HIV-associated CD8+ T-cell Skin Infiltrative Disease and EBV-associated Polymorphic B-cell Lymphoproliferative Disorder in an AIDS Patient Who Improved Dramatically with Antiretroviral Therapy Alone.

Human immunodeficiency virus (HIV)-associated CD8+ T-cell skin infiltrative disease with severe erythroderma has rarely been reported. While HIV-positive patients are prone to develop lymphoma, which is often associated with Epstein-Barr virus, polymorphic lymphoproliferative disorder is rare, accounting for <5% of cases. We herein report a 41-year-old HIV-positive man who presented with a fever, erythroderma, and lymphadenopathy and was diagnosed with the coexistence of both diseases. His condition improved significantly with continued antiretroviral therapy. This case suggests that HIV-induced immunodeficiency is central to the pathogenesis of both entities and that improvement of the immunodeficient state is an effective treatment.

Efficient detection of somatic UBA1 variants and clinical scoring system predicting patients with variants in VEXAS syndrome.

OBJECTIVES: To efficiently detect somatic UBA1 variants and establish a clinical scoring system predicting patients with pathogenic variants in VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome. METHODS: Eighty-nine Japanese patients with clinically suspected VEXAS syndrome were recruited [81 males and 8 females; median onset age (IQR) 69.3 years (62.1-77.6)]. Peptide nucleic acid-clamping PCR (PNA-PCR), regular PCR targeting exon 3 clustering UBA1 variants, and subsequent Sanger sequencing were conducted for variant screening. Partitioning digital PCR (pdPCR) or targeted amplicon deep sequencing (TAS) was also performed to evaluate the variant allele frequency (VAF). We developed our clinical scoring system to predict UBA1 variant-positive and ­negative patients and assessed the diagnostic value of our system using receiver operating characteristic (ROC) curve analysis. RESULTS: Forty patients with reported pathogenic UBA1 variants (40/89, 44.9%) were identified, including a case having a variant with VAF of 1.7%, using a highly sensitive method. Our clinical scoring system considering >50 years of age, cutaneous lesions, lung involvement, chondritis, and macrocytic anaemia efficiently predicted patients with UBA1 variants (the area under the curve for the scoring total was 0.908). CONCLUSIONS: Genetic screening with the combination of regular PCR and PNA-PCR detected somatic UBA1 variants with high sensitivity and specificity. Our scoring system could efficiently predict patients with UBA1 variants.

A Case of Recalcitrant Phaeohyphomycosis of the Face Caused by Exophiala lecanii-corni.

We describe a case of recalcitrant phaeohyphomycosis caused by Exophiala lecanii-corni, which was previously reported as Exophiala jeanselmei, infection. A 63-year-old Japanese woman presented with a 15-year history of multiple pruritic erythematous patches and plaques on the face. Histopathological examination and fungal culture revealed phaeohyphomycosis by E. jeanselmei. The attempted treatments included 6 g/day 5-flucytosine (5-FC), 100 mg/day itraconazole (ITCZ), and local hyperthermia. 5-FC was effective initially, but the patient deteriorated after discontinuation. Subsequently, she was referred to our hospital. Histopathological examination showed granuloma with multinucleated giant cells with infiltrating fungal hyphae in the dermis. The causative fungus was finally identified as E. lecanii-corni by ribosomal RNA gene analysis. The patient improved after receiving 200 mg/day ITCZ orally for 15 months with local hyperthermia. In the present case, we confirmed the identification of E. lecanii-corni as the causative agent by molecular methods. We also emphasize the importance of combination therapy with antimycotic agents and local hyperthermia in phaeohyphomycosis.

Development of functional agricultural products utilizing the new health claim labeling system in Japan.

In April 2015, Consumer Affairs Agency of Japan launched a new food labeling system known as "Foods with Function Claims (FFC)." Under this system, the food industry independently evaluates scientific evidence on foods and describes their functional properties. As of May 23, 2017, 1023 FFC containing 8 fresh foods have been launched. Meanwhile, to clarify the health-promoting effects of agricultural products, National Agriculture and Food Research Organization (NARO) implemented the "Research Project on Development of Agricultural Products" and demonstrated the risk reduction of osteoporosis of ß-cryptoxanthin rich Satsuma mandarins and the anti-allergic effect of the O-methylated catechin rich tea cultivar Benifuuki. These foods were subsequently released as FFC. Moreover, NARO elucidated the health-promoting effects of various functional agricultural products (ß-glucan rich barley, ß-conglycinin rich soybean, quercetin rich onion, etc.) and a healthy boxed lunch. This review focuses on new food labeling system or research examining functional aspects of agricultural products.

Preparation of Phi29 DNA polymerase free of amplifiable DNA using ethidium monoazide, an ultraviolet-free light-emitting diode lamp and trehalose.

We previously reported that multiply-primed rolling circle amplification (MRPCA) using modified random RNA primers can amplify tiny amounts of circular DNA without producing any byproducts. However, contaminating DNA in recombinant Phi29 DNA polymerase adversely affects the outcome of MPRCA, especially for negative controls such as non-template controls. The amplified DNA in negative control casts doubt on the result of DNA amplification. Since Phi29 DNA polymerase has high affinity for both single-strand and double-stranded DNA, some amount of host DNA will always remain in the recombinant polymerase. Here we describe a procedure for preparing Phi29 DNA polymerase which is essentially free of amplifiable DNA. This procedure is realized by a combination of host DNA removal using appropriate salt concentrations, inactivation of amplifiable DNA using ethidium monoazide, and irradiation with visible light from a light-emitting diode lamp. Any remaining DNA, which likely exists as oligonucleotides captured by the Phi29 DNA polymerase, is degraded by the 3'-5' exonuclease activity of the polymerase itself in the presence of trehalose, used as an anti-aggregation reagent. Phi29 DNA polymerase purified by this procedure has little amplifiable DNA, resulting in reproducible amplification of at least ten copies of plasmid DNA without any byproducts and reducing reaction volume. This procedure could aid the amplification of tiny amounts DNA, thereby providing clear evidence of contamination from laboratory environments, tools and reagents.

Karyotyping of barley chromosomes by a new fluorescence banding technique combined with scanning probe microscopy.

Fluorescence banding has been used to classify chromosomes, except those of barley. Four of the seven barley chromosomes are indistinguishable by length or arm ratio. C-banding has been used for classification; however, it requires a long aging period. Here, we describe a new fluorescence banding method for barley. The chromosomes are treated with warm acetate followed by staining with a fluorescent dye, YOYO-1. Using this method, all seven barley chromosomes can be clearly distinguished. Atomic force microscopy and scanning near-field microscopy analyses revealed that the surfaces of the banded chromosomes were flat, indicating that the fluorescence intensity reflected the internal DNA density or condensation of chromatin.

Effect of sectioning and water on resin-embedded sections of corn starch granules to analyze inner structure.

Resin-embedded sections and paired block surface of corn starch granules were observed using atomic force microscopy (AFM) and scanning electron microscopy to analyze the fine inner structure of starch granules and observe artifacts. Wrinkles were formed on the starch surfaces because of shear stress caused by the knife. Sectioned starches were isotropically expanded by water, and the growth rings and cracks between the growth rings were observed only on the sections. From this result, it was considered that the growth rings clearly showed shrinkage and/or corrosion of both edges of the ring during drying of the sections. Moreover, many small particles (width, ∼30 nm; height, several nanometers) were clearly observed on the growth rings. These particles could be single clusters (∼10 nm) of amylopectin molecules, considering the effect of the AFM tip radius.

A silanized mica substrate suitable for high-resolution fiber FISH analysis by scanning near-field optical/atomic force microscopy.

We applied a novel silanized mica substrate with an extremely flat surface constructed according to Sasou et al. (Langmuir 19, 9845-9849 (2003)) to high-resolution detection of a specific gene on a DNA fiber by scanning near-field optical/atomic force microscopy (SNOM/AFM). The interaction between the substrate and fluorescence-dye conjugated peptide nucleic acid (PNA) probes, which causes fluorescence noise signal, was minimal. By using the substrate, we successfully obtained a fluorescence in situ hybridization signal from the ea47 gene on a λphage DNA labeled with an Alexa 532-conjugated 15-base PNA probe. As the results, no fluorescence noises were observed, indicating that the surface adsorbed almost none of the PNA probe. The combination of the substrate and SNOM/AFM is an effective tool for visualizing DNA sequences at nanometer-scale resolution.

Cell-free cloning using multiply-primed rolling circle amplification with modified RNA primers.

The predominant method for DNA cloning is by propagation in biological hosts, but this method has limitations because certain sequences are difficult to clone using any combination of available hosts or vectors. Recently, multiply-primed rolling circle amplification (MPRCA) has been applied to overcome the problems of the DNA cloning via host cells. However, when MPRCA is used to amplify from minute quantities of DNA template, the products are mostly by-product DNA molecules generated by false priming and primer dimer formation. This study demonstrates that MPRCA using random RNA primers[#x02014]instead of DNA primers[#x02014]blocked the synthesis of by-products and succeeded in amplifying one copy of a circular DNA molecule more than 1012-fold to give microgram quantities of amplification product without using submicroliter reaction volumes. Furthermore, a ligation strategy was elaborated to circularize only the desired DNA sequence and eliminate undesired ligation-products. A combination of these methods was able to amplify and ligate a large construct without undesired DNA sequences and at microgram quantities within one day. Therefore, these methods have the possibility to improve DNA cloning techniques that have been restricted by the limitations of PCR methods or by the host cell.

Methods for reducing nonspecific interaction in antibody-antigen assay via atomic force microscopy.

We developed a method to measure the rupture forces between antibody and antigen by atomic force microscopy (AFM). Previous studies have reported that in the measurement of antibody-antigen interaction using AFM, the specific intermolecular forces are often obscured by nonspecific adhesive binding forces between antibody immobilized cantilever and substrate surfaces on which antigen or nonantigen are fixed. Here, we examined whether detergent and nonreactive protein, which have been widely used to reduce nonspecific background signals in ordinary immunoassay and immunoblotting, could reduce the nonspecific forces in the AFM measurement. The results showed that, in the presence of both nonreactive protein and detergent, the rupture forces between anti-ferritin antibodies immobilized on a tip of cantilever and ferritin (antigen) on the substrate could be successfully measured, distinguishing from nonspecific adhesive forces. In addition, we found that approach/retraction velocity of the AFM cantilever was also important in the reduction of nonspecific adhesion. These insights will contribute to the detection of specific molecules at nanometer scale region and the investigation of intermolecular interaction by the use of AFM.

Clinical effects of undershirts coated with borage oil on children with atopic dermatitis: a double-blind, placebo-controlled clinical trial.

It has been reported that gamma-linolenic acid contained in borage oil is effective against atopic dermatitis. The clinical effects of undershirts coated with borage oil rich in gamma-linolenic acid on atopic dermatitis were evaluated. Thirty-two children, aged 1-10 years, were involved in the clinical control study. Sixteen had worn undershirts coated with borage oil everyday for 2 weeks, and 16 had worn non-coated undershirts as a placebo. Their symptoms were assessed on a 4-point scale. Those children who had worn undershirts coated with borage oil for 2 weeks showed improvements in their erythema and itch, which were statistically significant. Transepidermal water loss from the back was decreased. In the placebo group, there were no statistically significant differences. The undershirts coated with borage oil were found to be statistically effective, and had no side-effects on children with mild atopic dermatitis.

Infectious mononucleosis-like syndrome induced by salazosulfapyridine in a patient with rheumatoid arthritis.

We describe a 43-year-old woman with rheumatoid arthritis (RA), who developed severe infectious mononucleosis (IM)-like syndrome during treatment with salazosulfapyridine (SASP). She presented with fever, skin rash, lymphadenopathy, and hepatosplenomegaly. Laboratory tests revealed a marked increase of atypical lymphocytes in the peripheral blood and biphasic hepatic dysfunction. IM-like syndrome can be caused by various drugs, including SASP, and the concept of drug-induced hypersensitivity syndrome has been proposed recently. IM-like syndrome due to SASP has been reported in patients taking higher dosages for the treatment of inflammatory bowel disease, but has not been reported earlier in patients with RA. The results of the drug-induced lymphocyte stimulation test tests suggested that 5-aminosalicylic acid was a possible causative metabolite. This severe type of drug-induced hypersensitivity reaction mimicking IM due to SASP should be granted wider awareness in the field of rheumatology, because the drug is widely used for the treatment of RA.

Induction of PDGF-B in TCA-treated epidermal keratinocytes.

Trichloroacetic acid (TCA) is one of the most widely used peeling agents, and induces full necrosis of the whole epidermis, followed by reconstitution of the epidermis and the matrix of the papillary dermis. The cytotoxic effects of TCA, such as suppressing proliferation of keratinocytes and fibroblasts and protein synthesis by fibroblasts, have already been reported. However, the entire biological mechanism responsible for TCA peeling has yet to be determined. Hypothetical activation effects of TCA treatment on epidermal cells to induce production of growth factors and cytokines are examined, and are compared with its cytotoxic effects in terms of time course and applied TCA concentrations. After various periods of incubation with TCA, viability of Pam212 murine keratinocytes was investigated with MTT assay and dye exclusion assay, and production of growth factors and cytokines with reverse transcription-polymerase chain reaction (RT-PCR). Changes in platelet-derived growth factor (PDGF)-B mRNA expression and protein production in the human skin specimens after TCA application were then examined by RT-PCR and immunohistochemistry, respectively. Incubation with TCA showed cytotoxicity and induced death of Pam212 cells, depending on the incubation period and the TCA concentration. In addition, expressions of PDGF-B, tumor growth factor (TGF)-alpha, TGF- beta1 and vascular endothelial growth factor, which are the growth factors reportedly secreted from keratinocytes during wound healing, were all detected in Pam212 cells after short-term treatment with TCA. Expressions of inflammatory cytokines such as interleukin (IL)-1 and IL-10 were also induced. In TCA-treated NIH-3T3 fibroblasts, in contrast, observed was upregulation of only keratinocyte growth factor, which is reportedly secreted from fibroblasts, as well as the similar cytotoxic effect. In human skin, PDGF-B mRNA expression became significantly upregulated after TCA application, and then immediately downregulated. Immunoreactive PDGF-B in the cytoplasm of keratinocytes became detectable throughout the epidermis after TCA application, reached maximum after the peak of mRNA expression, and then declined significantly over 24 h when the epidermis became completely necrotic. The TCA-treated epidermis acts as a major source of growth factors, including PDGF-B, before undergoing full necrosis. This effect might contribute to a promotion of re-epithelialization and dermal regeneration without wound contraction and scarring.

Chromatin dynamics of unfolding and refolding controlled by the nucleosome repeat length and the linker and core histones.

Chromatin is composed of genomic DNA and histones, forming a hierarchical architecture in the nucleus. The chromatin hierarchy is common among eukaryotes despite different intrinsic properties of the genome. To investigate an effect of the differences in genome organization, chromatin unfolding processes were comparatively analyzed using Schizosaccaromyces pombe, Saccharomyces cerevisiae, and chicken erythrocyte. NaCl titration showed dynamic changes of the chromatin. 400-1000 mM NaCl facilitated beads with approximately 115 nm in diameter in S. pombe chromatin. A similar transition was also observed in S. cerevisiae chromatin. This process did not involve core histone dissociation from the chromatin, and the persistence length after the transition was approximately 26 nm for S. pombe and approximately 28 nm for S. cerevisiae, indicating a salt-induced unfolding to "beads-on-a-string" fibers. Reduced salt concentration recovered the original structure, suggesting that electrostatic interaction would regulate this discrete folding-unfolding process. On the other hand, the linker histone was extracted from chicken chromatin at 400 mM NaCl, and AFM observed the "beads-on-a-string" fibers around a nucleus. Unlike yeast chromatin, therefore, this unfolding was irreversible because of linker histone dissociation. These results indicate that the chromatin unfolding and refolding depend on the presence and absence of the linker histone, and the length of the linker DNA.

Recessive dystrophic epidermolysis bullosa: case of non-Hallopeau-Siemens variant with premature termination codons in both alleles.

Dystrophic epidermolysis bullosa (DEB) is caused by mutations in the COL7A1 gene encoding collagen, the major component of anchoring fibrils. Premature termination codon (PTC) mutations in both alleles usually lead to the Hallopeau-Siemens variant that shows the most severe phenotype. We experienced a case of the non-Hallopeau-Siemens variant (nHS-RDEB), which had a mild clinical severity although it has PTC mutations in both alleles. Our patient was a compound heterozygote for a nonsense mutation (R669X) in exon 15 and a nonsense mutation (E2857X) in exon 116. But we confirmed the existence of some anchoring fibrils on electron micrograph. This suggested that a PTC close to the 3' end of COL7A1 does not completely abolish the collagen VII mRNA. We hypothesized that the truncated procollagen VII from the mutant allele with a nonsense mutation (E2857X) in exon 116 included two out of eight cysteines needed for disulfide bond formation, and hence a few functional anchoring fibrils could be formed.

Endotoxin shock due to Vibrio vulnificus infection.

A 54-year-old man with hepatitis C fell into shock with symptoms similar to enterocolitis after ingesting an undercooked barbecued mackerel. Most of his eruptions developed into annular erythema with small vesicles. He had taken high dose corticosteroids with intravenous cefotiam. His eruptions improved, but his shock state was exacerbated on Day 2. Treatment for endotoxin shock was initiated using piperacillin, intravenous immunoglobulin (IVIg), and hemoperfusion with Polymyxin B immobilized fiber (PMX-F), which resulted in shock reversal. The serum IL-6 value was 118,000 pg/mL on admission, and decreased to 2040 pg/mL on Day 3. On Day 6, the results from the culture of skin biopsy specimens showed the diagnosis as Vibrio vulnificus septic shock. Debridement was not needed, which is thought to be essential to Vibrio vulnificus sepsis. The changes in the serum IL-6 levels demonstrated that hemoperfusion with PMX-F and IVIg therapy was practical for Vibrio vulnificus septic shock.

Cyclophosphamide enhances TNF-alpha-induced apoptotic cell death in murine vascular endothelial cell.

Cyclophosphamide (CPA) is one of the therapeutic agents for systemic inflammatory disorders. In murine dermal endothelial cells (F-2), 4-hydroxycyclophosphamide (4-HC), which is active metabolite of CPA, enhanced TNF-alpha-induced DNA fragmentation. In addition, 4-HC was shown to elevate TNF-alpha-induced caspase-3 activation. Caspase-8 activation was identified by the treatment of TNF-alpha, whereas 4-HC was no effect. In contrast, only when treated with 4-HC, caspase-9 activation and the increase in the intracellular expression of Bax were detected. These results suggest that CPA may sensitize endothelial cells to TNF-alpha-induced apoptosis through a mitochondria-dependent pathway and clinically may contribute to the limitation of inflammatory process.

Comparative structural biology of the genome: nano-scale imaging of single nucleus from different kingdoms reveals the common physicochemical property of chromatin with a 40 nm structural unit.

Genome function is closely linked to the higher-order chromatin structures. To reveal a structural basis for the interphase chromatin organization, the 'on-substrate' lysis procedure was applied to nuclei isolated from human HeLa cells, chicken erythrocyte cells and yeast Schizosaccharomyces pombe, which possessed different intrinsic properties of the genomes such as histone composition and inter-nucleosomal distance. The isolated nuclei on a coverslip were successively treated with a detergent and a high-salt solution to extract the nuclear membrane and the nucleoplasm, and therefore, atomic force microscopy (AFM) visualized the structural changes in response to the lysis procedure. After the nucleoplasm was extracted, AFM clarified that chromatin fibers, approximately 40 nm in width, were partially released out of the nuclei and that the other chromatin still remaining in the nuclei was composed of granular structures with diameter of 80-100 nm. Thus, these results suggest that the approximately 40 nm fiber would be a stable structural unit and fold the 80-100 nm granules into a one-step higher unit. A common mechanism could be implied regardless of the intrinsic properties of the eukaryotic genomes.