Cutting edge: bacterial lipoprotein induces endotoxin-independent tolerance to septic shock

Tolerance to bacterial cell wall components is an adaptive host response. Endotoxin/LPS tolerance is characterized by a survival advantage against subsequent lethal LPS challenge. However, it is uncertain whether LPS tolerance can afford protection against other septic challenges. In this study, we show that tolerance induced by bacterial lipoprotein (BLP) protects mice against not only BLP-induced lethality, but also LPS-, live bacteria-, and polymicrobial sepsis-induced lethality. In contrast, LPS tolerance offers no survival benefit against the latter two challenges. Furthermore, induction of BLP tolerance results in overexpression of complement receptor type 3 and FcgammaIII/IIR on neutrophils (polymorphonuclear neutrophils) and peritoneal macrophages, with increased bacterial recognition and bactericidal activity, whereas LPS-tolerized mice exhibit an impaired ability to ingest and to kill bacteria. These results indicate that BLP tolerance is a novel adaptive host response associated with a unique protective effect during septic shock.     

Promoter DNA methylation couples genome-defence mechanisms to epigenetic reprogramming in the mouse germline

Mouse primordial germ cells (PGCs) erase global DNA methylation (5mC) as part of the comprehensive epigenetic reprogramming that occurs during PGC development. 5mC plays an important role in maintaining stable gene silencing and repression of transposable elements (TE) but it is not clear how the extensive loss of DNA methylation impacts on gene expression and TE repression in developing PGCs. Using a novel epigenetic disruption and recovery screen and genetic analyses, we identified a core set of germline-specific genes that are dependent exclusively on promoter DNA methylation for initiation and maintenance of developmental silencing. These gene promoters appear to possess a specialised chromatin environment that does not acquire any of the repressive H3K27me3, H3K9me2, H3K9me3 or H4K20me3 histone modifications when silenced by DNA methylation. Intriguingly, this methylation-dependent subset is highly enriched in genes with roles in suppressing TE activity in germ cells. We show that the mechanism for developmental regulation of the germline genome-defence genes involves DNMT3B-dependent de novo DNA methylation. These genes are then activated by lineage-specific promoter demethylation during distinct global epigenetic reprogramming events in migratory (～E8.5) and post-migratory (E10.5-11.5) PGCs. We propose that genes involved in genome defence are developmentally regulated primarily by promoter DNA methylation as a sensory mechanism that is coupled to the potential for TE activation during global 5mC erasure, thereby acting as a failsafe to ensure TE suppression and maintain genomic integrity in the germline.     

A PCR-directed cell-free approach to optimize protein expression using diverse fusion tags

N-terminal fusion tags that enhance translation initiation or protein solubility are often used to facilitate protein overexpression. As the optimal tag for a given target protein cannot be predicted a priori, valuable time can be lost in cloning and manipulating the corresponding gene to generate different fusion constructs for expression analysis. We have developed a cell-free strategy that consolidates these steps, enabling the utility of a panel of nine fusion-tags to be determined within one to two days. This approach exploits the fact that PCR-amplified DNA can be used as a template for cell-free protein synthesis. Overlap/extension PCR using the TEV protease site as the overlap region allows the fusion of different T7 promoter (T7p)-tag-TEV DNA fragments with a TEV-gene-T7 terminator (T7ter) fragment. For tag sequences where the TEV site is not compatible, a short C?G? repeat (CGr) sequence can be used as the overlap region. The resulting T7p-tag-TEV-gene-T7ter constructs are then used as templates for PCR-directed cell-free protein synthesis to identify which tag-TEV-gene fusion protein produces the highest amount of soluble protein. We have successfully applied this approach to the overexpression of the Adiponectin hypervariable domain (AHD). Five of the nine N-terminal fusion tags tested enabled the synthesis of soluble recombinant protein. The best of these was the Peptidyl-prolylcis-trans isomerise B (PpiB) fusion tag which produces 1mg/ml amounts of soluble fusion protein. PpiB is an example of a new class of fusion tag known as the "stress-responsive proteins". Our results suggest that this cell-free fusion-tag expression screen facilitates the rapid identification of suitable fusion-tags that overcome issues such as poor expression and insolubility, often encountered using conventional approaches.     

Identification of a new segment involved in cagA 3' region variation of Helicobacter pylori

The cagA 3' region shows marked variation among Helicobacter pylori strains. Two segments of 102 bp and 57 bp are reportedly responsible for this variation. We analysed the cagA 3' region in 70 H. pylori strains using polymerase chain reaction and sequencing. We found that another segment, namely beta segment, was also involved in the variation of this region. The beta segment was 105 bp long and located between the aforementioned two segments. Six genotypes were identified based on the structure of the cagA 3' region. No relationship was found between these genotypes and the clinical outcomes or vacA genotypes. The numbers of tyrosine phosphorylation sites within the cagA 3' region varied among strains, but this was not related to the cagA genotypes. Our data suggest that the cagA 3' region is significantly variable. It appears that the variation of the cagA 3' region might contribute to the modification of virulence.     

Magnetic core-shell nanoparticles for drug delivery by nebulization

Background

Aerosolized therapeutics hold great potential for effective treatment of various diseases including lung cancer. In this context, there is an urgent need to develop novel nanocarriers suitable for drug delivery by nebulization. To address this need, we synthesized and characterized a biocompatible drug delivery vehicle following surface coating of Fe₃O₄ magnetic nanoparticles (MNPs) with a polymer poly(lactic-co-glycolic acid) (PLGA). The polymeric shell of these engineered nanoparticles was loaded with a potential anti-cancer drug quercetin and their suitability for targeting lung cancer cells via nebulization was evaluated.

Results

Average particle size of the developed MNPs and PLGA-MNPs as measured by electron microscopy was 9.6 and 53.2 nm, whereas their hydrodynamic swelling as determined using dynamic light scattering was 54.3 nm and 293.4 nm respectively. Utilizing a series of standardized biological tests incorporating a cell-based automated image acquisition and analysis procedure in combination with real-time impedance sensing, we confirmed that the developed MNP-based nanocarrier system was biocompatible, as no cytotoxicity was observed when up to 100 μg/ml PLGA-MNP was applied to the cultured human lung epithelial cells. Moreover, the PLGA-MNP preparation was well-tolerated in vivo in mice when applied intranasally as measured by glutathione and IL-6 secretion assays after 1, 4, or 7 days post-treatment. To imitate aerosol formation for drug delivery to the lungs, we applied quercitin loaded PLGA-MNPs to the human lung carcinoma cell line A549 following a single round of nebulization. The drug-loaded PLGA-MNPs significantly reduced the number of viable A549 cells, which was comparable when applied either by nebulization or by direct pipetting.

Conclusion

We have developed a magnetic core-shell nanoparticle-based nanocarrier system and evaluated the feasibility of its drug delivery capability via aerosol administration. This study has implications for targeted delivery of therapeutics and poorly soluble medicinal compounds via inhalation route.     

Natural and modified (1-->3)-beta-D-glucans in health promotion and disease alleviation

A number of polysaccharides with beta-glycosidic linkage are widespread in nature in a variety of sources. All have a common structure and the (1-->3)-beta-D-glucan backbone is essential. They have attracted attention over the years because of their bioactive and medicinal properties. In many cases their functional role is a mystery, in others it is well established. Because of their insoluble chemical nature, particulate (1-->3)-beta-D-glucans are not suitable for many medical applications. Various methods of changing or modifying the beta-D-glucan chemical structure and transforming it to a soluble form have been published. The beta-D-glucan bioactive properties can be affected positively or negatively by such modifications. This review examines beta-glucan sources in nature, health effects and structure-activity relationships. It presents the current state of beta-D-glucan solubilization methods and discusses their effectiveness and application possibilities for the future.     

Investing in early human development: Timing and economic efficiency

Policy discussions to ameliorate socioeconomic (SES) inequalities are increasingly focused on investments in early childhood. Yet such interventions are costly to implement, and clear evidence on the optimal time to intervene to yield a high economic and social return in the future is meagre. The majority of successful early childhood interventions start in the preschool years. However socioeconomic gradients in cognitive skills, socio-emotional functioning and health can be observed by age three, suggesting that preventative programmes starting earlier in childhood may be even more effective. We discuss the optimal timing of early childhood intervention with reference to recent research in developmental neuroscience. We motivate the need for early intervention by providing an overview of the impact of adverse risk factors during the antenatal and early childhood periods on outcomes later in life. We provide a brief review of the economic rationale for investing early in life and propose the “antenatal investment hypothesis”. We conclude by discussing a suite of new European interventions that will inform this optimal timing debate.     

The p53 Codon 72 PRO/PRO Genotype May Be Associated with Initial Central Visual Field Defects in Caucasians with Primary Open Angle Glaucoma

Background

Loss of vision in glaucoma is due to apoptotic retinal ganglion cell loss. While p53 modulates apoptosis, gene association studies between p53 variants and glaucoma have been inconsistent. In this study we evaluate the association between a p53 variant functionally known to influence apoptosis (codon 72 Pro/Arg) and the subset of primary open angle glaucoma (POAG) patients with early loss of central visual field.

Methods

Genotypes for the p53 codon 72 polymorphism (Pro/Arg) were obtained for 264 POAG patients and 400 controls from the U.S. and in replication studies for 308 POAG patients and 178 controls from Australia (GIST). The glaucoma patients were divided into two groups according to location of initial visual field defect (either paracentral or peripheral). All cases and controls were Caucasian with European ancestry.

Results

The p53-PRO/PRO genotype was more frequent in the U.S. POAG patients with early visual field defects in the paracentral regions compared with those in the peripheral regions or control group (p = 2.7×10⁻⁵). We replicated this finding in the GIST cohort (p  = 7.3×10⁻³, and in the pooled sample (p = 6.6×10⁻⁷) and in a meta-analysis of both the US and GIST datasets (1.3×10⁻⁶, OR 2.17 (1.58–2.98 for the PRO allele).

Conclusions

These results suggest that the p53 codon 72 PRO/PRO genotype is potentially associated with early paracentral visual field defects in primary open-angle glaucoma patients.