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Taurine is a conditionally essential micronutrient and one of the most abundant amino acids in humans1-3. In endogenous taurine metabolism, dedicated enzymes are involved in the biosynthesis of taurine from cysteine and in the downstream metabolism of secondary taurine metabolites4,5. One taurine metabolite is N-acetyltaurine6. Levels of N-acetyltaurine are dynamically regulated by stimuli that alter taurine or acetate flux, including endurance exercise7, dietary taurine supplementation8 and alcohol consumption6,9. So far, the identities of the enzymes involved in N-acetyltaurine metabolism, and the potential functions of N-acetyltaurine itself, have remained unknown. Here we show that the body mass index associated orphan enzyme phosphotriesterase-related (PTER)10 is a physiological N-acetyltaurine hydrolase. In vitro, PTER catalyses the hydrolysis of N-acetyltaurine to taurine and acetate. In mice, PTER is expressed in the kidney, liver and brainstem. Genetic ablation of Pter in mice results in complete loss of tissue N-acetyltaurine hydrolysis activity and a systemic increase in N-acetyltaurine levels. After stimuli that increase taurine levels, Pter knockout mice exhibit reduced food intake, resistance to diet-induced obesity and improved glucose homeostasis. Administration of N-acetyltaurine to obese wild-type mice also reduces food intake and body weight in a GFRAL-dependent manner. These data place PTER into a central enzymatic node of secondary taurine metabolism and uncover a role for PTER and N-acetyltaurine in body weight control and energy balance.

Medium- and high-entropy alloys (M/HEAs) mix several principal elements with near-equiatomic composition and represent a model-shift strategy for designing previously unknown materials in metallurgy1-8, catalysis9-14 and other fields15-18. One of the core hypotheses of M/HEAs is lattice distortion5,19,20, which has been investigated by different numerical and experimental techniques21-26. However, determining the three-dimensional (3D) lattice distortion in M/HEAs remains a challenge. Moreover, the presumed random elemental mixing in M/HEAs has been questioned by X-ray and neutron studies27, atomistic simulations28-30, energy dispersive spectroscopy31,32 and electron diffraction33,34, which suggest the existence of local chemical order in M/HEAs. However, direct experimental observation of the 3D local chemical order has been difficult because energy dispersive spectroscopy integrates the composition of atomic columns along the zone axes7,32,34 and diffuse electron reflections may originate from planar defects instead of local chemical order35. Here we determine the 3D atomic positions of M/HEA nanoparticles using atomic electron tomography36 and quantitatively characterize the local lattice distortion, strain tensor, twin boundaries, dislocation cores and chemical short-range order (CSRO). We find that the high-entropy alloys have larger local lattice distortion and more heterogeneous strain than the medium-entropy alloys and that strain is correlated to CSRO. We also observe CSRO-mediated twinning in the medium-entropy alloys, that is, twinning occurs in energetically unfavoured CSRO regions but not in energetically favoured CSRO ones, which represents, to our knowledge, the first experimental observation of correlating local chemical order with structural defects in any material. We expect that this work will not only expand our fundamental understanding of this important class of materials but also provide the foundation for tailoring M/HEA properties through engineering lattice distortion and local chemical order.

Cell senescence deters the activation of various oncogenes. Induction of senescence is, therefore, a potentially effective strategy to interfere with vital processes in tumor cells. Sphingosine-1-phosphate receptor 1 (S1PR1) has been implicated in various cancer types, including ovarian cancer. The mechanism by which S1PR1 regulates ovarian cancer cell senescence is currently elusive. In this study, we demonstrate that S1PR1 was highly expressed in human ovarian cancer tissues and cell lines. S1PR1 deletion inhibited the proliferation and migration of ovarian cancer cells. S1PR1 deletion promoted ovarian cancer cell senescence and sensitized ovarian cancer cells to cisplatin chemotherapy. Exposure of ovarian cancer cells to sphingosine-1-phosphate (S1P) increased the expression of 3-phosphatidylinositol-dependent protein kinase 1 (PDK1), decreased the expression of large tumor suppressor 1/2 (LATS1/2), and induced phosphorylation of Yes-associated protein (p-YAP). Opposite results were obtained in S1PR1 knockout cells following pharmacological inhibition. After silencing LATS1/2 in S1PR1-deficient ovarian cancer cells, senescence was suppressed and S1PR1 expression was increased concomitantly with YAP expression. Transcriptional regulation of S1PR1 by YAP was confirmed by chromatin immunoprecipitation. Accordingly, the S1PR1-PDK1-LATS1/2-YAP pathway regulates ovarian cancer cell senescence and does so through a YAP-mediated feedback loop. S1PR1 constitutes a druggable target for the induction of senescence in ovarian cancer cells. Pharmacological intervention in the S1PR1-PDK1-LATS1/2-YAP signaling axis may augment the efficacy of standard chemotherapy.

Phosphatase and tensin homolog (PTEN) loss is associated with adverse outcomes in prostate cancer and can be measured via immunohistochemistry. The purpose of the study was to establish the clinical application of an in-house developed artificial intelligence (AI) image analysis workflow for automated detection of PTEN loss on digital images for identifying patients at risk of early recurrence and metastasis. Postsurgical tissue microarray sections from the Canary Foundation (n = 1264) stained with anti-PTEN antibody were evaluated independently by pathologist conventional visual scoring (cPTEN) and an automated AI-based image analysis pipeline (AI-PTEN). The relationship of PTEN evaluation methods with cancer recurrence and metastasis was analyzed using multivariable Cox proportional hazard and decision curve models. Both cPTEN scoring by the pathologist and quantification of PTEN loss by AI (high-risk AI-qPTEN) were significantly associated with shorter metastasis-free survival (MFS) in univariable analysis (cPTEN hazard ratio [HR], 1.54; CI, 1.07-2.21; P = .019; AI-qPTEN HR, 2.55; CI, 1.83-3.56; P < .001). In multivariable analyses, AI-qPTEN showed a statistically significant association with shorter MFS (HR, 2.17; CI, 1.49-3.17; P < .001) and recurrence-free survival (HR, 1.36; CI, 1.06-1.75; P = .016) when adjusting for relevant postsurgical clinical nomogram (Cancer of the Prostate Risk Assessment [CAPRA] postsurgical score [CAPRA-S]), whereas cPTEN does not show a statistically significant association (HR, 1.33; CI, 0.89-2; P = .2 and HR, 1.26; CI, 0.99-1.62; P = .063, respectively) when adjusting for CAPRA-S risk stratification. More importantly, AI-qPTEN was associated with shorter MFS in patients with favorable pathological stage and negative surgical margins (HR, 2.72; CI, 1.46-5.06; P = .002). Workflow also demonstrated enhanced clinical utility in decision curve analysis, more accurately identifying men who might benefit from adjuvant therapy postsurgery. This study demonstrates the clinical value of an affordable and fully automated AI-powered PTEN assessment for evaluating the risk of developing metastasis or disease recurrence after radical prostatectomy. Adding the AI-qPTEN assessment workflow to clinical variables may affect postoperative surveillance or management options, particularly in low-risk patients.

OBJECTIVE: High BMI is associated with many comorbidities and mortality. This study aimed to elucidate the overall clinical risk of obesity using a genome- and phenome-wide approach. METHODS: This study performed a phenome-wide association study of BMI using a clinical cohort of 736,726 adults. This was followed by genetic association studies using two separate cohorts: one consisting of 65,174 adults in the Electronic Medical Records and Genomics (eMERGE) Network and another with 405,432 participants in the UK Biobank. RESULTS: Class 3 obesity was associated with 433 phenotypes, representing 59.3% of all billing codes in individuals with severe obesity. A genome-wide polygenic risk score for BMI, accounting for 7.5% of variance in BMI, was associated with 296 clinical diseases, including strong associations with type 2 diabetes, sleep apnea, hypertension, and chronic liver disease. In all three cohorts, 199 phenotypes were associated with class 3 obesity and polygenic risk for obesity, including novel associations such as increased risk of renal failure, venous insufficiency, and gastroesophageal reflux. CONCLUSIONS: This combined genomic and phenomic systematic approach demonstrated that obesity has a strong genetic predisposition and is associated with a considerable burden of disease across all disease classes.

TAR DNA-binding protein-43 (TDP-43) proteinopathies are accompanied by the pathological hallmark of cytoplasmic inclusions in the neurodegenerative diseases, including frontal temporal lobar degeneration-TDP and amyotrophic lateral sclerosis. We found that transthyretin accumulates with TDP-43 cytoplasmic inclusions in frontal temporal lobar degeneration-TDP human patients and transgenic mice, in which transthyretin exhibits dramatic expression decline in elderly mice. The upregulation of transthyretin expression was demonstrated to facilitate the clearance of cytoplasmic TDP-43 inclusions through autophagy, in which transthyretin induces autophagy upregulation via ATF4. Of interest, transthyretin upregulated ATF4 expression and promoted ATF4 nuclear import, presenting physical interaction. Neuronal expression of transthyretin in frontal temporal lobar degeneration-TDP mice restored autophagy function and facilitated early soluble TDP-43 aggregates for autophagosome targeting, ameliorating neuropathology and behavioural deficits. Thus, transthyretin conducted two-way regulations by either inducing autophagy activation or escorting TDP-43 aggregates targeted autophagosomes, suggesting that transthyretin is a potential modulator therapy for neurological disorders caused by TDP-43 proteinopathy.

To develop and validate a new tissue-based biomarker that improves prediction of outcomes in localized prostate cancer by quantifying the host response to tumor. We use digital image analysis and machine learning to develop a biomarker of the prostate stroma called quantitative reactive stroma (qRS). qRS is a measure of percentage tumor area with a distinct, reactive stromal architecture. Kaplan Meier analysis was used to determine survival in a large retrospective cohort of radical prostatectomy samples. qRS was validated in two additional, distinct cohorts that include international cases and tissue from both radical prostatectomy and biopsy specimens. In the developmental cohort (Baylor College of Medicine, n = 482), patients whose tumor had qRS > 34% had increased risk of prostate cancer-specific death (HR 2.94; p = 0.039). This result was replicated in two validation cohorts, where patients with qRS > 34% had increased risk of prostate cancer-specific death (MEDVAMC; n = 332; HR 2.64; p = 0.02) and also biochemical recurrence (Canary; n = 988; HR 1.51; p = 0.001). By multivariate analysis, these associations were shown to hold independent predictive value when compared to currently used clinicopathologic factors including Gleason score and PSA. qRS is a new, validated biomarker that predicts prostate cancer death and biochemical recurrence across three distinct cohorts. It measures host-response rather than tumor-based characteristics, and provides information not represented by standard prognostic measurements.

Post-acute sequelae of COVID-19 (PASC) represent an emerging global crisis. However, quantifiable risk factors for PASC and their biological associations are poorly resolved. We executed a deep multi-omic, longitudinal investigation of 309 COVID-19 patients from initial diagnosis to convalescence (2-3 months later), integrated with clinical data and patient-reported symptoms. We resolved four PASC-anticipating risk factors at the time of initial COVID-19 diagnosis: type 2 diabetes, SARS-CoV-2 RNAemia, Epstein-Barr virus viremia, and specific auto-antibodies. In patients with gastrointestinal PASC, SARS-CoV-2-specific and CMV-specific CD8+ T cells exhibited unique dynamics during recovery from COVID-19. Analysis of symptom-associated immunological signatures revealed coordinated immunity polarization into four endotypes, exhibiting divergent acute severity and PASC. We find that immunological associations between PASC factors diminish over time, leading to distinct convalescent immune states. Detectability of most PASC factors at COVID-19 diagnosis emphasizes the importance of early disease measurements for understanding emergent chronic conditions and suggests PASC treatment strategies.

Rett syndrome (RTT) is a severe neurodevelopmental disorder primarily caused by heterozygous loss-of-function mutations in the X-linked gene MECP2 that is a global transcriptional regulator. Mutations in the methyl-CpG binding domain (MBD) of MECP2 disrupt its interaction with methylated DNA. Here, we investigate the effect of a novel MECP2 L124W missense mutation in the MBD of an atypical RTT patient with preserved speech in comparison to severe MECP2 null mutations. L124W protein had a limited ability to disrupt heterochromatic chromocenters due to decreased binding dynamics. We isolated two pairs of isogenic WT and L124W induced pluripotent stem cells. L124W induced excitatory neurons expressed stable protein, exhibited increased input resistance and decreased voltage-gated Na+ and K+ currents, and their neuronal dysmorphology was limited to decreased dendritic complexity. Three isogenic pairs of MECP2 null neurons had the expected more extreme morphological and electrophysiological phenotypes. We examined development and maturation of L124W and MECP2 null excitatory neural network activity using micro-electrode arrays. Relative to isogenic controls, L124W neurons had an increase in synchronous network burst frequency, in contrast to MECP2 null neurons that suffered a significant decrease in synchronous network burst frequency and a transient extension of network burst duration. A biologically motivated computational neural network model shows the observed changes in network dynamics are explained by changes in intrinsic Na+ and K+ currents in individual neurons. Our multilevel results demonstrate that RTT excitatory neurons show a wide spectrum of morphological, electrophysiological and circuitry phenotypes that are dependent on the severity of the MECP2 mutation.

We present a chemical approach to profile fatty acid uptake in single cells. We use azide-modified analogues to probe the fatty acid influx and surface-immobilized dendrimers with dibenzocyclooctyne (DBCO) groups for detection. A competition between the fatty acid probes and BHQ2-azide quencher molecules generates fluorescence signals in a concentration-dependent manner. By integrating this method onto a microfluidics-based multiplex protein analysis platform, we resolved the relationships between fatty acid influx, oncogenic signaling activities, and cell proliferation in single glioblastoma cells. We found that p70S6K and 4EBP1 differentially correlated with fatty acid uptake. We validated that cotargeting p70S6K and fatty acid metabolism synergistically inhibited cell proliferation. Our work provided the first example of studying fatty acid metabolism in the context of protein signaling at single-cell resolution and generated new insights into cancer biology.

Plasma lipids are known heritable risk factors for cardiovascular disease, but increasing evidence also supports shared genetics with diseases of other organ systems. We devised a comprehensive three-phase framework to identify new lipid-associated genes and study the relationships among lipids, genotypes, gene expression and hundreds of complex human diseases from the Electronic Medical Records and Genomics (347 traits) and the UK Biobank (549 traits). Aside from 67 new lipid-associated genes with strong replication, we found evidence for pleiotropic SNPs/genes between lipids and diseases across the phenome. These include discordant pleiotropy in the HLA region between lipids and multiple sclerosis and putative causal paths between triglycerides and gout, among several others. Our findings give insights into the genetic basis of the relationship between plasma lipids and diseases on a phenome-wide scale and can provide context for future prevention and treatment strategies.

Background & aimsIn contrast to most other common diseases, few genetic variants have been identified that impact risk of cirrhosis. We aimed to identify new genetic variants that predispose to cirrhosis, to test whether such variants, aggregated into a polygenic score, enable genomic risk stratification, and to test whether alcohol intake or body mass index interacts with polygenic predisposition.MethodsWe conducted a multi-trait genome-wide association study combining cirrhosis and alanine aminotransferase levels performed in 5 discovery studies (UK Biobank, Vanderbilt BioVU, Atherosclerosis Risk in Communities study, and 2 case-control studies including 4829 individuals with cirrhosis and 72,705 controls and 362,539 individuals with alanine aminotransferase levels). Identified variants were replicated in 3 studies (Partners HealthCare Biobank, FinnGen, and Biobank Japan including 3554 individuals with cirrhosis and 343,826 controls). A polygenic score was tested in Partners HealthCare Biobank.ResultsFive previously reported and 7 newly identified genetic variants were associated with cirrhosis in both the discovery studies multi-trait genome-wide association study (P < 5 × 10-8) and the replication studies (P < .05), including a missense variant in the APOE gene and a noncoding variant near EFN1A. These 12 variants were used to generate a polygenic score. Among Partners HealthCare Biobank individuals, high polygenic score-defined as the top quintile of the distribution-was associated with significantly increased risk of cirrhosis (odds ratio, 2.26; P < .001) and related comorbidities compared with the lowest quintile. Risk was even more pronounced among those with extreme polygenic risk (top 1% of the distribution, odds ratio, 3.16; P < .001). The impact of extreme polygenic risk was substantially more pronounced in those with elevated alcohol consumption or body mass index. Modeled as risk by age 75 years, probability of cirrhosis with extreme polygenic risk was 13.7%, 20.1%, and 48.2% among individuals with no or modest, moderate, and increased alcohol consumption, respectively (Pinteraction < .001). Similarly, probability among those with extreme polygenic risk was 6.5%, 10.3%, and 19.5% among individuals with normal weight, overweight, and obesity, respectively (Pinteraction < .001).ConclusionsTwelve independent genetic variants, 7 of which are newly identified in this study, conferred risk for cirrhosis. Aggregated into a polygenic score, these variants identified a subset of the population at substantially increased risk who are most susceptible to the hepatotoxic effects of excess alcohol consumption or obesity.

Unlike other epithelial cancer types, circulating tumor cells (CTCs) are less frequently detected in the peripheral blood of non-small cell lung cancer (NSCLC) patients using epithelial marker-based detection approaches despite the aggressive nature of NSCLC. Here, we demonstrate hexokinase-2 (HK2) as a metabolic function-associated marker for the detection of CTCs. In 59 NSCLC patients bearing cytokeratin-positive (CKpos) primary tumors, HK2 enables resolving cytokeratin-negative (HK2high/CKneg) CTCs as a prevalent population in about half of the peripheral blood samples with positive CTC counts. However, HK2high/CKneg tumor cells are a minority population in pleural effusions and cerebrospinal fluids. Single-cell analysis shows that HK2high/CKneg CTCs exhibit smaller sizes but consistent copy number variation profiles compared with CKpos counterparts. Single-cell transcriptome profiling reveals that CK expression levels of CTCs are independent of their epithelial-to-mesenchymal transition (EMT) status, challenging the long-standing association between CK expression and EMT. HK2high/CKneg CTCs display metastasis and EGFR inhibitor resistance-related molecular signatures and are selectively enriched in patients with EGFRL858R driver oncogene mutation as opposed to EGFR19Del , which is more frequently found in patients with prevalent CKpos CTCs in the blood. Consistently, treatment-naïve patients with a larger number or proportion of HK2high/CKneg CTCs in the blood exhibit poor therapy response and shorter progression-free survival. Collectively, our approach resolves a more complete spectrum of CTCs in NSCLC that can potentially be exploited to identify patient prognosis before therapy.

Secreted polypeptides are a fundamental axis of intercellular and endocrine communication. However, a global understanding of the composition and dynamics of cellular secretomes in intact mammalian organisms has been lacking. Here, we introduce a proximity biotinylation strategy that enables labeling, detection and enrichment of secreted polypeptides in a cell type-selective manner in mice. We generate a proteomic atlas of hepatocyte, myocyte, pericyte and myeloid cell secretomes by direct purification of biotinylated secreted proteins from blood plasma. Our secretome dataset validates known cell type-protein pairs, reveals secreted polypeptides that distinguish between cell types and identifies new cellular sources for classical plasma proteins. Lastly, we uncover a dynamic and previously undescribed nutrient-dependent reprogramming of the hepatocyte secretome characterized by the increased unconventional secretion of the cytosolic enzyme betaine-homocysteine S-methyltransferase (BHMT). This secretome profiling strategy enables dynamic and cell type-specific dissection of the plasma proteome and the secreted polypeptides that mediate intercellular signaling.

Background: Local protein synthesis and mRNA metabolism mediated by mRNP granules in the dendrites and the postsynaptic compartment is essential for synaptic remodeling and plasticity in neuronal cells. Dysregulation of these processes caused by TDP-43 proteinopathy leads to neurodegenerative diseases, such as frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Methods: Using biochemical analysis and imaging techniques, including super-resolution microscopy, we provide evidence, for the first time, for the postsynaptic localization of TDP-43 in mammalian synapses and we show that TDP-43 is a component of neuronal mRNP granules. Results: With activity stimulation and various molecular approaches, we further demonstrate activity-dependent mRNP granule dynamics involving disassembly of mRNP granules, release of mRNAs, activation of local protein translation, and the impairment of granule disassembly in cellular, animal and human models of TDP-43 proteinopathy. Conclusion: Our study elucidates the interplay between TDP-43 and neuronal mRNP granules in normal physiology and TDP-43 proteinopathy in the regulation of local protein translation and mRNA metabolism in the postsynaptic compartment.

We present an integrated analysis of the clinical measurements, immune cells, and plasma multi-omics of 139 COVID-19 patients representing all levels of disease severity, from serial blood draws collected during the first week of infection following diagnosis. We identify a major shift between mild and moderate disease, at which point elevated inflammatory signaling is accompanied by the loss of specific classes of metabolites and metabolic processes. Within this stressed plasma environment at moderate disease, multiple unusual immune cell phenotypes emerge and amplify with increasing disease severity. We condensed over 120,000 immune features into a single axis to capture how different immune cell classes coordinate in response to SARS-CoV-2. This immune-response axis independently aligns with the major plasma composition changes, with clinical metrics of blood clotting, and with the sharp transition between mild and moderate disease. This study suggests that moderate disease may provide the most effective setting for therapeutic intervention.

TDP-43 inclusions are found in many Alzheimer's disease (AD) patients presenting faster disease progression and greater brain atrophy. Previously, we showed full-length TDP-43 forms spherical oligomers and perturbs amyloid-β (Aβ) fibrillization. To elucidate the role of TDP-43 in AD, here, we examined the effect of TDP-43 in Aβ aggregation and the attributed toxicity in mouse models. We found TDP-43 inhibited Aβ fibrillization at initial and oligomeric stages. Aβ fibrillization was delayed specifically in the presence of N-terminal domain containing TDP-43 variants, while C-terminal TDP-43 was not essential for Aβ interaction. TDP-43 significantly enhanced Aβ's ability to impair long-term potentiation and, upon intrahippocampal injection, caused spatial memory deficit. Following injection to AD transgenic mice, TDP-43 induced inflammation, interacted with Aβ, and exacerbated AD-like pathology. TDP-43 oligomers mostly colocalized with intracellular Aβ in the brain of AD patients. We conclude that TDP-43 inhibits Aβ fibrillization through its interaction with Aβ and exacerbates AD pathology.

TAR DNA-binding protein 43 (TDP-43) has been implicated in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-TDP) and amyotrophic lateral sclerosis. Histone deacetylase 1 (HDAC1) is involved in DNA repair and neuroprotection in numerous neurodegenerative diseases. However, the pathological mechanisms of FTLD-TDP underlying TDP-43 proteinopathies are unclear, and the role of HDAC1 is also poorly understood. Here, we found that aberrant cell cycle activity and DNA damage are important pathogenic factors in FTLD-TDP transgenic (Tg) mice, and we further identified these pathological features in the frontal cortices of patients with FTLD-TDP. TDP-43 proteinopathies contributed to pathogenesis by inducing cytosolic mislocalization of HDAC1 and reducing its activity. Pharmacological recovery of HDAC1 activity in FTLD-TDP Tg mice ameliorated their cognitive and motor impairments, normalized their aberrant cell cycle activity, and attenuated their DNA damage and neuronal loss. Thus, HDAC1 deregulation is involved in the pathogenesis of TDP-43 proteinopathies, and HDAC1 is a potential target for therapeutic interventions in FTLD-TDP.

The determination of individual cell trajectories through a high-dimensional cell-state space is an outstanding challenge for understanding biological changes ranging from cellular differentiation to epigenetic responses of diseased cells upon drugging. We integrate experiments and theory to determine the trajectories that single BRAFV600E mutant melanoma cancer cells take between drug-naive and drug-tolerant states. Although single-cell omics tools can yield snapshots of the cell-state landscape, the determination of individual cell trajectories through that space can be confounded by stochastic cell-state switching. We assayed for a panel of signaling, phenotypic, and metabolic regulators at points across 5 days of drug treatment to uncover a cell-state landscape with two paths connecting drug-naive and drug-tolerant states. The trajectory a given cell takes depends upon the drug-naive level of a lineage-restricted transcription factor. Each trajectory exhibits unique druggable susceptibilities, thus updating the paradigm of adaptive resistance development in an isogenic cell population.

BACKGROUND:Chronic lesion activity driven by smoldering inflammation is a pathological hallmark of progressive forms of multiple sclerosis (MS). OBJECTIVE:To develop a method for automatic detection of slowly expanding/evolving lesions (SELs) on conventional brain magnetic resonance imaging (MRI) and characterize such SELs in primary progressive MS (PPMS) and relapsing MS (RMS) populations. METHODS:We defined SELs as contiguous regions of existing T2 lesions showing local expansion assessed by the Jacobian determinant of the deformation between reference and follow-up scans. SEL candidates were assigned a heuristic score based on concentricity and constancy of change in T2- and T1-weighted MRIs. SELs were examined in 1334 RMS patients and 555 PPMS patients. RESULTS:Compared with RMS patients, PPMS patients had higher numbers of SELs (p = 0.002) and higher T2 volumes of SELs (p

Chronic active and slowly expanding lesions with smouldering inflammation are neuropathological correlates of progressive multiple sclerosis pathology. T1 hypointense volume and signal intensity on T1-weighted MRI reflect brain tissue damage that may develop within newly formed acute focal inflammatory lesions or in chronic pre-existing lesions without signs of acute inflammation. Using a recently developed method to identify slowly expanding/evolving lesions in vivo from longitudinal conventional T2- and T1-weighted brain MRI scans, we measured the relative amount of chronic lesion activity as measured by change in T1 volume and intensity within slowly expanding/evolving lesions and non-slowly expanding/evolving lesion areas of baseline pre-existing T2 lesions, and assessed the effect of ocrelizumab on this outcome in patients with primary progressive multiple sclerosis participating in the phase III, randomized, placebo-controlled, double-blind ORATORIO study (n = 732, NCT01194570). We also assessed the predictive value of T1-weighted measures of chronic lesion activity for clinical multiple sclerosis progression as reflected by a composite disability measure including the Expanded Disability Status Scale, Timed 25-Foot Walk and 9-Hole Peg Test. We observed in this clinical trial population that most of total brain non-enhancing T1 hypointense lesion volume accumulation was derived from chronic lesion activity within pre-existing T2 lesions rather than new T2 lesion formation. There was a larger decrease in mean normalized T1 signal intensity and greater relative accumulation of T1 hypointense volume in slowly expanding/evolving lesions compared with non-slowly expanding/evolving lesions. Chronic white matter lesion activity measured by longitudinal T1 hypointense lesion volume accumulation in slowly expanding/evolving lesions and in non-slowly expanding/evolving lesion areas of pre-existing lesions predicted subsequent composite disability progression with consistent trends on all components of the composite. In contrast, whole brain volume loss and acute lesion activity measured by longitudinal T1 hypointense lesion volume accumulation in new focal T2 lesions did not predict subsequent composite disability progression in this trial at the population level. Ocrelizumab reduced longitudinal measures of chronic lesion activity such as T1 hypointense lesion volume accumulation and mean normalized T1 signal intensity decrease both within regions of pre-existing T2 lesions identified as slowly expanding/evolving and in non-slowly expanding/evolving lesions. Using conventional brain MRI, T1-weighted intensity-based measures of chronic white matter lesion activity predict clinical progression in primary progressive multiple sclerosis and may qualify as a longitudinal in vivo neuroimaging correlate of smouldering demyelination and axonal loss in chronic active lesions due to CNS-resident inflammation and/or secondary neurodegeneration across the multiple sclerosis disease continuum.

Allopurinol, which lowers uric acid (UA) concentration, is increasingly being recognized for its benefits in cardiovascular and renal disease. However, response to allopurinol is variable. We gathered samples from 4,446 multiethnic subjects for a genome-wide association study of allopurinol response. Consistent with previous studies, we observed that the Q141K variant in ABCG2 (rs2231142), which encodes the efflux pump breast cancer resistance protein (BCRP), associated with worse response to allopurinol. However, for the first time this association reached genome-wide level significance (P = 8.06 × 10-11 ). Additionally, we identified a novel association with a variant in GREM2 (rs1934341, P = 3.22 × 10-6 ). In vitro studies identified oxypurinol, the active metabolite of allopurinol, as an inhibitor of the UA transporter GLUT9, suggesting that oxypurinol may modulate UA reabsorption. These results provide strong evidence for a role of BCRP Q141K in allopurinol response, and suggest that allopurinol may have additional hypouricemic effects beyond xanthine oxidase inhibition.

Accurate prediction of chemo- or targeted therapy responses for patients with similar driver oncogenes through a simple and least-invasive assay represents an unmet need in the clinical diagnosis of non-small cell lung cancer. Using a single-cell on-chip metabolic cytometry and fluorescent metabolic probes, we show metabolic phenotyping on the rare disseminated tumor cells in pleural effusions across a panel of 32 lung adenocarcinoma patients. Our results reveal extensive metabolic heterogeneity of tumor cells that differentially engage in glycolysis and mitochondrial oxidation. The cell number ratio of the two metabolic phenotypes is found to be predictive for patient therapy response, physiological performance, and survival. Transcriptome analysis reveals that the glycolytic phenotype is associated with mesenchymal-like cell state with elevated expression of the resistant-leading receptor tyrosine kinase AXL and immune checkpoint ligands. Drug targeting AXL induces a significant cell killing in the glycolytic cells without affecting the cells with active mitochondrial oxidation.

The genetic variant rs72824905-G (minor allele) in the PLCG2 gene was previously associated with a reduced Alzheimer's disease risk (AD). The role of PLCG2 in immune system signaling suggests it may also protect against other neurodegenerative diseases and possibly associates with longevity. We studied the effect of the rs72824905-G on seven neurodegenerative diseases and longevity, using 53,627 patients, 3,516 long-lived individuals and 149,290 study-matched controls. We replicated the association of rs72824905-G with reduced AD risk and we found an association with reduced risk of dementia with Lewy bodies (DLB) and frontotemporal dementia (FTD). We did not find evidence for an effect on Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS) risks, despite adequate sample sizes. Conversely, the rs72824905-G allele was associated with increased likelihood of longevity. By-proxy analyses in the UK Biobank supported the associations with both dementia and longevity. Concluding, rs72824905-G has a protective effect against multiple neurodegenerative diseases indicating shared aspects of disease etiology. Our findings merit studying the PLCγ2 pathway as drug-target.

Phenotypic plasticity is associated with non-genetic drug tolerance in several cancers. Such plasticity can arise from chromatin remodeling, transcriptomic reprogramming, and/or protein signaling rewiring, and is characterized as a cell state transition in response to molecular or physical perturbations. This, in turn, can confound interpretations of drug responses and resistance development. Using BRAF-mutant melanoma cell lines as the prototype, we report on a joint theoretical and experimental investigation of the cell-state transition dynamics associated with BRAF inhibitor drug tolerance. Thermodynamically motivated surprisal analysis of transcriptome data was used to treat the cell population as an entropy maximizing system under the influence of time-dependent constraints. This permits the extraction of an epigenetic potential landscape for drug-induced phenotypic evolution. Single-cell flow cytometry data of the same system were modeled with a modified Fokker-Planck-type kinetic model. The two approaches yield a consistent picture that accounts for the phenotypic heterogeneity observed over the course of drug tolerance development. The results reveal that, in certain plastic cancers, the population heterogeneity and evolution of cell phenotypes may be understood by accounting for the competing interactions of the epigenetic potential landscape and state-dependent cell proliferation. Accounting for such competition permits accurate, experimentally verifiable predictions that can potentially guide the design of effective treatment strategies.

The Hippo pathway controls organ size and tissue homeostasis, and its dysregulation often contributes to tumorigenesis. Extensive studies have shown that the Hippo pathway inhibits cell proliferation, and survival in a cell-autonomous manner. We examined the function of the Hippo pathway kinases LATS1/2 (large tumor suppressor 1 and 2) in cancer cells. As expected, loss of LATS1/2 promotes cancer cell growth in most cell lines. Surprisingly, however, LATS1/2 deletion inhibits the growth of murine MC38 colon cancer cells, especially under detachment conditions. This growth inhibitory effect caused by LATS1/2 deletion is due to uncontrolled activation of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), the key downstream transcriptional coactivators inhibited by LATS1/2. We identified Wnt inducible signaling pathway protein 2 (Wisp2) and coiled-coil domain containing 80 (Ccdc80) as direct targets of YAP/TAZ. Their expression is selectively induced by LATS1/2 deletion in MC38 cells. Furthermore, deletion of WISP2 and CCDC80 prevents the growth inhibitory effect of LATS1/2 loss in MC38 cells. Our study demonstrates that the function of LATS1/2 in cell growth is cell context dependent, suggesting that LATS1/2 inhibition can be a therapeutic approach for some cancer types.

PurposePhosphatidylinositol 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.MethodsThis 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.ResultsSixty-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%]).ConclusionBuparlisib 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.

We present here a novel chemical method to continuously analyze intracellular AKT signaling activities at single-cell resolution, without genetic manipulations. A pair of cyclic peptide-based fluorescent probes were developed to recognize the phosphorylated Ser474 site and a distal epitope on AKT. A Förster resonance energy transfer signal is generated upon concurrent binding of the two probes onto the same AKT protein, which is contingent upon the Ser474 phosphorylation. Intracellular delivery of the probes enabled dynamic measurements of the AKT signaling activities. We further implemented this detection strategy on a microwell single-cell platform, and interrogated the AKT signaling dynamics in a human glioblastoma cell line. We resolved unique features of the single-cell signaling dynamics following different perturbations. Our study provided the first example of monitoring the temporal evolution of cellular signaling heterogeneities and unveiled biological information that was inaccessible to other methods.

BackgroundCoronary heart disease (CHD) is a leading cause of death globally. Although therapy with statins decreases circulating levels of low-density lipoprotein cholesterol and the incidence of CHD, additional events occur despite statin therapy in some individuals. The genetic determinants of this residual cardiovascular risk remain unknown.MethodsWe performed a 2-stage genome-wide association study of CHD events during statin therapy. We first identified 3099 cases who experienced CHD events (defined as acute myocardial infarction or the need for coronary revascularization) during statin therapy and 7681 controls without CHD events during comparable intensity and duration of statin therapy from 4 sites in the Electronic Medical Records and Genomics Network. We then sought replication of candidate variants in another 160 cases and 1112 controls from a fifth Electronic Medical Records and Genomics site, which joined the network after the initial genome-wide association study. Finally, we performed a phenome-wide association study for other traits linked to the most significant locus.ResultsThe meta-analysis identified 7 single nucleotide polymorphisms at a genome-wide level of significance within the LPA/PLG locus associated with CHD events on statin treatment. The most significant association was for an intronic single nucleotide polymorphism within LPA/PLG (rs10455872; minor allele frequency, 0.069; odds ratio, 1.58; 95% confidence interval, 1.35-1.86; P=2.6×10-10). In the replication cohort, rs10455872 was also associated with CHD events (odds ratio, 1.71; 95% confidence interval, 1.14-2.57; P=0.009). The association of this single nucleotide polymorphism with CHD events was independent of statin-induced change in low-density lipoprotein cholesterol (odds ratio, 1.62; 95% confidence interval, 1.17-2.24; P=0.004) and persisted in individuals with low-density lipoprotein cholesterol ≤70 mg/dL (odds ratio, 2.43; 95% confidence interval, 1.18-4.75; P=0.015). A phenome-wide association study supported the effect of this region on coronary heart disease and did not identify noncardiovascular phenotypes.ConclusionsGenetic variations at the LPA locus are associated with CHD events during statin therapy independently of the extent of low-density lipoprotein cholesterol lowering. This finding provides support for exploring strategies targeting circulating concentrations of lipoprotein(a) to reduce CHD events in patients receiving statins.

An analytical method is described for profiling lactate production in single cells via the use of coupled enzyme reactions on surface-grafted resazurin molecules. The immobilization of the redox-labile probes was achieved through chemical modifications on resazurin, followed by bio-orthogonal click reactions. The lactate detection was demonstrated to be sensitive and specific. The method was incorporated into a single-cell barcode chip for simultaneous quantification of aerobic glycolysis activities and oncogenic signaling phosphoproteins in cancer. The interplay between glycolysis and oncogenic signaling activities was interrogated on a glioblastoma cell line. Results revealed a drug-induced oncogenic signaling reliance accompanying shifted metabolic paradigms. A drug combination that exploits this induced reliance exhibited synergistic effects in growth inhibition.

Sudden cardiac death (SCD) is associated with both electrical and ischemic substrates, and is a major cause of ischemic heart disease mortality worldwide. Male sex predisposes to SCD but the underlying mechanisms are incompletely understood. KCNE4, a cardiac arrhythmia-associated potassium channel β-subunit, is upregulated by 5α-dihydrotestosterone (DHT). Thus, ventricular Kcne4 expression is low in young adult female mice, but high in males and postmenopausal (12+ months) females. Despite causing a sex-independent electrical substrate at 13 months of age (22% QT prolongation in both males and females; P

Continuous BRAF inhibition of BRAF mutant melanomas triggers a series of cell state changes that lead to therapy resistance and escape from immune control before establishing acquired resistance genetically. We used genome-wide transcriptomics and single-cell phenotyping to explore the response kinetics to BRAF inhibition for a panel of patient-derived BRAFV600 -mutant melanoma cell lines. A subset of plastic cell lines, which followed a trajectory covering multiple known cell state transitions, provided models for more detailed biophysical investigations. Markov modeling revealed that the cell state transitions were reversible and mediated by both Lamarckian induction and nongenetic Darwinian selection of drug-tolerant states. Single-cell functional proteomics revealed activation of certain signaling networks shortly after BRAF inhibition, and before the appearance of drug-resistant phenotypes. Drug targeting those networks, in combination with BRAF inhibition, halted the adaptive transition and led to prolonged growth inhibition in multiple patient-derived cell lines.

The decreasing cost of genomic technologies has enabled the molecular characterization of large-scale clinical disease samples and of molecular changes upon drug treatment in various disease models. Exploring methods to relate diseases to potentially efficacious drugs through various molecular features is critically important in the discovery of new therapeutics. Here we show that the potency of a drug to reverse cancer-associated gene expression changes positively correlates with that drug's efficacy in preclinical models of breast, liver and colon cancers. Using a systems-based approach, we predict four compounds showing high potency to reverse gene expression in liver cancer and validate that all four compounds are effective in five liver cancer cell lines. The in vivo efficacy of pyrvinium pamoate is further confirmed in a subcutaneous xenograft model. In conclusion, this systems-based approach may be complementary to the traditional target-based approach in connecting diseases to potentially efficacious drugs.

The ability to identify a specific cancer using minimally invasive biopsy holds great promise for improving the diagnosis, treatment selection, and prediction of prognosis in cancer. Using whole-genome methylation data from The Cancer Genome Atlas (TCGA) and machine learning methods, we evaluated the utility of DNA methylation for differentiating tumor tissue and normal tissue for four common cancers (breast, colon, liver, and lung). We identified cancer markers in a training cohort of 1,619 tumor samples and 173 matched adjacent normal tissue samples. We replicated our findings in a separate TCGA cohort of 791 tumor samples and 93 matched adjacent normal tissue samples, as well as an independent Chinese cohort of 394 tumor samples and 324 matched adjacent normal tissue samples. The DNA methylation analysis could predict cancer versus normal tissue with more than 95% accuracy in these three cohorts, demonstrating accuracy comparable to typical diagnostic methods. This analysis also correctly identified 29 of 30 colorectal cancer metastases to the liver and 32 of 34 colorectal cancer metastases to the lung. We also found that methylation patterns can predict prognosis and survival. We correlated differential methylation of CpG sites predictive of cancer with expression of associated genes known to be important in cancer biology, showing decreased expression with increased methylation, as expected. We verified gene expression profiles in a mouse model of hepatocellular carcinoma. Taken together, these findings demonstrate the utility of methylation biomarkers for the molecular characterization of cancer, with implications for diagnosis and prognosis.

Background & aimsDrug repositioning offers a shorter approval process than new drug development. We therefore searched large public datasets of drug-induced gene expression signatures to identify agents that might be effective against hepatocellular carcinoma (HCC).MethodsWe searched public databases of messenger RNA expression patterns reported from HCC specimens from patients, HCC cell lines, and cells exposed to various drugs. We identified drugs that might specifically increase expression of genes that are down-regulated in HCCs and reduce expression of genes up-regulated in HCCs using a nonparametric, rank-based pattern-matching strategy based on the Kolmogorov-Smirnov statistic. We evaluated the anti-tumor activity of niclosamide and its ethanolamine salt (NEN) in HCC cell lines (HepG2, Huh7, Hep3B, Hep40, and PLC/PRF/5), primary human hepatocytes, and 2 mouse models of HCC. In one model of HCC, liver tumor development was induced by hydrodynamic delivery of a sleeping beauty transposon expressing an activated form of Ras (v12) and truncated β-catenin (N90). In another mouse model, patient-derived xenografts were established by implanting HCC cells from patients into livers of immunocompromised mice. Tumor growth was monitored by bioluminescence imaging. Tumor-bearing mice were fed a regular chow diet or a chow diet containing niclosamide or NEN. In a separate experiment using patient-derived xenografts, tumor-bearing mice were given sorafenib (the standard of care for patients with advanced HCC), NEN, or niclosamide alone; a combination of sorafenib and NEN; or a combination sorafenib and niclosamide in their drinking water, or regular water (control), and tumor growth was monitored.ResultsBased on gene expression signatures, we identified 3 anthelmintics that significantly altered the expression of genes that are up- or down-regulated in HCCs. Niclosamide and NEN specifically reduced the viability of HCC cells: the agents were at least 7-fold more cytotoxic to HCCs than primary hepatocytes. Oral administration of NEN to mice significantly slowed growth of genetically induced liver tumors and patient-derived xenografts, whereas niclosamide did not, coinciding with the observed greater bioavailability of NEN compared with niclosamide. The combination of NEN and sorafenib was more effective at slowing growth of patient-derived xenografts than either agent alone. In HepG2 cells and in patient-derived xenografts, administration of niclosamide or NEN increased expression of 20 genes down-regulated in HCC and reduced expression of 29 genes up-regulated in the 274-gene HCC signature. Administration of NEN to mice with patient-derived xenografts reduced expression of proteins in the Wnt-β-catenin, signal transducer and activator of transcription 3, AKT-mechanistic target of rapamycin, epidermal growth factor receptor-Ras-Raf signaling pathways. Using immunoprecipitation assays, we found NEN to bind cell division cycle 37 protein and disrupt its interaction with heat shock protein 90.ConclusionsIn a bioinformatics search for agents that alter the HCC-specific gene expression pattern, we identified the anthelmintic niclosamide as a potential anti-tumor agent. Its ethanolamine salt, with greater bioavailability, was more effective than niclosamide at slowing the growth of genetically induced liver tumors and patient-derived xenografts in mice. Both agents disrupted interaction between cell division cycle 37 and heat shock protein 90 in HCC cells, with concomitant inhibition of their downstream signaling pathways. NEN might be effective for treatment of patients with HCC.

BackgroundType 2 diabetes mellitus (DM) and obesity independently increase the risk of heart failure by incompletely understood mechanisms. We propose that hyperinsulinemia might promote adverse consequences in the hearts of subjects with type-2 DM and obesity.MethodsHigh-fat diet feeding was used to induce obesity and DM in wild-type mice or mice lacking β2-adrenergic receptor (β2AR) or β-arrestin2. Wild-type mice fed with high-fat diet were treated with a β-blocker carvedilol or a GRK2 (G-protein-coupled receptor kinase 2) inhibitor. We examined signaling and cardiac contractile function.ResultsHigh-fat diet feeding selectively increases the expression of phosphodiesterase 4D (PDE4D) in mouse hearts, in concert with reduced protein kinase A phosphorylation of phospholamban, which contributes to systolic and diastolic dysfunction. The expression of PDE4D is also elevated in human hearts with DM. The induction of PDE4D expression is mediated by an insulin receptor, insulin receptor substrate, and GRK2 and β-arrestin2-dependent transactivation of a β2AR-extracellular regulated protein kinase signaling cascade. Thus, pharmacological inhibition of β2AR or GRK2, or genetic deletion of β2AR or β-arrestin2, all significantly attenuate insulin-induced phosphorylation of extracellular regulated protein kinase and PDE4D induction to prevent DM-related contractile dysfunction.ConclusionsThese studies elucidate a novel mechanism by which hyperinsulinemia contributes to heart failure by increasing PDE4D expression and identify β2AR or GRK2 as plausible therapeutic targets for preventing or treating heart failure in subjects with type 2 DM.

BackgroundIn addition to lowering low density lipoprotein cholesterol (LDL-C), statin therapy also raises high density lipoprotein cholesterol (HDL-C) levels. Inter-individual variation in HDL-C response to statins may be partially explained by genetic variation.Methods and resultsWe performed a meta-analysis of genome-wide association studies (GWAS) to identify variants with an effect on statin-induced high density lipoprotein cholesterol (HDL-C) changes. The 123 most promising signals with p

BackgroundRadiotherapy is of critical importance in the treatment of breast cancer. However, not all patients derive therapeutic benefit and some breast cancers are resistant to the treatment, and are thus evidenced with prospective distant metastatic spread and local recurrence. In this study, we investigated the potential therapeutic effects of all-trans retinoic acid (ATRA) on radiation-resistant breast cancer cells and the associated invasiveness.MethodsThe MCF7/C6 cells with gained radiation resistance after a long term treatment with fractionated ionizing radiation were derived from human breast cancer MCF7 cell line, and are enriched with cells expressing putative breast cancer stem cell biomarker CD44(+)/CD24(-/low)/ALDH(+). The enhanced invasiveness and the acquired resistances to chemotherapeutic treatments of MCF7/C6 cells were measured, and potential effects of all-trans retinoic acid (ATRA) on the induction of differentiation, invasion and migration, and on the sensitivities to chemotherapies in MCF7/C6 cells were investigated.ResultsMCF7/C6 cells are with enrichment of cancer stem-cell like cells with positive staining of CD44(+)/CD24(-/low), OCT3/4 and NANOG. MCF7/C6 cells showed an increased tumoregensis potential and enhanced aggressiveness of invasion and migration. Treatment with ATRA induces the differentiation in MCF7/C6 cells, resulting in reduced invasiveness and migration, and increased sensitivity to Epirubincin treatment.ConclusionOur study suggests a potential clinic impact for ATRA as a chemotherapeutic agent for treatment of therapy-resistant breast cancer especially for the metastatic lesions. The study also provides a rationale for ATRA as a sensitizer of Epirubincin, a first-line treatment option for breast cancer patients.

BackgroundGiven the uncertainties inherent in clinical measures of prostate cancer aggressiveness, clinically validated tissue biomarkers are needed. We tested whether Alpha-2-Glycoprotein 1, Zinc-Binding (AZGP1) protein levels, measured by immunohistochemistry, and RNA expression, by RNA in situ hybridization (RISH), predict recurrence after radical prostatectomy independent of clinical and pathological parameters.MethodsAZGP1 IHC and RISH were performed on a large multi-institutional tissue microarray resource including 1,275 men with 5 year median follow-up. The relationship between IHC and RISH expression levels was assessed using the Kappa analysis. Associations with clinical and pathological parameters were tested by the Chi-square test and the Wilcoxon rank sum test. Relationships with outcome were assessed with univariable and multivariable Cox proportional hazards models and the Log-rank test.ResultsAbsent or weak expression of AZGP1 protein was associated with worse recurrence free survival (RFS), disease specific survival, and overall survival after radical prostatectomy in univariable analysis. AZGP1 protein expression, along with pre-operative serum PSA levels, surgical margin status, seminal vesicle invasion, extracapsular extension, and Gleason score predicted RFS on multivariable analysis. Similarly, absent or low AZGP1 RNA expression by RISH predicted worse RFS after prostatectomy in univariable and multivariable analysis.ConclusionsIn our large, rigorously designed validation cohort, loss of AZGP1 expression predicts RFS after radical prostatectomy independent of clinical and pathological variables. Prostate 76:1409-1419, 2016. © 2016 Wiley Periodicals, Inc.

PTEN loss is a promising prognostic and predictive biomarker in prostate cancer. Because it occurs most commonly via PTEN gene deletion, we developed a clinical-grade, automated, and inexpensive immunohistochemical assay to detect PTEN loss. We studied the sensitivity and specificity of PTEN immunohistochemistry relative to four-color fluorescence in situ hybridization (FISH) for detection of PTEN gene deletion in a multi-institutional cohort of 731 primary prostate tumors. Intact PTEN immunostaining was 91% specific for the absence of PTEN gene deletion (549/602 tumors with two copies of the PTEN gene by FISH showed intact expression of PTEN by immunohistochemistry) and 97% sensitive for the presence of homozygous PTEN gene deletion (absent PTEN protein expression by immunohistochemistry in 65/67 tumors with homozygous deletion). PTEN immunohistochemistry was 65% sensitive for the presence of hemizygous PTEN gene deletion, with protein loss in 40/62 hemizygous tumors. We reviewed the 53 cases where immunohistochemistry showed PTEN protein loss and FISH showed two intact copies of the PTEN gene. On re-review, there was ambiguous immunohistochemistry loss in 6% (3/53) and failure to analyze the same tumor area by both methods in 34% (18/53). Of the remaining discordant cases, 41% (13/32) revealed hemizygous (n=8) or homozygous (n=5) PTEN gene deletion that was focal in most cases (11/13). The remaining 19 cases had two copies of the PTEN gene detected by FISH, representing truly discordant cases. Our automated PTEN immunohistochemistry assay is a sensitive method for detection of homozygous PTEN gene deletions. Immunohistochemistry screening is particularly useful to identify cases with heterogeneous PTEN gene deletion in a subset of tumor glands. Mutations, small insertions, or deletions and/or epigenetic or microRNA-mediated mechanisms may lead to PTEN protein loss in tumors with normal or hemizygous PTEN gene copy number.

Intratumoral heterogeneity of signaling networks may contribute to targeted cancer therapy resistance, including in the highly lethal brain cancer glioblastoma (GBM). We performed single-cell phosphoproteomics on a patient-derived in vivo GBM model of mTOR kinase inhibitor resistance and coupled it to an analytical approach for detecting changes in signaling coordination. Alterations in the protein signaling coordination were resolved as early as 2.5 days after treatment, anticipating drug resistance long before it was clinically manifest. Combination therapies were identified that resulted in complete and sustained tumor suppression in vivo. This approach may identify actionable alterations in signal coordination that underlie adaptive resistance, which can be suppressed through combination drug therapy, including non-obvious drug combinations.

BackgroundThe uncertainties inherent in clinical measures of prostate cancer (CaP) aggressiveness endorse the investigation of clinically validated tissue biomarkers. MUC1 expression has been previously reported to independently predict aggressive localized prostate cancer. We used a large cohort to validate whether MUC1 protein levels measured by immunohistochemistry (IHC) predict aggressive cancer, recurrence and survival outcomes after radical prostatectomy independent of clinical and pathological parameters.Material and methodsMUC1 IHC was performed on a multi-institutional tissue microarray (TMA) resource including 1,326 men with a median follow-up of 5 years. Associations with clinical and pathological parameters were tested by the Chi-square test and the Wilcoxon rank sum test. Relationships with outcome were assessed with univariable and multivariable Cox proportional hazard models and the Log-rank test.ResultsThe presence of MUC1 expression was significantly associated with extracapsular extension and higher Gleason score, but not with seminal vesicle invasion, age, positive surgical margins or pre-operative serum PSA levels. In univariable analyses, positive MUC1 staining was significantly associated with a worse recurrence free survival (RFS) (HR: 1.24, CI 1.03-1.49, P = 0.02), although not with disease specific survival (DSS, P>0.5). On multivariable analyses, the presence of positive surgical margins, extracapsular extension, seminal vesicle invasion, as well as higher pre-operative PSA and increasing Gleason score were independently associated with RFS, while MUC1 expression was not. Positive MUC1 expression was not independently associated with disease specific survival (DSS), but was weakly associated with overall survival (OS).ConclusionIn our large, rigorously designed validation cohort, MUC1 protein expression was associated with adverse pathological features, although it was not an independent predictor of outcome after radical prostatectomy.

AimTo investigate cyclo (Arg-Gly-Asp-D-Phe-Lys) peptide (RGD)-modified PEGylated dendrimer-entrapped gold nanoparticles (PEGylated Au DENPs-RGD) for targeted computed tomography (CT) imaging of breast carcinomas.Materials & methodsPEGylated Au DENPs-RGD were synthesized and characterized. Then, the PEGylated Au DENPs-RGD for targeted CT imaging were investigated using the MDA-MB-435 cell line, an integrin-rich breast carcinoma cells, and mice with MDA-MB-435 xenograft tumors. Finally, silver enhancement staining and integrin αvβ3 immunohistochemistry of the tumors were performed.ResultsThe synthesized PEGylated Au DENPs-RGD were spherical, water dispersible and biocompatible nanoprobes with a gold nanoparticle core size of 2.8 nm. Due to the presence of the Au nanoparticles, the PEGylated Au DENPs-RGD displayed a higher x-ray attenuation intensity than Omnipaque at the same Au or I concentrations. The conjugated RGD ligand can specifically identify and target overexpressed integrin receptors on MDA-MB-435 cells. After intravenous injection, these nanoprobes accumulated in the targeted area of mice with MDA-MB-435 xenograft tumors, which enabled the tumor to be detected by CT imaging. The histological results confirmed the imaging results.ConclusionThe PEGylated Au DENPs-RGD can be used as targeted nanoprobes with good biocompatibility for targeted CT imaging and diagnosis of integrin-positive tumors.

Although "histone" methyltransferases and demethylases are well established to regulate transcriptional programs and to use nonhistone proteins as substrates, their possible roles in regulation of heat-shock proteins in the nucleus have not been investigated. Here, we report that a highly conserved arginine residue, R469, in HSP70 (heat-shock protein of 70 kDa) proteins, an evolutionarily conserved protein family of ATP-dependent molecular chaperone, was monomethylated (me1), at least partially, by coactivator-associated arginine methyltransferase 1/protein arginine methyltransferase 4 (CARM1/PRMT4) and demethylated by jumonji-domain-containing 6 (JMJD6), both in vitro and in cultured cells. Functional studies revealed that HSP70 could directly regulate retinoid acid (RA)-induced retinoid acid receptor β2 (RARβ2) gene transcription through its binding to chromatin, with R469me1 being essential in this process. HSP70's function in gene transcriptional regulation appears to be distinct from its protein chaperon activity. R469me1 was shown to mediate the interaction between HSP70 and TFIIH, which involves in RNA polymerase II phosphorylation and thus transcriptional initiation. Our findings expand the repertoire of nonhistone substrates targeted by PRMT4 and JMJD6, and reveal a new function of HSP70 proteins in gene transcription at the chromatin level aside from its classic role in protein folding and quality control.

We describe chemical approaches for integrated metabolic and proteomic assays from single cells. Quantitative assays for intracellular metabolites, including glucose uptake and three other species, are designed as surface-competitive binding assays with fluorescence readouts. This enables integration into a microarray format with functional protein immunoassays, all of which are incorporated into the microchambers of a single-cell barcode chip (SCBC). By using the SCBC, we interrogate the response of human-derived glioblastoma cancer cells to epidermal growth factor receptor inhibition. We report, for the first time, on both the intercellular metabolic heterogeneity as well as the baseline and drug-induced changes in the metabolite-phosphoprotein correlation network.

Heparan sulfate (HS) is a complex and highly variable polysaccharide, expressed ubiquitously on the cell surface as HS proteoglycans (HSPGs), and found in the extracellular matrix as free HS fragments. Its heterogeneity due to various acetylation and sulfation patterns endows a multitude of functions. In animal tissues, HS interacts with a wide range of proteins to mediate numerous biological activities; given its multiple roles in inflammation processes, characterization of HS in human serum has significant potential for elucidating disease mechanisms. Historically, investigation of HS was limited by its low concentration in human serum, together with the complexity of the serum matrix. In this study, we used a modified mass spectrometry method to examine HS disaccharide profiles in the serum of 50 women with rheumatoid arthritis (RA), and compared our results to 51 sera from healthy women. Using various purification methods and online LC-MS/MS, we discovered statistically significant differences in the sulfation and acetylation patterns between populations. Since early diagnosis of RA is considered important in decelerating the disease's progression, identification of specific biomolecule characterizations may provide crucial information towards developing new therapies for suppressing the disease in its early stages. This is the first report of potential glycosaminoglycan biomarkers for RA found in human sera, while acknowledging the obvious fact that a larger population set, and more stringent collection parameters, will need to be investigated in the future.

Statins effectively lower LDL cholesterol levels in large studies and the observed interindividual response variability may be partially explained by genetic variation. Here we perform a pharmacogenetic meta-analysis of genome-wide association studies (GWAS) in studies addressing the LDL cholesterol response to statins, including up to 18,596 statin-treated subjects. We validate the most promising signals in a further 22,318 statin recipients and identify two loci, SORT1/CELSR2/PSRC1 and SLCO1B1, not previously identified in GWAS. Moreover, we confirm the previously described associations with APOE and LPA. Our findings advance the understanding of the pharmacogenetic architecture of statin response.

Quantities can be represented using either mathematical language (i.e., numbers) or natural language (i.e., quantifiers). Previous studies have shown that numerical processing elicits greater activation in the brain regions around the intraparietal sulcus (IPS) relative to other semantic processes. However, little research has been conducted to investigate whether the IPS is also critical for the semantic processing of quantifiers in natural language. In this study, 20 adults were scanned with functional magnetic resonance imaging while they performed semantic distance judgment involving six types of materials (i.e., frequency adverbs, quantity pronouns and nouns, animal names, Arabic digits, number words, and dot arrays). Conjunction analyses of brain activation showed that numbers and dot arrays elicited greater activation in the right IPS than did words (i.e., animal names) or quantifiers (i.e., frequency adverbs and quantity pronouns and nouns). Quantifiers elicited more activation in left middle temporal gyrus and inferior frontal gyrus than did numbers and dot arrays. No differences were found between quantifiers and animal names. These findings suggest that, although quantity processing for numbers and dot arrays typically relies on the right IPS region, quantity processing for quantifiers typically relies on brain regions for general semantic processing. Thus, the IPS does not appear to be the only brain region for quantity processing.

Hepatocellular carcinoma (HCC) is one of the most common and deadly human cancers and it remains poorly managed. Human HCC development is often associated both with elevated expression of inducible nitric oxide synthase (iNOS) and with genetic deletion of the major denitrosylase S-nitrosoglutathione reductase (GSNOR/ADH5). However, their causal involvement in human HCC is not established. In mice, GSNOR deficiency causes S-nitrosylation and depletion of the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (AGT) and increases rates of both spontaneous and DEN carcinogen-induced HCC. Here, we report that administration of 1400W, a potent and highly selective inhibitor of iNOS, blocked AGT depletion and rescued the repair of mutagenic O6-ethyldeoxyguanosines following DEN challenge in livers of GSNOR-deficient (GSNOR(-/-)) mice. Notably, short-term iNOS inhibition following DEN treatment had little effect on carcinogenesis in wild-type mice, but was sufficient to reduce HCC multiplicity, maximal size, and burden in GSNOR(-/-) mice to levels comparable with wild-type controls. Furthermore, increased HCC susceptibility in GSNOR(-/-) mice was not associated with an increase in interleukin 6, tumor necrosis factor-α, oxidative stress, or hepatocellular proliferation. These results suggested that GSNOR deficiency linked to defective DNA damage repair likely acts at the tumor initiation stage to promote HCC carcinogenesis. Together, our findings provide the first proof of principle that HCC development in the context of uncontrolled nitrosative stress can be blocked by pharmacologic inhibition of iNOS, possibly providing an effective therapy for patients with HCC.