Tumor suppressor gene methylation on the short arm of chromosome 1 in chronic myelogenous leukemia.

OBJECTIVES: We previously reported loss of heterozygosity on 1p in chronic myelogenous leukemia (CML). We analyzed promoter methylation and mutation of tumor suppressor genes on 1p36 in CML. METHODS: We performed methylation-specific PCR (MS-PCR) analysis of the PRDM2, RUNX3, and TP73 genes in 61 patients with CML (43 chronic phase, CP; two accelerated phase; and 16 blast crisis, BC). Oxidative MS-PCR, PCR-single-strand conformation polymorphism, and real-time reverse transcriptase PCR were also analyzed. K-562 cells were grown in the presence of 5-Aza-dC and trichostatin A. RESULTS: Methylation of the PRDM2, RUNX3, and TP73 genes was detected in 24/60 (40%), 21/61 (34%), and 28/60 (47%) patients, respectively. Methylation of all three genes was detected in 19/59 (32%) patients. Methylation was more frequent in BC than in CP. Oxidative MS-PCR analysis detected 5-mC in the PRDM2, RUNX3, and TP73 genes in 10/22 (45%), 15/21 (71%), and 16/26 (62%) samples with methylation detected by MS-PCR, respectively. Decreased expression was observed in several samples with methylation, while no mutations were found in the genes. Treatment of K-562 cells induced growth suppression, demethylation, and reexpression of the PRDM2 and RUNX3 genes. CONCLUSION: Multiple tumor suppressor genes on 1p were inactivated in CML by methylation.

Adaptive Spectral Model for abnormality detection based on physiological status monitoring of dairy cows.

This research study developed milk spectral data-driven approach, called Adaptive Spectral Model for Abnormality Detection - ASMAD, for detection of physiological abnormalities of individual dairy cows. The algorithm is based on the logic that milk spectra of each individual cow is highly animal-specific, which means it could be used as a respective individual marker for identification. When the algorithm fails to recognize the milk spectra as coming from a certain animal, instead of being treated as a mistake, this outcome is accepted as a deviation of the respective individual marker, and a potential indicator of abnormal physiological state. For the purpose of ASMAD development, near infrared spectra of milk of seven dairy cows have been collected daily during 1-year period. The abnormality detection model is built using supervised recognition method Soft Independent Modeling of Class Analogies - SIMCA, and optimized with respect to spectral pre-processing, choice of the wavelength region and size of the time-window when developing the adaptive model. The sensitivity and specificity of ASMAD were dependent on the animal, and in the ranges 40.00-64.29% and 87.23-98.86%, respectively. Considering significant level of day-to-day spectral variation and multitude of physiological and environmental factors influence on milk constituents and spectra, these results represent a significant potential for creating a health-status monitoring and detection of abnormal physiological states in dairy animals. The adaptive modeling based on the time series of spectral data collected from the individual organism utilized in this work for monitoring physiological status and abnormality detection in dairy cows, has a good potential to be used for similar purposes in other animals and humans.

CRMP5 (collapsin response mediator protein 5) regulates dendritic development and synaptic plasticity in the cerebellar Purkinje cells.

Collapsin response mediator protein 5 (CRMP5) is one of the CRMP members that expresses abundantly in the developing brain. To examine the in vivo function of CRMP5, we generated crmp5-deficient (crmp5(-/-)) mice. Anti-calbindin immunofluorescence studies of crmp5(-/-) mice revealed aberrant dendrite morphology; specifically, a decrease in the size of soma and diameter of primary dendrite of the cerebellar Purkinje cells at postnatal day 21 (P21) and P28, but not at P14. Coincidentally, CRMP5 is detected in Purkinje cells at P21 and P28 from crmp5(+/-) mice. In cerebellar slices of crmp5(-/-) mice, the induction of long-term depression of excitatory synaptic transmission between parallel fibers and Purkinje cells was deficient. Given that brain-derived neurotrophic factor (BDNF) plays major roles in dendritic development, we tried to elucidate the possible roles of CRMP5 in BDNF signaling. The effect of BDNF to induce dendritic branching was markedly attenuated in cultured crmp5(-/-) neurons. Furthermore, CRMP5 was tyrosine phosphorylated when coexpressed with neurotrophic tyrosine kinase receptor type 2 (TrkB), a receptor for BDNF, in HEK293T cells. These findings suggest that CRMP5 is involved in the development, maintenance and synaptic plasticity of Purkinje cells.

Genetic alterations in patients with chronic leucocytosis and persistent thrombocytosis.

To elucidate the relevance of genetic alterations, we analysed 17 genes known to be involved in haematological neoplasms in patients with chronic leucocytosis and patients with persistent thrombocytosis. Mutations of the JAK2, SETBP1 and ASXL1 genes were found in 1/13, 1/13, and 2/13 patients with leucocytosis, respectively. Mutations of the JAK2, CALR, SETBP1 and ASXL1 genes were found in 1/5, 1/5, 1/5 and 2/5 patients with thrombocytosis, respectively. One leucocytosis patient with a JAK2 V617F mutation developed polycythaemia vera. Another leucocytosis patient developed Philadelphia chromosome-negative chronic myeloid leukaemia (Ph(-) CML) accompanied by t(9;12)(q34.1;p13.?3) (Mori et al. 2016). Another leucocytosis patient with mutations of the SETBP1 and ASXL1 genes progressed to blast crisis of Ph(-) CML accompanied by i(17)(q10). Chronic leucocytosis patients who had genetic alterations tended to develop haematological neoplasms, while thrombocytosis unexpectedly resolved in two persistent thrombocytosis patients with genetic alterations.

Reduced PLCG1 expression is associated with inferior survival for myelodysplastic syndromes.

The PLCG1 gene, which encodes the phospholipase C γ1 isoform, is located within the commonly deleted region of the long arm of chromosome 20 (del(20q)) observed in myelodysplastic syndromes (MDS). Phospholipase C is involved in diverse physiological and pathological cellular processes through inositide signaling. We hypothesized that reduced PLCG1 expression because of haploinsufficiency by del(20q) plays a role in the molecular pathogenesis of MDS. Therefore, we analyzed PLCG1 expression in bone marrow mononuclear cells at diagnosis in 116 MDS patients with or without del(20q) by quantitative RT-PCR to evaluate its clinical significance. The expression level of PLCG1 was significantly lower not only in MDS patients with del(20q) but also in those without del(20q) compared to that of the controls, which suggests that reduced PLCG1 expression is a common molecular event in MDS. Patients in the lowest quartile (Q4) group for PLCG1 expression had lower overall survival (OS) compared to that of other patients (Q1-Q3) (log-rank test, P = .0004) with estimated median OS times of 22 in the Q4 group and 106 months in the Q1-3 group. Univariate and multivariate analysis indicated reduced PLCG1 expression (Q4) was associated with lower OS (hazard ratio 2.58, 95% CI 1.35-4.84, P = .0049), which suggests that reduced PLCG1 expression is an independent prognostic factor for OS. In addition, patients were well-stratified for OS by combining PLCG1 expression level (Q4 vs Q1-3) and bone marrow blast percentage (5% or more vs less than 5%). Thus, the level of PLCG1 expression at time of diagnosis is a prognostic biomarker for MDS.

Determination of amino acids in urine by cyclodextrin-modified capillary electrophoresis-laser-induced fluorescence detection.

Capillary electrophoresis (CE) combined with laser-induced fluorescence detection is applied to the determination of amino acids in urine samples. The urine samples are first ultrafiltered, to remove proteins and large peptides, and the filtrates are then directly labeled by reaction with fluorescein isothiocyanate (FITC). Cyclodextrin-modified CE using alpha-cyclodextrin is employed for the separation of the FITC-labeled amino acids. Seven amino acids are clearly separated from side reaction products produced during the labeling reaction, when an 80 mM borate buffer containing 45 mM alpha-cyclodextrin is used as the running buffer. For quantitative analysis, rhodamine B is added to the labeled urine samples as an internal standard. The calibration curves for phenylalanine, glutamine, proline, glycine, serine, alanine, and valine are linear in the range of 10 microM to 100 microM. The concentration limits of detection for all of the amino acids are estimated to be 160-330 nM. Conversely, the limit of quantitation (LOQ) was approximately 10 microM and the limitations are due to the labeling efficiency rather than the sensitivity of the detector. Three amino acids in urine samples, glutamine, glycine, and alanine, are readily quantitated, while the concentrations of the others are below the LOQ. The present method would permit the determination of seven amino acids in urine successfully.

Spectral pattern of urinary water as a biomarker of estrus in the giant panda.

Near infrared spectroscopy (NIRS) has been successfully used for non-invasive diagnosis of diseases and abnormalities where water spectral patterns are found to play an important role. The present study investigates water absorbance patterns indicative of estrus in the female giant panda. NIR spectra of urine samples were acquired from the same animal on a daily basis over three consecutive putative estrus periods. Characteristic water absorbance patterns based on 12 specific water absorbance bands were discovered, which displayed high urine spectral variation, suggesting that hydrogen-bonded water structures increase with estrus. Regression analysis of urine spectra and spectra of estrone-3-glucuronide standard concentrations at these water bands showed high correlation with estrogen levels. Cluster analysis of urine spectra grouped together estrus samples from different years. These results open a new avenue for using water structure as a molecular mirror for fast estrus detection.