenocarcinoma (AD), squamous cell carcinoma (SQ), large cell carcinoma (LC), and small cell carcinoma (SC). Molecular biological characterization of these subtypes has been performed mainly using DNA microarrays. In this study, we compared the gene expression profiles of these four subtypes using twelve human lung cancer cell lines and the more reliable quantitative real-time PCR (qPCR). RESULTS: We selected 100 genes from public DNA microarray data and examined them by DNA microarray analysis in eight test cell lines (A549, ABC-1, EBC-1, LK-2, LU65, LU99, STC 1, RERF-LC-MA) and a normal control lung cell line (MRC-9). From this, we extracted 19 candidate genes. We quantified the expression of the 19 genes and a housekeeping gene, GAPDH, with qPCR, using the same eight cell lines plus four additional validation lung cancer cell lines (RERF-LC-MS, LC-1/sq, 86-2, and MS-1-L). Finally, we characterized the four subtypes of lung cancer cell lines using principal component analysis (PCA) of gene expression profiling for 12 of the 19 genes (AMY2A, CDH1, FOXG1, IGSF3, ISL1, MALL, PLAU, RAB25, S100P, SLCO4A1, STMN1, and TGM2). The combined PCA and gene pathway analyses suggested that these genes were related to cell adhesion, growth, and invasion. S100P in AD cells and CDH1 in AD and SQ cells were identified as candidate markers of these lung cancer subtypes based on their upregulation and the results of PCA analysis. Immunohistochemistry for S100P and RAB25 was closely correlated to gene expression. CONCLUSIONS: These results show that the four subtypes, represented by 12 lung cancer cell lines, were well characterized using qPCR and PCA for the 12 genes examined. Certain genes, in particular S100P and CDH1, may be especially important for distinguishing the different subtypes. Our results confirm that qPCR and PCA analysis provide a useful tool for characterizing cancer cell subtypes, and we discuss the possible clinical applications of this approach.

Sentinel node mapping for node positive oral cancer: potential to predict multiple metastasis.

OBJECTIVE: The objective of this study is to evaluate lymph node mapping for clinically positive neck metastasis using a sentinel node navigation technique. METHODS: 99mTc-labeled rhenium sulfide was injected as a radiotracer in 10 patients with squamous cell carcinoma of the tongue. After surgery, lymph nodes were classified into two categories according to the radioactive accumulation: nodes with radioactivity and nodes without radioactivity. The ratio of the metastatic area (RMA) of pathologically metastatic lymph nodes was measured. RESULTS: In 5 of 10 cases, all of the metastatic nodes had radioactive accumulation. In one case with three metastatic nodes, radioactivity was not detected in one metastatic node, although it was detected in the other two nodes. In the other four cases, there were no radioactivities in any of the metastatic nodes. RMA of lymph nodes in which radioactivity was not detected was higher than that of lymph nodes in which radioactivity was detected. None of the nodes in which radioactivity was detected was fully occupied by metastatic carcinoma cells. In each case, in comparing the clinically positive lymph nodes, RMA of the nodes in which no radioactivity was detected was higher than that of the nodes in which radioactivity was detected. CONCLUSION: The principle behind the sentinel node technique is detection of the node that has the most lymph flow from the tumor through injection of the tracer into the circumference of the tumor. When no radioactive accumulation is found in clinically positive metastatic lymph nodes, the possibility of metastasis to other lymph nodes should be highly suspected.

[Marrow-tympanum connections in fetuses and infants].

The connections between hematopoietic bone marrow and the tympanum caused by bony dehiscences in immature middle ear were examined in 58 temporal bones of fetuses and infants. There were no anomalies in any of the cases, and they were divided into two groups: a group with inflammation of middle ear and a group without inflammation. The tympanic cavity was compartmentalized into thirteen portions to investigate the appearance of the connection, and the connections were classified into the mesenchymal type and the direct type dependent on residual mesenchyme in each portion. Marrow-tympanum connections were observed from 20 weeks of gestation until 14 months of age. In most cases the connections were the mesenchymal type. A large amount of mesenchyme remained in the inflammatory group. Some direct-type connections were observed in the non-inflammatory group from 1 month after birth onward and the mesenchyme was completely absorbed in these connections. Both types of the connections were frequently found in the antrum, facial recess and tympanic sinus. These results indicate that a marrow-tympanum connection is usually present in the temporal bone not only anomaly cases but also normal fetuses and infants. In addition, in normal cases without otitis media the connections first appear as mesenchymal type, and progress to direct type due to the absorption of mesenchyme. Presumably marrow-tympanum connections have structural disadvantage to induce osteomyelitis in cases with otitis media. And evoked osteomyelitis may cause complications such as acute mastoiditis and facial paralysis. The results of this study suggest that children under 2 years of age are at higher risk of complications of otitis media owing to marrow-tympanum connections than older children. Further study of the role of mesenchyme in otitis media is needed.

"Cannot ventilate, cannot intubate" situation after penetration of the tongue root through to the epipharynx by a surfboard: a case report.

BACKGROUND: Surfing is an increasingly popular activity and surfing-related injuries have increased accordingly. However, to the best of our knowledge, there are no reports of penetrating upper airway injuries in surfers. We present a "cannot ventilate, cannot intubate" situation following penetrating neck injury by a surfboard fin. CASE PRESENTATION: A previously healthy 29-year-old Japanese man was swept off his board by a large wave and his left mandible, tongue root, and right epipharynx were penetrated by the surfboard fin. He presented with severe hypovolemic shock because of copious bleeding from his mouth. Direct laryngoscopy failed, as did manual ventilation, because of the exacerbated upper airway bleeding and distorted upper airway anatomy. Open cricothyrotomy was immediately performed, followed by surgical exploration, which revealed extensive ablation of his tongue root and laceration of his lingual artery. After definitive hemostasis and intensive care, he returned home with no sequelae. CONCLUSIONS: The long, semi-sharp surfboard fin created both extensive crushing upper airway lesions and a sharp vascular injury, resulting in a difficult airway. This case illustrates that surfing injuries can prompt a life-threatening airway emergency and serves as a caution for both surfers and health care professionals.

Sequence analysis of canine and equine ferritin H and L subunit cDNAs.

Canine and equine ferritin H and L subunit cDNA clones were obtained using reverse transcriptase-polymerase chain reaction (RT-PCR) and TA cloning from various tissues. Canine liver and spleen ferritin H subunit cDNA clones contained an open reading frame for the same 182-amino acid protein as that reported in canine brain ferritin H subunit cDNA although there were substitutions in the 3'-noncoding regions. Ferritin L subunit cDNA clones from canine liver, spleen, and kidney showed identical coding sequences encoding the 174-amino acid protein except for a single nucleotide substitution in kidney (C474G). The H subunit nucleotide sequences of equine leukocyte and spleen were identical to the fragment encoding the 181-amino acid protein in equine peripheral blood mononuclear cells, with the exception of one substitution seen in both leukocyte and spleen sequences (C234T). The nucleotide sequence of equine leukocyte ferritin L subunit showed 7 substitutions compared with the published equine liver L subunit sequence with two substitutions at positions 281 and 282 resulting in an amino acid substitution of P94L. The amino acid residues involved in the ferroxidase center and in iron nucleation were perfectly conserved in H and L subunits of canine and equine ferritins, respectively.

Impact of sentinel node navigation technique for carcinoma of tongue with cervical node metastases.

We attempted lymph node mapping for clinically positive neck using sentinel node navigation technique. Technetium labeled rhenium sulfide was injected as a radiotracer in 11 patients with squamous cell carcinoma of the tongue. After surgery, the radioactivity and the ratio of metastatic area (RMA) of the removed nodes were measured. Average RMA (57%) of 18 high radioactive metastatic nodes was significantly lower than the RMA (90%) of 16 low radioactive metastatic nodes. Average number of metastatic nodes (4.7 nodes) in the five cases with low radioactive metastatic nodes was significantly larger than that (1.8 nodes) in the six cases with only high radioactive metastatic nodes. There is no accumulation of radioactive tracer if a lymph node is totally or predominantly occupied by metastatic cells. When the sentinel node was mostly occupied by malignant cells, the injected colloid could not flow to the sentinel node and flowed to a different lymph node through another basin. Sentinel node navigation technique can show the actual time of lymphatic flow at the operation of positive neck cases.