Biomimetic Vesicles with Designer Phospholipids Can Sense Environmental Redox Cues.

Cell-like materials that sense environmental cues can serve as next-generation biosensors and help advance the understanding of intercellular communication. Currently, bottom-up engineering of protocell models from molecular building blocks remains a grand challenge chemists face. Herein, we describe giant unilamellar vesicles (GUVs) with biomimetic lipid membranes capable of sensing environmental redox cues. The GUVs employ activity-based sensing through designer phospholipids that are fluorescently activated in response to specific reductive (hydrogen sulfide) or oxidative (hydrogen peroxide) conditions. These synthetic phospholipids are derived from 1,2-dipalmitoyl-rac-glycero-3-phosphocholine and they possess a headgroup with heterocyclic aromatic motifs. Despite their structural deviation from the phosphocholine headgroup, the designer phospholipids (0.5-1.0 mol %) mixed with natural lipids can vesiculate, and the resulting GUVs (7-20 µm in diameter) remain intact over the course of redox sensing. All-atom molecular dynamics simulations gave insight into how these lipids are positioned within the hydrophobic core of the membrane bilayer and at the membrane-water interface. This work provides a purely chemical method to investigate potential redox signaling and opens up new design opportunities for soft materials that mimic protocells.

Direct assessment of nitrative stress in lipid environments: Applications of a designer lipid-based biosensor for peroxynitrite.

Lipid membranes and lipid-rich organelles are targets of peroxynitrite (ONOO-), a highly reactive species generated under nitrative stress. We report a membrane-localized phospholipid (DPPC-TC-ONOO-) that allows the detection of ONOO- in diverse lipid environments: biomimetic vesicles, mammalian cell compartments, and within the lung lining. DPPC-TC-ONOO- and POPC self-assemble to membrane vesicles that fluorogenically and selectively respond to ONOO-. DPPC-TC-ONOO-, delivered through lipid nanoparticles, allowed for ONOO- detection in the endoplasmic reticulum upon cytokine-induced nitrative stress in live mammalian cells. It also responded to ONOO- within lung tissue murine models upon acute lung injury. We observed nitrative stress around bronchioles in precision cut lung slices exposed to nitrogen mustard and in pulmonary macrophages following intratracheal bleomycin challenge. Results showed that DPPC-TC-ONOO- functions specifically toward iNOS, a key enzyme modulating nitrative stress, and offers significant advantages over its hydrophilic analog in terms of localization and signal generation.

Filtered back projection vs. iterative reconstruction for CBCT: effects on image noise and processing time.

OBJECTIVES: To assess the effect of standard filtered back projection (FBP) and iterative reconstruction (IR) methods on CBCT image noise and processing time (PT), acquired with various acquisition parameters with and without metal artefact reduction (MAR). METHODS: CBCT scans using the Midmark EIOS unit of a human mandible embedded in soft tissue equivalent material with and without the presence of an implant at mandibular first molar region were acquired at various acquisition settings (milliamperages [4mA-14mA], FOV [5 × 5, 6 × 8, 9 × 10 cm], and resolutions [low, standard, high] and reconstructed using standard FBP and IR, and with and without MAR. The processing time was recorded for each reconstruction. ImageJ was used to analyze specific axial images. Radial transaxial fiducial lines were created relative to the implant site. Standard deviations of the gray density values (image noise) were calculated at fixed distances on the fiducial lines on the buccal and lingual aspects at specific axial levels, and mean values for FBP and IR were compared using paired t-tests. Significance was defined as p < 0.05. RESULTS: The overall mean for image noise (± SD) for FBP was 198.65 ± 55.58 and 99.84 ± 16.28 for IR. IR significantly decreased image noise compared to FBP at all acquisition parameters (p < 0.05). Noise reduction among different scanning protocols ranged between 29.7% (5 × 5 cm FOV) and 58.1% (5mA). IR increased processing time by an average of 35.1 s. CONCLUSIONS: IR significantly reduces CBCT image noise compared to standard FBP without substantially increasing processing time.

A Small-Molecule Approach to Bypass In Vitro Selection of New Aptamers: Designer Pre-Ligands Turn Baby Spinach into Sensors for Reactive Inorganic Targets.

Fluorescent light-up aptamer (FLAP) systems are promising biosensing platforms that can be genetically encoded. Here, we describe how a single FLAP that works with specific organic ligands can detect multiple, structurally unique, non-fluorogenic, and reactive inorganic targets. We developed 4-O-functionalized benzylidene imidazolinones as pre-ligands with suppressed fluorescent binding interactions with the RNA aptamer Baby Spinach. Inorganic targets, hydrogen sulfide (H2S) or hydrogen peroxide (H2O2), can specifically convert these pre-ligands into the native benzylidene imidazolinones, and thus be detected with Baby Spinach. Adaptation of this approach to live cells opened a new opportunity for top-down construction of whole-cell sensors: Escherichia coli transformed with a Baby Spinach-encoding plasmid and incubated with pre-ligands generated fluorescence in response to exogenous H2S or H2O2. Our approach eliminates the requirement of in vitro selection of a new aptamer sequence for molecular target detection, allows for the detection of short-lived targets, thereby advancing FLAP systems beyond their current capabilities. Leveraging the functional group reactivity of small molecules can lead to cell-based sensors for inorganic molecular targets, exploiting a new synergism between synthetic organic chemistry and synthetic biology.

Phosphatidic Acid Mediates the Nem1-Spo7/Pah1 Phosphatase Cascade in Yeast Lipid Synthesis.

In the yeast Saccharomyces cerevisiae, the PAH1-encoded Mg2+-dependent phosphatidate (PA) phosphatase Pah1 regulates the bifurcation of PA to diacylglycerol (DAG) for triacylglycerol (TAG) synthesis and to CDP-DAG for phospholipid synthesis. Pah1 function is mainly regulated via control of its cellular location by phosphorylation and dephosphorylation. Pah1 phosphorylated by multiple protein kinases is sequestered in the cytosol apart from its substrate PA in the membrane. The phosphorylated Pah1 is then recruited and dephosphorylated by the protein phosphatase complex Nem1 (catalytic subunit)-Spo7 (regulatory subunit) in the endoplasmic reticulum. The dephosphorylated Pah1 hops onto and scoots along the membrane to recognize PA for its dephosphorylation to DAG. Here, we developed a proteoliposome model system that mimics the Nem1-Spo7/Pah1 phosphatase cascade to provide a tool for studying Pah1 regulation. Purified Nem1-Spo7 was reconstituted into phospholipid vesicles prepared in accordance with the phospholipid composition of the nuclear/endoplasmic reticulum membrane. The Nem1-Spo7 phosphatase reconstituted in the proteoliposomes, which were measured 60 nm in an average diameter, was catalytically active on Pah1 phosphorylated by Pho85-Pho80, and its active site was located at the external side of the phospholipid bilayer. Moreover, we determined that PA stimulated the Nem1-Spo7 activity, and the regulatory effect was governed by the nature of the phosphate headgroup but not by the fatty acyl moiety of PA. The reconstitution system for the Nem1-Spo7/Pah1 phosphatase cascade, which starts with the phosphorylation of Pah1 by Pho85-Pho80 and ends with the production of DAG, is a significant advance to understand a regulatory cascade in yeast lipid synthesis.

Thermotropic Liquid-Crystalline and Light-Emitting Properties of Bis(4-aalkoxyphenyl) Viologen Bis(triflimide) Salts.

A series of bis(4-alkoxyphenyl) viologen bis(triflimide) salts with alkoxy chains of different lengths were synthesized by the metathesis reaction of respective bis(4-alkoxyphenyl) viologen dichloride salts, which were in turn prepared from the reaction of Zincke salt with the corresponding 4-n-alkoxyanilines, with lithium triflimide in methanol. Their chemical structures were characterized by 1H and 13C nuclear magnetic resonance spectra and elemental analysis. Their thermotropic liquid-crystalline (LC) properties were examined by differential scanning calorimetry, polarizing optical microscopy, and variable temperature X-ray diffraction. Salts with short length alkoxy chains had crystal-to-liquid transitions. Salts of intermediate length alkoxy chains showed both crystal-to-smectic A (SmA) transitions, Tms, and SmA-to-isotropic transitions, Tis. Those with longer length of alkoxy chains had relatively low Tms at which they formed the SmA phases that persisted up to the decomposition at high temperatures. As expected, all of them had excellent thermal stabilities in the temperature range of 330-370 °C. Their light-emitting properties in methanol were also included.

Molecular profiling of rheumatoid arthritis patients reveals an association between innate and adaptive cell populations and response to anti-tumor necrosis factor.

BACKGROUND: The goal of this study is to use comprehensive molecular profiling to characterize clinical response to anti-TNF therapy in a real-world setting and identify reproducible markers differentiating good responders and non-responders in rheumatoid arthritis (RA). METHODS: Whole-blood mRNA, plasma proteins, and glycopeptides were measured in two cohorts of biologic-naïve RA patients (n = 40 and n = 36) from the Corrona CERTAIN (Comparative Effectiveness Registry to study Therapies for Arthritis and Inflammatory coNditions) registry at baseline and after 3 months of anti-TNF treatment. Response to treatment was categorized by EULAR criteria. A cell type-specific data analysis was conducted to evaluate the involvement of the most common immune cell sub-populations. Findings concordant between the two cohorts were further assessed for reproducibility using selected NCBI-GEO datasets and clinical laboratory measurements available in the CERTAIN database. RESULTS: A treatment-related signature suggesting a reduction in neutrophils, independent of the status of response, was indicated by a high level of correlation (ρ = 0.62; p < 0.01) between the two cohorts. A baseline, response signature of increased innate cell types in responders compared to increased adaptive cell types in non-responders was identified in both cohorts. This result was further assessed by applying the cell type-specific analysis to five other publicly available RA datasets. Evaluation of the neutrophil-to-lymphocyte ratio at baseline in the remaining patients (n = 1962) from the CERTAIN database confirmed the observation (odds ratio of good/moderate response = 1.20 [95% CI = 1.03-1.41, p = 0.02]). CONCLUSION: Differences in innate/adaptive immune cell type composition at baseline may be a major contributor to response to anti-TNF treatment within the first 3 months of therapy.

Circulating Markers of Inflammation Persist in Children and Adults With Giant Aneurysms After Kawasaki Disease.

BACKGROUND: The sequelae of Kawasaki disease (KD) vary widely with the greatest risk for future cardiovascular events among those who develop giant coronary artery aneurysms (CAA). We sought to define the molecular signature associated with different outcomes in pediatric and adult KD patients. METHODS: Molecular profiling was conducted using mass spectrometry-based shotgun proteomics, transcriptomics, and glycomics methods on 8 pediatric KD patients at the acute, subacute, and convalescent time points. Shotgun proteomics was performed on 9 KD adults with giant CAA and matched healthy controls. Plasma calprotectin was measured by ELISA in 28 pediatric KD patients 1 year post-KD, 70 adult KD patients, and 86 healthy adult volunteers. RESULTS: A characteristic molecular profile was seen in pediatric patients during the acute disease, which resolved at the subacute and convalescent periods in patients with no coronary artery sequelae but persisted in 2 patients who developed giant CAA. We, therefore, investigated persistence of inflammation in KD adults with giant CAA by shotgun proteomics that revealed a signature of active inflammation, immune regulation, and cell trafficking. Correlating results obtained using shotgun proteomics in the pediatric and adult KD cohorts identified elevated calprotectin levels in the plasma of patients with CAA. Investigation of expanded pediatric and adult KD cohorts revealed elevated levels of calprotectin in pediatric patients with giant CAA 1 year post-KD and in adult KD patients who developed giant CAA in childhood. CONCLUSIONS: Complex patterns of biomarkers of inflammation and cell trafficking can persist long after the acute phase of KD in patients with giant CAA. Elevated levels of plasma calprotectin months to decades after acute KD and infiltration of cells expressing S100A8 and A9 in vascular tissues suggest ongoing, subclinical inflammation. Calprotectin may serve as a biomarker to inform the management of KD patients following the acute illness.

A proteome-scale map of the human interactome network.

Just as reference genome sequences revolutionized human genetics, reference maps of interactome networks will be critical to fully understand genotype-phenotype relationships. Here, we describe a systematic map of ?14,000 high-quality human binary protein-protein interactions. At equal quality, this map is ?30% larger than what is available from small-scale studies published in the literature in the last few decades. While currently available information is highly biased and only covers a relatively small portion of the proteome, our systematic map appears strikingly more homogeneous, revealing a "broader" human interactome network than currently appreciated. The map also uncovers significant interconnectivity between known and candidate cancer gene products, providing unbiased evidence for an expanded functional cancer landscape, while demonstrating how high-quality interactome models will help "connect the dots" of the genomic revolution.

Independently evolved virulence effectors converge onto hubs in a plant immune system network.

Plants generate effective responses to infection by recognizing both conserved and variable pathogen-encoded molecules. Pathogens deploy virulence effector proteins into host cells, where they interact physically with host proteins to modulate defense. We generated an interaction network of plant-pathogen effectors from two pathogens spanning the eukaryote-eubacteria divergence, three classes of Arabidopsis immune system proteins, and ~8000 other Arabidopsis proteins. We noted convergence of effectors onto highly interconnected host proteins and indirect, rather than direct, connections between effectors and plant immune receptors. We demonstrated plant immune system functions for 15 of 17 tested host proteins that interact with effectors from both pathogens. Thus, pathogens from different kingdoms deploy independently evolved virulence proteins that interact with a limited set of highly connected cellular hubs to facilitate their diverse life-cycle strategies.