Non-invasive transcriptomic analysis using mRNAs in skin surface lipids obtained from children with mild-to-moderate atopic dermatitis.

BACKGROUND: Specimens for analysing the molecular pathology of skin disease are generally obtained through invasive methods, such as biopsy. However, less burdensome methods are desirable for paediatric patients. We recently established a method that comprehensively analyses RNA present in sebum (skin surface lipid-RNAs: SSL-RNAs) using a next-generation sequencer. Using this method, biological information can be obtained from the skin in a completely non-invasive manner. OBJECTIVES: To verify the applicability of the SSL-RNA method for analysis of paediatric skin and analyse the molecular pathology of mild-to-moderate atopic dermatitis (AD) in children. METHODS: We collected sebum specimens from the whole faces of 23 healthy children and 16 children with mild-to-moderate AD (eczema area and severity index (EASI) score: 5.9 ± 2.6) ranging in age from 6 months to 5 years, using an oil-blotting film. We then extracted SSL-RNAs from the samples and performed an AmpliSeq transcriptomic analysis. RESULTS: The expressions of genes related to keratinization (LCE, PSORS1C2, IVL and KRT17), triglyceride synthesis and storage (PLIN2, DGAT2 and CIDEA), wax synthesis (FAR2), ceramide synthesis (GBA2, SMPD3 and SPTLC3), antimicrobial peptides (DEFB1) and intercellular adhesion (CDSN), all of which are related to the skin barrier, are lower in children with AD than in healthy children. The children with AD also have higher expression of CCL17, a Th2-cytokine and an increased Th2-immune response as demonstrated by a gene set variation analysis. Moreover, KRT17 and CCL17 expression levels are significantly correlated with the EASI score. CONCLUSIONS: Molecular changes associated with abnormal immune responses and the epidermal barrier in children with mild-to-moderate AD can be determined using the SSL-RNA method. This non-invasive method could therefore be a useful means for understanding the molecular pathology of paediatric AD.

Endoscopic ultrasound-guided celiac ganglia neurolysis vs. celiac plexus neurolysis: a randomized multicenter trial.

BACKGROUND AND STUDY AIMS: No prospective comparison of endoscopic ultrasonography-guided direct celiac ganglia neurolysis (EUS - CGN) vs. EUS-guided celiac plexus neurolysis (EUS - CPN) has been reported. The aim of the current study was to compare the effectiveness of EUS - CGN and EUS - CPN in providing pain relief from upper abdominal cancer pain in a multicenter randomized controlled trial. PATIENTS AND METHODS: Patients with upper abdominal cancer pain were randomly assigned to treatment using either EUS - CGN or EUS - CPN. Evaluation was performed at Day 7 postoperatively using a pain scale of 0 to 10. Patients for whom pain decreased to ≤ 3 were considered to have a positive response, and those experiencing a decrease in pain to ≤ 1 were considered to be completely responsive. Comparison between the two groups was performed using intention-to-treat analysis. The primary endpoint was the difference in treatment response rates between EUS - CGN and EUS - CPN at postoperative Day 7. Secondary endpoints included differences in complete response rates, pain scores, duration of pain relief, and incidence of adverse effects. RESULTS: A total of 34 patients were assigned to each group. Visualization of ganglia was possible in 30 cases (88 %) in the EUS - CGN group. The positive response rate was significantly higher in the EUS - CGN group (73.5 %) than in the EUS - CPN group (45.5 %; P = 0.026). The complete response rate was also significantly higher in the EUS - CGN group (50.0 %) than in the EUS - CPN group (18.2 %; P = 0.010). There was no difference in adverse events or duration of pain relief between the two groups. CONCLUSIONS: EUS - CGN is significantly superior to conventional EUS - CPN in cancer pain relief. CLINICAL TRIAL REGISTRATION: http://www.umin.ac.jp/ctr/index.htm (ID: UMIN-000002536).

Effects of surgery and chronic disease states on the concentrations and phenotype distribution of α1-acid glycoprotein: studies in patients with breast cancer and patients with chronic inflammatory disease.

OBJECTIVE: Although the concentration of α1-acid glycoprotein (AGP) in serum increases under some conditions, the behavior of the individual genetic variants is not well understood. Therefore, we studied the relative changes in AGP variants pre- and postoperatively in patients with cancer and patients with chronic inflammatory disease states, as well as the distribution of AGP phenotypes in a Japanese population. METHODS: Serum samples were taken before and after surgery from 25 female patients with early breast cancer. Serum samples were also obtained from 134 patients with rheumatoid arthritis (RA) and 33 with systemic lupus erythematosus (SLE), and from 103 healthy subjects. The relative concentrations of the individual genetic variants in the serum samples were determined by isoelectric focusing after desialylation with neuraminidase. RESULTS: The postoperative AGP concentrations in patients with early breast cancer were 2-fold higher than before surgery. The relative concentrations of the F1 and S variants were significantly increased, whereas that of the A variant was not changed significantly. The relative concentrations of all the AGP variants in patients with RA and SLE were significantly higher than those in healthy subjects. The distribution of the AGP phenotypes did not differ significantly among the groups examined in this study. CONCLUSIONS: The F1/S variants of AGP, but not the A variant, were significantly increased after early breast cancer surgery, but all the variants were increased in patients with chronic inflammatory states such as RA and SLE. The distribution of the AGP phenotypes did not differ significantly among the disease groups studied.

BLM has early and late functions in homologous recombination repair in mouse embryonic stem cells.

BLM is a RecQ family helicase that is defective in individuals with the cancer predisposition disorder, Bloom's syndrome (BS). At the cellular level, BS is characterized by hyper-recombination manifested as excessive sister chromatid exchange and loss of heterozygosity. However, the precise function of BLM remains unclear. Multiple roles have been proposed for BLM in the homologous recombination (HR) repair pathway, including 'early' functions, such as the stimulation of resection of DNA double-strand break ends or displacement of the invading strand of DNA displacement loops, and 'late' roles, such as dissolution of double Holliday junctions. However, most of the evidence for these putative roles comes from in vitro biochemical data. In this study, we report the characterization of mouse embryonic stem cells with disruption of Blm and/or Rad54 genes. We show that Blm has roles both upstream and downstream of the Rad54 protein, a core HR factor. Disruption of Rad54 in the Blm-mutant background reduced the elevated level of gene targeting and of sister chromatid exchanges, implying that Blm primarily functions downstream of Rad54 in the HR pathway. Conversely, however, mutation of Blm in Rad54(-/-) cells rescued their mitomycin C (MMC) sensitivity, and decreased both the level of DNA damage and cell cycle perturbation induced by MMC, suggesting an early role for Blm. Our data are consistent with Blm having at least two roles in HR repair in mammalian cells.

Unique grouping of the Far East Asian begomovirus complex based on sequence analyses of the DNA-A genome and associated DNAbeta satellite molecules isolated from tomato, honeysuckle and Eupatorium plants in Japan.

Nucleotide (nt) sequencing has contributed to the identification of virus species and has also proved diagnostically useful in the control of tomato-infecting begomoviruses disease. We determined the complete nt sequences of the DNA-A genome and its cognate DNAbeta satellite molecules in isolates of Tobacco leaf curl Japan virus, Honeysuckle yellow vein mosaic virus, Eupatorium yellow vein virus in Japan. Pairwise comparison analyses based on the nt sequences of DNA-A from the genetic group of these viruses tentatively named as TbLCJV, HYVMV and EpYVV (TbJV/HYV/EpV) revealed that this group had a significance threshold of 84 % identity. Phylogenetic relationship analyses of the nt sequences of DNA-A and DNAbeta revealed that their isolates were separated into a discrete Far East Asian clade, distinct from all other begomoviruses. This clade was divided into two distinct clusters comprising the subgroups TbJV/HYV and EpV. Furthermore, recombination analysis revealed that members of the TbJV/HYV/EpV group had the genetic variation indicative of many recombination events. Our study demonstrates that this group forms a unique species complex, but that members have discrete lineages depending on their natural perennial host plants.

Molecular genetics of RecQ helicase disorders.

The RecQ helicases belong to the Superfamily II group of DNA helicases, and are defined by amino acid motifs that show sequence similarity to the catalytic domain of Escherichia coli RecQ. RecQ helicases have crucial roles in the maintenance of genome stability. In humans, there are five RecQ helicases and deficiencies in three of them cause genetic disorders characterised by cancer predisposition, premature aging and/or developmental abnormalities. RecQ helicase-deficient cells exhibit aberrant genetic recombination and/or DNA replication, which result in chromosomal instability and a decreased potential for proliferation. Here, we review the current knowledge of the molecular genetics of RecQ helicases, focusing on the human RecQ helicase disorders and mouse models of these conditions.

Genetic variation of the prevailing porcine respiratory and reproductive syndrome viruses occurring on a pig farm upon vaccination.

Recurrence of porcine respiratory and reproductive syndrome (PRRS) was observed on a pig farm after introducing two PRRS live virus vaccines to combat preceding outbreaks. The phylogenetic analysis of the nucleotide sequence encoding the GP5 envelope glycoprotein and the nucleocapsid protein coding sequences (ORF5 and ORF7, respectively) showed a close genetic relationship between the new outbreak-related and one of the vaccine viruses, while the prevailing PRRS virus genetic variants disappeared from the farm. These findings, supported by the epidemiological data, indicate that the new variant PRRS viruses might originate from a vaccine virus and demonstrate the limited efficacy of modified live vaccines against heterologous PRRS virus strains.

Konjak mosaic virus: the complete nucleotide sequence of the genomic RNA and its comparison with other potyviruses.

Konjak mosaic virus (KoMV) belongs to the genus Potyvirus, family Potyviridae. The complete nucleotide sequence of KoMV F isolate (KoMV F) was determined. The genome is 9,544 nucleotides long excluding the 3' terminal poly A tail and encodes a typical potyviral 350-kDa polyprotein of 3,087 amino acids. Phylogenetic analysis using known potyvirus polyproteins shows that KoMV constitutes a branch with yam mosaic virus, close to another branch including Japanese yam mosaic virus, turnip mosaic virus, scallion mosaic virus and lettuce mosaic virus. The 3' terminal 1,842 nucleotides of a different isolate of KoMV, K-2, was also determined, covering the C-terminal 292 amino acids of the nuclear inclusion protein b (NIb), coat protein (CP), and the 3' untranslated region. The amino acid sequences of the KoMV F CP and the nucleotide sequences of the KoMV F 3' untranslated region showed 92.5 and 90.5% identity to the corresponding genes of K-2, 88.7-96.8 and 92.7-94.4% to those of Zantedeschia mosaic virus (ZaMV) isolates, 87.5-89.7% and 85.5-90.3% to those of Japanese hornwort mosaic virus (JHMV) isolates. These results showed that KoMV is a distinct potyvirus and that KoMV, ZaMV, and JHMV are members of the same potyvirus species. Considering that KoMV was the first of these to be described, ZaMV and JHMV may be considered isolates of KoMV.

Enhancement of ultraviolet-induced apoptosis by NF-kappaB decoy oligonucleotides.

BACKGROUND: A decoy strategy utilizing oligonucleotides (ODN) containing the specific binding sequence of a certain transcription factor has been developed and is considered to be a potential new class of antigene therapy. However, the application of this new therapeutic modality to skin diseases has not been fully documented. OBJECTIVES: The aim of this work was to examine the effects of the nuclear factor (NF)-kappaB decoy ODN on UV-elicited skin change. METHODS: Mouse keratinocyte Pam 212 cells were transfected with NF-kappaB decoy ODN to examine the effects of the decoy ODN on ultraviolet (UV) B-induced apoptosis. Tape-stripped rat dorsal skin was treated with an ointment containing NF-kappaB decoy ODN for the examination of the in vivo impact of the decoy ODN on sunburned cell (SBC) formation and UVB erythema. RESULTS: NF-kappaB decoy ODN specifically induced apoptosis of Pam 212 cells and SBC formation was significantly enhanced by topical NF-kappaB decoy ODN ointment, while UV-induced erythema was not affected. CONCLUSIONS: These data suggest that enhancement of UV-induced apoptosis by NF-kappaB decoy ODN may play a cancer-preventive role by further eliminating photodamaged keratinocytes.

Morphology of the articular eminence in temporomandibular joints and condylar bone change.

The purpose of the present study was to investigate the relationship between the inclination of the articular eminence and temporomandibular joint (TMJ) pathology in orthognathic surgery patients with signs and symptoms of TMJ disorders. Twenty-one female orthognathic surgery patients with signs and symptoms of TMJ disorders were examined using pre-treatment helical computed tomography scans. The slope of the eminence in the medial, central and lateral sections of the subjects with osteophyte formation was significantly less than in the subjects with no bone change, and the medial section of the subjects with osteophyte formation was also significantly less steep than in the subjects with erosion. The central and lateral sections in the subjects with anterior disc displacement with reduction were significantly steeper than in subjects with anterior disc displacement without reduction. These results suggest that eminence flattening might occur during changes from erosion to osteophyte formation and from anterior disc displacement with reduction to anterior disc displacement without reduction. This appears to represent adaptation of the condyle, articular disc and articular eminence to changes in loading.

Photodynamic therapy may be useful in debulking cutaneous lymphoma prior to radiotherapy.

Photodynamic therapy (PDT) with topical 5-aminolaevulinic acid (5-ALA) is a promising new treatment for superficial malignant nonmelanoma tumours, including cutaneous malignant lymphoma. Here, we report a case of cutaneous anaplastic large cell lymphoma effectively treated by PDT with topical 5-ALA in combination with radiotherapy.

Thickness of the roof of the glenoid fossa and condylar bone change: a CT study.

OBJECTIVES: The aim of this study is to investigate the relationship between the thickness of the roof of the glenoid fossa in the temporomandibular joint (TMJ) and the existence and types of condylar bone change. MATERIALS AND METHODS: Helical CT was used to measure the thickness of the roof of the glenoid fossa at its thinnest part in 37 orthodontic patients with temporomandibular disorders. Condylar bone changes were classified into four types: no bone change (24 joints); flattening (19 joints); osteophyte formation (13 joints); and erosion (18 joints). RESULTS: The roof of the glenoid fossa was significantly thicker in joints with bone change than in joints with no bone change (Mann-Whitney U-test, P<0.05). There was also a significant difference in relation to the type of condylar bone change: the thickness of the roof of the glenoid fossa in the erosion group was significantly greater than in the no bone change (P<0.01), flattening (P<0.05) and osteophyte formation (P<0.05) groups (Kruskal-Wallis and Games-Howell tests). CONCLUSION: Compensative bone formation in the roof of the glenoid fossa might help to withstand the increased stress in the TMJ accompanying condylar bone change, especially erosion.

Mandibular movement and frontal craniofacial morphology in orthognathic surgery patients with mandibular deviation and protrusion.

The present study was conducted to investigate the relationship between mandibular movement (lateral excursion and masticatory movements) and craniofacial morphology in 16 patients with mandibular deviation, using a six degrees-of-freedom measuring device. (i) Mandibular deviation was found to be significantly related to frontal maxillary and occlusal plane angles. (ii) Three-dimensional non-working condylar and incisal path lengths were longer during the lateral excursion to the non-deviated side than to the deviated side, and the incisal path moved antero-inferior. (iii) The lateral motion range of the incisal path was wider during masticatory movement on the non-deviated side than on the deviated side, and the molar and non-working condylar path lengths corresponding to the lateral range of the incisal path were also longer on the non-deviated side. The group with posterior crossbite showed a significantly smaller horizontal range of incisal path, and also significantly smaller frontal projected incisal and molar path angles during masticatory movement on the deviated side than on the non-deviated side. These results suggest that lateral excursion and masticatory movements could be related to craniofacial morphology and posterior crossbite.

Human cytomegalovirus glycoprotein N (gpUL73-gN) genomic variants: identification of a novel subgroup, geographical distribution and evidence of positive selective pressure.

Human cytomegalvirus (HCMV) ORF UL73 is a polymorphic locus, encoding the viral glycoprotein gpUL73-gN, a component of the gC-II envelope complex. The previously identified gN genomic variants, denoted gN-1, gN-2, gN-3 and gN-4, were further investigated in this work by analysing a large panel of HCMV clinical isolates collected from all over the world (223 samples). Sequencing and phylogenetic analysis confirmed the existence of the four gN genotypes, but also allowed the identification of a novel subgroup belonging to the gN-3 genotype, which was designated gN-3b. The number of non-synonymous (d(N)) and synonymous (d(S)) nucleotide substitutions and their ratio (d(N)/d(S)) were estimated among the gN genotypes to evaluate the possibility of positive selection. Results showed that the four variants evolved by neutral (random) selection, but that the gN-3 and gN-4 genotypes are maintained by positive selective pressure. The 223 HCMV clinical isolates were subdivided according to their geographical origin, and four main regions of gN prevalence were identified: Europe, China, Australia and Northern America. The gN variants were found to be widespread and represented within the regions analysed without any significant difference, and no new genotype was detected. Finally, for clinical and epidemiological purposes, a rapid and low-cost method for genetic grouping of the HCMV clinical isolates was developed based on the RFLP revealed by SacI, ScaI and SalI digestion of the PCR-amplified UL73 sequence. This technique enabled us to distinguish all four gN genomic variants and also their subtypes.

Tamoxifen agonism and estrogen antagonism of c-fos gene promoter activity through non-consensus-responsive elements in MC3T3-E1 osteoblasts.

We find that the activity of a 0.4-kb human c-fos gene promoter (-404/+41), which lacks consensus estrogen-responsive elements (EREs), is regulated by estrogen receptor (ER) ligands in MC3T3-E1 osteoblastic cells through ERs in a manner distinct from ERE-mediated regulation. When ERalpha is coexpressed, both estrogens and antiestrogens upregulate promoter activity. When ERbeta is coexpressed, however, three tested antiestrogens affect c-fos promoter activity, with tamoxifen exerting the greatest effect, while estrogens have no such effect. The tamoxifen agonism through ERbeta is antagonized by 17beta-estradiol, while the 17beta-estradiol agonism through ERalpha is canceled by excess-level coexpression of ERbeta. Deletion analysis revealed that the sequence -206/-110 plays a crucial role in the ERbeta-mediated tamoxifen agonism. Interestingly, there is no ERbeta-mediated tamoxifen agonism when nonosteoblastic cells are tested. Taken together, these results suggest that the transcription of the c-fos gene is regulated by ER ligands possibly through non-ERE elements in ligand structure-, cell type-, and ER subtype-dependent manners.

Role of stromal cells in osteoclast differentiation in bone marrow.

Bone marrow stromal cells have been considered to play an important role in osteoclast differentiation. However, the interaction of these cells in vivo has not been clearly demonstrated. To clarify this, we examined the distribution of alkaline phosphatase (ALPase) and tartrate-resistant acid phosphatase (TRAPase) activities as markers of osteoblastic and osteoclastic cells, respectively. Rat tibiae were fixed and embedded in Technovit 8100 or paraffin. ALPase and TRAPase activities were detected simultaneously on a plastic section by the azo-dye method. ALPase activity was detected on the plasma membranes of osteoblasts and some bone marrow fibroblastic stromal cells. These ALPase-positive cells were connected to each other by cytoplasmic processes, forming a cellular network in bone marrow. The ALPase activity of fibroblastic stromal cells tended to be stronger in those cells close to the bone surface than in the cells in the center of bone marrow. Reticular fibers in bone marrow were found to form a network. The ALPase-positive fibroblastic stromal cells may be reticular cells, because the localization of those cells was in accord with the localization of reticular fibers. The TRAPase-positive mononuclear cells and osteoclasts were mostly observed to be associated with the intensely ALPase-positive fibroblastic stromal cells. Immunoreactivity of osteoclast differentiation factor (ODF) was found in the fibroblastic stromal cells. These findings suggest that the network of ALPase-positive fibroblastic stromal cells in bone marrow serves as a guide for the migration of osteoclast precursor cells toward the bone surface, and may control the differentiation and activity of osteoclasts.

Condylar bony change and self-reported parafunctional habits in prospective orthognathic surgery patients with temporomandibular disorders.

OBJECTIVE: The purpose of this study was to investigate the relationship between self-reported parafunctional habits and condylar bony change and disk displacement in orthognathic surgery patients with signs and symptoms of temporomandibular joint disorders. STUDY DESIGN: This is a cross-sectional retrospective study of pretreatment helical computed tomography scans and questionnaires of 94 female orthognathic surgery patients. RESULTS: Condylar bony change, unilaterally or bilaterally, was found in 56.4% of the subjects, or 43.6% of the joints. Disk displacement, unilaterally or bilaterally, was seen in 59.6% of the subjects, or 45.7% of the joints. Bruxism and clenching was significantly associated with condylar bony change and disk displacement. Subjects with 3 or more parafunctional habits showed a significantly higher rate of bilateral condylar bony change. CONCLUSION: Our results suggest that bruxism and clenching might be related to deterioration of the temporomandibular joint and that the greater the number of parafunctional habits a subject has, the higher the risk of condylar bony change.