High-plex immunofluorescence imaging and traditional histology of the same tissue section for discovering image-based biomarkers.

Precision medicine is critically dependent on better methods for diagnosing and staging disease and predicting drug response. Histopathology using hematoxylin and eosin (H&E)-stained tissue (not genomics) remains the primary diagnostic method in cancer. Recently developed highly multiplexed tissue imaging methods promise to enhance research studies and clinical practice with precise, spatially resolved single-cell data. Here, we describe the 'Orion' platform for collecting H&E and high-plex immunofluorescence images from the same cells in a whole-slide format suitable for diagnosis. Using a retrospective cohort of 74 colorectal cancer resections, we show that immunofluorescence and H&E images provide human experts and machine learning algorithms with complementary information that can be used to generate interpretable, multiplexed image-based models predictive of progression-free survival. Combining models of immune infiltration and tumor-intrinsic features achieves a 10- to 20-fold discrimination between rapid and slow (or no) progression, demonstrating the ability of multimodal tissue imaging to generate high-performance biomarkers.

Assessment of Protective Action Decision Making during Nuclear Power Plant Biennial Exercises.

ABSTRACT: Radiological emergency preparedness for commercial nuclear power plants provides planning for implementing predetermined, prompt protective actions such as evacuation and shelter-in-place. In the event of a significant radiological release, onsite emergency response organizations will notify offsite response organizations and provide a protective action recommendation. The cognizant offsite authority will then make a protective action decision and inform the public of the need to act. Both the protective action recommendation and decision are driven by US Environmental Protection Agency protective action guides. Protective action strategies contain conservatisms and are intended to balance protection against other factors to ensure that actions result in more benefit than harm. But added conservatism can potentially shift the risks to those inherent to the protective action with no added benefit of protection. Protective action recommendations and protective action decisions made during biennial exercises were analyzed to assess how well they comport with the protective action guides. Trends in precautionary actions and the use of potassium iodide were also investigated. The analysis shows that protective action decisions typically exceed the protective action recommendation, resulting in an increase in the number of potential evacuees. However, exercise dose projection data do not appear to support such extensive initial evacuation decisions based on consideration of the protective action guides.

Stable binding to phosphatidylserine-containing membranes requires conserved arginine residues in tandem C domains of blood coagulation factor VIII.

At sites of vascular damage, factor VIII (fVIII) is proteolytically activated by thrombin and binds to activated platelet surfaces with activated factor IX (fIXa) to form the intrinsic "tenase" complex. Previous structural and mutational studies of fVIII have identified the C1 and C2 domains in binding to negatively charged membrane surfaces through ß-hairpin loops with solvent-exposed hydrophobic residues and a ring of positively charged basic residues. Several hemophilia A-associated mutations within the C domains are suggested to disrupt lipid binding, preventing formation of the intrinsic tenase complex. In this study, we devised a novel platform for generating recombinant C1, C2, and C1C2 domain constructs and performed mutagenesis of several charged residues proximal to the putative membrane binding region of each C domain. Binding measurements between phosphatidylserine (PS)-containing lipid membrane surfaces and fVIII C domains demonstrated an ionic strength dependence on membrane binding affinity. Mutations to basic residues adjacent to the surface-exposed hydrophobic regions of C1 and C2 differentially disrupted membrane binding, with abrogation of binding occurring for mutations to conserved arginine residues in the C1 (R2163) and C2 (R2320) domains. Lastly, we determined the X-ray crystal structure of the porcine fVIII C2 domain bound to o-phospho-L-serine, the polar headgroup of PS, which binds to a basic cleft and makes charge-charge contact with R2320. We conclude that basic clefts in the fVIII C domains bind to PS-containing membranes through conserved arginine residues via a C domain modularity, where each C domain possesses modest electrostatic-dependent affinity and tandem C domains are required for high affinity binding.

A novel TEM contrasting technique for extracellular polysaccharides in in vitro biofilms.

In the past, the visualization of the extracellular matrix of biofilms on an ultrastructural level was hampered by shrinkage artifacts. In addition, the reproducible contrasting of extracellular polysaccharides (EPS) has not satisfactorily been solved. Here we describe a method overcoming these difficulties, which produces artifact-free transmission electron microscopic (TEM) images using multispecies biofilms grown in vitro. Sufficient contrast was achieved by replacing Schiff's reagent with the osmiophilic amino acid methionine. In addition, shrinkage was avoided by replacing the classical dehydration agents with ethylene glycol and 1,2-pentanediol. Applying this method provided images of biofilms with an intact matrix in which differentially contrasted bacteria were embedded. All six members of the biofilm consortium (Streptococcus sobrinus, Streptococcus oralis, Veillonella dispar, Fusobacterium nucleatum, Actinomyces naeslundii, and Candida albicans) could be distinguished. Within the matrix, structural differences of EPS, probably due to different proportions of alpha-1,3 and alpha-1,6 linkages, were apparent. Fibrilar polysaccharides were evident around microcolonies of S. sobrinus, and fluffy polysaccharides were detected in the vicinity of S. oralis microcolonies. The ultrastructure of biofilms prepared for TEM using this method allows the imaging of undistorted EPS as well as the differentiated contrasting of the six microbial species of the in vitro biofilm model. This is a major step forward in determining the spatial arrangement of microorganisms in biofilms on an ultrastructural level.