LPS Unmasking of Shigella flexneri Reveals Preferential Localisation of Tagged Outer Membrane Protease IcsP to Septa and New Poles

The Shigella flexneri outer membrane (OM) protease IcsP (SopA) is a member of the enterobacterial Omptin family of proteases which cleaves the polarly localised OM protein IcsA that is essential for Shigella virulence. Unlike IcsA however, the specific localisation of IcsP on the cell surface is unknown. To determine the distribution of IcsP, a haemagglutinin (HA) epitope was inserted into the non-essential IcsP OM loop 5 using Splicing by Overlap Extension (SOE) PCR, and IcsP^HA was characterised. Quantum Dot (QD) immunofluorescence (IF) surface labelling of IcsP^HA was then undertaken. Quantitative fluorescence analysis of S. flexneri 2a 2457T treated with and without tunicaymcin to deplete lipopolysaccharide (LPS) O antigen (Oag) showed that IcsP^HA was asymmetrically distributed on the surface of septating and non-septating cells, and that this distribution was masked by LPS Oag in untreated cells. Double QD IF labelling of IcsP^HA and IcsA showed that IcsP^HA preferentially localised to the new pole of non-septating cells and to the septum of septating cells. The localisation of IcsP^HA in a rough LPS S. flexneri 2457T strain (with no Oag) was also investigated and a similar distribution of IcsP^HA was observed. Complementation of the rough LPS strain with rmlD resulted in restored LPS Oag chain expression and loss of IcsP^HA detection, providing further support for LPS Oag masking of surface proteins. Our data presents for the first time the distribution for the Omptin OM protease IcsP, relative to IcsA, and the effect of LPS Oag masking on its detection.     

Genomic and signalling pathway characterization of the NZM panel of melanoma cell lines: A valuable model for studying the impact of genetic diversity in melanoma

Melanoma is a disease associated with a very high mutation burden and thus the possibility of a diverse range of oncogenic mechanisms that allow it to evade therapeutic interventions and the immune system. Here, we describe the characterization of a panel of 102 cell lines from metastatic melanomas (the NZM lines), including using whole‐exome and RNA sequencing to analyse genetic variants and gene expression changes in a subset of this panel. Lines possessing all major melanoma genotypes were identified, and hierarchical clustering of gene expression profiles revealed four broad subgroups of cell lines. Immunogenotyping identified a range of HLA haplotypes as well as expression of neoantigens and cancer–testis antigens in the lines. Together, these characteristics make the NZM panel a valuable resource for cell‐based, immunological and xenograft studies to better understand the diversity of melanoma biology and the responses of melanoma to therapeutic interventions.     

Supply- and Demand-Side Factors Influencing Utilization of Infant and Young Child Feeding Counselling Services in Viet Nam

Adequate utilization of services is critical to maximize the impact of counselling on infant and young child feeding (IYCF), but little is known about factors affecting utilization. Our study examined supply- and demand-side factors associated with the utilization of IYCF counselling services in Viet Nam. We used survey data from mothers with children <2y (n = 1,008) and health staff (n = 60) from the evaluation of a program that embedded IYCF counseling into the existing government health system. The frequency of never users, one-time users, repeat users, and achievers of the recommended minimum number of visits at health facilities were 45.1%, 13.0%, 28.4% and 13.5%, respectively. Poisson regression showed that demand-generation strategies, especially invitation cards, were the key factors determining one-time use (Prevalence ratio, PR 3.0, 95% CI: 2.2–4.2), repeated use (PR 3.2, 95% CI: 2.4–4.2), and achievement of minimum visits (PR 5.5, 95% CI: 3.6–8.4). Higher maternal education was associated with higher utilization both for one-time and repeated use. Being a farmer, belonging to an ethnic minority, and having a wasted child were associated with greater likelihood of achieving the minimum recommended number of visits, whereas child stunting or illness were not. Distance to health center was a barrier to repeated visits. Among supply-side factors, good counselling skills (PR: 1.3–1.8) was the most important factor associated with any service use, whereas longer employment duration and greater work pressure of health center staff were associated with lower utilization. Population attributable risk estimations showed that an additional 25% of the population would have achieved the minimum number of visits if exposed to three demand-generation strategies, and further increased to 49% if the health staff had good counseling skills and low work pressure. Our study provides evidence that demand-generation strategies are essential to increase utilization of facility-based IYCF counselling services in Viet Nam, and may be relevant for increasing and sustaining use of nutrition services in similar contexts.     

Concentrations of high-mobility-group proteins in the nucleus and cytoplasm of several rat tissues

Nuclear and cytoplasmic fractions were isolated from various tissues of the rat by a nonaqueous technique. The high-mobility-group (HMG) proteins were extracted from these fractions with acid and separated by one- and two-dimensional PAGE. The concentrations of high-mobility-group proteins HMG1, HMG2, and HMG17 in the nucleus and cytoplasm were then estimated from the staining intensities of the electrophoretic bands. The cytoplasmic concentrations of these proteins were very low--usually less than 1/30 of those present in the corresponding nuclear fractions. For the tissues studied (liver, kidney, heart, and lung), the concentrations of HMG proteins in the nucleus did not differ significantly from one tissue to another. Averaged over the four tissues investigated, there were 0.28 molecule of HMG1, 0.18 molecule of HMG2, and 0.46 molecule of HMG17 per nucleosome. These values are considerably higher than those that have been reported previously.     

Design and optimisation of orally active TLR7 agonists for the treatment of hepatitis C virus infection

The synthesis and structure-activity relationships of a series of novel interferon inducers are described. Pharmacokinetic studies and efficacy assessment of a series of 8-oxo-3-deazapurine analogues led to the identification of compound 33, a potent and selective agonist of the TLR7 receptor with an excellent in vivo efficacy profile in a mouse model.     

Combined Inactivation of MYC and K-Ras oncogenes reverses tumorigenesis in lung adenocarcinomas and lymphomas

Background

Conditional transgenic models have established that tumors require sustained oncogene activation for tumor maintenance, exhibiting the phenomenon known as “oncogene-addiction.” However, most cancers are caused by multiple genetic events making it difficult to determine which oncogenes or combination of oncogenes will be the most effective targets for their treatment.

Methodology/Principal Findings

To examine how the MYC and K-ras^G12D oncogenes cooperate for the initiation and maintenance of tumorigenesis, we generated double conditional transgenic tumor models of lung adenocarcinoma and lymphoma. The ability of MYC and K-ras^G12D to cooperate for tumorigenesis and the ability of the inactivation of these oncogenes to result in tumor regression depended upon the specific tissue context. MYC-, K-ras^G12D- or MYC/K-ras^G12D-induced lymphomas exhibited sustained regression upon the inactivation of either or both oncogenes. However, in marked contrast, MYC-induced lung tumors failed to regress completely upon oncogene inactivation; whereas K-ras^G12D-induced lung tumors regressed completely. Importantly, the combined inactivation of both MYC and K-ras^G12D resulted more frequently in complete lung tumor regression. To account for the different roles of MYC and K-ras^G12D in maintenance of lung tumors, we found that the down-stream mediators of K-ras^G12D signaling, Stat3 and Stat5, are dephosphorylated following conditional K-ras^G12D but not MYC inactivation. In contrast, Stat3 becomes dephosphorylated in lymphoma cells upon inactivation of MYC and/or K-ras^G12D. Interestingly, MYC-induced lung tumors that failed to regress upon MYC inactivation were found to have persistent Stat3 and Stat5 phosphorylation.

Conclusions/Significance

Taken together, our findings point to the importance of the K-Ras and associated down-stream Stat effector pathways in the initiation and maintenance of lymphomas and lung tumors. We suggest that combined targeting of oncogenic pathways is more likely to be effective in the treatment of lung cancers and lymphomas.     

Shiga toxin production and translocation during microaerobic human colonic infection with Shiga toxin‐producing E. coli O157:H7 and O104:H4

Haemolytic uraemic syndrome caused by Shiga toxin‐producing E. coli (STEC) is dependent on release of Shiga toxins (Stxs) during intestinal infection and subsequent absorption into the bloodstream. An understanding of Stx‐related events in the human gut is limited due to lack of suitable experimental models. In this study, we have used a vertical diffusion chamber system with polarized human colon carcinoma cells to simulate the microaerobic (MA) environment in the human intestine and investigate its influence on Stx release and translocation during STEC O157:H7 and O104:H4 infection. Stx2 was the major toxin type released during infection. Whereas microaerobiosis significantly reduced bacterial growth as well as Stx production and release into the medium, Stx translocation across the epithelial monolayer was enhanced under MA versus aerobic conditions. Increased Stx transport was dependent on STEC infection and occurred via a transcellular pathway other than macropinocytosis. While MA conditions had a similar general effect on Stx release and absorption during infection with STEC O157:H7 and O104:H4, both serotypes showed considerable differences in colonization, Stx production, and Stx translocation which suggest alternative virulence strategies. Taken together, our study suggests that the MA environment in the human colon may modulate Stx‐related events and enhance Stx absorption during STEC infection.