Awareness of children's developmental problems and sharing of concerns with parents by preschool teachers and childcare workers: The Japanese context.

BACKGROUND: This study aims to determine the extent to which preschool teachers and childcare workers are aware of the presence of developmental problems among children and to what extent they share information with parents about their concerns regarding a child's development or diagnosis of neurodevelopmental disorders (NDDs). METHODS: We wrote to all 924 preschools and childcare centres in Japan's Nagano and Yamanashi prefectures to request participants. We then sent survey forms to the preschools and childcare centres that agreed to cooperate for three grades comprising 3-, 4- and 5-year-olds in the school year 2020. We asked the staff member in charge of each child to complete the survey. The survey included questions about the teacher's concerns regarding the possibility of an NDD and whether the matter had been shared with the children's parents. RESULTS: We obtained data for 10 354 children from 206 preschools and childcare centres (response rate = 22.3%). Among these children, 457 (4.4%) had an NDD diagnosis that their parents shared with the teachers. However, the teachers of 1274 children (12.3%) had concerns regarding their development but were not informed by the parents about the diagnosis, if any. These 1274 children included 775 (60.8%) cases where the teachers failed to share their concerns with parents because (1) the teachers could not communicate with parents (n = 119), (2) the teachers were not sure if there was a neurodevelopmental problem (n = 360) and (3) the parents were not aware of the problem (n = 296). CONCLUSIONS: Preschool teachers and childcare workers had concerns about the development of a substantial proportion of children in their charge. However, teachers and childcare workers did not share their concerns regarding many children's developmental problems with their parents. The findings suggest that there are challenges in information-sharing between teachers/childcare workers and parents.

[A case of oldest-old patient with chronic renal failure successfully treated with peritoneal dialysis].

He was a 92-year-old male patient with mild cognitive impairment while preserved activity of daily life. His renal dysfunction gradually increased due to the nephrosclerosis accompanied by systemic edema and water retention. We eventually decided to initiate peritoneal dialysis instead of standard hemodialysis for his end-stage renal dysfunction refractory to optimal medical therapy, given his preserved cognitive function and family support. He underwent an established therapeutic program for the peritoneal dialysis at home with an Information and Communication Technology (ICT).Given the increase in age of the patients with renal dysfunction, peritoneal dialysis has been receiving great attention as a home care strategy. The recent improvement in the device technology and ICT that enables us remote monitoring would reduce patients' effort in the management of peritoneal dialysis. Collaboration with home nursing and care workers would also be warranted for successful home care.

Generation of a novel artificial TrkB agonist, BM17d99, using T7 phage-displayed random peptide libraries.

Tropomyosin receptor kinase B (TrkB) is a known receptor of brain-derived neurotrophic factor (BDNF). Because it plays a critical role in the regulation of neuronal development, maturation, survival, etc., TrkB is a good target for drugs against central nervous system diseases. In this study, we aimed to generate peptidic TrkB agonists by applying random peptide phage display technology. After the phage panning against recombinant Fc-fused TrkB (TrkB-Fc), agonistic phages were directly screened against TrkB-expressing HEK293 cells. Through subsequent screening of the first-hit BM17 peptide-derived focus library, we successfully obtained the BM17d99 peptide, which had no sequence similarity with BDNF but had TrkB-binding capacity. We then synthesized a dimeric BM17d99 analog peptide that could phosphorylate or activate TrkB by facilitating receptor homodimerization. Treatment of TrkB-expressing HEK293 cells with the dimeric BM17d99 analog peptide significantly induced the phosphorylation of TrkB, suggesting that homodimerization of TrkB was enhanced by the dimeric peptide. This report demonstrates that our approach is useful for the generation of artificial peptidic agonists of cell surface receptors.

A potent neuromedin U receptor 2-selective alkylated peptide.

Neuromedin U (NMU) mediates various physiological functions via NMUR1 and NMUR2 receptors. NMUR2 has been considered a promising treatment option for diabetes and obesity. Although NMU-8, a shorter peptide, has potent agonist activity for both receptors, it is metabolically unstable. Therefore, NMU-8 analogs modified with long-chain alkyl moieties via a linker were synthesized. An octadecanoyl analog (17) with amino acid substitutions [αMePhe19, Nle21, and Arg(Me)24] and a linker [Tra-γGlu-PEG(2)] dramatically increased NMUR2 selectivity, with retention of high agonist activity. Subcutaneous administration of 17 induced anorectic activity in C57BL/6J mice. Owing to its high metabolic stability, 17 would be useful in clarifying the physiological role and therapeutic application of NMU.

A potent and selective natriuretic peptide receptor-3 blocker 11-mer peptide created by hybridization of musclin and atrial natriuretic peptide.

The natriuretic peptide (NP) system is a critical endocrine, autocrine, and paracrine system and has been investigated for potential use against cardiovascular and metabolic diseases. The clearance of NPs is regulated by the proteolysis of neutral endopeptidase (NEP) and by endocytosis via natriuretic peptide receptor-3 (NPR3). A linear NPR3-selective peptide, [Cha8]-ANP(7-16)-NH2 (1), showed potent binding affinity for NPR3 but poor predicted chemical stability due to its free thiol group. A 12-mer peptide (9) without a thiol group was designed by the hybridization of two NPR3-binding peptides: a linear ANP fragment peptide analog and musclin, a murine member of the bHLH family of transcription factors, possessed high binding affinity and strict selectivity for NPR3. To increase the proteolytic resistance of 9, amino acid substitutions at the cleavage sites led to hydroxyacetyl-[d-Phe5,d-Hyp7,Cha8,d-Ser9,Hyp11,Arg(Me)14]-ANP(5-15)-NHCH3 (23), showing high and selective binding affinity for NPR3 over NPR1 and excellent stability in mouse serum. Compound 23 increased intracellular cGMP concentrations in primary cultured adipocytes, and continuous administration induced substantial plasma cGMP elevation in mice, suggesting its potential to clarify the physiological role of NPR3 and its therapeutic application.

Anti-proliferative effect of Fe(III) complexed with 1-(2-hydroxy-3-methoxybenzaldehyde)-4-aminosalicylhydrazone in HepG2 cells.

We previously developed a chelating ligand, 1-(2-hydroxy-3-methoxybenzaldehyde)-4-aminosalicylhydrazone (HMB-ASH), which can chelate Fe(III) to form a complex. The HMB-ASH-Fe(III) complex exhibits a dose-dependent anti-proliferative effect in HepG2 cells, whereas the ligand, HMB-ASH, and Fe(III) alone had no considerable effect. The HMB-ASH-Fe(III) complex was composed of Fe(III):HMB-ASH (1:2), as determined by high-performance liquid chromatography with high-resolution mass spectrometry. The IC50 value was approximately 20 µM, which was comparable to those of the anti-cancer drugs oxaliplatin (OXP) and etoposide (ETP) under the same conditions. Similar to OXP and ETP, HMB-ASH-Fe(III) induced apoptosis in HepG2 cells, as revealed by terminal deoxynucleotidyl transferase fluorescein-12-dUTP nick end labeling assay.

Three school-age cases of xeroderma pigmentosum variant type.

BACKGROUND: Xeroderma pigmentosum (XP) is a photosensitive genodermatosis with increased susceptibility to skin cancers. Patients are typically diagnosed with XP when they consult a dermatologist for skin cancers. CASE/METHODS: The genetic analysis and 2-8 years of follow-up for three school-age patients with XP-V is described. The patients were referred to us because of increased pigmented freckles; they had not experienced abnormal sunburn or developed skin cancer at their first visit. All patients harbored a genetic mutation in the POLH gene. XPV9KO was diagnosed at age 13 with a homozygous del1661A that creates a stop codon in the non-catalytic domain of POLH. The patient practiced sun protection, effectively preventing the development of skin cancer by age 21. XPV19KO was diagnosed at age 11 with a compound heterozygous mutation of G490T and C1066T, causing POLH truncation in the catalytic domain. This patient developed basal cell carcinoma at ages 12 and 13. XPV18KO was referred to us at age 11 and diagnosed with compound heterozygous variants of c.1246_1311del66 (exon 9 skipping), a novel mutation, and c.661_764 del104 (exon 6 skipping). CONCLUSION: Freckle-like pigmentation on sun-exposed skin is sometimes the only sign of XP-V, and early diagnosis is extremely important for children.

Acute Kidney Injury with Severe Metabolic Alkalosis Caused by Habitual Vomiting in an Alcohol Abuser with Pyloric Stenosis.

A 47-year-old man was complaining of consciousness disorder. He had acute kidney injury, hypokalemia, and severe metabolic alkalosis. Initial treatment using intravenous infusion of 0.9% saline and potassium chloride improved his consciousness. It was clarified that he was a severe alcohol abuser who habitually self-vomited. We diagnosed him with volume depletion and pseudo-Bartter's syndrome due to loss of chloride by habitual vomiting. Gastrointestinal endoscopy demonstrated pyloric stenosis, which was ameliorated by Helicobacter pylori eradication therapy. We should consider volume depletion and pseudo-Bartter's syndrome as differential diagnoses when we encounter patients with acute kidney injury and severe metabolic alkalosis.

Pituicytoma with pleomorphism: A case report with cytological findings.

Pituicytoma is a rare neoplasm, arising in the posterior pituitary or in the hypophyseal stalk, and its cytological findings have not yet been well-described. We have experienced a case of pituicytoma, which was difficult to diagnose intraoperatively, because of its cellular pleomorphism. A tumor measuring 18 mm in maximum diameter was found at the sella turcica in a Japanese woman in her forties. Both intraoperative crush cytology and histology of the resected tumor showed pleomorphic spindle or round cells, including multinucleated cells. Tumor cells were positive for TTF-1, S-100 protein, and vimentin, partially positive for glial fibrillary acidic protein and epithelial membrane antigen, and negative for synaptophysin, hormones of the anterior pituitary gland, CD34, Olig2, PAX8, and napsin A. Ki-67 labeling index was 2.0%. Tumors included in the differential diagnosis in general are pituitary adenoma, craniopharyngioma, germinoma, and metastatic tumor on the radiological standpoint, and pilocytic astrocytoma and meningioma on the cytological standpoint. However, our case was difficult to differentiate especially from high-grade glioma only by morphology, and immunohistochemistry including TTF-1 was helpful.

Loss of myeloid related protein-8/14 exacerbates cardiac allograft rejection.

BACKGROUND: The calcium-binding proteins myeloid-related protein (MRP)-8 (S100A8) and MRP-14 (S100A9) form MRP-8/14 heterodimers (S100A8/A9, calprotectin) that regulate myeloid cell function and inflammatory responses and serve as early serum markers for monitoring acute allograft rejection. Despite functioning as a proinflammatory mediator, the pathophysiological role of MRP-8/14 complexes in cardiovascular disease is incompletely defined. This study investigated the role of MRP-8/14 in cardiac allograft rejection using MRP-14(-/-) mice that lack MRP-8/14 complexes. METHODS AND RESULTS: We examined parenchymal rejection after major histocompatibility complex class II allomismatched cardiac transplantation (bm12 donor heart and B6 recipients) in wild-type (WT) and MRP-14(-/-) recipients. Allograft survival averaged 5.9±2.9 weeks (n=10) in MRP-14(-/-) recipients compared with >12 weeks (n=15; P<0.0001) in WT recipients. Two weeks after transplantation, allografts in MRP-14(-/-) recipients had significantly higher parenchymal rejection scores (2.8±0.8; n=8) than did WT recipients (0.8±0.8; n=12; P<0.0001). Compared with WT recipients, allografts in MRP-14(-/-) recipients had significantly increased T-cell and macrophage infiltration and increased mRNA levels of interferon-γ and interferon-γ-associated chemokines (CXCL9, CXCL10, and CXCL11), interleukin-6, and interleukin-17 with significantly higher levels of Th17 cells. MRP-14(-/-) recipients also had significantly more lymphocytes in the adjacent para-aortic lymph nodes than did WT recipients (cells per lymph node: 23.7±0.7×10(5) for MRP-14(-/-) versus 6.0±0.2×10(5) for WT; P<0.0001). The dendritic cells (DCs) of the MRP-14(-/-) recipients of bm12 hearts expressed significantly higher levels of the costimulatory molecules CD80 and CD86 than did those of WT recipients 2 weeks after transplantation. Mixed leukocyte reactions with allo-endothelial cell-primed MRP-14(-/-) DCs resulted in significantly higher antigen-presenting function than reactions using WT DCs. Ovalbumin-primed MRP-14(-/-) DCs augmented proliferation of OT-II (ovalbumin-specific T cell receptor transgenic) CD4(+) T cells with increased interleukin-2 and interferon-γ production. Cardiac allografts of B6 major histocompatibility complex class II(-/-) hosts and of B6 WT hosts receiving MRP-14(-/-) DCs had significantly augmented inflammatory cell infiltration and accelerated allograft rejection compared with WT DCs from transferred recipient allografts. Bone marrow-derived MRP-14(-/-) DCs infected with MRP-8 and MRP-14 retroviral vectors showed significantly decreased CD80 and CD86 expression compared with controls, indicating that MRP-8/14 regulates B7-costimulatory molecule expression. CONCLUSIONS: Our results indicate that MRP-14 regulates B7 molecule expression and reduces antigen presentation by DCs and subsequent T-cell priming. The absence of MRP-14 markedly increased T-cell activation and exacerbated allograft rejection, indicating a previously unrecognized role for MRP-14 in immune cell biology.

Severe anaphylaxis caused by intravenous anti-cancer drugs.

BACKGROUND: The incidence and risk factors of severe anaphylaxis by intravenous anti-cancer drugs are unclear, whereas those of milder reactions have been reported. STUDY DESIGN: Electronic medical charts of cancer patients who have undergone intravenous chemotherapy between January 2013 and October 2020 in a university hospital were retrospectively reviewed. Non-epithelial malignancies were also included in the analysis. "Severe anaphylaxis" was judged using Brown's criteria: typical presentation of anaphylaxis and one or more of hypoxia, shock, and neurologic compromise. (UMIN000042887). RESULTS: Among 5584 patients (2964 males [53.1%], 2620 females [46.9%], median age 66 years), 88,200 person-day anti-cancer drug administrations were performed intravenously, and 27 severe anaphylaxes were observed. The causative drugs included carboplatin (14 cases), paclitaxel (9 cases), and cisplatin, docetaxel, trastuzumab, and cetuximab (1 case each). The person-based lifetime incidence of severe anaphylaxis for patients who received at least one intravenous chemotherapy was 0.48% (27/5584, 95% confidence interval (CI) 0.30%-0.67%) and the administration-based incidence was 0.031% (27/88,200, 95% CI 0.019%-0.043%). Among 124 patients who received at least 10 carboplatin administrations, 10 patients experienced carboplatin-induced severe anaphylaxis (10/124, 8.1%, 95% CI 3.0%-13.1%). Carboplatin caused severe anaphylaxis after at least 9-min interval since the drip started. Thirteen out of 14 patients experienced carboplatin-induced severe anaphylaxis within a 75-day interval from the previous treatment. Paclitaxel infusion caused severe anaphylaxis after a median of 5 min after the first drip of the day at a life-long incidence of 0.93% (9/968, 95% CI 0.27%-1.59%). CONCLUSION: We elucidated the high-risk settings of chemotherapy-induced severe anaphylaxis.

Genital abnormalities in Pallister-Hall syndrome: Report of two patients and review of the literature.

We describe two patients with Pallister-Hall syndrome (PHS) with genital abnormalities: a female with hydrometrocolpos secondary to vaginal atresia and a male with micropenis, hypoplastic scrotum, and bilateral cryptorchidism. Nonsense mutations in GLI3 were identified in both patients. Clinical and molecular findings of 12 previously reported patients who had GLI3 mutations and genital abnormalities were reviewed. Genital features in the male patients included hypospadias, micropenis, and bifid or hypoplastic scrotum, whereas all the females had hydrometrocolpos and/or vaginal atresia. No hotspot for GLI3 mutations has been found. The urogenital and anorectal abnormalities associated with PHS might be related to dysregulation of SHH signaling caused by GLI3 mutations rather than hormonal aberrations. We recommend that clinical investigations of genital abnormalities are considered in patients with PHS, even those without hypopituitarism.

Prevalence of Salmonella in green anoles (Anolis Carolinensis), an invasive alien species in Naha and Tomigusuku Cities, Okinawa Main Island, Japan.

Here, we investigated the prevalence of Salmonella enterica, with and without resistance to 17 common antimicrobial agents, in 706 green anoles (Anolis carolinensis) that were collected in Naha and Tomigusuku Cities, Okinawa Main Island, Japan, between 2009 and 2014. Salmonella strains, including S. enterica Weltevreden and Enteritidis serovars, were identified in the large intestinal content samples extracted from 15 (2.1%) of the analyzed green anoles. No antimicrobial resistance was detected. Thus, the present study demonstrates that although the prevalence of Salmonella and the risk of its transmission from the green anoles to humans or other animals on Okinawa Main Island are relatively low, the green anole population nevertheless represents a potential source of Salmonella infection that could affect human health in this region.

Gamified Mobile Computerized Cognitive Behavioral Therapy for Japanese University Students With Depressive Symptoms: Protocol for a Randomized Controlled Trial.

BACKGROUND: Evidence shows that computerized self-help interventions are effective for reducing symptoms of depression. One such intervention, SPARX, is a gamified mobile computerized cognitive behavioral therapy (cCBT) developed for adolescents in New Zealand, which was shown to be as effective as usual care for young people with mild-to-moderate symptoms of depression. However, gamified cCBT has not yet been tested in Japan. OBJECTIVE: This trial is designed to investigate whether a Japanese-adapted version of SPARX improves depressive symptoms in Japanese university students with mild-to-moderate depressive symptoms. METHODS: In this 7-week, multicenter, stratified, parallel-group, superiority randomized trial, participants will be allocated to either a treatment condition (SPARX) or a wait-list control condition. SPARX is a fully automated program, which will be delivered to the mobile phone or tablet device of the participants. SPARX is designed as an interactive fantasy game to guide the user through seven modules that teach key CBT strategies. All participants will be recruited from universities via advertisements on online bulletin boards, the campus newspaper, and posters. Participants in the treatment condition will use the SPARX program weekly. The primary outcome is the reduction of depressive symptoms (using Patient Health Questionnaires-9) measured at baseline and weekly: once after the 7-week intervention and once at a 1-month follow-up. Secondary outcomes include satisfaction with the program and satisfaction with life, measured by the Satisfaction With Life Scale; positive and negative moods, measured by the Profile of Mood States Second Edition; social functioning, measured by the EuroQol Instrument; rumination, measured by the Ruminative Responses Scale; and coping, measured by the Brief Coping Orientation to Problem Experienced Inventory. RESULTS: This study received funding from The Research Institute of Personalized Health Sciences, Health Sciences University of Hokkaido, and obtained institutional review board approval in September 2019. Data collection began in April 2019. CONCLUSIONS: Results of this trial may provide further evidence for the efficacy of gamified cCBT for the treatment of depression and, specifically, provide support for using SPARX with Japanese university students. TRIAL REGISTRATION: Japan Primary Registries Network UMIN000034354; https://tinyurl.com/uu7xd77. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/15164.

Sex steroid hormone receptors in bladder cancer: Usefulness in differential diagnosis and implications in histogenesis of bladder cancer.

OBJECTIVE: In rare cases, differential diagnosis between bladder cancer (BC) and gynecological tract cancer (GTC) is difficult because of anatomical proximity and morphological similarity. We analyzed expression status of sex steroid hormone receptors in BC in this study. First, we investigated their usefulness as a histological marker for differential diagnosis. Second, we considered their roles in BC histogenesis. METHODS: Estrogen receptor α (ERα) and progesterone receptor (PgR) expression was investigated by immunohistochemistry in 125 BCs obtained by transurethral resection or biopsy, then in nonneoplastic background mucosa (trigone, fundus, and dome) of 33 total cystectomy samples. They were evaluated as positive when ≥ 1% of 500 subject cells were immunoreactive with moderate or strong intensities. RESULTS: ERα and PgR were positive in 38.4% and 3.2% of BCs, respectively, suggesting that ERα status alone could not definitely differentiate between BC and GTC. ERα expression was not significantly associated with age and sex of BC patients and histopathology of BCs. Although not significant, ERα expression was more frequent in higher grade (G1/G2 vs. G3/G4; P = 0.143) and marginally associated with advanced stage of BCs (pTis/pTa/pT1 vs. pT2/pT3, P = 0.056). ERα expression was significantly more frequent in background mucosa with ERα-positive BC (In the epithelium and stroma; both P < 0.001). ERα expression was continuously observed from normal to malignant epithelium in some cases. Although not significant, Brunn's nest or cystitis glandularis was more frequent in background mucosa with ERα-positive BC (P = 0.218). Analyses of nonneoplastic mucosa in cystectomy revealed that ERα was more frequently positive in urothelium of trigone, a predilection site for cystitis glandularis, than those of fundus and dome, with a significant difference between trigone and dome (P = 0.034). These data suggest that chronic inflammation may up-regulate ERα in the background epithelium, especially in trigone, and ERα expression in BC might be the reflection of bladder epithelium from which BC arose. CONCLUSIONS: Usefulness of ERα was limited in differential diagnosis between BC and GTC. ERα up-regulation might not play a critical role in the development of BC because it was already noted in the background bladder mucosa.

A new melanoma antigen fatty acid-binding protein 7, involved in proliferation and invasion, is a potential target for immunotherapy and molecular target therapy.

The identification of molecules that are preferentially expressed in melanoma cells and involved in their malignant phenotypes is important for understanding melanoma biology and the development of new diagnostic and therapeutic methods. By comparing the expression profile of a melanoma cell line with those of various normal tissues using GeneChip and by confirming the actual expression of the selected genes by reverse transcription-PCR and Northern and Western blot analyses, fatty acid-binding protein 7 (FABP7), which is frequently expressed in melanomas, was identified. Immunohistochemical examination revealed that FABP7 was expressed in 11 of 15 melanoma tissues. By down-regulating the FABP7 expression with FABP7-specific small interfering RNAs, in vitro cell proliferation and Matrigel invasion were suppressed in two of six melanoma cell lines. Overexpression of FABP7 in a FABP7-negative embryonic kidney cell line 293T by transfecting with the FABP7 cDNA resulted in enhanced cell proliferation and Matrigel invasion, indicating that FABP7 plays a role in the malignant phenotype of some melanoma cell lines. IgG antibodies specific for the phage or bacterial recombinant FABP7 protein were detected in 14 of 25 (56%) or in 8 of 31 (26%) sera from melanoma patients, respectively, but not in sera from healthy individuals, indicating that FABP7 is an immunogenic antigen in melanoma patients. These results showed that FABP7 is frequently expressed in melanoma, may be involved in cell proliferation and invasion, and may be a potential target for development of diagnostic and therapeutic methods.

[Usefulness of transesophageal echocardiography to evaluate the completeness of pediatric patent ductus arteriosus ligation].

Transesophageal echocardiography (TEE) is being utilized with increasing frequency in the operating room both as a monitor and as a diagnostic tool, and its usefulness for determining the adequacy of congenital heart disease repair intraoperatively and postoperatively has been addressed previously. This report describes TEE monitoring in a 4-month-old baby undergoing patent ductus arteriosus (PDA) re-operation. After application of a first double ligation to the ductus via thoracotomy, several investigations in intensive care unit including chest X-ray, transthoracic echocardiography and TEE, detected incomplete ligation, and reoperation was scheduled immediately. Re-operation, a clipping to the ductus, was successfully performed under real-time TEE monitoring. We conclude that the use of intraoperative TEE monitoring during a PDA ligation enables us to avoid residual ductal flows.

Relationship between colors of carious dentin and laser fluorescence evaluations in caries diagnosis.

This in vitro study investigated the relationship between assessments of dentin caries using a laser fluorescence device (DIAGNOdent) and a caries detector dye during caries removal. The dentin of eight extracted carious molars was removed at 300-microm interval points from the dentin surface toward the pulp chamber. Before and after each removal, images of the carious surfaces were taken in association with color-matching stickers (for color correction) and the surfaces were evaluated by DIAGNOdent based on fluorescence emission from the tooth surface. For the L* values (CIE 1976 L*a*b* color system), there was a strong negative correlation between DIAGNOdent results and the corrected L* values of the carious surfaces (Pearson's correlation coefficient: -0.853); additionally, there was a significant correlation between them (p<0.05). However, there were no significant correlations between the DIAGNOdent results and the corrected a* and b* values of the carious surfaces (Pearson's correlation coefficients: 0.108 and 0.018 respectively). In conclusion, DIAGNOdent was shown to be applicable for caries diagnosis during caries removal.

Inhibition of growth and invasive ability of melanoma by inactivation of mutated BRAF with lentivirus-mediated RNA interference.

Oncogenic mutations of molecules involved in the mitogen-activated protein kinase (MAPK) pathways provide signals mediating both tumor growth and invasion in various cancers including melanomas. BRAF somatic mutations, found in 66% of melanomas, have NIH3T3 transforming ability with the elevated kinase activity in vitro. We attempted to mediate RNA interference (RNAi) with HIV lentiviral vectors specific for either wild type or the most frequently mutated form of BRAF (V599E) in 10 melanoma cell lines, and found that RNAi inhibited the growth of most melanoma cell lines in vitro as well as in vivo, which was accompanied by decrease of both BRAF protein and ERK phosphorylation. Interestingly, the mutated BRAF (V599E)-specific siRNA inhibited the growth and MAPK activity of only melanoma cell lines with this mutation. Furthermore, BRAF RNAi inhibited matrigel invasion of melanoma cells accompanied with a decrease of matrix metalloproteinase activity and beta(1) integrin expression. These results clarify that the mutated BRAF (V599E) is essentially involved in malignant phenotype of melanoma cells through the MAPK activation and is an attractive molecular target for melanoma treatment. The lentivirus-mediated RNAi specific for oncogenic mutations may be a powerful technique for gene therapy of cancer.

Three-dimensional evaluation of gap formation of cervical restorations.

OBJECTIVES: In some studies gap formation has been evaluated in just one section of the restorative. This in vitro study aimed to design a quantitative three-dimensional method for evaluation of the contraction gap in restoratives. METHODS: Cervical cavities were prepared on buccal, palatal or lingual surfaces in human extracted molars and were then filled with resin composites. Specimens were reduced every 100 microm in a direction parallel to the tooth axis, and perpendicular to the cavity floor from one proximal side to the other. The sequence of reducing the sections by 100 microm, image taking (250x) and observation of these images (maximal 2500x) were repeated. Three-dimensional images of the contraction gap were made using analytical software and the proportions of the interface with gap formation calculated. RESULTS: The mean proportions of the interface with gap formation of the self-etching system (Clearfil Liner Bond II Sigma) was 41.7+/-6.3% and that of the self-priming system (Single Bond) was 38.2+/-3.9%; there was no significant difference. CONCLUSIONS: Approximate three-dimensional images of the in vitro contraction gap could be drawn and the mean proportions of the interface with gap formation could be more precisely calculated than by previous methods.