Single Protein Level Research Articles

Shedding Light on Intracellular Proteins using Flow Cytometry.

Intracellular protein abundance is routinely measured in mammalian cells using population-based techniques such as western blotting which fail to capture single cell protein levels or using fluorescence microscopy which is although suitable for single cell protein detection but not for rapid analysis of large no. of cells. Flow cytometry offers rapid, high-throughput, multiparameter-based analysis of intracellular protein expression in statistically significant no. of cells at single cell resolution. In past few decades, customized assays have been developed for flow cytometric detection of specific intracellular proteins. This review discusses the scope of flow cytometry for intracellular protein detection in mammalian cells along with specific applications. Technological advancements to overcome the limitations of traditional flow cytometry for the same are also discussed.

Quantification of absolute labeling efficiency at the single-protein level

State-of-the-art super-resolution microscopy allows researchers to spatially resolve single proteins in dense clusters. However, accurate quantification of protein organization and stoichiometries requires a general method to evaluate absolute binder labeling efficiency, which is currently unavailable. Here we introduce a universally applicable approach that uses a reference tag fused to a target protein of interest. By attaching high-affinity binders, such as antibodies or nanobodies, to both the reference tag and the target protein, and then employing DNA-barcoded sequential super-resolution imaging, we can correlate the location of the reference tag with the target molecule binder. This approach facilitates the precise quantification of labeling efficiency at the single-protein level.

Studying the O-GlcNAcome of human placentas using banked tissue samples.

O-GlcNAcylation is a dynamic modulator of signaling pathways, equal in magnitude to the widely studied phosphorylation. With the rapid development of tools for its detection at the single protein level, the O-GlcNAc modification rapidly emerged as a novel diagnostic and therapeutic target in human diseases. Yet, mapping the human O-GlcNAcome in various tissues is essential for generating relevant biomarkers. In this study, we used human banked tissue as a sample source to identify O-GlcNAcylated protein targets relevant to human diseases. Using human term placentas, we propose (1) a method to clean frozen banked tissue of blood proteins; (2) an optimized protocol for the enrichment of O-GlcNAcylated proteins using immunoaffinity purification; and (3) a bioinformatic workflow to identify the most promising O-GlcNAc targets. As a proof-of-concept, we used 45mg of banked placental samples from two pregnancies to generate intracellular protein extracts depleted of blood protein. Then, antibody-based O-GlcNAc enrichment on denatured samples yielded over 2000 unique HexNAc PSMs and 900 unique sites using 300μg of protein lysate. Due to efficient sample cleanup, we also captured 82 HexNAc proteins with high placental expression. Finally, we provide a bioinformatic tool (CytOVS) to sort the HexNAc proteins based on their cellular localization and extract the most promising O-GlcNAc targets to explore further. To conclude, we provide a simple 3-step workflow to generate a manageable list of O-GlcNAc proteins from human tissue and improve our understanding of O-GlcNAcylation's role in health and diseases.

Visualizing HIV-1 Assembly at the T-Cell Plasma Membrane Using Single-Molecule Localization Microscopy.

The 20-year revolution in optical fluorescence microscopy, supported by the optimization of both spatial resolution and timely acquisition, allows the visualization of nanoscaled objects in cell biology. Currently, the use of a recent generation of super-resolution fluorescence microscope coupled with improved fluorescent probes gives the possibility to study the replicative cycle of viruses in living cells, at the single-virus particle or protein level. Here, we highlight the protocol for visualizing HIV-1 Gag assembly at the host T-cell plasma membrane using super-resolution light microscopy. Total internal reflection fluorescence microscopy (TIRF-M) coupled with single-molecule localization microscopy (SMLM) enables the detection and characterization of the assembly of viral proteins at the plasma membrane of infected host cells at the single protein level. Here, we describe the TIRF equipment, the T-cell culture for HIV-1, the sample preparation for single-molecule localization microscopies such as PALM and STORM, acquisition protocols, and Gag assembling cluster analysis.

In Situ Observation of Chemically Induced Protein Denaturation at Solvated Interfaces.

Proteins unfold in chaotropic salt solutions, a process that is difficult to observe at the single protein level. The work presented here demonstrates that a liquid-based atomic force microscope and graphene liquid-cell-based scanning transmission electron microscope make it possible to observe chemically induced protein unfolding. To illustrate this capability, ferritin proteins were deposited on a graphene surface, and the concentration-dependent urea- or guanidinium-induced changes of morphology were monitored for holo-ferritin with its ferrihydrite core as well as apo-ferritin without this core. Depending on the chaotropic agent the liquid-based imaging setup captured an unexpected transformation of natively folded holo-ferritin proteins into rings after urea treatment but not after guanidinium treatment. Urea treatment of apo-ferritin did not result in nanorings, confirming that nanorings are a specific signature of denaturation of holo-ferritins after exposture to sufficiently high urea concentrations. Mapping the in situ images with molecular dynamics simulations of ferritin subunits in urea solutions suggests that electrostatic destabilization triggers denaturation of ferritin as urea makes direct contact with the protein and also disrupts the water H-bonding network in the ferritin solvation shell. Our findings deepen the understanding of protein denaturation studied using label-free techniques operating at the solid-liquid interface.

Conformational Selection and Induced Fit: The Behavior of Two Homologous Proteases

AbstractProtein‐ligand interactions are crucial for many cellular processes, with details of the binding mechanism being discussed as essential for biological functions. Interestingly, protein binding often involves conformational changes between two or more states, whereby different binding mechanisms are possible even with a simple two‐state description. Two models are widely used to portray protein‐ligand interactions: Induced fit and conformational selection. However, distinguishing them experimentally is challenging. Single‐molecule Förster resonance energy transfer (smFRET) has emerged as a powerful tool to resolve structural dynamics at the level of single proteins. Here, we investigated immobilized Zika virus (ZIKV) and dengue virus (DENV2) NS2B‐NS3 proteases using smFRET to compare their conformational changes upon binding to competitive small molecule inhibitors. The analysis of the smFRET data allowed us to distinguish between induced fit and conformational selection models and assign the binding mechanism from the kinetic parameters obtained. Although DENV and ZIKV protease are proteins with high structural similarities, our results reveal that they have opposite binding mechanisms for competitive ligands. While the protein‐ligand interaction in the ZIKV protease follows an induced fit mechanism, the DENV protease follows the conformational selection mechanism.

#5668 PERITONEAL DIALYSIS ADDITIVE REDUCES TRANSPERITONEAL LOSS OF BLOOD MICROPARTICLE AND EXTRACELLULAR MATRIX PROTEINS IN A CONTROLLED CROSSOVER TRIAL

Abstract Background and Aims About 11% of patients requiring dialysis worldwide are treated with peritoneal dialysis (PD). With each PD exchange high amounts of protein are removed from the peritoneal cavity stemming from the systemic circulation (reflecting toxin removal, loss of serum proteins, inflammatory processes) and local inflammatory processes. Thereby, the clinically measured peritoneal protein loss (PPL) is the result of transperitoneal serum loss, lymphatic drainage, transmesothelial reabsorption, local production, and cellular components. Although high PPL has been found to be predictive of mortality and cardiovascular morbidity, the pathophysiological concept is still poorly understood. Method Peritoneal dialysis effluent (PDE) samples were obtained from peritoneal equilibrium tests during a prospective, multicenter, double-blinded, controlled, randomized, dual-period, 2-treatment, crossover, phase II, proof-of-concept study in Austria. PDE samples were submitted to proteomic analysis by tandem mass tag (TMT) labeling and 2D reversed phase liquid chromatography mass spectrometry. To disentangle transmembrane serum protein loss from local protein production we developed a novel and unique analytical approach. Briefly, partitioning around medoids and proximity in the Euclidean space for between visit (non-treatment visits only) single-protein kinetics was utilized to achieve this separation. A multivariate mixed-effects model was calculated to identify treatment (intraperitoneal alanyl-glutamine administration) effects. Results We identified 2,624 different proteins within the PDE of 12 patients across two time-points. A large cluster of proteins with stable abundances in collinearity with well-established clinical characteristics of transperitoneal transport (e.g., dialysate-to-plasma-albumin) in accordance with the three-pore-model was identified. After clustering and separation, the proteins resembling similar time-course kinetics to clinical transperitoneal membrane transport characteristics (one of five clusters) were all identified to be able to theoretically traverse through the small peritoneal pore (< 4-6 nm). Of these 549 proteins lost from the systemic circulation via transperitoneal small pore diffusion, the appearance of 51 in the PD effluent was significantly reduced by intraperitoneal administration of alanyl-glutamine. The affected proteins are mainly involved in extracellular matrix organization, part of extracellular exosome signaling and blood microparticles, e.g. high density lipoprotein (HDL) formation (Apolipoprotein A1). Conclusion PPL is an important but poorly understood undesired effect of PD associated with cardiovascular morbidity and mortality. Our novel analytical approach enables us to disentangle the complexity of transperitoneal serum protein loss and local protein production on single-protein level across the peritoneal proteome. Furthermore, we were able to delineate the potential beneficial effects of intraperitoneal alanyl-glutamine treatment on PPL and peritoneal health which might even suggest potential beneficial effects on cardiovascular morbidity (e.g., HDL formation).

The brain reacting to COVID-19: analysis of the cerebrospinal fluid proteome, RNA and inflammation

Patients with COVID-19 can have a variety of neurological symptoms, but the active involvement of central nervous system (CNS) in COVID-19 remains unclear. While routine cerebrospinal fluid (CSF) analyses in patients with neurological manifestations of COVID-19 generally show no or only mild inflammation, more detailed data on inflammatory mediators in the CSF of patients with COVID-19 are scarce. We studied the inflammatory response in paired CSF and serum samples of patients with COVID-19 (n = 38). Patients with herpes simplex virus encephalitis (HSVE, n = 10) and patients with non-inflammatory, non-neurodegenerative neurological diseases (n = 28) served as controls. We used proteomics, enzyme-linked immunoassays, and semiquantitative cytokine arrays to characterize inflammatory proteins. Autoantibody screening was performed with cell-based assays and native tissue staining. RNA sequencing of long-non-coding RNA and circular RNA was done to study the transcriptome. Proteomics on single protein level and subsequent pathway analysis showed similar yet strongly attenuated inflammatory changes in the CSF of COVID-19 patients compared to HSVE patients with, e.g., downregulation of the apolipoproteins and extracellular matrix proteins. Protein upregulation of the complement system, the serpin proteins pathways, and other proteins including glycoproteins alpha-2 and alpha-1 acid. Importantly, calculation of interleukin-6, interleukin-16, and CXCL10 CSF/serum indices suggest that these inflammatory mediators reach the CSF from the systemic circulation, rather than being produced within the CNS. Antibody screening revealed no pathological levels of known neuronal autoantibodies. When stratifying COVID-19 patients into those with and without bacterial superinfection as indicated by elevated procalcitonin levels, inflammatory markers were significantly (p < 0.01) higher in those with bacterial superinfection. RNA sequencing in the CSF revealed 101 linear RNAs comprising messenger RNAs, and two circRNAs being significantly differentially expressed in COVID-19 than in non-neuroinflammatory controls and neurodegenerative patients. Our findings may explain the absence of signs of intrathecal inflammation upon routine CSF testing despite the presence of SARS-CoV2 infection-associated neurological symptoms. The relevance of blood-derived mediators of inflammation in the CSF for neurological COVID-19 and post-COVID-19 symptoms deserves further investigation.

Comprehensive fluorescence lifetime fluctuation spectroscopy to investigate protein mobility during aggregation processes in living cells.

How proteins move and interact in the complicated, crowded cellular environment is still unknown. The formation of membrane-less condensates, caused by interacting proteins and RNA molecules, in the so-called liquid-to-liquid phase transition has recently raised scientific interest. Primarily related to protein mutations, the condensates can undergo a liquid-to-solid transition often associated with neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Here, we introduce a novel method based on fluorescence fluctuation spectroscopy (FFS) as a tool to study proteins interactions, movement, and aggregate formation comprehensively. FFS is a family of analytical methods that allow quantifying molecular processes by studying the fluorescence intensity fluctuations generated by a population of fluorescent molecules entering and leaving a confined microscope focal volume. We enhance conventional FFS methods by employing a novel single-photon-avalanche-diode array detector in an ISM confocal microscope, which allows us to spatially and temporally tag single photons. Within the same system, we can systematically, and with high throughput, access a wide range of spatial and temporal scales, leading to a comprehensive knowledge of the process. In particular, we can follow slow changes in the overall cell organization by performing time-lapse super-resolved image scanning microscopy, fast-slow molecular movements on the level of single proteins, e.g., time-resolved FFS, and interaction with dual-color cross-correlation methods. Moreover, we introduce fluorescence lifetime fluctuation spectroscopy, a novel paradigm where the molecule's mobility and diffusion mode are simultaneously investigated with fluorescence lifetime, allowing us to directly correlate changes in diffusion mode/mobility with structural changes in the molecule's environment. We validated our methods by measuring the time evolution of stress granules formation, focusing on G3BP1 and FUS proteins, in an in vitro cellular system that recapitulates several features of the ALS pathology.

Feeding gilthead sea bream with increasing dietary bacterial single cell protein level: Implication on growth, plasma biochemistry, gut histology, and gut microbiota

Bacterial single cell protein (SCP) is considered a promising circular protein ingredient for aquafeed, due to the high protein content, and for the possibilities to grow them on different substrates such as organic waste thus leading to low environmental footprint and affordable production costs. Their use as raw material has been assessed in several farmed species, however, research on Mediterranean ones is still scarce. Hence, a study was undertaken to evaluate growth, plasma biochemistry, gut histology and gut microbiota (GM) response of gilthead sea bream (Sparus aurata) fed diets with increasing levels of bacterial SCP in comparison to a control without SCP. Three isonitrogenous and isolipidic extruded diets (44% protein; 19% lipid) were formulated with different bacterial SCP (derived from Corinebacterium glutamicum) level (10% SCP, SPC10; 15% SCP, SCP15; 20% SCP, SCP20) to replace vegetable protein ingredients (total replacement of soy protein concentrate and partial replacement of corn gluten), while a control diet (CTRL) was formulated without SCP. Fish groups of 45 individuals (initial weight: 75 g) were fed to visual satiation over 108 days. At the end of the trial there were no significant differences on growth, feed intake, feed conversion rate, protein efficiency ratio and protein apparent digestibility. Most of plasma parameters were found to be equal for all treatments, except for those related to nucleic acids molecules degradation such as phosphorus and urea which were higher in SCP10 and SCP20 compared to CTRL, respectively. No morphological alterations were found in the intestines of any fish analysed. Different responses of the overall GM structure in relation to the bacterial SCP inclusion level were detected. Specifically, SCP exerted a positive effect on GM internal diversity which increased at increasing dietary SCP inclusion level. In addition, SCP inclusion lead to increase in the abundance of Bacillus spp., taxa which can potentially support nutrition, immune system, and disease resistance. In conclusion, it seems feasible to include up to 20% of SCP dietary level for gilthead sea bream without compromising growth, feed efficiency and health parameters.

Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET.

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the environment, a cell must be able to recognize and process it. This task mainly relies on the function of membrane receptors, whose role is to convert signals into the biochemical language of the cell. G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptor proteins in humans. Among GPCRs, metabotropic glutamate receptors (mGluRs) are a unique subclass that function as obligate dimers and possess a large extracellular domain that contains the ligand-binding site. Recent advances in structural studies of mGluRs have improved the understanding of their activation process. However, the propagation of large-scale conformational changes through mGluRs during activation and modulation is poorly understood. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to visualize and quantify the structural dynamics of biomolecules at the single-protein level. To visualize the dynamic process of mGluR2 activation, fluorescent conformational sensors based on unnatural amino acid (UAA) incorporation were developed that allowed site-specific protein labeling without perturbation of the native structure of receptors. The protocol described here explains how to perform these experiments, including the novel UAA labeling approach, sample preparation, and smFRET data acquisition and analysis. These strategies are generalizable and can be extended to investigate the conformational dynamics of a variety of membrane proteins.

Distinctive Adsorption Mechanism and Kinetics of Immunoglobulin G on a Nanoscale Polymer Surface.

Elucidation of protein adsorption beyond simple polymer surfaces to those presenting greater chemical complexity and nanoscopic features is critical to developing well-controlled nanobiomaterials and nanobiosensors. In this study, we repeatedly and faithfully track individual proteins on the same nanodomain areas of a block copolymer (BCP) surface and monitor the adsorption and assembly behavior of a model protein, immunoglobulin G (IgG), over time into a tight surface-packed structure. With discrete protein adsorption events unambiguously visualized at the biomolecular level, the detailed assembly and packing states of IgG on the BCP nanodomain surface are subsequently correlated to various regimes of IgG adsorption kinetic plots. Intriguing features, entirely different from those observed from macroscopic homopolymer templates, are identified from the IgG adsorption isotherms on the nanoscale, chemically varying BCP surface. They include the presence of two Langmuir-like adsorption segments and a nonmonotonic regime in the adsorption plot. Via correlation to time-corresponding topographic data, the unique isotherm features are explained with single biomolecule level details of the IgG adsorption pathway on the BCP. This work not only provides much needed, direct experimental evidence for time-resolved, single protein level, adsorption events on nanoscale polymer surfaces but also signifies mutual linking between specific topographic states of protein adsorption and assembly to particular segments of adsorption isotherms. From the fundamental research viewpoint, the correlative ability to examine the nanoscopic surface organizations of individual proteins and their local as well as global adsorption kinetic profiles will be highly valuable for accurately determining protein assembly mechanisms and interpreting protein adsorption kinetics on nanoscale surfaces. Application-wise, such knowledge will also be important for fundamentally guiding the design and development of biomaterials and biomedical devices that exploit nanoscale polymer architectures.

Expanding Direct Coupling Analysis to Identify Heterodimeric Interfaces from Limited Protein Sequence Data.

Direct coupling analysis (DCA) is a global statistical approach that uses information encoded in protein sequence data to predict spatial contacts in a three-dimensional structure of a folded protein. DCA has been widely used to predict the monomeric fold at amino acid resolution and to identify biologically relevant interaction sites within a folded protein. Going beyond single proteins, DCA has also been used to identify spatial contacts that stabilize the interaction in protein complex formation. However, extracting this higher order information necessary to predict dimer contacts presents a significant challenge. A DCA evolutionary signal is much stronger at the single protein level (intraprotein contacts) than at the protein-protein interface (interprotein contacts). Therefore, if DCA-derived information is to be used to predict the structure of these complexes, there is a need to identify statistically significant DCA predictions. We propose a simple Z-score measure that can filter good predictions despite noisy, limited data. This new methodology not only improves our prediction ability but also provides a quantitative measure for the validity of the prediction.

SARS-CoV-2 Receptor Binding Domain as a Stable-Potential Target for SARS-CoV-2 Detection by Surface-Enhanced Raman Spectroscopy.

In this work, we report a new approach for detecting SARS-CoV-2 RBD protein (RBD) using the surface-enhanced Raman spectroscopy (SERS) technique. The optical enhancement was obtained thanks to the preparation of nanostructured Ag/Au substrates. Fabricated Au/Ag nanostructures were used in the SERS experiment for RBD protein detection. SERS substrates show higher capabilities and sensitivity to detect RBD protein in a short time (3 s) and with very low power. We were able to push the detection limit of proteins to a single protein detection level of 1 pM. The latter is equivalent to 1 fM as a detection limit of viruses. Additionally, we have shown that the SERS technique was useful to figure out the presence of RBD protein on antibody functionalized substrates. In this case, the SERS detection was based on protein-antibody recognition, which led to shifts in the Raman peaks and allowed signal discrimination between RBD and other targets such as Bovine serum albumin (BSA) protein. A perfect agreement between a 3D simulated model based on finite element method and experiment was reported confirming the SERS frequency shift potential for trace proteins detection. Our results could open the way to develop a new prototype based on SERS sensitivity and selectivity for rapid detection at a very low concentration of virus and even at a single protein level.

Controlling Photosynthetic Light Harvesting at the Single Protein Level

The light-harvesting complexes of oxygenic photosynthetic organisms have to balance two vital functions: efficient transport of photoexcitations to the photochemical reaction center under low levels of solar radiation and efficient photoprotection under intense solar radiation and other stress conditions, during which photoexcitations are rapidly dissipated as heat. Most light-harvesting proteins bind a dense arrangement of chromophores, ensuring that subtle protein conformational changes are often sufficient to switch between the complexes’ light-harvesting and photoprotective functions. Single-molecule spectroscopy allows one to follow these switches in real time. I will show how this technique has led to the discovery of new light-harvesting and photoprotective states in various photosynthetic light-harvesting complexes and how the population equilibrium between these states can be finely tuned at the single protein level by the incident photon flux, the pH, the chromophore arrangement, and by controlling the interaction with small, accessory proteins. I will also demonstrate how the light-harvesting efficiency of these complexes can be actively controlled through interactions with localized surface plasmons from metallic nanostructures to dramatically alter the radiative and nonradiative deexcitation rates.

Practical Guide to Single-Protein AFM Nanomechanical Spectroscopy Mapping: Insights and Pitfalls As Unraveled by All-Atom MD Simulations on Immunoglobulin G.

Atomic force microscopy is an invaluable characterization tool in almost every biophysics laboratory. However, obtaining atomic/sub-nanometer resolution on single proteins has thus far remained elusive-a feat long achieved on hard substrates. In this regard, nanomechanical spectroscopy mapping may provide a viable approach to overcome this limitation. By complementing topography with mechanical properties measured locally, one may thus enhance spatial resolution at the single-protein level. In this work, we perform all-atom molecular dynamics simulations of the indentation process on a single immunoglobulin G (IgG) adsorbed on a graphene slab. Our simulations reveal three different stages as a function of strain: a noncontact regime-where the mechanical response is linked to the presence of the water environment- followed by an elastic response and a final plastic deformation regime. In the noncontact regime, we are able to identify hydrophobic/hydrophilic patches over the protein. This regime provides the most local mechanical information that allows one to discern different regions with similar height/topography and leads to the best spatial resolution. In the elastic regime, we conclude that the Young modulus is a well-defined property only within mechanically decoupled domains. This is caused by the fact that the elastic deformation is associated with a global reorganization of the domain. Differences in the mechanical response are large enough to clearly resolve domains within a single protein, such as the three subunits forming the IgG. Two events, unfolding or protein slipping, are observed in the plastic regime. Our simulations allow us to characterize these two processes and to provide a strategy to identify them in the force curves. Finally, we elaborate on possible challenges that could hamper the interpretation of such experiments/simulations and how to overcome them. All in all, our simulations provide a detailed picture of nanomechanical spectroscopy mapping on single proteins, showing its potential and the challenges that need to be overcome to unlock its full potential.

Pioglitazone Ameliorates Gentamicin Ototoxicity by Affecting the TLR and STAT Pathways in the Early Postnatal Organ of Corti.

Noise trauma, infection, and ototoxic drugs are frequent external causes of hearing loss. With no pharmacological treatments currently available, understanding the mechanisms and pathways leading to auditory hair cell (HC) damage and repair is crucial for identifying potential pharmacological targets. Prior research has implicated increased reactive oxygen species (ROS) and inflammation as general mechanisms of hearing loss common to diverse causes. Novel targets of these two key mechanisms of auditory damage may provide new paths toward the prevention and treatment of hearing loss. Pioglitazone, an oral antidiabetic drug from the class of thiazolidinediones, acts as an agonist of the peroxisome proliferator-activated receptor-gamma (PPAR-γ) and is involved in the regulation of lipid and glucose metabolism. PPAR-γ is an important player in repressing the expression of inflammatory cytokines and signaling molecules. We evaluated the effects of pioglitazone in the mouse Organ of Corti (OC) explants to characterize its influence on signaling pathways involved in auditory HC damage. The OC explants was cultured with pioglitazone, gentamicin, or a combination of both agents. Pioglitazone treatment resulted in significant repression of interferon (IFN)-α and -gamma pathways and downstream cytokines, as assessed by RNA sequencing and quantitative PCR gene expression assays. More detailed investigation at the single gene and protein level showed that pioglitazone mediated its anti-inflammatory effects through alterations of the Toll-like receptor (TLR) and STAT pathways. Together, these results indicate that pioglitazone significantly represses IFN and TLR in the cochlea, dampening the activity of gentamicin-induced pathways. These data support our previous results demonstrating significant protection of auditory HCs in the OC explants exposed to pioglitazone and other PPAR-targeted agents.

Periostin: contributor to abnormal airway epithelial function in asthma?

Periostin (POSTN) may serve as a biomarker for Type-2 mediated eosinophilic airway inflammation in asthma. We hypothesised that a Type-2 cytokine, interleukin (IL)-13, induces airway epithelial expression of POSTN, which in turn contributes to epithelial changes observed in asthma.We studied the effect of IL-13 on POSTN expression in BEAS-2B and air-liquid interface differentiated primary bronchial epithelial cells (PBECs). Additionally, the effects of recombinant human POSTN on epithelial-to-mesenchymal transition (EMT) markers and mucin genes were assessed. POSTN single cell gene expression and protein levels were analysed in bronchial biopsies and induced sputum from asthma patients and healthy controls.IL-13 increased POSTN expression in both cell types and this was accompanied by EMT-related features in BEAS-2B. In air-liquid interface differentiated PBECs, IL-13 increased POSTN basolateral and apical release. Apical administration of POSTN increased the expression of MMP-9, MUC5B and MUC5AC In bronchial biopsies, POSTN expression was mainly confined to basal epithelial cells, ionocytes, endothelial cells and fibroblasts, showing higher expression in basal epithelial cells from asthma patients versus those from controls. A higher level of POSTN protein expression in epithelial and subepithelial layers was confirmed in bronchial biopsies from asthma patients when compared to healthy controls. Although sputum POSTN levels were not higher in asthma, levels correlated with eosinophil numbers and with the coughing-up of mucus.POSTN expression is increased by IL-13 in bronchial epithelial cells and is higher in bronchial biopsies from asthma patients. This may have important consequences, as administration of POSTN increases epithelial expression of mucin genes, supporting the relationship of POSTN with Type-2 mediated asthma and mucus secretion.

Abstract 1145: Assessing the diagnostic value of the combination of Matrix Metalloproteinase 2 (MMP2) and Progranulin (PGRN) preoperative plasma levels for colorectal cancer (CRC)

Abstract Introduction: MMP 2, also known as Gelatinase A, is an extracellular matrix remodeling enzymes. MMP-2 activity is regulated by various oncogenes, cytokines and growth factors. MMP 2 functions in the breakdown of extracellular matrix (ECM) and participates in tumor neoangiogensis. It is also associated with an angiogenic and metastatic tumor cell phenotype, in vivo. Up-regulation of MMP-2 aggravates the loss of basement membrane type IV collagen, breaks down ECM and promotes tumor progression and invasion. Progranulin (PGRN) is a ubiquitously expressed secreted glycoprotein that activates both the PI3k and ERK pathways, and promotes cyclin D1 and cyclin B expression which supports proliferation and survival of mesenchymal and epithelial origin cells. PGRN stimulates VEGF expression in breast cancer cells, in vitro, and stimulates inflammation, fibroblast accumulation and angiogenesis. Over-expression of MMP2 and PGRN has been reported in many cancers including CRC. The aim of this study was to compare preoperative (preop) plasma MMP2 and PGRN levels in CRC and benign colonic disease (BCD) patients (pts), alone and in combination, as a means of detecting CRC. Method: Preop plasma samples were obtained from consenting CRC and BCD pts undergoing elective resection for whom adequate preop plasma was available from an IRB approved tissue bank. Demographic, clinical, and pathologic data were collected. PreOp plasma levels of MMP2 and PGRN (ng/ml) were determined via ELISA in duplicate and reported as median + 95%CI. The receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) were used to assess single or multiple plasma protein levels as a diagnostic tool for CRC. The Mann-Whitney test was used for statistical analysis (significance p&amp;lt;0.05). Results: A total of 172 CRC pts (70% colon, 30% rectal) and 102 BCD pts (adenoma 33%, diverticulitis 54%, other 15%) were eligible for the study. The CRC pathologic stage distributions were: Stage-I, 25%; Stage-2, 32%, Stage-3, 31%, Stage- 4, 12%. Median preop plasma protein levels in CRC pts were significantly higher than BCD pts. [MMP2; 205.3,CI 198.2,217.5 vs.165.3,CI 156.8,179.1: PGRN; 54.7,CI 51.8,57.1 vs.43.3,CI 40.1,46.8,P&amp;lt;0.001) The AUC value for ROC curve for MMP2 and PGRN were 0.721 and 0.724 respectively with 73% and 71% specificity. AUC for the combination of these 2 proteins was 0.757 with 97% specificity. Conclusion: Median MMP2 and PGRN levels were higher in CRC pts and the CRC median values were 24% and 26% higher than BCD median levels respectively. ROC curve analysis showed that the two protein combination improved specificity and AUC values. MMP2 and PGRN are potential candidates for a larger diagnostic panel to include other proteins found to be elevated in CRC pts. Further study is warranted. Citation Format: Chandana S K Herath Mudiyanselage, Neil Mitra, Dasuni Niyagama Gamage, Hiromichi Miyagaki, Abhinit Shah, Xiaohong Yan, Vesna Cekic, Richard L. Whelan. Assessing the diagnostic value of the combination of Matrix Metalloproteinase 2 (MMP2) and Progranulin (PGRN) preoperative plasma levels for colorectal cancer (CRC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1145.

Recent advances towards single biomolecule level understanding of protein adsorption phenomena unique to nanoscale polymer surfaces with chemical variations

Protein adsorption onto polymer surfaces is a very complex and ubiquitous phenomenon whose integrated process impacts essential applications in our daily lives such as food packaging materials, health devices, diagnostic tools, and medical products. Increasingly, novel polymer materials with greater chemical intricacy and reduced dimensionality are used for various applications involving adsorbed proteins on their surfaces. Hence, the nature of protein-surface interactions to consider is becoming much more complicated than before. A large body of literature exists for protein adsorption. However, most of these investigations have focused on collectively measured, ensemble-averaged protein behaviors that occur on macroscale and chemically unvarying polymer surfaces instead of direct measurements at the single protein or sub-protein level. In addition, interrogations of protein-polymer adsorption boundaries in these studies were typically carried out by indirect methods, whose insights may not be suitably applied for explaining individual protein adsorption processes occurring onto nanostructured, chemically varying polymer surfaces. Therefore, an important gap in our knowledge still exists that needs to be systematically addressed via direct measurement means at the single protein and sub-protein level. Such efforts will require multifaceted experimental and theoretical approaches that can probe multilength scales of protein adsorption, while encompassing both single proteins and their collective ensemble behaviors at the length scale spanning from the nanoscopic all the way to the macroscopic scale. In this review, key research achievements in nanoscale protein adsorption to date will be summarized. Specifically, protein adsorption studies involving polymer surfaces with their defining feature dimensions and associated chemical partitions comparable to the size of individual proteins will be discussed in detail. In this regard, recent works bridging the crucial knowledge gap in protein adsorption will be highlighted. New findings of intriguing protein surface assembly behaviors and adsorption kinetics unique to nanoscale polymer templates will be covered. Single protein and sub-protein level approaches to reveal unique nanoscale protein-polymer surface interactions and protein surface assembly characteristics will be also emphasized. Potential advantages of these research endeavors in laying out fundamentally guided design principles for practical product development will then be discussed. Lastly, important research areas still needed to further narrow the knowledge gap in nanoscale protein adsorption will be identified.