Members Event Screening Room

Event participants

Nigol Bezjian, Director, Broken Dinners, Postponed Kisses
Chair: Rima Maktabi, Bureau Chief, Al Arabiya (UK)

7 April 2020

Research Event

Event participants

¹⁸F-PI-2620 is a next generation tau positron emission tomography (PET)-tracer that has demonstrated ability to image the spatial distribution of suspected tau pathology. The objective of this study was to assess the tracer biodistribution, dosimetry and quantitative methods of ¹⁸F-PI-2620 in the human brain. Full kinetic modelling approaches to quantify tau load were investigated. Non-invasive kinetic modeling approaches and semi-quantitative methods were evaluated against the full tracer kinetics. Finally, the reproducibility of PET measurements from test and retest scans was assessed. Methods: Three healthy controls (HC) and 4 Alzheimer disease (AD) subjects underwent two dynamic PET scans including arterial sampling. Distribution volume ratio (DVR) was estimated using full tracer kinetics (2 Tissue Compartment (2TC) models, Logan Graphical Analysis (LGA)) and non-invasive kinetic models (Non-Invasive Logan Graphical Analysis (NI-LGA) and the multilinear reference tissue model (MRTM2)). Standardized uptake value ratio (SUVR) was determined at different imaging windows after injection. Correlation between DVR and SUVR, effect size (Cohen’s d) and test-retest variability (TRV) were evaluated. Additionally, 6 HC subjects received one tracer administration and underwent whole-body PET for dosimetry calculation. Organ doses and the whole-body effective dose were calculated using OLINDA 2.0. Results: Strong correlation was found across different kinetic models (R2 >0.97) and between DVR(2TC) and SUVRs between 30 to 90 min with R2>0.95. Secular equilibrium was reached around 40 min post injection (p.i.) in most regions and subjects. The TRV and effect size for the SUVR across different regions was similar at 30-60 min (TRV=3.8%, d=3.80), 45-75 min (TRV=4.3%, d=3.77) and 60-90 min (TRV=4.9%, d=3.73) and increased at later time points. Elimination was via the hepatobiliary and urinary system. The whole-body effective dose was determined to be 33.3±2.1 μSv/MBq for an adult female and 33.1±1.4 μSv/MBq for an adult male with a 1.5 hour urinary bladder voiding interval. Conclusion: ¹⁸F-PI-2620 exhibits fast kinetics, suitable dosimetry and low TRV. DVR measured using the 2TC model with arterial sampling correlated strongly with DVR measured by NI-LGA, MRTM2 and SUVR. SUVR can be used for ¹⁸F-PI-2620 PET quantification of tau deposits avoiding arterial blood sampling. Static ¹⁸F-PI-2620 PET scans between 45-75min p.i. provide excellent quantification accuracy, large effect size and low TRV.

Purpose: Although the incidence of de novo neuroendocrine prostate cancer (NEPC) is rare, recent data suggests that low expression of prostate-specific membrane antigen (PSMA) is associated with a spectrum of neuroendocrine (NE) hallmarks and androgen receptor (AR)-suppression in prostate cancer (PC). Previous clinical reports indicate that PCs with a phenotype similar to NE tumors can be more amenable to imaging by ¹⁸F-Fluorodeoxyglucose (FDG) rather than PSMA-targeting radioligands. In this study, we evaluated the association between NE gene signature and FDG uptake-associated genes including glucose transporters (GLUTs) and hexokinases, with the goal of providing a genomic signature to explain the reported FDG-avidity of PSMA-suppressed tumors. Methods: Data mining approaches, cell lines and patient-derived xenograft (PDX) models were used to study the levels of 14 members of the SLC2A family (encoding GLUT proteins), 4 members of the hexokinase family (genes: HK1 to 3 and GCK) and PSMA (FOLH1 gene) following AR-inhibition and in correlation with NE hallmarks. Also, we characterize a NE-like PC (NELPC) subset among a cohort of primary and metastatic PC samples with no NE histopathology. We measured glucose uptake in a NE-induced in vitro model and a zebrafish model by non-radioactive imaging of glucose uptake using fluorescent glucose bioprobe, GB2-Cy3. Results: This work demonstrates that a NE gene signature associates with differential expression of genes encoding GLUT and hexokinase proteins. In NELPC, elevated expression of GCK (encoding glucokinase protein) and decreased expression of SLC2A12 correlated with earlier biochemical recurrence. In tumors treated with AR-inhibitors, high expression of GCK and low expression of SLC2A12 correlated with NE histopathology and PSMA gene suppression. GLUT12-suppression and amplification of glucokinase was observed in NE-induced PC cell lines and PDX models. A higher glucose uptake was confirmed in low-PSMA tumors using a GB2-Cy3 probe in a zebrafish model. Conclusion: NE gene signature in NEPC and NELPC associates with a distinct transcriptional profile of GLUTs and HKs. PSMA-suppression correlates with GLUT12-suppression and glucokinase-amplification. Alteration of FDG uptake-associated genes correlated positively with higher glucose uptake in AR and PSMA-suppressed tumors. Zebrafish xenograft tumor models are an accurate and efficient pre-clinical method for monitoring non-radioactive glucose uptake.

Introduction: The aim of this study was to analyze patterns of persistent versus recurrent or new PET lesions in a selected patient cohort with PSA persistence following salvage lymph node dissection (SLND) and pre/post procedure prostate-specific membrane antigen ligand positron emission tomography (PSMA-PET). Material and Methods: 16 patients were included in this multicenter study. Inclusion criteria were: a) PSMA-PET performed for biochemical recurrence before SLND (pre-SLND PET) and b) repeat PSMA-PET performed for persistently elevated PSA level (≥0.1 ng/mL) ≥6 weeks after SLND (post-SLND PET). Image analysis was performed by three independent nuclear medicine physicians applying the molecular imaging TNM system PROMISE. Lesions were confirmed by histopathology, presence on correlative CT/MRI/bone scan or PSA response after focal therapy. Results: post-SLND PET identified PCa-lesions in 88% (14/16) of patients with PSA persistence after SLND. Median PSA was 1.2 ng/mL (IQR, 0.6-2.8 ng/mL). Disease was confined to the pelvis in 56% of patients (9/16) and most of these men had common iliac (6/16, 38%) and internal iliac lymph node metastases (6/16, 38%). Extrapelvic disease was detected in 31% of patients (5/16). In pre- and post-SLND PET comparison, 10/16 had at least one lesion already detected at baseline (63% PET persistence); 4/16 had new lesions only (25% PET recurrence); 2 had no disease on post-SLND PET. All validated regions (11 regions in 9 patients) were true positive. 9/14 (64%) patients underwent repeat local therapies after SLND (7/14 radiotherapy, 2/14 surgery). Conclusion: SLND of pelvic nodal metastases was often not complete according to PSMA-PET. About two thirds of patients had PET positive nodal disease after SLND already seen on pre-SLND PSMA-PET. Notably, about one quarter of patients had new lesions, not detected by pre-surgical PSMA-PET.

⁶⁸Ga-DOTATOC-PET/MRI (⁶⁸Gallium-DOTATOC-positron emission tomography/magnetic resonance imaging) combines the advantages of PET in the acquisition of metabolic-functional information with the high soft tissue contrast of MRI. Standardized uptake values (SUV) in tumors were suggested as a measure of somatostatin receptor expression. A challenge with receptor ligands is, that the distribution volume is confined to tissues with tracer-uptake, potentially limiting SUV quantification. In this study, different functional, three-dimensional (3D) SUV, apparent diffusion coefficient (ADC) parameters and arterial tumor enhancement were tested for the characterization of gastroendopancreatic neuroendocrine tumors (GEP-NET). Methods: For this single-center, cross-sectional study, 22 patients with 24 histologically confirmed GEP-NET lesions (15 men/7 women; median, 61 years, range, 43-81 years), who received hybrid ⁶⁸Ga-DOTA-PET/MRI examinations at 3T between January 2017 and July 2019 met eligibility criteria. SUVs, tumor-to-background ratios (TBR), the total functional tumor volume (TFTV), ADCmean and ADCmin were measured based on volumes of interest (VOI) and examined with receiver operating characteristic analysis to determine cut-off values for differentiation between low and intermediate grade GEP-NET. Spearman’s rank correlation coefficients were used to assess correlations between functional imaging parameters. Results: The ratio of PET-derived SUVmean and diffusion-weighted imaging (DWI)-derived ADCmin was introduced as a combined variable to predict tumor grade, outperforming single predictors. Based on a threshold ratio of 0.03 to be exceeded, tumors could be classified as grade 2 with a sensitivity of 86% and specificity of 100%. SUV and functional ADC values as well as arterial contrast enhancement parameters showed non-significant and mostly negligible correlations. Conclusion: As receptor density and tumor cellularity appear to be independent, potentially complementary phenomena, the combined PET/MRI ratio SUVmean/ADCmin may be used as a novel biomarker, allowing to differentiate between grade 1 and 2 GEP-NET.

Cancer Survival is related to tumor volume. FDG PET measurement of tumor volume holds promise but is not yet a clinical tool. Measurements come in two forms: the total lesion volume (TLV) based on the number of voxels in the tumor and secondly the total lesion glycolysis (TLG) which is the TLV multiplied by the average SUL per voxel of the tumor (SUL is the standardize uptake value normalized for lean mass). In this study we measured tumor volume in patients with malignant pleural mesothelioma (MPM). METHODS: A threshold-based program in IDL was developed to measure tumor volume in FDG PET images. 19 patients with malignant pleural mesothelioma (MPM) were studied before and after two cycles (6 weeks) of chemo-immunotherapy. Measurements included the total lesion volume (TLV), Total Lesion Glycolysis (TLG), the sum of the SULs in the tumor (SUL- total), a measure of total FDG uptake, and the average SUL per voxel. RESULTS: Baseline MPM volumes (TLV) ranged from 11 to 2610 cc. TLG values ranged from 32 to 8552 SUL-cc and were strongly correlated with TLV. While tumor volumes ranged over 3 orders of magnitude, the average SUL per voxel, SUL-average, stayed within a narrow range of 2.4 to 5.3 units. Thus, TLV was the major component of TLG while SUL-average was a minor component and was essentially constant. Further evaluation of SUL-average showed that in this cohort it’s two components SUL-total and tumor volume changed in parallel and were strongly correlated, r= 0.99, p<.01. Thus, whether the tumors were large or small, the FDG uptake as measured by SUL-total was proportional to the total tumor volume. Conclusion: TLG equals TLV multiplied by the average SUL per voxel, essentially TLV multiplied by a constant. Thus TLG, commonly considered a measure of "metabolic activity" in tumors, is also in this cohort a measure of tumor volume. The constancy of SUL per voxel is due to FDG uptake being proportional to tumor volume. Thus, in this study, the FDG uptake was also a measure of volume.

Multidrug resistance-associated protein 1 (ABCC1) is abundantly expressed at the lung epithelial barrier, where it may influence the pulmonary disposition of inhaled drugs and contribute to variability in therapeutic response. Aim of this study was to assess the impact of ABCC1 on the pulmonary disposition of 6-bromo-7-¹¹C-methylpurine (¹¹C-BMP), a prodrug radiotracer which is intracellularly conjugated with glutathione to form the ABCC1 substrate S-(6-(7-¹¹C-methylpurinyl))glutathione (¹¹C-MPG). Methods: Groups of Abcc1(-/-) rats, wild-type rats pretreated with the ABCC1 inhibitor MK571 and wild-type control rats underwent dynamic PET scans after administration of ¹¹C-BMP intravenously (i.v.) or by intratracheal aerosolization (i.t.). In vitro transport experiments were performed with unlabeled BMP in the human distal lung epithelial cell line NCI-H441. Results: Pulmonary kinetics of radioactivity were significantly different between wild-type and Abcc1(-/-) rats, but differences were more pronounced after i.t. than after i.v. administration. After i.v. administration lung exposure (AUClung) was 77% higher and the elimination slope of radioactivity washout from the lungs (kE,lung) was 70% lower, whereas after i.t. administration AUClung was 352% higher and kE,lung was 86% lower in Abcc1(-/-) rats. Pretreatment with MK571 decreased kE,lung by 20% after i.t. radiotracer administration. Intracellular accumulation of MPG in NCI-H441 cells was significantly higher and extracellular efflux was lower in presence than in absence of MK571. Conclusion: PET with pulmonary administered ¹¹C-BMP can measure ABCC1 activity at the lung epithelial barrier and may be applicable in humans to assess the effects of disease, genetic polymorphisms or concomitant drug intake on pulmonary ABCC1 activity.

Bone is the most common site of distant metastatic spread in prostate adenocarcinoma. Prostate-specific membrane antigen uptake has been described in both benign and malignant bone lesions, which can lead to false-positive findings on ⁶⁸Ga-prostate-specific membrane antigen-11 positron emission tomography (⁶⁸Ga-PSMA-11 PET). The purpose of this study was to evaluate the diagnostic accuracy of ⁶⁸Ga-PSMA-11 PET for osseous prostate cancer metastases and improve bone uptake interpretation using semi-quantitative metrics. METHODS: 56 prostate cancer patients (18 pre-prostatectomy, 38 biochemical recurrence) who underwent ⁶⁸Ga-PSMA-11 PET/MRI or PET/CT examinations with osseous PSMA-ligand uptake were included in the study. Medical records were reviewed retrospectively by board-certified nuclear radiologists to determine true or false positivity based on a composite endpoint. For each avid osseous lesion, biological volume, size, PSMA-RADS rating, maximum standardized uptake value (SUV_max), and ratio of lesion SUV_max to liver, blood pool, and background bone SUV_max were measured. Differences between benign and malignant lesions were evaluated for statistical significance, and cut-off values for these parameters were determined to maximize diagnostic accuracy. RESULTS: Among 56 participants, 13 patients (22.8%) had false-positive osseous ⁶⁸Ga-PSMA-11 findings and 43 patients (76.8%) had true-positive osseous ⁶⁸Ga-PSMA-11 findings. Twenty-two patients (39%) had 1 osseous lesion, 18 (32%) had 2-4 lesions, and 16 (29%) had 5 or more lesions. Cut-off values resulting in statistically significant (p<0.005) differences between benign and malignant lesions were: PSMA-RADS ≥4, SUV_max ≥4.1, SUV_max ratio of lesion to blood pool ≥2.11, to liver ≥0.55, and to bone ≥4.4. These measurements corresponded to lesion-based ⁶⁸Ga-PSMA-11 PET lesion detection rate for malignancy of 80%, 93%, 89%, 21%, 89%, and a specificity of 73%, 73%, 73%, 93%, 60%, respectively. CONCLUSION: PSMA-RADS rating, SUV_max, and SUV_max ratio of lesion to blood pool can help differentiate benign from malignant lesions on ⁶⁸Ga-PSMA-11 PET. SUV_max ratio to blood pool above 2.2 is a reasonable parameter to support image interpretation and presented superior lesion detection rate and specificity when compared to visual interpretation by PSMA RADS. These parameters hold clinical value by improving diagnostic accuracy for metastatic prostate cancer on ⁶⁸Ga-PSMA-11 PET/MRI and PET/CT.

Invitation Only Research Event

Event participants

Professor Nick Binedell, Founding Director and Sasol Chair of Strategic Management, Gordon Institute of Business Science (GIBS), University of Pretoria

Research Event

Event participants

Alan Doss, President, Kofi Annan Foundation
 

Laser-capture microdissection (LCM) allows the visualization and isolation of morphologically distinct subpopulations of cells from heterogeneous tissue specimens. In combination with formalin-fixed and paraffin-embedded (FFPE) tissue it provides a powerful tool for retrospective and clinically relevant studies of tissue proteins in a healthy and diseased context. We first optimized the protocol for efficient LCM analysis of FFPE tissue specimens. The use of SDS containing extraction buffer in combination with the single-pot solid-phase-enhanced sample preparation (SP3) digest method gave the best results regarding protein yield and protein/peptide identifications. Microdissected FFPE human substantia nigra tissue samples (~3,000 cells) were then analyzed, using tandem mass tag (TMT) labeling and LC-MS/MS, resulting in the quantification of >5,600 protein groups. Nigral proteins were classified and analyzed by abundance, showing an enrichment of extracellular exosome and neuron-specific gene ontology (GO) terms among the higher abundance proteins. Comparison of microdissected samples with intact tissue sections, using a label-free shotgun approach, revealed an enrichment of neuronal cell type markers, such as tyrosine hydroxylase and alpha-synuclein, as well as proteins annotated with neuron-specific GO terms. Overall, this study provides a detailed protocol for laser-capture proteomics using FFPE tissue and demonstrates the efficiency of LCM analysis of distinct cell subpopulations for proteomic analysis using low sample amounts.

Accumulating evidence suggests that brown adipose tissue (BAT) is a potential therapeutic target for managing obesity and related diseases. PGAM family member 5, mitochondrial serine/threonine protein phosphatase (PGAM5), is a protein phosphatase that resides in the mitochondria and regulates many biological processes, including cell death, mitophagy, and immune responses. Because BAT is a mitochondria-rich tissue, we have hypothesized that PGAM5 has a physiological function in BAT. We previously reported that PGAM5-knockout (KO) mice are resistant to severe metabolic stress. Importantly, lipid accumulation is suppressed in PGAM5-KO BAT, even under unstressed conditions, raising the possibility that PGAM5 deficiency stimulates lipid consumption. However, the mechanism underlying this observation is undetermined. Here, using an array of biochemical approaches, including quantitative RT-PCR, immunoblotting, and oxygen consumption assays, we show that PGAM5 negatively regulates energy expenditure in brown adipocytes. We found that PGAM5-KO brown adipocytes have an enhanced oxygen consumption rate and increased expression of uncoupling protein 1 (UCP1), a protein that increases energy consumption in the mitochondria. Mechanistically, we found that PGAM5 phosphatase activity and intramembrane cleavage are required for suppression of UCP1 activity. Furthermore, utilizing a genome-wide siRNA screen in HeLa cells to search for regulators of PGAM5 cleavage, we identified a set of candidate genes, including phosphatidylserine decarboxylase (PISD), which catalyzes the formation of phosphatidylethanolamine at the mitochondrial membrane. Taken together, these results indicate that PGAM5 suppresses mitochondrial energy expenditure by down-regulating UCP1 expression in brown adipocytes and that its phosphatase activity and intramembrane cleavage are required for UCP1 suppression.

Gemma Cadby
Apr 1, 2020; 61:537-545
Patient-Oriented and Epidemiological Research

Kotaro Hama
Apr 1, 2020; 61:523-536
Patient-Oriented and Epidemiological Research

Xia Meng
May 1, 2020; 61:591-591
Images in Lipid Research

7 April 2020

The histidine kinase Hik33 plays important roles in mediating cyanobacterial response to divergent types of abiotic stresses including cold, salt, high light (HL), and osmotic stresses. However, how these functions are regulated by Hik33 remains to be addressed. Using a hik33-deficient strain (hik33) of Synechocystis sp. PCC 6803 (Synechocystis) and quantitative proteomics, we found that Hik33 depletion induces differential protein expression highly similar to that induced by divergent types of stresses. This typically includes downregulation of proteins in photosynthesis and carbon assimilation that are necessary for cell propagation, and upregulation of heat shock proteins, chaperons, and proteases that are important for cell survival. This observation indicates that depletion of Hik33 alone mimics divergent types of abiotic stresses, and that Hik33 could be important for preventing abnormal stress response in the normal condition. Moreover, we found the majority of proteins of plasmid origin were significantly upregulated in hik33, though their biological significance remains to be addressed. Together, the systematically characterized Hik33-regulated cyanobacterial proteome, which is largely involved in stress responses, builds the molecular basis for Hik33 as a general regulator of stress responses.

Accumulation and propagation of hyperphosphorylated tau (p-tau) is a common neuropathological hallmark associated with neurodegeneration of Alzheimer's disease (AD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), and related tauopathies. Extracellular vesicles, specifically exosomes, have recently been demonstrated to participate in mediating tau propagation in brain. Exosomes produced by human induced pluripotent stem cell (iPSC)-derived neurons expressing mutant Tau (mTau), containing the P301L and V337M Tau mutations of FTDP-17, possess the ability to propagate p-tau pathology after injection into mouse brain.  To gain an understanding of the mTau exosome cargo involved in tau pathogenesis, these pathogenic exosomes were analyzed by proteomics and bioinformatics. The data showed that mTau expression dysregulates the exosome proteome to result in (1) proteins uniquely present only in mTau, and not control exosomes, (2) the absence of proteins in mTau exosomes, uniquely present in control exosomes, and (3) shared proteins which were significantly up-regulated or down-regulated in mTau compared to control exosomes. Notably, mTau exosomes (not control exosomes) contain ANP32A (also known as I1PP2A), an endogenous inhibitor of the PP2A phosphatase which regulates the phosphorylation state of p-tau.  Several of the mTau exosome-specific proteins have been shown to participate in AD mechanisms involving lysosomes, inflammation, secretases, and related processes.  Furthermore, the mTau exosomes lacked a substantial portion of proteins present in control exosomes involved in pathways of localization, vesicle transport, and protein binding functions. The shared proteins present in both mTau and control exosomes represented exosome functions of vesicle-mediated transport, exocytosis, and secretion processes. These data illustrate mTau as a dynamic regulator of the biogenesis of exosomes to result in acquisition, deletion, and up- or down-regulation of protein cargo to result in pathogenic mTau exosomes capable of in vivo propagation of p-tau neuropathology in mouse brain. 

Assembly factors play a critical role in the biogenesis of mitochondrial respiratory chain complexes I-IV where they assist in the membrane insertion of subunits, attachment of co-factors, and stabilization of assembly intermediates. The major fraction of complexes I, III and IV are present together in large molecular structures known as respiratory chain supercomplexes. A number of assembly factors have been proposed as required for supercomplex assembly, including the hypoxia inducible gene 1 domain family member HIGD2A. Using gene-edited human cell lines and extensive steady state, translation and affinity enrichment proteomics techniques we show that loss of HIGD2A leads to defects in the de novo biogenesis of mtDNA-encoded COX3, subsequent accumulation of complex IV intermediates and turnover of COX3 partner proteins. Deletion of HIGD2A also leads to defective complex IV activity. The impact of HIGD2A loss on complex IV was not altered by growth under hypoxic conditions, consistent with its role being in basal complex IV assembly. While in the absence of HIGD2A we show that mitochondria do contain an altered supercomplex assembly, we demonstrate it to harbor a crippled complex IV lacking COX3. Our results redefine HIGD2A as a classical assembly factor required for building the COX3 module of complex IV.

Atherosclerosis-related CVD causes nearly 20 million deaths annually. Most patients are treated after plaques develop, so therapies must regress existing lesions. Current therapies reduce plaque volume, but targeting all apoB-containing lipoproteins with intensive combinations that include alirocumab or evinacumab, monoclonal antibodies against cholesterol-regulating proprotein convertase subtilisin/kexin type 9 and angiopoietin-like protein 3, may provide more benefit. We investigated the effect of such lipid-lowering interventions on atherosclerosis in APOE*3-Leiden.CETP mice, a well-established model for hyperlipidemia. Mice were fed a Western-type diet for 13 weeks and thereafter matched into a baseline group (euthanized at 13 weeks) and five groups that received diet alone (control) or with treatment [atorvastatin; atorvastatin and alirocumab; atorvastatin and evinacumab; or atorvastatin, alirocumab, and evinacumab (triple therapy)] for 25 weeks. We measured effects on cholesterol levels, plaque composition and morphology, monocyte adherence, and macrophage proliferation. All interventions reduced plasma total cholesterol (37% with atorvastatin to 80% with triple treatment; all P < 0.001). Triple treatment decreased non-HDL-C to 1.0 mmol/l (91% difference from control; P < 0.001). Atorvastatin reduced atherosclerosis progression by 28% versus control (P < 0.001); double treatment completely blocked progression and diminished lesion severity. Triple treatment regressed lesion size versus baseline in the thoracic aorta by 50% and the aortic root by 36% (both P < 0.05 vs. baseline), decreased macrophage accumulation through reduced proliferation, and abated lesion severity. Thus, high-intensive cholesterol-lowering triple treatment targeting all apoB-containing lipoproteins regresses atherosclerotic lesion area and improves lesion composition in mice, making it a promising potential approach for treating atherosclerosis.

apoB exists as apoB100 and apoB48, which are mainly found in hepatic VLDLs and intestinal chylomicrons, respectively. Elevated plasma levels of apoB-containing lipoproteins (Blps) contribute to coronary artery disease, diabetes, and other cardiometabolic conditions. Studying the mechanisms that drive the assembly, intracellular trafficking, secretion, and function of Blps remains challenging. Our understanding of the intracellular and intraorganism trafficking of Blps can be greatly enhanced, however, with the availability of fusion proteins that can help visualize Blp transport within cells and between tissues. We designed three plasmids expressing human apoB fluorescent fusion proteins: apoB48-GFP, apoB100-GFP, and apoB48-mCherry. In Cos-7 cells, transiently expressed fluorescent apoB proteins colocalized with calnexin and were only secreted if cells were cotransfected with microsomal triglyceride transfer protein. The secreted apoB-fusion proteins retained the fluorescent protein and were secreted as lipoproteins with flotation densities similar to plasma HDL and LDL. In a rat hepatoma McA-RH7777 cell line, the human apoB100 fusion protein was secreted as VLDL- and LDL-sized particles, and the apoB48 fusion proteins were secreted as LDL- and HDL-sized particles. To monitor lipoprotein trafficking in vivo, the apoB48-mCherry construct was transiently expressed in zebrafish larvae and was detected throughout the liver. These experiments show that the addition of fluorescent proteins to the C terminus of apoB does not disrupt their assembly, localization, secretion, or endocytosis. The availability of fluorescently labeled apoB proteins will facilitate the exploration of the assembly, degradation, and transport of Blps and help to identify novel compounds that interfere with these processes via high-throughput screening.

CVD is the leading cause of death worldwide, and genetic investigations into the human lipidome may provide insight into CVD risk. The aim of this study was to estimate the heritability of circulating lipid species and their genetic correlation with CVD traits. Targeted lipidomic profiling was performed on 4,492 participants from the Busselton Family Heart Study to quantify the major fatty acids of 596 lipid species from 33 classes. We estimated narrow-sense heritabilities of lipid species/classes and their genetic correlations with eight CVD traits: BMI, HDL-C, LDL-C, triglycerides, total cholesterol, waist-hip ratio, systolic blood pressure, and diastolic blood pressure. We report heritabilities and genetic correlations of new lipid species/subclasses, including acylcarnitine (AC), ubiquinone, sulfatide, and oxidized cholesteryl esters. Over 99% of lipid species were significantly heritable (h²: 0.06–0.50) and all lipid classes were significantly heritable (h²: 0.14–0.50). The monohexosylceramide and AC classes had the highest median heritabilities (h² = 0.43). The largest genetic correlation was between clinical triglycerides and total diacylglycerol (r_g = 0.88). We observed novel positive genetic correlations between clinical triglycerides and phosphatidylglycerol species (r_g: 0.64–0.82), and HDL-C and alkenylphosphatidylcholine species (r_g: 0.45–0.74). Overall, 51% of the 4,768 lipid species-CVD trait genetic correlations were statistically significant after correction for multiple comparisons. This is the largest lipidomic study to address the heritability of lipids and their genetic correlation with CVD traits. Future work includes identifying putative causal genetic variants for lipid species and CVD using genome-wide SNP and whole-genome sequencing data.

X-linked adrenoleukodystrophy (X-ALD) is an inherited disorder caused by deleterious mutations in the ABCD1 gene. The ABCD1 protein transports very long-chain FAs (VLCFAs) from the cytosol into the peroxisome where the VLCFAs are degraded through β-oxidation. ABCD1 dysfunction leads to VLCFA accumulation in individuals with X-ALD. FAs are activated by esterification to CoA before metabolic utilization. However, the intracellular pools and metabolic profiles of individual acyl-CoA esters have not been fully analyzed. In this study, we profiled the acyl-CoA species in fibroblasts from X-ALD patients and in ABCD1-deficient HeLa cells. We found that hexacosenoyl (26:1)-CoA, but not hexacosanoyl (26:0)-CoA, was the most abundantly concentrated among the VLCFA-CoA species in these cells. We also show that 26:1-CoA is mainly synthesized from oleoyl-CoA, and the metabolic turnover rate of 26:1-CoA was almost identical to that of oleoyl-CoA in both WT and ABCD1-deficient HeLa cells. The findings of our study provide precise quantitative and metabolic information of each acyl-CoA species in living cells. Our results suggest that VLCFA is endogenously synthesized as VLCFA-CoA through a FA elongation pathway and is then efficiently converted to other metabolites, such as phospholipids, in the absence of ABCD1.

When you run the Copy Files task in SAS Enterprise Guide and there is no connection to a SAS server, it fails with the following error: "ERROR: Target folder does not exist or cannot be accessed."

α-Klotho is a circulating factor with well-documented anti-aging properties; however, the central role of α-klotho in metabolism remains largely unexplored. The current study investigated the potential role of central α-klotho to modulate NPY/AgRP neurons, energy balance, and glucose homeostasis. Intracerebroventricular (ICV) administration of α-klotho suppressed food intake, improved glucose profiles, and reduced body weight in mouse models of Type I and II diabetes. Furthermore, central α-klotho inhibition via an anti-α-klotho antibody impaired glucose tolerance. Ex vivo patch clamp electrophysiology and immunohistochemical analysis revealed that α-klotho suppresses NPY/AgRP neuron activity, at least in part, by enhancing mIPSC’s. Experiments in hypothalamic GT1-7 cells observed α-klotho induces phosphorylation of AKT^ser473, ERK^{thr202/tyr204}, and FOXO1^ser256, as well as blunts AgRP gene transcription. Mechanistically, fibroblast growth factor 1 (FGFR1) inhibition abolished the downstream signaling of α-klotho, negated its ability to modulate NPY/AgRP neurons, and blunted its therapeutic effects. PI3 kinase inhibition also abolished α-klotho’s ability to suppress food intake and improve glucose clearance. These results indicate a prominent role of hypothalamic α-klotho/FGFR1/PI3K signaling in the modulation of NPY/AgRP neuron activity and maintenance of energy homeostasis, thus providing new insight into the pathophysiology of metabolic disease.

Type 2 diabetes mellitus predicts outcome following acute myocardial infarction (AMI). Since underlying mechanics are incompletely understood, we investigated left ventricular (LV) and atrial (LA) pathophysiological changes and their prognostic implications using cardiovascular magnetic resonance (CMR). Consecutive patients (n=1147, n=265 diabetic; n=882 non-diabetic) underwent CMR 3 days after AMI. Analyses included LV ejection fraction (LVEF), global longitudinal, circumferential and radial strains (GLS, GCS and GRS), LA reservoir, conduit and booster pump strains, as well as infarct size, edema and microvascular obstruction. Predefined endpoints were major adverse cardiovascular events (MACE) within 12 months. Diabetic patients had impaired LA reservoir (19.8 vs. 21.2%, p<0.01) and conduit strains (7.6 vs. 9.0%, p<0.01) but not ventricular function or myocardial damage. They were at higher risk of MACE than non-diabetic patients (10.2% vs. 5.8%, p<0.01) with most MACE occurring in patients with LVEF≥35%. Whilst LVEF (p=0.045) and atrial reservoir strain (p=0.024) were independent predictors of MACE in non-diabetic patients, GLS was in diabetic patients (p=0.010). Considering patients with diabetes and LVEF≥35% (n=237), GLS and LA reservoir strain below median were significantly associated with MACE. In conclusion, in patients with diabetes, LA and LV longitudinal strain permit optimized risk assessment early after reperfused AMI with incremental prognostic value over and above LVEF.

The actin cytoskeleton is extremely dynamic and supports diverse cellular functions in many physiological and pathological processes, including tumorigenesis. However, the mechanisms that regulate the actin-related protein 2/3 (ARP2/3) complex and thereby promote actin polymerization and organization in cancer cells are not well-understood. We previously implicated the proline-rich 11 (PRR11) protein in lung cancer development. In this study, using immunofluorescence staining, actin polymerization assays, and siRNA-mediated gene silencing, we uncovered that cytoplasmic PRR11 is involved in F-actin polymerization and organization. We found that dysregulation of PRR11 expression results in F-actin rearrangement and nuclear instability in non-small cell lung cancer cells. Results from molecular mechanistic experiments indicated that PRR11 associates with and recruits the ARP2/3 complex, facilitates F-actin polymerization, and thereby disrupts the F-actin cytoskeleton, leading to abnormal nuclear lamina assembly and chromatin reorganization. Inhibition of the ARP2/3 complex activity abolished irregular F-actin polymerization, lamina assembly, and chromatin reorganization due to PRR11 overexpression. Notably, experiments with truncated PRR11 variants revealed that PRR11 regulates F-actin through different regions. We found that deletion of either the N or C terminus of PRR11 abrogates its effects on F-actin polymerization and nuclear instability and that deletion of amino acid residues 100–184 or 100–200 strongly induces an F-actin structure called the actin comet tail, not observed with WT PRR11. Our findings indicate that cytoplasmic PRR11 plays an essential role in regulating F-actin assembly and nuclear stability by recruiting the ARP2/3 complex in human non-small cell lung carcinoma cells.

Over the last several years it has become clear that higher order assemblies on membranes, exemplified by signalosomes, are a paradigm for the regulation of many membrane signaling processes. We have recently combined two-color direct stochastic optical reconstruction microscopy (dSTORM) with the (Clus-DoC) algorithm that combines cluster detection and colocalization analysis to observe the organization of 5-lipoxygenase (5-LO) and 5-lipoxygenase–activating protein (FLAP) into higher order assemblies on the nuclear envelope of mast cells; these assemblies were linked to leukotriene (LT) C4 production. In this study we investigated whether higher order assemblies of 5-LO and FLAP included cytosolic phospholipase A2 (cPLA2) and were linked to LTB4 production in murine neutrophils. Using two- and three-color dSTORM supported by fluorescence lifetime imaging microscopy we identified higher order assemblies containing 40 molecules (median) (IQR: 23, 87) of 5-LO, and 53 molecules (62, 156) of FLAP monomer. 98 (18, 154) molecules of cPLA2 were clustered with 5-LO, and 77 (33, 114) molecules of cPLA2 were associated with FLAP. These assemblies were tightly linked to LTB4 formation. The activation-dependent close associations of cPLA2, FLAP, and 5-LO in higher order assemblies on the nuclear envelope support a model in which arachidonic acid is generated by cPLA2 in apposition to FLAP, facilitating its transfer to 5-LO to initiate LT synthesis.

Accumulating evidence suggests that brown adipose tissue (BAT) is a potential therapeutic target for managing obesity and related diseases. PGAM family member 5, mitochondrial serine/threonine protein phosphatase (PGAM5), is a protein phosphatase that resides in the mitochondria and regulates many biological processes, including cell death, mitophagy, and immune responses. Because BAT is a mitochondria-rich tissue, we have hypothesized that PGAM5 has a physiological function in BAT. We previously reported that PGAM5-knockout (KO) mice are resistant to severe metabolic stress. Importantly, lipid accumulation is suppressed in PGAM5-KO BAT, even under unstressed conditions, raising the possibility that PGAM5 deficiency stimulates lipid consumption. However, the mechanism underlying this observation is undetermined. Here, using an array of biochemical approaches, including quantitative RT-PCR, immunoblotting, and oxygen consumption assays, we show that PGAM5 negatively regulates energy expenditure in brown adipocytes. We found that PGAM5-KO brown adipocytes have an enhanced oxygen consumption rate and increased expression of uncoupling protein 1 (UCP1), a protein that increases energy consumption in the mitochondria. Mechanistically, we found that PGAM5 phosphatase activity and intramembrane cleavage are required for suppression of UCP1 activity. Furthermore, utilizing a genome-wide siRNA screen in HeLa cells to search for regulators of PGAM5 cleavage, we identified a set of candidate genes, including phosphatidylserine decarboxylase (PISD), which catalyzes the formation of phosphatidylethanolamine at the mitochondrial membrane. Taken together, these results indicate that PGAM5 suppresses mitochondrial energy expenditure by down-regulating UCP1 expression in brown adipocytes and that its phosphatase activity and intramembrane cleavage are required for UCP1 suppression.

The developing nervous system is remarkably sensitive to environmental signals, including disruptive toxins, such as polybrominated diphenyl ethers (PBDEs). PBDEs are an environmentally pervasive class of brominated flame retardants whose neurodevelopmental toxicity mechanisms remain largely unclear. Using dissociated cortical neurons from embryonic Rattus norvegicus, we found here that chronic exposure to 6-OH–BDE-47, one of the most prevalent hydroxylated PBDE metabolites, suppresses both spontaneous and evoked neuronal electrical activity. On the basis of our previous work on mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK) (MEK) biology and our observation that 6-OH–BDE-47 is structurally similar to kinase inhibitors, we hypothesized that certain hydroxylated PBDEs mediate neurotoxicity, at least in part, by impairing the MEK–ERK axis of MAPK signal transduction. We tested this hypothesis on three experimental platforms: 1) in silico, where modeling ligand–protein docking suggested that 6-OH–BDE-47 is a promiscuous ATP-competitive kinase inhibitor; 2) in vitro in dissociated neurons, where 6-OH–BDE-47 and another specific hydroxylated BDE metabolite similarly impaired phosphorylation of MEK/ERK1/2 and activity-induced transcription of a neuronal immediate early gene; and 3) in vivo in Drosophila melanogaster, where developmental exposures to 6-OH–BDE-47 and a MAPK inhibitor resulted in offspring displaying similarly increased frequency of mushroom-body β–lobe midline crossing, a metric of axonal guidance. Taken together, our results support that certain ortho-hydroxylated PBDE metabolites are promiscuous kinase inhibitors and can cause disruptions of critical neurodevelopmental processes, including neuronal electrical activity, pre-synaptic functions, MEK–ERK signaling, and axonal guidance.

Kindlins are focal adhesion proteins that regulate integrin activation and outside-in signaling. The kindlin family consists of three members, kindlin-1, -2, and -3. Kindlin-2 is widely expressed in multiple cell types, except those from the hematopoietic lineage. A previous study has reported that the Drosophila Fit1 protein (an ortholog of kindlin-2) prevents abnormal spindle assembly; however, the mechanism remains unknown. Here, we show that kindlin-2 maintains spindle integrity in mitotic human cells. The human neuroblastoma SH-SY5Y cell line expresses only kindlin-2, and we found that when SH-SY5Y cells are depleted of kindlin-2, they exhibit pronounced spindle abnormalities and delayed mitosis. Of note, acetylation of α-tubulin, which maintains microtubule flexibility and stability, was diminished in the kindlin-2–depleted cells. Mechanistically, we found that kindlin-2 maintains α-tubulin acetylation by inhibiting the microtubule-associated deacetylase histone deacetylase 6 (HDAC6) via a signaling pathway involving AKT Ser/Thr kinase (AKT)/glycogen synthase kinase 3β (GSK3β) or paxillin. We also provide evidence that prolonged hypoxia down-regulates kindlin-2 expression, leading to spindle abnormalities not only in the SH-SY5Y cell line, but also cell lines derived from colon and breast tissues. The findings of our study highlight that kindlin-2 regulates mitotic spindle assembly and that this process is perturbed in cancer cells in a hypoxic environment.

Sperm head shaping is a key event in spermiogenesis and is tightly controlled via the acrosome–manchette network. Linker of nucleoskeleton and cytoskeleton (LINC) complexes consist of Sad1 and UNC84 domain–containing (SUN) and Klarsicht/ANC-1/Syne-1 homology (KASH) domain proteins and form conserved nuclear envelope bridges implicated in transducing mechanical forces from the manchette to sculpt sperm nuclei into a hook-like shape. However, the role of LINC complexes in sperm head shaping is still poorly understood. Here we assessed the role of SUN3, a testis-specific LINC component harboring a conserved SUN domain, in spermiogenesis. We show that CRISPR/Cas9-generated Sun3 knockout male mice are infertile, displaying drastically reduced sperm counts and a globozoospermia-like phenotype, including a missing, mislocalized, or fragmented acrosome, as well as multiple defects in sperm flagella. Further examination revealed that the sperm head abnormalities are apparent at step 9 and that the sperm nuclei fail to elongate because of the absence of manchette microtubules and perinuclear rings. These observations indicate that Sun3 deletion likely impairs the ability of the LINC complex to transduce the cytoskeletal force to the nuclear envelope, required for sperm head elongation. We also found that SUN3 interacts with SUN4 in mouse testes and that the level of SUN4 proteins is drastically reduced in Sun3-null mice. Altogether, our results indicate that SUN3 is essential for sperm head shaping and male fertility, providing molecular clues regarding the underlying pathology of the globozoospermia-like phenotype.

2 May 2019 , Volume 95, Number 3

In the latest issue a collection of articles explore how international norms are increasingly contested by both state and non-state actors.

Just last year, one could find a variety of deals and promotions for Mother’s Day with ease, but for 2020, the opposite is true. This change could be connected with the coronavirus pandemic and a number of businesses having to close their doors for...

Research Event

Invitation Only Research Event

Event participants

Shane Tomlinson, Senior Research Fellow, Energy, Environment and Resources Department, Chatham House

There has been an outpouring of grief following the death of legendary Jamaican singer Millicent Dolly May Small, popularly known as Millie Small. She died in the United Kingdom today at the age of 73 after suffering a stroke. The voice...