Membership and behavior of ultra-low-diversity pathogen communities present in the gut of humans during prolonged critical illness.

UnlabelledWe analyzed the 16S rRNA amplicon composition in fecal samples of selected patients during their prolonged stay in an intensive care unit (ICU) and observed the emergence of ultra-low-diversity communities (1 to 4 bacterial taxa) in 30% of the patients. Bacteria associated with the genera Enterococcus and Staphylococcus and the family Enterobacteriaceae comprised the majority of these communities. The composition of cultured species from stool samples correlated to the 16S rRNA analysis and additionally revealed the emergence of Candida albicans and Candida glabrata in ~75% of cases. Four of 14 ICU patients harbored 2-member pathogen communities consisting of one Candida taxon and one bacterial taxon. Bacterial members displayed a high degree of resistance to multiple antibiotics. The virulence potential of the 2-member communities was examined in C. elegans during nutrient deprivation and exposure to opioids in order to mimic local conditions in the gut during critical illness. Under conditions of nutrient deprivation, the bacterial members attenuated the virulence of fungal members, leading to a "commensal lifestyle." However, exposure to opioids led to a breakdown in this commensalism in 2 of the ultra-low-diversity communities. Application of a novel antivirulence agent (phosphate-polyethylene glycol [Pi-PEG]) that creates local phosphate abundance prevented opioid-induced virulence among these pathogen communities, thus rescuing the commensal lifestyle. To conclude, the gut microflora in critically ill patients can consist of ultra-low-diversity communities of multidrug-resistant pathogenic microbes. Local environmental conditions in gut may direct pathogen communities to adapt to either a commensal style or a pathogenic style.ImportanceDuring critical illness, the normal gut microbiota becomes disrupted in response to host physiologic stress and antibiotic treatment. Here we demonstrate that the community structure of the gut microbiota during prolonged critical illness is dramatically changed such that in many cases only two-member pathogen communities remain. Most of these ultra-low-membership communities display low virulence when grouped together (i.e., a commensal lifestyle); individually, however, they can express highly harmful behaviors (i.e., a pathogenic lifestyle). The commensal lifestyle of the whole community can be shifted to a pathogenic one in response to host factors such as opioids that are released during physiologic stress and critical illness. This shift can be prevented by using compounds such as Pi-PEG15-20 that interrupt bacterial virulence expression. Taking the data together, this report characterizes the plasticity seen with respect to the choice between a commensal lifestyle and a pathogenic lifestyle among ultra-low-diversity pathogen communities that predominate in the gut during critical illness and offers novel strategies for prevention of sepsis

Colocalization of neurons in optical coherence microscopy and Nissl-stained histology in Brodmann’s area 32 and area 21

Published in final edited form as: Brain Struct Funct. 2019 January ; 224(1): 351–362. doi:10.1007/s00429-018-1777-z.Optical coherence tomography is an optical technique that uses backscattered light to highlight intrinsic structure, and when applied to brain tissue, it can resolve cortical layers and fiber bundles. Optical coherence microscopy (OCM) is higher resolution (i.e., 1.25 µm) and is capable of detecting neurons. In a previous report, we compared the correspondence of OCM acquired imaging of neurons with traditional Nissl stained histology in entorhinal cortex layer II. In the current method-oriented study, we aimed to determine the colocalization success rate between OCM and Nissl in other brain cortical areas with different laminar arrangements and cell packing density. We focused on two additional cortical areas: medial prefrontal, pre-genual Brodmann area (BA) 32 and lateral temporal BA 21. We present the data as colocalization matrices and as quantitative percentages. The overall average colocalization in OCM compared to Nissl was 67% for BA 32 (47% for Nissl colocalization) and 60% for BA 21 (52% for Nissl colocalization), but with a large variability across cases and layers. One source of variability and confounds could be ascribed to an obscuring effect from large and dense intracortical fiber bundles. Other technical challenges, including obstacles inherent to human brain tissue, are discussed. Despite limitations, OCM is a promising semi-high throughput tool for demonstrating detail at the neuronal level, and, with further development, has distinct potential for the automatic acquisition of large databases as are required for the human brain.Accepted manuscrip

Liposome Encapsulation Of A Photochemical No Precursor For Controlled Nitric Oxide Release And Simultaneous Fluorescence Imaging

Described are photochemical studies of the nitric oxide precursors, trans-Cr(L)(ONO)(2)(+) (L = cyclam = 1,4,8,11-tetraazacyclotetradecane, CrONO, or L = mac = 5,7-dimethyl-6-anthracenylcyclam, mac-CrONO) encapsulated in phosphatidylcholine liposomes. The liposomes provide a means to maintain a localized high concentration of NO releasing complexes and are easily modified for in vivo targeting through self-assembly. Steady, controlled release of NO is seen after photolysis of the liposome-encapsulated CrONO as compared to the burst of NO release seen by the unencapsulated complex in oxygenated solutions. The quantum yields for photochemical NO release from liposome-encapsulated CrONO and mac-CrONO were determined in both oxygenated and anoxic solutions. The quantum yield for NO release in oxygenated solution for encapsulated CrONO was more than 5 times larger than that of unencapsulated CrONO, thus the net NO released after photolysis in oxygenated solutions is enhanced by encapsulation of CrONO in liposomes. Encapsulated mac-CrONO shows NO release after photolysis with low-intensity blue light. Furthermore, the fluorescence of mac-CrONO can be detected through the liposomes, thus allowing for development of theranostic NO delivery vessels where tracking and imaging can occur simultaneously with therapeutic NO release. This work provides insight into the development of multifunctional liposome constructs for disease theranostics

Structure and Dynamics of Hybrid Colloid-Polyelectrolyte Coacervates: Insights from Molecular Simulations

Electrostatic interactions in polymeric systems are responsible for a wide range of liquid-liquid phase transitions that are of importance for biology and materials science. Such transitions are referred to as complex coacervation, and recent studies have sought to understand the underlying physics and chemistry. Most theoretical and simulation efforts to date have focused on oppositely charged linear polyelectrolytes, which adopt nearly ideal-coil conformations in the condensed phase. However, when one of the coacervate components is a globular protein, a better model of complexation should replace one of the species with a spherical charged particle or colloid. In this work, we perform coarse-grained simulations of colloid-polyelectrolyte coacervation using a spherical model for the colloid. Simulation results indicate that the electroneutral cell of the resulting (hybrid) coacervates consists of a polyelectrolyte layer adsorbed on the colloid. Power laws for the structure and the density of the condensed phase, which are extracted from simulations, are found to be consistent with the adsorption-based scaling theory of coacervation. The coacervates remain amorphous (disordered) at a moderate colloid charge, $Q$, while an intra-coacervate colloidal crystal is formed above a certain threshold, at $Q > Q^{*}$. In the disordered coacervate, if $Q$ is sufficiently low, colloids diffuse as neutral non-sticky nanoparticles in the semidilute polymer solution. For higher $Q$, adsorption is strong and colloids become effectively sticky. Our findings are relevant for the coacervation of polyelectrolytes with proteins, spherical micelles of ionic surfactants, and solid organic or inorganic nanoparticles

Multivalent ions induce lateral structural inhomogeneities in polyelectrolyte brushes

Subtle details about a polyelectrolyte's surrounding environment can dictate its structural features and potential applications. Atomic force microscopy (AFM), surface forces apparatus (SFA) measurements, and coarse-grained molecular dynamics simulations are combined to study the structure of planar polyelectrolyte brushes [poly(styrenesulfonate), PSS] in a variety of solvent conditions. More specifically, AFM images provide a first direct visualization of lateral inhomogeneities on the surface of polyelectrolyte brushes collapsed in solutions containing trivalent counterions. These images are interpreted in the context of a coarse-grained molecular model and are corroborated by accompanying interaction force measurements with the SFA. Our findings indicate that lateral inhomogeneities are absent from PSS brush layers collapsed in a poor solvent without multivalent ions. Together, AFM, SFA, and our molecular model present a detailed picture in which solvophobic and multivalent ion-induced effects work in concert to drive strong phase separation, with electrostatic bridging of polyelectrolyte chains playing an essential role in the collapsed structure formation

De Novo Design of Bioactive Protein-Resembling Nanospheres via Dendrimer-Templated Peptide Amphiphile Assembly

Self-assembling peptide amphiphiles (PAs) have been extensively used in the development of novel biomaterials. Because of their propensity to form cylindrical micelles, their use is limited in applications where small spherical micelles are desired. Here we present a platform method for controlling the self-assembly of biofunctional PAs into spherical 50 nm particles using dendrimers as shape-directing scaffolds. This templating approach results in biocompatible, stable protein-like assemblies displaying peptides with native secondary structure and biofunctionality

Live-Cell Imaging of Cellular Proteins by a Strain-Promoted Azide–Alkyne Cycloaddition

Live and let dye: Three coumarin-cyclooctyne conjugates have been used to label proteins tagged with azidohomoalanine in Rat-1 fibroblasts. All three fluorophores labeled intracellular proteins with fluorescence enhancements ranging from eight- to 20-fold. These conjugates are powerful tools for visualizing biomolecule dynamics in living cells

De Novo Design of Bioactive Protein-Resembling Nanospheres via Dendrimer-Templated Peptide Amphiphile Assembly

Self-assembling peptide amphiphiles (PAs) have been extensively used in the development of novel biomaterials. Because of their propensity to form cylindrical micelles, their use is limited in applications where small spherical micelles are desired. Here we present a platform method for controlling the self-assembly of biofunctional PAs into spherical 50 nm particles using dendrimers as shape-directing scaffolds. This templating approach results in biocompatible, stable protein-like assemblies displaying peptides with native secondary structure and biofunctionality

Tracking transcription factor complexes on DNA using total internal reflectance fluorescence protein binding microarrays

We have developed a high-throughput protein binding microarray (PBM) assay to systematically investigate transcription regulatory protein complexes binding to DNA with varied specificity and affinity. Our approach is based on the novel coupling of total internal reflectance fluorescence (TIRF) spectroscopy, swellable hydrogel double-stranded DNA microarrays and dye-labeled regulatory proteins, making it possible to determine both equilibrium binding specificities and kinetic rates for multiple protein:DNA interactions in a single experiment. DNA specificities and affinities for the general transcription factors TBP, TFIIA and IIB determined by TIRF–PBM are similar to those determined by traditional methods, while simultaneous measurement of the factors in binary and ternary protein complexes reveals preferred binding combinations. TIRF–PBM provides a novel and extendible platform for multi-protein transcription factor investigation