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Three-dimensional brain organoids have emerged as a valuable model system for studies of human brain development and pathology. Here we establish a midbrain organoid culture system to study the developmental trajectory from pluripotent stem cells to mature dopamine neurons. Using single cell RNA sequencing, we identify the presence of three molecularly distinct subtypes of human dopamine neurons with high similarity to those in developing and adult human midbrain. However, despite significant advancements in the field, the use of brain organoids can be limited by issues of reproducibility and incomplete maturation which was also observed in this study. We therefore designed bioengineered ventral midbrain organoids supported by recombinant spider-silk microfibers functionalized with full-length human laminin. We show that silk organoids reproduce key molecular aspects of dopamine neurogenesis and reduce inter-organoid variability in terms of cell type composition and dopamine neuron formation., 3D brain organoids have been used to investigate human brain development and pathology. Here the authors establish human ventral midbrain organoids coupled with single cell sequencing to study developing and mature dopamine neurons and use silk scaffolding to generate bioengineered brain organoids

Characterization of gene expression in pancreatic islets and its alteration in type 2 diabetes (T2D) are vital in understanding islet function and T2D pathogenesis. We leveraged RNA sequencing and genome-wide genotyping in islets from 188 donors to create the Islet Gene View (IGW) platform to make this information easily accessible to the scientific community. Expression data were related to islet phenotypes, diabetes status, other islet-expressed genes, islet hormone-encoding genes and for expression in insulin target tissues. The IGW web application produces output graphs for a particular gene of interest. In IGW, 284 differentially expressed genes (DEGs) were identified in T2D donor islets compared with controls. Forty percent of DEGs showed cell-type enrichment and a large proportion significantly co-expressed with islet hormone-encoding genes; glucagon (GCG, 56%), amylin (IAPP, 52%), insulin (INS, 44%), and somatostatin (SST, 24%). Inhibition of two DEGs,UNC5DandSERPINE2, impaired glucose-stimulated insulin secretion and impacted cell survival in a human β-cell model. The exploratory use of IGW could help designing more comprehensive functional follow-up studies and serve to identify therapeutic targets in T2D.

Huntington's disease is a neurodegenerative disorder caused by CAG expansions in the huntingtin (HTT) gene. Modelling Huntington's disease is challenging, as rodent and cellular models poorly recapitulate the disease as seen in ageing humans. To address this, we generated induced neurons through direct reprogramming of human skin fibroblasts, which retain age-dependent epigenetic characteristics. Huntington's disease induced neurons (HD-iNs) displayed profound deficits in autophagy, characterized by reduced transport of late autophagic structures from the neurites to the soma. These neurite-specific alterations in autophagy resulted in shorter, thinner and fewer neurites specifically in HD-iNs. CRISPRi-mediated silencing of HTT did not rescue this phenotype but rather resulted in additional autophagy alterations in control induced neurons, highlighting the importance of wild-type HTT in normal neuronal autophagy. In summary, our work identifies a distinct subcellular autophagy impairment in adult patient derived Huntington's disease neurons and provides a new rationale for future development of autophagy activation therapies.

Peer reviewed: True, Funder: New York Stem Cell Foundation; Id: http://dx.doi.org/10.13039/100003194, Funder: Parkinsonfonden; Id: http://dx.doi.org/10.13039/100008444, Funder: Lund University; Id: http://dx.doi.org/10.13039/501100003252, Funder: HORIZON EUROPE Marie Sklodowska-Curie Actions, Significant efforts are ongoing to develop refined differentiation protocols to generate midbrain dopamine (DA) neurons from pluripotent stem cells for application in disease modeling, diagnostics, drug screening and cell-based therapies for Parkinson's disease. An increased understanding of the timing and molecular mechanisms that promote the generation of distinct subtypes of human midbrain DA during development will be essential for guiding future efforts to generate molecularly defined and subtype-specific DA neurons from pluripotent stem cells. Here, we use droplet-based single-cell RNA sequencing to transcriptionally profile the developing human ventral midbrain (VM) when the DA neurons are generated (6-11 weeks post-conception) and their subsequent differentiation into functional mature DA neurons in primary fetal 3D organoid-like cultures. This approach reveals that 3D cultures are superior to monolayer conditions for their ability to generate and maintain mature DA neurons; hence, they have the potential to be used for studying human VM development. These results provide a unique transcriptional profile of the developing human fetal VM and functionally mature human DA neurons that can be used to guide stem cell-based therapies and disease modeling approaches in Parkinson's disease.

Partially unfolded alpha-lactalbumin forms the oleic acid complex HAMLET, with potent tumoricidal activity. Here we define a peptide-based molecular approach for targeting and killing tumor cells, and evidence of its clinical potential (ClinicalTrials.gov NCT03560479). A 39-residue alpha-helical peptide from alpha-lactalbumin is shown to gain lethality for tumor cells by forming oleic acid complexes (alpha1-oleate). Nuclear magnetic resonance measurements and computational simulations reveal a lipid core surrounded by conformationally fluid, alpha-helical peptide motifs. In a single center, placebo controlled, double blinded Phase I/II interventional clinical trial of non-muscle invasive bladder cancer, all primary end points of safety and efficacy of alpha1-oleate treatment are reached, as evaluated in an interim analysis. Intra-vesical instillations of alpha1-oleate triggers massive shedding of tumor cells and the tumor size is reduced but no drug-related side effects are detected (primary endpoints). Shed cells contain alpha1-oleate, treated tumors show evidence of apoptosis and the expression of cancer-related genes is inhibited (secondary endpoints). The results are especially encouraging for bladder cancer, where therapeutic failures and high recurrence rates create a great, unmet medical need., Partially unfolded alpha-lactalbumin forms an oleic acid complex with antitumorigenic properties. Here, the authors define a structurally flexible, peptide-based oleate complex and report a phase I/II clinical trial where this complex is used to treat patients with bladder cancer.

Diabetes & Metabolism - In Press.Proof corrected by the author Available online since jeudi 12 janvier 2017

Dysregulation of gene expression in islets from patients with type 2 diabetes (T2D) might be causally involved in the development of hyperglycemia, or it could develop as a consequence of hyperglycemia (i.e., glucotoxicity). To separate the genes that could be causally involved in pathogenesis from those likely to be secondary to hyperglycemia, we exposed islets from human donors to normal or high glucose concentrations for 24 h and analyzed gene expression. We compared these findings with gene expression in islets from donors with normal glucose tolerance and hyperglycemia (including T2D). The genes whose expression changed in the same direction after short-term glucose exposure, as in T2D, were considered most likely to be a consequence of hyperglycemia. Genes whose expression changed in hyperglycemia but not after short-term glucose exposure, particularly those that also correlated with insulin secretion, were considered the strongest candidates for causal involvement in T2D. For example, ERO1LB, DOCK10, IGSF11, and PRR14L were downregulated in donors with hyperglycemia and correlated positively with insulin secretion, suggesting a protective role, whereas TMEM132C was upregulated in hyperglycemia and correlated negatively with insulin secretion, suggesting a potential pathogenic role. This study provides a catalog of gene expression changes in human pancreatic islets after exposure to glucose.

Summary The current view of type 1 diabetes (T1D) is that it is an immune-mediated disease where lymphocytes infiltrate the pancreatic islets, promote killing of beta cells and cause overt diabetes. Although tissue resident immune cells have been demonstrated in several organs, the composition of lymphocytes in human healthy pancreatic islets have been scarcely studied. Here we aimed to investigate the phenotype of immune cells associated with human islets of non-diabetic organ donors. A flow cytometry analysis of isolated islets from perfused pancreases (n = 38) was employed to identify alpha, beta, T, natural killer (NK) and B cells. Moreover, the expression of insulin and glucagon transcripts was evaluated by RNA sequencing. Up to 80% of the lymphocytes were CD3+ T cells with a remarkable bias towards CD8+ cells. Central memory and effector memory phenotypes dominated within the CD8+ and CD4+ T cells and most CD8+ T cells were positive for CD69 and up to 50–70% for CD103, both markers of resident memory cells. The frequency of B and NK cells was low in most islet preparations (12 and 3% of CD45+ cells, respectively), and the frequency of alpha and beta cells varied between donors and correlated clearly with insulin and glucagon mRNA expression. In conclusion, we demonstrated the predominance of canonical tissue resident memory CD8+ T cells associated with human islets. We believe that these results are important to understand more clearly the immunobiology of human islets and the disease-related phenotypes observed in diabetes.

Latent autoimmune diabetes in adults (LADA) usually refers to GAD65 autoantibodies (GADAb)–positive diabetes with onset after 35 years of age and no insulin treatment within the first 6 months after diagnosis. However, it is not always easy to distinguish LADA from type 1 or type 2 diabetes. In this study, we examined whether metabolite profiling could help to distinguish LADA (n = 50) from type 1 diabetes (n = 50) and type 2 diabetes (n = 50). Of 123 identified metabolites, 99 differed between the diabetes types. However, no unique metabolite profile could be identified for any of the types. Instead, the metabolome varied along a C-peptide–driven continuum from type 1 diabetes via LADA to type 2 diabetes. LADA was more similar to type 2 diabetes than to type 1 diabetes. In a principal component analysis, LADA patients overlapping with type 1 diabetes progressed faster to insulin therapy than those overlapping with type 2 diabetes. In conclusion, we could not find any unique metabolite profile distinguishing LADA from type 1 and type 2 diabetes. Rather, LADA was metabolically an intermediate of type 1 and type 2 diabetes, with those patients closer to the former showing a faster progression to insulin therapy than those closer to the latter.

To study prevalence of diabetic retinopathy (DR) at diagnosis (DRAD) and to estimate contributing risk by sociodemographic, cardiovascular and metabolic characteristics present in patients recently diagnosed with type 2 diabetes (T2D) or latent autoimmune diabetes in the adult (LADA).Patients (n=2174) recently diagnosed T2D (93%) or LADA (7%) were included upon arrival for their baseline DR screening. Fundus photographs of 4902 eyes were graded by a senior ophthalmologist according to the International Diabetic Retinopathy Disease Severity Scale. Official registers held by Statistics Sweden provided sociodemographic variables. The National Patient Register and Swedish Prescribed Drug Register were used to assess cardiovascular risk. Beta cell function (HOMA2%b) and insulin sensitivity (HOMA2%s) were estimated from fasting (f) C-Peptide using the homeostasis model assessment (HOMA) 2 calculator. Odds ratios (OR) for DRAD were estimated using generalized estimating equation models.The prevalence of DRAD was 12% (7% mild and 5% moderate) and of diabetic macular edema it was 11% (all within vascular arch). The prevalence did not significantly differ between T2D and LADA. Due to sample size, the regression analysis of LADA patients did not yield any significant estimates. In T2D low educational level (≤9years) increased risk for DRAD by 44% (OR 1.44; 95% CI 1.07-1.93) and50% beta-cell function adjusted for HbA1c and insulin sensitivity at diagnosis increased the risk by 77% (OR 1.77; 95% CI 1.28-2.44). For every unit increase in BMI, risk for DRAD decreased by 3% (OR 0.97; 95% CI 0.95-0.99).DRAD prevalence in patients recently diagnosed with T2D or is 12%. Low educational level and low beta cell function at diagnosis are risk factors for DRAD. Estimation of beta cell function from (f)C-Peptide and (f)P-Glucose may be a valuable tool in identifying patients at risk for DRAD.

C4BP (C4b-binding protein) is a polymer of seven identical α chains and one unique β chain synthesized in liver and pancreas. We showed previously that C4BP enhances islet amyloid polypeptide (IAPP) fibril formation in vitro. Now we report that polymeric C4BP strongly inhibited lysis of human erythrocytes incubated with monomeric IAPP, whereas no lysis was observed after incubation with preformed IAPP fibrils. In contrast, incubation with the monomeric α-chain of C4BP was less effective. These data indicate that polymeric C4BP with multiple binding sites for IAPP neutralizes lytic activity of IAPP. Furthermore, addition of monomeric IAPP to a rat insulinoma cell line (INS-1) resulted in decreased cell viability, which was restored in the presence of physiological concentrations of C4BP. Treatment of INS-1 cells and primary rat islets with IAPP also diminished their ability to secrete insulin upon stimulation with glucose, which was reversed in the presence of C4BP. Further, C4BP was internalized together with IAPP into INS-1 cells. Pathway analyses of mRNA expression microarray data indicated that cells exposed to C4BP and IAPP in comparison with IAPP alone increased expression of genes involved in cholesterol synthesis. Depletion of cholesterol through methyl-β-cyclodextrin or cholesterol oxidase abolished the protective effect of C4BP on IAPP cytotoxicity of INS-1 cells. Also, inhibition of phosphoinositide 3-kinase but not NF-κB had a similar effect. Taken together, C4BP protects β-cells from IAPP cytotoxicity by modulating IAPP fibril formation extracellularly and also, after uptake by the cells, by enhancing cholesterol synthesis.

OBJECTIVE Smoking is an established risk factor for type 2 diabetes. In contrast, it has been proposed that smoking may reduce the risk of latent autoimmune diabetes in adults (LADA), but studies are scarce. We aimed to study the impact of smoking on LADA and type 2 diabetes risks. RESEARCH DESIGN AND METHODS We used data from a Swedish case-control study including incident case patients with LADA (GAD antibody [GADA] positive, n = 377) and type 2 diabetes (GADA negative, n = 1,188) and control subjects randomly selected from the population (n = 1,472). We calculated odds ratios (ORs) with 95% CIs by logistic regression, adjusted for age, sex, BMI, family history of diabetes, and alcohol consumption. RESULTS There was no indication of reduced risk of LADA in smokers; instead, heavy smoking was associated with an increased risk of LADA (OR 1.37, 95% CI 1.02–1.84). Heavy smokers had higher levels of HOMA of insulin resistance (9.89 vs. 4.38, P = 0.0479) and HOMA of β-cell function (55.7 vs. 42.5, P = 0.0204), but lower levels of GADA (75 vs. 250, P = 0.0445), compared with never smokers. Smokers also displayed an increased risk of type 2 diabetes (OR in ever smokers 1.53, 95% CI 1.25–1.88). CONCLUSIONS In this large population of LADA patients, we did not observe a protective effect of smoking on autoimmunity and the risk of LADA. A protective effect could possibly be masked by a smoking-induced aggravation of insulin resistance, akin to the diabetogenic effect seen in individuals with type 2 diabetes.

Cartilage oligomeric matrix protein (COMP) is a soluble pentameric protein expressed in cartilage and involved in collagen organization. Tissue microarrays derived from two cohorts of patients with breast cancer (n=122 and n=498) were immunostained, revealing varying expression of COMP, both in the tumor cells and surrounding stroma. High levels of COMP in tumor cells correlated, independently of other variables, with poor survival and decreased recurrence-free survival. Breast cancer cells, MDA-MB-231, stably expressing COMP were injected into the mammary fat pad of SCID (CB-17/Icr-Prkdcscid/Rj) mice. Tumors expressing COMP were significantly larger and were more prone to metastasize as compared with control, mock-transfected, tumors. In vitro experiments confirmed that COMP-expressing cells had a more invasive phenotype, which could in part be attributed to an upregulation of matrix metalloprotease-9. Furthermore, microarray analyses of gene expression in tumors formed in vivo showed that COMP expression induced higher expression of genes protecting against endoplasmic reticulum stress. This observation was confirmed in vitro as COMP-expressing cells showed better survival as well as a higher rate of protein synthesis when treated with brefeldin A, compared with control cells. Further, COMP-expressing cells appeared to undergo a metabolic switch, that is, a Warburg effect. Thus, in vitro measurement of cell respiration indicated decreased mitochondrial metabolism. In conclusion, COMP is a novel biomarker in breast cancer, which contributes to the severity of the disease by metabolic switching and increasing invasiveness and tumor cell viability, leading to reduced survival in animal models and human patients.

SUMMARY Monoamine and acetylcholine neurotransmitters from the autonomic nervous system (ANS) regulate insulin secretion in pancreatic islets. The molecular mechanisms controlling neurotransmitter signaling in islet β cells and their impact on diabetes development are only partially understood. Using a glucose-intolerant, MafA-deficient mouse model, we demonstrate that MAFA controls ANS-mediated insulin secretion by activating the transcription of nicotinic (ChrnB2 and ChrnB4) and adrenergic (Adra2A) receptor genes, which are integral parts of acetylcholine-and monoamine-signaling pathways. We show that acetylcholine-mediated insulin secretion requires nicotinic signaling and that nicotinic receptor expression is positively correlated with insulin secretion and glycemic control in human donor islets. Moreover, polymorphisms spanning MAFA-binding regions within the human CHRNB4 gene are associated with type 2 diabetes. Our data show that MAFA transcriptional activity is required for establishing β cell sensitivity to neurotransmitter signaling and identify nicotinic signaling as a modulator of insulin secretion impaired in type 2 diabetes., Graphical abstract

Summary Background Diabetes is presently classified into two main forms, type 1 and type 2 diabetes, but type 2 diabetes in particular is highly heterogeneous. A refined classification could provide a powerful tool to individualise treatment regimens and identify individuals with increased risk of complications at diagnosis. Methods We did data-driven cluster analysis (k-means and hierarchical clustering) in patients with newly diagnosed diabetes (n=8980) from the Swedish All New Diabetics in Scania cohort. Clusters were based on six variables (glutamate decarboxylase antibodies, age at diagnosis, BMI, HbA 1c , and homoeostatic model assessment 2 estimates of β-cell function and insulin resistance), and were related to prospective data from patient records on development of complications and prescription of medication. Replication was done in three independent cohorts: the Scania Diabetes Registry (n=1466), All New Diabetics in Uppsala (n=844), and Diabetes Registry Vaasa (n=3485). Cox regression and logistic regression were used to compare time to medication, time to reaching the treatment goal, and risk of diabetic complications and genetic associations. Findings We identified five replicable clusters of patients with diabetes, which had significantly different patient characteristics and risk of diabetic complications. In particular, individuals in cluster 3 (most resistant to insulin) had significantly higher risk of diabetic kidney disease than individuals in clusters 4 and 5, but had been prescribed similar diabetes treatment. Cluster 2 (insulin deficient) had the highest risk of retinopathy. In support of the clustering, genetic associations in the clusters differed from those seen in traditional type 2 diabetes. Interpretation We stratified patients into five subgroups with differing disease progression and risk of diabetic complications. This new substratification might eventually help to tailor and target early treatment to patients who would benefit most, thereby representing a first step towards precision medicine in diabetes. Funding Swedish Research Council, European Research Council, Vinnova, Academy of Finland, Novo Nordisk Foundation, Scania University Hospital, Sigrid Juselius Foundation, Innovative Medicines Initiative 2 Joint Undertaking, Vasa Hospital district, Jakobstadsnejden Heart Foundation, Folkhalsan Research Foundation, Ollqvist Foundation, and Swedish Foundation for Strategic Research.

Islet produced 5-hydroxy tryptamine (5-HT) is suggested to regulate islet hormone secretion in a paracrine and autocrine manner in rodents. Hitherto, no studies demonstrate a role for this amine in human islet function, nor is it known if 5-HT signaling is involved in the development of beta cell dysfunction in type 2 diabetes (T2D). To clarify this, we performed a complete transcriptional mapping of 5-HT receptors and processing enzymes in human islets and investigated differential expression of these genes in non-diabetic and T2D human islet donors. We show the expression of fourteen 5-HT receptors as well as processing enzymes involved in the biosynthesis of 5-HT at the mRNA level in human islets. Two 5-HT receptors (HTR1D and HTR2A) were over-expressed in T2D islet donors. Both receptors (5-HT1d and 5-HT2a) were localized to human alpha, beta and delta cells. 5-HT inhibited both insulin and glucagon secretion in non-diabetic islet donors. In islets isolated from T2D donors the amine significantly increased release of insulin in response to glucose. Our results suggest that 5-HT signaling participates in regulation of overall islet hormone secretion in non- diabetic individuals and over-expression of HTR1D and HTR2A may either contribute to islet dysfunction in T2D or arise as a consequence of an already dysfunctional islet.

Background. - A family history of diabetes (FHD) is a strong predictor of diabetes risk, yet has rarely been investigated in latent autoimmune diabetes in adults (LADA). This study therefore investigated the risk of LADA and type 2 diabetes (T2D) in relation to FHD, taking into account the type of diabetes in relatives. Methods. - Data from a population-based study were used, including incident cases of LADA [glutamic acid decarboxylase antibody (GADA)-positive, n = 378] and T2D (GADA-negative, n = 1199), and their matched controls (n = 1484). First-degree relatives with disease onset at age

Ca 2+ ‐sensor proteins are generally implicated in insulin release through SNARE interactions. Here, secretagogin, whose expression in human pancreatic islets correlates with their insulin content and the incidence of type 2 diabetes, is shown to orchestrate an unexpectedly distinct mechanism. Single‐cell RNA‐seq reveals retained expression of the TRP family members in β‐cells from diabetic donors. Amongst these, pharmacological probing identifies Ca 2+ ‐permeable transient receptor potential vanilloid type 1 channels (TRPV1) as potent inducers of secretagogin expression through recruitment of Sp1 transcription factors. Accordingly, agonist stimulation of TRPV1s fails to rescue insulin release from pancreatic islets of glucose intolerant secretagogin knock‐out( −/− ) mice. However, instead of merely impinging on the SNARE machinery, reduced insulin availability in secretagogin −/− mice is due to β‐cell loss, which is underpinned by the collapse of protein folding and deregulation of secretagogin‐dependent USP9X deubiquitinase activity. Therefore, and considering the desensitization of TRPV1s in diabetic pancreata, a TRPV1‐to‐secretagogin regulatory axis seems critical to maintain the structural integrity and signal competence of β‐cells.

Genetic variation can modulate gene expression, and thereby phenotypic variation and susceptibility to complex diseases such as type 2 diabetes (T2D). Here we harnessed the potential of DNA and RNA sequencing in human pancreatic islets from 89 deceased donors to identify genes of potential importance in the pathogenesis of T2D. We present a catalog of genetic variants regulating gene expression (eQTL) and exon use (sQTL), including many long noncoding RNAs, which are enriched in known T2D-associated loci. Of 35 eQTL genes, whose expression differed between normoglycemic and hyperglycemic individuals, siRNA of tetraspanin 33 (TSPAN33), 5'-nucleotidase, ecto (NT5E), transmembrane emp24 protein transport domain containing 6 (TMED6), and p21 protein activated kinase 7 (PAK7) in INS1 cells resulted in reduced glucose-stimulated insulin secretion. In addition, we provide a genome-wide catalog of allelic expression imbalance, which is also enriched in known T2D-associated loci. Notably, allelic imbalance in paternally expressed gene 3 (PEG3) was associated with its promoter methylation and T2D status. Finally, RNA editing events were less common in islets than previously suggested in other tissues. Taken together, this study provides new insights into the complexity of gene regulation in human pancreatic islets and better understanding of how genetic variation can influence glucose metabolism.

Diabetes mellitus is a lifelong, incapacitating disease affecting multiple organs. Presently, type 2 diabetes (T2D) can neither be prevented nor cured and the disease is associated with devastating chronic complications. These complications impose an immense burden on the quality of life of patients and account for about 12% of direct health care costs in Europe. Genetic analysis will increase our understanding of this heterogeneous disease and may help offer more personalized treatment.

Adhesion G protein-coupled receptor (GPCR)-G1 (ADGRG1) is the most abundant GPCR in human pancreatic islets, but its role in islet function is unclear.Investigate how ADGRG1 expression and activation by its ligand, collagen III, impacts β-cell function in normal and type 2 diabetic (T2D) islets.Genes associated with the ADGRG1 in human islets was probed by RNA-sequencing of human pancreatic islet isolated from cadaveric donors, followed by functional studies on β-cell proliferation, apoptosis, and insulin secretion in human and mouse islets and in INS-1 cells.Changes in β-cell gene expression, proliferation, apoptosis, and insulin secretion were quantified by RNA-sequencing, qPCR, Thymidine incorporation, Western blotting, and RIA, respectively.ADGRG1 is the most abundant GPCR mRNA in both human and mouse islets, and its expression in human islets strongly correlates with genes important for β-cell function and T2D risk. The expression of ADGRG1 was reduced in islets of T2D donors, in db/db mouse islets, and in isolated human islets exposed to chronic hyperglycemia. Beneficial effects of collagen type III on β-cell function via activation of the cAMP/protein kinase A pathway, suppression of RhoA and caspase-3 activity, increased β-cell viability, and proliferation were abolished when ADGRG1 was down-regulated in β-cells.We demonstrate a mechanistic link between ADGRG1 expression and β-cell function. Pharmacological agents that promote expression or activation of the ADGRG1 receptor may represent a novel approach for the treatment of T2D.

Human α-lactalbumin made lethal to tumor cells (HAMLET) is the first member in a new family of protein–lipid complexes that kills tumor cells with high selectivity. The protein component of HAMLET is α-lactalbumin, which in its native state acts as a substrate specifier in the lactose synthase complex, thereby defining a function essential for the survival of lactating mammals. In addition, α-lactalbumin acquires tumoricidal activity after partial unfolding and binding to oleic acid. The lipid cofactor serves the dual role as a stabilizer of the altered fold of the protein and a coactivator of specific steps in tumor cell death. HAMLET is broadly tumoricidal, suggesting that the complex identifies conserved death pathways suitable for targeting by novel therapies. Sensitivity to HAMLET is defined by oncogene expression including Ras and c-Myc and by glycolytic enzymes. Cellular targets are located in the cytoplasmic membrane, cytoskeleton, mitochondria, proteasomes, lysosomes and nuclei, and specific signaling pathways are rapidly activated, first by interactions of HAMLET with the cell membrane and subsequently after HAMLET internalization. Therapeutic effects of HAMLET have been demonstrated in human skin papillomas and bladder cancers, and HAMLET limits the progression of human glioblastomas, with no evidence of toxicity for normal brain or bladder tissue. These findings open up new avenues for cancer therapy and the understanding of conserved death responses in tumor cells.

To access publisher's full text version of this article click on the hyperlink at the bottom of the page We performed fine mapping of 39 established type 2 diabetes (T2D) loci in 27,206 cases and 57,574 controls of European ancestry. We identified 49 distinct association signals at these loci, including five mapping in or near KCNQ1. 'Credible sets' of the variants most likely to drive each distinct signal mapped predominantly to noncoding sequence, implying that association with T2D is mediated through gene regulation. Credible set variants were enriched for overlap with FOXA2 chromatin immunoprecipitation binding sites in human islet and liver cells, including at MTNR1B, where fine mapping implicated rs10830963 as driving T2D association. We confirmed that the T2D risk allele for this SNP increases FOXA2-bound enhancer activity in islet- and liver-derived cells. We observed allele-specific differences in NEUROD1 binding in islet-derived cells, consistent with evidence that the T2D risk allele increases islet MTNR1B expression. Our study demonstrates how integration of genetic and genomic information can define molecular mechanisms through which variants underlying association signals exert their effects on disease. cademy of Finland 77299 102318 10493 118065 123885 124243 129293 129680 136895 139635 211119 213506 251217 263836 Agence National de la Recherche Association de Langue Francaise pour l'Etude du Diabete et des Maladies Metaboliques Association Diabeete Risque Vasculaire Association Francaise des Diabetiques Association of Danish Pharmacies Augustinus Foundation Becket Foundation British Diabetes Association (BDA) Research British Heart Foundation Central Norway Health Authority Central Finland Hospital District Center for Inherited Disease Research (CIDR) City of Kuopio City of Leutkirch Copenhagen County Danish Centre for Evaluation and Health Technology Assessment Danish Council for Independent Research Danish Heart Foundation Danish Research Councils Deutsche Forschungsgemeinschaft ER 155/6-2 Diabetes Research Foundation Diabetes UK Doris Duke Charitable Foundation Erasmus Medical Center Erasmus University Estonian government SF0180142s08 European Commission ENGAGE HEALTH-F4-2007-201413 FP7-201413 FP7-245536 EXGENESIS LSHM-CT-2004-005272 FP6 LSHM_CT_2006_037197 LSHM-CT-2007-037273 C-Public Health 2004310 European Regional Development Fund Federal Ministry of Education and Research, Germany FKZ 01GI1128 FKZ 01EO1001 Federal Ministry of Health, Germany Finnish Diabetes Association Finnish Diabetes Research Foundation Finnish Foundation for Cardiovascular Research Finnish Medical Society Folkhalsan Research Foundation Foundation for Life and Health in Finland Foundation for Old Servants Fredrick och Ingrid Thuring Foundation French region of Nord-Pas-de-Calais (Contrat de Projets Etat-Region) German Center for Diabetes Research German Research Council GRK1041 German National Genome Research Network Groupe d'Etude des Maladies Metaboliques et Systemiques Health Care Centers in Vasa, Narpes and Korsholm, Finland Health Foundation Heinz Nixdorf Foundation Helmholtz Zentrum Munchen Helsinki University Central Hospital Research Foundation Hospital District of Southwest Finland Ib Henriksens Foundation IngaBritt and Arne Lundberg's Research Foundation 359 Karolinska Institutet Knut and Alice Wallenberg Foundation KAW 2009.0243 Kuopio University Hospital Lundbeck Foundation Magnus Bergvall Foundation Medical Faculty of University Duisburg-Essen Medical Research Council, UK G0000649 G0601261 Ministry for Health, Welfare and Sports, the Netherlands Ministry of Education and Culture, Finland 722 627 Ministry of Education, Culture and Science, the Netherlands Ministry of Health and Prevention, Denmark Ministry of Social Affairs and Health, Finland Ministry of Innovation, Science, Research and Technology of North Rhine-Westphalia, Germany Munich Center of Health Sciences Municipal Health Care Center and Hospital in Jakobstad, Finland municipality of Rotterdam, the Netherlands Narpes Health Care Foundation National Health Screening Service of Norway National Heart, Lung, and Blood Institute, USA HHSN268201100005C HHSN268201100006C HHSN268201100007C HHSN268201100008C HHSN268201100009C HHSN268201100010C HHSN268201100011C HHSN268201100012C N01HC25195 N02HL64278 R01HL087641 R01HL59367 R01HL086694 National Human Genome Research Institute, USA U01HG004402 N01HG65403 National Institute for Diabetes and Digestive and Kidney Diseases, USA R01DK078616 U01DK085526 K24DK080140 R01DK073490 National Institute for Health and Welfare, Finland National Institutes of Health, USA HHSN268200625226C UL1RR025005 R01DK062370 R01DK072193 1Z01HG000024 AG028555 AG08724 AG04563 AG10175 AG08861 U01HG004399 DK58845 CA055075 DK085545 DK098032 Netherlands Genomics Initiative Netherlands Organisation for Health Research and Development Netherlands Organisation of Scientific Research NOW Investments 175.010.2005.011 911-03-012 050-060-810 Nord-Trondelag County Council Nordic Center of Excellence in Disease Genetics Norwegian Institute of Public Health Norwegian Research Council Novo Nordisk Foundation Ollquist Foundation Oxford National Institute for Health Research (NIHR) Biomedical Research Centre Paavo Nurmi Foundation Paivikki and Sakari Sohlberg Foundation Perklen Foundation Pirkanmaa Hospital District, Finland Programme Hospitalier de Recherche Clinique Programme National de Recherche sur la Diabete Research Institute for Diseases in the Elderly 014-93-015 Robert Dawson Evans Endowment, Department of Medicine, Boston University School of Medicine and Boston Medical Center Royal Swedish Academy of Sciences Sarstedt, Germany Signe and Ane Gyllenberg Foundation Sigrid Juselius Foundation Slottery Machine Association, Finland Social Insurance Institution of Finland South OstroBothnia Hospital District state of Baden-Wurttemberg, Germany Stockholm County Council 560183 Swedish Cultural Foundation, Finland Swedish Diabetes Foundation Swedish e-science Research Center Swedish Foundation for Strategic Research Swedish Heart-Lung Foundation Swedish Research Council SFO EXODIAB 2009-1039 521-2010-3490 521-2007-4037 521-2008-2974 ANDIS 825-2010-5983 LUDC 349-2008-6589 8691 Swedish Society of Medicine Tore Nilsson Foundation Torsten and Ragnar Soderbergs Stiftelser MT33/09 University Hospital Essen University of Tromso University College London NIHR Biomedical Research Centre UK NIHR Cambridge Biomedical Research Centre Uppsala University Uppsala University Hospital Vaasa Hospital District Velux Foundation Wellcome Trust GR072960 076113 083948 090367 090532 083270 086596 098017 095101 098051 098381

Our aim was to investigate the association between birthweight and latent autoimmune diabetes in adults (LADA), a common diabetes form with features of both type 1 and type 2 diabetes. We used data from the Epidemiological Study of Risk Factors for LADA and Type 2 Diabetes (ESTRID), a Swedish population-based study. Eligible for the analysis were 134 incident LADA cases (glutamic acid decarboxylase antibody [GADA] positive), 350 incident type 2 diabetes cases (GADA negative) and 603 randomly selected controls. We present ORs and 95% CIs for LADA and type 2 diabetes in relation to birthweight, adjusted for sex, age, BMI and family history of diabetes. Low birthweight increased the risk of LADA as well as the risk of type 2 diabetes; OR per kg reduction was estimated as 1.52 (95% CI 1.12, 2.08) and 1.58 (1.23, 2.04), respectively. The OR for participants weighing

Although several molecular pathways have been linked to type 2 diabetes (T2D) pathogenesis, it is uncertain which pathway has the most implication on the disease. Changes in the expression of an entire pathway might be more important for disease pathogenesis than changes in the expression of individual genes. To identify the molecular alterations in T2D, DNA microarrays of human pancreatic islets from donors with hyperglycemian=20and normoglycemian=58were subjected to Gene Set Enrichment Analysis (GSEA). About 178 KEGG pathways were investigated for gene expression changes between hyperglycemic donors compared to normoglycemic. Pathway enrichment analysis showed that type II diabetes mellitus (T2DM) and maturity onset diabetes of the young (MODY) pathways are downregulated in hyperglycemic donors, while proteasome and spliceosome pathways are upregulated. The mean centroid of gene expression of T2DM and MODY pathways was shown to be associated positively with insulin secretion and negatively with HbA1c level. To conclude, downregulation of T2DM and MODY pathways is involved in islet function and might be involved in T2D. Also, the study demonstrates that gene expression profiles from pancreatic islets can reveal some of the biological processes related to regulation of glucose hemostats and diabetes pathogenesis.

We have previously identified transcription factor B1 mitochondrial (TFB1M) as a type 2 diabetes (T2D) risk gene, using human and mouse genetics. To further understand the function of TFB1M and how it is associated with T2D, we created a beta-cell-specific knockout of Tfb1m, which gradually developed diabetes. Prior to the onset of diabetes, beta-Tfb1m(-/-) mice exhibited retarded glucose clearance owing to impaired insulin secretion. beta-Tfb1m(-/-) islets released less insulin in response to fuels, contained less insulin and secretory granules and displayed reduced beta-cell mass. Moreover, mitochondria in Tfb1m-deficient beta-cells were more abundant with disrupted architecture. TFB1M is known to control mitochondrial protein translation by adenine dimethylation of 12S ribosomal RNA (rRNA). Here, we found that the levels of TFB1M and mitochondrial-encoded proteins, mitochondrial 12S rRNA methylation, ATP production and oxygen consumption were reduced in beta-Tfb1m(-/-) islets. Furthermore, the levels of reactive oxygen species (ROS) in response to cellular stress were increased whereas induction of defense mechanisms was attenuated. We also show increased apoptosis and necrosis as well as infiltration of macrophages and CD4(+) cells in the islets. Taken together, our findings demonstrate that Tfb1m-deficiency in beta-cells caused mitochondrial dysfunction and subsequently diabetes owing to combined loss of beta-cell function and mass. These observations reflect pathogenetic processes in human islets: using RNA sequencing, we found that the TFB1M risk variant exhibited a negative gene-dosage effect on islet TFB1M mRNA levels, as well as insulin secretion. Our findings highlight the role of mitochondrial dysfunction in impairments of beta-cell function and mass, the hallmarks of T2D.

The normal flora furnishes the host with ecological barriers that prevent pathogen attack while maintaining tissue homeostasis. Urinary tract infections (UTIs) constitute a highly relevant model of microbial adaptation in which some patients infected with Escherichia coil develop acute pyelonephritis, while other patients with bacteriuria exhibit an asymptomatic carrier state similar to bacterial commensalism. It remains unclear if the lack of destructive inflammation merely reflects low virulence or if carrier strains actively inhibit disease-associated responses in the host. Here, we identify a new mechanism of bacterial adaptation through broad suppression of RNA polymerase II-dependent (Pol II-dependent) host gene expression. Over 60% of all genes were suppressed 24 hours after human inoculation with the prototype asymptomatic bacteriuria (ABU) strain E. coil 83972, and inhibition was verified by infection of human cells. Specific repressors and activators of Pol II-dependent transcription were modified, Pol II phosphorylation was inhibited, and pathogen-specific signaling was suppressed in cell lines and inoculated patients. An increased frequency of strains inhibiting Pol II was epidemiologically verified in ABU and fecal strains compared with acute pyelonephritis, and a Pol II antagonist suppressed the disease-associated host response. These results suggest that by manipulating host gene expression, ABU strains promote tissue integrity while inhibiting pathology. Such bacterial modulation of host gene expression may be essential to sustain asymptomatic bacterial carriage by ensuring that potentially destructive immune activation will not occur.

Long-chain fatty acids are internalized by receptor-mediated mechanisms or receptor-independent diffusion across cytoplasmic membranes and are utilized as nutrients, building blocks, and signaling intermediates. Here we describe how the association of long-chain fatty acids to a partially unfolded, extracellular protein can alter the presentation to target cells and cellular effects. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin and oleic acid (OA). As OA lacks independent tumoricidal activity at concentrations equimolar to HAMLET, the contribution of the lipid has been debated. We show by natural abundance (13)C NMR that the lipid in HAMLET is deprotonated and by chromatography that oleate rather than oleic acid is the relevant HAMLET constituent. Compared with HAMLET, oleate (175 μm) showed weak effects on ion fluxes and gene expression. Unlike HAMLET, which causes metabolic paralysis, fatty acid metabolites were less strongly altered. The functional overlap increased with higher oleate concentrations (500 μm). Cellular responses to OA were weak or absent, suggesting that deprotonation favors cellular interactions of fatty acids. Fatty acids may thus exert some of their essential effects on host cells when in the deprotonated state and when presented in the context of a partially unfolded protein.

Ion channels and ion fluxes control many aspects of tissue homeostasis. During oncogenic transformation, critical ion channel functions may be perturbed but conserved tumor specific ion fluxes remain to be defined. Here we used the tumoricidal protein-lipid complex HAMLET as a probe to identify ion fluxes involved in tumor cell death. We show that HAMLET activates a non-selective cation current, which reached a magnitude of 2.74±0.88 nA within 1.43±0.13 min from HAMLET application. Rapid ion fluxes were essential for HAMLET-induced carcinoma cell death as inhibitors (amiloride, BaCl2), preventing the changes in free cellular Na(+) and K(+) concentrations also prevented essential steps accompanying carcinoma cell death, including changes in morphology, uptake, global transcription, and MAP kinase activation. Through global transcriptional analysis and phosphorylation arrays, a strong ion flux dependent p38 MAPK response was detected and inhibition of p38 signaling delayed HAMLET-induced death. Healthy, differentiated cells were resistant to HAMLET challenge, which was accompanied by innate immunity rather than p38-activation. The results suggest, for the first time, a unifying mechanism for the initiation of HAMLET's broad and rapid lethal effect on tumor cells. These findings are particularly significant in view of HAMLET's documented therapeutic efficacy in human studies and animal models. The results also suggest that HAMLET offers a two-tiered therapeutic approach, killing cancer cells while stimulating an innate immune response in surrounding healthy tissues.

HAMLET is the first member of a new family of tumoricidal protein-lipid complexes that kill cancer cells broadly, while sparing healthy, differentiated cells. Many and diverse tumor cell types are sensitive to the lethal effect, suggesting that HAMLET identifies and activates conserved death pathways in cancer cells. Here we investigated the molecular basis for the difference in sensitivity between cancer cells and healthy cells. Using a combination of small hairpin RNA inhibition, proteomic and metabolomic technology we identified the c-Myc oncogene as one essential determinant of HAMLET sensitivity. Increased c-Myc expression levels promoted the sensitivity to HAMLET and shRNA knockdown of c-Myc suppressed the lethal response, suggesting that oncogenic transformation with c-Myc creates a HAMLET-sensitive phenotype. Furthermore, the HAMLET sensitivity was modified by the glycolytic state of the tumor cells. Glucose deprivation sensitized tumor cells to HAMLET-induced cell death and in the shRNA screen Hexokinase 1, PFKFB1 and HIF1α modified HAMLET sensitivity. Hexokinase 1 was shown to bind HAMLET in a protein array containing approximately 8000 targets and Hexokinase activity decreased within 15 minutes of HAMLET treatment, prior to morphological signs of tumor cell death. In parallel, HAMLET triggered rapid metabolic paralysis in carcinoma cells. The glycolytic machinery was modified and glycolysis was shifted towards the pentose phosphate pathway. Tumor cells were also shown to contain large amounts of oleic acid and its derivatives already after 15 minutes. The results identify HAMLET as a novel anti-cancer agent that kills tumor cells by exploiting unifying features of cancer cells such as oncogene-addiction or the Warburg effect.

The mucosal immune system identifies and fights invading pathogens, while allowing non-pathogenic organisms to persist. Mechanisms of pathogen/non-pathogen discrimination are poorly understood, as is the contribution of human genetic variation in disease susceptibility. We describe here a new, IRF3-dependent signaling pathway that is critical for distinguishing pathogens from normal flora at the mucosal barrier. Following uropathogenic E. coli infection, Irf3−/− mice showed a pathogen-specific increase in acute mortality, bacterial burden, abscess formation and renal damage compared to wild type mice. TLR4 signaling was initiated after ceramide release from glycosphingolipid receptors, through TRAM, CREB, Fos and Jun phosphorylation and p38 MAPK-dependent mechanisms, resulting in nuclear translocation of IRF3 and activation of IRF3/IFNβ-dependent antibacterial effector mechanisms. This TLR4/IRF3 pathway of pathogen discrimination was activated by ceramide and by P-fimbriated E. coli, which use ceramide-anchored glycosphingolipid receptors. Relevance of this pathway for human disease was supported by polymorphic IRF3 promoter sequences, differing between children with severe, symptomatic kidney infection and children who were asymptomatic bacterial carriers. IRF3 promoter activity was reduced by the disease-associated genotype, consistent with the pathology in Irf3−/− mice. Host susceptibility to common infections like UTI may thus be strongly influenced by single gene modifications affecting the innate immune response., Author Summary The host immune system must identify pathogens and defeat them through TLR-dependent signaling pathway activation, while distinguishing them from commensal flora. Contrary to current dogma, the host cannot solely use “pattern recognition” since the microbial molecules involved in such recognition are present on pathogens and commensals alike. We identify here a pathogen-specific mechanism of TLR4 activation and signaling intermediates in this pathway, leading to IRF3-dependent transcription of innate immune response genes. We show in knockout mice that Irf3 deficiency causes severe tissue pathology and that effector functions controlled by IFNβ are involved. Finally, in highly disease-prone pyelonephritis patients we found a high frequency of IRF3 promoter polymorphism compared to asymptomatic bacterial carriers or controls. The polymorphisms influenced promoter activity in reporter assays, suggesting that they are functionally important. Urinary tract infections are among the most common bacterial infections in man, and are a major cause of morbidity and mortality. A subset of disease-prone individuals is at risk for recurrent disease, severe renal dysfunction and end-stage renal disease. At present, there is no method to identify disease-prone infants and to prevent future morbidity and renal damage. The genetic and functional studies described here indicate that genetic variation in IRF3 influences individual susceptibility to kidney infection and might serve as a new tool for future risk assessment in this patient group.

By changing the three-dimensional structure, a protein can attain new functions, distinct from those of the native protein. Amyloid-forming proteins are one example, in which conformational change may lead to fibril formation and, in many cases, neurodegenerative disease. We have proposed that partial unfolding provides a mechanism to generate new and useful functional variants from a given polypeptide chain. Here we present HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) as an example where partial unfolding and the incorporation of cofactor create a complex with new, beneficial properties. Native alpha-lactalbumin functions as a substrate specifier in lactose synthesis, but when partially unfolded the protein binds oleic acid and forms the tumoricidal HAMLET complex. When the properties of HAMLET were first described they were surprising, as protein folding intermediates and especially amyloid-forming protein intermediates had been regarded as toxic conformations, but since then structural studies have supported functional diversity arising from a change in fold. The properties of HAMLET suggest a mechanism of structure-function variation, which might help the limited number of human protein genes to generate sufficient structural diversity to meet the diverse functional demands of complex organisms.

In situ synthesis of oestrogens is of great importance in the development and progression of breast cancer. 17beta-hydroxysteroid dehydrogenase (17HSD) type 2 catalyses oxidation from oestradiol to oestrone, and thereby protects the breast epithelial cells from oestradiol. Low expression of 17HSD type 2 has been associated with decreased survival in breast cancer, but no studies have investigated the mechanism behind the low expression. The 17HSD type 2 gene (HSD17B2) was screened for mutations with Single Stranded Conformation Polymorphism (SSCP)-DNA sequencing in 59 sporadic breast cancer cases, 19 hereditary breast cancer cases and seven breast cancer cell lines. DNA samples from 226 healthy individuals were used to identify if changes were previously unknown polymorphisms. No mutation was detected and therefore mutations in HSD17B2 do not explain why some breast tumours exhibit low 17HSD type 2 expression. However, a previously unknown polymorphism was found in exon four (Met226Val). Using molecular modelling, we found that the substituted residue is located at the outer part of the steroid binding site, probably causing minor alterations in the substrate binding. We further studied if the polymorphism contributes to breast cancer susceptibility in a larger material, but did not find an increased risk in the group of 317 sporadic breast cancer patients, 188 breast cancer patients with two close relatives with breast cancer or 122 hereditary breast cancer patients, compared to the healthy control group. We suggest that the detected polymorphism does not contribute to a higher risk of developing breast cancer.

Infection remains a significant cause of morbidity and mortality especially in newborn infants. Analytical methods for diagnosing infection are severely limited in terms of sensitivity and specificity and require relatively large samples. It is proposed that stringent regulation of the human transcriptome affords a new molecular diagnostic approach based on measuring a highly specific systemic inflammatory response to infection, detectable at the RNA level. This proposition raises a number of as yet poorly characterised technical and biological variation issues that urgently need to be addressed. Here we report a quantitative assessment of methodological approaches for processing and extraction of RNA from small samples of infant whole blood and applying analysis of variation from biochip measurements. On the basis of testing and selection from a battery of assays we show that sufficient high quality RNA for analysis using multiplex array technology can be obtained from small neonatal samples. These findings formed the basis of implementing a set of robust clinical and experimental standard operating procedures for whole blood RNA samples from 58 infants. Modelling and analysis of variation between samples revealed significant sources of variation from the point of sample collection to processing and signal generation. These experiments further permitted power calculations to be run indicating the tractability and requirements of using changes in RNA expression profiles to detect different states between patient groups. Overall the results of our investigation provide an essential first step toward facilitating an alternative way for diagnosing infection from very small neonatal blood samples, providing methods and requirements for future chip-based studies.

SummaryType 2 diabetes is triggered by reduced insulin production, caused by genetic and environmental factors such as inflammation originating from the innate immune system. Complement proteins are a component of innate immunity and kill non-self cells by perforating the plasma membrane, a reaction prevented by CD59. Human pancreatic islets express CD59 at very high levels. CD59 is primarily known as a plasma membrane protein in membrane rafts, but most CD59 protein in pancreatic β cells is intracellular. Removing extracellular CD59 disrupts membrane rafts and moderately stimulates insulin secretion, whereas silencing intracellular CD59 markedly suppresses regulated secretion by exocytosis, as demonstrated by TIRF imaging. CD59 interacts with the exocytotic proteins VAMP2 and Syntaxin-1. CD59 expression is reduced by glucose and in rodent diabetes models but upregulated in human diabetic islets, potentially reflecting compensatory reactions. This unconventional action of CD59 broadens the established view of innate immunity in type 2 diabetes.