Quantitative methanol-burning lung model for validating gas-exchange measurements over wide ranges of FIO2

Themethanol-burning lung model has been used as a technique for generatinga predictable ratio of carbon dioxide production (CO₂) to oxygen consumption(O₂) or respiratoryquotient (RQ). Although an accurate RQ can be generated, quantitativelypredictable and adjustableO₂ andCO₂ cannot be generated. Wedescribe a new burner device in which the combustion rate of methanolis always equal to the infusion rate of fuel over an extended range ofO₂ concentrations. This permitsthe assembly of a methanol-burning lung model that is usable withO₂ concentrations up to 100% and provides continuously adjustable and quantitativeO₂ (69-1,525 ml/min)and CO₂ (46-1,016ml/min) at a RQ of 0.667.

     

Dithiothreitol reactivates desacetoxycephalosporin C synthetase after inactivation

Dithiothreitol (DTT) has been found to stimulate the desacetoxycephalosporin C synthetase (expandase) activity of a frozen crude extract of Streptomyces clavuligerus, even in the presence of an optimum concentration of ascorbate. Catalase, both native and heat-denatured, and bovine serum albumin (BSA) also stimulated the enzyme activity but were less active than DTT. Although fresh extracts were sporadically stimulated, frozen extracts or extracts inactivated by shaking at 37°C in air were consistently activated. It is felt that DTT functions as a reactivating, rather than an activaing, agent. Similar effects were observed with extracts of Cephalosporium acremonium.     

Anti-Peptide Monoclonal Antibodies Generated for Immuno-Multiple Reaction Monitoring-Mass Spectrometry Assays Have a High Probability of Supporting Western blot and ELISA

Immunoaffinity enrichment of peptides coupled to targeted, multiple reaction monitoring-mass spectrometry (immuno-MRM) has recently been developed for quantitative analysis of peptide and protein expression. As part of this technology, antibodies are generated to short, linear, tryptic peptides that are well-suited for detection by mass spectrometry. Despite its favorable analytical performance, a major obstacle to widespread adoption of immuno-MRM is a lack of validated affinity reagents because commercial antibody suppliers are reluctant to commit resources to producing anti-peptide antibodies for immuno-MRM while the market is much larger for conventional technologies, especially Western blotting and ELISA. Part of this reluctance has been the concern that affinity reagents generated to short, linear, tryptic peptide sequences may not perform well in traditional assays that detect full-length proteins. In this study, we test the feasibility and success rates of generating immuno-MRM monoclonal antibodies (mAbs) (targeting tryptic peptide antigens) that are also compatible with conventional, protein-based immuno-affinity technologies. We generated 40 novel, peptide immuno-MRM assays and determined that the cross-over success rates for using immuno-MRM monoclonals for Western blotting is 58% and for ELISA is 43%, which compare favorably to cross-over success rates amongst conventional immunoassay technologies. These success rates could most likely be increased if conventional and immuno-MRM antigen design strategies were combined, and we suggest a workflow for such a comprehensive approach. Additionally, the 40 novel immuno-MRM assays underwent fit-for-purpose analytical validation, and all mAbs and assays have been made available as a resource to the community via the Clinical Proteomic Tumor Analysis Consortium''s (CPTAC) Antibody (http://antibodies.cancer.gov) and Assay Portals (http://assays.cancer.gov), respectively. This study also represents the first determination of the success rate (92%) for generating mAbs for immuno-MRM using a recombinant B cell cloning approach, which is considerably faster than the traditional hybridoma approach.The ability to measure specific proteins of interest is critical to the basic sciences and clinical research. To this end, immunoaffinity-based assays such as Western blotting, immunohistochemistry, and ELISAs have been in use for decades, but have several shortcomings including difficulty in multiplexing, a lack of standardization, and a semi-quantitative nature (e.g. Western blotting and immunohistochemistry) (1). Recently, there has been tremendous growth in using the sensitive, specific, multiplexable, and quantitative technology, multiple reaction monitoring-mass spectrometry, to measure tryptic peptides as stoichiometric surrogates for the detection of proteins from complex samples (2–7). The sensitivity of targeted multiple reaction monitoring (MRM)¹ is enhanced 10³–10⁴-fold by coupling it upstream with immunoaffinity enrichment of tryptic peptides in a peptide immuno-MRM assay (8–14). Advantages of immuno-MRM include high specificity, multiplexability (15, 16), and standardization, enabling high inter-laboratory reproducibility (17).The extent to which antibodies generated for immuno-MRM could support widely-used conventional immunoassay formats has not been investigated. This question is important because a lack of validated affinity reagents is a major obstacle to widespread implementation of immuno-MRM, which has considerable analytical advantages over traditional methods. Because the market for immuno-MRM is at present small relative to that for widely adopted conventional immunoassay formats (e.g. Western blotting and ELISA), commercial antibody suppliers are not incentivized to develop content specifically for immuno-MRM assays. Thus, we reasoned that if antibodies could be generated that are capable of supporting both conventional technologies as well as the emerging MRM platform, this might spark commercial interest by increasing the value of the antibodies, ultimately providing reagents to foster widespread implementation of immuno-MRM.Antigens used for antibody generation in conventional assays typically consist of either purified proteins, protein segments of 100–150 amino acids, or synthetic peptide sequences (18, 19). Antigenic prediction algorithms are often used to identify regions of target proteins that are most likely to be exposed on the surface of the protein and, thus, accessible for antibody binding. In contrast, proteotypic peptide antigens are selected for development of antibodies for immuno-MRM based on their uniqueness in the genome and their robust detectability by mass spectrometry, without regard to protein structure (because the protein will be proteolyzed during the assay). Because some widely used conventional immunoassay formats (e.g. Western blotting and indirect ELISA) detect proteins in their denatured form, it was reasonable to ask whether antibodies raised against short, linear, tryptic peptides would also work in these alternative formats.Here, we develop, characterize, and make publicly available 40 novel immuno-MRM assays and the associated monoclonals, and report the success rate of generating recombinant monoclonal antibodies (mAbs) that work in immuno-MRM assays. Furthermore, we determine the cross-over success rates of applying the mAbs in Western blotting and indirect ELISA assays.     

An O Antigen Capsule Modulates Bacterial Pathogenesis in Shigella sonnei

Shigella is the leading cause for dysentery worldwide. Together with several virulence factors employed for invasion, the presence and length of the O antigen (OAg) of the lipopolysaccharide (LPS) plays a key role in pathogenesis. S. flexneri 2a has a bimodal OAg chain length distribution regulated in a growth-dependent manner, whereas S. sonnei LPS comprises a monomodal OAg. Here we reveal that S. sonnei, but not S. flexneri 2a, possesses a high molecular weight, immunogenic group 4 capsule, characterized by structural similarity to LPS OAg. We found that a galU mutant of S. sonnei, that is unable to produce a complete LPS with OAg attached, can still assemble OAg material on the cell surface, but a galU mutant of S. flexneri 2a cannot. High molecular weight material not linked to the LPS was purified from S. sonnei and confirmed by NMR to contain the specific sugars of the S. sonnei OAg. Deletion of genes homologous to the group 4 capsule synthesis cluster, previously described in Escherichia coli, abolished the generation of the high molecular weight OAg material. This OAg capsule strongly affects the virulence of S. sonnei. Uncapsulated knockout bacteria were highly invasive in vitro and strongly inflammatory in the rabbit intestine. But, the lack of capsule reduced the ability of S. sonnei to resist complement-mediated killing and to spread from the gut to peripheral organs. In contrast, overexpression of the capsule decreased invasiveness in vitro and inflammation in vivo compared to the wild type. In conclusion, the data indicate that in S. sonnei expression of the capsule modulates bacterial pathogenesis resulting in balanced capabilities to invade and persist in the host environment.     

Molecular modeling studies demonstrate key mutations that could affect the ligand recognition by influenza AH1N1 neuraminidase

Journal of Molecular Modeling - There is a wide variety of ion channel types with various types of blockers, making research in this field very complicated. To reduce this complexity, it is...     

Perceived Impact of Dental Pain on the Quality of Life of Preschool Children and Their Families

The aim of the present study was to evaluate the perceived impact of dental caries and dental pain on oral health-related quality of life (OHRQoL) among preschool children and their families. A cross-sectional study was conduct with 843 preschool children in Campina Grande, Brazil. Parents/caregivers answered a questionnaire on socio-demographic information, their child’s general/oral health and history of dental pain. The Brazilian version of the Early Childhood Oral Health Impact Scale was administered to determine the perceived impact of caries and dental pain on OHRQoL. The children underwent an oral examination. Logistic regression for complex sample was used to determine associations between the dependent and independent variables (OR: Odds ratio, α = 5%). The independents variables that had a p-value <0.20 in the bivariate analysis were selected for the multivariate model. The prevalence of dental caries and dental pain was 66.3% and 9.4%, respectively. Order of birth of the child, being the middle child (OR: 10.107, 95%CI: 2.008-50.869) and youngest child (OR: 3.276, 95%CI: 1.048-10.284) and dental pain (OR: 84.477, 95%CI: 33.076-215.759) were significant predictors of the perceived impact on OHRQOL for children. Poor perception of oral health was significant predictor of the perceived impact on OHRQOL for family (OR=7.397, 95%CI: 2.190-24.987). Dental caries was not associated with a perceived impact on the ORHQoL of either the children or their families. However, order of child birth and dental pain were indicators of impact of OHRQoL on preschool children and poor perception of oral health was indicators of impact on families.