Nuclear factor 1 X-type-associated regulation of myogenesis in developing mouse tongue.

OBJECTIVES: The tongue contains skeletal myofibers that differ from those in the trunk, limbs, and other orofacial muscles. However, the molecular basis of myogenic differentiation in the tongue muscles remains unclear. In this study, we conducted comprehensive gene expression profiling of the developing murine tongue. METHODS: Tongue primordia were dissected from mouse embryos at embryonic day (E)10.5-E18.5, while myogenic markers were detected via microarray analysis and quantitative polymerase chain reaction (PCR). In addition to common myogenic regulatory factors such as Myf5, MyoD, myogenin, and Mrf4, we focused on Nfix, which acts as a unique molecular switch triggering the shift from embryonic to fetal myoblast lineage during limb myogenesis. Nfix inhibition was performed using a specific antisense oligonucleotide in the organ culture of tongue primordia. RESULTS: Microarray and ingenuity pathway analyses confirmed the significant upregulation of myogenic signaling molecules, including Nfix, associated with the differentiation of myoblasts from myogenic progenitor cells during E10.5-E11.5. Quantitative PCR confirmed that Nfix expression started at E10.5 and peaked at E14.5. Fetal myoblast-specific genes, such as Mck and Myh8, were upregulated after E14.5, whereas embryonic myoblast-specific genes, such as Myh3 and Myh7, were downregulated. When Nfix was inhibited in the organ culture of tongue primordia, subtle morphological differences were noted in the tongue. Such an observation was only noted in the cultures of E10.5-derived tongue primordia. CONCLUSIONS: These results reveal the contribution of Nfix to tongue myogenesis. Nfix expression during early tongue development may play a vital role in tongue muscle development.

An investigation into nutritional methods at the fifth day after birth of infants in association with cleft type and laterality.

In patients with clefts, the affection of other congenital malformations on the feeding is unclear. We investigated the other congenital malformations and nutritional intake of neonates with cleft lip and/or palate and examined their relationships associated with cleft type and laterality. The participants included 126 infants under treatment with a presurgical naso-alveolar molding (PNAM) or a Hotz-type plate. The survey items were gender, cleft type and side, presence and nature of other congenital malformations, birth weight and nutritional method at age of the fifth day. The number of infants was 36 (28.6%) of cleft lip and alveolus, 82 (65.1%) of cleft lip and palate, and 8 (6.3%) of cleft palate only. Forty-three patients (34.1%) had other various congenital malformations. The nutritional method included oral intake in 78.6% (n = 99) of cases and tube feeding with/without oral intake in 21.4% (n = 27) of cases. The rate of tube feeding was higher for right-sided clefts than that for left-sided clefts. This observation was consistent with the fact that right-sided clefts were associated with more significant other congenital malformations than those on the left-side. The nutritional method for infants with cleft lip and/or palate was related to the presence of other congenital malformations, not to cleft laterality or oral cleft itself under early treatment with PNAM plate. These results proposed that screening the general condition is essential for neonates with right-sided cleft lip with/without cleft palate compared to left-sided clefts, which should be conducted immediately after birth for planning the appropriate nutritional method.

Early response to erythropoiesis-stimulating agents in non-dialysis chronic kidney disease patients.

BACKGROUND: Renal anemia complicated with chronic kidney disease is usually treated with erythropoiesis-stimulating agents (ESAs). However, few studies have compared the early response of hemoglobin (Hb) to different kinds of ESAs. METHODS: The effects of three types of ESAs-epoetin alfa or beta (EPO), darbepoetin alfa (DPO), and epoetin beta pegol (EPObp)-on renal anemia were followed in 416 pre-dialysis chronic kidney disease (CKD) patients. After the initial 12-week administration of ESAs, ΔHb/ESA dose/kg was calculated as an index of efficacy of each ESA. Furthermore, independent variables associated with ΔHb/ESA dose/kg (dependent variable) were determined using multiple linear regression analysis. The ten independent variables selected for analysis were: presence of diabetic nephropathy, estimated glomerular filtration rate (eGFR), Hb, albumin, iron (Fe), transferrin saturation (TSAT), ferritin, phosphate (P), intact parathyroid hormone (iPTH), and C-reactive protein. RESULTS: The efficacy of DPO and EPObp were similar and higher than EPO. TSAT was most strongly correlated with ΔHb/EPO dose/kg in all three types of ESAs. Other significant independent factors were Hb, albumin, P, iPTH, and diabetic nephropathy in the EPO group, eGFR in the DPO group, and Fe in the EPObp group. The adjusted coefficient of determination (R (2)) ranged from 0.415 to 0.520 in the three ESA groups. CONCLUSIONS: The study results suggest that TSAT is the best predictor of the initial 12-week responsiveness to ESA, irrespective of the type. Variables not investigated in this study also affect responsiveness to ESA in Japanese pre-dialysis CKD patients.

Effects of Cardiovascular Events on End-Stage Renal Disease and Mortality in Patients With Chronic Kidney Disease Before Dialysis.

Cardiovascular events (CVEs) are major complications in patients with chronic kidney disease (CKD). However, few studies have investigated the effects of CVEs on end-stage renal disease (ESRD) and mortality of pre-dialysis patients. We followed 377 CKD patients who were at stage ≥G3 at first clinic visit in the Shuuwa General Hospital between April 2005 and July 2014. After taking baseline patient data, we evaluated renal survival rates and all-cause and CVE-related mortality in patients with CVEs [(+)CVEs] and without CVEs [(-)CVEs]. A total of 99 CVEs occurred in 93 study patients (57.0% cardiac events, 43.0% cerebrovascular events, and 6.5% peripheral artery disease events). During the study period, 127 patients reached ESRD over a median of 4.51 years' follow-up. Kaplan-Meier analysis found longer renal survival rates in the (-)CVEs group compared with the (+)CVEs group. Forty patients died during the study period over a median of 5.43 years' follow-up. Survival rates for all-cause and CVE-related mortality of (-)CVEs patients were higher than in (+)CVEs patients. After adjustment for sex, age, current smoking, blood pressure, diabetes, estimated glomerular filtration rate, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, left ventricular hypertrophy, body mass index, albumin, hemoglobin, calcium, phosphate, C-reactive protein, and spot urine protein, the occurrence of CVEs was still a significant risk factor for ESRD (HR 1.516, P = 0.017) and all-cause mortality (HR 7.871, P < 0.001). Our findings suggest that the occurrence of CVEs is a potent risk factor for ESRD and mortality in CKD patients before dialysis.

Myhre syndrome: a rare craniofacial disorder.

AIMS: Myhre syndrome is a rare disorder characterized by abnormal growth of the skeleton, muscles, and joints. The relationship of this syndrome to craniofacial growth and development is unknown. To the authors' knowledge, this is the first Japanese case ever studied. METHODOLOGY: At 10 years and 7 months of age, the patient was referred to the Department of Pediatric Dentistry in the authors' hospital, complaining of a dental problem. RESULTS: The craniofacial region exhibited a long lower face, high and narrowed palate with submucous cleft palate, maxillary constriction, prognathism, open bite, and crowding of the dental arch. Some of these morphological disorders could be affected by the functional manifestations of muscular hypertrophy of the cheek region, muscle tenseness, or the low position of the tongue. General disorders of muscular hypertrophy, thickened bones, and limited joint mobility are consistent with craniofacial findings of muscle tension in the cheek region, thickened calvarium, and limitation of temporomandibular joint movement. The submucous cleft palate and crown deformation of the mandibular central incisor may be affected by dysfunctions of SMAD4 signaling. CONCLUSIONS: Craniofacial growth and development is affected by the general characteristics of Myhre syndrome, and could be important in its diagnosis.

Molecular contribution to cleft palate production in cleft lip mice.

Cleft palate following cleft lip may include a developmental disorder during palatogenesis. CL/Fr mice fetuses, which develop cleft lip and palate spontaneously, have less capability for in vivo cell proliferation in palatal mesenchyme compared with CL/Fr normal fetuses. In order to know the changes of signaling molecules contributing to cleft palate morphogenesis following cleft lip, the mRNA expression profiles were compared in palatal shelves oriented vertically (before elevation) in CL/Fr fetuses with or without cleft lip. The changes in mRNA profile of cleft palate morphogenesis were presented in a microarray analysis, and genes were restricted to lists contributing to cleft palate development in CL/Fr fetuses with cleft lip. Four candidate genes (Ywhab, Nek2, Tacc1 and Frk) were linked in a gene network that associates with cell proliferation (cell cycle, MAPK, Wnt and Tgf beta pathways). Quantitative real-time RT-PCR highlighted the candidate genes that significantly changed in CL/Fr fetuses with cleft lip (Ywhab, Nek2 and Tacc1). The results of these molecular contributions will provide useful information for a better understanding of palatogenesis in cleft palate following cleft lip. Our data indicated the genetic contribution to cleft palate morphogenesis following cleft lip.

The P561T polymorphism of the growth hormone receptor gene has an inhibitory effect on mandibular growth in young children.

P561T heterozygous missense mutation in the growth hormone receptor (GHR) is a candidate genetic polymorphism (single-nucleotide polymorphism) for human mandibular growth. The purpose of this study was to assess whether this mutation affects mandibular growth during early childhood. The difference in mandibular growth between P561T heterozygous and wild-type individuals was analysed by cephalometric measurements during childhood. The subjects included 33 children with mandibular protrusion (aged 3-12 years, 16 males and 17 females) and 27 normal children (aged 3-13 years, 14 males and 13 females). Genomic DNA extracted from buccal epithelial cells was genotyped for the P561T heterozygous mutation with a molecular analysis (polymerase chain reaction--restriction fragment length polymorphism method). Two of the patients with normal occlusion and five with mandibular protrusion were heterozygous for the mutation. Chi-square analysis showed that the frequency of this mutation did not differ statistically between the normal and mandibular protrusion subjects. Multilevel model analysis of the 101 cephalograms showed that the mutation reduced the linear measurements of the mandible. These findings suggest that P561T heterozygous mutation affects mandibular growth during early childhood, and this mutation in the GHR gene is hypothesized to function as an inhibitory factor in the process of mandibular growth.

Temporal and spatial expression of Pax9 and Sonic hedgehog during development of normal mouse palates and cleft palates in TGF-beta3 null embryos.

Transforming growth factor-beta (TGF-beta3) gene disruption causes cleft secondary palate. Pax9 and Sonic hedgehog (Shh) genes are involved in the patterning of vertebrate embryonic tissues, including the facial skeleton. We investigated the expression of Pax9 and Shh genes during normal mouse palate development and in the developing cleft palates of TGF-beta3 null embryos. Whole mount in situ hybridization was conducted with use of Pax9 and Shh riboprobes for TGF-beta3 null, heterozygous and wild type mice at E12.5-E16.5. Histological analysis was processed by section in situ hybridization. In the wild type, Pax9 and Shh were expressed in the palate between E12.5-E15.5. Shh expression in the secondary palate was restricted to the rugae and the soft palate. Pax9 expression was predominantly in the palatal medial edge between E14.5 and E15.5. These patterns suggest that Shh and Pax9 may have different functions during palate development. In TGF-beta3 null mice, both genes expression patterns in the palate were different to those in wild type mice. In TGF-beta3 null mice, Pax9 expression was much reduced in the palatal medial edge at the critical time of palatal fusion (E14.5-E15.5). Shh expression in the palates of TGF-beta3 null mice was reduced throughout E12.5-E15.5, whilst Shh expression in heterozygous did not appear down regulated compared with the wild type. These results indicate that Pax9 and Shh expression are altered when the TGF-beta3 gene is deleted and suggest that Pax9 and Shh may be involved in the TGF-beta3 regulation of normal palatal fusion.

Association between palatal morphogenesis and Pax9 expression pattern in CL/Fr embryos with clefting during palatal development.

The CL/Fr mouse strain develops cleft lip and palate (CLP) spontaneously. In this study, Pax9 mRNA expression was investigated in the palatal shelves during palatal morphogenesis to assess the correlation between secondary palatal morphogenesis and Pax9 expression of CL/Fr embryos with spontaneous cleft lip and palate. The expression of Pax9 mRNA was characterised using whole mount in situ hybridisation with a digoxygenin-labelled probe. In the control strain of C57BL/6 and CL/Fr normal embryos, Pax9 was expressed in the palate, especially along the medial edge (ME), on embryonic day 13.5 (E13.5) and E14.5 when the palatal shelves grew vertically down the side of the tongue and subsequently elevated to a horizontal position, and was down regulated on E15.5 when the palatal shelves met and began fusing. In the cleft embryo, Pax9 was expressed in the ME region but was not down regulated on E15.5. Furthermore, whole mount in situ hybridisation was performed after organ culture, using CL/Fr-N and CL/Fr-BCL palatal shelves dissected and approximated by pairs on E13.5. This showed that Pax9 was still expressed in the ME region in separated palatal shelves of CL/Fr-N and CL/Fr-BCL embryos, while Pax9 expression was down regulated in paired palatal shelves. These expression patterns of Pax9 in normal and cleft embryos during palatal fusion indicate that Pax9 expression is altered in spontaneous cleft lip and palate, and concludes that there is a direct correlation between Pax9 expression and palatal fusion.