Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost

Seamless cloning methods, such as co-transformation cloning, sequence- and ligation-independent cloning (SLIC) or the Gibson assembly, are essential tools for the precise construction of plasmids. The efficiency of co-transformation cloning is however low and the Gibson assembly reagents are expensive. With the aim to improve the robustness of seamless cloning experiments while keeping costs low, we examined the importance of complementary single-stranded DNA ends for co-transformation cloning and the influence of single-stranded gaps in circular plasmids on SLIC cloning efficiency. Most importantly, our data show that single-stranded gaps in double-stranded plasmids, which occur in typical SLIC protocols, can drastically decrease the efficiency at which the DNA transforms competent E. coli bacteria. Accordingly, filling-in of single-stranded gaps using DNA polymerase resulted in increased transformation efficiency. Ligation of the remaining nicks did not lead to a further increase in transformation efficiency. These findings demonstrate that highly efficient insert-plasmid assembly can be achieved by using only T5 exonuclease and Phusion DNA polymerase, without Taq DNA ligase from the original Gibson protocol, which significantly reduces the cost of the reactions. We successfully used this modified Gibson assembly protocol with two short insert-plasmid overlap regions, each counting only 15 nucleotides.     

Renal Transplant Recipients Treated with Calcineurin-Inhibitors Lack Circulating Immature Transitional CD19+CD24hiCD38hi Regulatory B-Lymphocytes

BackgroundCD19⁺CD24^hiCD38^hi transitional immature B-lymphocytes have been demonstrated to play an important role in regulating the alloimmune response in transplant recipients. Here, we analyzed the effect of calcineurin inhibition on these peripherally circulating regulatory B-cells (Breg) in renal transplant recipients receiving cyclosporine A (CsA) or tacrolimus.MethodsPBMCs from healthy subjects (HS) (n = 16) and renal transplant recipients (n = 46) were isolated. Flow cytometry was performed for CD19, CD24, CD38 and IL-10 either after isolation or after 72 hours of co-culture in presence of PMA/Ionomycin and TLR9-ligand in presence or absence of increasing concentrations of tacrolimus or CsA.ResultsThe amount of CD19⁺ B-cells among lymphocytes was ∼9.1% in HS, ∼3.6% in CsA (n = 11, p<0.05) and ∼6.4% in TAC (n = 35, p<0.05) treated patients. Among B-cells, a distinct subset of Breg was found to be 4.7% in HS, 1.4% in tacrolimus treated patients and almost blunted in patients receiving CsA. Similarily, ∼4% of B-cells in HS and even fewer in CsA or tacrolimus treated patients produced IL-10 (0.5% and 1.5%, p<0.05) and this was confirmed both in non-transplanted CsA-treated healthy subjects and in in vitro co-culture experiments. Among 29 patients with <1% of Breg, 9 cases (31%) displayed an allograft rejection in contrast to only one case of rejection (6%) among 17 patients with >1%.ConclusionCalcineurin inhibitors reduce number and IL-10 production of Bregs in the peripheral circulation of both renal transplant recipients and non-transplanted healthy subjects. CNI induced Breg reduction is not restricted to a solid organ transplant setting and is not mediated by co-medication with steroids or MPA. A low proportion of Breg cells is associated with an elevated frequency of allograft rejection events.     

α‐Hemolysin enhances Staphylococcus aureus internalization and survival within mast cells by modulating the expression of β1 integrin

Mast cells (MCs) are important sentinels of the host defence against invading pathogens. We previously reported that Staphylococcus aureus evaded the extracellular antimicrobial activities of MCs by promoting its internalization within these cells via β1 integrins. Here, we investigated the molecular mechanisms governing this process. We found that S. aureus responded to the antimicrobial mediators released by MCs by up‐regulating the expression of α‐hemolysin (Hla), fibronectin‐binding protein A and several regulatory systems. We also found that S. aureus induced the up‐regulation of β1 integrin expression on MCs and that this effect was mediated by Hla‐ADAM10 (a disintegrin and metalloproteinase 10) interaction. Thus, deletion of Hla or inhibition of Hla‐ADAM10 interaction significantly impaired S. aureus internalization within MCs. Furthermore, purified Hla but not the inactive Hla_H35L induced up‐regulation of β1 integrin expression in MCs in a dose‐dependent manner. Our data support a model in which S. aureus counter‐reacts the extracellular microbicidal mechanisms of MCs by increasing expression of fibronectin‐binding proteins and by inducing Hla‐ADAM10‐mediated up‐regulation of β1 integrin in MCs. The up‐regulation of bacterial fibronectin‐binding proteins, concomitantly with the increased expression of its receptor β1 integrin on the MCs, resulted in enhanced S. aureus internalization through the binding of fibronectin‐binding proteins to integrin β1 via fibronectin.     

Neocortical Expansion Due to Increased Proliferation of Basal Progenitors Is Linked to Changes in Their Morphology

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Glycomics-driven discoveries in schistosome research

Schistosome glycans and glycoconjugates play a prominent role in the parasite's biology, in particular in the interaction with the human host. A large amount of structural data regarding glycosylation of different schistosome life stages and glycoconjugate subsets has been collected in the last decade, but many significant gaps in our knowledge of the schistosomal glycome remain. Here we will present a concise review of the already available data guided by a selection of recently generated stage-specific glycan profiles, and discuss implications and prospects of glycomics studies of schistosomes.