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Transcription factor CP2c (also known as TFCP2, α-CP2, LSF, and LBP-1c) is involved in diverse ubiquitous and tissue/stage-specific cellular processes and in human malignancies such as cancer. Despite its importance, many fundamental regulatory mechanisms of CP2c are still unclear. Here, we uncover an unprecedented mechanism of CP2c degradation via a previously unidentified SUMO1/PSME3/20 S proteasome pathway and its biological meaning. CP2c is SUMOylated in a SUMO1-dependent way, and SUMOylated CP2c is degraded through the ubiquitin-independent PSME3 (also known as REGγ or PA28)/20 S proteasome system. SUMOylated PSME3 could also interact with CP2c to degrade CP2c via the 20 S proteasomal pathway. Moreover, precisely timed degradation of CP2c via the SUMO1/PSME3/20 S proteasome axis is required for accurate progression of the cell cycle. Therefore, we reveal a unique SUMO1-mediated uncanonical 20 S proteasome degradation mechanism via the SUMO1/PSME3 axis involving mutual SUMO-SIM interaction of CP2c and PSME3, providing previously unidentified mechanistic insights into the roles of dynamic degradation of CP2c in cell cycle progression.

The primary aims of this study were to determine the correlation between absolute quantitative 99mTc-pyrophosphate metrics and traditional measures of cardiac amyloid burden and to measure the intraobserver repeatability of the quantitative metrics. Methods: We studied 72 patients who underwent 99mTc-pyrophosphate SPECT/CT using a novel general-purpose cadmium-zinc-telluride-based SPECT/CT system. The clinical standard for these studies is visual grading (with grades of 0, 1, 2, and 3 indicating myocardial uptake absent, less than rib uptake, equal to rib uptake, or more than rib uptake, respectively). A visual grade of 2 or more was considered positive. For 72 patients, SUVmax, SUVmean, cardiac amyloid activity (CAA; i.e., SUVmean × left ventricular [LV] volume), and percentage injected dose (%ID) were calculated, and visual grading was performed. The correlation was determined between the 4 quantitative metrics or visual grades and the LV mass index (LVMI) (indexed to body surface area on echocardiography, 67 patients). For a subset of 11 patients, the correlation was determined between the visual or quantitative metrics and the extracellular volume (ECV) on cardiac MRI. Normal linear regression was used to compare the standardized association of each of the 4 quantitative metrics with LVMI, as a surrogate for amyloid burden. Receiver-operating-characteristic curve analysis was used to determine the diagnostic accuracy of quantitative metrics, using visual grading as the reference standard. The intraobserver repeatability of generating quantitative metrics was also determined. Results: All 4 quantitative metrics were highly accurate, with an area under the receiver-operating-characteristic curve of more than 0.96 for diagnosis of transthyretin cardiac amyloidosis. SUVmax, SUVmean, CAA, %ID, and visual grade were moderately positively correlated with LVMI (r = 0.485 for %ID) and strongly positively correlated, albeit in a small cohort, with ECV (r = 0.873, SUVmax). Intraobserver repeatability was excellent, with less than a 2% coefficient of variation for SUVmax, %ID, and CAA and 3.8% for SUVmean All 4 quantitative metrics had a standardized effect of more than 0.324 on LVMI; the largest standardized effect was 0.485, for %ID. Conclusion: In this first (to our knowledge) study of 99mTc-pyrophosphate cardiac imaging using a novel cadmium-zinc-telluride SPECT/CT scanner, SUVmax, SUVmean, CAA, and %ID measured by absolute quantitation of 99mTc-pyrophosphate were moderately correlated with LVMI and strongly correlated, albeit in a small cohort, with ECV. The intraobserver repeatability of generating the quantitative metrics was excellent.

Objective Early detection and proper management of mental illness can help to prevent severe deterioration. However, with limited financial and human resources of community mental health services, it is not practical to carry out all conventional screening tools simultaneously. In this study, we aimed to develop and validate a brief but comprehensive screening questionnaire for four common mental illnesses of the elderly.Methods The brief screening for four mental illnesses of elderly (BS4MI-elderly) is a 14-item binary response questionnaire that covers dementia, depressive disorder, sleep disorder, and hwa-byung. To test validity, we compared conventional scale scores for three groups of participants classified using the BS4MI-elderly. The sensitivity, specificity, predictive value of positive test, likelihood ratio of positive test and internal consistency of the BS4MI-elderly were assessed. Finally, a correlation analysis between the BS4MI-elderly and general mental health scales was conducted.Results A total of 254 participants aged over 65 years were recruited. The BS4MI-elderly showed moderate to high sensitivity for the test that distinguishes the normal group from the risk and disorder groups (dementia: 0.61, depressive disorder: 0.88, sleep disorder: 0.85, hwa-byung: 0.94) and high specificity for the test that distinguishes the disorder group from the normal and risk groups (dementia: 0.91, depressive disorder: 0.93, hwa-byung: 0.84, sleep disorder: 0.84). The BS4MI-elderly also exhibited good internal consistency and significant correlations with general mental health scales.Conclusion The BS4MI-elderly, a brief but comprehensive screening tool, could be a useful instrument for screening the elderly in community mental health services.

Background: Single photon emission computed tomography (SPECT) has limited ability to identify multivessel and microvascular coronary artery disease. Gamma cameras with cadmium zinc telluride detectors allow the quantification of absolute myocardial blood flow (MBF) and myocardial flow reserve (MFR). However, evidence of its accuracy is limited, and of its reproducibility is lacking. We aimed to validate 99m Tc-sestamibi SPECT MBF and MFR using standard and spline-fitted reconstruction algorithms compared with 13 N-ammonia positron emission tomography in a cohort of patients with known or suspected coronary artery disease and to evaluate the reproducibility of this technique. Methods: Accuracy was assessed in 34 participants who underwent dynamic 99m Tc-sestamibi SPECT and 13 N-ammonia positron emission tomography and reproducibility in 14 participants who underwent 2 99m Tc-sestamibi SPECT studies, all within 2 weeks. A rest/pharmacological stress single-day SPECT protocol was performed. SPECT images were reconstructed using a standard ordered subset expectation maximization (OSEM) algorithm with (N=21) and without (N=30) application of spline fitting. SPECT MBF was quantified using a net retention kinetic model‚ and MFR was derived as the stress/rest MBF ratio. Results: SPECT global MBF with splines showed good correlation with 13 N-ammonia positron emission tomography (r=0.81, P P P P =0.07, for MBF and MFR, respectively). Reproducibility of global MBF estimates with splines in paired SPECT scans was good (r=0.77, P P P =0.3). There were no significant differences in MBF or MFR between the 2 reproducibility scans independently of the reconstruction algorithm ( P >0.05 for all). Conclusions: MBF and MFR quantification using 99m Tc-sestamibi cadmium zinc telluride SPECT with spatiotemporal spline fitting improved the correlation with 13 N-ammonia positron emission tomography flow estimates and test/retest reproducibility. The use of splines may represent an important step toward the standardization of SPECT flow estimation.

Background Pyroglutamate-3 Aβ (pGlu-3 Aβ) is an N-terminally truncated and post-translationally modified Aβ species found in Alzheimer’s disease (AD) brain. Its increased peptide aggregation propensity and toxicity make it an attractive emerging treatment strategy for AD. We address the question of how the effector function of an anti-pGlu-3 Aβ antibody influences the efficacy of immunotherapy in mouse models with AD-like pathology. Methods We compared two different immunoglobulin (Ig) isotypes of the same murine anti-pGlu-3 Aβ mAb (07/1 IgG1 and 07/2a IgG2a) and a general N-terminal Aβ mAb (3A1 IgG1) for their ability to clear Aβ and protect cognition in a therapeutic passive immunotherapy study in aged, plaque-rich APPSWE/PS1ΔE9 transgenic (Tg) mice. We also compared the ability of these antibodies and a CDC-mutant form of 07/2a (07/2a-k), engineered to avoid complement activation, to clear Aβ in an ex vivo phagocytosis assay and following treatment in APPSLxhQC double Tg mice, and to activate microglia using longitudinal microPET imaging with TSPO-specific 18F-GE180 tracer following a single bolus antibody injection in young and old Tg mice. Results We demonstrated significant cognitive improvement, better plaque clearance, and more plaque-associated microglia in the absence of microhemorrhage in aged APPSWE/PS1ΔE9 Tg mice treated with 07/2a, but not 07/1 or 3A1, compared to PBS in our first in vivo study. All mAbs cleared plaques in an ex vivo assay, although 07/2a promoted the highest phagocytic activity. Compared with 07/2a, 07/2a-k showed slightly reduced affinity to Fcγ receptors CD32 and CD64, although the two antibodies had similar binding affinities to pGlu-3 Aβ. Treatment of APPSLxhQC mice with 07/2a and 07/2a-k mAbs in our second in vivo study showed significant plaque-lowering with both mAbs. Longitudinal 18F-GE180 microPET imaging revealed different temporal patterns of microglial activation for 3A1, 07/1, and 07/2a mAbs and no difference between 07/2a-k and PBS-treated Tg mice. Conclusion Our results suggest that attenuation of behavioral deficits and clearance of amyloid is associated with strong effector function of the anti-pGlu-3 Aβ mAb in a therapeutic treatment paradigm. We present evidence that antibody engineering to reduce CDC-mediated complement binding facilitates phagocytosis of plaques without inducing neuroinflammation in vivo. Hence, the results provide implications for tailoring effector function of humanized antibodies for clinical development.

The clinical diagnosis of pulmonary involvement in individuals with systemic AL amyloidosis remains challenging. [18F]florbetapir imaging has previously identified AL amyloid deposits in the heart and extra-cardiac organs. The aim of this study is to determine quantitative [18F]florbetapir pulmonary kinetics to identify pulmonary involvement in individuals with systemic AL amyloidosis. We prospectively enrolled 58 subjects with biopsy-proven AL amyloidosis and 9 control subjects (5 without amyloidosis and 4 with ATTR cardiac amyloidosis). Pulmonary [18F]florbetapir uptake was evaluated visually and quantified as distribution volume of specific binding (Vs) derived from compartmental analysis and simpler semiquantitative metrics of maximum standardized uptake values (SUVmax), retention index (RI), and target-to-blood ratio (TBR). On visual analysis, pulmonary tracer uptake was absent in most AL subjects (40/58, 69%); 12% (7/58) of AL subjects demonstrated intense bilateral homogeneous tracer uptake. In this group, compared to the control group, Vs (median Vs 30-fold higher, 9.79 vs. 0.26, p 3-fold higher Vs compared to the control group (median 0.99 vs. 0.26, p < 0.001). Vs was independently related to left ventricular SUVmax, a marker of cardiac AL deposition, but not to ejection fraction, a marker of cardiac dysfunction. Also, intense [18F]florbetapir lung uptake was not related to [11C]acetate lung uptake, suggesting that intense [18F]florbetapir lung uptake represents AL amyloidosis rather than heart failure. [18F]florbetapir PET/CT offers the potential to noninvasively identify pulmonary AL amyloidosis, and its clinical relevance warrants further study.

This study sought to test whether relative apical sparing (RELAPS) of left ventricular (LV) longitudinal strain (LS) in cardiac amyloidosis (CA) is explained by regional differences in markers of amyloid burden (Further knowledge of the pathophysiological basis for RELAPS can help understand the adverse outcomes associated with apical LS impairment.This was a prospective study of 32 subjects (age 62 ± 7 years; 50% males) with light chain CA. All subjects underwent two-dimensional echocardiography for LS estimation andThere was a significant base-to-apex gradient in LS (-7.4 ± 3.2% vs. -8.6 ± 4.0% vs. -20.8 ± 6.6%; p 0.0001), maximal LV wall thickness (15.7 ± 1.9 cm vs. 15.4 ± 2.9 cm vs. 10.1 ± 2.4 cm; p 0.0001), and LV mass (74.8 ± 21.2 g vs. 60.8 ± 17.3 g vs. 23.4 ± 6.2 g; p 0.0001). In contrast, florbetapir RI (0.089 ± 0.03 μmol/min/g vs. 0.097 ± 0.03 μmol/min/g vs. 0.085 ± 0.03 μmol/min/g; p = 0.45) and ECV (0.53 ± 0.08 vs. 0.49 ± 0.08 vs. 0.49 ± 0.07; p = 0.15) showed no significant base-to-apex gradient in the tissue concentration or proportion of amyloid infiltration, whereas markers of total amyloid load, such as total florbetapir binding (3.4 ± 1.7 μmol/min vs. 2.8 ± 1.5 μmol/min vs. 0.93 ± 0.49 μmol/min; p 0.0001) and extracellular LV mass (40.0 ± 15.6 g vs. 30.2 ± 10.9 g vs. 11.6 ± 3.9 g; p 0.0001), did show a marked base-to-apex gradient.Segmental differences in the distribution of the total amyloid mass, rather than the proportion of amyloid deposits, appear to explain the marked regional differences in LS in CA. Although these 2 matrices are clearly related concepts, they should not be used interchangeably.

BACKGROUND: Gallium-68 Dotatate binds preferentially to somatostatin receptor (sstr) subtype-2 (sstr-2) on inflammatory cells. We aimed at investigating the potential clinical use of sstr-targeted imaging for the detection of myocardial inflammation. METHODS: 13 patients with suspected cardiac sarcoidosis (CS) based on clinical history and myocardial uptake on recent fluorine-18 fluorodeoxyglucose (FDG) PET, were enrolled to undergo Dotatate PET after FDG PET (median time 37 days [IQR 25 – 55]). Additionally, we investigated ex-vivo the immunohistochemistry expression of sstr-2 in 3 explanted sarcoid hearts. RESULTS: All FDG scans showed cardiac uptake (focal/multifocal = 6, focal on diffuse/heterogeneous = 7), and 46% (n=6) extra-cardiac uptake (mediastinal/hilar). In comparison, Dotatate scans showed definite abnormal cardiac uptake (focal/multifocal) in 4 patients, probably abnormal (heterogenous/patchy) in 3, and negative uptake in 6 cases. Similarly, 6 patients had increased mediastinal/hilar Dotatate uptake. Overall concordance of FDG and Dotatate uptake was 54% in the heart and 100% for thoracic nodal activity. Quantitatively, FDG maximum standardized uptake value was 5.0 times [3.8 – 7.1] higher in the heart, but only 2.25 times [1.7 – 3.0; P=0.019] higher in thoracic nodes relative to Dotatate. Ex-vivo, sstr-2 immunostaining was weakly seen within well-formed granulomas in all 3 examined sarcoid heart specimens with no significant staining of background myocardium or normal myocardium. CONCLUSION: Our preliminary data suggests that, compared to FDG imaging, somatostatin receptor-targeted imaging may be less sensitive for the detection of myocardial inflammation, but comparable for detecting extra-cardiac inflammation.

Hypercholesterolemia is becoming a problem with increasing significance. Dietary vegetable oils may help to improve this condition due to presence of phytonutrients with potentially synergistic cholesterol-lowering effects. The objective of this 8-week double-blinded randomized clinical trial was to investigate the effects of consuming 30 g of two different blended cooking oils, rich in omega-3 alpha-linolenic acid and phytonutrients, or refined olive oil on the intestinal microbiota in 126 volunteers with borderline hypercholesterolemia. Multi-factor analysis of relationships between the gut microbiota composition at various taxonomic ranks and the clinical trial parameters revealed the association between beneficial effects of the dietary intervention on the blood lipid profile with abundance of Clostridia class of the gut microbiota. This microbiota feature was upregulated in the course of the dietary intervention and associated with various plasma markers of metabolic health status, such as Triglycerides, Apolipoprotein B and Total Cholesterol to HDL ratio in a beneficial way. The relative abundance of a single species—Clostridium leptum—highly increased during the dietary intervention in all the three study groups. The oil blend with the highest concentration of omega-3 PUFA is associated with faster and more robust responses of the intestinal microbiota, including elevation of alpha-diversity. Butyrate production is being discussed as a plausible process mediating the observed beneficial influence on the plasma lipid profile. Causal mediation analysis suggested that Clostridium genus rather than the higher rank of the phylogeny—Clostridia class—may be involved in the diet-induced improvements of the blood lipid profile.

Adoptive therapies with genetically modified somatic T cells rendered HIV resistant have shown promise for AIDS therapy. A renewable source of HIV resistant human T cells from induced pluripotent stem cells (iPSCs) would further facilitate and broaden the applicability of these therapies. Here, we report successful targeting of the CCR5 locus in iPSCs generated from peripheral blood T cells (T-iPSCs) or fibroblasts (fib-iPSCs) from Mauritian Cynomolgus macaques (MCM), using CRISPR/Cas9 technology. We found that CCR5 editing does not affect pluripotency or hematopoietic and T cell differentiation potentials of fib-iPSCs. However, deletion of CCR5 in T-iPSCs leads to selective loss of their T cell redifferentiation potential without affecting myeloid development. T cells and macrophages produced from CCR5-edited MCM- iPSCs did not support replication of the CCR5-tropic simian immunodeficiency viruses SIVmac239 (T-cell tropic) and SIVmac316 (macrophage-tropic). Overall, these studies provide a platform for further exploration of AIDS therapies based on gene-edited iPSCs in a nonhuman primate preclinical model.

The discovery that CCR5 serves as an R5-HIV-1 co-receptor, coupled with findings of protection from HIV infection in individuals lacking CCR5, led to the exploration of novel therapeutic strategies for HIV infection based on genome editing of CCR5. Advancing translation of CCR5-mutant-based cellular therapies for HIV requires development of novel physiologically relevant animal models. Mauritian cynomolgus macaques (MCMs), with high degree of MHC allele sharing, are valuable models for HIV-1 research and stem cell therapies. To facilitate the generation of a CCR5-mutant MHC-defined MCM model, we explored editing the CCR5 gene in MCM embryos via CRISPR-Cas9. We refined ovarian stimulation and in vitro fertilization (IVF) methods established for Chinese cynomolgus macaques to generate in vitro MCM embryos. Time-lapse embryo imaging was performed to assess the timing of MCM embryonic developmental events in control and CRISPR-Cas9 microinjected embryos. Using a dual-guide gene targeting approach, biallelic deletions in the CCR5 gene were introduced into ~ 23–37% of MCM embryos. In addition, single blastomere PCR analysis revealed mosaicism in CCR5 editing within the same embryo. Successful development of IVF and CCR5 editing protocols in MCM embryos lays a foundation for the creation of CCR5-mutant MCMs to assess novel stem cell-based HIV therapeutics.

ObjectiveThe goal of our study is to assess the role of microglial activation in MS-associated fatigue (MSAF) using [F-18]PBR06-PET.MethodsFatigue severity was measured using the Modified Fatigue Impact Scale (MFIS) in 12 subjects with MS (7 relapsing-remitting and 5 secondary progressive) and 10 healthy control participants who underwent [F-18]PBR06-PET. The MFIS provides a total fatigue score as well as physical, cognitive, and psychosocial fatigue subscale scores. Standardized Uptake Value (SUV) 60–90 minute frame PET maps were coregistered to 3T MRI. Voxel-by-voxel analysis using Statistical Parametric Mapping and atlas-based regional analyses were performed. SUV ratios (SUVRs) were global brain normalized.ResultsPeak voxel-based level of significance for correlation between total fatigue score and PET uptake was localized to the right substantia nigra (T-score 4.67, p = 0.001). Similarly, SUVRs derived from atlas-based segmentation of the substantia nigra showed significant correlation with MFIS (r = 0.76, p = 0.004). On multiple regression, the right substantia nigra was an independent predictor of total MFIS (p = 0.02) and cognitive MFIS subscale values (p = 0.007), after adjustment for age, disability, and depression. Several additional areas of significant correlations with fatigue scores were identified, including the right parahippocampal gyrus, right precuneus, and juxtacortical white matter (all p < 0.05). There was no correlation between fatigue scores and brain atrophy and lesion load in patients with MS.ConclusionSubstantia nigra microglial activation is linked to fatigue in MS. Microglial activation across key brain regions may represent a unifying mechanism for MSAF, and further evaluation of neuroimmunologic basis of MSAF is warranted.

OBJECTIVES: The purpose of this study was to determine phenotypes characterizing cardiac involvement in AL amyloidosis by using direct (fluorine-18-labeled florbetapir {[(18)F]florbetapir} positron emission tomography [PET]/computed tomography) and indirect (echocardiography and cardiac magnetic resonance [CMR]) imaging biomarkers of AL amyloidosis. BACKGROUND: Cardiac involvement in systemic light chain amyloidosis (AL) is the main determinant of prognosis and, therefore, guides management. The hypothesis of this study was that myocardial AL deposits and expansion of extracellular volume (ECV) could be identified before increases in N-terminal pro-B-type natriuretic peptide or wall thickness. METHODS: A total of 45 subjects were prospectively enrolled in 3 groups: 25 with active AL amyloidosis with cardiac involvement (active-CA), 10 with active AL amyloidosis without cardiac involvement by conventional criteria (active-non-CA), and 10 with AL amyloidosis with cardiac involvement in remission for at least 1 year (remission-CA). All subjects underwent echocardiography, CMR, and [(18)F]florbetapir PET/CT to evaluate cardiac amyloid burden. RESULTS: The active-CA group demonstrated the largest myocardial AL amyloid burden, quantified by [(18)F]florbetapir retention index (RI) 0.110 (interquartile range [IQR]: 0.078 to 0.139) min(−1), and the lowest cardiac function by global longitudinal strain (GLS), median GLS −1 1% (IQR: −8% to −13%). The remission-CA group had expanded extracellular volume (ECV) and [(18)F]florbetapir RI of 0.097 (IQR: 0.070 to 0.124 min(−1)), and abnormal GLS despite hematologic remission for >1 year. The active-non-CA cohort had evidence of cardiac amyloid deposition by advanced imaging metrics in 50% of the subjects; cardiac involvement was identified by late gadolinium enhancement in 20%, elevated ECV in 20%, and elevated [(18)F]florbetapir RI in 50%. CONCLUSIONS: Evidence of cardiac amyloid infiltration was found based on direct and indirect imaging biomarkers in subjects without CA by conventional criteria. The findings from [(18)F]florbetapir PET imaging provided insight into the preclinical disease process and on the basis of interpretation of expanded ECV on CMR and have important implications for future research and clinical management of AL amyloidosis. (Molecular Imaging of Primary Amyloid Cardiomyopathy [MICA]; NCT02641145)

The primary aims of this study were to determine the correlation between absolute quantitative (99m)Tc-pyrophosphate metrics and traditional measures of cardiac amyloid burden and to measure the intraobserver repeatability of the quantitative metrics. Methods: We studied 72 patients who underwent (99m)Tc-pyrophosphate SPECT/CT using a novel general-purpose cadmium-zinc-telluride–based SPECT/CT system. The clinical standard for these studies is visual grading (with grades of 0, 1, 2, and 3 indicating myocardial uptake absent, less than rib uptake, equal to rib uptake, or more than rib uptake, respectively). A visual grade of 2 or more was considered positive. For 72 patients, SUV(max), SUV(mean), cardiac amyloid activity (CAA; i.e., SUV(mean) × left ventricular [LV] volume), and percentage injected dose (%ID) were calculated, and visual grading was performed. The correlation was determined between the 4 quantitative metrics or visual grades and the LV mass index (LVMI) (indexed to body surface area on echocardiography, 67 patients). For a subset of 11 patients, the correlation was determined between the visual or quantitative metrics and the extracellular volume (ECV) on cardiac MRI. Normal linear regression was used to compare the standardized association of each of the 4 quantitative metrics with LVMI, as a surrogate for amyloid burden. Receiver-operating-characteristic curve analysis was used to determine the diagnostic accuracy of quantitative metrics, using visual grading as the reference standard. The intraobserver repeatability of generating quantitative metrics was also determined. Results: All 4 quantitative metrics were highly accurate, with an area under the receiver-operating-characteristic curve of more than 0.96 for diagnosis of transthyretin cardiac amyloidosis. SUV(max), SUV(mean), CAA, %ID, and visual grade were moderately positively correlated with LVMI (r = 0.485 for %ID) and strongly positively correlated, albeit in a small cohort, with ECV (r = 0.873, SUV(max)). Intraobserver repeatability was excellent, with less than a 2% coefficient of variation for SUV(max), %ID, and CAA and 3.8% for SUV(mean). All 4 quantitative metrics had a standardized effect of more than 0.324 on LVMI; the largest standardized effect was 0.485, for %ID. Conclusion: In this first (to our knowledge) study of (99m)Tc-pyrophosphate cardiac imaging using a novel cadmium-zinc-telluride SPECT/CT scanner, SUV(max), SUV(mean), CAA, and %ID measured by absolute quantitation of (99m)Tc-pyrophosphate were moderately correlated with LVMI and strongly correlated, albeit in a small cohort, with ECV. The intraobserver repeatability of generating the quantitative metrics was excellent.

Karthik Ananthasubramaniam, E. Gordon DePuey, John J. Mahmarian, Raymond Russell III, Randall C. Thompson, and R. Glenn Wells are section lead authors.

Background and ObjectivesThis [18F]fluorodeoxyglucose (FDG) PET study evaluates the accuracy of semiquantitative measurement of putaminal hypermetabolism in identifying anti–leucine-rich, glioma–inactivated-1 (LGI1) protein autoimmune encephalitis (AE). In addition, the extent of brain dysmetabolism, their association with clinical outcomes, and longitudinal metabolic changes after immunotherapy in LGI1-AE are examined.MethodsFDG-PET scans from 49 age-matched and sex-matched subjects (13 in LGI1-AE group, 15 in non–LGI1-AE group, 11 with Alzheimer disease [AD], and 10 negative controls [NCs]) and follow-up scans from 8 patients with LGI1 AE on a median 6 months after immunotherapy were analyzed. Putaminal standardized uptake value ratios (SUVRs) normalized to global brain (P-SUVRg), thalamus (P/Th), and midbrain (P/Mi) were evaluated for diagnostic accuracy. SUVRg was applied for all other analyses.ResultsP-SUVRg, P/Th, and P/Mi were higher in LGI1-AE group than in non–LGI1-AE group, AD group, and NCs (all p < 0.05). P/Mi and P-SUVRg differentiated LGI1-AE group robustly from other groups (areas under the curve 0.84–0.99). Mediotemporal lobe (MTL) SUVRg was increased in both LGI1-AE and non–LGI1-AE groups when compared with NCs (both p < 0.05). SUVRg was decreased in several frontoparietal regions and increased in pallidum, caudate, pons, olfactory, and inferior occipital gyrus in LGI1-AE group when compared with that in NCs (all p < 0.05). In LGI1-AE group, both MTL and putaminal hypermetabolism were reduced after immunotherapy. Normalization of regional cortical dysmetabolism associated with clinical improvement at the 6- and 20-month follow-up.DiscussionSemiquantitative measurement of putaminal hypermetabolism with FDG-PET may be used to distinguish LGI1-AE from other pathologies. Metabolic abnormalities in LGI1-AE extend beyond putamen and MTL into other subcortical and cortical regions. FDG-PET may be used in evaluating disease evolution in LGI1-AE.Classification of EvidenceThis study provides Class II evidence that semiquantitative measures of putaminal metabolism on PET can differentiate patients with LGI1-AE from patients without LGI1-AE, patients with AD, or NCs.

Space radiation presents a substantial threat to travel beyond Earth. Relatively low doses of high-energy particle radiation cause physiological and behavioral impairments in rodents and may pose risks to human spaceflight. There is evidence that 56Fe irradiation, a significant component of space radiation, may be more harmful to males than to females and worsen Alzheimer’s disease pathology in genetically vulnerable models. Yet, research on the long-term, sex- and genotype-specific effects of 56Fe irradiation is lacking. Here, we irradiated 4-month-old male and female, wild-type and Alzheimer’s-like APP/PS1 mice with 0, 0.10, or 0.50 Gy of 56Fe ions (1GeV/u). Mice underwent microPET scans before and 7.5 months after irradiation, a battery of behavioral tests at 11 months of age and were sacrificed for pathological and biochemical analyses at 12 months of age. 56Fe irradiation worsened amyloid-beta (Aβ) pathology, gliosis, neuroinflammation and spatial memory, but improved motor coordination, in male transgenic mice and worsened fear memory in wild-type males. Although sham-irradiated female APP/PS1 mice had more cerebral Aβ and gliosis than sham-irradiated male transgenics, female mice of both genotypes were relatively spared from radiation effects 8 months later. These results provide evidence for sex-specific, long-term CNS effects of space radiation.

Funding Acknowledgements Spectrum Dynamics Background Traditional relative assessment of regional myocardial perfusion by single photon emission computed tomography (SPECT) has limited ability to identify multivessel coronary artery disease, as well as diffuse atherosclerosis and coronary microvascular dysfunction. Current gamma cameras with cadmium-zinc-telluride (CZT) detectors have higher temporal resolution and sensitivity and allow the acquisition of multi-frame images and quantification of absolute myocardial blood flow (MBF) and coronary flow reserve (CFR Purpose The aim of this study was to assess the accuracy and reproducibility of quantitative measurements of MBF and CFR obtained with a CZT SPECT system compared to PET in a cohort of patients with known or suspected coronary artery disease. Methods Accuracy was assessed in 22 patients who underwent dynamic rest/stress 99mTc-sestamibi-SPECT and 13N-ammonia PET myocardial perfusion imaging within two weeks of each other. Fourteen patients comprised the reproducibility cohort and underwent two dynamic 99mTc-sestamibi SPECT scans within two weeks. A rest/pharmacological stress single-day SPECT protocol was performed, using a 1:3 dose ratio. SPECT image reconstruction was performed using a spline-fitting method and SPECT MBF was quantified using a net retention kinetic model in commercially available software. Rest MBF and CFR were adjusted for heart-rate pressure product. Results Global MBF at rest and stress showed a good correlation between SPECT and PET (r = 0.814, p Conclusion MBF and CFR quantification is feasible using a CZT gamma camera and provides accurate and reproducible results that correlate with 13N-ammonia PET. This may be of special relevance in sites where PET is not available, enabling MBF and CFR quantification with CZT SPECT cameras. Abstract P361 Figure.

Pluripotent stem cells (PSCs) have the potential to provide homogeneous cell populations of T cells that can be grown at a clinical scale and genetically engineered to meet specific clinical needs. OP9-DLL4, a stromal line ectopically expressing the Notch ligand Delta-like 4 (DLL4) is used to support differentiation of PSCs to T-lymphocytes. This article outlines several protocols related to generation of T cells from human and non-human primate (NHP) PSCs, including initial hematopoietic differentiation of PSC on OP9 feeders or defined conditions, followed by coculture of the OP9-DLL4 cells with the PSC-derived hematopoietic progenitors (HPs), leading to efficient differentiation to T lymphocytes. In addition, we describe a protocol for robust T cell generation from hPSCs conditionally expressing ETS1. The presented protocols provide a platform for T cell production for disease modeling and evaluating their use for immunotherapy in large animal models.

BACKGROUND. (18)F-florbetapir PET is emerging as an excellent quantitative tool to quantify cardiac light chain (AL) amyloidosis burden. The primary aim of this study was to determine interobserver reproducibility and intraobserver repeatability, defined per the recommendations of the Quantitative Imaging Biomarker Alliance technical performance group, of PET (18)F-florbetapir retention index (RI) in patients with cardiac AL amyloidosis. METHODS. The study cohort comprised 37 subjects with systemic AL amyloidosis enrolled in the prospective study: Molecular Imaging of Primary Amyloid Cardiomyopathy (clinicaltrials.gov NCT02641145). Using 10 mCi of (18)F-florbetapir, a 60-minute dynamic cardiac scan was acquired. Global and segmental left ventricular estimates of retention index (RI) of (18)F-florbetapir were calculated (Carimas 2.9 software, Turku, Finland). RI was analyzed twice, at least 24 hours apart, by two independent observers. Intraobserver repeatability and interobserver reproducibility were evaluated using Bland–Altman plots and scatter plots with fitted linear regression curves. RESULTS. All reproducibility (interobserver, r = 0.98) and repeatability (intraobserver, R=0.99 for each observer) measures of (18)F-florbetapir RI are excellent. On the Bland–Altman plots, the agreement limits for global (18)F-florbetapir RI were high and ranged for reproducibility (interobserver) from − 9.3 to + 9.4% (Fig. 1), and for repeatability (observer 1 from − 10.8 to + 10.7% and from − 9.2 to + 11.4%, for observer 2). CONCLUSIONS. The present study showed excellent interobserver reproducibility and intraobserver repeatability of (18)F-florbetapir PET retention index in patients with cardiac AL amyloidosis.

PURPOSE: The clinical diagnosis of pulmonary involvement in individuals with systemic AL amyloidosis remains challenging. [(18)F]florbetapir imaging has previously identified AL amyloid deposits in the heart and extra-cardiac organs. The aim of this study is to determine quantitative [(18)F]florbetapir pulmonary kinetics to identify pulmonary involvement in individuals with systemic AL amyloidosis. METHODS: We prospectively enrolled 58 subjects with biopsy proven AL amyloidosis and 9 control subjects (5 without amyloidosis and 4 with ATTR cardiac amyloidosis). Pulmonary [(18)F]florbetapir uptake was evaluated visually and quantified as distribution volume of specific binding (Vs) derived from compartmental analysis; and, simpler semi-quantitative metrics of maximum standardized uptake values (SUVmax), retention index (RI), and target-to-blood ratio (TBR). RESULTS: On visual analysis, pulmonary tracer uptake was absent in most AL subjects (40/58, 69%); 12% (7/58) of AL subjects demonstrated intense bilateral homogeneous tracer uptake. In this group, compared to the control group, Vs (median Vs 30-fold higher, 9.79 vs. 0.26, p < 0.001), TBR (median TBR 12.0 vs. 1.71, p < 0.001), and RI (median RI 0.310 vs. 0.033, p < 0.001) were substantially higher. Notably, the AL group without visually apparent pulmonary [(18)F]florbetapir uptake also demonstrated a >3-fold higher Vs compared to the control group (median 0.99 vs. 0.26, p

Purpose: One approach to preclinical single-photon emission computed tomography (SPECT) imaging that provides both high resolution and high sensitivity is based on imaging a mouse inside a collimating tube; many magnified pinhole projection images from a small target region, e.g., the heart, can be recorded simultaneously on multiple detectors with little multiplexing since each pinhole aperture’s opening angle is restricted to view mostly the target organ. However, to obtain complete data for reconstruction, it may be necessary to scan the mouse through the target region of the tube. The authors are developing a different approach based on acquisition and reconstruction of both low-resolution and high-resolution projection data acquired sequentially through many pinholes embedded in two tungsten tube sections of different diameters, a “scout” section and a high-resolution section, placed end-to-end along the axis of a triple-head clinical SPECT scanner. This paper describes the design procedures used to determine the geometric parameters of two new collimator-tube sections, as well as one approach for joint reconstruction of data acquired from both sections. Methods: The high-resolution section was designed by projecting as many pinhole views of a simulated mouse heart as possible over each detector’s camera, with no overlapping of heart projections and minimal overlapping between adjacent “hot” organ and cardiac projections. The authors then jointly optimized the geometric design of the scout section for a triple-detector camera system, as well as the number of maximum-likelihood expectation maximization (MLEM) iterations required to provide minimum mean-squared error of reconstructed voxel counts throughout a 7-cm axial range, with the constraints of fixed, 2.4-mm scout system resolution at the tube center for all apertures, limited multiplexing, and no detector motion. Simulated mouse projection data from both tube sections were then reconstructed to illustrate a simple approach for using high-resolution data to improve the whole-body scout images within a cylindrical region surrounding the heart. Results: The 2-cm-inner-radius high-resolution tube section accommodated 87 platinum–iridium pinhole inserts, each with a 0.3-mm square aperture; their radial distances from the centerline of the system ranged from 2.2 to 3.0 cm. The optimal radial distance to the closest scout pinhole and optimal number of MLEM iterations were 4.4 cm and 35 iterations, respectively, and the radial distances of the 39 scout pinholes ranged from 4.4 to 4.8 cm; aperture sizes ranged from 1.1 to 1.7 mm transaxially and 0.9–1.5 mm axially. After including data from the high-resolution section viewing the heart region into whole-body mouse reconstructions from scout data, the authors obtained high-resolution images of the heart, embedded within lower resolution images of the body, with minimal artifacts. Conclusions: The authors have optimized a dual-resolution collimator tube that provides both whole-body projections of a mouse and more targeted projections centered on the heart that can be jointly reconstructed to obtain high-resolution images of the heart embedded within lower-resolution whole-body images.

Human embryonic stem cell line WA0 1 was genetically modified using zinc-finger nucleases and the PiggyBac/transponson system to introduce a fluorescence reporter for VE-cadherin (VEC; tdTomato) and CD43 (eGFP). Phenotypic and functional assays for pluripotency revealed the modified hES cell reporter lines remained normal. When the cells were differentiated into hematoendothelial lineages, either by directed differentiation or direct reprogramming, flow cytometric and fluorescence microscopy showed that VEC + endothelial cells express tdTomato and CD43 + hematopoietic progenitors express eGFP.

Purpose Noise levels of brain SPECT images are highest in central regions, due to preferential attenuation of photons emitted from deep structures. To address this problem, the authors have designed a novel collimator for brain SPECT imaging that yields greatly increased sensitivity near the center of the brain without loss of resolution. This hybrid collimator consisted of ultrashort cone-beam holes in the central regions and slant-holes in the periphery (USCB). We evaluated this collimator for quantitative brain imaging tasks. Methods Owing to the uniqueness of the USCB collimation, the hole pattern required substantial variations in collimator parameters. To utilize the lead-casting technique, the authors designed two supporting plates to position about 37 000 hexagonal, slightly tapered pins. The holes in the supporting plates were modeled to yield the desired focal length, hole length, and septal thickness. To determine the properties of the manufactured collimator and to compute the system matrix, the authors prepared an array of point sources that covered the entire detector area. Each point source contained 32 μCi of Tc-99m at the first scan time. The array was imaged for 5 min at each of the 64 shifted locations to yield a 2-mm sampling distance, and hole parameters were calculated. The sensitivity was also measured using a point source placed along the central ray at several distances from the collimator face. High-count projection data from a five-compartment brain phantom were acquired with the three collimators on a dual-head SPECT/CT system. The authors calculated Cramer-Rao bounds on the precision of estimates of striatal and background activity concentration. In order to assess the new collimation system to detect changes in striatal activity, the authors evaluated the precision of measuring a 5% decrease in right putamen activity. The authors also reconstructed images of projection data obtained by summing data from the individual phantom compartments. Results The sensitivity of the novel cone-beam collimator varied with distance from the detector face; it was higher than that of the fan-beam collimator by factors ranging from 2.7 to 162. Examination of the projections of the point sources revealed that only a few holes were distorted or partially blocked, indicating that the intensive manual fabrication process was very successful. Better reconstructed phantom images were obtained from the USCB+FAN collimator pair than from either LEHR or FAN collimation. For the left caudate, located near the center of the brain, the detected counts were 9.8 (8.3) times higher for UCSB compared with LEHR (FAN), averaged over 60 views. The task-specific SNR for detecting a 5% decrease in putamen uptake was 7.4 for USCB and 3.2 for LEHR. Conclusions The authors have designed and manufactured a novel collimator for brain SPECT imaging. The sensitivity is much higher than that of a fan-beam collimator. Because of differences between the manufactured collimator and its design, reconstruction of the data requires a measured system matrix. The authors have demonstrated the potential of USCB collimation for improved precision in estimating striatal uptake. The novel collimator may be useful for early detection of Parkinson's disease, and for monitoring therapy response and disease progression.

SUMMARY SOX17 has been implicated in arterial specification and the maintenance of hematopoietic stem cells (HSCs) in the murine embryo. However, knowledge about molecular pathways and stage-specific effects of SOX17 in humans remains limited. Here, using SOX17-knockout and SOX17-inducible human pluripotent stem cells (hPSCs), paired with molecular profiling studies, we reveal that SOX17 is a master regulator of HOXA and arterial programs in hemogenic endothelium (HE) and is required for the specification of HE with robust lympho-myeloid potential and DLL4+CXCR4+ phenotype resembling arterial HE at the sites of HSC emergence. Along with the activation of NOTCH signaling, SOX17 directly activates CDX2 expression, leading to the upregulation of the HOXA cluster genes. Since deficiencies in HOXA and NOTCH signaling contribute to the impaired in vivo engraftment of hPSC-derived hematopoietic cells, the identification of SOX17 as a key regulator linking arterial and HOXA programs in HE may help to program HSC fate from hPSCs., Graphical Abstract, In Brief Jung et al. report that SOX17 is a critical upstream factor that is required for the activation and linkage of HOXA and arterial programs in the hemogenic endothelium and for establishing DLL4+CXCR4+ arterial hemogenic endothelium with definitive lympho-myeloid potential. These SOX17 effects are mediated through the activation of CDX2 and NOTCH signaling.

PURPOSE Phosphatidylinositol 3-kinase (PI3K) signaling is highly active in glioblastomas. We assessed pharmacokinetics, pharmacodynamics, and efficacy of the pan-PI3K inhibitor buparlisib in patients with recurrent glioblastoma with PI3K pathway activation. METHODS This study was a multicenter, open-label, multi-arm, phase II trial in patients with PI3K pathway–activated glioblastoma at first or second recurrence. In cohort 1, patients scheduled for re-operation after progression received buparlisib for 7 to 13 days before surgery to evaluate brain penetration and modulation of the PI3K pathway in resected tumor tissue. In cohort 2, patients not eligible for re-operation received buparlisib until progression or unacceptable toxicity. Once daily oral buparlisib 100 mg was administered on a continuous 28-day schedule. Primary end points were PI3K pathway inhibition in tumor tissue and buparlisib pharmacokinetics in cohort 1 and 6-month progression-free survival (PFS6) in cohort 2. RESULTS Sixty-five patients were treated (cohort 1, n = 15; cohort 2, n = 50). In cohort 1, reduction of phosphorylated AKTS473 immunohistochemistry score was achieved in six (42.8%) of 14 patients, but effects on phosphoribosomal protein S6S235/236 and proliferation were not significant. Tumor-to-plasma drug level was 1.0. In cohort 2, four (8%) of 50 patients reached 6-month PFS6, and the median PFS was 1.7 months (95% CI, 1.4 to 1.8 months). The most common grade 3 or greater adverse events related to treatment were lipase elevation (n = 7 [10.8%]), fatigue (n = 4 [6.2%]), hyperglycemia (n = 3 [4.6%]), and elevated ALT (n = 3 [4.6%]). CONCLUSION Buparlisib had minimal single-agent efficacy in patients with PI3K-activated recurrent glioblastoma. Although buparlisib achieved significant brain penetration, the lack of clinical efficacy was explained by incomplete blockade of the PI3K pathway in tumor tissue. Integrative results suggest that additional study of PI3K inhibitors that achieve more-complete pathway inhibition may still be warranted.

Background: Microcalcifications in atherosclerotic plaques are destabilizing, predict adverse cardiovascular events, and are associated with increased morbidity and mortality. 18 F-fluoride positron emission tomography (PET)/computed tomography (CT) imaging has demonstrated promise as a useful clinical diagnostic tool in identifying high-risk plaques; however, there is confusion as to the underlying mechanism of signal amplification seen in PET-positive, CT-negative image regions. This study tested the hypothesis that 18 F-fluoride PET/CT can identify early microcalcifications. Methods: 18 F-fluoride signal amplification derived from microcalcifications was validated against near-infrared fluorescence molecular imaging and histology using an in vitro 3-dimensional hydrogel collagen platform, ex vivo human specimens, and a mouse model of atherosclerosis. Results: Microcalcification size correlated inversely with collagen concentration. The 18 F-fluoride ligand bound to microcalcifications formed by calcifying vascular smooth muscle cell derived extracellular vesicles in the in vitro 3-dimensional collagen system and exhibited an increasing signal with an increase in collagen concentration (0.25 mg/mL collagen −33.8×10 2 ±12.4×10 2 counts per minute; 0.5 mg/mL collagen −67.7×10 2 ±37.4×10 2 counts per minute; P =0.0014), suggesting amplification of the PET signal by smaller microcalcifications. We further incubated human atherosclerotic endarterectomy specimens with clinically relevant concentrations of 18 F-fluoride. The 18 F-fluoride ligand labeled microcalcifications in PET-positive, CT-negative regions of explanted human specimens as evidenced by 18 F-fluoride PET/CT imaging, near-infrared fluorescence, and histological analysis. Additionally, the 18 F-fluoride ligand identified micro and macrocalcifications in atherosclerotic aortas obtained from low-density lipoprotein receptor-deficient mice. Conclusions: Our results suggest that 18 F-fluoride PET signal in PET-positive, CT-negative regions of human atherosclerotic plaques is the result of developing microcalcifications, and high surface area in regions of small microcalcifications may amplify PET signal.

The Quality assurance of ultrasound clinical imaging systems is essential for maintaining their performance to the highest level and for complying with the requirements by various regulatory and accrediting agencies. Although there is no standardization yet, most of the quality assessment procedures available in literature are proposed for B‐mode and Doppler imaging. However, ultrasound imaging systems offer a variety of advanced imaging modes, besides B‐mode and Doppler, which are primarily aimed at improving image quality. This study presents computer‐based methods for evaluating image quality for the advanced imaging modes of ultrasound imaging systems: harmonic imaging, spatial compounding imaging, adaptive speckle reduction, and tissue aberration correction. The functions and parameters proposed for evaluating image quality are: grayscale mapping function, image contrast, contrast‐to‐noise ratio (CNR), and high‐contrast spatial resolution. We present our computer‐based methods for evaluating image quality of these modes with a number of probe and scanner combinations, which were employed to image targets in ultrasound phantoms. The functions and parameters here proposed in image quality performance evaluation are: grayscale mapping function, image contrast, CNR, and high‐contrast spatial resolution. We show that these quantities could be useful in developing standardized methods for evaluating the advanced ultrasound imaging modes, especially when the advanced mode resulted in subtle visual differences.

NOTCH signaling is required for the arterial specification and formation of hematopoietic stem cells (HSCs) and lympho-myeloid progenitors in the embryonic aorta-gonad-mesonephros region and extraembryonic vasculature from a distinct lineage of vascular endothelial cells with hemogenic potential. However, the role of NOTCH signaling in hemogenic endothelium (HE) specification from human pluripotent stem cell (hPSC) has not been studied. Here, using a chemically defined hPSC differentiation system combined with the use of DLL1-Fc and DAPT to manipulate NOTCH, we discover that NOTCH activation in hPSC-derived immature HE progenitors leads to formation of CD144+CD43−CD73−DLL4+Runx1 + 23-GFP+ arterial-type HE, which requires NOTCH signaling to undergo endothelial-to-hematopoietic transition and produce definitive lympho-myeloid and erythroid cells. These findings demonstrate that NOTCH-mediated arterialization of HE is an essential prerequisite for establishing definitive lympho-myeloid program and suggest that exploring molecular pathways that lead to arterial specification may aid in vitro approaches to enhance definitive hematopoiesis from hPSCs., It is unclear whether arterial specification is required for hematopoietic stem cell formation. Here, the authors use a chemically defined human pluripotent stem cell (hPSC) differentiation system to show the role of NOTCH signaling in forming arterial-type hemogenic endothelial cells.

Alzheimer's disease (AD) is the most common cause of dementia. Neuroinflammation appears to play an important role in AD pathogenesis. Ligands of the 18 kDa translocator protein (TSPO), a marker for activated microglia, have been used as positron emission tomography (PET) tracers to reflect neuroinflammation in humans and mouse models. Here, we used the novel TSPO-targeted PET tracer18F-GE180 (flutriciclamide) to investigate differences in neuroinflammation between young and old WT and APP/PS1dE9 transgenic (Tg) mice.In vivoPET scans revealed an overt age-dependent elevation in whole-brain uptake of18F-GE180 in both WT and Tg mice, and a significant increase in whole-brain uptake of18F-GE180 (peak-uptake and retention) in old Tg mice compared with young Tg mice and all WT mice. Similarly, the18F-GE180 binding potential in hippocampus was highest to lowest in old Tg > old WT > young Tg > young WT mice using MRI coregistration.Ex vivoPET and autoradiography analysis further confirmed ourin vivoPET results: enhanced uptake and specific binding (SUV75%) of18F-GE180 in hippocampus and cortex was highest in old Tg mice followed by old WT, young Tg, and finally young WT mice.18F-GE180 specificity was confirmed by anin vivocold tracer competition study. We also examined18F-GE180 metabolites in 4-month-old WT mice and found that, although total radioactivity declined over 2 h, of the remaining radioactivity, ∼90% was due to parent18F-GE180. In conclusion,18F-GE180 PET scans may be useful for longitudinal monitoring of neuroinflammation during AD progression and treatment.SIGNIFICANCE STATEMENTMicroglial activation, a player in Alzheimer's disease (AD) pathogenesis, is thought to reflect neuroinflammation. Usingin vivomicroPET imaging with a novel TSPO radioligand,18F-GE180, we detected significantly enhanced neuroinflammation during normal aging in WT mice and in response to AD-associated pathology in APP/PS1dE9 Tg mice, an AD mouse model. Increased uptake and specific binding of18F-GE180 in whole brain and hippocampus were confirmed byex vivoPET and autoradiography. The binding specificity and stability of18F-GE180 was further confirmed by a cold tracer competition study and a metabolite study, respectively. Therefore,18F-GE180 PET imaging may be useful for longitudinal monitoring of neuroinflammation during AD progression and treatment and may also be useful for other neurodegenerative diseases.

Purpose: Recently, a multipinhole collimator with inserts that have both rectangular apertures and rectangular fields of view (FOVs) has been proposed for SPECTimaging since it can tile the projection onto the detector efficiently and the FOVs in transverse and axial directions become separable. The purpose of this study is to investigate the image properties of rectangular-aperture pinholes with submillimeter apertures sizes. Methods: In this work, the authors have conducted sensitivity and FOV experiments for 18 replicates of a prototype insert fabricated in platinum/iridium (Pt/Ir) alloy with submillimeter square-apertures. A sinqθ fit to the experimental sensitivity has been performed for these inserts. For the FOV measurement, the authors have proposed a new formula to calculate the projection intensity of a flood image on the detector, taking into account the penumbra effect. By fitting this formula to the measured projection data, the authors obtained the acceptance angles. Results: The mean (standard deviation) of fitted sensitivity exponents q and effective edge lengths we were, respectively, 10.8 (1.8) and 0.38 mm (0.02 mm), which were close to the values, 7.84 and 0.396 mm, obtained from Monte Carlo calculations using the parameters of the designed inserts. For the FOV measurement, the mean (standard deviation) of the transverse and axial acceptances were 35.0° (1.2°) and 30.5° (1.6°), which are in good agreement with the designed values (34.3° and 29.9°). Conclusions: These results showed that the physical properties of the fabricated inserts with submillimeter aperture size matched our design well.

SummaryThe mammalian target of rapamycin complex 1 (mTORC1) regulates activation of immune cells and cellular energy metabolism. Although glycolysis has been linked to immune functions, the mechanisms by which glycolysis regulates NLRP3 inflammasome activation remain unclear. Here, we demonstrate that mTORC1-induced glycolysis provides an essential mechanism for NLRP3 inflammasome activation. Moreover, we demonstrate that hexokinase 1 (HK1)-dependent glycolysis, under the regulation of mTORC1, represents a critical metabolic pathway for NLRP3 inflammasome activation. Downregulation of glycolysis by inhibition of Raptor/mTORC1 or HK1 suppressed both pro-IL-1β maturation and caspase-1 activation in macrophages in response to LPS and ATP. These results suggest that upregulation of HK1-dependent glycolysis by mTORC1 regulates NLRP3 inflammasome activation.

Radionuclide myocardial perfusion imaging (MPI) plays a vital role in the evaluation and management of patients with coronary artery disease. However, because of a steep growth in MPI in the mid 2000s, concerns about inappropriate use of MPI and imaging-related radiation exposure increased. In response, the professional societies developed appropriate-use criteria for MPI. Simultaneously, novel technology, image-reconstruction software for traditional scanners, and dedicated cardiac scanners emerged and facilitated the performance of MPI with low-dose and ultra-low-dose radiotracers. This paper provides a practical approach to performing low-radiation-dose MPI using traditional and novel technologies.

Purpose: Triple coincidences in positron emission tomography (PET) are events in which three γ-rays are detected simultaneously. These events, though potentially useful for enhancing the sensitivity of PET scanners, are discarded or processed without special consideration in current systems, because there is not a clear criterion for assigning them to a unique line-of-response (LOR). Methods proposed for recovering such events usually rely on the use of highly specialized detection systems, hampering general adoption, and/or are based on Compton-scatter kinematics and, consequently, are limited in accuracy by the energy resolution of standard PET detectors. In this work, the authors propose a simple and general solution for recovering triple coincidences, which does not require specialized detectors or additional energy resolution requirements. Methods: To recover triple coincidences, the authors’ method distributes such events among their possible LORs using the relative proportions of double coincidences in these LORs. The authors show analytically that this assignment scheme represents the maximum-likelihood solution for the triple-coincidence distribution problem. The PET component of a preclinical PET/CT scanner was adapted to enable the acquisition and processing of triple coincidences. Since the efficiencies for detecting double and triple events were found to be different throughout the scanner field-of-view, a normalization procedure specific for triple coincidences was also developed. The effect of including triple coincidences using their method was compared against the cases of equally weighting the triples among their possible LORs and discarding all the triple events. The authors used as figures of merit for this comparison sensitivity, noise-equivalent count (NEC) rates and image quality calculated as described in the NEMA NU-4 protocol for the assessment of preclinical PET scanners. Results: The addition of triple-coincidence events with the authors’ method increased peak NEC rates of the scanner by 26.6% and 32% for mouse- and rat-sized objects, respectively. This increase in NEC-rate performance was also reflected in the image-quality metrics. Images reconstructed using double and triple coincidences recovered using their method had better signal-to-noise ratio than those obtained using only double coincidences, while preserving spatial resolution and contrast. Distribution of triple coincidences using an equal-weighting scheme increased apparent system sensitivity but degraded image quality. The performance boost provided by the inclusion of triple coincidences using their method allowed to reduce the acquisition time of standard imaging procedures by up to ∼25%. Conclusions: Recovering triple coincidences with the proposed method can effectively increase the sensitivity of current clinical and preclinical PET systems without compromising other parameters like spatial resolution or contrast.

During hematopoiesis, red blood cells originate from the hematopoietic stem cell reservoir. Although the regulation of erythropoiesis and globin expression has been intensively investigated, the underlining mechanisms are not fully understood, including the interplay between transcription factors and epigenetic factors. Here, we uncover that the Mbd2-free NuRD chromatin remodeling complex potentiates erythroid differentiation of proerythroblasts via managing functions of the CP2c complexes. We found that both Mbd2 and Mbd3 expression is downregulated during differentiation of MEL cells in vitro and in normal erythropoiesis in mouse bone marrow, and Mbd2 downregulation is crucial for erythropoiesis. In uninduced MEL cells, the Mbd2-NuRD complex is recruited to the promoter via Gata1/Fog1, and, via direct binding through p66α, it acts as a transcriptional inhibitor of the CP2c complexes, preventing their DNA binding and promoting degradation of the CP2c family proteins to suppress globin gene expression. Conversely, during erythropoiesis in vitro and in vivo, the Mbd2-free NuRD does not dissociate from the chromatin and acts as a transcriptional coactivator aiding the recruitment of the CP2c complexes to chromatin, and thereby leading to the induction of the active hemoglobin synthesis and erythroid differentiation. Our study highlights the regulation of erythroid differentiation by the Mbd2-CP2c loop.

This prospective study aimed to evaluate the clinical values ofSeventy-three patients with surgically confirmed cerebral gliomas (19 grade II, 21 grade III, and 33 grade IV) who underwentMedian TCR was 1.20 (interquartile range [IQR], 1.14 to 1.4) in grade II, 1.65 (IQR, 1.26 to 1.79) in grade III, and 2.53 (IQR, 1.93 to 3.30) in grade IV gliomas. Significant differences in TCR were seen among the three WHO grade groups (P 0.001). In Cox regression analysis including TCR, MTV, molecular markers, and other clinical factors, TCR was prognostic for PFS (P = 0.016) and TCR and MTV were prognostic for OS (P = 0.024 [TCR], P = 0.030 [MTV]). PFS and OS were significantly shorter in patients with a TCR ≥ 1.6 than in those with a TCR 1.6. OS were significantly shorter in patients with a MTV ≥ 1 than in those with a TCR 1.TCR on

Recent advances in understanding the major bottlenecks in derivation of engraftable HSCs and lymphoid cells from pluripotent stem cells (PSC), have identified deficiencies in NOTCH and HOXA signaling as contributing factors to the observed functional deficits of PSC-derived hematopoietic progenitors. However, little is known about the mechanisms that are essential for establishing these pathways during PSC differentiation. Here, we revealed the critical role of SOX17 in linking HOXA and NOTCH-mediated arterial programs in hemogenic endothelium (HE) and specification of definitive lympho-myeloid hematopoiesis. Using SOX17-knockout (SOX17-/-) and SOX17 DOX-inducible (iSOX17) hESCs, we found that SOX17-deficiency substantially reduces formation of CD144+CD43-CD73-DLL4+CXCR4+/- arterial HE and definitive lympho-myeloid hematopoiesis, while SOX17 upregulation at mesodermal stage of development causes the opposite effect. Molecular profiling of HE generated from iSOX17 hESCs in DOX+ and DOX- conditions using RNAseq, SOX17 ChIPseq and ATACseq, revealed that SOX17 overexpression upregulates 522 genes enriched in NOTCH, TGFb, HEDGEHOG and WNT signaling, including DLL1, DLL4, NOTCH4, LFNG, WNT5a, WNT5b, GLI3, and genes associated with HSC development, CXCR4,KITLG and ALDH1A2. In addition, we noted significant upregulation of HOXA7,HOXA9, HOXA10, HOXB8, HOXC4 and CDX2 homeobox genes in SOX17-induced cultures, with no expression of HOXA genes observed in HE from SOX17-/- cells. ChIPSeq analysis revealed DOX+ specific SOX17 binding at transcriptional start sites (TSS) of 316 significantly upregulated genes, including ALDH1A2, CDX2, DLL1, DLL4, HEY1, HOXA7, HOXB8, HOXC4 and KITLG, suggesting that upregulation of these genes could be explained by their direct activation by SOX17. Since ALDH1A2 and CDX2 are known to play a role in the activation of HOXA genes, we investigated whether SOX17's effect on HOXA expression could also be mediated by ALDH1A2 and CDX2. We found that adding ALDH1 inhibitor to DOX+ cultures had no effect on arterial HE development and HOXA expression. In contrast, transfection of iSOX17 hPSCs cultures with CDX2 shRNA significantly decreased arterial HE formation and downregulated HOXA7, HOXA9, and HOXA10 expression. Overall, our studies indicate that SOX17 plays a critical role in the activation and integration of arterial and HOXA programs in HE, which is mediated by CDX2. These findings will be important for designing a strategy for direct HSC fate programming from hPSCs. Disclosures Uenishi: Casebia Therapeutics: Employment. Slukvin:Cynata Therapeutics: Consultancy, Other: Founder and Stockholder.

This study investigated purplish Washington clam, Saxidomus purpuratus on its gonadal development, reproductive cycle and shell length (age) at 50% female group maturity from January to December 2011 in Jinhae Bay, Korea. This species is dioecious. The gonad index (GI) began to increase in January, reached the maximum value in May and to decrease in June, reached the minimum value in September then to increase slowly in October again. The reproductive cycle of the clams can be divided into five successive stages: the early active stage (Ea, from February to March), the late active stage (La, from January to March and October to December), the ripe stage (R, from January to May), the spawning stage (S, from June to December) and the degenerative and inactive stage (Ia, from January to February and November to December). The spawning periods were from June to December and the main spawning periods were from July to October. The shell length at 50% female group maturity (SL50) was estimated to be 63.6 mm, which can be converted as sexual maturity age of 2.2 year.

This survey was conducted to evaluate the microbial contamination level of butcher's shops in Seoul, Korea. For microbial inspections, a total of 584 samples (146 cotton work gloves, 146 utensils and equipments, 154 beef samples, 138 pork samples) were collected from butcher's shops. E. coli and pathogenic bacteria such as Staphylococcus aureus , Yersinia enterocolitica , Listeria monocytogenes , Salmonella spp., and E. coli O157:H7 were tested in the samples. As a result, the level of aerobic plate count (APC) ranged ≤10 4 CFU/cm 2 from utensils and equipments, 10 1 ∼10 9 CFU/glove from cotton work gloves and ≤10 6 CFU/g from meat. The APC level of E. coli ranged ≤10 1 CFU/cm 2 from utensils and equipments, ≤10 5 CFU/glove from cotton work gloves, and ≤10 3 CFU/g from meat, respectively. Staphylococcus aureus was detected in 2 beef samples, 1 pork sample, and 10 used cotton work gloves. Yersinia enterocolitica was detected in 3 beef samples, 1 pork sample, and 3 used cotton work gloves.