Establishing protein expression profiles involved in tooth development using a proteomic approach.

Various growth and transcription factors are involved in tooth development and developmental abnormalities; however, the protein dynamics do not always match the mRNA expression level. Using a proteomic approach, this study comprehensively analyzed protein expression in epithelial and mesenchymal tissues of the tooth germ during development. First molar tooth germs from embryonic day 14 and 16 Crlj:CD1 (ICR) mouse embryos were collected and separated into epithelial and mesenchymal tissues by laser microdissection. Mass spectrometry of the resulting proteins was carried out, and three types of highly expressed proteins [ATP synthase subunit beta (ATP5B), receptor of activated protein C kinase 1 (RACK1), and calreticulin (CALR)] were selected for immunohistochemical analysis. The expression profiles of these proteins were subsequently evaluated during all stages of amelogenesis using the continuously growing incisors of 3-week-old male ICR mice. Interestingly, these three proteins were specifically expressed depending on the stage of amelogenesis. RACK1 was highly expressed in dental epithelial and mesenchymal tissues during the proliferation and differentiation stages of odontogenesis, except for the pigmentation stage, whereas ATP5B and CALR immunoreactivity was weak in the enamel organ during the early stages, but became intense during the maturation and pigmentation stages, although the timing of the increased protein expression was different between the two. Overall, RACK1 plays an important role in maintaining the cell proliferation and differentiation in the apical end of incisors. In contrast, ATP5B and CALR are involved in the transport of minerals and the removal of organic materials as well as matrix deposition for CALR.

Extracellular Phosphate Induces the Expression of Dentin Matrix Protein 1 Through the FGF Receptor in Osteoblasts.

Dentin matrix protein 1 (Dmp1) is an extracellular matrix protein involved in phosphate metabolism and biomineralization, and its expression markedly increases during the maturation of osteoblasts into osteocytes. We previously reported that an increased level of inorganic phosphate (Pi) in media up-regulated the expression of Dmp1 in primary osteocytes isolated from mouse bones. In the present study, we found that elevated extracellular Pi strongly induced the expression of Dmp1 in osteoblasts and explored its underlying mechanism of action. In an osteoblastic cell line MC3T3-E1, increases in extracellular Pi induced the phosphorylation of ERK1/2 and up-regulated the expression of Dmp1, fibroblast growth factor 2 (Fgf2), and Fgf receptor 1 (Fgfr1). A co-treatment with the MEK inhibitor U0126 abolished the increase in the expression of Dmp1 and Fgfr1 by elevated Pi, suggesting the involvement of the MEK/ERK pathway in this up-regulation. Elevated extracellular Pi also resulted in the phosphorylation of FGF receptor substrate 2α (FRS2α), which was diminished by knockdown of Slc20a1 encoding Pit1 sodium-phosphate co-transporter. The co-treatment with an inhibitor against FGFR (SU5402) abolished the up-regulation of Dmp1 induced by elevated extracellular Pi. In primary osteoblasts, a treatment with 4 mM Pi transiently increased the expression of early growth response 1 (Egr1) before the up-regulation of Dmp1. These results indicate that FGFR mediates the direct effects of extracellular Pi on the expression of Dmp1 in osteoblasts and enhance the close relationship between the signaling evoked by elevated extracellular Pi and FGF/FGFR signaling. J. Cell. Biochem. 118: 1151-1163, 2017. © 2016 Wiley Periodicals, Inc.

Glass syndrome derived from chromosomal breakage downstream region of SATB2.

BACKGROUND: Glass syndrome, derived from chromosomal 2q33.1 microdeletions, manifests with intellectual disability, microcephaly, epilepsy, and distinctive features, including micrognathia, down-slanting palpebral fissures, cleft palate, and crowded teeth. Recently, SATB2 located within the deletion region, was identified as the causative gene responsible for Glass syndrome. Numerous disease-causing variants within the SATB2 coding region have been reported. OBJECTIVE: Given the presentation of intellectual disability and multiple congenital anomalies in a patient with a de novo reciprocal translocation between chromosomes 1 and 2, disruption of the causative gene(s) was suspected. This study sought to identify the causative gene in the patient. METHODS: Long-read whole-genome sequencing was performed, and the expression level of the candidate gene was analyzed. RESULTS: The detection of breakpoints was successful. While the breakpoint on chromosome 1 disrupted RNF220, it was not deemed to be a genetic cause. Conversely, SATB2 is located in the approximately 100-kb telomeric region of the breakpoint on chromosome 2. The patient's clinical features resembled those of previously reported cases of Glass syndrome, despite the lack of confirmed reduced SATB2 expression. CONCLUSION: The patient was diagnosed with Glass syndrome due to the similarity in clinical features. This led us to hypothesize that disruption in the downstream region of SATB2 could result in Glass syndrome. The microhomologies identified in the breakpoint junctions indicate a potential molecular mechanism involving microhomology-mediated break-induced repair mechanism or template switching.

Mass spectrometry-based proteomic analysis of proteins adsorbed by hexadecyl-immobilized cellulose bead column for the treatment of dialysis-related amyloidosis.

BACKGROUND: Dialysis-related amyloidosis (DRA) is a severe complication in end-stage kidney disease (ESKD) patients undergoing long-term dialysis treatment, characterized by the deposition of ß2-microglobulin-related amyloids (Aß2M amyloid). To inhibit DRA progression, hexadecyl-immobilized cellulose bead (HICB) columns are employed to adsorb circulating ß2-microglobulin (ß2M). However, it is possible that the HICB also adsorbs other molecules involved in amyloidogenesis. METHODS: We enrolled 14 ESKD patients using HICB columns for DRA treatment; proteins were extracted from HICBs following treatment and identified using liquid chromatography-linked mass spectrometry. We measured the removal rate of these proteins and examined the effect of those molecules on Aß2M amyloid fibril formation in vitro. RESULTS: We identified 200 proteins adsorbed by HICBs. Of these, 21 were also detected in the amyloid deposits in the carpal tunnels of patients with DRA. After passing through the HICB column and hemodialyzer, the serum levels of proteins such as ß2M, lysozyme, angiogenin, complement factor D and matrix Gla protein were reduced. These proteins acted in the Aß2M amyloid fibril formation. CONCLUSIONS: HICBs adsorbed diverse proteins in ESKD patients with DRA, including those detected in amyloid lesions. Direct hemoperfusion utilizing HICBs may play a role in acting Aß2M amyloidogenesis by reducing the amyloid-related proteins.

Rare mosaic variant of GJA1 in a patient with a neurodevelopmental disorder.

GJA1 is the causative gene for oculodentodigital dysplasia (ODDD). A novel de novo GJA1 variant, NM 000165:c263C > T [p.P88L], was identified in a mosaic state in a patient with short stature, seizures, delayed myelination, mild hearing loss, and tooth enamel hypoplasia. Although the patient exhibited severe neurodevelopmental delay, other clinical features of ODDD, including limb anomalies, were mild. This may be due to differences in the mosaic ratios in different organs.

Risk assessment of metachronous squamous cell carcinoma after endoscopic resection for esophageal carcinoma based on the genetic polymorphisms of alcoholdehydrogense-1B aldehyde dehydrogenase-2: temperance reduces the risk.

BACKGROUND: Metachronous multiple squamous cell carcinoma (SCC) of the esophagus and the head and neck is commonly observed in patients who have previously undergone endoscopic resection (ER) for SCC of the esophagus (ESCC). We evaluated the risk for developing metachronous SCC following ER for ESCC based on the genetic polymorphisms for alcohol dehydrogenase-1B (ADH1B) and aldehyde dehydrogenase-2 (ALDH2) as well as the alcohol consumption and smoking habits. METHODS: We studied 158 patients who underwent ER for ESCC (median follow-up 80 months). Genotyping of ADH1B/ALDH2 was performed using saliva sampling. The alcohol consumption and smoking histories of the patients before and after the ER were documented. RESULTS: Multivariate analyses revealed that inactive heterozygous ALDH2 [hazard ratio (HR) 2.25] and alcohol consumption after ER (HR 1.94) were independently associated with the risk of developing secondary SCC. Moreover, inactive heterozygous ALDH2 (HR 4.39) and alcohol consumption after the ER (HR 2.82) were independently associated with the risk of a third SCC. We analyzed 110 patients who had a history of moderate or heavy alcohol consumption before the ER. The 3-year cumulative incidence rates of secondary SCC in the temperance (n = 65) and non-temperance groups (n = 45) were 14.0 and 42.1% (p = 0.0002). Further, the 5-year cumulative incidence rates of a third SCC in the temperance and non-temperance groups were 0 and 15.6% (p = 0.0011), respectively. In addition, the 7-year cumulative incidence rates of a fourth SCC in the temperance and non-temperance groups were 0 and 15.3% (p = 0.0015), respectively. CONCLUSIONS: Continued alcohol consumption is an important risk factor for the onset of metachronous SCC and is a risk factor for the third and subsequent SCCs. Strict advice in favor of temperance is crucial.

Phase I study of pegylated interferon-alpha-2b as an adjuvant therapy in Japanese patients with malignant melanoma.

In the adjuvant setting for malignant melanoma, interferon (IFN)-α-2b and pegylated (PEG) IFN-α-2b were approved in several countries including the USA before these were approved in Japan. To resolve the "drug-lag" issue, this phase I study was designed to evaluate the safety and tolerability in Japanese patients with stage II or III malignant melanoma who had undergone surgery, by treating with PEG IFN-α-2b. As with a previously reported phase III study, patients were to receive PEG IFN-α-2b 6 µg/kg per week s.c. during an 8-week induction phase, followed by a maintenance phase at a dose of 3 µg/kg per week up to 5 years. Dose-limiting toxicity and pharmacokinetics were assessed during the initial 8 weeks. Of the nine patients enrolled, two patients had dose-limiting toxicities that resolved after discontinuation of treatment. The most frequently reported drug-related adverse events (DRAE) included pyrexia, decreased neutrophil and white blood cell counts, and arthralgia. Grade 3 DRAE included decreased neutrophil count. No deaths, serious adverse events and grade 4 adverse events were reported. Distant metastasis occurred in one patient. No apparent differences in area under the concentration-time curve and maximum observed serum concentration were observed between Japanese and historical non-Japanese pharmacokinetic data, suggesting no marked racial differences. No neutralizing antibody was detected in these patient samples. PEG IFN-α-2b was tolerated in Japanese patients, and eventually approved in Japan in May 2015 for adjuvant therapy in patients with stage III malignant melanoma. Because the number of patients was limited, further investigation would be crucial.

Genomic structure and mutational analysis of the human KIF1Balpha gene located at 1p36.2 in neuroblastoma.

KIF1a is a member of the kinesin superfamily proteins that are microtubule-dependent molecular motors involved in important intracellular functions such as organelle transport and cell division. We previously determined the structure of the human KIF1Bbeta gene, which was found to be a homologue of the murine Kif1bbeta, and demonstrated that the human KIF1Bbeta is a causative gene of Charcot-Marie-Tooth disease type 2A although we did not prove that it is a tumor suppressor gene of neuroblastoma. Here, we identified another isoform of the human KIF1B gene, KIF1Balpha. The KIF1Balpha and KIF1Bbeta are alternative splicing products of the KIF1B gene located on 1p36.2. The KIF1Balpha is distinct from KIF1Bbeta in the C-terminal cargo-binding domain; however, they have the same N-terminal motor domain. We found that the transcript of approximately 7.8 kb of KIF1Balpha was expressed in several tissues, especially in skeletal muscle, by Northern blot analysis. To determine whether this gene is one of the candidate tumor suppressor genes for neuroblastoma (NB) or other pediatric solid tumors, we performed mutational screening of KIF1Balpha in 25 NB, 9 rhabdomyosarcoma, 12 Ewing sarcoma and 24 other pediatric solid tumor cell lines. Using RT-PCR single-strand conformation polymorphism analysis and direct sequencing we detected a missense mutation (M807I) in 1 NB cell line (SK-N-SH), 3 silent mutations in 2 NB cell lines and 1 primitive neuroectodermal tumor cell line, respectively. RT-PCR analysis revealed that KIF1Balpha was obviously expressed in almost all of the tumor cell lines examined except NB-1. Furthermore, real-time quantitative RT-PCR showed that there was no significant difference in KIF1Balpha expression between 14 early-stage (stage I and II) and 14 advanced-stage (stage III and IV) NB fresh tumor specimens. These results suggest that KIF1Ba in addition to KIF1Bbeta may not be a candidate tumor suppressor gene for NB.

Physiological and psychological evaluation of the wrapped warm footbath as a complementary nursing therapy to induce relaxation in hospitalized patients with incurable cancer: a pilot study.

BACKGROUND: The wrapped warm footbath (WW-footbath) was originally designed as a means to promote relaxation, positive emotion, comfort, pleasure, and enjoyment in hospitalized patients with incurable stomach cancer during palliative care. OBJECTIVES: This study examined the physiological and psychological effects of the WW-footbath as complementary nursing intervention to induce relaxation in hospitalized patients with incurable cancer. METHODS: Eighteen subjects were divided into a footbath group (n=9) and a control group (n=9). Heart rate variability analysis (per second) with wavelet transformation was used for evaluation of autonomic, sympathetic, and parasympathetic activities. Salivary secretory immunoglobulin A and salivary cortisol levels were used as neuroimmunological parameters. Pain and psychological conditions were measured using a visual analog scale and Face Scale (FS). RESULTS: The high-frequency component of heart rate variability was not significantly increased in either group. However, the low-frequency component versus high-frequency ratio was significantly decreased in the footbath group at periods after footbath (P=.017, P=.000, P=.000, P=.003). In the footbath group, the salivary secretory immunoglobulin A level was significantly increased (P=.019), and the cortisol level tended to be reduced. Changes in visual analog scale and FS scores showed that the footbath group experienced significant pain relief and relaxation (pain, P=.047; relaxation, P=.042; comfortability, P=.008; FS, P=.013). CONCLUSIONS: The WW-footbath significantly decreased sympathetic activity in hospitalized patients with incurable cancer and may have secondary benefit for pain relief. IMPLICATIONS FOR PRACTICE: The WW-footbath can promote relaxation in hospitalized patients with incurable cancer. Moreover, the WW-footbath appears to afford pain relief associated with enhanced overall comfort.

Computed tomography morphology of the mandibular ramus in prognathism: effect on the medial osteotomy of the sagittal split ramus osteotomy.

PURPOSE: The purpose of this study was to investigate the distribution of cancellous bone relative to performance of the medial osteotomy of a sagittal split ramus osteotomy using computed tomography scanning. PATIENTS AND METHODS: Skeletal Class III patients consisted of 11 males and 22 females with a mean age of 23 years. The control patients without dentofacial deformity consisted of 10 males and 20 females with a mean age of 25 years. Scans were taken parallel to the mandibular occlusal plane, and morphologic measurements, including distribution of cancellous bone, were obtained. RESULTS: Compared with the control patients, the mandibular ramus of Class III patients was thinner with a more complicated and varied cancellous bone pattern, such as separate regions of cancellous bone and lack of cancellous bone posterior and anterior to the mandibular foramen. CONCLUSIONS: It was apparent that the safest region in which to establish the medial osteotomy line was just superior and 5 to 6 mm posterior to the lingula, directing the line slightly inferior.