Successful use of differential target multiplexed spinal cord stimulation for chronic postsurgical abdominal pain.

Introduction: Recent advances in stimulation techniques have improved the efficacy and expanded the applicability of spinal cord stimulation (SCS). Among these techniques, there are no reports on the efficacy of differential target multiplexed (DTM) SCS for chronic postsurgical pain (CPSP) after abdominal surgery. Therefore, we present the successful use of DTM SCS for CPSP after distal pancreatectomy. Methods: A 49-year-old man with hypertension and severe chronic low back pain presented with neuropathic CPSP involving the left abdomen in the area of a laparotomy incision. His pain was refractory to conservative treatment and was rated 10 on a numerical rating scale (NRS). He underwent permanent implantation of a pulse generator after a 14-day trial stimulation. Results: Chronic postsurgical pain was well controlled (NRS 1-2) at a 3-month follow-up with DTM SCS. Conclusion: Differential target multiplexed SCS can be a new treatment option for neuropathic CPSP that is resistant to conservative treatment. It is important to further examine the characteristics of CPSP and identify appropriate candidates for the successful use of DTM SCS.

Low humidity altered the gene expression profile of keratinocytes in a three-dimensional skin model.

BACKGROUND: The skin is constantly exposed to various external stimuli including humidity variations. Low humidity affects skin properties such as decreased water content of the stratum corneum, reduced skin elasticity, and itching. However, the effects of humidity on the skin cells are not completely understood. This study aimed to investigate how low humidity affects keratinocytes of the skin. METHODS AND RESULTS: In the present study, the effects of dry environment on the gene expression profile of epidermal keratinocytes were demonstrated using a three-dimensional skin model (3D-skin), composed of keratinocytes. Exposure of 3D-skin to low humidity (relative humidity ~ 10%) increased the expression levels of various genes, including those related to signal transduction and immune system. Accordingly, p38 mitogen-activated protein kinase (MAPK) signaling in keratinocytes of the 3D-skin was activated in response to low humidity for 30 min. Additionally, several chemokines, such as chemokine (C-X-C motif) ligand 1 (CXCL1) and Chemokine (C-C motif) ligand 20 (CCL20), were up regulated after 3 h of exposure to low humidity. CONCLUSIONS: We hypothesize that increased chemokine production may affect the immune system of the whole skin through chemoattractants. Our findings imply that keratinocytes sense low humidity and resultant activation of some cell-signaling pathways leads to variations in gene expression profiles including various chemokines. We provide evidence that keratinocytes adapt to external humidity variations.

[Successful detection of MYD88 L265P mutation in Waldenström's macroglobulinemia complicated with myelofibrosis using highly sensitive digital PCR].

A 78-year-old man with anemia (Hb 9.6 g/dl) and elevated serum immunoglobulin M (IgM 3,577 mg/dl) levels was referred to our hospital. Bone marrow aspiration yielded a dry tap, and bone marrow biopsy revealed the infiltration of CD20 positive lymphoplasmacytic lymphoma cells and myelofibrosis. The patient was diagnosed with Waldenström's macroglobulinemia complicated with myelofibrosis. TGF-ß plasma concentration was elevated. Further, after chemotherapy with bendamustine and rituximab, remission of both Waldenström's macroglobulinemia and myelofibrosis was achieved, and TGF-ß levels normalized. MYD88 L265P mutation was detected using highly sensitive digital PCR, which compared with currently used direct PCR product sequencing, has a superior sensitivity. The use of digital PCR has additional advantages toward MYD88 L265P detection, particularly when the available amount of sample DNA is limited owing to myelofibrosis.

Laser-assisted cell removing (LACR) technology contributes to the purification process of the undifferentiated cell fraction during pluripotent stem cell culture.

Purification of undifferentiated cells by removing differentiated parts is an essential step in pluripotent stem cell culture. This process has been traditionally performed manually using a fine glass capillary or plastic tip under a microscope, or by culturing in a selective medium supplemented with anti-differentiation inhibitors. However, there are several inevitable problems associated with these methods, such as contamination or biological side-effects. Here, we developed a laser-assisted cell removing (LACR) technology that enables precise, fast, and contact-less cell removal. Using LACR combined with computational image recognition/identification-discriminating technology, we achieved automatic cell purification (A-LACR). Practicability of A-LACR was evaluated by two demonstrations: selective removal of trophoblast stem (TS) cells from human iPS and TS cell co-cultures, and purification of undifferentiated iPS cells by targeting differentiated cells that spontaneously developed. Our results suggested that LACR technology is a novel approach for stem cell processing in regenerative medicine.

[A Case of Essential Thrombocythemia with a JAK2V617F Mutation andPolymyalgia Rheumatica].

A patient with polymyalgia rheumatica(PMR)had increasing thrombocytosis. CRP levels, the ESR, and serum interleukin (IL)-6 levels were slightly elevated, and the patient tested negative for RF and anti CCP antibodies. Muscle pain was ameliorated with the administration of corticosteroids. Genetic analysis of the peripheral white blood cells demonstrated the presence of a JAK2V617F mutation. The muscle pain experienced by the patient was considered to be due to essential thrombocythemia( ET)of myeloproliferative neoplasms(MPNs)along with an inflammatory reaction. Unfortunately, the patient died suddenly because of cerebral infarction.

[A Case of Severe, Idiopathic Cytopenia of Undetermined Significance(ICUS)in an Elderly Patient].

Severe idiopathic cytopenia(initially neutropenia)of undetermined significance(ICUS)was identified in an elderly patient. Immature blasts and dysplastic cells were not observed, and the karyotypic analysis was normal. These hematological findings did not meet the minimal diagnostic criteria for myelodysplastic syndrome(MDS). Causative disease and drugs were excluded. Cases of ICUS that have the possibility of evolving into MDS are expected to increase in the elderly.

Identification of a hydrolyzable tannin, oenothein B, as an aluminum-detoxifying ligand in a highly aluminum-resistant tree, Eucalyptus camaldulensis.

Eucalyptus camaldulensis is a tree species in the Myrtaceae that exhibits extremely high resistance to aluminum (Al). To explore a novel mechanism of Al resistance in plants, we examined the Al-binding ligands in roots and their role in Al resistance of E. camaldulensis. We identified a novel type of Al-binding ligand, oenothein B, which is a dimeric hydrolyzable tannin with many adjacent phenolic hydroxyl groups. Oenothein B was isolated from root extracts of E. camaldulensis by reverse-phase high-performance liquid chromatography and identified by nuclear magnetic resonance and mass spectrometry analyses. Oenothein B formed water-soluble or -insoluble complexes with Al depending on the ratio of oenothein B to Al and could bind at least four Al ions per molecule. In a bioassay using Arabidopsis (Arabidopsis thaliana), Al-induced inhibition of root elongation was completely alleviated by treatment with exogenous oenothein B, which indicated the capability of oenothein B to detoxify Al. In roots of E. camaldulensis, Al exposure enhanced the accumulation of oenothein B, especially in EDTA-extractable forms, which likely formed complexes with Al. Oenothein B was localized mostly in the root symplast, in which a considerable amount of Al accumulated. In contrast, oenothein B was not detected in three Al-sensitive species, comprising the Myrtaceae tree Melaleuca bracteata, Populus nigra, and Arabidopsis. Oenothein B content in roots of five tree species was correlated with their Al resistance. Taken together, these results suggest that internal detoxification of Al by the formation of complexes with oenothein B in roots likely contributes to the high Al resistance of E. camaldulensis.

Demonstration of genome-wide association studies for identifying markers for wood property and male strobili traits in Cryptomeria japonica.

Genome-wide association studies (GWAS) are an alternative to bi-parental QTL mapping in long-lived perennials. In the present study, we examined the potential of GWAS in conifers using 367 unrelated plus trees of Cryptomeria japonica D. Don, which is the most widely planted and commercially important tree species in Japan, and tried to detect significant associations between wood property traits and quantity of male strobili on the one hand, and 1,032 single nucleotide polymorphisms (SNPs) assigned to 1,032 genes on the other. Association analysis was performed with the mixed linear model taking into account kinship relationships and subpopulation structure. In total, 6 SNPs were found to have significant associations with the variations in phenotype. These SNPs were not associated with the positions of known genes and QTLs that have been reported to date, thus they may identify novel QTLs. These 6 SNPs were all found in sequences showing similarities with known genes, although further analysis is required to dissect the ways in which they affect wood property traits and abundance of male strobili. These presumptive QTL loci provide opportunities for improvement of C. japonica, based on a marker approach. The results suggest that GWAS has potential for use in future breeding programs in C. japonica.

JAK2 46/1 haplotype is associated with JAK2 V617F-positive myeloproliferative neoplasms in Japanese patients.

Myeloproliferative neoplasms (MPNs) constitute a group of phenotypically diverse chronic myeloid malignancies, characterized by clonal hematopoiesis and excessive production of terminally differentiated myeloid blood cells. The MPNs include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), most of which are characterized by a somatic point mutation, V617F, in the janus kinase 2 (JAK2) gene. This mutation was recently shown to occur more frequently in a specific JAK2 haplotype, JAK2 46/1, in North American and European MPN patients. Little is known, however, about JAK2 haplotypes in Japanese MPN patients. Therefore, we examined 108 Japanese patients with MPN, including 19 with PV, 61 with ET, 10 with PMF, and 17 with unclassifiable MPN, as well as 104 control individuals for the JAK2 rs10974944(C/G) single nucleotide polymorphism, in which the G allele indicates the 46/1 haplotype. We found that the JAK2 46/1 haplotype was significantly more frequent in patients with V617F-positive MPN than in controls (odds ratio [OR], 3.6; 95 % confidence interval [CI], 2.2-5.8, p < 0.001), and in PV patients than in controls (OR, 6.3; 95 % CI, 3.0-29.4, p < 0.001). In conclusion, we demonstrated that the JAK2 46/1 haplotype is associated with JAK2 V617F-positive MPNs in Japanese patients.

Highly fluctuating thrombocytopenia developing in a patient with immune thrombocytopenia (ITP) while administering romiplostim.

A patient with immune thrombocytopenia (ITP) was administered a thrombopoietin (TPO) receptor agonist, romiplostim. After a step-wise increase the dose of romiplostim, the platelet count was highly fluctuated; it initially markedly increased, but then it subsequently steeply dropped. These fluctuations were observed three consecutive times. The administration of romipolostin was then discontinued. After the re-administration of romiplostim, the dose was gradually increased, and the platelet count rose steadily. The highly fluctuating platelet count in the present patient indicates the need for careful monitoring of the platelet count at the beginning of romiplostim administration.

A second generation framework for the analysis of microsatellites in expressed sequence tags and the development of EST-SSR markers for a conifer, Cryptomeria japonica.

BACKGROUND: Microsatellites or simple sequence repeats (SSRs) in expressed sequence tags (ESTs) are useful resources for genome analysis because of their abundance, functionality and polymorphism. The advent of commercial second generation sequencing machines has lead to new strategies for developing EST-SSR markers, necessitating the development of bioinformatic framework that can keep pace with the increasing quality and quantity of sequence data produced. We describe an open scheme for analyzing ESTs and developing EST-SSR markers from reads collected by Sanger sequencing and pyrosequencing of sugi (Cryptomeria japonica). RESULTS: We collected 141,097 sequence reads by Sanger sequencing and 1,333,444 by pyrosequencing. After trimming contaminant and low quality sequences, 118,319 Sanger and 1,201,150 pyrosequencing reads were passed to the MIRA assembler, generating 81,284 contigs that were analysed for SSRs. 4,059 SSRs were found in 3,694 (4.54%) contigs, giving an SSR frequency lower than that in seven other plant species with gene indices (5.4-21.9%). The average GC content of the SSR-containing contigs was 41.55%, compared to 40.23% for all contigs. Tri-SSRs were the most common SSRs; the most common motif was AT, which was found in 655 (46.3%) di-SSRs, followed by the AAG motif, found in 342 (25.9%) tri-SSRs. Most (72.8%) tri-SSRs were in coding regions, but 55.6% of the di-SSRs were in non-coding regions; the AT motif was most abundant in 3' untranslated regions. Gene ontology (GO) annotations showed that six GO terms were significantly overrepresented within SSR-containing contigs. Forty-four EST-SSR markers were developed from 192 primer pairs using two pipelines: read2Marker and the newly-developed CMiB, which combines several open tools. Markers resulting from both pipelines showed no differences in PCR success rate and polymorphisms, but PCR success and polymorphism were significantly affected by the expected PCR product size and number of SSR repeats, respectively. EST-SSR markers exhibited less polymorphism than genomic SSRs. CONCLUSIONS: We have created a new open pipeline for developing EST-SSR markers and applied it in a comprehensive analysis of EST-SSRs and EST-SSR markers in C. japonica. The results will be useful in genomic analyses of conifers and other non-model species.

The construction of a high-density linkage map for identifying SNP markers that are tightly linked to a nuclear-recessive major gene for male sterility in Cryptomeria japonica D. Don.

BACKGROUND: High-density linkage maps facilitate the mapping of target genes and the construction of partial linkage maps around target loci to develop markers for marker-assisted selection (MAS). MAS is quite challenging in conifers because of their large, complex, and poorly-characterized genomes. Our goal was to construct a high-density linkage map to facilitate the identification of markers that are tightly linked to a major recessive male-sterile gene (ms1) for MAS in C. japonica, a species that is important in Japanese afforestation but which causes serious social pollinosis problems. RESULTS: We constructed a high-density saturated genetic linkage map for C. japonica using expressed sequence-derived co-dominant single nucleotide polymorphism (SNP) markers, most of which were genotyped using the GoldenGate genotyping assay. A total of 1261 markers were assigned to 11 linkage groups with an observed map length of 1405.2 cM and a mean distance between two adjacent markers of 1.1 cM; the number of linkage groups matched the basic chromosome number in C. japonica. Using this map, we located ms1 on the 9th linkage group and constructed a partial linkage map around the ms1 locus. This enabled us to identify a marker (hrmSNP970_sf) that is closely linked to the ms1 gene, being separated from it by only 0.5 cM. CONCLUSIONS: Using the high-density map, we located the ms1 gene on the 9th linkage group and constructed a partial linkage map around the ms1 locus. The map distance between the ms1 gene and the tightly linked marker was only 0.5 cM. The identification of markers that are tightly linked to the ms1 gene will facilitate the early selection of male-sterile trees, which should expedite C. japonica breeding programs aimed at alleviating pollinosis problems without harming productivity.

[Severe immune thrombocytopenia caused by interferon-beta in a patient with malignant melanoma].

A 57-year-old woman was diagnosed with malignant melanoma three years ago. She received pre- and post-operative therapy with DAV chemotherapy and Feron (Feron®, interferon-b, IFN-b). Thereafter, she received a monthly local injection of Feron®. Severe thrombocytopenia developed three years later. Immune pathogenesis was suspected since platelet-associated immunoglobulin G (PAIGg) was increased and the administration of prednisolone (PSL) quickly ameliorated the thrombocytopenia. This is the first report of severe immune thrombocytopenia caused by IFN-b. Local injections of Feron® were resumed; however, thrombocytopenia was not observed.

[Acute leukemia of ambiguous lineage with monosomy 7 and Philadelphia chromosome].

A 67-year-old female was admitted with a diagnosis of acute leukemia. Immature blasts did not show cytoplasmic granules and were POX(-), ES(-), and PAS(+). Flow cytometry of leukemic cells demonstrated positivity for CD7, CD10, CD19, CD13, CD34, HLA-DR, and coexpression of CD7 and CD34, CD10 and HLA-DR, and CD19 and CD13. Cytogenetic analysis demonstrated -7 and t(9;22)(q34;q11.2), and genomic studies demonstrated minor BCR/ABL chimeric mRNA and rearrangements of IgH and TCR. These findings indicated the clonal proliferation of leukemic blasts that expressed a mixed phenotype. Acute leukemia of ambiguous lineage was diagnosed, although the significance of the specificity of lineage markers remains unclear. The differential diagnosis included CML and B-ALL. The patient was treated according to Ph+ALL. However, the hematological response was poor, with persistent residual blasts and severe pancytopenia. The subsequent administration of imatinib mesylate led to a complication of heart failure, and the patient died on the 19th hospital day.

[Neurological disturbance of the lower extremities by an extramedullary hematopoietic mass complicated with primary myelofibrosis].

A 59-year-old man with primary myelofibrosis developed motor and sensory neurological disturbance of the legs. Magnetic resonance imaging (MRI) demonstrated a mass lesion of the thoracic vertebra at Th4-6, and in the thoracic vertebral canal at Th4-9, which compressed the spinal cord. Needle biopsy of the mass lesion demonstrated extramedullary hematopoiesis. Initial treatment with bolus methylprednisolone was ineffective and, after subsequent radiation therapy, the mass lesion disappeared and the neurological symptoms ameliorated; however, regrowth of the extramedullary lesion was observed one month later. Surgical resection of the extramedullary lesion, laminectomy, and subsequent radiation were performed. The clinical course after the final treatment was good with no neurological symptoms, although the follow-up period is still short.