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OBJECTIVE: Abnormal expression of the hepatic gluconeogenic genes (glucose-6-phosphatase [G6Pase] and PEPCK) contributes to hyperglycemia. These genes are repressed by insulin,but this process is defective in diabetic subjects. Protein kinase B (PKB) is implicated in this action of insulin. An inhibitor of PKB,Akt inhibitor (Akti)-1/2,was recently reported; however,the specificity and efficacy against insulin-induced PKB was not reported. Our aim was to characterize the specificity and efficacy of Akti-1/2 in cells exposed to insulin and then establish whether inhibition of PKB is sufficient to prevent regulation of hepatic gene expression by insulin. RESEARCH DESIGN AND METHODS: Akti-1/2 was assayed against 70 kinases in vitro and its ability to block PKB activation in cells exposed to insulin fully characterized. RESULTS: Akti-1/2 exhibits high selectivity toward PKBalpha and PKBbeta. Complete inhibition of PKB activity is achieved in liver cells incubated with 1-10 mumol/l Akti-1/2,and this blocks insulin regulation of PEPCK and G6Pase expression. Our data demonstrate that only 5-10% of maximal insulin-induced PKB is required to fully repress PEPCK and G6Pase expression. Finally,we demonstrate reduced insulin sensitivity of these gene promoters in cells exposed to submaximal concentrations of Akti-1/2; however,full repression of the genes can still be achieved by high concentrations of insulin. CONCLUSIONS: This work establishes the requirement for PKB activity in the insulin regulation of PEPCK,G6Pase,and a third insulin-regulated gene,IGF-binding protein-1 (IGFBP1); suggests a high degree of functional reserve; and identifies Akti-1/2 as a useful tool to delineate PKB function in the liver. View Publication

Murine hybridomas producing neutralizing mAbs specific to the pandemic influenza virus A/California/07/2009 haemagglutinin (HA) were isolated. These antibodies recognized at least two different but overlapping new epitopes that were conserved in the HA of most Spanish pandemic isolates. However,one of these isolates (A/Extremadura/RR6530/2010) lacked reactivity with the mAbs and carried two unique mutations in the HA head (S88Y and K136N) that were required simultaneously to eliminate reactivity with the murine antibodies. This unusual requirement directly illustrates the phenomenon of enhanced antigenic change proposed previously for the accumulation of simultaneous amino acid substitutions at antigenic sites of the influenza A virus HA during virus evolution (Shih et al.,Proc Natl Acad Sci USA,104,6283-6288,2007). The changes found in the A/Extremadura/RR6530/2010 HA were not found in escape mutants selected in vitro with one of the mAbs,which contained instead nearby single amino acid changes in the HA head. Thus,either single or double point mutations may similarly alter epitopes of the new antigenic site identified in this work in the 2009 H1N1 pandemic virus HA. Moreover,this site is relevant for the human antibody response,as shown by competition of mAbs and human post-infection sera for virus binding. The results are discussed in the context of the HA antigenic structure and challenges posed for identification of sequence changes with possible antigenic impact during virus surveillance.

AbstractChlamydia genital infection caused by Chlamydia trachomatis is the most common bacterial sexually transmitted disease worldwide. A mouse model has been developed in our laboratory to better understand the effect of cold-induced stress on chlamydia genital infection and immune response. However,the stress mechanism affecting the host response to Chlamydia muridarum genital infection remains unclear. Here,we demonstrate a role for the beta2-adrenergic receptor (β2-AR),which binds noradrenaline and modulates the immune response against chlamydia genital infection in a mouse model. A successful β2-AR homozygous knockout (KO) mouse model was used to study the infection and analyze the immune response. Our data show that stressed mice lacking the β2-AR are less susceptible to C. muridarum genital infection than controls. A correlation was obtained between lower organ load and higher interferon-gamma production by CD4+ and CD8+ cells of the KO mice. Furthermore,exposure of CD4+ T cells to noradrenaline alters the production of cytokines in mice during C. muridarum genital infection. This study suggests that the blockade of β2-AR signaling could be used to increase resistance to chlamydia genital infection. We value the β2-AR KO as a viable model that can provide reproducible results in investigating medical research,including chlamydia genital infection. Deficiency in a receptor leads to a reduced disease of chlamydia in a mouse model. View Publication

Bipolar disorder (BD) is a common neuropsychiatric disorder characterized by chronic recurrent episodes of depression and mania. Despite evidence for high heritability of BD,little is known about its underlying pathophysiology. To develop new tools for investigating the molecular and cellular basis of BD,we applied a family-based paradigm to derive and characterize a set of 12 induced pluripotent stem cell (iPSC) lines from a quartet consisting of two BD-affected brothers and their two unaffected parents. Initially,no significant phenotypic differences were observed between iPSCs derived from the different family members. However,upon directed neural differentiation,we observed that CXCR4 (CXC chemokine receptor-4) expressing central nervous system (CNS) neural progenitor cells (NPCs) from both BD patients compared with their unaffected parents exhibited multiple phenotypic differences at the level of neurogenesis and expression of genes critical for neuroplasticity,including WNT pathway components and ion channel subunits. Treatment of the CXCR4(+) NPCs with a pharmacological inhibitor of glycogen synthase kinase 3,a known regulator of WNT signaling,was found to rescue a progenitor proliferation deficit in the BD patient NPCs. Taken together,these studies provide new cellular tools for dissecting the pathophysiology of BD and evidence for dysregulation of key pathways involved in neurodevelopment and neuroplasticity. Future generation of additional iPSCs following a family-based paradigm for modeling complex neuropsychiatric disorders in conjunction with in-depth phenotyping holds promise for providing insights into the pathophysiological substrates of BD and is likely to inform the development of targeted therapeutics for its treatment and ideally prevention. View Publication

The purpose of the present study was to characterize primitive epithelial progenitor populations present in adult normal human mammary tissue using a combination of flow cytometry and in vitro colony assay procedures. Three types of human breast epithelial cell (HBEC) progenitors were identified: luminal-restricted,myoepithelial-restricted and bipotent progenitors. The first type expressed epithelial cell adhesion molecule (EpCAM),alpha6 integrin and MUC1 and generated colonies composed exclusively of cells positive for the luminal-associated markers keratin 8/18,keratin 19,EpCAM and MUC1. Bipotent progenitors produced colonies containing a central core of cells expressing luminal markers surrounded by keratin 14+ myoepithelial-like cells. Single cell cultures confirmed the bipotentiality of these progenitors. Their high expression of alpha6 integrin and low expression of MUC1 suggests a basal position of these cells in the mammary epithelium in vivo. Serial passage in vitro of an enriched population of bipotent progenitors demonstrated that only myoepithelial-restricted progenitors could be readily generated under the culture conditions used. These results support a hierarchical branching model of HBEC progenitor differentiation from a primitive uncommitted cell to luminal- and myoepithelial-restricted progenitors. View Publication

PurposeLittle is known about the development of Bruch's membrane (BrM),the structure separating and supporting the retina and choroid,nor whether differentiation of human pluripotent stem cell (hPSC)-derived retinal pigment epithelium (RPE) accurately replicates BrM. This has relevance for tissue engineering strategies,both in the development of accurate in vitro models,and effective RPE transplant strategies. Here,we investigated BrM-associated protein production in human fetal tissue and hPSC-derived RPE.MethodsThe presence of laminin,elastin,fibronectin,and types I/III/IV collagen was examined in human fetal eyes at 6 to 21 post-conception weeks (PCWs) and hPSC-derived RPE cultures at 1 to 6 weeks in culture using immunohistochemistry/immunocytochemistry and quantitative PCR (qPCR).ResultsIn human fetal retina,laminin and fibronectin were present from 6 PCW,type IV collagen from 8 PCW,elastin from 12 PCW,type I collagen by 17 PCW,and type III collagen from 21 PCW. BrM layering was discernible from 12 PCW,becoming distinct by 17 PCW. In hPSC-derived RPE cultures,basement membranes containing laminin and fibronectin were present from week 1,type IV collagen from week 2,and type I collagen from week 4. Type III collagen was present at all timepoints,although not localized as a basement membrane. Elastin was absent at all timepoints.ConclusionsBrM-like membrane synthesis in hPSC-derived RPE largely recapitulates the temporal sequence seen in human development,excluding elastin. These support the utility of hPSC-derived RPE in in vitro systems to model RPE/retina interactions in health and disease,and inform cell therapy approaches,as de novo BrM-like membrane has the potential to support transplanted donor RPE. View Publication

BACKGROUND Identification of species-specific trypanosome molecules is important for laboratory- and field-based research into epidemiology and disease diagnosis. Although Trypanosoma congolense is the most important trypanosome pathogen of cattle in Africa,no species-specific molecules found in infective bloodstream forms (BSF) of the parasites have been identified,thus limiting development of diagnostic tests. METHODS Immuno-mass spectrometric methods were used to identify a protein that is recognized by a T. congolense-specific monoclonal antibody (mAb) Tc6/42.6.4. The identified molecule was expressed as a recombinant protein in E. coli and was tested in several immunoassays for its ability to interact with the mAb. The three dimensional structure of the protein was modeled and compared to crystal- and NMR-structures of the homologous proteins from T. cruzi and T. brucei respectively,in order to examine structural differences leading to the different immunoreactivity of the T. congolense molecule. Enzyme-linked immunosorbent assays (ELISA) were used to measure antibodies produced by trypanosome-infected African cattle in order to assess the potential for use of T. congolense calflagin in a serodiagnostic assay. RESULTS The antigen recognized by the T. congolense-specific mAb Tc6/42.6.4 was identified as a flagellar calcium-binding protein,calflagin. The recombinant molecule showed immunoreactivity with the T. congolense-specific mAb confirming that it is the cognate antigen. Immunofluorescence experiments revealed that Ca2+ modulated the localization of the calflagin molecule in trypanosomes. Structural modelling and comparison with calflagin homologues from other trypanosomatids revealed four non-conserved regions on the surface of the T. congolense molecule that due to differences in surface chemistry and structural topography may form species-specific epitopes. ELISAs using the recombinant calflagin as antigen to detect antibodies in trypanosome-infected cattle showed that the majority of cattle had antibody responses. Area under the Receiver-Operating Characteristic (ROC) curves,associated with host IgG and IgM,were calculated to be 0.623 and 0.709 respectively,indicating a positive correlation between trypanosome infection and the presence of anti-calflagin antibodies. CONCLUSIONS While calflagin is conserved among different species of African trypanosomes,our results show that T. congolense calflagin possesses unique epitopes that differentiate this protein from homologues in other trypanosome species. MAb Tc6/42.6.4 has clear utility as a laboratory tool for identifying T. congolense. T. congolense calflagin has potential as a serodiagnostic antigen and should be explored further for its utility in antigen-detection assays for diagnosis of cattle infections. View Publication

BACKGROUND: The presence of chromosomal abnormalities could have a negative impact for human embryonic stem cell (hESC) applications both in regenerative medicine and in research. A biomarker that allows the identification of chromosomal abnormalities induced in hESC in culture before they take over the culture would represent an important tool for defining optimal culture conditions for hESC. Here we investigate the expression of CD30,reported to be a biomarker of hESCs with abnormal karyotype,in undifferentiated and spontaneously differentiated hESC.backslashnbackslashnMETHODS AND RESULTS: hESC were derived and cultured on mouse fibroblasts in KO-SR containing medium (serum free media) and passaged mechanically. Our results based on analysis at mRNA (RT-PCR) and protein (fluorescence-activated cell sorting and immunocytochemistry) level show that CD30 is expressed in undifferentiated hESC,even at very early passages,without any correlation with the presence of chromosomal anomalies. We also show that the expression of CD30 is rapidly lost during early spontaneous differentiation of hESC.backslashnbackslashnCONCLUSION: We conclude that CD30 expression in hESC cultures is probably a consequence of culture conditions,and that KO-SR may play a role. In addition,the expression of so-called 'stemness' markers does not change in undifferentiated hESC during long-term culture or when cells acquire chromosomal abnormalities. View Publication

Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme that is responsible for the oxidation of intracellular aldehydes. Elevated levels of ALDH have been demonstrated in murine and human progenitor cells compared with other hematopoietic cells,and this is thought to be important in chemoresistance. A method for the assessment of ALDH activity in viable cells recently has been developed and made commercially available in a kit format. In this study,we confirmed the use of the ALDH substrate kit to identify cord blood stem/progenitor cells. Via multicolor flow cytometry of cord blood ALDH+ cells,we have expanded on their phenotypic analysis. We then assessed the incidence,morphology,phenotype,and nonobese diabetic/ severe combined immunodeficiency engraftment ability of ALDH+ cells from acute myeloid leukemia (AML) samples. AML samples had no ALDH+ cells at all,an extremely rare nonmalignant stem/progenitor cell population,or a less rare,leukemic stem cell population. Hence,in addition to identifying nonmalignant stem cells within some AML samples,a high ALDH activity also identifies some patients' CD34+/ CD38- leukemic stem cells. The incidence of normal or leukemic stem cells with an extremely high ALDH activity may have important implications for resistance to chemotherapy. Identification and isolation of leukemic cells on the basis of ALDH activity provides a tool for their isolation and further analysis. View Publication

RNA sequencing and genetic data support spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) as putative targets to be modulated for Alzheimer’s disease (AD) therapy. FCER1G is a component of Fc receptor complexes that contain an immunoreceptor tyrosine-based activation motif (ITAM). SYK interacts with the Fc receptor by binding to doubly phosphorylated ITAM (p-ITAM) via its two tandem SH2 domains (SYK-tSH2). Interaction of the FCER1G p-ITAM with SYK-tSH2 enables SYK activation via phosphorylation. Since SYK activation is reported to exacerbate AD pathology,we hypothesized that disruption of this interaction would be beneficial for AD patients. Herein,we developed biochemical and biophysical assays to enable the discovery of small molecules that perturb the interaction between the FCER1G p-ITAM and SYK-tSH2. We identified two distinct chemotypes using a high-throughput screen (HTS) and orthogonally assessed their binding. Both chemotypes covalently modify SYK-tSH2 and inhibit its interaction with FCER1G p-ITAM,however,these compounds lack selectivity and this limits their utility as chemical tools. View Publication

Tissue engineering strategies,based on solid/porous scaffolds,suffer from several limitations,such as ineffective vascularization,poor cell distribution and organization within scaffold,in addition to low final cell density,among others. Therefore,the search for other tissue engineering approaches constitutes an active area of investigation. Decellularized matrices (DM) present major advantages compared to solid scaffolds,such as ideal chemical composition,the preservation of vascularization structure and perfect three-dimensional structure. In the present study,we aimed to characterize and investigate murine heart decellularized matrices as biomaterials for regular and whole organ tissue engineering. Heart decellularized matrices were characterized according to: 1. DNA content,through DNA quantificationo and PCR of isolated genomic DNA; 2. Histological structure,assessed after Hematoxylin and Eosin,as well as Masson's Trichrome stainings; 3. Surface nanostructure analysis,performed,using SEM. Those essays allowed us to conclude that DM was indeed decellularized,with preserved extracellular matrix structure. Following characterization,decellularized heart slices were seeded with induced Pluripotent Stem cells (iPS). As expected,but - to the best of our knowledge - never shown before,decellularization of murine heart matrices maintained matrix biocompatibility,as iPS cells rapidly attached to the surface of the material and proliferated. Strikingly though,heart DM presented a differentiation induction effect over those cells,which lost their pluripotency markers after 7 days of culture in the DM. Such loss of differentiation markers was observed,even though bFGF containing media mTSR was used during such period. Gene expression of iPS cells cultured on DM will be further analyzed,in order to assess the effects of culturing pluripotent stem cells in decellularized heart matrices. View Publication

VEXAS syndrome is a clonal hematopoietic disorder characterized by hyperinflammation,bone marrow failure,and high mortality. The molecular hallmark of VEXAS is somatic mutations at methionine 41 (M41) in the E1 ubiquitin enzyme,UBA1. These mutations induce a protein isoform switch,but the mechanisms underlying disease pathogenesis remain unclear. Here,we developed a human cell model of VEXAS syndrome by engineering the male monocytic THP1 cell line to express the common UBA1 M41V mutation. We found that mutant UBA1 M41V cells exhibit aberrant UBA1 isoform expression,increased vacuolization,and upregulation of the unfolded protein response,recapitulating key features of VEXAS. Moreover,proteomic analyses revealed dysregulated ubiquitination and proteotoxic stress in UBA1 M41V cells,with alterations in inflammatory and stress-response pathways. Functional studies demonstrated that UBA1 M41V cells were highly sensitive to genetic or pharmacological inhibition of E1 ubiquitin enzymes. Treatment with the E1 enzyme inhibitor TAK-243 preferentially suppressed colony formation of UBA1 M41V cells as compared to WT cells. Moreover,UBA1 M41V cells exhibited greater sensitivity to TAK-243 in competition assays and showed increased apoptosis. Interestingly,TAK-243 preferentially inhibited UBA6 activity over UBA1,suggesting that UBA6 may compensate for UBA1 dysfunction in UBA1 M41V cells. Targeting UBA6 using shRNA or the UBA6-specific inhibitor phytic acid further revealed an acquired dependency on UBA6 in UBA1 M41V cells. Phytic acid selectively impaired growth and colony formation in UBA1 M41V cells while sparing WT cells,highlighting a potential therapeutic vulnerability. Together,these findings establish a novel human model of VEXAS syndrome,identify key roles for UBA1 and UBA6 in disease pathogenesis,and demonstrate that UBA6 inhibition represents a promising therapeutic strategy for selectively targeting UBA1 mutant clones. Subject terms: Haematological cancer,Cell signalling View Publication