👤 Jissy Thomas

Lymphoplasmacytic lymphoma (LPL) is an incurable low-grade lymphoma with no standard therapy. Nine asymptomatic patients treated with a first-in-human, neoantigen DNA vaccine experienced no dose limiting toxicities (primary endpoint, NCT01209871). All patients achieve stable disease or better, with one minor response, and median time to progression of 72+ months. Post-vaccine single-cell transcriptomics reveal dichotomous antitumor responses, with reduced tumor B-cells (tracked by unique B cell receptor) and their survival pathways, but no change in clonal plasma cells. Downregulation of human leukocyte antigen (HLA) class II molecules and paradoxical upregulation of insulin-like growth factor (IGF) by the latter suggest resistance mechanisms. Vaccine therapy activates and expands bone marrow T-cell clonotypes, and functional neoantigen-specific responses (secondary endpoint), but not co-inhibitory pathways or Treg, and reduces protumoral signaling by myeloid cells, suggesting favorable perturbation of the tumor immune microenvironment. Future strategies may require combinations of vaccines with agents targeting plasma cell subpopulations, or blockade of IGF-1 signaling or myeloid cell checkpoints. Show less

PP2A B55α, encoded by PPP2R2A, acts as a regulatory subunit of the serine/threonine phosphatase PP2A. Despite a frequent loss of heterozygosity of PPP2R2A in cases of non-small cell lung cancer (NSCLC), research on PP2A B55α's functions remains limited and controversial. To investigate the biological roles of PP2A B55α, we conducted bulk RNA-sequencing to assess the impact of PPP2R2A knockdown using two shRNAs in a NSCLC cell line. Gene set enrichment analysis (GSEA) of the RNA-sequencing data revealed significant enrichment of the epithelial-mesenchymal transition (EMT) pathway, with SNAI2 (the gene encoding Slug) emerging as one of the top candidates. Our findings demonstrate that PP2A B55α suppresses EMT, as PPP2R2A deficiency through knockdown or homozygous or hemizygous depletion promotes EMT and metastatic behavior in NSCLC cells, as evidenced by changes in EMT biomarkers, invasion and migration abilities, as well as metastasis in a tail vein assay. Mechanistically, PP2A B55α inhibits EMT by downregulating SNAI2 expression via the GSK3β-β-catenin pathway. Importantly, PPP2R2A deficiency also slows cell proliferation by disrupting DNA replication, particularly in PPP2R2A Show less

Proteomics provides an opportunity for detection and monitoring of anorexia nervosa (AN) and its related variant, atypical-AN (atyp-AN). However, research to date has been limited by the small number of proteins explored, exclusive focus on adults with AN, and lack of replication across studies. This study performed Olink Proseek Multiplex profiling of 92 proteins involved in inflammation among females with AN and atyp-AN (N = 64), all < 90% of expected body weight, and age-matched healthy controls (HC; N=44). After correction for multiple testing, nine proteins differed significantly in the AN/atyp-AN group relative to HC group ( Show less

Amyloid-beta (Aβ) is produced from amyloid precursor protein (APP) primarily after APP is internalised by endocytosis and clathrin-mediated endocytic processes are altered in Alzheimer's disease (AD). There is also evidence that cholesterol and flotillin affect APP endocytosis. We hypothesised that endocytic protein expression would be altered in the brains of people with AD compared to non-diseased subjects which could be linked to increased Aβ generation. We compared protein expression in frontal cortex samples from men with AD compared to age-matched, non-diseased controls. Soluble and insoluble Aβ40 and Aβ42, the soluble Aβ42/Aβ40 ratio, βCTF, BACE1, presenilin-1 and the ratio of phosphorylated:total GSK3β were significantly increased while the insoluble Aβ42:Aβ40 ratio was significantly decreased in AD brains. Total and phosphorylated tau were markedly increased in AD brains. Significant increases in clathrin, AP2, PICALM isoform 4, Rab-5 and caveolin-1 and 2 were seen in AD brains but BIN1 was decreased. However, using immunohistochemistry, caveolin-1 and 2 were decreased. The results obtained here suggest an overall increase in endocytosis in the AD brain, explaining, at least in part, the increased production of Aβ during AD. Show less

Variations in the Dlg2 gene have been linked to increased risk for psychiatric disorders, including schizophrenia, autism spectrum disorders, intellectual disability, bipolar disorder, attention deficit hyperactivity disorder, and pubertal disorders. Recent studies have reported disrupted brain circuit function and behaviour in models of Dlg2 knockout and haploinsufficiency. Specifically, deficits in hippocampal synaptic plasticity were found in heterozygous Dlg2+/- rats suggesting impacts on hippocampal dependent learning and cognitive flexibility. Here, we tested these predicted effects with a behavioural characterisation of the heterozygous Dlg2+/- rat model. Dlg2+/- rats exhibited a specific, mild impairment in reversal learning in a substrate deterministic bowl-digging reversal learning task. The performance of Dlg2+/- rats in other bowl digging task, visual discrimination and reversal, novel object preference, novel location preference, spontaneous alternation, modified progressive ratio, and novelty-suppressed feeding test were not impaired. These findings suggest that despite altered brain circuit function, behaviour across different domains is relatively intact in Dlg2+/- rats, with the deficits being specific to only one test of cognitive flexibility. The specific behavioural phenotype seen in this Dlg2+/- model may capture features of the clinical presentation associated with variation in the Dlg2 gene. Show less

The discovery of frequent 8p11-p12 amplifications in squamous cell lung cancer (SQLC) has fueled hopes that FGFR1, located inside this amplicon, might be a therapeutic target. In a clinical trial, only 11% of patients with 8p11 amplification (detected by FISH) responded to FGFR kinase inhibitor treatment. To understand the mechanism of FGFR1 dependency, we performed deep genomic characterization of 52 SQLCs with 8p11-p12 amplification, including 10 tumors obtained from patients who had been treated with FGFR inhibitors. We discovered somatically altered variants of FGFR1 with deletion of exons 1-8 that resulted from intragenic tail-to-tail rearrangements. These ectodomain-deficient FGFR1 variants (ΔEC-FGFR1) were expressed in the affected tumors and were tumorigenic in both in vitro and in vivo models of lung cancer. Mechanistically, breakage-fusion-bridges were the source of 8p11-p12 amplification, resulting from frequent head-to-head and tail-to-tail rearrangements. Generally, tail-to-tail rearrangements within or in close proximity upstream of FGFR1 were associated with FGFR1 dependency. Thus, the genomic events shaping the architecture of the 8p11-p12 amplicon provide a mechanistic explanation for the emergence of FGFR1-driven SQLC. Specifically, we believe that FGFR1 ectodomain-deficient and FGFR1-centered amplifications caused by tail-to-tail rearrangements are a novel somatic genomic event that might be predictive of therapeutically relevant FGFR1 dependency. Show less

FGFR alterations are reported across various malignancies and might act as oncogenic drivers in multiple histologies. Erdafitinib is an oral, selective pan-FGFR tyrosine kinase inhibitor with activity in FGFR-altered advanced urothelial carcinoma. We aimed to evaluate the safety and activity of erdafitinib in previously treated patients with FGFR-altered advanced solid tumours. The single-arm, phase 2 RAGNAR study was conducted at 156 investigative centres (hospitals or oncology practices that are qualified oncology study centres) across 15 countries. The study consisted of four cohorts based on tumour histology and patient age; the results reported in this Article are for the primary cohort of the study, defined as the Broad Panel Cohort, which was histology-agnostic. We recruited patients aged 12 years or older with advanced or metastatic tumours of any histology (except urothelial cancer) with predefined FGFR1-4 alterations (mutations or fusions according to local or central testing). Eligible patients had disease progression on at least one previous line of systemic therapy and no alternative standard therapy available to them, and an Eastern Cooperative Oncology Group performance status of 0-1 (or equivalent for adolescents aged 12-17 years). Patients received once-daily oral erdafitinib (8 mg/day with provision for pharmacodynamically guided up-titration to 9 mg/day) on a continuous 21-day cycle until disease progression or intolerable toxicity. The primary endpoint was objective response rate by independent review committee according to Response Evaluation Criteria In Solid Tumors (RECIST), version 1.1, or Response Assessment In Neuro-Oncology (RANO). The primary analysis was conducted on the treated population of the Broad Panel Cohort. This ongoing study is registered with ClinicalTrials.gov, number NCT04083976. Patients were recruited between Dec 5, 2019, and Feb 15, 2022. Of 217 patients treated with erdafitinib, 97 (45%) patients were female and 120 (55%) were male. The data cutoff was Aug 15, 2022. At a median follow-up of 17·9 months (IQR 13·6-23·9), an objective response was observed in 64 (30% [95% CI 24-36]) of 217 patients across 16 distinct tumour types. The most common grade 3 or higher treatment-emergent adverse events related to erdafitinib were stomatitis (25 [12%]), palmar-plantar erythrodysaesthesia syndrome (12 [6%]), and hyperphosphataemia (11 [5%]). The most commonly occurring serious treatment-related adverse events (grade 3 or higher) were stomatitis in four (2%) patients and diarrhoea in two (1%). There were no treatment-related deaths. RAGNAR results show clinical benefit for erdafitinib in the tumour-agnostic setting in patients with advanced solid tumours with susceptible FGFR alterations who have exhausted other treatment options. These results support the continued development of FGFR inhibitors in patients with advanced solid tumours. Janssen Research & Development. Show less

Lymphoplasmacytic lymphoma (LPL) is an incurable low-grade B-cell lymphoma of the bone marrow. Despite a cumulative risk of progression, there is no approved therapy for patients in the asymptomatic phase. We conducted a first-in-human clinical trial of a novel therapeutic DNA idiotype neoantigen vaccine in nine patients with asymptomatic LPL. Treatment was well tolerated with no dose limiting toxicities. One patient achieved a minor response, and all remaining patients experienced stable disease, with median time to disease progression of 61+ months. Direct interrogation of the tumor microenvironment by single-cell transcriptome analysis revealed an unexpected dichotomous antitumor response, with significantly reduced numbers of clonal tumor mature B-cells, tracked by their unique BCR, and downregulation of genes involved in signaling pathways critical for B-cell survival post-vaccine, but no change in clonal plasma cell subpopulations. Downregulation of HLA class II molecule expression suggested intrinsic resistance by tumor plasma cell subpopulations and cell-cell interaction analyses predicted paradoxical upregulation of IGF signaling post vaccine by plasma cell, but not mature B-cell subpopulations, suggesting a potential mechanism of acquired resistance. Vaccine therapy induced dynamic changes in bone marrow T-cells, including upregulation of signaling pathways involved in T-cell activation, expansion of T-cell clonotypes, increased T-cell clonal diversity, and functional tumor antigen-specific cytokine production, with little change in co-inhibitory pathways or Treg. Vaccine therapy also globally altered cell-cell communication networks across various bone marrow cell types and was associated with reduction of protumoral signaling by myeloid cells, principally non-classical monocytes. These results suggest that this prototype neoantigen vaccine favorably perturbed the tumor immune microenvironment, resulting in reduction of clonal tumor mature B-cell, but not plasma cell subpopulations. Future strategies to improve clinical efficacy may require combinations of neoantigen vaccines with agents which specifically target LPL plasma cell subpopulations, or enable blockade of IGF-1 signaling or myeloid cell checkpoints. Show less

Cerebral arteriovenous malformations (cAVM) are a significant cause of intracranial hemorrhagic stroke and brain damage. The arteriovenous junctions in AVM nidus are known to have hemodynamic disturbances such as altered shear stress, which could lead to endothelial dysfunction. The molecular mechanisms coupling shear stress and endothelial dysfunction in cAVMs are poorly understood. We speculated that disturbed blood flow in artery-vein junctions activates Notch receptors and promotes endothelial mesenchymal plasticity during cAVM formation. We investigated the expression profile of endothelial mesenchymal transition (EndMT) and cell adhesion markers, as well as activated Notch receptors, in 18 human cAVM samples and 15 control brain tissues, by quantitative real-time PCR (qRT-PCR) and immunohistochemical evaluation. Employing a combination of a microfluidic system, qRT-PCR, immunofluorescence, as well as invasion and inhibitor assays, the effects of various shear stress conditions on Notch-induced EndMT and invasive potential of human cerebral microvascular endothelial cells (hCMEC/d3) were analyzed. We found evidence for EndMT and enhanced expression of activated Notch intracellular domain (NICD3 and NICD4) in human AVM nidus samples. The expression of transmembrane adhesion receptor integrin α9/β1 is significantly reduced in cAVM nidal vessels. Cell-cell adhesion proteins such as VE-cadherin and N-cadherin were differentially expressed in AVM nidus compared with control brain tissues. Using well-characterized hCMECs, we show that altered fluid shear stress steers Notch3 nuclear translocation and promotes SNAI1/2 expression and nuclear localization. Oscillatory flow downregulates integrin α9/β1 and VE-cadherin expression, while N-cadherin expression and endothelial cell invasiveness are augmented. Gamma-secretase inhibitor RO4929097, and to a lesser level DAPT, prevent the mesenchymal transition and invasiveness of cerebral microvascular endothelial cells exposed to oscillatory fluid flow. Our study provides, for the first time, evidence for the role of oscillatory shear stress in mediating the EndMT process and dysregulated expression of cell adhesion molecules, especially multifunctional integrin α9/β1 in human cAVM nidus. Concomitantly, our findings indicate the potential use of small-molecular inhibitors such as RO4929097 in the less-invasive therapeutic management of cAVMs. Show less

With an increasing prevalence of obesity, there is a need for new therapies to improve body weight management and metabolic health. Multireceptor agonists in development may provide approaches to fulfill this unmet medical need. LY3437943 is a novel triple agonist peptide at the glucagon receptor (GCGR), glucose-dependent insulinotropic polypeptide receptor (GIPR), and glucagon-like peptide-1 receptor (GLP-1R). In vitro, LY3437943 shows balanced GCGR and GLP-1R activity but more GIPR activity. In obese mice, administration of LY3437943 decreased body weight and improved glycemic control. Body weight loss was augmented by the addition of GCGR-mediated increases in energy expenditure to GIPR- and GLP-1R-driven calorie intake reduction. In a phase 1 single ascending dose study, LY3437943 showed a safety and tolerability profile similar to other incretins. Its pharmacokinetic profile supported once-weekly dosing, and a reduction in body weight persisted up to day 43 after a single dose. These findings warrant further clinical assessment of LY3437943. Show less

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are closely related progressive disorders with no available disease-modifying therapy, neuropathologically characterized by intraneuronal aggregates of misfolded α-synuclein. To explore the role of DNA methylation changes in PD and DLB pathogenesis, we performed an epigenome-wide association study (EWAS) of 322 postmortem frontal cortex samples and replicated results in an independent set of 200 donors. We report novel differentially methylated replicating loci associated with Braak Lewy body stage near TMCC2, SFMBT2, AKAP6 and PHYHIP. Differentially methylated probes were independent of known PD genetic risk alleles. Meta-analysis provided suggestive evidence for a differentially methylated locus within the chromosomal region affected by the PD-associated 22q11.2 deletion. Our findings elucidate novel disease pathways in PD and DLB and generate hypotheses for future molecular studies of Lewy body pathology. Show less

Despite the success of LDL-lowering drugs in reducing cardiovascular disease (CVD), there remains a large burden of residual disease due in part to persistent dyslipidemia characterized by elevated levels of triglyceride-rich lipoproteins (TRLs) and reduced levels of HDL. This form of dyslipidemia is increasing globally as a result of the rising prevalence of obesity and metabolic syndrome. Accumulating evidence suggests that impaired hepatic clearance of cholesterol-rich TRL remnants leads to their accumulation in arteries, promoting foam cell formation and inflammation. Low levels of HDL may associate with reduced cholesterol efflux from foam cells, aggravating atherosclerosis. While fibrates and fish oils reduce TRL, they have not been uniformly successful in reducing CVD, and there is a large unmet need for new approaches to reduce remnants and CVD. Rare genetic variants that lower triglyceride levels via activation of lipolysis and associate with reduced CVD suggest new approaches to treating dyslipidemia. Apolipoprotein C3 (APOC3) and angiopoietin-like 3 (ANGPTL3) have emerged as targets for inhibition by antibody, antisense, or RNAi approaches. Inhibition of either molecule lowers TRL but respectively raises or lowers HDL levels. Large clinical trials of such agents in patients with high CVD risk and elevated levels of TRL will be required to demonstrate efficacy of these approaches. Show less

Identification of cancer-predisposing germline variants in childhood cancer patients is important for therapeutic decisions, disease surveillance and risk assessment for patients, and potentially, also for family members. We investigated the spectrum and prevalence of pathogenic germline variants in selected childhood cancer patients with features suggestive of genetic predisposition to cancer. Germline DNA was subjected to exome sequencing to filter variants in 1048 genes of interest including 176 known cancer predisposition genes (CPGs). An enrichment burden analysis compared rare deleterious germline CPG variants in the patient cohort with those in a healthy aged control population. A subset of predicted deleterious variants in novel candidate CPGs was investigated further by examining matched tumor samples, and the functional impact of AXIN1 variants was analyzed in cultured cells. Twenty-two pathogenic/likely pathogenic (P/LP) germline variants detected in 13 CPGs were identified in 19 of 76 patients (25.0%). Unclear association with the diagnosed cancer types was observed in 11 of 19 patients carrying P/LP CPG variants. The burden of rare deleterious germline variants in autosomal dominant CPGs was significantly higher in study patients versus healthy aged controls. A novel AXIN1 frameshift variant (Ser321fs) may impact the regulation of β-catenin levels. Selection of childhood cancer patients for germline testing based on features suggestive of an underlying genetic predisposition could help to identify carriers of clinically relevant germline CPG variants, and streamline the integration of germline genomic testing in the pediatric oncology clinic. Show less

Skeletal muscle exhibits remarkable plasticity under both physiological and pathological conditions. One major manifestation of this plasticity is muscle atrophy that is an adaptive response to catabolic stimuli. Because the heterogeneous transcriptome responses to catabolism in different types of muscle cells are not fully characterized, we applied single-nucleus RNA sequencing (snRNA-seq) to unveil muscle atrophy related transcriptional changes at single nucleus resolution. Using a sciatic denervation mouse model of muscle atrophy, snRNA-seq was performed to generate single-nucleus transcriptional profiles of the gastrocnemius muscle from normal and denervated mice. Various bioinformatics analyses, including unsupervised clustering, functional enrichment analysis, trajectory analysis, regulon inference, metabolic signature characterization and cell-cell communication prediction, were applied to illustrate the transcriptome changes of the individual cell types. A total of 29 539 muscle nuclei (normal vs. denervation: 15 739 vs. 13 800) were classified into 13 nuclear types according to the known cell markers. Among these, the type IIb myonuclei were further divided into two subgroups, which we designated as type IIb1 and type IIb2 myonuclei. In response to denervation, the proportion of type IIb2 myonuclei increased sharply (78.12% vs. 38.45%, P < 0.05). Concomitantly, trajectory analysis revealed that denervated type IIb2 myonuclei clearly deviated away from the normal type IIb2 myonuclei, indicating that this subgroup underwent robust transcriptional reprogramming upon denervation. Signature genes in denervated type IIb2 myonuclei included Runx1, Gadd45a, Igfn1, Robo2, Dlg2, and Sh3d19 (P < 0.001). The gene regulatory network analysis captured a group of atrophy-related regulons (Foxo3, Runx1, Elk4, and Bhlhe40) whose activities were enhanced (P < 0.01), especially in the type IIb2 myonuclei. The metabolic landscape in the myonuclei showed that most of the metabolic pathways were down-regulated by denervation (P < 0.001), while some of the metabolic signalling, such as glutathione metabolism, was specifically activated in the denervated type IIb2 myonulei. We also investigated the transcriptomic alterations in the type I myofibres, muscle stem cells, fibro-adipogenic progenitors, macrophages, endothelial cells and pericytes and characterized their signature responses to denervation. By predicting the cell-cell interactions, we observed that the communications between myofibres and muscle resident cells were diminished by denervation. Our results define the myonuclear transition, metabolic remodelling, and gene regulation networks reprogramming associated with denervation-induced muscle atrophy and illustrate the molecular basis of the heterogeneity and plasticity of muscle cells in response to catabolism. These results provide a useful resource for exploring the molecular mechanism of muscle atrophy. Show less

Mutations affecting DLG2 are emerging as a genetic risk factor associated with neurodevelopmental psychiatric disorders including schizophrenia, autism spectrum disorder, and bipolar disorder. Discs large homolog 2 (DLG2) is a member of the membrane-associated guanylate kinase protein superfamily of scaffold proteins, a component of the post-synaptic density in excitatory neurons and regulator of synaptic function and plasticity. It remains an important question whether and how haploinsuffiency of DLG2 contributes to impairments in basic behavioural and cognitive functions that may underlie symptomatic domains in patients that cross diagnostic boundaries. Using a heterozygous Dlg2 mouse model we examined the impact of reduced Dlg2 expression on functions commonly impaired in neurodevelopmental psychiatric disorders including motor co-ordination and learning, pre-pulse inhibition and habituation to novel stimuli. The heterozygous Dlg2 mice exhibited behavioural impairments in long-term motor learning and long-term habituation to a novel context, but not motor co-ordination, initial responses to a novel context, PPI of acoustic startle or anxiety. We additionally showed evidence for the reduced regulation of the synaptic plasticity-associated protein cFos in the motor cortex during motor learning. The sensitivity of selective behavioural and cognitive functions, particularly those dependent on synaptic plasticity, to reduced expression of DLG2 give further credence for DLG2 playing a critical role in specific brain functions but also a mechanistic understanding of symptom expression shared across psychiatric disorders. Show less

Copy number variants indicating loss of function in the DLG2 gene have been associated with markedly increased risk for schizophrenia, autism spectrum disorder, and intellectual disability. DLG2 encodes the postsynaptic scaffolding protein DLG2 (PSD93) that interacts with NMDA receptors, potassium channels, and cytoskeletal regulators but the net impact of these interactions on synaptic plasticity, likely underpinning cognitive impairments associated with these conditions, remains unclear. Here, hippocampal CA1 neuronal excitability and synaptic function were investigated in a novel clinically relevant heterozygous Dlg2+/- rat model using ex vivo patch-clamp electrophysiology, pharmacology, and computational modelling. Dlg2+/- rats had reduced supra-linear dendritic integration of synaptic inputs resulting in impaired associative long-term potentiation. This impairment was not caused by a change in synaptic input since NMDA receptor-mediated synaptic currents were, conversely, increased and AMPA receptor-mediated currents were unaffected. Instead, the impairment in associative long-term potentiation resulted from an increase in potassium channel function leading to a decrease in input resistance, which reduced supra-linear dendritic integration. Enhancement of dendritic excitability by blockade of potassium channels or activation of muscarinic M1 receptors with selective allosteric agonist 77-LH-28-1 reduced the threshold for dendritic integration and 77-LH-28-1 rescued the associative long-term potentiation impairment in the Dlg2+/- rats. These findings demonstrate a biological phenotype that can be reversed by compound classes used clinically, such as muscarinic M1 receptor agonists, and is therefore a potential target for therapeutic intervention. Show less

Genetic studies implicate disruption to the DLG2 gene in copy number variants as increasing risk for schizophrenia, autism spectrum disorders and intellectual disability. To investigate psychiatric endophenotypes associated with DLG2 haploinsufficiency (and concomitant PSD-93 protein reduction) a novel clinically relevant Dlg2 Show less

Migraine affects over a billion individuals worldwide but its genetic underpinning remains largely unknown. Here, we performed a genome-wide association study of 102,084 migraine cases and 771,257 controls and identified 123 loci, of which 86 are previously unknown. These loci provide an opportunity to evaluate shared and distinct genetic components in the two main migraine subtypes: migraine with aura and migraine without aura. Stratification of the risk loci using 29,679 cases with subtype information indicated three risk variants that seem specific for migraine with aura (in HMOX2, CACNA1A and MPPED2), two that seem specific for migraine without aura (near SPINK2 and near FECH) and nine that increase susceptibility for migraine regardless of subtype. The new risk loci include genes encoding recent migraine-specific drug targets, namely calcitonin gene-related peptide (CALCA/CALCB) and serotonin 1F receptor (HTR1F). Overall, genomic annotations among migraine-associated variants were enriched in both vascular and central nervous system tissue/cell types, supporting unequivocally that neurovascular mechanisms underlie migraine pathophysiology. Show less

We describe a precision medicine workflow, the integrated single nucleotide polymorphism network platform (iSNP), designed to determine the mechanisms by which SNPs affect cellular regulatory networks, and how SNP co-occurrences contribute to disease pathogenesis in ulcerative colitis (UC). Using SNP profiles of 378 UC patients we map the regulatory effects of the SNPs to a human signalling network containing protein-protein, miRNA-mRNA and transcription factor binding interactions. With unsupervised clustering algorithms we group these patient-specific networks into four distinct clusters driven by PRKCB, HLA, SNAI1/CEBPB/PTPN1 and VEGFA/XPO5/POLH hubs. The pathway analysis identifies calcium homeostasis, wound healing and cell motility as key processes in UC pathogenesis. Using transcriptomic data from an independent patient cohort, with three complementary validation approaches focusing on the SNP-affected genes, the patient specific modules and affected functions, we confirm the regulatory impact of non-coding SNPs. iSNP identified regulatory effects for disease-associated non-coding SNPs, and by predicting the patient-specific pathogenic processes, we propose a systems-level way to stratify patients. Show less

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) regulate glucose and energy homeostasis. Targeting both pathways with GIP receptor (GIPR) antagonist antibody (GIPR-Ab) and GLP-1 receptor (GLP-1R) agonist, by generating GIPR-Ab/GLP-1 bispecific molecules, is an approach for treating obesity and its comorbidities. In mice and monkeys, these molecules reduce body weight (BW) and improve many metabolic parameters. BW loss is greater with GIPR-Ab/GLP-1 than with GIPR-Ab or a control antibody conjugate, suggesting synergistic effects. GIPR-Ab/GLP-1 also reduces the respiratory exchange ratio in DIO mice. Simultaneous receptor binding and rapid receptor internalization by GIPR-Ab/GLP-1 amplify endosomal cAMP production in recombinant cells expressing both receptors. This may explain the efficacy of the bispecific molecules. Overall, our GIPR-Ab/GLP-1 molecules promote BW loss, and they may be used for treating obesity. Show less

Worldwide demand for petroleum products has resulted in increased oil and gas activities in many countries. Conventional and unconventional oil and gas extraction, production, and transport lead to increased levels of petroleum-derived polycyclic aromatic hydrocarbons (PAHs) in the environment. PAH exposure has profound effects on reproduction by affecting pathways involved in placental trophoblast cell function and impairing normal placental development and function-key contributors to reproductive success. However, other components found in petroleum and wastewaters from oil and gas extraction, including the sulfur-containing heterocyclic aromatic compounds such as dibenzothiophene (DBT) and its alkylated derivatives, may also impact reproductive success. The goal of this study was to examine the effect of exposure to DBT, a compound commonly detected in the environment, and one of its alkylated analogues, 2,4,7-trimethyldibenzothiophene (2,4,7-DBT), on steroidogenic and angiogenic pathways critical for mammalian development in placental trophoblast cells (HTR-8/SVneo cells). 2,4,7-DBT but not DBT increased estradiol output in association with increased tube-like formation (surrogate for angiogenesis). These changes in angiogenesis did not appear to be related to altered expression of the key placental angiogenic gene targets (ANGPTL4, VEGFA, and PGF). Neither compound showed a concentration related effect on progesterone synthesis or its receptor expression. Our results suggest that 2,4,7-DBT can disrupt key pathways important for placental trophoblast function and highlight the importance of determining the impact of exposure to both parent and alkylated compounds. Further, these data suggest that exposure to sulfur-containing heterocyclic aromatic compounds may lead to placental dysfunction and impact reproductive success at environmentally relevant levels. Show less

Head and neck squamous cell carcinoma (HNSCC), which includes cancers of the oral cavity and oropharynx, is a cause of substantial global morbidity and mortality. Strategies to reduce disease burden include discovery of novel therapies and repurposing of existing drugs. Statins are commonly prescribed for lowering circulating cholesterol by inhibiting HMG-CoA reductase (HMGCR). Results from some observational studies suggest that statin use may reduce HNSCC risk. We appraised the relationship of genetically-proxied cholesterol-lowering drug targets and other circulating lipid traits with oral (OC) and oropharyngeal (OPC) cancer risk using two-sample Mendelian randomization (MR). For the primary analysis, germline genetic variants in HMGCR, NPC1L1, CETP, PCSK9 and LDLR were used to proxy the effect of low-density lipoprotein cholesterol (LDL-C) lowering therapies. In secondary analyses, variants were used to proxy circulating levels of other lipid traits in a genome-wide association study (GWAS) meta-analysis of 188,578 individuals. Both primary and secondary analyses aimed to estimate the downstream causal effect of cholesterol lowering therapies on OC and OPC risk. The second sample for MR was taken from a GWAS of 6,034 OC and OPC cases and 6,585 controls (GAME-ON). Analyses were replicated in UK Biobank, using 839 OC and OPC cases and 372,016 controls and the results of the GAME-ON and UK Biobank analyses combined in a fixed-effects meta-analysis. We found limited evidence of a causal effect of genetically-proxied LDL-C lowering using HMGCR, NPC1L1, CETP or other circulating lipid traits on either OC or OPC risk. Genetically-proxied PCSK9 inhibition equivalent to a 1 mmol/L (38.7 mg/dL) reduction in LDL-C was associated with an increased risk of OC and OPC combined (OR 1.8 95%CI 1.2, 2.8, p = 9.31 x10-05), with good concordance between GAME-ON and UK Biobank (I2 = 22%). Effects for PCSK9 appeared stronger in relation to OPC (OR 2.6 95%CI 1.4, 4.9) than OC (OR 1.4 95%CI 0.8, 2.4). LDLR variants, resulting in genetically-proxied reduction in LDL-C equivalent to a 1 mmol/L (38.7 mg/dL), reduced the risk of OC and OPC combined (OR 0.7, 95%CI 0.5, 1.0, p = 0.006). A series of pleiotropy-robust and outlier detection methods showed that pleiotropy did not bias our findings. We found limited evidence for a role of cholesterol-lowering in OC and OPC risk, suggesting previous observational results may have been confounded. There was some evidence that genetically-proxied inhibition of PCSK9 increased risk, while lipid-lowering variants in LDLR, reduced risk of combined OC and OPC. This result suggests that the mechanisms of action of PCSK9 on OC and OPC risk may be independent of its cholesterol lowering effects; however, this was not supported uniformly across all sensitivity analyses and further replication of this finding is required. Show less

Synapse loss is an early event in late-onset Alzheimer's disease (LOAD). In this study, we have assessed the capacity of a polygenic risk score (PRS) restricted to synapse-encoding loci to predict LOAD. We used summary statistics from the International Genetics of Alzheimer's Project genome-wide association meta-analysis of 74,046 patients for model construction and tested the "synaptic PRS" in 2 independent data sets of controls and pathologically confirmed LOAD. The mean synaptic PRS was 2.3-fold higher in LOAD than that in controls (p < 0.0001) with a predictive accuracy of 72% in the target data set (n = 439) and 73% in the validation data set (n = 136), a 5%-6% improvement compared with the APOE locus (p < 0.00001). The model comprises 8 variants from 4 previously identified (BIN1, PTK2B, PICALM, APOE) and 2 novel (DLG2, MINK1) LOAD loci involved in glutamate signaling (p = 0.01) or APP catabolism or tau binding (p = 0.005). As the simplest PRS model with good predictive accuracy to predict LOAD, we conclude that synapse-encoding genes are enriched for LOAD risk-modifying loci. The synaptic PRS could be used to identify individuals at risk of LOAD before symptom onset. Show less

Increases in the red blood cell (RBC) degree of fatty acid desaturation are reported in response to exercise, aging, or diseases associated with systemic oxidant stress. However, no studies have focused on the presence and activity of fatty acid desaturases (FADS) in the mature RBC. Steady state metabolomics and isotope-labeled tracing experiments, immunofluorescence approaches, and pharmacological interventions were used to determine the degree of fatty acid unsaturation, FADS activity as a function of storage, oxidant stress, and G6PD deficiency in human and mouse RBCs. In 250 blood units from the REDS III RBC Omics recalled donor population, we report a storage-dependent accumulation of free mono-, poly-(PUFAs), and highly unsaturated fatty acids (HUFAs), which occur at a faster rate than saturated fatty acid accumulation. Through a combination of immunofluorescence, pharmacological inhibition, tracing experiments with stable isotope-labeled fatty acids, and oxidant challenge with hydrogen peroxide, we demonstrate the presence and redox-sensitive activity of FADS2, FADS1, and FADS5 in the mature RBC. Increases in PUFAs and HUFAs in human and mouse RBCs correlate negatively with storage hemolysis and positively with posttransfusion recovery. Inhibition of these enzymes decreases accumulation of free PUFAs and HUFAs in stored RBCs, concomitant to increases in pyruvate/lactate ratios. Alterations of this ratio in G6PD deficient patients or units supplemented with pyruvate-rich rejuvenation solutions corresponded to decreased PUFA and HUFA accumulation. Fatty acid desaturases are present and active in mature RBCs. Their activity is sensitive to oxidant stress, storage duration, and alterations of the pyruvate/lactate ratio. Show less

Interleukin-27 (IL-27) is a cytokine that suppresses human immunodeficiency virus (HIV)-1 infection in macrophages and is considered as an immunotherapeutic reagent for infectious diseases. It is reported that IL-27 suppresses autophagy in Mycobacterium tuberculosis-infected macrophages; however, a role for IL-27 on autophagy induction has been less studied. In this study, we investigated the impact of IL-27 in both autophagy induction and HIV-1 infection in macrophages. Primary human monocytes were differentiated into macrophages using human AB serum (huAB) alone, macrophage-colony stimulating factor (M-CSF) alone, or a combination of IL-27 with huAB or M-CSF. Electron microscopy and immunofluorescence staining demonstrated that a 20-fold increase in autophagosome formation was only detected in IL-27 + huAB-induced macrophages. Western blot analysis indicated that the autophagosome induction was not linked to either dephosphorylation of the mammalian target of rapamycin (mTOR) or lipidation of microtubule-associated protein 1A/1B-light chain 3 (LC3), an autophagosomal marker, implying that IL-27 can induce autophagy through a novel non-canonical pathway. Here we show for the first time that IL-27 induces autophagy during monocyte-to-macrophage differentiation in a subtype-dependent manner. Show less

Skeletal muscles are composed of hundreds of multinucleated muscle fibers (myofibers) whose myonuclei are regularly positioned all along the myofiber's periphery except the few ones clustered underneath the neuromuscular junction (NMJ) at the synaptic zone. This precise myonuclei organization is altered in different types of muscle disease, including centronuclear myopathies (CNMs). However, the molecular machinery regulating myonuclei position and organization in mature myofibers remains largely unknown. Conversely, it is also unclear how peripheral myonuclei positioning is lost in the related muscle diseases. Here, we describe the microtubule-associated protein, MACF1, as an essential and evolutionary conserved regulator of myonuclei positioning and maintenance, in cultured mammalian myotubes, in Show less

PromarkerD is a novel proteomics derived blood test for predicting diabetic kidney disease (DKD). The test is based on an algorithm that combines the measurement of three plasma protein biomarkers (CD5L, APOA4, and IBP3) with three clinical variables (age, HDL-cholesterol, and eGFR). The initial format of the assay used immunodepletion of plasma samples followed by targeted mass spectrometry (MRM-LCMS). The aim of this study was to convert the existing assay into an immunoaffinity approach compatible with higher throughput and robust clinical application. A newly optimised immunoaffinity-based assay was developed in a 96 well format with MRM measurements made using a low-flow LCMS method. The stability, reproducibility and precision of the assay was evaluated. A direct comparison between the immunoaffinity method and the original immunodepletion method was conducted on a 100-person cohort. Subsequently, an inter-lab study was performed of the optimised immunoaffinity method in two independent laboratories. Processing of plasma samples was greatly simplified by switching to an immunoaffinity bead capture method, coupled to a faster and more robust microflow LCMS system. Processing time was reduced from seven to two days and the chromatography reduced from 90 to 8 min. Biomarker stability by temperature and time difference treatments passed acceptance criteria. Intra/Inter-day test reproducibility and precision were within 11% CV for all biomarkers. PromarkerD test results from the new immunoaffinity method demonstrated excellent correlation (R = 0.96) to the original immunodepletion method. The immunoaffinity assay was successfully transferred to a second laboratory (R = 0.98) demonstrating the robustness of the methodology and ease of method transfer. An immunoaffinity capture targeted mass spectrometry assay was developed and optimised. It showed statistically comparable results to those obtained from the original immunodepletion method and was also able to provide comparable results when deployed to an independent laboratory. Taking a research grade assay and optimising to a clinical grade workflow provides insights into the future of multiplex biomarker measurement with an immunoaffinity mass spectrometry foundation. In the current format the PromarkerD immunoaffinity assay has the potential to make a significant impact on prediction of diabetic kidney disease with consequent benefit to patients. Show less

The palmitoyl acyltransferase (PAT) ZDHHC14 is highly expressed in the hippocampus and is the only PAT predicted to bind Type-I PDZ domain-containing proteins. However, ZDHHC14's neuronal roles are unknown. Here, we identify the PDZ domain-containing Membrane-associated Guanylate Kinase (MaGUK) PSD93 as a direct ZDHHC14 interactor and substrate. PSD93, but not other MaGUKs, localizes to the axon initial segment (AIS). Using lentiviral-mediated shRNA knockdown in rat hippocampal neurons, we find that ZDHHC14 controls palmitoylation and AIS clustering of PSD93 and also of Kv1 potassium channels, which directly bind PSD93. Neurodevelopmental expression of ZDHHC14 mirrors that of PSD93 and Kv1 channels and, consistent with ZDHHC14's importance for Kv1 channel clustering, loss of ZDHHC14 decreases outward currents and increases action potential firing in hippocampal neurons. To our knowledge, these findings identify the first neuronal roles and substrates for ZDHHC14 and reveal a previously unappreciated role for palmitoylation in control of neuronal excitability. Show less

Reduced insulin secretion results in hyperglycaemia and diabetes involving a complex aetiology that is yet to be fully elucidated. Genetic susceptibility is a key factor in beta cell dysfunction and hyperglycaemia but the responsible genes have not been defined. The Collaborative Cross (CC) is a recombinant inbred mouse panel with diverse genetic backgrounds allowing the identification of complex trait genes that are relevant to human diseases. The aim of this study was to identify and characterise genes associated with hyperglycaemia. Using an unbiased genome-wide association study, we examined random blood glucose and insulin sensitivity in 53 genetically unique mouse strains from the CC population. The influences of hyperglycaemia susceptibility quantitative trait loci (QTLs) were investigated by examining glucose tolerance, insulin secretion, pancreatic histology and gene expression in the susceptible mice. Expression of candidate genes and their association with insulin secretion were examined in human islets. Mechanisms underlying reduced insulin secretion were studied in MIN6 cells using RNA interference. Wide variations in blood glucose levels and the related metabolic traits (insulin sensitivity and body weight) were observed in the CC population. We showed that elevated blood glucose in the CC strains was not due to insulin resistance nor obesity but resulted from reduced insulin secretion. This insulin secretory defect was demonstrated to be independent of abnormalities in islet morphology, beta cell mass and pancreatic insulin content. Gene mapping identified the E2f8 (p = 2.19 × 10 Collectively, these findings suggest that E2F transcription factor 8 (E2F8) and discs large homologue 2 (DLG2) regulate insulin secretion. The CC resource enables the identification of E2f8 and Dlg2 as novel genes associated with hyperglycaemia due to reduced insulin secretion in pancreatic beta cells. Taken together, our results provide better understanding of the molecular control of insulin secretion and further support the use of the CC resource to identify novel genes relevant to human diseases. Show less