Nfix expression critically modulates early B lymphopoiesis and myelopoiesis

The commitment of stem and progenitor cells toward specific hematopoietic lineages is tightly controlled by a number of transcription factors that regulate differentiation programs via the expression of lineage restricting genes. Nuclear factor one (NFI) transcription factors are important in regulating hematopoiesis and here we report an important physiological role of NFIX in B- and myeloid lineage commitment and differentiation. We demonstrate that NFIX acts as a regulator of lineage specification in the haematopoietic system and the expression of Nfix was transcriptionally downregulated as B cells commit and differentiate, whilst maintained in myeloid progenitor cells. Ectopic Nfix expression in vivo blocked early B cell development stage, coincident with the stage of its downregulation. Furthermore, loss of Nfix resulted in the perturbation of myeloid and lymphoid cell differentiation, and a skewing of gene expression involved in lineage fate determination. Nfix was able to promote myeloid differentiation of total bone marrow cells under B cell specific culture conditions but not when expressed in the hematopoietic stem cell (HSPC), consistent with its role in HSPC survival. The lineage choice determined by Nfix correlated with transcriptional changes in a number of genes, such as E2A, C/EBP, and Id genes. These data highlight a novel and critical role for NFIX transcription factor in hematopoiesis and in lineage specification

Impact of Environmental Factors on Bacteriocin Promoter Activity in Gut-Derived Lactobacillus salivarius

peer-reviewedBacteriocin production is regarded as a desirable probiotic trait that aids in colonization and persistence in the gastrointestinal tract (GIT). Strains of Lactobacillus salivarius, a species associated with the GIT, are regarded as promising probiotic candidates and have a number of associated bacteriocins documented to date. These include multiple class IIb bacteriocins (salivaricin T, salivaricin P, and ABP-118) and the class IId bacteriocin bactofencin A, which show activity against medically important pathogens. However, the production of a bacteriocin in laboratory media does not ensure production under stressful environmental conditions, such as those encountered within the GIT. To allow this issue to be addressed, the promoter regions located upstream of the structural genes encoding the L. salivarius bacteriocins mentioned above were fused to a number of reporter proteins (green fluorescent protein [GFP], red fluorescent protein [RFP], and luciferase [Lux]). Of these, only transcriptional fusions to GFP generated signals of sufficient strength to enable the study of promoter activity in L. salivarius. While analysis of the class IIb bacteriocin promoter regions indicated relatively weak GFP expression, assessment of the promoter of the antistaphylococcal bacteriocin bactofencin A revealed a strong promoter that is most active in the absence of the antimicrobial peptide and is positively induced in the presence of mild environmental stresses, including simulated gastric fluid. Taken together, these data provide information on factors that influence bacteriocin production, which will assist in the development of strategies to optimize in vivo and in vitro production of these antimicrobials.This work was funded by a SFI PI award “Obesibiotics” (11/PI/1137) to PD

Trib2 expression in granulocyte-monocyte progenitors drives a highly drug resistant acute myeloid leukaemia linked to elevated Bcl2.

Trib2 pseudokinase has oncogenic and tumour suppressive functions depending on the cellular context. We investigated the ability of Trib2 to transform different haemopoietic stem and progenitor cells (HSPCs). Our study identified the granulocyte-macrophage progenitor (GMP) subpopulation as a potent leukaemia initiating cell of Trib2-driven AML in vivo. Trib2 transformed GMPs generated a fully penetrant and short latency AML. AML cells expressing elevated Trib2 led to a chemoresistant phenotype following chemotherapy treatment. We show that Trib2 overexpression results in an increase in BCL2 expression, and high Trib2 expressing cells are highly sensitive to cell killing by BCL2 inhibition (ABT199). Combined treatment with chemotherapeutic agents and BCL2 inhibition resulted in synergistic killing of Trib2+ AML cells. Trib2 transformed GMP AML cells showed more chemoresistance compared with HSPC derived Trib2 AML cells associated with higher Bcl2 expression. There is significant correlation of high TRIB2 and BCL2 expression in patient derived human AML cells. These data demonstrate that the cell of origin influences the leukaemic profile and chemotherapeutic response of Trib2+ AML. Combined TRIB2 and BCL2 expression in AML cells may have clinical utility relevant for monitoring drug resistance and disease relapse

Age-specific biological and molecular profiling distinguishes paediatric from adult acute myeloid leukaemias

Acute myeloid leukaemia (AML) affects children and adults of all ages. AML remains one of the major causes of death in children with cancer and for children with AML relapse is the most common cause of death. Here, by modelling AML in vivo we demonstrate that AML is discriminated by the age of the cell of origin. Young cells give rise to myeloid, lymphoid or mixed phenotype acute leukaemia, whereas adult cells give rise exclusively to AML, with a shorter latency. Unlike adult, young AML cells do not remodel the bone marrow stroma. Transcriptional analysis distinguishes young AML by the upregulation of immune pathways. Analysis of human paediatric AML samples recapitulates a paediatric immune cell interaction gene signature, highlighting two genes, RGS10 and FAM26F as prognostically significant. This work advances our understanding of paediatric AML biology, and provides murine models that offer the potential for developing paediatric specific therapeutic strategies

Whey protein effects on energy balance link the intestinal mechanisms of energy absorption with adiposity and hypothalamic neuropeptide gene expression

peer-reviewedWe tested the hypothesis that dietary whey protein isolate (WPI) affects the intestinal mechanisms related to energy absorption and that the resulting energy deficit is compensated by changes in energy balance to support growth. C57BL/6 mice were provided a diet enriched with WPI with varied sucrose content, and the impact on energy balance-related parameters was investigated. As part of a high-sucrose diet, WPI reduced the hypothalamic expression of pro-opiomelanocortin gene expression and increased energy intake. The energy expenditure was unaffected, but epididymal weight was reduced, indicating an energy loss. Notably, there was a reduction in the ileum gene expression for amino acid transporter SLC6a19, glucose transporter 2, and fatty acid transporter 4. The composition of the gut microbiota also changed, where Firmicutes were reduced. The above changes indicated reduced energy absorption through the intestine. We propose that this mobilized energy in the adipose tissue and caused hypothalamic changes that increased energy intake, acting to counteract the energy deficit arising in the intestine. Lowering the sucrose content in the WPI diet increased energy expenditure. This further reduced epididymal weight and plasma leptin, whereupon hypothalamic ghrelin gene expression and the intestinal weight were both increased. These data suggest that when the intestine-adipose-hypothalamic pathway is subjected to an additional energy loss (now in the adipose tissue), compensatory changes attempt to assimilate more energy. Notably, WPI and sucrose content interact to enable the component mechanisms of this pathway

The microbiota-gut-brain axis

The importance of the gut-brain axis in maintaining homeostasis has long been appreciated. However, the past 15 yr have seen the emergence of the microbiota (the trillions of microorganisms within and on our bodies) as one of the key regulators of gut-brain function and has led to the appreciation of the importance of a distinct microbiota-gut-brain axis. This axis is gaining ever more traction in fields investigating the biological and physiological basis of psychiatric, neurodevelopmental, age-related, and neurodegenerative disorders. The microbiota and the brain communicate with each other via various routes including the immune system, tryptophan metabolism, the vagus nerve and the enteric nervous system, involving microbial metabolites such as short-chain fatty acids, branched chain amino acids, and peptidoglycans. Many factors can influence microbiota composition in early life, including infection, mode of birth delivery, use of antibiotic medications, the nature of nutritional provision, environmental stressors, and host genetics. At the other extreme of life, microbial diversity diminishes with aging. Stress, in particular, can significantly impact the microbiota-gut-brain axis at all stages of life. Much recent work has implicated the gut microbiota in many conditions including autism, anxiety, obesity, schizophrenia, Parkinson's disease, and Alzheimer's disease. Animal models have been paramount in linking the regulation of fundamental neural processes, such as neurogenesis and myelination, to microbiome activation of microglia. Moreover, translational human studies are ongoing and will greatly enhance the field. Future studies will focus on understanding the mechanisms underlying the microbiota-gut-brain axis and attempt to elucidate microbial-based intervention and therapeutic strategies for neuropsychiatric disorders

The effects of progressive taxation on risk-taking

SIGLECopy held by FIZ Karlsruhe; available from UB/TIB Hannover / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekDEGerman

NeuroChip: a microfluidic electrophysiological device for genetic and chemical biology screening of Caenorhabditis elegans adult and larvae

Genetic and chemical biology screens of C. elegans have been of enormous benefit in providing fundamental insight into neural function and neuroactive drugs. Recently the exploitation of microfluidic devices has added greater power to this experimental approach providing more discrete and higher throughput phenotypic analysis of neural systems. Here we make a significant addition to this repertoire through the design of a semi-automated microfluidic device, NeuroChip, which has been optimised for selecting worms based on the electrophysiological features of the pharyngeal neural network. We demonstrate this device has the capability to sort mutant from wild-type worms based on high definition extracellular electrophysiological recordings. NeuroChip resolves discrete differences in excitatory, inhibitory and neuromodulatory components of the neural network from individual animals. Worms may be fed into the device consecutively from a reservoir and recovered unharmed. It combines microfluidics with integrated electrode recording for sequential trapping, restraining, recording, releasing and recovering of C. elegans. Thus mutant worms may be selected, recovered and propagated enabling mutagenesis screens based on an electrophysiological phenotype. Drugs may be rapidly applied during the recording thus permitting compound screening. For toxicology, this analysis can provide a precise description of sub-lethal effects on neural function. The chamber has been modified to accommodate L2 larval stages showing applicability for small size nematodes including parasitic species which otherwise are not tractable to this experimental approach. We also combine NeuroChip with optogenetics for targeted interrogation of the function of the neural circuit. NeuroChip thus adds a new tool for exploitation of C. elegans and has applications in neurogenetics, drug discovery and neurotoxicology

TRIB2 Transformed GMP is the Myeloid Leukaemia Initiating Cell

serine/threonine pseudokinases. When ectopically expressed in hematopoietic stem and progenitor cell (HSPC) enriched bone marrow cells, TRIB2 was shown to induce acute myeloid leukemia (AML) in a murine transplant model dependent on C/EBPα degradation, showing it to be a potent myeloid oncogene. The cell of origin or leukemia initiating cell (LIC) refers to the cell from which a specific leukemia normally arises, and it is hypothesised that the LIC may influence the progression, disease phenotype and response to therapy. It remains unclear whether the LIC is a HSC or a more committed progenitor cell in TRIB2-driven AML. Aims: Our current study focuses on identifying the LIC in TRIB2-driven AML and characterizing its role in disease potency and maintenance. Methods: FACS sorted CD45.2+ stem and progenitor cell populations (HSC, MPP, CMP, GMP and MEP) transduced with a lentiviral vector encoding TRIB2 were cultured in methocult supplemented with cytokines that support myeloid cell growth and differentiation. Following the first plating (P1), GFP+ cells were sorted and serially replated. Cells which still formed colonies by the third replating (P3) implied acquisition of self-renewal ability and increased proliferation characteristic of myeloid transformation. These CD45.2+ cells from P3 were transplanted into sublethally irradiated CD45.1+ C57BL/6 recipient mice and chimeric animals were monitored for 1 year. Chemoresistance experiments were performed on the bulk bone marrow population (CD45.2+ cells >95%) from the TRIB2 AML mice. Cells were treated with a range of concentrations of daunorubicin (DNR), followed by trypan blue cell counts to assess viability. Results: Our study identified that while lentivirally transduced TRIB2 can transform all stem and progenitor cell populations of the hematopoietic system with variable efficiencies in vitro, the GMP subpopulation was identified as the LIC of TRIB2-driven AML. TRIB2 transformed GMP cells generated a more potent AML with complete penetrance and shortened latency compared to all other HSPC populations analysed. Indeed, phenotypically different diseases were propagated from TRIB2 expression in the HSC and GMP, with the former having a weakly penetrant, longer latency AML with a mixed lineage phenotype, whereas the later was a dominantly myeloid disease phenotype with a short latency. We next addressed the chemotherapeutic response of TRIB2 positive AML cells. We show that GMP-TRIB2 AML and bulk TRIB2 AML cells are chemoresistant. TRIB2 overexpression decreases DNR induced apoptosis, and knockdown of TRIB2 expression in AML cells leads to an increase in apoptotic gene expression. Our studies illustrate that TRIB2 expression is key in mediating the anti-apoptotic signals following DNR treatment. Summary/Conclusions:We identify the GMP as the LIC in TRIB2 driven AML. Our findings are further supported by our previous work showing that degradation of C/EBPa is required for TRIB2-driven AML and the GMP population expresses the highest level of C/EBPa in hematopoiesis. We provide evidence for TRIB2 role in chemoresistance, and that the TRIB2 LIC is a highly chemoresistant cell. Our findings provide insight into the molecular events contributing to AML, and provide potential for novel avenues for therapeutic targeting

Investigation of the role of TRIB2 in normal murine hematopoiesis

TRIB2 is a member of the mammalian Tribbles family of serine/threonine pseudokinases (TRIB1, 2, 3). Pathologically, TRIB2 induces potent murine acute myeloid leukemia and is associated with acute lymphoblastic leukemia. However, the normal hematopoietic role of TRIB2 remains elusive. Here, we studied murine hematopoiesis after Trib2 ablation. At the steady state, Trib2 loss did not adversely affect peripheral blood cell counts and populations. Trib2-/- mice had similar bone marrow cellularity compared to wild type (WT) mice and no significant differences in the populations of hematopoietic stem and progenitor cells. However, Trib2-/- mice had significantly higher thymic cellularity. Both Trib2-/- and WT mice had similar numbers of immature double negative (DN)1-4 thymic subsets. However, Trib2-/- DN cells proliferated faster as indicated by the higher expression of Ki-67, which contributed to the increased number of double and single positive (DP and SP) mature thymic subsets. To evaluate the impact of Trib2-/- on stress hematopoiesis, we treated both groups of mice with 5-fluorouracil (5-FU) in vivo and compared their hematopoietic recovery at different time points. Trib2 loss did not impair the bone marrow multilineage hematopoietic recovery but increased SP T cells detected in the peripheral blood. At 16 and 24 hours post treatment, Trib2-/- mice had significantly reduced numbers of DN1 c-Kithi precursors, DN3L and DPbl cycling subsets, and increased apoptosis, respectively. At day 4 and 14 post treatment, Trib2-/- mice had significantly higher thymic cellularity, frequency and number of thymic subsets compared to WT mice. DN1 c-Kit- precursors expanded significantly more in Trib2-/- compared to WT mice. Our results demonstrate that, in the absence of TRIB2, thymocytes are more sensitive to 5-FU induced cell death and thymopoietic recovery is accelerated. These data suggest TRIB2 regulates the differentiation and survival of intrathymic precursors and potentially has a role in the checkpoints of thymopoiesis