MerA functions as a hypothiocyanous acid reductase and defense mechanism in Staphylococcus aureus

The major pathogen Staphylococcus aureus has to cope with host-derived oxidative stress to cause infections in humans. Here, we report that S. aureus tolerates high concentrations of hypothiocyanous acid (HOSCN), a key antimicrobial oxidant produced in the respiratory tract. We discovered that the flavoprotein disulfide reductase (FDR) MerA protects S. aureus from this oxidant by functioning as a HOSCN reductase, with its deletion sensitizing bacteria to HOSCN. Crystal structures of homodimeric MerA (2.4 Å) with a Cys43–Cys48 intramolecular disulfide, and reduced MerACys43S (1.6 Å) showed the FAD cofactor close to the active site, supporting that MerA functions as a group I FDR. MerA is controlled by the redox-sensitive repressor HypR, which we show to be oxidized to intermolecular disulfides under HOSCN stress, resulting in its inactivation and derepression of merA transcription to promote HOSCN tolerance. Our study highlights the HOSCN tolerance of S. aureus and characterizes the structure and function of MerA as a major HOSCN defense mechanism. Crippling the capacity to respond to HOSCN may be a novel strategy for treating S. aureus infections

Combined use of steady-state fluorescence emission and anisotropy of merocyanine 540 to distinguish crystalline, gel, ripple, and liquid crystalline phases in dipalmitoylphosphatidylcholine bilayers

The various lamellar phases of dipalmitoylphosphadtidylcholine bilayers with and without cholesterol were used to assess the versatility of the fluorescent probe merocyanine 540 through simultaneous measurements of emission intensity, spectral shape, and steady-state anisotropy. Induction of the crystalline phase (Lc') by pre-incubation at 4°C produced a wavelength dependence of anisotropy which was strong at 15 and 25°C, weak at 38°C, and minimal above the main transition (>~41.5°C) or after returning the temperature from 46 to 25°C. The profile of anisotropy values across this temperature range revealed the ability of the probe to detect crystalline, gel (Lβ'), and liquid crystalline (Lα) phases. The temperature dependence of fluorescence intensity was additionally able to distinguish between the ripple (Pβ') and gel phases. In contrast, the shape of the emission spectrum, quantified as the ratio of merocyanine monomer and dimer peaks (585 and 621 nm), was primarily sensitive to the crystalline and gel phases because dimer fluorescence requires a highly-ordered environment. This requirement also explained the diminution of anisotropy wavelength dependence above 25°C. Repetition of experiments with vesicles containing cholesterol allowed creation of a phase map. Superimposition of data from the three simultaneous measurements provided details about the various phase regions in the map not discernible from any one of the three alone. The results were applied to assessment of calcium-induced membrane changes in living cells

Type I Interferon Potentiates IgA Immunity to Respiratory Syncytial Virus Infection During Infancy

© 2018, The Author(s). Respiratory syncytial virus (RSV) infection is the most frequent cause of hospitalization in infants and young children worldwide. Although mucosal RSV vaccines can reduce RSV disease burden, little is known about mucosal immune response capabilities in children. Neonatal or adult mice were infected with RSV; a subset of neonatal mice received interferon alpha (IFN-α) (intranasal) prior to RSV infection. B cells, B cell activating factor (BAFF) and IgA were measured by flow cytometry. RSV specific IgA was measured in nasal washes. Nasal associated lymphoid tissue (NALT) and lungs were stained for BAFF and IgA. Herein, we show in a mouse model of RSV infection that IFN-α plays a dual role as an antiviral and immune modulator and age-related differences in IgA production upon RSV infection can be overcome by IFN-α administration. IFN-α administration before RSV infection in neonatal mice increased RSV-specific IgA production in the nasal mucosa and induced expression of the B-cell activating factor BAFF in NALT. These findings are important, as mucosal antibodies at the infection site, and not serum antibodies, have been shown to protect human adults from experimental RSV infection

Systems biology approach in the development of chemically-defined media for production of protein therapeutics in Chinese hamster ovary cells

Cell culture medium plays a critical role on mammalian cell growth, protein expression and quality. Typical cell culture medium formulations consist of \u3e50 components which include amino acids, vitamins, trace metals, lipids and proteins. Chinese Hamster Ovary (CHO) cells that produce biotherapeutics are propagated in specific cell culture media to ensure robust productivity and product quality. Systems biology has been applied to multiple areas of biological research to gain a better understanding of disease origins and to identify potential new drug targets. Although CHO cells are simpler systems, they share similar biochemistry and cellular pathways. Therefore, leveraging the systems biology knowledge from animal systems and applying these strategic systems biological tools to bioprocess development can be valuable in gaining better understanding of CHO cell culture performance, optimizing cell culture media, and subsequently resulting in better control of the overall production processes. In this presentation, we will present several case studies of various ‘omics tools applied to (1) optimize cell culture medium formulation for improve cell growth and productivity via metabolomics, (2) understand effects of medium components on cellular gene expression via transcriptomics, and on product quality via glycomics, and (3) identify potential cellular protein targets that are affected by stress imposed during production process via proteomics. The development of a statistical model that aims to highlight key metabolites and a machine learning model that identifies significantly important genes which are involved in monoclonal antibody production will also be discussed

Participatory logic modeling in a multi-site initiative to advance implementation science

BACKGROUND: Logic models map the short-term and long-term outcomes that are expected to occur with a program, and thus are an essential tool for evaluation. Funding agencies, especially in the United States (US), have encouraged the use of logic models among their grantees. They also use logic models to clarify expectations for their own funding initiatives. It is increasingly recognized that logic models should be developed through a participatory approach which allows input from those who carry out the program being evaluated. While there are many positive examples of participatory logic modeling, funders have generally not engaged grantees in developing the logic model associated with their own initiatives. This article describes an instance where a US funder of a multi-site initiative fully engaged the funded organizations in developing the initiative logic model. The focus of the case study is Implementation Science Centers in Cancer Control (ISC METHODS: The reflective case study was collectively constructed by representatives of the seven centers funded under ISC RESULTS: The initial logic model for ISC CONCLUSIONS: The IS

Collaboration networks of the implementation science centers for cancer control: A social network analysis

BACKGROUND: Multi-center research initiatives offer opportunities to develop and strengthen connections among researchers. These initiatives often have goals of increased scientific collaboration which can be examined using social network analysis. METHODS: The National Cancer Institute (NCI)-funded Implementation Science Centers in Cancer Control (ISC RESULTS: Of the 192 invitees, 182 network members completed the survey (95%). The most prevalent roles were faculty (60%) and research staff (24%). Almost one-quarter (23%) of members reported advanced expertise in IS, 42% intermediate, and 35% beginner. Most members were female (69%) and white (79%). One-third (33%) of collaboration ties were among members from different centers. Across all collaboration activities, the network had a density of 14%, suggesting moderate cohesion. Degree centralization (0.33) and betweenness centralization (0.07) measures suggest a fairly dispersed network (no single or few central member(s) holding all connections). The most prevalent and densely connected collaboration was in planning/conducting research (1470 ties; 8% density). Practice/policy dissemination had the fewest collaboration, lowest density (284 ties\u27 3% density), and the largest number of non-connected members (n=43). Access to the ISC CONCLUSIONS: Results establish a baseline for assessing the growth of cross-center collaborations, highlighting specific areas in need of particular growth in network collaborations such as increasing engagement of racial and ethnic minorities and trainees or those with less expertise in IS

Multiple Wolbachia strains provide comparative levels of protection against dengue virus infection in Aedes aegypti.

The insect bacterium Wolbachia pipientis is being introgressed into Aedes aegypti populations as an intervention against the transmission of medically important arboviruses. Here we compare Ae. aegypti mosquitoes infected with wMelCS or wAlbB to the widely used wMel Wolbachia strain on an Australian nuclear genetic background for their susceptibility to infection by dengue virus (DENV) genotypes spanning all four serotypes. All Wolbachia-infected mosquitoes were more resistant to intrathoracic DENV challenge than their wildtype counterparts. Blocking of DENV replication was greatest by wMelCS. Conversely, wAlbB-infected mosquitoes were more susceptible to whole body infection than wMel and wMelCS. We extended these findings via mosquito oral feeding experiments, using viremic blood from 36 acute, hospitalised dengue cases in Vietnam, additionally including wMel and wildtype mosquitoes on a Vietnamese nuclear genetic background. As above, wAlbB was less effective at blocking DENV replication in the abdomen compared to wMel and wMelCS. The transmission potential of all Wolbachia-infected mosquito lines (measured by the presence/absence of infectious DENV in mosquito saliva) after 14 days, was significantly reduced compared to their wildtype counterparts, and lowest for wMelCS and wAlbB. These data support the use of wAlbB and wMelCS strains for introgression field trials and the biocontrol of DENV transmission. Furthermore, despite observing significant differences in transmission potential between wildtype mosquitoes from Australia and Vietnam, no difference was observed between wMel-infected mosquitoes from each background suggesting that Wolbachia may override any underlying variation in DENV transmission potential

Spatial Organization and Molecular Correlation of Tumor-Infiltrating Lymphocytes Using Deep Learning on Pathology Images

Beyond sample curation and basic pathologic characterization, the digitized H&E-stained images of TCGA samples remain underutilized. To highlight this resource, we present mappings of tumorinfiltrating lymphocytes (TILs) based on H&E images from 13 TCGA tumor types. These TIL maps are derived through computational staining using a convolutional neural network trained to classify patches of images. Affinity propagation revealed local spatial structure in TIL patterns and correlation with overall survival. TIL map structural patterns were grouped using standard histopathological parameters. These patterns are enriched in particular T cell subpopulations derived from molecular measures. TIL densities and spatial structure were differentially enriched among tumor types, immune subtypes, and tumor molecular subtypes, implying that spatial infiltrate state could reflect particular tumor cell aberration states. Obtaining spatial lymphocytic patterns linked to the rich genomic characterization of TCGA samples demonstrates one use for the TCGA image archives with insights into the tumor-immune microenvironment