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.     

Fatty Acid‐Derived Pro‐Toxicants of the Rat Selective Toxicant Norbormide

Norbormide [5‐(α‐hydroxy‐α‐2‐pyridylbenzyl)‐7‐(α‐2‐pyridylbenzylidene)‐5‐norbornene‐2,3‐dicarboximide] (NRB), an existing but infrequently used rodenticide, is known to be uniquely toxic to rats but relatively harmless to other rodents and mammals. However, as an acute vasoactive, NRB has a rapid onset of action which makes it relatively unpalatable to rats, often leading to sublethal uptake and accompanying bait shyness. A series of NRB‐derived pro‐toxicants ( 3a  –  i , 4a  –  i , and 5a  –  i ) were prepared in an effort to ‘mask’ this acute response and improve both palatability and efficacy. Their synthesis, in vitro biological evaluation (vasocontractile response in rat vasculature, stability in selected rat media) and palatability/efficacy in Sprague–Dawley, wild Norway, and wild ship rats is described. Most notably, pro‐toxicant 3d was revealed to be free of all pre‐cleavage vasoconstrictory activity in rat caudal artery and was subsequently demonstrated to release NRB in the presence of rat blood, liver, and pancreatic enzymes. Moreover, it consistently displayed a high level of acceptance by rats in a two‐choice bait‐palatability and efficacy trial, with accompanying high mortality. On this evidence, fatty acid ester prodrugs would appear to offer a promising platform for the further development of NRB‐derived toxicants with enhanced palatability and efficacy profiles.     

Evidence for conformationally different states of interleukin‐10: binding of a neutralizing antibody enhances accessibility of a hidden epitope

We present the mapping of two anti‐human interleukin‐10 (hIL‐10) antibodies (CB/RS/2 and CB/RS/11) which have been described as binding their antigen cooperatively. The epitopes were identified using hIL‐10‐derived overlapping peptide scans prepared by spot synthesis. To identify residues essential for binding within the two epitopes, each position was replaced by all other L ‐amino acids. The epitope‐derived peptides were further characterized with respect to antibody affinity and their inhibition of the antibody–hIL‐10 interaction. One antibody (CB/RS/11) binds to residues which are completely buried in the X‐ray structure of IL‐10. Accessibility of this hidden epitope is enhanced upon binding of the antibody CB/RS/2, which recognizes a discontinuous epitope located nearby. The recognition of the hidden CB/RS/11 epitope, as well as the cooperative binding behaviour of the two antibodies, provides evidence that IL‐10 can adopt a conformational state other than that observed in the crystal structure. Copyright © 1999 John Wiley & Sons, Ltd.     

Large-scale candidate gene analysis of HDL particle features

Background

HDL cholesterol (HDL-C) is an established marker of cardiovascular risk with significant genetic determination. However, HDL particles are not homogenous, and refined HDL phenotyping may improve insight into regulation of HDL metabolism. We therefore assessed HDL particles by NMR spectroscopy and conducted a large-scale candidate gene association analysis.

Methodology/Principal Findings

We measured plasma HDL-C and determined mean HDL particle size and particle number by NMR spectroscopy in 2024 individuals from 512 British Caucasian families. Genotypes were 49,094 SNPs in >2,100 cardiometabolic candidate genes/loci as represented on the HumanCVD BeadChip version 2. False discovery rates (FDR) were calculated to account for multiple testing. Analyses on classical HDL-C revealed significant associations (FDR<0.05) only for CETP (cholesteryl ester transfer protein; lead SNP rs3764261: p = 5.6*10⁻¹⁵) and SGCD (sarcoglycan delta; rs6877118: p = 8.6*10⁻⁶). In contrast, analysis with HDL mean particle size yielded additional associations in LIPC (hepatic lipase; rs261332: p = 6.1*10⁻⁹), PLTP (phospholipid transfer protein, rs4810479: p = 1.7*10⁻⁸) and FBLN5 (fibulin-5; rs2246416: p = 6.2*10⁻⁶). The associations of SGCD and Fibulin-5 with HDL particle size could not be replicated in PROCARDIS (n = 3,078) and/or the Women''s Genome Health Study (n = 23,170).

Conclusions

We show that refined HDL phenotyping by NMR spectroscopy can detect known genes of HDL metabolism better than analyses on HDL-C.     

Sonic hedgehog shedding results in functional activation of the solubilized protein

All Hedgehog (Hh) proteins are released from producing cells despite being synthesized as N- and C-terminally lipidated, membrane-tethered molecules. Thus, a cellular mechanism is needed for Hh solubilization. We previously suggested that a disintegrin and metalloprotease (ADAM)-mediated shedding of Sonic hedgehog (ShhNp) from its lipidated N and C termini results in protein solubilization. This finding, however, seemed at odds with the established role of N-terminal palmitoylation for ShhNp signaling activity. We now resolve this paradox by showing that N-palmitoylation of ShhNp N-terminal peptides is required for their proteolytic removal during solubilization. These peptides otherwise block ShhNp zinc coordination sites required for ShhNp binding to its receptor Patched (Ptc), explaining the essential yet indirect role of N-palmitoylation for ShhNp function. We suggest a functional model in which membrane-tethered multimeric ShhNp is at least partially autoinhibited in trans but is processed into fully active, soluble multimers upon palmitoylation-dependent cleavage of inhibitory N-terminal peptides.     

DMSO Represses Inflammatory Cytokine Production from Human Blood Cells and Reduces Autoimmune Arthritis

Dimethyl sulfoxide (DMSO) is currently used as an alternative treatment for various inflammatory conditions as well as for cancer. Despite its widespread use, there is a paucity of data regarding its safety and efficacy as well as its mechanism of action in human cells. Herein, we demonstrate that DMSO has ex-vivo anti-inflammatory activity using Escherichia coli- (E. coli) and herpes simplex virus-1 (HSV-1)-stimulated whole human blood. Specifically, we found that between 0.5%– 2%, DMSO significantly suppressed the expression of many pro-inflammatory cytokines/chemokines and prostaglandin E₂ (PGE₂). However, a significant reduction in monocyte viability was also observed at 2% DMSO, suggesting a narrow window of efficacy. Anti-inflammatory concentrations of DMSO suppressed E. coli-induced ERK1/2, p38, JNK and Akt phosphorylation, suggesting DMSO acts on these signaling pathways to suppress inflammatory cytokine/chemokine production. Although DMSO induces the differentiation of B16/F10 melanoma cells in vitro, topical administration of DMSO to mice subcutaneously implanted with B16 melanoma cells was ineffective at reducing tumor growth, DMSO was also found to block mouse macrophages from polarizing to either an M1- or an M2-phenotype, which may contribute to its inability to slow tumor growth. Topical administration of DMSO, however, significantly mitigated K/BxN serum-induced arthritis in mice, and this was associated with reduced levels of pro-inflammatory cytokines in the joints and white blood cell levels in the blood. Thus, while we cannot confirm the efficacy of DMSO as an anti-cancer agent, the use of DMSO in arthritis warrants further investigation to ascertain its therapeutic potential.     

Advanced genetic strategies for recombinant protein expression in Escherichia coli

Preparations enriched by a specific protein are rarely easily obtained from natural host cells. Hence, recombinant protein production is frequently the sole applicable procedure. The ribosomal machinery, located in the cytoplasm is an outstanding catalyst of recombinant protein biosynthesis. Escherichia coli facilitates protein expression by its relative simplicity, its inexpensive and fast high-density cultivation, the well-known genetics and the large number of compatible tools available for biotechnology. Especially the variety of available plasmids, recombinant fusion partners and mutant strains have advanced the possibilities with E. coli. Although often simple for soluble proteins, major obstacles are encountered in the expression of many heterologous proteins and proteins lacking relevant interaction partners in the E. coli cytoplasm. Here we review the current most important strategies for recombinant expression in E. coli. Issues addressed include expression systems in general, selection of host strain, mRNA stability, codon bias, inclusion body formation and prevention, fusion protein technology and site-specific proteolysis, compartment directed secretion and finally co-overexpression technology. The macromolecular background for a variety of obstacles and genetic state-of-the-art solutions are presented.     

Cryopreservation of <Emphasis Type="Italic">Robinia</Emphasis><Emphasis Type="Italic">pseudoacacia</Emphasis>

Cryopreservation of Robinia pseudoacacia explants by vitrification achieved 78% survival following the stepwise preculture of shoot tips in (0.3 + 0.5 + 0.7 M) sucrose with a 80 min incubation in PVS2; compared to 87% survival after desiccation of explants to 30% water content, following 3 days alginate bead (with glycerol and sucrose treatments) preculture in 0.7 M sucrose.     

Linkage disequilibrium in European elite maize germplasm investigated with SSRs

Information about the extent and genomic distribution of linkage disequilibrium (LD) is of fundamental importance for association mapping. The main objectives of this study were to (1) investigate genetic diversity within germplasm groups of elite European maize (Zea mays L.) inbred lines, (2) examine the population structure of elite European maize germplasm, and (3) determine the extent and genomic distribution of LD between pairs of simple sequence repeat (SSR) markers. We examined genetic diversity and LD in a cross section of European and US elite breeding material comprising 147 inbred lines genotyped with 100 SSR markers. For gene diversity within each group, significant (P<0.05) differences existed among the groups. The LD was significant (P<0.05) for 49% of the SSR marker pairs in the 80 flint lines and for 56% of the SSR marker pairs in the 57 dent lines. The ratio of linked to unlinked loci in LD was 1.1 for both germplasm groups. The high incidence of LD suggests that the extent of LD between SSR markers should allow the detection of marker-phenotype associations in a genome scan. However, our results also indicate that a high proportion of the observed LD is generated by forces, such as relatedness, population stratification, and genetic drift, which cause a high risk of detecting false positives in association mapping.