Identification of novel BCL11A variants in patients with epileptic encephalopathy: Expanding the phenotypic spectrum.

BCL11A encodes a zinc finger protein that is highly expressed in hematopoietic tissues and the brain, and that is known to function as a transcriptional repressor of fetal hemoglobin (HbF). Recently, de novo variants in BCL11A have been reported in individuals with intellectual disability syndrome without epilepsy. In this study, we performed whole-exome sequencing of 302 patients with epileptic encephalopathies (EEs), and identified 2 novel BCL11A variants, c.577delC (p.His193Metfs*3) and c.2351A>C (p.Lys784Thr). Both the patients shared major physical features characteristic of BCL11A-related intellectual disability syndrome, suggesting that characteristic physical features and the persistence of HbF should lead clinicians to suspect EEs caused by BCL11A pathogenic variants. Patient 1, with a frameshift variant, presented with Lennox-Gastaut syndrome, which expands the phenotypic spectrum of BCL11A haploinsufficiency. Patient 2, with a p.Lys784Thr variant, presented with West syndrome followed by drug-resistant focal seizures and more severe developmental disability. These 2 newly described patients contribute to delineating the associated, yet uncertain phenotypic characteristics of BCL11A disease-causing variants.

Resting salivary flow independently associated with oral malodor.

BACKGROUND: Dryness of the oral cavity is considered one cause of oral malodor. However, it is unclear which of the factors regulating the wetness of the oral cavity are involved in oral malodor development. This study investigated the effects of salivary flow and oral mucosal moisture on oral malodor. METHODS: The study population comprised 119 patients (48 men and 71 women, mean age of 50.6 ± 15.4 years) with complaint of oral malodor. After the oral malodor level had been evaluated by the organoleptic test and gas chromatography, the rates of stimulated saliva and resting saliva and the moisture levels of the tongue and buccal mucosa were measured. The plaque index, bleeding on pocket probing, probing pocket depth, and tongue coating score were also assessed. Strong oral malodor was defined as an organoleptic test score of ≥3. RESULTS: The flow rate of resting saliva in women was significantly lower than in men. The flow rate of resting saliva and the moisture levels of the tongue and buccal mucosa showed significant negative correlations with age. The flow rate of resting saliva was significantly lower in patients with strong oral malodor than in those with no or weak oral malodor. The flow rate of stimulated saliva and the moisture levels of the tongue and buccal mucosa had no relationship with strong oral malodor. Logistic regression analysis showed that a ≥5-mm probing pocket depth with bleeding on pocket probing, an increased tongue coating score, and decreased resting salivary flow were strong explanatory factors in clinical findings for oral malodor. CONCLUSION: This study suggests that the flow rate of resting saliva is a significant modulating factor for oral malodor.

Functional studies for the TRAF6 mutation associated with hypohidrotic ectodermal dysplasia.

BACKGROUND: Hypohidrotic ectodermal dysplasia (HED) is a rare condition characterized by hypotrichosis, hypohidrosis and hypodontia. A de novo heterozygous mutation in the tumour necrosis factor receptor-associated factor 6 gene (TRAF6) was recently identified in a patient with HED, while functional consequences resulting from the mutation remained unknown. OBJECTIVES: To determine the mechanism by which the TRAF6 mutation results in HED. METHODS: We performed coimmunoprecipitation (co-IP) studies to determine whether the mutation would affect the interaction of TRAF6 with transforming growth factor ß-activated kinase 1 (TAK1), TAK1-binding protein 2 (TAB 2) and ectodysplasin-A receptor-associated death domain protein (EDARADD). We then performed co-IP and glutathione S-transferase-pulldown assays to determine the TRAF6 binding sequences in EDARADD. In addition, we analysed the effect of the mutant TRAF6 protein on the affinity between wild-type TRAF6 and EDARADD, as well as on EDARADD-mediated nuclear factor (NF)-κB activation. RESULTS: The mutant TRAF6 protein was capable of forming a complex with TAK1 and TAB 2 in a similar way to wild-type TRAF6. However, the mutant TRAF6 protein completely lost the affinity to EDARADD, while the wild-type TRAF6 bound to the N-terminal domain of EDARADD. Furthermore, the mutant TRAF6 inhibited the interaction between the wild-type TRAF6 and EDARADD, and also potentially reduced the EDARADD-mediated NF-κB activity. CONCLUSIONS: We conclude that the mutant TRAF6 protein shows a dominant negative effect against the wild-type TRAF6 protein, which is predicted to affect the EDARADD-mediated activation of NF-κB during the development of ectoderm-derived organs, and to lead to the HED phenotype.

Novel Functional Peptide for Next-Generation Vital Pulp Therapy.

Although vital pulp therapy should be performed by promoting the wound-healing capacity of dental pulp, existing pulp-capping materials were not developed with a focus on the pulpal repair process. In previous investigations of wound healing in dental pulp, we found that organic dentin matrix components (DMCs) were degraded by matrix metalloproteinase-20, and DMC degradation products containing protein S100A7 (S100A7) and protein S100A8 (S100A8) promoted the pulpal wound-healing process. However, the direct use of recombinant proteins as pulp-capping materials may cause clinical problems or lead to high medical costs. Thus, we hypothesized that functional peptides derived from recombinant proteins could solve the problems associated with direct use of such proteins. In this study, we identified functional peptides derived from the protein S100 family and investigated their effects on dental pulp tissue. We first performed amino acid sequence alignments of protein S100 family members from several mammalian sources, then identified candidate peptides. Next, we used a peptide array method that involved human dental pulp stem cells (hDPSCs) to evaluate the mineralization-inducing ability of each peptide. Our results supported the selection of 4 candidate functional peptides derived from proteins S100A8 and S100A9. Direct pulp-capping experiments in a rat model demonstrated that 1 S100A8-derived peptide induced greater tertiary dentin formation compared with the other peptides. To investigate the mechanism underlying this induction effect, we performed liquid chromatography-tandem mass spectrometry analysis using hDPSCs and the S100A8-derived peptide; the results suggested that this peptide promotes tertiary dentin formation by inhibiting inflammatory responses. In addition, this peptide was located in a hairpin region on the surface of S100A8 and could function by direct interaction with other molecules. In summary, this study demonstrated that a S100A8-derived functional peptide promoted wound healing in dental pulp; our findings provide insights for the development of next-generation biological vital pulp therapies.

Effectiveness of high-dose barium enema filling for colonic diverticular bleeding.

AIM: Therapeutic barium enema was first reported in 1970. The long-term recurrence rate of colonic diverticular bleeding after therapeutic barium enema was compared with that of endoscopic haemostasis. METHOD: This study included 57 consecutive patients admitted between 2003 and 2008 with colonic diverticular bleeding in whom conservative treatment failed to stop bleeding within 3 h of hospital admission. Lower gastrointestinal endoscopy was performed immediately after admission. In 75% of patients, bleeding was from the right colon, and any identifiable source of bleeding was treated by endoscopic haemostasis. Cases with an undetectable source received high-dose barium impaction therapy. RESULTS: Treatment was as follows: Group A (n = 37) solely by endoscopic haemostasis; Group B (n = 11) solely by therapeutic barium enema group, and Group C (n = 9) by endoscopic haemostasis and therapeutic barium enema. At a follow up of seven (median; range: 1-56) months, recurrent bleeding rates were 18/37 (48.6%), 6/11 (54.5%) and 2/9 (22.2%) (P = 0.3930). CONCLUSION: High-dose barium enema is as effective as endoscopic haemostasis for the prevention of recurrent diverticular bleeding.

Effects of respiratory muscle training on parasympathetic activity in diabetes mellitus.

This study verified the effects of respiratory muscle training (RMT) on hemodynamics, heart rate (HR) variability, and muscle morphology in rats with streptozotocin-induced diabetes mellitus (DM). Thirty-six male Wistar rats were randomized into 4 groups and 34 completed the study: i) sham-sedentary (Sham-ST; n=9); ii) sham-RMT (Sham-RMT; n=9); iii) DM-sedentary (DM-ST; n=8); and iv) DM-RMT (DM-RMT; n=8). Hemodynamics were assessed by central cannulation, and R-R intervals were measured by electrocardiogram. In addition, the effects of RMT on the cross-sectional area of the diaphragm, anterior tibial, and soleus muscles were analyzed. The induction of DM by streptozotocin resulted in weight loss, hyperglycemia, reduced blood pressure, and attenuated left ventricular contraction and relaxation (P<0.05). We also observed a decrease in root mean square of successive differences between adjacent RR intervals (RMSSD) index and in the cross-sectional area of the muscles assessed, specifically the diaphragm, soleus, and anterior tibial muscles in diabetic rats (P<0.05). Interestingly, RMT led to an increase in RMSSD in rats with DM (P<0.05). The induction of DM produced profound deleterious changes in the diaphragmatic and peripheral muscles, as well as impairments in cardiovascular hemodynamics and autonomic control. Nevertheless, RMT may beneficially attenuate autonomic changes and improve parasympathetic modulation.

Enrichment of marine anammox bacteria from seawater-related samples and bacterial community study.

Anaerobic ammonium oxidation (anammox) is a novel nitrogen pathway catalyzed by anammox bacteria which are obligate anaerobic chemoautotrophs. In this study, enrichment culture of marine anammox bacteria (MAAOB) from the samples related to seawater was conducted. Simultaneous removal of ammonium and nitrite was confirmed in continuous culture inoculated with sediment of a sea-based waste disposal site within 50 days. However, no simultaneous nitrogen removal was observed in cultures inoculated with seawater-acclimated denitrifying sludge or with muddy sediment of tideland even during 200 days. Nitrogen removal rate of 0.13 kg/m(3)/day was achieved at nitrogen loading rate of 0.16 kg/m(3)/day after 320th days in the culture inoculated with the sediment of waste disposal site. The nitrogen removal ratio between ammonium nitrogen and nitrite nitrogen was 1:1.07. Denaturing gradient gel electrophoresis (DGGE) analysis indicated that an abundance of the bacteria close to MAAOB and coexistence of ammonium oxidizing bacteria and denitrifying bacteria in the culture.

Molecular ordering in self-organized dye particles: polarized evanescent-field studies.

Self-organized rhodamine 6G particles prepared by wetting/dewetting process of an ethanol solution on a hydrophilic glass surface did show fluorescence without quenching. Polarized evanescent-field excitation showed that the molecule's transition moment within dye particles was oriented unidirectionally and parallel to the substrate surface. The deduced dye orientation showed correlation between adjacent particles, which implies a simultaneous aggregate growth from a common crystal seed grown in a possible 'arm' region connecting the adjacent droplets just before these droplets were disconnected upon solvent evaporation. The dye orientation of most particles pointed about 45 degrees off the dewetting direction. By contrast, the particles of another pi-conjugated NK1420 dye, J-aggregates of which grow easily from an oversaturated solution, showed dye orientation along the dewetting direction preferably, still indicating the effect of self-organization, however based on a different mechanism.

Deficit of state-dependent risk attitude modulation in gambling disorder.

Gambling disorder (GD) is often considered as a problem of trait-like risk preference. However, the symptoms of GD cannot be fully understood by this trait view. In the present study, we hypothesized that GD patients also had problem with a flexible control of risk attitude (state-dependent strategy optimization), and aimed to investigate the mechanisms underlying abnormal risk-taking of GD. To address this issue, we tested GD patients without comorbidity (GD group: n=21) and age-matched healthy control participants (HC group: n=29) in a multi-step gambling task, in which participants needed to clear 'block quota' (required units to clear a block, 1000-7000 units) in 20 choices, and conducted a task-functional magnetic resonance imaging (fMRI) experiment. Behavioral analysis indeed revealed a less flexible risk-attitude change in the GD group; the GD group failed to avoid risky choice in a specific quota range (low-quota condition), in which risky strategy was not optimal to solve the quota. Accordingly, fMRI analysis highlighted diminished functioning of the dorsolateral prefrontal cortex (dlPFC), which has been heavily implicated in cognitive flexibility. To our knowledge, the present study provided the first empirical evidence of a deficit of state-dependent strategy optimization in GD. Focusing on flexible control of risk attitude under quota may contribute to a better understanding of the psychopathology of GDs.

Effects of respiratory muscle training on parasympathetic activity in diabetes mellitus

This study verified the effects of respiratory muscle training (RMT) on hemodynamics, heart rate (HR) variability, and muscle morphology in rats with streptozotocin-induced diabetes mellitus (DM). Thirty-six male Wistar rats were randomized into 4 groups and 34 completed the study: i) sham-sedentary (Sham-ST; n=9); ii) sham-RMT (Sham-RMT; n=9); iii) DM-sedentary (DM-ST; n=8); and iv) DM-RMT (DM-RMT; n=8). Hemodynamics were assessed by central cannulation, and R-R intervals were measured by electrocardiogram. In addition, the effects of RMT on the cross-sectional area of the diaphragm, anterior tibial, and soleus muscles were analyzed. The induction of DM by streptozotocin resulted in weight loss, hyperglycemia, reduced blood pressure, and attenuated left ventricular contraction and relaxation (P<0.05). We also observed a decrease in root mean square of successive differences between adjacent RR intervals (RMSSD) index and in the cross-sectional area of the muscles assessed, specifically the diaphragm, soleus, and anterior tibial muscles in diabetic rats (P<0.05). Interestingly, RMT led to an increase in RMSSD in rats with DM (P<0.05). The induction of DM produced profound deleterious changes in the diaphragmatic and peripheral muscles, as well as impairments in cardiovascular hemodynamics and autonomic control. Nevertheless, RMT may beneficially attenuate autonomic changes and improve parasympathetic modulation.

p16INK4a inactivation is not frequent in uncultured sporadic primary cutaneous melanoma.

In an attempt to examine whether the inactivation of p16INK4a is an important early event in the development of sporadic melanoma in vivo, we have systematically analysed 46 uncultured primary cutaneous melanomas. Loss of heterozygosity (LOH) of chromosome region 9p21-22 (where the p16INK4a resides) was detected in 11 tumours (24%) by PCR-based LOH analyses. Direct sequencing of all three exons of the p16INK4a gene in these 11 tumours revealed no somatic mutation although germline mutations which have not been reported previously as common polymorphisms were detected in two patients. Further sequencing analyses of the p16INK4a gene exon 2 in 19 additional tumours with no evidence of LOH on 9p21-22 identified only one heterozygous C- >T mutation at codon 81 altering a proline to a leucine. A sensitive methylation-specific PCR assay did not reveal de novo methylation of the 5'CpG island in exon 1 of the p16INK4a gene in any of the tumours showing 9p21-22 allelic loss or a heterozygous p16INK4a mutation. Complete loss of p16INK4a protein, most likely due to homozygous deletion of the p16INK4a gene, was observed in 6 (15%) out of 39 evaluable cases by immunohistochemical analyses on frozen sections using two different anti-p16INK4a antibodies. The results show that inactivation of p16INK4a is not as frequent in primary melanoma as has been reported in cell lines, and warrant further search for another tumour suppressor on 9p21-22. This study also emphasizes the importance of examining uncultured primary tumours rather than cell lines to define early events in tumorigenesis.

Cell-cycle-dependent and ATM-independent expression of human Chk1 kinase.

Checkpoint genes cause cell cycle arrest when DNA is damaged or DNA replication is blocked. Although a human homolog of Chk1 (hChk1) has recently been reported to be involved in the DNA damage checkpoint through phosphorylation of Cdc25A, B, and C, it is not known at which phase(s) of the cell cycle hChk1 functions and how hChk1 causes cell cycle arrest in response to DNA damage. In the present study, we demonstrate that in normal human fibroblasts (MJ90), hChk1 is expressed specifically at the S to M phase of the cell cycle at both the RNA and protein levels and that it is localized to the nucleus at this time. hChk1 activity, as determined by phosphorylation of Cdc25C, is readily detected at the S to M phase of the cell cycle, and DNA damage induced by UV or ionizing radiation does not enhance the expression of hChk1 or its activity. Furthermore, hChk1 exists in an active form at the S to M phase in fibroblasts derived from patients with ataxia telangiectasia (AT) which lack the functional AT mutated (ATM) gene product, suggesting that hChk1 expression is independent of functional ATM. Taken together with the findings that phosphorylation of Cdc25C on serine 216 is increased at the S to M phase, it is suggested that at this particular phase of the cell cycle, even in the absence of DNA damage, hChk1 phosphorylates Cdc25C on serine 216, which is considered to be a prerequisite for the G2/M checkpoint. Thus, hChk1 may play an important role in keeping Cdc25C prepared for responding to DNA damage by phosphorylating its serine residue at 216 during the S to M phase.

Mouse Brachyury the Second (T2) is a gene next to classical T and a candidate gene for tct.

The mouse Brachyury the Second (T2) gene is 15 kb away from classical Brachyury (T). A mutation in T2 disrupts notochord development, pointing to the existence of a second T/t complex gene involved in axis development. T2 encodes a novel protein that is disrupted by an insertion in T2(Bob) mice. Sequence analysis of T2 from several t haplotypes shows that they all share the same changed stop codon, and, thus, T2 is a candidate gene for the t complex tail interaction factor. T1, T2, and the unlinked t-int are distinct and unrelated loci, and mutations in these genes do not complement one another genetically. Either their products interact in the same pathway during the genesis of the embryonic axis, or the T/t region itself is truly complex.

Cancer whole-genome sequencing: present and future.

Recent explosive advances in next-generation sequencing technology and computational approaches to massive data enable us to analyze a number of cancer genome profiles by whole-genome sequencing (WGS). To explore cancer genomic alterations and their diversity comprehensively, global and local cancer genome-sequencing projects, including ICGC and TCGA, have been analyzing many types of cancer genomes mainly by exome sequencing. However, there is limited information on somatic mutations in non-coding regions including untranslated regions, introns, regulatory elements and non-coding RNAs, and rearrangements, sometimes producing fusion genes, and pathogen detection in cancer genomes remain widely unexplored. WGS approaches can detect these unexplored mutations, as well as coding mutations and somatic copy number alterations, and help us to better understand the whole landscape of cancer genomes and elucidate functions of these unexplored genomic regions. Analysis of cancer genomes using the present WGS platforms is still primitive and there are substantial improvements to be made in sequencing technologies, informatics and computer resources. Taking account of the extreme diversity of cancer genomes and phenotype, it is also required to analyze much more WGS data and integrate these with multi-omics data, functional data and clinical-pathological data in a large number of sample sets to interpret them more fully and efficiently.

Comparison of genetic profiles between primary melanomas and their metastases reveals genetic alterations and clonal evolution during progression.

To examine for the genetic basis of metastatic progression in cutaneous melanoma, we have compared loss of heterozygosity (LOH) of several selected chromosome regions that are implicated in the initiation and progression of melanoma, and alterations of the p16INK4a gene in 14 pairs of primary tumor and synchronous or asynchronous metastasis excised from the same patients. The most frequent genetic alteration during metastatic progression detected was the loss of p16INK4a protein expression (four of 14 cases), whereas no somatic p16INK4a gene mutations were found in any primary or metastatic tumors. LOH analyses showed that most of the chromosome losses including 6q, 8p, 9p, 9q, and 18q were shared between primary tumors and their metastases. Nevertheless, LOH of 6q and 11q and LOH of 7q not detected in primary tumors were, respectively, observed in two lymph node metastases. These results suggest that loss of p16INK4a protein expression (but not p16INK4a gene mutation) and the losses of chromosome arms 6q, 7q, and 11q play an important role in the acquisition of metastatic potential in sporadic melanoma. Furthermore, comparison of genetic profiles between the primary tumor and its metastasis revealed in several cases that heterogenous tumor cell populations might already exist at the early stage of tumorigenesis and evolve independently in the primary tumor and its metastasis, strongly suggesting that metastatic progression of sporadic melanoma is not accounted for by a linear progression model.

erbB-2 overexpression but no activation of beta-Catenin gene in extramammary Paget's disease.

Our previous study in extramammary Paget's disease showed neither p53 mutations nor allelic loss at selected loci implicated in other cancers, suggesting a pathogenesis of this skin cancer different from other common epithelial malignancies. To examine further the genetic defects in extramammary Paget's disease, we carried out molecular genetic analyses in 31 tumor samples obtained from 27 cases of extramammary Paget's disease without underlying malignancies. Immunohistochemistry using CB-11 monoclonal antibody revealed either membrane or cytoplasmic erbB-2 oncoprotein overexpression in none of the 13 primary in situ tumors, but in one recurrent in situ tumor, 10 of 13 invasive primary tumors and two of four lymph node metastases. Sensitive dual color fluorescence in situ hybridization analysis using probes for erbB-2 gene locus and chromosome 17 pericentromere, however, revealed different erbB-2 gene status in the erbB-2 overexpressing tumors. One recurrent in situ tumor and one lymph node metastasis showed definite gene amplification characterized by multiple scattered signals or a few large clustered erbB-2 signals, whereas four tumors with predominantly cytoplasmic erbB-2 overexpression were thought to have low-grade gene amplification. The remaining six tumors overexpressing erbB-2 showed no increase of erbB-2 copy numbers. No evidence of abnormal activation of the beta-catenin gene, a critical mediator of Wnt signaling pathway, in any tumor by immunohistochemical staining and by direct sequencing and reverse transcription-polymerase chain reaction analysis was found. Frequent overexpression of erbB-2 by either gene amplification or possible transcriptional activation in invasive primary tumors and metastases suggests an important part for this oncogene in the progression of extramammary Paget's disease.