A Guide to Simple and Informative Binding Assays

The aim of binding assays is to measure interactions between two molecules, such as a protein binding another protein, a small molecule, or a nucleic acid. Hard work is required to prepare reagents, but flaws in the design of many binding experiments limit the information obtained. In particular many experiments fail to measure the affinity of the reactants for each other. This essay describes simple methods to get the most out of valuable reagents in binding experiments

Role of circulating T follicular helper subsets following Ty21a immunization and oral challenge with wild type S. Typhi in humans

Despite decades of intense research, our understanding of the correlates of protection against Salmonella Typhi (S. Typhi) infection and disease remains incomplete. T follicular helper cells (TFH), an important link between cellular and humoral immunity, play an important role in the development and production of high affinity antibodies. While traditional TFH cells reside in germinal centers, circulating TFH (cTFH) (a memory subset of TFH) are present in blood. We used specimens from a typhoid controlled human infection model whereby participants were immunized with Ty21a live attenuated S. Typhi vaccine and then challenged with virulent S. Typhi. Some participants developed typhoid disease (TD) and some did not (NoTD), which allowed us to assess the association of cTFH subsets in the development and prevention of typhoid disease. Of note, the frequencies of cTFH were higher in NoTD than in TD participants, particularly 7 days after challenge. Furthermore, the frequencies of cTFH2 and cTFH17, but not cTFH1 subsets were higher in NoTD than TD participants. However, we observed that ex-vivo expression of activation and homing markers were higher in TD than in NoTD participants, particularly after challenge. Moreover, cTFH subsets produced higher levels of S. Typhi-specific responses (cytokines/chemokines) in both the immunization and challenge phases. Interestingly, unsupervised analysis revealed unique clusters with distinct signatures for each cTFH subset that may play a role in either the development or prevention of typhoid disease. Importantly, we observed associations between frequencies of defined cTFH subsets and anti-S. Typhi antibodies. Taken together, our results suggest that circulating TFH2 and TFH17 subsets might play an important role in the development or prevention of typhoid disease. The contribution of these clusters was found to be distinct in the immunization and/or challenge phases. These results have important implications for vaccines aimed at inducing long-lived protective T cell and antibody responses

The type III secretion system effector SeoC of salmonella enterica subsp. salamae and S. enterica subsp. arizonae ADP-Ribosylates Src and inhibits opsonophagocytosis

Salmonella species utilize type III secretion systems (T3SSs) to translocate effectors into the cytosol of mammalian host cells, subverting cell signaling and facilitating the onset of gastroenteritis. In this study, we compared a draft genome assembly of Salmonella enterica subsp. salamae strain 3588/07 against the genomes of S. enterica subsp. enterica serovar Typhimurium strain LT2 and Salmonella bongori strain 12419. S. enterica subsp. salamae encodes the Salmonella pathogenicity island 1 (SPI-1), SPI-2, and the locus of enterocyte effacement (LEE) T3SSs. Though several key S Typhimurium effector genes are missing (e.g., avrA, sopB, and sseL), S. enterica subsp. salamae invades HeLa cells and contains homologues of S. bongori sboK and sboC, which we named seoC SboC and SeoC are homologues of EspJ from enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), which inhibit Src kinase-dependent phagocytosis by ADP-ribosylation. By screening 73 clinical and environmental Salmonella isolates, we identified EspJ homologues in S. bongori, S. enterica subsp. salamae, and Salmonella enterica subsp. arizonae The β-lactamase TEM-1 reporter system showed that SeoC is translocated by the SPI-1 T3SS. All the Salmonella SeoC/SboC homologues ADP-ribosylate Src E310 in vitro Ectopic expression of SeoC/SboC inhibited phagocytosis of IgG-opsonized beads into Cos-7 cells stably expressing green fluorescent protein (GFP)-FcγRIIa. Concurrently, S. enterica subsp. salamae infection of J774.A1 macrophages inhibited phagocytosis of beads, in a seoC-dependent manner. These results show that S. bongori, S. enterica subsp. salamae, and S. enterica subsp. arizonae share features of the infection strategy of extracellular pathogens EPEC and EHEC and shed light on the complexities of the T3SS effector repertoires of Enterobacteriaceae

Exploring the relationships between International Classification of Functioning, Disability and Health (ICF) constructs of Impairment, Activity Limitation and Participation Restriction in people with osteoarthritis prior to joint replacement

<p>Abstract</p> <p>Background</p> <p>The International Classification of Functioning, Disability and Health (ICF) proposes three main constructs, impairment (I), activity limitation (A) and participation restriction (P). The ICF model allows for all paths between the constructs to be explored, with significant paths likely to vary for different conditions. The relationships between I, A and P have been explored in some conditions but not previously in people with osteoarthritis prior to joint replacement. The aim of this paper is to examine these relationships using separate measures of each construct and structural equation modelling.</p> <p>Methods</p> <p>A geographical cohort of 413 patients with osteoarthritis about to undergo hip and knee joint replacement completed the Aberdeen measures of Impairment, Activity Limitation and Participation Restriction (Ab-IAP). Confirmatory factor analysis was used to test the three factor (I, A, P) measurement model. Structural equation modelling was used to explore the I, A and P pathways in the ICF model.</p> <p>Results</p> <p>There was support from confirmatory factor analysis for the three factor I, A, P measurement model. The structural equation model had good fit [S-B Chi-square = 439.45, df = 149, CFI robust = 0.91, RMSEA robust = 0.07] and indicated significant pathways between I and A (standardised coefficient = 0.76 p < 0.0001) and between A and P (standardised coefficient = 0.75 p < 0.0001). However, the path between I and P was not significant (standardised coefficient = 0.01).</p> <p>Conclusion</p> <p>The significant pathways suggest that treatments and interventions aimed at reducing impairment, such as joint replacement, may only affect P indirectly, through A, however, longitudinal data would be needed to establish this.</p

Young children's research: children aged 4-8 years finding solutions at home and at school

Children's research capacities have become increasingly recognised by adults, yet children remain excluded from the academy, with reports of their research participation generally located in adults' agenda. Such practice restricts children's freedom to make choices in matters affecting them, underestimates children’s capabilities and denies children particular rights. The present paper reports on one aspect of a small-scale critical ethnographic study adopting a constructivist grounded approach to conceptualise ways in which children's naturalistic behaviours may be perceived as research. The study builds on multi-disciplinary theoretical perspectives, embracing 'new' sociology, psychology, economics, philosophy and early childhood education and care (ECEC). Research questions include: 'What is the nature of ECEC research?' and 'Do children’s enquiries count as research?' Initially, data were collected from the academy: professional researchers (n=14) confirmed 'finding solutions' as a research behaviour and indicated children aged 4-8 years, their practitioners and primary carers as 'theoretical sampling'. Consequently, multi-modal case studies were constructed with children (n=138) and their practitioners (n=17) in three ‘good’ schools, with selected children and their primary carers also participating at home. This paper reports on data emerging from children aged 4-8 years at school (n=17) and at home (n=5). Outcomes indicate that participating children found diverse solutions to diverse problems, some of which they set themselves. Some solutions engaged children in high order thinking, whilst others did not; selecting resources and trialing activities engaged children in 'finding solutions'. Conversely, when children's time, provocations and activities were directed by adults, the quality of their solutions was limited, they focused on pleasing adults and their motivation to propose solutions decreased. In this study, professional researchers recognised 'finding solutions' as research behaviour and children aged 4-8 years naturalistically presented with capacities for finding solutions; however, the children's encounters with adults affected the solutions they found

Chemotaxis: a feedback-based computational model robustly predicts multiple aspects of real cell behaviour

The mechanism of eukaryotic chemotaxis remains unclear despite intensive study. The most frequently described mechanism acts through attractants causing actin polymerization, in turn leading to pseudopod formation and cell movement. We recently proposed an alternative mechanism, supported by several lines of data, in which pseudopods are made by a self-generated cycle. If chemoattractants are present, they modulate the cycle rather than directly causing actin polymerization. The aim of this work is to test the explanatory and predictive powers of such pseudopod-based models to predict the complex behaviour of cells in chemotaxis. We have now tested the effectiveness of this mechanism using a computational model of cell movement and chemotaxis based on pseudopod autocatalysis. The model reproduces a surprisingly wide range of existing data about cell movement and chemotaxis. It simulates cell polarization and persistence without stimuli and selection of accurate pseudopods when chemoattractant gradients are present. It predicts both bias of pseudopod position in low chemoattractant gradients and-unexpectedly-lateral pseudopod initiation in high gradients. To test the predictive ability of the model, we looked for untested and novel predictions. One prediction from the model is that the angle between successive pseudopods at the front of the cell will increase in proportion to the difference between the cell's direction and the direction of the gradient. We measured the angles between pseudopods in chemotaxing Dictyostelium cells under different conditions and found the results agreed with the model extremely well. Our model and data together suggest that in rapidly moving cells like Dictyostelium and neutrophils an intrinsic pseudopod cycle lies at the heart of cell motility. This implies that the mechanism behind chemotaxis relies on modification of intrinsic pseudopod behaviour, more than generation of new pseudopods or actin polymerization by chemoattractant

Individual Actin Filaments in a Microfluidic Flow Reveal the Mechanism of ATP Hydrolysis and Give Insight Into the Properties of Profilin

A novel microfluidic approach allows the analysis of the dynamics of individual actin filaments, revealing both their local ADP/ADP-Pi-actin composition and that Pi release is a random mechanism

Combinatorial CRISPR-Cas9 screens for de novo mapping of genetic interactions.

We developed a systematic approach to map human genetic networks by combinatorial CRISPR-Cas9 perturbations coupled to robust analysis of growth kinetics. We targeted all pairs of 73 cancer genes with dual guide RNAs in three cell lines, comprising 141,912 tests of interaction. Numerous therapeutically relevant interactions were identified, and these patterns replicated with combinatorial drugs at 75% precision. From these results, we anticipate that cellular context will be critical to synthetic-lethal therapies