Self-Assembled Block Copolymers as a Facile Pathway to Create Functional Nanobiosensor and Nanobiomaterial Surfaces.

Block copolymer (BCP) surfaces permit an exquisite level of nanoscale control in biomolecular assemblies solely based on self-assembly. Owing to this, BCP-based biomolecular assembly represents a much-needed, new paradigm for creating nanobiosensors and nanobiomaterials without the need for costly and time-consuming fabrication steps. Research endeavors in the BCP nanobiotechnology field have led to stimulating results that can promote our current understanding of biomolecular interactions at a solid interface to the never-explored size regimes comparable to individual biomolecules. Encouraging research outcomes have also been reported for the stability and activity of biomolecules bound on BCP thin film surfaces. A wide range of single and multicomponent biomolecules and BCP systems has been assessed to substantiate the potential utility in practical applications as next-generation nanobiosensors, nanobiodevices, and biomaterials. To this end, this Review highlights pioneering research efforts made in the BCP nanobiotechnology area. The discussions will be focused on those works particularly pertaining to nanoscale surface assembly of functional biomolecules, biomolecular interaction properties unique to nanoscale polymer interfaces, functionality of nanoscale surface-bound biomolecules, and specific examples in biosensing. Systems involving the incorporation of biomolecules as one of the blocks in BCPs, i.e., DNA-BCP hybrids, protein-BCP conjugates, and isolated BCP micelles of bioligand carriers used in drug delivery, are outside of the scope of this Review. Looking ahead, there awaits plenty of exciting research opportunities to advance the research field of BCP nanobiotechnology by capitalizing on the fundamental groundwork laid so far for the biomolecular interactions on BCP surfaces. In order to better guide the path forward, key fundamental questions yet to be addressed by the field are identified. In addition, future research directions of BCP nanobiotechnology are contemplated in the concluding section of this Review.

Preparation, Properties, and Bioapplications of Block Copolymer Nanopatterns.

Block copolymer (BCP) self-assembly has emerged as a feasible method for large-scale fabrication with remarkable precision - features that are not common for most of the nanofabrication techniques. In this review, recent advancements in the molecular design of BCP along with state-of-the-art processing methodologies based on microphase separation alone or its combination with different lithography methods are presented. Furthermore, the bioapplications of the generated nanopatterns in the development of protein arrays, cell-selective surfaces, and antibacterial coatings are explored. Finally, the current challenges in the field are outlined and the potential breakthroughs that can be achieved by adopting BCP approaches already applied in the fabrication of electronic devices are discussed.

The Marine Natural Compound Dragmacidin D Selectively Induces Apoptosis in Triple-Negative Breast Cancer Spheroids.

Cancer cells grown in 3D spheroid cultures are considered more predictive for clinical efficacy. The marine natural product dragmacidin D induces apoptosis in MDA-MB-231 and MDA-MB-468 triple-negative breast cancer (TNBC) spheroids within 24 h of treatment while showing no cytotoxicity against the same cells grown in monolayers and treated for 72 h. The IC50 for cytotoxicity based on caspase 3/7 cleavage in the spheroid assay was 8 ± 1 µM in MDA-MB-231 cells and 16 ± 0.6 µM in MDA-MB-468 cells at 24 h. No cytotoxicity was seen at all in 2D, even at the highest concentration tested. Thus, the IC50 for cytotoxicity in the MTT assay (2D) in these cells was found to be >75 µM at 72 h. Dragmacidin D exhibited synergy when used in conjunction with paclitaxel, a current treatment for TNBC. Studies into the signaling changes using a reverse-phase protein array showed that treatment with dragmacidin D caused significant decreases in histones. Differential protein expression was used to hypothesize that its potential mechanism of action involves acting as a protein synthesis inhibitor or a ribonucleotide reductase inhibitor. Further testing is necessary to validate this hypothesis. Dragmacidin D also caused a slight decrease in an invasion assay in the MDA-MB-231 cells, although this failed to be statistically significant. Dragmacidin D shows intriguing selectivity for spheroids and has the potential to be a treatment option for triple-negative breast cancer, which merits further research into understanding this activity.

Comparative Analysis of Antimicrobial Antibodies between Mild and Severe COVID-19.

Patients with 2019 coronavirus disease (COVID-19) exhibit a broad spectrum of clinical presentations. A person's antimicrobial antibody profile, as partially shaped by past infection or vaccination, can reflect the immune system health that is critical to control and resolve the infection. We performed an explorative immunoproteomics study using microbial protein arrays displaying 318 full-length antigens from 77 viruses and 3 bacteria. We compared antimicrobial antibody profiles between 135 patients with mild COVID-19 disease and 215 patients with severe disease in 3 independent cohorts from Mexico and Italy. Severe disease patients were older with higher prevalence of comorbidities. We confirmed that severe disease patients elicited a stronger anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) response. We showed that antibodies against HCoV-229E and HcoV-NL63 but not against HcoV-HKU1 and HcoV-OC43 were also higher in those who had severe disease. We revealed that for a set of IgG and IgA antibodies targeting coronaviruses, herpesviruses, and other respiratory viruses, a subgroup of patients with the highest reactivity levels had a greater incidence of severe disease compared to those with mild disease across all three cohorts. On the contrary, fewer antibodies showed consistent greater prevalence in mild disease in all 3 cohorts. IMPORTANCE The clinical presentations of COVID-19 range from asymptomatic to critical illness that may lead to intensive care or even death. The health of the immune system, as partially shaped by past infections or vaccinations, is critical to control and resolve the infection. Using an innovative protein array platform, we surveyed antibodies against hundreds of full-length microbial antigens from 80 different viruses and bacteria in COVID-19 patients from different geographic regions with mild or severe disease. We not only confirmed the association of severe COVID-19 disease with higher reactivity of antibody responses to SARS-CoV-2 but also uncovered known and novel associations with antibody responses against herpesviruses and other respiratory viruses. Our study represents a significant step forward in understanding the factors contributing to COVID-19 disease severity. We also demonstrate the power of comprehensive antimicrobial antibody profiling in deciphering risk factors for severe COVID-19. We anticipate that our approach will have broad applications in infectious diseases.

Protein Microarrays and their Fabrication.

Protein microarrays are an important tool when analyzing multiple analytes simultaneously. As the human genome contains approximately 20,000 genes, examining the interactions of even just one representative protein for each gene requires a high-throughput technique. For instance, the interaction between glycosaminoglycans (GAGs), a form of polysaccharide, and chemokines, small chemoattractant proteins, is critical for local inflammation. GAGs present in the glycocalyx on the surface of the cell bind to chemokines, which are released in response to injury. These chemokines can then form concentration gradients that direct the migration and recruitment of leucocytes via leukocyte receptors which in turn leads to immune cell responses, inflammation, or innate immunity and cell or antibody-mediated immune responses. Discovering the novel interactions between the GAGs and chemokines can help in designing drugs which can alter cellular binding to organ tissues, thereby potentially reducing damaging innate immune (inflammation) or acquired immune (antibody-mediated) responses.

Cytokines and Lymphoid Populations as Potential Biomarkers in Locally and Borderline Pancreatic Adenocarcinoma.

Despite its relative low incidence, PDAC is one of the most aggressive and lethal types of cancer, being currently the seventh leading cause of cancer death worldwide, with a 5-year survival rate of 10.8%. Taking into consideration the necessity to improve the prognosis of these patients, this research has been focused on the discovery of new biomarkers. For this purpose, patients with BL and resectable disease were recruited. Serum cytokines and growth factors were monitored at different time points using protein arrays. Immune cell populations were determined by flow cytometry in peripheral blood as well as by immunohistochemistry (IHC) in tumor tissues. Several cytokines were found to be differentially expressed between the study subgroups. In the BL disease setting, two different scores were proven to be independent prognostic factors for progression-free survival (PFS) (based on IL-10, MDC, MIF, and eotaxin-3) and OS (based on eotaxin-3, NT-3, FGF-9, and IP10). In the same context, CA19-9 was found to play a role as independent prognostic factor for OS. Eotaxin-3 and MDC cytokines for PFS, and eotaxin-3, NT-3, and CKß8-1 for OS, were shown to be predictive biomarkers for nab-paclitaxel and gemcitabine regimen. Similarly, oncostatin, BDNF, and IP10 cytokines were proven to act as predictive biomarkers regarding PFS, for FOLFIRINOX regimen. In the resectable cohort, RANTES, TIMP-1, FGF-4, and IL-10 individually differentiated patients according to their cancer-associated survival. Regarding immune cell populations, baseline high levels of circulating B lymphocytes were related to a significantly longer OS, while these levels significantly decreased as progression occurred. Similarly, baseline high levels of helper lymphocytes (CD4+), low levels of cytotoxic lymphocytes (CD8+), and a high CD4/CD8 ratio, were related to a significantly longer PFS. Finally, high levels of CD4+ and CD8+ intratumoural infiltration was associated with significantly longer PFS. In conclusion, in this study we were able to identify several prognostic and predictive biomarker candidates in patients diagnosed of resectable or BL PDAC.

Circulating Protein Biomarkers for Prognostic Use in Patients with Advanced Pancreatic Ductal Adenocarcinoma Undergoing Chemotherapy.

Patients with advanced pancreatic ductal adenocarcinoma (PDAC) have a dismal prognosis. We aimed to find a prognostic protein signature for overall survival (OS) in patients with advanced PDAC, and to explore whether early changes in circulating-protein levels could predict survival. We investigated 92 proteins using the Olink Immuno-Oncology panel in serum samples from 363 patients with advanced PDAC. Protein panels for several survival cut-offs were developed independently by two bioinformaticians using LASSO and Ridge regression models. Two panels of proteins discriminated patients with OS < 90 days from those with OS > 2 years. Index I (CSF-1, IL-6, PDCD1, TNFRSF12A, TRAIL, TWEAK, and CA19-9) had AUCs of 0.99 (95% CI: 0.98−1) (discovery cohort) and 0.89 (0.74−1) (replication cohort). For Index II (CXCL13, IL-6, PDCD1, and TNFRSF12A), the corresponding AUCs were 0.97 (0.93−1) and 0.82 (0.68−0.96). Four proteins (ANGPT2, IL-6, IL-10, and TNFRSF12A) were associated with survival across all treatment groups. Longitudinal samples revealed several changes, including four proteins that were also part of the prognostic signatures (CSF-1, CXCL13, IL-6, TNFRSF12A). This study identified two circulating-protein indices with the potential to identify patients with advanced PDAC with very short OS and with long OS.

Multilayered Ordered Protein Arrays Self-Assembled from a Mixed Population of Virus-like Particles.

Biology shows many examples of spatially controlled assembly of cells and biomacromolecules into hierarchically organized structures, to which many of the complex biological functions are attributed. While such biological structures have inspired the design of synthetic materials, it is still a great challenge to control the spatial arrangement of individual building blocks when assembling multiple types of components into bulk materials. Here, we report self-assembly of multilayered, ordered protein arrays from mixed populations of virus-like particles (VLPs). We systematically tuned the magnitude of the surface charge of the VLPs via mutagenesis to prepare four different types of VLPs for mixing. A mixture of up to four types of VLPs selectively assembled into higher-order structures in the presence of oppositely charged dendrimers during a gradual lowering of the ionic strength of the solution. The assembly resulted in the formation of three-dimensional ordered VLP arrays with up to four distinct layers including a central core, with each layer comprising a single type of VLP. A coarse-grained computational model was developed and simulated using molecular dynamics to probe the formation of the multilayered, core-shell structure. Our findings establish a simple and versatile bottom-up strategy to synthesize multilayered, ordered materials by controlling the spatial arrangement of multiple types of nanoscale building blocks in a one-pot fabrication.

Proteomic and Transcriptomic Profiling Reveals Mitochondrial Oxidative Phosphorylation as Therapeutic Vulnerability in Androgen Receptor Pathway Active Prostate Tumors.

Metastatic prostate cancer (PC) is the second leading cause of cancer deaths in males and has limited therapeutic options. The lack of preclinical models for advanced stage PC represents one of the primary barriers in understanding the key genetic drivers of aggressive subsets, including androgen receptor (AR) pathway active and AR-null castration-resistant prostate cancers (CRPC). In our studies, we described a series of LuCaP patient-derived xenograft (PDX) models representing the major genomic and phenotypic features of human disease. To fully exploit the potential of these preclinical models, we carried out a comprehensive transcriptomic and proteomic profiling of 42 LuCaP PDX prostate tumors. The collected proteomic data (~6000 data points) based on 71 antibodies revealed many of the previously known molecular markers associated with AR-positive and AR-null CRPC. Genomic analysis indicated subtype-specific activation of pathways such as Wnt/beta-catenin signaling, mTOR, and oxidative phosphorylation for AR-positive CRPC and upregulation of carbohydrate metabolism and glucose metabolism for AR-null CRPC. Of these, we functionally confirmed the role of mitochondrial metabolism in AR-positive CRPC cell lines. Our data highlight how the integration of transcriptomic and proteomic approaches and PDX systems as preclinical models can potentially map the connectivity of poorly understood signaling pathways in metastatic prostate cancer.

Proteomics technologies for cancer liquid biopsies.

Alterations in DNAs could not reveal what happened in proteins. The accumulated alterations of DNAs would change the manifestation of proteins. Therefore, as is the case in cancer liquid biopsies, deep proteome profiling will likely provide invaluable and clinically relevant information in real-time throughout all stages of cancer progression. However, due to the great complexity of proteomes in liquid biopsy samples and the limitations of proteomic technologies compared to high-plex sequencing technologies, proteomic discoveries have yet lagged behind their counterpart, genomic technologies. Therefore, novel protein technologies are in urgent demand to fulfill the goals set out for biomarker discovery in cancer liquid biopsies.Notably, conventional and innovative technologies are being rapidly developed for proteomic analysis in cancer liquid biopsies. These advances have greatly facilitated early detection, diagnosis, prognosis, and monitoring of cancer evolution, adapted or adopted in response to therapeutic interventions. In this paper, we review the high-plex proteomics technologies that are capable of measuring at least hundreds of proteins simultaneously from liquid biopsy samples, ranging from traditional technologies based on mass spectrometry (MS) and antibody/antigen arrays to innovative technologies based on aptamer, proximity extension assay (PEA), and reverse phase protein arrays (RPPA).

High ETV6 Levels Support Aggressive B Lymphoma Cell Survival and Predict Poor Outcome in Diffuse Large B-Cell Lymphoma Patients.

The identification of prognostic factors for aggressive B-cell lymphomas still represents an unmet clinical need. We used forward phase protein arrays (FFPA) to identify proteins associated with overall survival (OS) from diagnostic formalin-fixed paraffin-embedded material of diffuse large B-cell lymphoma (DLBCL) patients (n = 47). Univariate Cox regression analysis identified numerous proteins, including immune check-point molecules (PDCD1, PDCD2 and PD1L2) and BCL2 to be significantly associated with OS. However, only ETV6 and PIM2 proteins persisted following multivariate Cox analysis. Independent validation studies by immunohistochemistry and analysis of public gene expression profiles of DLBCL confirmed a prognostic role for high ETV6 and ETV6/PIM2 ratios in DLBCL. ETV6 is a recurrently mutated/deleted gene in DLBCL for which its function in this disease entity is currently unknown. We find that ETV6 is upregulated during oncogenic transformation of germinal center B-cells and that it regulates DLBCL survival, as its acute loss results in marked apoptosis. Fluctuations in survivin (BIRC5) expression levels were associated with this phenomenon. Furthermore, an inverse correlation between ETV6 and BIRC5 expression levels was found and correlated with a response to the BIRC5 inhibitor, YM155. In conclusion, we present evidence for an oncogenic function of ETV6 in DLBCL.

Evaluation of Signaling Pathways Profiling in Human Dermal Endothelial Cells Treated by Snake Venom Cysteine-Rich Secretory Proteins (svCRiSPs) from North American Snakes Using Reverse Phase Protein Array (RPPA).

Cysteine-Rich Secretory Proteins (CRiSPs) are typically found in many snake venoms; however, the role that these toxins play in the pathophysiology of snakebites is still unclear. Herein, we compared the effects of snake venom CRiSPs (svCRiSPs) from the most medically important species of North American snakes on endothelial cell permeability and vascular permeability. We used reverse phase protein array (RPPA) to identify key signaling molecules on human dermal lymphatic (HDLECs) and blood (HDBECs) endothelial cells treated with svCRiSPs. The results showed that Css-CRiSP isolated from Crotalus scutulatus scutulatus and App-CRiSP from Agkistrodon piscivorus piscivorus are the most potent causes of increase vascular and endothelial permeability in comparison with other svCRiSPs used in this study. We examined the protein expression levels and their activated phosphorylation states in HDLECs and HDBECs induced by App-CRiSP and Css-CRiSP using RPPA. Interestingly, both App-CRiSP and Css-CRiSP induced caveolin-1 expression in HDBECs. We also found that stimulating HDBECs with Css-CRiSP and App-CRiSP significantly induced the phosphorylation of mTOR and Src, respectively. In HDLECs, Css-CRiSP significantly downregulated the expression of N-Cadherin and phospholipase C-gamma, while App-CRiSP significantly enhanced Akt and JNK phosphorylation. These results suggest that the increased endothelial permeability in HDLECs and HDBECs by Css-CRiSP and App-CRiSP may occur through different pathways.

Protein Arrays for the Identification of Seroreactive Protein Markers for Infectious Diseases.

The protein array is a powerful platform to study humoral responses to infectious agents using small sample volumes [<3 µL]. Its success can be largely attributed to the development of new strategies for high-throughput cloning and expression, and improved manufacturing techniques for the construction of arrays. Here, we describe a method to hybridize protein arrays with malaria patients' sera in order to identify seroreactive antigens, some of which may have a high potential of conferring protection from severe forms of malaria.

Human apoptosis antibody array-membranes studying the apoptotic effect of marine bacterial exopolysaccharides in HepG2 cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is considered as the third leading cause of cancer-related deaths, in spite of great advances in its treatment. The carbohydrate polymers, exopolysaccharides (EPSs), showed anticancer activity in diverse cancers. OBJECTIVE: The purpose of this study is to investigate a panel of 43 apoptotic proteins to assess the possible apoptotic induction effect of bacterial EPSs showing promising cytotoxic effects in HepG2 cells in our previous study, in an attempt to introduce exopolysaccharides as new source for cancer treatment. MATERIALS AND METHODS: Apoptosis-related proteins panel were examined through the analysis of Human Apoptosis Antibody Array-Membrane (43 targets). RESULTS: EPS-6 induces apoptosis through upregulation of different pro-apoptotic proteins as cytochrome C (9.52 fold) and tumor necrosis factor-related apoptosis-inducing ligand receptor (TRAIL-R1) (153.49 fold). EPS-RS induces apoptosis through up regulation of second mitochondria-derived activator of caspases (SMAC) (15.75 fold) and the six insulin-like growth factors binding proteins (IGFBP-1 through - 6) (76.81 fold, 7.68 fold, 55.15 fold, 4.9 × 107 fold, 29.69 fold, and 28.92 fold), respectively. CONCLUSION: Our results suggested that EPS-6 and EPS-RS could be considered as promising agents in hepatocellular carcinoma treatment.

[New antibodies in diagnostics]. / Neue Antikörper in der Diagnostik.

Rheumatologists were pivotal in the development of using autoantibodies to diagnose chronic inflammatory diseases. Rheumatoid factors were already discovered in 1940 and antinuclear antibodies and their target structures in the 1950s and 1960s. Even though now a vast array of autoantibodies can be routinely measured, we still need more diagnostic markers for chronic inflammatory diseases. Nowadays novel autoantibodies can be easily discovered using new technologies which are described in this article, Therefore we can expect, that new diagnostic autoantibodies will be available soon.

Current Techniques to Accurately Measure Anti-retinal Autoantibodies.

INTRODUCTION: Autoimmune retinopathy (AR) is a sight-threating retinal disorder that is mediated by autoantibodies (AAbs) against retinal proteins. The visual paraneoplastic syndromes, including cancer-associated retinopathy (CAR) and melanoma-associated retinopathy (MAR) are mediated by anti-retinal AAbs. A number of immunochemical techniques have been used to detect serum anti-retinal autoantibodies in patients to help with autoimmune diagnosis. AREA COVERED: We review techniques used for serum autoantibody evaluation in patients with suspected autoimmune retinopathy. EXPERT OPINION: Detection of serum AAbs have served as the standard diagnostic tool for autoimmune retinopathies and for management of retinal disorders. An identification of anti-retinal autoantibody or multiple autoantibodies can be useful for not only for diagnosis of autoimmune retinopathies but also for management of retinal disorders. We propose that the line-blotting technique used in conjunction with immunohistochemistry are the best and most reliable assays for detection of serum anti-retinal AAb in the context of clinical history and findings. Clinician should recognize that the majority of antigenic targets identified to date in retinal autoimmunity are ubiquitously expressed proteins (e.g. enolase), which may be difficult to reconcile with the specific patterns of retinal damage observed in CAR, MAR, or AR.

Comparative Advantages and Limitations of Quantum Dots in Protein Array Applications.

Antibody microarrays have become a powerful tool in multiplexed immunoassay technologies. The advantage of microarray technology is the possibility of rapid analysis of multiple targets in a single sample with a high sensitivity, which makes them ideal for high throughput screening. Usually these microarrays contain biological recognition molecules, such as full-size antibodies, antigen-binding fragments, and single-domain antibodies, and a label for detection. Organic fluorophores are the most popular labels, but they suffer from low sensitivity and instability due to their photodegradation. Here, we describe a protocol for fabricating an antibody microarray with highly fluorescent semiconductor nanocrystals or quantum dots (QDs) as the source of fluorescent signals, which may significantly improve the properties of microarrays, including their sensitivity and specificity. Our approach to analyte detection is based on the use of sandwich approach with streptavidin-biotin to assess and monitor the fluorescence signal instead of direct labeling of samples, which helps improve the reproducibility of results and sensitivity of the microarrays. The antibody microarray developed has been tested for its capacity of detecting DNA-PKcs in glial cell lines and measuring cell protein phosphorylation changes caused by camptothecin-induced DNA damage with different protein kinase inhibitors in HeLa cells.

Circulating Retinol-Binding Protein 4 as a Possible Biomarker of Treatment Response for Ankylosing Spondylitis: An Array-Based Comparative Study.

OBJECTIVE: To explore proteins associated with ankylosing spondylitis (AS) and to investigate potential proteins that may predict treatment response of adalimumab (ADA) in AS patients. METHODS: In the discovery cohort, 39 AS patients and 20 healthy controls (HCs) were included, and 16 AS patients received ADA treatment for 24 weeks after included. In the validation cohort, 43 AS patients and 39 HCs were enrolled, and all 43 patients received ADA treatment after enrollment. Blood samples and clinical information were collected from two cohorts at baseline from all participants and week 24 from patients received ADA treatment. A human antibody array containing 1,000 proteins was used in the discovery phase, and Elisa kits were used for protein validation. RESULTS: Compared with HCs, we identified 53 differentially expressed proteins (DEPs) in AS patients. Bioinformatics analysis revealed they were mostly enriched in coagulation function-related pathways, acute response signaling, and LXR/RXR activation. Bone metabolism pathways were also associated. Comparison between samples of pre- and post-ADA treatment revealed 42 DEPs. They were mostly associated with bone metabolism and inflammation response pathways. Significant enrichment was also found in LXR/RXR activation but not the coagulation function-related pathways. Upstream regulator analysis suggested that most regulators also significantly functioned under usage of ADA. Precisely, seven proteins were abnormally expressed in AS and restored after ADA treatment. Retinol-binding protein 4 (RBP4), one of the seven proteins, was validated that its baseline levels were inversely correlated with improvements in Ankylosing Spondylitis Disease Activity Score-C-reactive protein (ASDAS-CRP). Likewise, percentage changes in RBP4 levels were inversely correlated with changes in ASDAS-CRP score. CONCLUSION: A dysregulated serum protein profile existed in AS. ADA exerted a considerable but not entire alteration toward the dysregulation. RBP4 could be a biomarker for predicting and monitoring ADA treatment response.