Arsenicicoccus cauae sp. nov., isolated from the blood of a pediatric gastroenteritis patient.

A Gram-stain-positive coccus was isolated from the blood of a paediatric patient suffering from gastroenteritis. The taxonomic position of this catalase-positive, non-motile, non-spore-forming facultative anaerobe designated as strain MKL-02T was investigated using a polyphasic approach. Colonies grown on tryptic soy agar with 10 % sheep blood were circular, creamy yellow, and convex. Phylogenetic analysis based on 16S rRNA gene and whole-genome sequences revealed that this strain was most closely related to Arsenicicoccus bolidensis CCUG 47306T within the cluster of the genus Arsenicicoccus. Average nucleotide identity and digital DNA-DNA hybridization values between strain MKL-02T and A. bolidensis DSM 15745T, A. dermatophillus DSM 25571T and A. piscis DSM 22760T were 89.5 and 37.0 %, 79.6 and 22.4 %, and 75.9 and 21.0 %, respectively. The genomic size of strain MKL-02T was 3 423 857 bp with a 72.7 mol% G+C content. Growth was observed at 10-45 °C (optimum, 37-40 °C) and pH 6.0-10.0 (optimum, pH 7.0), in the presence of 0-10 % (w/v) NaCl (optimum, 0.5 %). Cells of strain MKL-02T were non-motile cocci and 0.50-0.60 µm long, as determined by transmission electron microscopy. The strain was catalase-positive and oxidase-negative. The major fatty acid type (>10 % of total) was C15 : 0. The polar lipid profile consisted of two unidentified phospholipids, three unidentified lipids and an unidentified aminophospholipid. The strain contained MK-8 (H4) as the predominant menaquinone. Based on phylogenetic and phenotypic considerations, it is proposed that strain MKL-02T be classified as a new species, named Arsenicicoccus cauae sp. nov. The type strain is MKL-02T (=NCCP 16967T=JCM 34624T).

miR-542-3p Contributes to the HK2-Mediated High Glycolytic Phenotype in Human Glioma Cells.

(1) Background: The elevation of glucose metabolism is linked to high-grade gliomas such as glioblastoma multiforme (GBM). The high glycolytic phenotype is associated with cellular proliferation and resistance to treatment with chemotherapeutic agents in GBM. MicroRNA-542-3p (miR-542-3p) has been implicated in several tumors including gliomas. However, the role of miR-542-3p in glucose metabolism in human gliomas remains unclear; (2) Methods: We measured the levels of cellular proliferation in human glioma cells. We measured the glycolytic activity in miR-542-3p knockdown and over-expressed human glioma cells. We measured the levels of miR-542-3p and HK2 in glioma tissues from patients with low- and high-grade gliomas using imaging analysis; (3) Results: We show that knockdown of miR-542-3p significantly suppressed cellular proliferation in human glioma cells. Knockdown of miR-542-3p suppressed HK2-induced glycolytic activity in human glioma cells. Consistently, over-expression of miR-542-3p increased HK2-induced glycolytic activity in human glioma cells. The levels of miR-542-3p and HK2 were significantly elevated in glioma tissues of patients with high-grade gliomas relative to that in low-grade gliomas. The elevation of HK2 levels in patients with high-grade gliomas were positively correlated with the high levels of miR-542-3p in GBM and low-grade gliomas (LGG) based on the datasets from the Cancer Genome Atlas (TCGA) database. Moreover, the high levels of miR-542-3p were associated with poor survival rate in the TCGA database; (4) Conclusions: miR-542-3p contributes to the HK2-mediated high glycolytic phenotype in human glioma cells.

PF-04620110, a Potent Antidiabetic Agent, Suppresses Fatty Acid-Induced NLRP3 Inflammasome Activation in Macrophages.

BACKGROUND: Chronic inflammation has been linked to insulin resistance and type 2 diabetes mellitus (T2DM). High-fat diet (HFD)-derived fatty acid is associated with the activation of chronic inflammation in T2DM. PF-04620110, which is currently in phase 1 clinical trials as a selective acyl-CoA:diacylglycerol acyltransferase-1 (DGAT1) inhibitor, is a potent anti-diabetic agent that may be important for the regulation of chronic inflammation in T2DM. However, the mechanisms by which PF-04620110 regulates fatty acid-induced chronic inflammation remain unclear. METHODS: PF-04620110 was used in vitro and in vivo. DGAT1-targeting gRNAs were used for deletion of mouse DGAT1 via CRISPR ribonucleoprotein (RNP) system. The activation of NLRP3 inflammasome was measured by immunoblot or cytokine analysis in vitro and in vivo. RESULTS: Here we show that PF-04620110 suppressed fatty acid-induced nucleotide-binding domain, leucine-rich-repeat-containing receptor (NLR), pyrin-domain-containing 3 (NLRP3) inflammasome activation in macrophages. In contrast, PF-04620110 did not change the activation of the NLR family, CARD-domain-containing 4 (NLRC4), or the absent in melanoma 2 (AIM2) inflammasomes. Moreover, PF-04620110 inhibited K⁺ efflux and the NLRP3 inflammasome complex formation, which are required for NLRP3 inflammasome activation. PF-04620110 reduced the production of interleukin 1ß (IL-1ß) and IL-18 and blood glucose levels in the plasma of mice fed HFD. Furthermore, genetic inhibition of DGAT1 suppressed fatty acid-induced NLRP3 inflammasome activation. CONCLUSION: Our results suggest that PF-04620110 suppresses fatty acid-induced NLRP3 inflammasome activation.

DROSHA-Dependent AIM2 Inflammasome Activation Contributes to Lung Inflammation during Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) has been linked to chronic lung inflammation. Drosha ribonuclease III (DROSHA), a class 2 ribonuclease III enzyme, plays a key role in microRNA (miRNA) biogenesis. However, the mechanisms by which DROSHA affects the lung inflammation during idiopathic pulmonary fibrosis (IPF) remain unclear. Here, we demonstrate that DROSHA regulates the absent in melanoma 2 (AIM2) inflammasome activation during idiopathic pulmonary fibrosis (IPF). Both DROSHA and AIM2 protein expression were elevated in alveolar macrophages of patients with IPF. We also found that DROSHA and AIM2 protein expression were increased in alveolar macrophages of lung tissues in a mouse model of bleomycin-induced pulmonary fibrosis. DROSHA deficiency suppressed AIM2 inflammasome-dependent caspase-1 activation and interleukin (IL)-1ß and IL-18 secretion in primary mouse alveolar macrophages and bone marrow-derived macrophages (BMDMs). Transduction of microRNA (miRNA) increased the formation of the adaptor apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) specks, which is required for AIM2 inflammasome activation in BMDMs. Our results suggest that DROSHA promotes AIM2 inflammasome activation-dependent lung inflammation during IPF.

Circulating Cell-Free mtDNA Contributes to AIM2 Inflammasome-Mediated Chronic Inflammation in Patients with Type 2 Diabetes.

Mitochondrial dysfunction has been implicated in the pathogenesis of insulin resistance and type 2 diabetes. Damaged mitochondria DNA (mtDNA) may have a role in regulating hyperglycemia during type 2 diabetes. Circulating cell-free mitochondria DNA (ccf-mtDNA) was found in serum and plasma from patients and has been linked to the prognosis factors in various human diseases. However, the role of ccf-mtDNA in chronic inflammation in type 2 diabetes is unclear. In this study, we hypothesized that the ccf-mtDNA levels are associated with chronic inflammation in patients with type 2 diabetes. The mtDNA levels were elevated in the plasma from patients with type 2 diabetes compared to healthy subjects. The elevated mtDNA levels were associated with interleukin-1 (IL-1)ß levels in patients with type 2 diabetes. The mtDNA, from patients with type 2 diabetes, induced absent in melanoma 2 (AIM2) inflammasome-dependent caspase-1 activation and IL-1ß and IL-18 secretion in macrophages. Our results suggest that the ccf-mtDNA might contribute to AIM2 inflammasome-mediated chronic inflammation in type 2 diabetes.

Carbon monoxide regulates glycolysis-dependent NLRP3 inflammasome activation in macrophages.

Low dose of carbon monoxide (CO) has anti-inflammatory role through various signaling pathways. Cellular metabolism has been implicated in the activation of inflammation in immune cells. However, the mechanisms by which CO-dependent metabolic regulation affect the immune response remain unclear. Here we show that CO-dependent metabolic pathway regulates the activation of the nucleotide-binding domain, leucine-rich-repeat-containing receptor (NLR), pyrin-domain-containing 3 (NLRP3) inflammasome. CO-releasing molecule-3 (CORM-3) resulted in reduced glycolysis-dependent NLRP3 inflammasome activation in macrophages. The reduced mTORC1 activation by CORM-3 resulted in less glycolysis during NLRP3 inflammasome activation. CORM-3 suppressed caspase-1 activation and the secretion of interleukin (IL)-1ß and IL-18 in macrophages in response to lipopolysaccharide (LPS) and ATP. Moreover, CORM-3 inhibits the oligomerization of the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which is required for NLRP3-dependent caspase-1 activation. Furthermore, CORM-3-treated mice showed substantial reduction in IL-1ß production by hyperglycemia in a mouse model of streptozotocin (STZ)-induced diabetes. Our results suggest that CO regulates glycolysis-dependent NLRP3 inflammasome activation and may provide a therapeutic approach for inflammation in metabolic diseases.

Posterior Deltoid-to-Triceps Tendon Transfer for Elbow Extension in a Tetraplegia Patient: A Case Report.

In tetraplegia patients, activities of daily living are highly dependent on the remaining upper limb functions. In other countries, upper limb reconstruction surgery to improve function has been applied to diverse cases, but few cases have been reported in Korea. The current authors experienced a case of posterior deltoid-to-triceps tendon transfer and rehabilitation in a complete spinal cord injury with a C6 neurologic level, and we introduce the case-a 36-year-old man-with a literature review. The patient's muscle strength in C5 C6 muscles were normal, but C7 muscles were trace, and the Spinal Cord Independence Measure III (SCIM III) score was 24. The tendon of the posterior deltoid was transferred to the triceps brachii muscle, and then the patient received comprehensive rehabilitative treatment. His C7 muscle strength in the right upper extremity was enhanced from trace to fair, and his SCIM III score improved to 29.

Screening PCR Versus Sanger Sequencing: Detection of CALR Mutations in Patients With Thrombocytosis.

BACKGROUND: Mutations in calreticulin (CALR) have been reported to be key markers in the molecular diagnosis of myeloid proliferative neoplasms. In most previous reports, CALR mutations were analyzed by using Sanger sequencing. Here, we report a new, rapid, and convenient system for screening CALR mutations without sequencing. METHODS: Eighty-three bone marrow samples were obtained from 81 patients with thrombocytosis. PCR primers were designed to detect wild-type CALR (product: 357 bp) and CALR with type 1 (product: 302 bp) and type 2 mutations (product: 272 bp) in one reaction. The results were confirmed by Sanger sequencing and compared with results from fragment analysis. RESULTS: The minimum detection limit of the screening PCR was 10 ng for type 1, 1 ng for type 2, and 0.1 ng for cases with both mutations. CALR type 1 and type 2 mutants were detected with screening PCR with a maximal analytical sensitivity of 3.2% and <0.8%, respectively. The screening PCR detected 94.1% (16/17) of mutation cases and showed concordant results with sequencing in the cases of type 1 and type 2 mutations. Sanger sequencing identified one novel mutation (c.1123_1132delinsTGC). Compared with sequencing, the screening PCR showed 94.1% sensitivity, 100.0% specificity, 100.0% positive predictive value, and 98.5% negative predictive value. Compared with fragment analysis, the screening PCR presented 88.9% sensitivity and 100.0% specificity. CONCLUSIONS: This screening PCR is a rapid, sensitive, and cost-effective method for the detection of major CALR mutations.

A t(11;14)(p13;q11.2) in myelofibrosis following polycythemia vera.

Chromosomal abnormalities at 14q11, which encodes the T-cell receptor α and δ chain genes, are generally specific for T-cell malignancies, and are rarely reported in other malignancies. We report a novel t(11;14)(p13;q11.2) in a patient with myelofibrosis (MF) following polycythemia vera (PV). This 55-year-old male developed post-PV MF 12 years after the initial diagnosis of PV. He had a normal karyotype at polycythemic disease stage, t(11;14)(p13;q11.2) was newly detected at the time of fibrotic transformation. Therefore, it is likely that this clonal chromosomal abnormality was associated with progression of disease.

Prognostic impact of concordant and discordant bone marrow involvement and cell-of-origin in Korean patients with diffuse large B-cell lymphoma treated with R-CHOP.

AIMS: Previous studies have suggested many prognostic factors in diffuse large B-cell lymphoma (DLBCL), but the prognostic importance of cell-of-origin and discordant bone marrow involvement remains unclear. The aim of this study was to evaluate the prognostic impact of bone marrow involvement histological subtype, cell-of-origin subtype and international prognostic index (IPI) scores in patients with DLBCL. METHODS: Patients who were newly diagnosed with DLBCL and treated with rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) were analysed. Clinical information was reviewed retrospectively. Patients were classified into negative, concordant and discordant bone marrow involvement by histological review. The cell-of-origin types were defined using immunohistochemical analysis. RESULTS: Both concordant and discordant bone marrow involvement had a negative prognostic impact on progression-free survival, independent of the standard and National Comprehensive Cancer Network (NCCN) IPI scores and cell-of-origin. Patients with non-germinal centre B-cell type showed significantly shorter progression-free survival than those with germinal centre B-cell type. However, non-germinal centre B-cell type did not have a prognostic impact on progression-free survival or overall survival after controlling for the standard and NCCN-IPI and bone marrow involvement. CONCLUSIONS: Both concordant and discordant bone marrow involvement had an adverse prognostic impact on progression-free survival and overall survival; this was independent of the standard and NCCN-IPI and cell-of-origin (non-germinal centre B-cell type). The NCCN-IPI had more powerful prognostic value than the standard IPI (sIPI). The non-germinal centre B-cell type lost significant prognostic impact on progression-free survival after adjustment for standard and NCCN-IPI and bone marrow involvement.

Comparison of sputum and nasopharyngeal swabs for detection of respiratory viruses.

Diagnostic tests for respiratory viral infections use traditionally either nasopharyngeal washes or swabs. Sputum is representative of the lower respiratory tract but is used rarely for viral testing. The aim of this study was to compare the detection rates of respiratory viruses from nasopharyngeal swabs and sputum using a multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR). Adults who were admitted or presented to the clinics of Gil Medical Center with acute respiratory symptoms were recruited from 1 November 2012 to 31 March 2013. Paired specimens of nasopharyngeal swabs and sputum were obtained from 154 subjects, and RNA was extracted and tested for 16 different respiratory viruses using the Anyplex II RV16 Detection kit (Seegene, Seoul, Korea). The positive rate was 53% (81/154) for nasopharyngeal swabs and 68% (105/154) for sputum (P < 0.001). One hundred thirty-four viruses were identified for 107 illnesses. Influenza A virus, RSV A, HRV, coronavirus OC43, and adenovirus were detected more frequently in sputum samples than in nasopharyngeal swabs (P < 0.001). Importantly, 12 of 44 (27%) influenza A infections and 11 of 27 (41%) RSV infections were positive in only sputum samples. The detection rates of respiratory viruses from sputum samples were significantly higher than those from nasopharyngeal swabs in adults using real-time multiplex RT-PCR. These findings suggest that sputum would benefit for the detection of respiratory viruses by nucleic acid amplification tests (NAATs) in patients who produce sputum. Further studies are needed to establish standardized RNA extraction methods from sputum samples.

Comparison of platelet parameters in thrombocytopenic patients associated with acute myeloid leukemia and primary immune thrombocytopenia.

Thrombocytopenia is caused by insufficient production and excessive destruction of platelets. Recent improvement of automated blood cell analyzers has allowed measurement of several platelet parameters, providing better understanding of the underlying mechanisms of thrombocytopenia. We investigated the significance of platelet parameters in thrombocytopenic patients. Thrombocytopenic patients (platelet <100 × 10/µl) who were newly diagnosed with acute myeloid leukemia and primary immune thrombocytopenia were enrolled, and platelet, mean platelet volume, platelet distribution width, platelet crit, mean platelet component, mean platelet mass, and large platelet count were measured, and the percentages of large platelets were calculated. The parameters were also measured in the reference population. The mean values of each parameter were as follows: platelet, 259 × 10/µl; mean platelet volume, 7.9 fl; platelet distribution width, 51.3%; platelet crit, 0.20%; mean platelet component, 26.0 g/dl; mean platelet mass, 1.9 pg; large platelet, 4.7 × 10/µl; large platelet percentage, 1.7%. In comparison with acute myeloid leukemia patients, patients with primary immune thrombocytopenia showed significantly higher mean platelet volume, platelet distribution width, mean platelet component, mean platelet mass, large platelet, and large platelet % (P < 0.05). Because of increased destruction of platelets, primary immune thrombocytopenia patients have increased mean platelet volume, platelet distribution width, mean platelet component, mean platelet mass, large platelet, and large platelet percentage compared with acute myeloid leukemia patients who have ineffective platelet production. Parameters measured by automated analyzer provide better understanding of thrombopoiesis in the bone marrow and the status of the peripheral blood in the clinical field.

Nocardia brain abscess in an immunocompetent patient.

Nocardia cerebral abscess is rare, constituting approximately 1-2% of all cerebral abscesses. Mortality for a cerebral abscess of Nocardia is three times higher than that of other bacterial cerebral abscesses, therefore, early diagnosis and therapy is important. Nocardia cerebral abscess is generally occur among immunocompromised patients, and critical infection in immunocompetent patients is extremely rare. We report on a case of a brain abscess by Nocardia farcinica in an immunocompetent patient who received treatment with surgery and antibiotics. This is the second case of a brain abscess caused by N. farcinica in an immunocompetent patient in Korea.

Frequency of KRAS mutations in adult Korean patients with acute myeloid leukemia.

Mutation of KRAS genes occurs with a frequency of 0.5-32 % in AML. In the present study, mutations of KRAS codon 12, 13, and 61 were detected by pyrosequencing and direct sequencing in AML. Seven KRAS mutations (7/123, 5.7 %) were detected. The most common mutation was a G-to-A transition in the second base of KRAS codon 13. No mutations were detected in KRAS codon 61. Combinations of KRAS and FLT3 mutation were not found in the same patient. There was no statistically significant difference between patients with KRAS mutations and patients with wild-type KRAS in terms of sex, age, CBC at diagnosis, CD34 positivity, MPO positivity, FLT3 mutation, karyotype, progression-free survival, and overall survival, although this may be attributable to the small sample size. To our knowledge, this is the first report of the detection of KRAS mutation in Asian AML patients using pyrosequencing and direct sequencing. These two methods showed identical efficiencies in their ability to detect KRAS mutations in 84 patients.

N-ras mutation detection by pyrosequencing in adult patients with acute myeloid leukemia at a single institution.

BACKGROUND: N-ras mutations are one of the most commonly detected abnormalities of myeloid origin. N-ras mutations result in a constitutively active N-ras protein that induces uncontrolled cell proliferation and inhibits apoptosis. We analyzed N-ras mutations in adult patients with AML at a particular institution and compared pyrosequencing analysis with a direct sequencing method for the detection of N-ras mutations. METHODS: We analyzed 90 bone marrow samples from 83 AML patients. We detected N-ras mutations in codons 12, 13, and 61 using the pyrosequencing method and subsequently confirmed all data by direct sequencing. Using these methods, we screened the N-ras mutation quantitatively and determined the incidence and characteristic of N-ras mutation. RESULTS: The incidence of N-ras mutation was 7.2% in adult AML patients. The patients with N-ras mutations showed significant higher hemoglobin levels (P=0.022) and an increased incidence of FLT3 mutations (P=0.003). We observed 3 cases with N-ras mutations in codon 12 (3.6%), 2 cases in codon 13 (2.4%), and 1 case in codon 61 (1.2%). All the mutations disappeared during chemotherapy. CONCLUSIONS: There is a low incidence (7.2%) of N-ras mutations in AML patients compared with other populations. Similar data is obtained by both pyrosequencing and direct sequencing. This study showed the correlation between the N-ras mutation and the therapeutic response. However, pyrosequencing provides quantitative data and is useful for monitoring therapeutic responses.

The relationship between iron parameters and platelet parameters in women with iron deficiency anemia and thrombocytosis.

Changes of platelet count (PLT) and platelet parameters have been reported in iron deficiency anemia (IDA). However, the relationship between iron metabolism and thrombopoiesis is not yet fully known. We studied the relationship between iron and platelet parameters in women with IDA and thrombocytosis. Forty-one adult women with IDA and thrombocytosis were enrolled. The relationship between iron parameters (such as serum iron, serum ferritin, total iron-binding capacity (TIBC)C, and transferrin saturation (Tfsat)), and platelet parameters (PLT, platelet crit (PCT), mean platelet volume (MPV), mean platelet component (MPC), mean platelet mass, platelet distribution width, and large platelet (LPLT)), which measured with CBC on ADVIA, were investigated. In addition, the difference in platelet and iron parameters between severe IDA (Hb < 7 g/dl) and non-severe IDA were compared. PLT inversely correlated with serum iron and Tfsat (p < 0.05). Serum iron and TIBC revealed no significant relationships with any platelet parameters. PLT, PCT, and MPV inversely correlated with mean corpuscular hemoglobin concentration (MCHC) but MPC exhibited linear correlation with Hb, hematocrit, and MCHC (p < 0.05). PCT had linear correlation with PLT and MPV (p < 0.001), whereas PCT, MPV, and LPLT (p < 0.001 for two formers, p < 0.05) inversely correlated with MPC. In this study, the important iron parameters affecting PLT were serum iron and Tfsat. In addition, patients with more severe and hypochromic anemia had higher PLT, PCT, and MPV.

A case of therapy-related acute myeloid leukemia with inv(16)(p13.1q22) after single low-dose iodine-131 treatment for thyroid cancer.

Radioiodine is regularly used in the treatment of thyroid cancer to eliminate residual malignant tissue after thyroidectomy and to treat metastasis. Because of the low dose of radioiodine used to treat thyroid cancer patients, leukemia is an uncommon complication of exposure to radioiodine. Here, we present a patient who developed therapy-related acute myeloid leukemia with inv(16)(p13.1q22);CBFß-MYH11, eosinophilia, and K-ras mutation and who had been treated with very low-dose radioiodine following total thyroidectomy.