Specific HLA genotypes confer susceptibility to acute necrotizing encephalopathy.

Acute necrotizing encephalopathy (ANE) is a rare and severe syndrome of acute encephalopathy triggered by viral infections. Cytokine storm is considered as the main pathogenetic mechanism of ANE. ANE is prevalent in East Asia, suggesting the association of host genetic factors. To elucidate the genetic background of Japanese ANE, we examined genotypes of human leukocyte antigen (HLA)-A, C, B, DRB1, DQB1 and DPB1 in 31 patients. Significant positive association was observed in both the allele frequency and positivity of DRB1*09:01 (P=0.043 and 0.025, respectively), as well as those of DQB1*03:03 (P=0.034 and 0.026, respectively). The carrier frequency of DRB1*09:01 and DQB1*03:03 alleles was higher in the patients (45.16%) than in controls (28.57%). These alleles are more common in East Asian than in European populations, and are reportedly associated with various autoimmune diseases in Japanese patients. Our data provide further evidence that altered immune response based on individual HLA genotypes may contribute to ANE pathogenesis.

Hyaluronan metabolism in overloaded temporomandibular joint.

This study aimed to examine hyaluronan (HA) metabolism in relation to the onset and progression of temporomandibular joint osteoarthritis (TMJ-OA) induced by mechanical overloading. Two-month-old and 6-month-old C57BL/6N mice were divided into experimental and untreated control groups (n = 5/group). A sliding plate was attached to the maxillary incisors of the experimental mice for 10 days to overload the condylar cartilage in TMJ. In experimental group, profound cartilage degradation was detected in haematoxylin-eosin, Safranin-O-Fast Green-stained sections. It was also shown that the cartilage degradation was greater in older mice in both the control and the experimental groups. The number of HABP-positive cells was decreased by mechanical overloading and with age. The reduction of HA expression was correlated with the progression of cartilage degradation induced by mechanical overloading. The absolute quantification of the mRNA expression related to HA synthesis and HA degradation was also performed in each group. The mRNA expression levels of HA synthase (HAS) 2 and 3 were lower in the experimental group compared with the control group in the younger mice. In contrast, the mRNA expression levels of the HA degradation gene, HYAL2 and KIAA1199, were higher in the experimental group compared with the control group in the older mice. Thus, mechanical overload differently affected the balance of HA degradation and HA synthesis in the older and younger mice, respectively. In conclusion, mechanical overloading affects HA metabolism and it might initiate or amplify the condylar cartilage degradation.

HLA-DRB1, -DRB3, -DRB4 and -DRB5 genotyping at a super-high resolution level by long range PCR and high-throughput sequencing.

Super high-resolution single molecule sequence-based typing (SS-SBT) is a human leukocyte antigen (HLA) DNA typing method to the field 4 level of allelic resolution (formerly known as eight-digit typing) to efficiently detect new and null alleles without phase ambiguity by combination of long ranged polymerase chain reaction (PCR) amplification and next-generation sequencing (NGS) technologies. We previously reported the development and application of the SS-SBT method for the eight classical HLA loci, A, B, C, DRB1, DQA1, DQB1, DPA1 and DPB1. In this article, we describe the development of the SS-SBT method for three DRB1 linked loci, DRB3, DRB4 and DRB5 (DRB3/4/5) and characterization of DRB1-DRB3/4/5 haplotype structures to the field 4 level. Locus specific PCR primers for DRB3/4/5 were designed to amplify the gene regions from intron 1 to exon 6 [3' untranslated region (3'UTR)]. In total 20 DRB1 and 13 DRB3/4/5 allele sequences were determined by the SS-SBT to the field 4 level without phase ambiguity using 19 DR51, DR52 and DR53 positive genomic DNA samples obtained from Japanese. Moreover, 18 DRB1-DRB3/4/5 haplotypes were estimated to the field 4 level by the SS-SBT method in contrast to 10 haplotypes estimated by conventional methods to the field 1 level (formerly known as two digit typing). Therefore, DRB1-DRB3/4/5 haplotyping by SS-SBT is expected to provide informative data for improved HLA matching in medical research, transplantation procedures, HLA-related disease studies and human population diversity studies.

Systemic lupus erythematosus complicated with liver cirrhosis in a patient with Papillon-Lefèvre syndrome.

We report the first case of a girl who presented with Papillon-Lefèvre syndrome (PLS) and subsequently developed systemic lupus erythematosus and liver cirrhosis. This indicates that autoimmune diseases can be a complication in patients with PLS. Cathepsin C gene mutations were not found in our patient or her mother. Thus, other genetic factors may have been involved in this patient.

Super high resolution for single molecule-sequence-based typing of classical HLA loci at the 8-digit level using next generation sequencers.

Current human leukocyte antigen (HLA) DNA typing methods such as the sequence-based typing (SBT) and sequence-specific oligonucleotide (SSO) methods generally yield ambiguous typing results because of oligonucleotide probe design limitations or phase ambiguity for HLA allele assignment. Here we describe the development and application of the super high-resolution single-molecule sequence-based typing (SS-SBT) of HLA loci at the 8-digit level using next generation sequencing (NGS). NGS which can determine an HLA allele sequence derived from a single DNA molecule is expected to solve the phase ambiguity problem. Eight classical HLA loci-specific polymerase chain reaction (PCR) primers were designed to amplify the entire gene sequences from the enhancer-promoter region to the 3' untranslated region. Phase ambiguities of HLA-A, -B, -C, -DRB1 and -DQB1 were completely resolved and unequivocally assigned without ambiguity to single HLA alleles. Therefore, the SS-SBT method described here is a superior and effective HLA DNA typing method to efficiently detect new HLA alleles and null alleles without ambiguity.

Requirement for matrix metalloproteinase stromelysin-3 in cell migration and apoptosis during tissue remodeling in Xenopus laevis.

The matrix metalloproteinase (MMP) stromelysin-3 (ST3) was originally discovered as a gene whose expression was associated with human breast cancer carcinomas and with apoptosis during organogenesis and tissue remodeling. It has been shown previously, in our studies as well as those by others, that ST3 mRNA is highly upregulated during apoptotic tissue remodeling during Xenopus laevis metamorphosis. Using a function-blocking antibody against the catalytic domain of Xenopus ST3, we demonstrate here that ST3 protein is specifically expressed in the cells adjacent to the remodeling extracellular matrix (ECM) that lies beneath the apoptotic larval intestinal epithelium in X. laevis in vivo, and during thyroid hormone-induced intestinal remodeling in organ cultures. More importantly, addition of this antibody, but not the preimmune antiserum or unrelated antibodies, to the medium of intestinal organ cultures leads to an inhibition of thyroid hormone-induced ECM remodeling, apoptosis of the larval epithelium, and the invasion of the adult intestinal primodia into the connective tissue, a process critical for adult epithelial morphogenesis. On the other hand, the antibody has little effect on adult epithelial cell proliferation. Furthermore, a known MMP inhibitor can also inhibit epithelial transformation in vitro. These results indicate that ST3 is required for cell fate determination and cell migration during morphogenesis, most likely through ECM remodeling.

ARR1, a transcription factor for genes immediately responsive to cytokinins.

Cytokinins are a class of phytohormones involved in various physiological events of plants. The Arabidopsis sensor histidine kinase CRE1 was recently reported to be a cytokinin receptor. We used a steroid-inducible system to show that the transcription factor-type response regulator ARR1 directs transcriptional activation of the ARR6 gene, which responds to cytokinins without de novo protein synthesis. This fact, together with characteristics of ARR1-overexpressing plants and arr1 mutant plants, indicates that the phosphorelay to ARR1, probably from CRE1, constitutes an intracellular signal transduction occurring immediately after cytokinin perception.

A genome-wide association scan for asthma in a general Australian population.

To date, almost every chromosome has been implicated in genetic susceptibility to asthma to some degree. When compared with single nucleotide polymorphism, microsatellite markers exhibit high levels of heterozygosity and therefore provide higher statistical power in association. The objective of this study was to perform a genome-wide association study using 23,465 in-house microsatellite markers to detect asthma susceptibility regions in the Busselton population. In this study, three separate pooled DNA screenings yielded 18 markers with significantly different estimated frequencies in the three separate "case and control" pools: each pool consisting of 60 males and 60 females. These markers were evaluated by individual typing in 360 cases and 360 controls. Two markers showed significant differences between cases and controls (P = 0.001 and P = 0.003). Regions surrounding the two markers were subjected to high-density association mapping with a total of 14 additional markers. We were able to confirm and fine map the association in these two regions by typing 14 additional microsatellite markers (1805A09 (D18S0325i), P = 0.002; 1806D05 (D18S0181i), P = 0.001). Each region contains a predicted gene that showed strong associations with asthma. Further studies are underway to characterize the novel candidate asthma susceptibility genes identified in this genome-wide study.

Identification of critical members in a sulfidogenic benzene-degrading consortium by DNA stable isotope probing.

Stable isotope probing (SIP) was used to identify the active members in a benzene-degrading sulfidogenic consortium. SIP-terminal restriction fragment length polymorphism analysis indicated that a 270-bp peak incorporated the majority of the (13)C label and is a sequence closely related to that of clone SB-21 (GenBank accession no. AF029045). This target may be an important biomarker for anaerobic benzene degradation in the field.

Pathological Profile, Early Complications, Functional and Oncological Outcome after Radical Cystectomy - Ileal Conduit for Bladder Cancer Patients in Sanglah General Hospital between January 2013 and December 2016.

BACKGROUND: Radical cystectomy is the standard treatment for nonmetastatic bladder cancer (muscle-invasive and selective superficial bladder cancer). There are many types of urinary diversion after this procedure; the ileal conduit is the most and simplest one. AIM: To asses clinical, pathological profile, early complication, functional and oncological outcome after radical cystectomy and ileal conduit for muscle-invasive bladder cancer patients. METHOD: Between January 2013 and December 2016, there were 68 patients diagnosed with bladder cancer. From those patients, 24 (35.29%) patients had been performed radical cystectomy with ileal conduit type for urinary diversion (100%). Patients demographic, clinical and pathological profile, early postoperative complication, functional and oncological outcome were collected from the medical record. RESULT: Among the 24 patients who underwent radical cystectomy, 20 patients were male (83.3%) with the mean age was 57.3 y.o (33-77 y.o). Twelve patients (50%) showed pT4 and pT2 respectively. Based on pathological result 20 patient (83.34%) had the urothelial carcinoma, three patients (12.5%) had squamous cell carcinoma, and one patient (4.1%) had adenocarcinoma. Two patients (8.3%) got neoadjuvant chemotherapy, and nine patient (37.5%) of patients followed adjuvant chemotherapy after surgery. Wound dehiscence, fistula enterocutan, prolong ileus, leakage anastomosis and sepsis were kind of complication after surgery. One year's survival rate is 84%, mortality rate 20.8% and a recurrence rate of 20.8% in 4 years follow up. CONCLUSION: Radical cystectomy and ileal conduit type of urinary diversion still become the preferable procedure for nonmetastatic bladder cancer with good functional and oncological outcome.

Thyroid hormone regulation of apoptotic tissue remodeling: implications from molecular analysis of amphibian metamorphosis.

Organogenesis and tissue remodeling are critical processes during postembryonic animal development. Anuran metamorphosis has for nearly a century served as an excellent model to study these processes in vertebrates. Frogs not only have essentially the same organs with the same functions as higher vertebrates such as humans, but also employ similar organogenic processes involving highly conserved genes. Development of frog organs takes place during metamorphosis, which is free of any maternal influences but absolutely dependent on the presence of thyroid hormone. Furthermore, this process can be easily manipulated both in intact tadpoles and in organ cultures by controlling the availability of thyroid hormone. These interesting properties have led to extensive morphological, cellular, and biochemical studies on amphibian metamorphosis. More recently, the cloning of thyroid hormone receptors and the demonstration that they are transcription factors have encouraged enormous interest in the molecular pathways controlling tissue remodeling induced by thyroid hormone during metamorphosis. This article summarizes some of the recent studies on the mechanisms of gene regulation by thyroid hormone receptors and isolation and functional characterization of genes induced by thyroid hormone during Xenopus metamorphosis. Particular focus is placed on the remodeling of the animal intestine, which involves both apoptosis (programmed cell death) of larval cells and de novo development of adult tissues, and the roles of thyroid hormone-induced genes that encode matrix metalloproteinases during this process.

A nationwide survey of pediatric acquired demyelinating syndromes in Japan.

OBJECTIVE: To investigate the clinical and epidemiologic features of pediatric acquired demyelinating syndromes (ADS) of the CNS in Japan. METHODS: We conducted a nationwide survey and collected clinical data on children with ADS aged 15 years or younger, who visited hospitals between 2005 and 2007. RESULTS: Among 977 hospitals enrolled, 723 (74.0%) responded to our inquiries and reported a total of 439 patients as follows: 244 with acute disseminated encephalomyelitis (ADEM), 117 with multiple sclerosis (MS), 14 with neuromyelitis optica (NMO), and 64 with other ADS. We collected and analyzed detailed data from 204 cases, including those with ADEM (66), MS (58), and NMO (10). We observed the following: (1) the estimated annual incidence rate of pediatric ADEM in Japan was 0.40 per 100,000 children (95% confidence interval [CI], 0.34-0.46), with the lowest prevalence in the north; (2) the estimated prevalence rate of MS was 0.69 per 100,000 children (95% CI, 0.58-0.80), with the lowest prevalence in the south; (3) NMO in Japan was rare, with an estimated prevalence of 0.06 per 100,000 children (95% CI, 0.04-0.08); and (4) the sex ratio and mean age at onset varied by ADS type, and (5) male/female ratios correlated with ages at onset in each ADS group. CONCLUSIONS: Our results clarify the characteristic clinical features of pediatric ADS in the Japanese population.

Accumulation of glycolipids in mutant Chinese hamster ovary cells (Z65) with defective peroxisomal assembly and comparison of the metabolic rate of glycosphingolipids between Z65 cells and wild-type CHO-K1 cells.

The influence of peroxisomal dysfunction on glycosphingolipid metabolism was investigated using mutant Chinese hamster ovary (CHO) cells (Z65) with defective assembly of the peroxisomal membranes. In accordance with previous observations, the concentration of very long chain fatty acid (C24:0) was shown to be higher in Z65 cells than in control cells. We then compared the composition of glycolipids in Z65 cells with that in CHO-K1 cells, which are wild-type Chinese hamster ovary cells with intact peroxisomes, and found significantly increased concentrations of ceramide monohexoside (CMH) and ganglioside GM3 in Z65 cells. However, there were no differences in the concentrations of glycerophospholipids, triglycerides, free fatty acids and cholesterol between Z65 and CHO-K1 cells. Further, to investigate the metabolic rate of the major lipids, Z65 and CHO-K1 cells were pulse-labeled with [3-14C]serine. [3-14C]Serine was incorporated into phosphatidylserine, phosphatidylethanolamine and sphingomyelin more quickly in CHO-K1 than in Z65 cells. However, after 48 h, the radioactivity incorporated into those lipids, including CMH, was greater in Z65 cells than in CHO-K1 cells. Thus, the altered metabolism of glycosphingolipids, probably due to peroxisomal dysfunction, was thought to be responsible for the change in glycosphingolipid composition in Z65 cells.

Sequence organization of replication origin of the Escherichia coli K-12 chromosome.

A sequence of 245 base-pairs (oriC) in the replication origin of the Escherichia coli K-12 chromosome has been shown to provide all the information essential for initiation of bidirectional replication. In order to elucidate the sequence organization of oriC, numerous mutants carrying a single-to-multiple transitions from G X C to A X T base-pair were constructed by localized mutagenesis in vitro, which uses sodium bisulfite, and the correlation between the mutation sites and replicating ability (Ori function) was systematically analyzed. By isolating non-defective (Ori+) mutants with multiple base changes, transitions at 71 positions among 101 G X C pairs in oriC were found to have no effect on Ori function. Investigation of defective (Ori-) mutants, on the other hand, showed that individual replacements at 18 positions were detrimental to Ori function to some extent. These irreplaceable G X C pairs fell in the positions where no substitution was detected in the Ori+ mutants. The defect of the Ori- mutants with a single base substitution was generally weaker than that of the previously constructed Ori- mutants lacking a part of oriC. The addition of two or more base changes each giving a faint Ori- phenotype, however, resulted in a more intensive Ori- phenotype. We have previously demonstrated that oriC contains several regions where deletion or insertion of oligonucleotides leads to strong Ori- phenotypes. Transitions in those areas did not cause any defect of Ori function. Combining present results on base substitution mutants with the previous observations together, we assumed that the oriC sequence provides multiple interaction sites with replication initiation factors, and the precise arrangement of these sites are required for Ori function.

Binding of the regulatory protein VirG to the phased signal sequences upstream from virulence genes on the hairy-root-inducing plasmid.

The VirG protein is a positive regulator for the virulence genes of which expression is induced by a plant factor, and is essential for Agrobacterium pathogenicity on dicotyledonous plants. The VirG protein of the hairy-root-inducing plasmid A4 was overproduced in Escherichia coli cells, and purified to homogeneity. DNase I footprinting experiments revealed that the purified VirG protein was bound to the upstream region of virulence genes including the phased vir box sequences, which had been presumed to be the VirG recognition signal from the sequence analysis. In dimethyl sulfate footprinting, the VirG protein specifically protected the guanine residues within every vir box sequence. It was concluded that the VirG protein was bound to the phased vir box sequences from the major groove along one side of double-helical DNA.