Standardization of Synthetic Biology Tools and Assembly Methods for and Emerging Yeast Species.

As redesigning organisms using engineering principles is one of the purposes of synthetic biology (SynBio), the standardization of experimental methods and DNA parts is becoming increasingly a necessity. The synthetic biology community focusing on the engineering of has been in the foreground in this area, conceiving several well-characterized SynBio toolkits widely adopted by the community. In this review, the molecular methods and toolkits developed for are discussed in terms of their contributions to the required standardization efforts.

Developmental cell programs are co-opted in inflammatory skin disease.

The skin confers biophysical and immunological protection through a complex cellular network established early in embryonic development. We profiled the transcriptomes of more than 500,000 single cells from developing human fetal skin, healthy adult skin, and adult skin with atopic dermatitis and psoriasis. We leveraged these datasets to compare cell states across development, homeostasis, and disease.

Decoding human fetal liver haematopoiesis.

Definitive haematopoiesis in the fetal liver supports self-renewal and differentiation of haematopoietic stem cells and multipotent progenitors (HSC/MPPs) but remains poorly defined in humans. Here, using single-cell transcriptome profiling of approximately 140,000 liver and 74,000 skin, kidney and yolk sac cells, we identify the repertoire of human blood and immune cells during development. We infer differentiation trajectories from HSC/MPPs and evaluate the influence of the tissue microenvironment on blood and immune cell development.

The impact of single-cell RNA sequencing on understanding the functional organization of the immune system.

Application of single-cell genomics technologies has revolutionized our approach to study the immune system. Unravelling the functional diversity of immune cells and their coordinated response is key to understanding immunity. Single-cell transcriptomics technologies provide high-dimensional assessment of the transcriptional states of immune cells and have been successfully applied to discover new immune cell types, reveal haematopoietic lineages, identify gene modules dictating immune responses and investigate lymphocyte antigen receptor diversity.

MicroRNA profiling of the bovine alveolar macrophage response to Mycobacterium bovis infection suggests pathogen survival is enhanced by microRNA regulation of endocytosis and lysosome trafficking.

Mycobacterium bovis, the causative agent of bovine tuberculosis, a major problem for global agriculture, spreads via an airborne route and is taken up by alveolar macrophages (AM) in the lung. Here, we describe the first next-generation sequencing (RNA-seq) approach to temporally profile miRNA expression in primary bovine AMs post-infection with M. bovis.

The Role of microRNAs in Bovine Infection and Immunity.

MicroRNAs (miRNAs) are a class of small, non-coding RNAs that are recognized as critical regulators of immune gene expression during infection. Many immunologically significant human miRNAs have been found to be conserved in agriculturally important species, including cattle. Discovering how bovine miRNAs mediate the immune defense during infection is critical to understanding the etiology of the most prevalent bovine diseases.

Profiling microRNA expression in bovine alveolar macrophages using RNA-seq.

MicroRNAs (miRNAs) are important regulators of gene expression and are known to play a key role in regulating both adaptive and innate immunity. Bovine alveolar macrophages (BAMs) help maintain lung homeostasis and constitute the front line of host defense against several infectious respiratory diseases, such as bovine tuberculosis. Little is known, however, about the role miRNAs play in these cells.

Immune complex signatures of patients with active and inactive SLE revealed by multiplex protein binding analysis on antigen microarrays.

Systemic lupus erythematosus is characterized by dysfunctional clearance of apoptotic debris and the development of pathogenic autoantibodies. While the complement system is also involved in the disease no attempt has been made to generate a comprehensive view of immune complex formation from various autoantigens. We increased the complexity of autoantibody profiles by measuring the binding of two complement proteins, C3 and C4, in addition to two antibody classes, IgG and IgM, to a collection of autoantigens.

Characterization of factors influencing on-chip complement activation to optimize parallel measurement of antibody and complement proteins on antigen microarrays.

Binding of immunoglobulins and complement fragments to targets of adaptive immune responses can be monitored using collections of arrayed antigens and is used to generate profiles of antibody binding and function. The collection of reliable data on these reactions on a large scale requires the establishment of criteria from sample collection through reaction conditions to normalization strategies. We characterized the detection of IgG, complement C3 and C4 under conditions that better resemble in vivo events than most serological assays and are also relevant for in vitro diagnostic purposes.

Lineage divergence at the first TCR-dependent checkpoint: preferential γδ and impaired αβ T cell development in nonobese diabetic mice.

The first TCR-dependent checkpoint in the thymus determines αβ versus γδ T lineage fate and sets the stage for later T cell differentiation decisions. We had previously shown that early T cells in NOD mice that are unable to rearrange a TCR exhibit a defect in checkpoint enforcement at this stage. To determine if T cell progenitors from wild-type NOD mice also exhibit cell-autonomous defects in development, we investigated their differentiation in the Notch-ligand-presenting OP9-DL1 coculture system, as well as by analysis of T cell development in vivo.

Progression of lupus-like disease drives the appearance of complement-activating IgG antibodies in MRL/lpr mice.

Objectives: Nucleic acids are known to induce complement activation, which results in the masking and removal of apoptotic cells exposing nuclear components. Dysregulation of these events is characteristic of SLE, a systemic autoimmune disease characterized by the appearance of ANAs. In this study, we aimed to investigate the relationship between development of ANAs and their effect on complement activation by nucleic acids.

Conventional chromogenic heparin assays are influenced by patient's endogenous plasma Antithrombin levels.

Objectives: To determine, in vitro, the influence of plasma AT concentrations on heparin levels as measured by commercially available chromogenic kits.

Methods: Purified AT was added to plasma that was immune depleted of AT at the following final concentrations: 0, 0.5, 1.

Long-term "in vitro" proliferating mouse hematopoietic progenitor cell lines.

Long-term proliferating hematopoietic progenitor cell lines have been established from mouse bone marrow in tissue cultures on the M-CSF-deficient stromal cell line OP9. In the presence of stem cell factor (SCF), thrombopoietin, IL-3 and IL-6 pluripotent hematopoietic stem cells (pHSC) initiate proliferation. For 2-3 weeks they maintain long-term reconstitution capacity, as tested in adoptive transfer experiments into sublethally irradiated hosts, but later loose this capacity.

Detection of complement activation on antigen microarrays generates functional antibody profiles and helps characterization of disease-associated changes of the antibody repertoire.

Humoral immune responses are traditionally characterized by determining the presence and quality of Abs specific for certain Ags. Arraying of large numbers of Ags allows the parallel measurement of Abs, generating patterns called Ab profiles. Functional characterization of these Abs could help draw an even more informative map of an immune response.

B lymphocytes and macrophages release cell membrane deposited C3-fragments on exosomes with T cell response-enhancing capacity.

Recently exosomes have been shown to play important roles in several immune phenomena. These small vesicles contain MHC proteins along with co-stimulatory and adhesion molecules, and mediate antigen presentation to T cells. In the present study we show that upon incubation with autologous serum, murine macrophages and B cells--but not T lymphocytes--fix C3-fragments covalently to the cell membrane and release them on exosomes in a time dependent fashion.

Lack of correlation between heparin dose and standard clinical monitoring tests in treatment with unfractionated heparin in critically ill children.

Unlabelled: The activated partial thromboplastin time (aPTT) and anti-Xa activity are used for monitoring unfractionated heparin (UFH) therapy in children and may not be optimal.

Objective: Determine correlations of aPTT, anti-Xa and UFH dose in children. Single centre prospective cohort study in children receiving UFH.

A clinically significant incidence of bleeding in critically ill children receiving therapeutic doses of unfractionated heparin: a prospective cohort study.

Unfractionated heparin (UFH) is frequently prescribed for children for the prevention and treatment of thrombosis; however, its safety and efficacy have not been assessed. The aim of this single center, prospective cohort study was to determine the incidence of major bleeding and recurrent thrombosis in children receiving UFH. Major bleeding was defined a priori as: central nervous system or retroperitoneal bleeding, bleeding resulting in UFH being stopped or overt bleeding causing a drop in hemoglobin >20 g/dL in less than 24 h.

Comparison of the anticoagulant effect of a direct thrombin inhibitor and a low molecular weight heparin in an acquired antithrombin deficiency in children with acute lymphoblastic leukaemia treated with L-asparaginase: an in vitro study.

Thrombosis occurs in 37% of children with acute lymphoblastic leukaemia (ALL) and is related to an L-asparaginase-induced acquired antithrombin (AT) deficiency. The incidence dictates the need for anticoagulant prophylaxis. Direct thrombin inhibitors (DTI) are independent of AT for effect and may thus have advantages in this population.

Dose-finding and pharmacokinetics of therapeutic doses of tinzaparin in pediatric patients with thromboembolic events.

In children, there is an increasing off-label use of low molecular weight heparin (LMWH). However, there is an absence of information on dosing and pharmacokinetics of LMWH over all age groups. The objectives of the current study were to determine i) the once daily dose required to achieve anti-Xa levels of 0.

Predictive value of persistent versus transient antiphospholipid antibody subtypes for the risk of thrombotic events in pediatric patients with systemic lupus erythematosus.

Study objectives were to determine, in children with systemic lupus erythematosus (SLE), (1) the association of antiphosholipid antibody (APLA) subtypes with thrombotic events (TEs) and (2) the predictive value of persistent versus transient antibodies for TEs. This is a cohort study of 58 SLE children in whom lupus anticoagulants (LAs), anticardiolipin antibodies (ACLAs), anti-beta2-glycoprotein-I (anti-beta2-GPI), and antiprothrombin (anti-PT) were assessed on at least 2 occasions (more than 3 months apart). Antibodies were classified as persistent (positive on at least 2 occasions) or transient (positive once).

Acetyl:succinate CoA-transferase in procyclic Trypanosoma brucei. Gene identification and role in carbohydrate metabolism.

Acetyl:succinate CoA-transferase (ASCT) is an acetate-producing enzyme shared by hydrogenosomes, mitochondria of trypanosomatids, and anaerobically functioning mitochondria. The gene encoding ASCT in the protozoan parasite Trypanosoma brucei was identified as a new member of the CoA transferase family. Its assignment to ASCT activity was confirmed by 1) a quantitative correlation of protein expression and activity upon RNA interference-mediated repression, 2) the absence of activity in homozygous Deltaasct/Deltaasct knock out cells, 3) mitochondrial colocalization of protein and activity, 4) increased activity and acetate excretion upon transgenic overexpression, and 5) depletion of ASCT activity from lysates upon immunoprecipitation.