Normal specification of the extraembryonic lineage after somatic nuclear transfer

To examine the establishment and maintenance of trophectoderm (TE) lineage in somatic cloned blastocysts, the expression of Cdx2, a key molecule for specification of TE fate, was immunohistochemically examined simultaneously with Oct4 expression. Cloned mouse embryos were made by nuclear transfer using cumulus cells, tail-tip fibroblasts, and embryonic stem cells. After 96 h of culture, the rates of Oct4-expressing blastocysts were as low as 50% and 60% for cumulus and fibroblast clones, respectively. However, regardless of Oct4 expression, the majority of those cloned blastocysts (> 90%) normally expressed Cdx2. Thus, even though somatic cloned embryos have reduced potential to produce the inner cell mass lineage, the TE lineage can be established and maintained.     

IAP antagonists target cIAP1 to induce TNFalpha-dependent apoptosis

XIAP prevents apoptosis by binding to and inhibiting caspases, and this inhibition can be relieved by IAP antagonists, such as Smac/DIABLO. IAP antagonist compounds (IACs) have therefore been designed to inhibit XIAP to kill tumor cells. Because XIAP inhibits postmitochondrial caspases, caspase 8 inhibitors should not block killing by IACs. Instead, we show that apoptosis caused by an IAC is blocked by the caspase 8 inhibitor crmA and that IAP antagonists activate NF-kappaB signaling via inhibtion of cIAP1. In sensitive tumor lines, IAP antagonist induced NF-kappaB-stimulated production of TNFalpha that killed cells in an autocrine fashion. Inhibition of NF-kappaB reduced TNFalpha production, and blocking NF-kappaB activation or TNFalpha allowed tumor cells to survive IAC-induced apoptosis. Cells treated with an IAC, or those in which cIAP1 was deleted, became sensitive to apoptosis induced by exogenous TNFalpha, suggesting novel uses of these compounds in treating cancer.     

Effect of volume of oocyte cytoplasm on embryo development after parthenogenetic activation, intracytoplasmic sperm injection, or somatic cell nuclear transfer

Animal cloning methods are now well described and are becoming routine. Yet, the frequency at which live cloned offspring are produced remains below 5%, irrespective of the nuclear donor species or cell type. One possible explanation is that the reprogramming factor(s) of each oocyte is insufficient or not properly adapted for the receipt of a somatic cell nucleus, because it is naturally prepared only for the receipt of a gamete. Here, we have increased the oocyte volume by oocyte fusion and examined its subsequent development. We constructed oocytes with volumes two to nine times greater than the normal volume by the electrofusion or mechanical fusion of intact and enucleated oocytes. We examined their in vitro and in vivo developmental potential after parthenogenetic activation, intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). When the fused oocytes were activated parthenogenetically, most developed to morulae or blastocysts, regardless of their original size. Diploid fused oocytes were fertilized by ICSI and developed normally and after embryo transfer, we obtained 12 (4-15%) healthy and fertile offspring. However, enucleated fused oocytes could not support the development of mice cloned by SCNT. These results suggest that double fused oocytes have normal potential for development after fertilization, but oocytes with extra cytoplasm do not have enhanced reprogramming potential.     

A Randomised Trial Evaluating the Safety and Immunogenicity of the Novel Single Oral Dose Typhoid Vaccine M01ZH09 in Healthy Vietnamese Children

Background

The emergence of drug resistant typhoid fever is a major public health problem, especially in Asia. An oral single dose typhoid vaccine would have major advantages. M01ZH09 is a live oral single dose candidate typhoid vaccine containing Salmonella enterica serovar Typhi (Ty2 aroC ⁻ ssaV ⁻) ZH9 with two independently attenuating deletions. Studies in healthy adults demonstrated immunogenicity and an acceptable safety profile.

Objectives

We conducted a randomised placebo controlled, single-blind trial to evaluate the safety and immunogenicity of M01ZH09 in healthy Vietnamese children aged 5 to 14 years.

Methods

Subjects were randomly assigned to receive either a nominal dose of 5×10⁹ CFU of M01ZH09 or placebo and were followed up for 28 days. The primary safety outcome was the proportion of subjects with any adverse event attributed to M01ZH09. The primary immunogenicity endpoint was the proportion of subjects who showed a positive immune response to M01ZH09 in the Salmonella Typhi lipopolysaccharide (LPS) specific serum IgA and IgG ELISA.

Principal Findings

One hundred and fifty-one children were enrolled, 101 subjects received M01ZH09 and 50 subjects received placebo. An intention to treat analysis was conducted. There were no serious adverse events and no bacteraemias. In the M01ZH09 group, 26 (26%; 95% CI, 18–5%) of 101 subjects experienced adverse events compared to 11 (22%; 95% CI, 12–36%) of 50 subjects in the placebo group (odds ratio (OR) [95%CI]  = 1.23 [0.550–2.747]; p = 0.691). Faecal shedding of S. Typhi (Ty2 aroC ⁻ ssaV ⁻) ZH9 was detected in 51 (51%; 95% CI, 41–61%) of 100 M01ZH09 subjects. No shedding was detected beyond day 3. A positive immune response, defined as 70% increase (1.7 fold change) in LPS specific serum IgG (day 14 or 28) and/or 50% increase (1.5 fold change) in LPS specific serum IgA (day 7 or 14) from baseline was detected in 98 (97%; 95% CI, 92–99%) of 101 M01ZH09 recipients and 8 (16%; 95% CI, 7–29%) of 50 placebo recipients. Twenty-eight (100%; 95% CI, 88–100%) of 28 vaccine recipients who were evaluated in the LPS specific IgA ELISPOT assay showed a positive response compared to none of the 14 placebo recipients tested.

Conclusions

This was the first phase II trial of a novel oral candidate typhoid vaccine in children in an endemic country. M01ZH09 had an appropriate safety profile and was immunogenic in children.

Trial Registration

Controlled-trials.comISRCTN91111837     

Novel Staphylococcal Glycosyltransferases SdgA and SdgB Mediate Immunogenicity and Protection of Virulence-Associated Cell Wall Proteins

Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR). The prototype member of this family is clumping factor A (ClfA), a key virulence factor that mediates adhesion to host tissues by binding to extracellular matrix proteins such as fibrinogen. However, the biological siginificance of the SDR-domain and its implication for pathogenesis remain poorly understood. Here, we identified two novel bacterial glycosyltransferases, SdgA and SdgB, which modify all SDR-proteins in these two bacterial species. Genetic and biochemical data demonstrated that these two glycosyltransferases directly bind and covalently link N-acetylglucosamine (GlcNAc) moieties to the SDR-domain in a step-wise manner, with SdgB appending the sugar residues proximal to the target Ser-Asp repeats, followed by additional modification by SdgA. GlcNAc-modification of SDR-proteins by SdgB creates an immunodominant epitope for highly opsonic human antibodies, which represent up to 1% of total human IgG. Deletion of these glycosyltransferases renders SDR-proteins vulnerable to proteolysis by human neutrophil-derived cathepsin G. Thus, SdgA and SdgB glycosylate staphylococcal SDR-proteins, which protects them against host proteolytic activity, and yet generates major eptopes for the human anti-staphylococcal antibody response, which may represent an ongoing competition between host and pathogen.     

Toward the production of flavone-7-O-β-d-glucopyranosides using Arabidopsis glycosyltransferase in Escherichia coli

Flavonoid glycosides are highly attractive targets due to their dominant roles in clinical, cosmetic production and in the food industry. In this research, an Escherichia coli strain bearing the reconstructed uridine-diphosphate glucose (UDP-glucose) pathway cassette and a putative glycosyltransferase from Arabidopsis thaliana, was developed as a host for the production of apigenin-7-O-β-d-glucoside (APG) and baicalein-7-O-β-d-glucoside (BCG) from exogenously supplied flavone aglycones (apigenin and baicalein, respectively). In order to improve the yield, genetic engineering of E. coli strains for optimization of intracellular UDP-glucose generation, as well as media optimization were carried out. The production was scaled up using a fed batch fermentation, and the maximal yield of products reached 90.88 μM (39.28 mg L^?1) and 76.82 μM (33.19 mg L^?1) of APG and BCG, respectively. And, the maximum bioconversion rate corresponded to 90.88% and 76.82% of apigenin and baicalein, respectively.     

Engineering co-culture system for production of apigetrin in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Microbial cells have extensively been utilized to produce value-added bioactive compounds. Based on advancement in protein engineering, DNA recombinant technology, genome engineering, and metabolic remodeling, the microbes can be re-engineered to produce industrially and medicinally important platform chemicals. The emergence of co-culture system which reduces the metabolic burden and allows parallel optimization of the engineered pathway in a modular fashion restricting the formation of undesired byproducts has become an alternative way to synthesize and produce bioactive compounds. In this study, we present genetically engineered E. coli-based co-culture system to the de novo synthesis of apigetrin (APG), an apigenin-7-O-β-d-glucopyranoside of apigenin. The culture system consists of an upstream module including 4-coumarate: CoA ligase (4CL), chalcone synthase, chalcone flavanone isomerase (CHS, CHI), and flavone synthase I (FNSI) to synthesize apigenin (API) from p-coumaric acid (PCA). Whereas, the downstream system contains a metabolizing module to enhance the production of UDP-glucose and expression of glycosyltransferase (PaGT3) to convert API into APG. To accomplish this improvement in titer, the initial inoculum ratio of strains for making the co-culture system, temperature, and media component was optimized. Following large-scale production, a yield of 38.5 µM (16.6 mg/L) of APG was achieved. In overall, this study provided an efficient tool to synthesize bioactive compounds in microbial cells.