Expression of collagen-related piRNA is dysregulated in cultured dermal fibroblasts derived from patients with scleroderma.

PIWI-interacting RNA (piRNA) is a class of recently discovered small non-coding RNAs. piRNAs derive from an initial transcript encompassing a piRNA cluster via a unique biosynthesis process, interact with PIWI proteins, bind to specific targets, and recruit chromatin modifiers to enable transcriptional repression. Abnormal expression of PIWI proteins and piRNAs has been reported in some human cancers, with participation of some PIWI/piRNAs complexes in tumorigenesis and association with cancer prognosis. Their expression in patients with systemic sclerosis (SSc) has not been widely elucidated. PIWI/piRNAs and their role in the pathogenesis of collagen accumulation in SSc was therefore investigated; no difference was found in the PIWIL1-4 levels between normal and cultured SSc dermal fibroblasts. Among piRNAs predicted to target SSc-related molecules, we first found significant piR-32364 up-regulation in SSc dermal fibroblasts, likely due to intrinsic TGF-ß signaling. Forced piR-32364 overexpression in normal fibroblasts significantly reduced COL1A1 expression both at mRNA and protein levels, but not COL1A2. Thus, piR-32364 overexpression in SSc fibroblasts may be the negative feedback against collagen up-regulation, which could suggest the potential of piRNAs as a therapeutic target.

Glycoprotein M6A upregulation detected by transcriptome analysis controls the proliferation of keloidal fibroblasts.

Hypertrophic scars and keloids are fibroproliferative disorders caused by abnormal wound healing. Their exact cause has not been found, but abnormalities during the wound healing process including inflammatory, immune, genetic, and other factors are thought to predispose an individual to excessive scarring. In the present study, we performed transcriptome analysis of established keloid cell lines (KEL FIB), focusing on gene expression analysis and fusion gene detection for the first time. For gene expression analysis, fragments per kilobase per million map read values were calculated, which were validated by real-time PCR and immunohistochemistry. Fusion genes were predicted by transcriptome sequence, and validated by Sanger sequence and G-banding. As a result, GPM6A was shown in the expression analysis to be upregulated in KEL FIB compared with normal fibroblasts. The GPM6A upregulation in KEL FIB was confirmed by real-time PCR, and GPM6A messenger ribonucleic acid expression was consistently significantly elevated in the tissues of hypertrophic scar and keloid compared to normal skin. Immunohistochemistry also revealed that the number of fibroblast-like spindle-shaped cells positive for GPM6A was significantly increased in keloidal tissues. GPM6A inhibition by small interfering ribonucleic acid significantly reduced the number of KEL FIB. On the other hand, although we hypothesized that fusion genes are involved in the pathogenesis of keloids, the transcriptome analysis could not prove the presence of fusion genes in KEL FIB. Taken together, GPM6A upregulation may have an inducible effect on cell proliferation in keloidal fibroblasts. GPM6A can be a novel therapeutic target in hypertrophic scars and keloids. The inflammatory nature may be more prominent in the pathogenesis of keloids, rather than being skin tumors, as proposed by Ogawa et al. Future studies using several cell lines will be required.

A rapid and facile preparation of APTS-labeled N-glycans by combination of ion pair-assisted extraction and HILIC-SPE for routine glycan analysis.

Glycoanalytical technology is required for a wide variety of scientific research, including basic glycobiological pharmaceutical, and biomarker research. Although several innovative analytical techniques have been developed for these purposes, quantitative glycan analysis based on electrophoretic separation, has often been impeded by the lack of cost-effective and facile sample preparation approaches. Here, we developed a rapid and facile sample preparation workflow for cost-effective glycan analysis and demonstrated its use with fully automated microchip electrophoresis (ME). Purification of 8-aminopyrene-1,3,6-trisulfonate (APTS)-labeled glycans was based on the combination of ion-pair assisted extraction (IPAE) with hydrophilic interaction chromatography-solid phase extraction (HILIC-SPE). Compared to commonly used sample preparation methods, the IPAE/HILIC-SPE method undergoes minimal nonspecific loss and undesirable degradation of N-glycans during the purification step. Furthermore, our method required only 10 min, and the entire workflow, including glycan release, labeling, and concentration processes was completed within 4 h. Although the present system should be improved to enable analysis of more complex mixtures, ME-based separation of APTS-labeled N-glycans offers a fully automated operation including conditioning, sample loading, separation, and can be analyzed with a sample-to-sample throughput of 120 s in parallel processes. The present workflow is easy to implement, does not require expensive reagents and instruments and may be useful for glycoscientists across disciplines.

The effect of hydroxychloroquine on lupus erythematosus-like skin lesions in MRL/lpr mice.

OBJECTIVES: To evaluate the effect and safety of hydroxychloroquine (HCQ) on lupus erythematosus (LE)-like skin lesions in the MRL/lpr mouse, a model for systemic LE (SLE). METHODS: We divided the MRL/lpr mice into three groups that were given: (1) drinking water, (2) HCQ at a dose of 4 mg/kg/d, or (3) HCQ at a dose of 40 mg/kg/d. The HCQ was administered to examine the effect and safety of HCQ on skin lesions and the number of infiltrating cells including mast cells in the dermis. RESULTS: Six of 13 mice in the group given drinking water, 3 of 11 mice in the group administered low-dose HCQ (4 mg/kg/d), and 1 of 10 mice in the group administered high-dose HCQ (40 mg/kg/d) presented the skin lesions. The average number of mast cells was 81, 50, and 12 (magnification, ×100), the mortality rate was 24%, 8%, and 9% and the mean body weight gain was 4.6 g, 8.0 g and 5.1 g, respectively. CONCLUSIONS: HCQ was demonstrated to decrease the appearance of LE-like lesions and the number of mast cells in the dermis. Furthermore, there were no obvious systemic adverse effects. This study provides evidence that suggests benefits in human patients.

Application of online preconcentration affinity capillary electrophoresis method to glycans labeled with 8-aminonaphthalene-1,3,6-trisulfonic acid using blue light emitting diode-induced fluorescence detection.

An online preconcentration technique, large volume sample stacking with an electroosmotic flow pump, was combined with partial filling affinity capillary electrophoresis (PFACE) to create a highly sensitive analysis of the interaction of glycoprotein-derived oligosaccharides with plant lectins. Oligosaccharides were derivatized with 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) for use in a blue light emitting diode-induced fluorescence detection capillary electrophoresis system. ANTS-labeled oligosaccharides were delivered to an entire neutrally coated capillary, and lectin solution was then hydrodynamically introduced from the outlet of the capillary as a short plug. When negative voltage was then applied, a low concentration sample solution caused a significant flow by electroosmosis from anode to cathode and the ANTS-labeled oligosaccharides moved quickly towards the anode and concentrated in the lectin phase. Finally, when the electroosmotic flow became negligible, ANTS-labeled saccharides passed through the lectin plug and were detected at the anodic end. The sensitivity was enhanced by a factor of roughly 200 compared to typical hydrodynamic injection (13.8 kPa, 5 s).

Partial filling affinity capillary electrophoresis using large-volume sample stacking with an electroosmotic flow pump for sensitive profiling of glycoprotein-derived oligosaccharides.

An online preconcentration technique, large-volume sample stacking with an electroosmotic flow pump (LVSEP) was combined with partial filling affinity capillary electrophoresis (PFACE) to realize highly sensitive analysis of the interaction of glycoprotein-derived oligosaccharides with some plant lectins. Oligosaccharides derivatized with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) were delivered to an entire neutrally-coated capillary and then lectin solution was hydrodynamically introduced from the outlet of the capillary as a short plug. A negative voltage was then applied after immersion of both ends of the capillary in 100 mM Tris-acetate buffer, pH 7.0 containing 0.5% hydroxypropylcellulose as electrophoresis buffers. A low concentration of electrolytes in the sample solution causes a significant flow by electroendosmosis from anode to cathode and the APTS-labeled oligosaccharides move quickly towards the anode and concentrate in the lectin phase. Finally, electroosmotic flow becomes negligible when the capillary is filled with the background electrolyte delivered from the anodic reservoir and APTS-labeled saccharides pass through the lectin plug and are detected at the anodic end. If the APTS-labeled oligosaccharides are recognized by the lectin, the migration profiles should be altered. The sensitivity was enhanced by a factor of ca. 900 compared to typical hydrodynamic injection (3.45 kPa, 10s). By this method, increased residence time of APTS-saccharides in the lectin plug indicates highly efficient interaction with lectins, which differs completely from the results obtained by ordinary lectin PFACE. The run-to-run repeatability (n=18) of the migration time and peak area was high, with relative standard deviations of less than 0.7% and 6.1%, respectively.

[Genetic involvement of bacterial sensor molecules in Japanese leprosy].

Occurrence of new patients of leprosy, caused by Mycobacterium leprae infection, is now almost absent in Japan but is still uncontrolled in developing countries. As one factor affecting the disease development, genetic predisposition of a host has been considered to be associated. Actually, various gene mutations have been reported to be associated at two stages of the disease progression, not only establishment of the disease but also determination of the phenotype, such as lepromatous (L)-type, tuberculoid (T)-type and reversal reaction. On the basis of recent progress of the research on innate immunity, here we analyzed single nucleotide polymorphisms (SNPs) of the genes of major bacterial sensor molecules expressed in antigen-presenting cells, TLR2, DC-SIGN, NOD1 and NOD2, in Japanese leprosy patients. As a result, frequency of polymorphisms in DC-SIGN -336 showed significant difference between the leprosy patients and the healthy controls, reflecting its role in establishment of the disease. Especially, among those with a particular TLR2 -16934 genotype, frequency of the polymorphisms in DC-SIGN -336 showed significant difference between the patients and the controls, suggesting any cooperation of these SNPs.