Parameterization of metabolite and macromolecule contributions in interrelated MR spectra of human brain using multidimensional modeling.

Macromolecular signals are crucial constituents of short echo-time 1 H MR spectra with potential clinical implications in themselves as well as essential ramifications for the quantification of the usually targeted metabolites. Their parameterization, needed for general fitting models, is difficult because of their unknown composition. Here, a macromolecular signal parameterization together with metabolite signal quantification including relaxation properties is investigated by multidimensional modeling of interrelated 2DJ inversion-recovery (2DJ-IR) datasets. Simultaneous and iterative procedures for defining the macromolecular background (MMBG) as mono-exponentially or generally decaying signals over TE are evaluated. Varying prior knowledge and restrictions in the metabolite evaluation are tested to examine their impact on results and fitting stability for two sets of three-dimensional spectra acquired with metabolite-cycled PRESS from cerebral gray and white matter locations. One dataset was used for model optimization, and also examining the influence of prior knowledge on estimated parameters. The most promising model was applied to a second dataset. It turned out that the mono-exponential decay model appears to be inadequate to represent TE-dependent signal features of the MMBG. TE-adapted MMBG spectra were therefore determined. For a reliable overall quantification of implicated metabolite concentrations and relaxation times, a general fitting model had to be constrained in terms of the number of fitting variables and the allowed parameter space. With such a model in place, fitting precision for metabolite contents and relaxation times was excellent, while fitting accuracy is difficult to judge and bias was likely influenced by the type of fitting constraints enforced. In summary, the parameterization of metabolite and macromolecule contributions in interrelated MR spectra has been examined by using multidimensional modeling on complex 2DJ-IR datasets. A tightly restricted model allows fitting of individual subject data with high fitting precision documented in small Cramér-Rao lower bounds, good repeatability values and a relatively small spread of estimated concentration and relaxation values for a healthy subject cohort.

Targeting levels or oligomerization of nucleophosmin 1 induces differentiation and loss of survival of human AML cells with mutant NPM1.

Nucleophosmin 1 (NPM1) is an oligomeric, nucleolar phosphoprotein that functions as a molecular chaperone for both proteins and nucleic acids. NPM1 is mutated in approximately one-third of patients with AML. The mutant NPM1c+ contains a 4-base insert that results in extra C-terminal residues encoding a nuclear export signal, which causes NPM1c+ to be localized in the cytoplasm. Here, we determined the effects of targeting NPM1 in cultured and primary AML cells. Treatment with siRNA to NPM1 induced p53 and p21, decreased the percentage of cells in S-phase of the cell cycle, as well as induced differentiation of the AML OCI-AML3 cells that express both NPMc+ and unmutated NPM1. Notably, knockdown of NPM1 by shRNA abolished lethal AML phenotype induced by OCI-AML3 cells in NOD/SCID mice. Knockdown of NPM1 also sensitized OCI-AML3 to all-trans retinoic acid (ATRA) and cytarabine. Inhibition of NPM1 oligomerization by NSC348884 induced apoptosis and sensitized OCI-AML3 and primary AML cells expressing NPM1c+ to ATRA. This effect was significantly less in AML cells coexpressing FLT3-ITD, or in AML or normal CD34+ progenitor cells expressing wild-type NPM1. Thus, attenuating levels or oligomerization of NPM1 selectively induces apoptosis and sensitizes NPM1c+ expressing AML cells to treatment with ATRA and cytarabine.

Pan-histone deacetylase inhibitor panobinostat depletes CXCR4 levels and signaling and exerts synergistic antimyeloid activity in combination with CXCR4 antagonists.

Stromal cell derived factor-1 (SDF-1 or CXCL12) and its receptor CXCR4 are involved in the directional homing to the bone marrow niches and in peripheral mobilization of normal and transformed hematopoietic stem and myeloid progenitor cells. Elevated CXCR4 expression confers poor prognosis, whereas inhibition of CXCR4 signaling overcomes stroma-mediated chemoresistance in acute myeloid leukemia (AML). Here, we demonstrate that treatment with the pan-histone deacetylase inhibitor panobinostat (PS) depleted the mRNA and protein levels of CXCR4 in the cultured and primary AML cells. PS-induced acetylation of the heat shock protein (hsp) 90 reduced the chaperone association between CXCR4 and hsp90, directing CXCR4 to degradation by the 20S proteasome. PS treatment also depleted G protein-coupled receptor kinase 3, as well as attenuated the phosphorylation of AKT and ERK1/2 in AML cells, which was not affected by cotreatment with CXCL12. Compared with each agent alone, cotreatment with PS and CXCR4 antagonist AMD3100 or FC-131 synergistically induced apoptosis of cultured and primary AML cells. PS and FC-131 exerted more lethal effects on primary AML versus normal CD34(+) bone marrow progenitor cells. These findings support the rationale to test the in vivo efficacy of PS in enhancing the lethal effects of CXCR4 antagonists against AML cells.

Magnetization exchange with water and T1 relaxation of the downfield resonances in human brain spectra at 3.0 T.

The use of water suppression for in vivo proton MR spectroscopy diminishes the signal intensities from resonances that undergo magnetization exchange with water, particularly those downfield of water. To investigate these exchangeable resonances, an inversion transfer experiment was performed using the metabolite cycling technique for non-water-suppressed MR spectroscopy from a large brain voxel in 11 healthy volunteers at 3.0 T. The exchange rates of the most prominent peaks downfield of water were found to range from 0.5 to 8.9 s(-1), while the T(1) relaxation times in absence of exchange were found to range from 175 to 525 ms. These findings may help toward the assignments of the downfield resonances and a better understanding of the sources of contrast in chemical exchange saturation transfer imaging.

Cotreatment with panobinostat and JAK2 inhibitor TG101209 attenuates JAK2V617F levels and signaling and exerts synergistic cytotoxic effects against human myeloproliferative neoplastic cells.

The mutant JAK2V617F tyrosine kinase (TK) is present in the majority of patients with BCR-ABL-negative myeloproliferative neoplasms (MPNs). JAK2V617F activates downstream signaling through the signal transducers and activators of transcription (STAT), RAS/mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3 (PI3)/AKT pathways, conferring proliferative and survival advantages in the MPN hematopoietic progenitor cells (HPCs). Treatment with the pan-histone deacetylase (HDAC) inhibitor panobinostat (PS) is known to inhibit the chaperone function of heat shock protein 90, as well as induce growth arrest and apoptosis of transformed HPCs. Here, we demonstrate that PS treatment depletes the autophosphorylation, expression, and downstream signaling of JAK2V617F. Treatment with PS also disrupted the chaperone association of JAK2V617F with hsp90, promoting proteasomal degradation of JAK2V617F. PS also induced apoptosis of the cultured JAK2V617F-expressing human erythroleukemia HEL92.1.7 and Ba/F3-JAK2V617F cells. Treatment with the JAK2 TK inhibitor TG101209 attenuated JAK2V617F autophosphorylation and induced apoptosis of HEL92.1.7 and Ba/F3-JAK2V617F cells. Cotreatment with PS and TG101209 further depleted JAK/STAT signaling and synergistically induced apoptosis of HEL92.1.7 and Ba/F3-JAK2V617F cells. Cotreatment with TG101209 and PS exerted greater cytotoxicity against primary CD34(+) MPN cells than normal CD34(+) HPCs. These in vitro findings suggest combination therapy with HDAC and JAK2V617F inhibitors is of potential value for the treatment of JAK2V617F-positive MPN.

Two-dimensional linear-combination model fitting of magnetic resonance spectra to define the macromolecule baseline using FiTAID, a Fitting Tool for Arrays of Interrelated Datasets.

OBJECT: To propose the determination of the macromolecular baseline (MMBL) in clinical 1H MR spectra based on T(1) and T(2) differentiation using 2D fitting in FiTAID, a general Fitting Tool for Arrays of Interrelated Datasets. MATERIALS AND METHODS: Series of localized inversion-recovery (IR) and 2DJ separation spectra of the brain were recorded at 3T. The MMBL was determined by three 2D evaluation methods based on (1) IR spectra only, (2) 2DJ spectra only, (3) both IR and 2DJ spectra (2DJ-IR). Their performance was compared using synthetic spectra and based on variability and reproducibility as obtained in vivo from 12 subjects in 20 examinations. RESULTS: All methods performed well for synthetic data. In vivo, 2DJ-only yielded larger variations than the other methods. IR-only and 2DJ-IR yielded similar performance. FiTAID is illustrated with further applications where linear-combination model fitting of interrelated arrays of spectra is advantageous. CONCLUSION: 2D-Fitting offers the possibility to determine the MMBL based on a range of complementary experimental spectra not relying on smoothness criteria or global assumptions on T(1). Since 2DJ-IR includes information from spectra with different inversion and echo times, it is expected to be more robust in cases with more variable data quality and overlap with lipid resonances.

Reporting of alcohol as a contributor to death in Australian national suicide statistics and its relationship to post-mortem alcohol concentrations.

AIM: To describe the assignment of International Classification of Disease (ICD)-10 alcohol codes as underlying or contributory causes of death by the Australian Bureau of Statistics during mortality coding for suicides according to the blood alcohol concentration (BAC) detected at autopsy. DESIGN: Population-based case-series descriptive analysis. SETTING AND PARTICIPANTS: Data for all alcohol-related (Alc+) suicide deaths (aged 15+) in Australia from 2010-2015 (n = 3132) from the National Coronial Information System. MEASUREMENTS: Alc+ suicides were categorised as those with a post-mortem BAC ≥0.05 g/100 mL. The outcome variable was whether the case was assigned an ICD-10 alcohol code (F10.0-F10.9, R78.0, T51, X45 and/or X65). We estimated OR for the assignment of codes in Alc+ suicides using BAC as the key predictor. We also examined several covariates that have been implicated in the risk of Alc+ suicides. FINDINGS: An ICD-10 alcohol code was assigned during the mortality coding process in 47.6% (n = 1491) of Alc+ suicides. Higher BAC was associated with higher odds of having a code assigned; cases with a BAC over 0.20 g/100 mL over were twice as likely to have an alcohol code assigned (adjusted OR [AOR] = 2.06, 95% CI = 1.59, 2.67) compared with cases with a BAC of 0.050-0.075 g/100 mL. Compared with New South Wales, higher likelihood of code assignment was found in Northern Territory (AOR = 3.85, 95% CI = 2.32, 6.63) and Western Australia (AOR = 2.89, 95% CI = 2.27, 3.68). Compared with 15-24 year olds, 25-44 (AOR = 0.79, 95% CI = 0.63, 0.99) and 65-84 year olds (AOR = 0.63, 95% CI = 0.43, 0.93) were less likely to have a code assigned. CONCLUSIONS: An ICD-10 alcohol code was not assigned as an underlying or contributory cause of death in over half of suicides in Australia (2010-2015) with a BAC ≥0.05 g/100 mL. The higher the BAC detected at autopsy, the more likely cases were to be assigned an alcohol code during the mortality coding process.

Muscle contraction, but not insulin, increases microvascular blood volume in the presence of free fatty acid-induced insulin resistance.

OBJECTIVE: Insulin and contraction each increase muscle microvascular blood volume (MBV) and glucose uptake. Inhibiting nitric oxide synthase blocks insulin's but not contraction's effects. We examined whether contraction could augment the MBV increase seen with physiologic hyperinsulinemia and whether free fatty acid (FFA)-induced insulin resistance differentially affects contraction- versus insulin-mediated increases in MBV. RESEARCH DESIGN AND METHODS: Rats were fasted overnight. Plasma FFAs were increased by intralipid/heparin infusion (3 h), insulin was increased with a euglycemic clamp (3 mU x min(-1) x kg(-1)), and hindlimb muscle contraction was electrically stimulated. Muscle MBV was measured using contrast-enhanced ultrasound. Insulin transport into muscle was measured using (125)I-insulin. BQ-123 (0.4 mg/h) was used to block the endothelin-1 (ET-1) receptor A. RESULTS: Superimposing contraction on physiologic hyperinsulinemia increased MBV within 10 min by 37 and 67% for 0.1 or 1 Hz, respectively (P < 0.01). FFA elevation alone did not affect MBV, whereas 0.1 Hz stimulation doubled MBV (P < 0.05) and increased muscle insulin uptake (P < 0.05) despite high FFA. Physiologic hyperinsulinemia during FFA elevation paradoxically decreased MBV (P < 0.05). This MBV decrease was reversed by either 0.1 Hz contraction or ET-1 receptor A antagonism, and the combination raised MBV above basal. CONCLUSIONS: Contraction recruits microvasculature beyond that seen with physiologic hyperinsulinemia by a distinct mechanism that is not blocked by FFA-induced vascular insulin resistance. The paradoxical MBV decline seen with insulin plus FFA may result from differential inhibition of insulin-stimulated nitric oxide-dependent vasodilation relative to ET-1 vasoconstriction. Our results implicate ET-1 as a potential mediator of FFA-induced vascular insulin resistance.