Development of a fluorescent monoclonal antibody‐based assay to measure the allosteric effects of synthetic peptides on self‐oligomerization of AGR2 protein

Many regulatory proteins are homo‐oligomeric and designing assays that measure self‐assembly will provide novel approaches to study protein allostery and screen for novel small molecule modulators of protein interactions. We present an assay to begin to define the biochemical determinants that regulate dimerization of the cancer‐associated oncoprotein AGR2. A two site‐sandwich microtiter assay (^2SMTA) was designed using a DyLight800‐labeled monoclonal antibody that binds to an epitope in AGR2 to screen for synthetic self‐peptides that might regulate dimer stability. Peptides derived from the intrinsically disordered N‐terminal region of AGR2 increase in trans oligomer stability as defined using the ^2SMTA assay. A DSS‐crosslinking assay that traps the AGR2 dimer through K95‐K95 adducts confirmed that Δ45‐AGR2 was a more stable dimer using denaturing gel electrophoresis. A titration of wt‐AGR2, Δ45‐AGR2 (more stable dimer), and monomeric AGR2^E60A revealed that Δ45‐AGR2 was more active in binding to Reptin than either wt‐AGR2 or the AGR2^E60A mutant. Our data have defined a functional role for the AGR2 dimer in the binding to its most well characterized interacting protein, Reptin. The ability to regulate AGR2 oligomerization in trans opens the possibility for developing small molecules that regulate its' biochemical activity as potential cancer therapeutics. The data also highlight the utility of this oligomerization assay to screen chemical libraries for ligands that could regulate AGR2 dimer stability and its' oncogenic potential.     

Massively Parallel Sequencing Reveals the Complex Structure of an Irradiated Human Chromosome on a Mouse Background in the Tc1 Model of Down Syndrome

Down syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21) and presents a complex phenotype that arises from abnormal dosage of genes on this chromosome. However, the individual dosage-sensitive genes underlying each phenotype remain largely unknown. To help dissect genotype – phenotype correlations in this complex syndrome, the first fully transchromosomic mouse model, the Tc1 mouse, which carries a copy of human chromosome 21 was produced in 2005. The Tc1 strain is trisomic for the majority of genes that cause phenotypes associated with DS, and this freely available mouse strain has become used widely to study DS, the effects of gene dosage abnormalities, and the effect on the basic biology of cells when a mouse carries a freely segregating human chromosome. Tc1 mice were created by a process that included irradiation microcell-mediated chromosome transfer of Hsa21 into recipient mouse embryonic stem cells. Here, the combination of next generation sequencing, array-CGH and fluorescence in situ hybridization technologies has enabled us to identify unsuspected rearrangements of Hsa21 in this mouse model; revealing one deletion, six duplications and more than 25 de novo structural rearrangements. Our study is not only essential for informing functional studies of the Tc1 mouse but also (1) presents for the first time a detailed sequence analysis of the effects of gamma radiation on an entire human chromosome, which gives some mechanistic insight into the effects of radiation damage on DNA, and (2) overcomes specific technical difficulties of assaying a human chromosome on a mouse background where highly conserved sequences may confound the analysis. Sequence data generated in this study is deposited in the ENA database, Study Accession number: ERP000439.     

Spry1 and Spry4 Differentially Regulate Human Aortic Smooth Muscle Cell Phenotype via Akt/FoxO/Myocardin Signaling

Background

Changes in the vascular smooth muscle cell (VSMC) contractile phenotype occur in pathological states such as restenosis and atherosclerosis. Multiple cytokines, signaling through receptor tyrosine kinases (RTK) and PI3K/Akt and MAPK/ERK pathways, regulate these phenotypic transitions. The Spry proteins are feedback modulators of RTK signaling, but their specific roles in VSMC have not been established.

Methodology/Principal Findings

Here, we report for the first time that Spry1, but not Spry4, is required for maintaining the differentiated state of human VSMC in vitro. While Spry1 is a known MAPK/ERK inhibitor in many cell types, we found that Spry1 has little effect on MAPK/ERK signaling but increases and maintains Akt activation in VSMC. Sustained Akt signaling is required for VSMC marker expression in vitro, while ERK signaling negatively modulates Akt activation and VSMC marker gene expression. Spry4, which antagonizes both MAPK/ERK and Akt signaling, suppresses VSMC differentiation marker gene expression. We show using siRNA knockdown and ChIP assays that FoxO3a, a downstream target of PI3K/Akt signaling, represses myocardin promoter activity, and that Spry1 increases, while Spry4 decreases myocardin mRNA levels.

Conclusions

Together, these data indicate that Spry1 and Spry4 have opposing roles in VSMC phenotypic modulation, and Spry1 maintains the VSMC differentiation phenotype in vitro in part through an Akt/FoxO/myocardin pathway.     

Inducing Acute Traumatic Coagulopathy In Vitro: The Effects of Activated Protein C on Healthy Human Whole Blood

Introduction

Acute traumatic coagulopathy has been associated with shock and tissue injury, and may be mediated via activation of the protein C pathway. Patients with acute traumatic coagulopathy have prolonged PT and PTT, and decreased activity of factors V and VIII; they are also hypocoagulable by thromboelastometry (ROTEM) and other viscoelastic assays. To test the etiology of this phenomenon, we hypothesized that such coagulopathy could be induced in vitro in healthy human blood with the addition of activated protein C (aPC).

Methods

Whole blood was collected from 20 healthy human subjects, and was “spiked” with increasing concentrations of purified human aPC (control, 75, 300, 2000 ng/mL). PT/PTT, factor activity assays, and ROTEM were performed on each sample. Mixed effect regression modeling was performed to assess the association of aPC concentration with PT/PTT, factor activity, and ROTEM parameters.

Results

In all subjects, increasing concentrations of aPC produced ROTEM tracings consistent with traumatic coagulopathy. ROTEM EXTEM parameters differed significantly by aPC concentration, with stepwise prolongation of clotting time (CT) and clot formation time (CFT), decreased alpha angle (α), impaired early clot formation (a10 and a20), and reduced maximum clot firmness (MCF). PT and PTT were significantly prolonged at higher aPC concentrations, with corresponding significant decreases in factor V and VIII activity.

Conclusion

A phenotype of acute traumatic coagulopathy can be induced in healthy blood by the in vitro addition of aPC alone, as evidenced by viscoelastic measures and confirmed by conventional coagulation assays and factor activity. This may lend further mechanistic insight to the etiology of coagulation abnormalities in trauma, supporting the central role of the protein C pathway. Our findings also represent a model for future investigations in the diagnosis and treatment of acute traumatic coagulopathy.     

Into the deep: the functionality of mesopelagic excursions by an oceanic apex predator

Comprehension of ecological processes in marine animals requires information regarding dynamic vertical habitat use. While many pelagic predators primarily associate with epipelagic waters, some species routinely dive beyond the deep scattering layer. Actuation for exploiting these aphotic habitats remains largely unknown. Recent telemetry data from oceanic whitetip sharks (Carcharhinus longimanus) in the Atlantic show a strong association with warm waters (>20°C) less than 200 m. Yet, individuals regularly exhibit excursions into the meso‐ and bathypelagic zone. In order to examine deep‐diving behavior in oceanic whitetip sharks, we physically recovered 16 pop‐up satellite archival tags and analyzed the high‐resolution depth and temperature data. Diving behavior was evaluated in the context of plausible functional behavior hypotheses including interactive behaviors, energy conservation, thermoregulation, navigation, and foraging. Mesopelagic excursions (n = 610) occurred throughout the entire migratory circuit in all individuals, with no indication of site specificity. Six depth‐versus‐time descent and ascent profiles were identified. Descent profile shapes showed little association with examined environmental variables. Contrastingly, ascent profile shapes were related to environmental factors and appear to represent unique behavioral responses to abiotic conditions present at the dive apex. However, environmental conditions may not be the sole factors influencing ascents, as ascent mode may be linked to intentional behaviors. While dive functionality remains unconfirmed, our study suggests that mesopelagic excursions relate to active foraging behavior or navigation. Dive timing, prey constituents, and dive shape support foraging as the most viable hypothesis for mesopelagic excursions, indicating that the oceanic whitetip shark may regularly survey extreme environments (deep depths, low temperatures) as a foraging strategy. At the apex of these deep‐water excursions, sharks exhibit a variable behavioral response, perhaps, indicating the presence or absence of prey.     

Streptococcus pneumoniae capsule determines disease severity in experimental pneumococcal meningitis

Streptococcus pneumoniae bacteria can be characterized into over 90 serotypes according to the composition of their polysaccharide capsules. Some serotypes are common in nasopharyngeal carriage whereas others are associated with invasive disease, but when carriage serotypes do invade disease is often particularly severe. It is unknown whether disease severity is due directly to the capsule type or to other virulence factors. Here, we used a clinical pneumococcal isolate and its capsule-switch mutants to determine the effect of capsule, in isolation from the genetic background, on severity of meningitis in an infant rat model. We found that possession of a capsule was essential for causing meningitis. Serotype 6B caused significantly more mortality than 7F and this correlated with increased capsule thickness in the cerebrospinal fluid (CSF), a stronger inflammatory cytokine response in the CSF and ultimately more cortical brain damage. We conclude that capsule type has a direct effect on meningitis severity. This is an important consideration in the current era of vaccination targeting a subset of capsule types that causes serotype replacement.     

The Burden of Drug-Resistant Tuberculosis in Papua New Guinea: Results of a Large Population-Based Survey

Background

Reliable estimates of the burden of multidrug-resistant tuberculosis (MDR-TB) are crucial for effective control and prevention of tuberculosis (TB). Papua New Guinea (PNG) is a high TB burden country with limited information on the magnitude of the MDR-TB problem.

Methods

A cross-sectional study was conducted in four PNG provinces: Madang, Morobe, National Capital District and Western Province. Patient sputum samples were tested for rifampicin resistance by the Xpert MTB/RIF assay and those showing the presence of resistance underwent phenotypic susceptibility testing to first- and second-line anti-TB drugs including streptomycin, isoniazid, rifampicin, ethambutol, pyrazinamide, ofloxacin, amikacin, kanamycin and capreomycin.

Results

Among 1,182 TB patients enrolled in the study, MDR-TB was detected in 20 new (2.7%; 95% confidence intervals [CI] 1.1–4.3%) and 24 previously treated (19.1%; 95%CI: 8.5–29.8%) TB cases. No case of extensively drug-resistant TB (XDR-TB) was detected. Thirty percent (6/20) of new and 33.3% (8/24) of previously treated cases with MDR-TB were detected in a single cluster in Western Province.

Conclusion

In PNG the proportion of MDR-TB in new cases is slightly lower than the regional average of 4.4% (95%CI: 2.6–6.3%). A large proportion of MDR-TB cases were identified from a single hospital in Western Province, suggesting that the prevalence of MDR-TB across the country is heterogeneous. Future surveys should further explore this finding. The survey also helped strengthening the use of smear microscopy and Xpert MTB/RIF testing as diagnostic tools for TB in the country.     

A New Metric for Quantifying the Relative Impact of Risk Factors on Loss of Working Life Illustrated in a Population of Working Dogs

In a resource-limited world, organisations attempting to reduce the impact of health or behaviour issues need to choose carefully how to allocate resources for the highest overall impact. However, such choices may not always be obvious. Which has the biggest impact? A large change to a small number of individuals, or a small change to a large number of individuals? The challenge is identifying the issues that have the greatest impact on the population so potential interventions can be prioritised. We addressed this by developing a score to quantify the impact of health conditions and behaviour problems in a population of working guide dogs using data from Guide Dogs, UK. The cumulative incidence of different issues was combined with information about their impact, in terms of reduction in working life, to create a work score. The work score was created at population-level to illustrate issues with the greatest impact on the population and to understand contributions of breeds or crossbreeds to the workforce. An individual work deficit score was also created and means of this score used to illustrate the impact on working life within a subgroup of the population such as a breed, or crossbreed generation. The work deficit scores showed that those removed for behavioural issues had a greater impact on the overall workforce than those removed for health reasons. Additionally trends over time illustrated the positive influence of interventions Guide Dogs have made to improve their workforce. Information highlighted by these scores is pertinent to the effort of Guide Dogs to ensure partnerships are lasting. Recognising that the scores developed here could be transferable to a wide variety of contexts and species, most notably human work force decisions; we discuss possible uses and adaptations such as reduction in lifespan, quality of life and yield in production animals.     

Land–Ocean Connectivity Through Subsidies of Terrestrially Derived Organic Matter to a Nearshore Marine Consumer

Ecosystems - Land–ocean coupling in the form of riverine inputs of terrestrial matter can constitute an energetic subsidy to food webs in nearshore coastal areas. In regions with distinctly...