Microarray Scanner Research Articles

Immunoblot Analysis from Single Cells Using Milo™ Single-Cell Western Platform.

In this new era of precision medicine, characterization of single-cell subpopulations to better understand disease etiology is paramount. It is thus an opportune time to explore techniques that allow molecular analysis of single cells and to better understand the basis of pathogenesis of diseases like cancer. Single-cell western blotting is one such method that allows analysis of single cells at the protein level. In contrast to traditional western blotting, which relies heavily on bulk analysis of lysates generated from tissues and is often indicative of the population average, this technique allows analysis of lysates from single-cell subpopulations thereby providing a glimpse into cell heterogeneity. The method entails the use of a chip containing 30μm thick photoactivated polyacrylamide gel spotted with nearly 6400 microwells. Single cells loaded on the chip are captured in the microwells by passive gravity and are then lysed and electrophoresed using the MILO™ single-cell western platform. This method forgoes the use of transfer of proteins on a PVDF and a nitrocellulose membrane, as performed in traditional western blotting, and all other steps including probing of primary and fluorescent secondary antibodies against the protein of interest are performed directly on the chip. The proteins of interest can then be visualized by scanning a chip with the use of a microarray scanner. The entire procedure can be performed in as less as 4-6h, and thus this method provides several advantages over traditional western blotting.

A-271 Verification of a multiplexed STI Pathogen Microarray Test with Saliva Collected With SDNA-1000 Kit

Abstract Background Sexually transmitted infections (STI) spread through oral sex potentially cause infections of the mouth, throat, genitals, or rectum. Saliva may be an important source of transmission of STI following oral sex. Multiplexed STI assays are useful diagnostically for patients with risk factors for STI as co-infections occur frequently. Methods Alimetrix STI is a multiplexed laboratory developed test for the identification of STI pathogens present in clinical specimens. DNA was collected in SDNA-1000 devices, extracted, purified, underwent multiplex PCR, and labeled amplicons were hybridized to a microarray with gene-specific 50mer capture probes. Fluorescently tagged amplicons were detected on a microarray scanner. Verification included analytical sensitivity (Limit of Detection (LOD), competition, and inclusivity), stability, and analytical specificity (exclusivity, precision and accuracy). Pathogens included in the multiplexed microarray included N. gonorrhea, C. trachomatis, T. vaginalis, T. pallidum , herpes 1 and 2, M. genitalium, U. urealyticum, and monkeypox. Results LOD for each pathogen in the Spectrum Solutions SDNA Collection System, throat swab, and first catch urine is found in Table 1. Competition of organisms showed the ability to co-detect up to 5 organisms from a single sample. Saliva collected using Spectrum Solutions SDNA Collection System was stable for 35 days at room temperature enabling discreet at home collection. Inclusivity showed related strains were detectable at 1× LOD, while maintaining 100% precision, 100% accuracy, and 97% exclusivity for high concentrations of closely related analytes in normal flora. Conclusion Control of STI associated with oral sex is dependent upon the demonstration of all pathogens present. Saliva collected in the SDNA-1000 device a high degree of sensitivity, precision, and accuracy, as well as the ability to detect multiple analytes (co-infection) within a given sample. Saliva is a clinically informative specimen source useful for the diagnosis, monitoring, and management of patients with oral and systemic disease.

Auto-immunoproteomics analysis of COVID-19 ICU patients revealed increased levels of autoantibodies related to the male reproductive system.

Background: Coronavirus disease (COVID-19) manifests many clinical symptoms, including an exacerbated immune response and cytokine storm. Autoantibodies in COVID-19 may have severe prodromal effects that are poorly understood. The interaction between these autoantibodies and self-antigens can result in systemic inflammation and organ dysfunction. However, the role of autoantibodies in COVID-19 complications has yet to be fully understood. Methods: The current investigation screened two independent cohorts of 97 COVID-19 patients [discovery (Disc) cohort from Qatar (case = 49 vs. control = 48) and replication (Rep) cohort from New York (case = 48 vs. control = 28)] utilizing high-throughput KoRectly Expressed (KREX) Immunome protein-array technology. Total IgG autoantibody responses were evaluated against 1,318 correctly folded and full-length human proteins. Samples were randomly applied on the precoated microarray slides for 2h. Cy3-labeled secondary antibodies were used to detect IgG autoantibody response. Slides were scanned at a fixed gain setting using the Agilent fluorescence microarray scanner, generating a 16-bit TIFF file. Group comparisons were performed using a linear model and Fisher's exact test. Differentially expressed proteins were used for KEGG and WIKIpathway annotation to determine pathways in which the proteins of interest were significantly over-represented. Results and conclusion: Autoantibody responses to 57 proteins were significantly altered in the COVID-19 Disc cohort compared to healthy controls (p ≤ 0.05). The Rep cohort had altered autoantibody responses against 26 proteins compared to non-COVID-19 ICU patients who served as controls. Both cohorts showed substantial similarities (r 2 = 0.73) and exhibited higher autoantibody responses to numerous transcription factors, immunomodulatory proteins, and human disease markers. Analysis of the combined cohorts revealed elevated autoantibody responses against SPANXN4, STK25, ATF4, PRKD2, and CHMP3 proteins in COVID-19 patients. The sequences for SPANXN4 and STK25 were cross-validated using sequence alignment tools. ELISA and Western blot further verified the autoantigen-autoantibody response of SPANXN4. SPANXN4 is essential for spermiogenesis and male fertility, which may predict a potential role for this protein in COVID-19-associated male reproductive tract complications, and warrants further research.

Blood Group A Enhances SARS-CoV-2 Infection

BACKGROUND: Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) continues to infect millions of individuals worldwide. However, not all individuals are equally susceptible to infection. Many studies have demonstrated that the first polymorphism described in the human population, ABO(H) blood group antigens, are associated with increased risk of SARS-CoV-2 infection. While conflicting data exist, the most common findings suggest that individuals with blood group A exhibit an increased risk of infection when compared to blood group O individuals. However, the mechanism whereby ABO(H) blood group status influences SARS-CoV-2 infection has remained unknown. The receptor binding domain (RBD) of SARS-CoV-2, which facilitates host cell engagement, bears significant similarity to galectins, an ancient family of carbohydrate binding proteins previously shown to recognize ABO(H) blood group antigens. As a result, we hypothesized that SARS-CoV-2 may recognize blood group A directly through its RBD and that this may in part account for increased susceptibility of blood group A individuals for SARS-CoV-2 infection. Methods: The SARS-CoV-2 RBD from the parent strain (Wuhan-Hu-1) and variants (Delta and Omicron), in addition to individual human galectins (1, 2, 3, 4, 7 and 9) were cloned, purified and subjected to glycan binding specificity analysis using a glycan microarray populated with hundreds of distinct glycans, including blood group antigens. This was accomplished by incubating each fluorescently labeled RBD or galectin on the array, followed by detection of bound fluorescence using a microarray scanner (GenePix 4000 B, Molecular devices) and integrated spot intensity analysis using Imagene software (GenePix Pro 7). SARS-CoV-2 with distinct spike proteins (Wuhan-Hu-1, Delta, and Omicron) were used to assess infection using Chinese hamster ovary (CHO) cells engineered to express angiotensin converting enzyme 2 (ACE2) and type 1 blood group A or H (blood group O antigen). A one-way ANOVA with a Tukey's post hoc with a p value <0.05 was considered significant. Results: Sequence analysis of galectins demonstrates that several galectins possess up to 11% sequence identity with the RBDs of SARS-CoV-2 WT (Wuhan-Hu-1) and variants (Delta and Omicron). Each RBD (Wuhan, Delta and Omicron) exhibited glycan binding specificity that overlapped with distinct members of the galectin family, with the highest similarity observed toward the C terminal domain of galectin-4 (Gal-4C). Direct comparison of binding toward ABO(H) antigens demonstrated that each RBD and Gal-4C displayed high specificity for the type 1 blood group A structure uniquely found on respiratory epithelial cells when compared to other blood group antigens (p<0.0001). To specifically examine the impact of blood group A on SARS-CoV-2 infection, CHO cells were engineered to express ACE2 and the type 1 blood group A or H antigen (the antigen expressed in blood group O individuals) normally found on respiratory epithelial cells. Consistent with the increased binding of the RBD to blood group A on the glycan microarray, enhanced binding was observed by each RBD toward blood group A expressing cells (p<0.001). Blood group A cells were also significantly more likely to be infected with Wuhan-Hu-1, Delta and Omicron variants of SARS-CoV-2 when compared to blood group O cells, while pre-incubation of cells with Gal-4C specifically inhibited infection of blood group A expressing cells, while failing to impact infection of blood group O cells (p<0.001). Conclusions: These results demonstrate a direct effect of blood group A on viral infection and suggest that blood group A individuals may exhibit an increased susceptibility to SARS-CoV-2 as a result of direct engagement of the blood group A antigen. However, variations in ACE2 levels, blood group A expression and many other factors likely influence the overall risk of SARS-CoV-2 infection following exposure in a given individual. The present results provide one mechanism whereby blood group A itself may directly influence SARS-CoV-2 infectious risk.

Preparation of substrates for microarray protein chips with different ending functional groups

Protein microarray chips are composed of three components, these are pre-treatment substrates, surface chemical modification, and immobilizing protein on substrate surfaces. In this study, self-assembly monolayers are used for surface chemical modification. Using this method, silanization on a glass and silicon chip is achieved, forming the terminal group substrates. Modification of the substrate surface to provide COOH and NH2 terminal functional groups provides a mechanism to proteins to immobilize on the substrate surface. To observe immobilized proteins on the substrate surface, they are first labeled with Cy5 fluorescent dye before analysis using a GenePix 4000B Microarray Scanner. The scanner induces fluorescence in the labelling dye and the resulting light is analyzed to provide information concerning both the quantity of immobilized protein, and the orientation of attachment. The antigen of the HSV-1 virus, a common human virus, was used in this study, performing an antigen-antibody analysis to determine the efficacy of the method under test for clinical diagnosis.

PSIX-13 Activation of cell signaling pathways by galectins in cow blood

Abstract Galectins are part of a conserved family of β-galactoside-binding proteins that contribute to critical biological events during mammalian gestation and increasingly recognized for a possible role in the immune response of the cow. The objective of this study was to evaluate the effect of Galectins on signal transduction and cell activation in cow blood. Whole blood was collected aseptically from the jugular vein of healthy Holstein Friesian dairy cows (N=3). Blood samples (2.5ml) in duplicate were treated with 150µl of the four different type of recombinant galectins (1, 3, 4, and 9) respectively and untreated samples were served as control. The concentration of total plasma protein was determined using the Pierce BCA kit. Protein expression profiling was performed using1,358 antibodies on the Full Moon BioSystems’ Signaling Explorer antibody array covering 20 cell signaling pathways, as recommended by the manufacturer using an Agilent microarray scanner. Data normalization was performed using GeneSpring GX software to generate fold changes in gene expression and then filtered to obtain a list of significantly upregulated and downregulated genes. Features were extracted from protein array images of samples treated with Galectin 1, 3, 4, 9, and untreated sample as a control group. Treatment with all four Galectins increased the concentration of total plasma protein. Average increases due to treatment with Gal1, Gal3, Gal4, and Gal9 were 27%, 10%, 20%, and 14% respectively. ANOVA test showed significance difference among the groups (p &amp;lt; 0.05). Dunnett option was used to compare each of the treated samples to the control group as a baseline. The results also showed that there was significant difference between the control group and any of the treated group (p &amp;lt; 0.05). Distinct signaling pathways are activated in response to Galectin exposure. Further studies are needed to define their regulation and functional impact on cow health.

Select DNA Replication And Repair Genes Are Transcriptionally Modified By Acute Exercise In Hypoxia

DNA replication and repair in eukaryote cells are critical processes that ensures the correct transfer of DNA material through mitosis. Errors in the replication and repair of DNA during and following cell division may lead to genome instability. PURPOSE: To investigate the regulation of select gene transcripts aligned to DNA replication and repair following exercise in hypoxia. METHODS: Twenty-four (n = 24) apparently healthy male participants were recruited (age 28 ± 5 years; mass 74 ± 8 kg; stature 177 ± 6 cm; VO2max hypoxia 45 ± 2 ml·kg-1·min-1: normoxia 60 ± 9 ml·kg-1·min-1), and completed 1 hr of exercise at a workload corresponding to 75% of pre-determined VO2max in hypoxia (Fio2 = 0.16%), and repeated in normoxia (Fio2 = 0.21%). RNA was extracted from whole blood. Two-colour oligonucleotide microarrays [for DNA primase subunit 1 - PRIM1; Chromatin assembly factor 1 subunit B - CHAF1B; and Werner syndrome RecQ like helicase - WRN] were processed on an Agilent DNA microarray scanner, while mRNA analysis was conducted using GeneSpring software incorporating checks for data normalisation, quality control, filtering grouping (by experimental condition) and statistical analysis (paired t-test with Benjamini & Hochberg False Discovery Rate corrections for multiple comparisons). RESULTS: Exercise performed in hypoxia per se downregulated PRIM1 and CHAF1B (p = 0.03 and p = 0.01 respectively vs. pre-exercise), with no change observed in normoxia (p > 0.05). Compared with normoxia, CHAF1B and WRN were differentially downregulated following exercise in hypoxia (p = 0.05 and p = 0.05 respectively vs. pre-exercise). CONCLUSION: Acute exercise in moderate hypoxia interferes with gene transcription aligned to blood cell DNA replication and repair. Further work is necessary to ascertain the consequence of these select modifications on genome stability.

Fabrication of a Fluorophore/Liquid‐Crystal‐Based Oligopeptide Biosensor for the Detection of Cu (II) Ions

AbstractA new sensing methodology has been developed for the detection of Cu(II) ions in water by using biotinylated oligopeptide as a probe whereas liquid crystal (LC) and fluorophore were used as signal reporters. The multidentate oligopeptide ligand was used to investigate Cu(II) as a model metal ion. The alkyl terminal group of the coated dimethyl octadecyl [3‐(trimethoxysilyl) propyl] ammonium chloride (DMOAP) was first activated to aldehydic functional group by UV irridiation for 50 seconds. The activated aldehydic functional group was covalent linked with biotinylated oligopeptide via thiolation reaction. As fabricated oligopeptide probe spotted with metal ions was incubated for 30 minutes and followed by tagging with streptavidin‐cyanine‐3 as a fluorophore. Polarized optical microscopy (POM) and fluorescence microarray scanner (FMS) were used as characterization techniques. The developed sensing methodology results were reproducible for different concentrations of model Cu (II) ions as confirmed from the characterization techniques. Our methodology highlighted the detection of Cu(II) ions in water as low as 100 nM, and is the lowest concentration so far reported.

Single-Cell Multiplex Proteomics Reveals Synergistic Impact of Antigen and Rimiducid-Dependent Stimulatory Signals on Promoting Polyfunctional GoCAR-T Cells Targeting Prostate Stem Cell Antigen (PSCA)

Background: The GoCAR-T cell products targeting tumor antigens and incorporated with a rimiducid-controlled switch for T cell full activation have emerged as next generation CAR-T cell therapy in solid tumor. To fully characterize the cytokine/chemokine profile of pre-infusion GoCAR-T cell products, we employed a single-cell detection and bioinformatic platform to deeply assess functional attributes of GoCAR-T cells by simultaneous detection of the 32 proteins secreted from single GoCAR-T cells upon various stimulations.Methods: GoCAR-T cells were manufactured from human peripheral blood mononuclear cells (PBMCs) of healthy donors transduced with a rimiducid-inducible costimulatory unit, MyD88/CD40 (iMC), and a first-generation CAR targeting PSCA. CD4+ and CD8+ GoCAR-T cells were enriched by anti-CD4 or anti-CD8 microbeads and stimulated with PSCA-expressing human pancreatic adenocarcinoma cell line (HPAC) in the presence or absence of rRimiducid at 37°C, 5% CO2. After 24-hour coculture, CD4+ and CD8+ GoCAR-T cells were collected and loaded into a single-cell barcode chip (SCBC) containing ~12,000 microchambers. Each chamber (~1.2 nl) was pre-patterned with a complete copy of a 32-plex antibody array. Cells on the SCBC were imaged and incubated for 16 hrs at 37°C, 5% CO2; single-cell cytokine signals were captured with a microarray scanner. The polyfunctional profile (2+ cytokines per cell) of single GoCAR-T cells was evaluated across 5 functional groups: Effector: Granzyme B, TNF-α, IFN-γ, MIP-1α, Perforin, TNF-β; Stimulatory: GM-CSF, IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, IL-15, IL-21; Chemoattractive: CCL11, IP-10, MIP-1β, RNATES; Regulatory: IL-4, IL-10, IL-13, IL-22, sCD137, sCD40L, TGF-β1; Inflammatory: IL-6, IL-17A, IL-17F, MCP-1, MCP-4, IL-1β.Results: Single-cell multiplex proteomic analysis demonstrates the synergistic effects of PSCA-specific and rimiducid-induced stimulation on polyfunctional upregulation in both CD4+ and CD8+ GoCAR-T cells across donors. Polyfunctional strength index (PSI) reveals the PSCA-induced polyfunctional increase in GoCAR-T cells was predominated by antitumor-associated proteomics including Granzyme B, IFN-γ, TNF-α, Perforin, MIP-1α, IL-2, IL-8, IP-10, MIP-1β, sCD137 and sCD40L, and further elevated in GM-CSF by the costimulatory signals induced by rimiducid. In addition, the polyfunctional heatmap uncovers the upregulated polyfunctional cell subsets of single GoCAR-T cells that co-secreted combinatorial proteins of Granzyme B, IFN-γ, TNF-α, Perforin, MIP-1α, IL-2, IL-8, IP-10, MIP-1β, sCD137 and sCD40L in response to PSCA and/or rimiducid.Conclusions: Single-cell multiplexed precision profiling reveals antitumor polyfunctional upregulation and heterogeneity of GoCAR-T cell products with the stimulation of PSCA+ tumor cells and/or iMC activation by rimiducid. The presented polyfunctional metrics may provide insights into quality check of pre-infusion GoCAR-T cell products and potential biomarker discovery to predict efficacy, persistence and safety of GoCAR-T cell therapy in solid tumor. DisclosuresMackay:IsoPlexis: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Speakers Bureau. Flynn:IsoPlexis: Employment, Equity Ownership. Morse:IsoPlexis: Employment, Equity Ownership. Ng:IsoPlexis: Employment, Equity Ownership. Paczkowski:IsoPlexis: Employment, Equity Ownership. Mahendravada:Bellicum Pharmaceuticals: Employment. Shinners:Bellicum Pharmaceuticals: Employment. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership, Other: stockholders . Foster:Bellicum Pharmaceuticals: Employment, Other: stockholders . Zhou:IsoPlexis: Employment, Equity Ownership.

ProtGear: A protein microarray data pre-processing suite

Protein microarrays are versatile tools for high throughput study of the human proteome, but systematic and non-systematic sources of bias constrain optimal interpretation and the ultimate utility of the data. Published guidelines to limit technical variability whilst maintaining important biological variation favour DNA-based microarrays that often differ fundamentally in their experimental design. Rigorous tools to guide background correction, the quantification of within-sample variation, normalisation, and batch correction specifically for protein microarrays are limited, require extensive investigation and are not centrally accessible.Here, we develop a generic one-stop-shop pre-processing suite for protein microarrays that is compatible with data from the major protein microarray scanners. Our graphical and tabular interfaces facilitate a detailed inspection of data and are coupled with supporting guidelines that enable users to select the most appropriate algorithms to systematically address bias arising in customized experiments. The localization and distribution of background signal intensities determine the optimal correction strategy. A novel function overcomes the limitations in the interpretation of the coefficient of variation when signal intensities are at the lower end of the detection threshold. We demonstrate essential considerations in the experimental design and their impact on a range of algorithms for normalization and minimization of batch effects.Our user-friendly interactive web-based platform eliminates the need for prowess in programming. The open-source R interface includes illustrative examples, generates an auditable record, enables reproducibility, and can incorporate additional custom scripts through its online repository. This versatility will enhance its broad uptake in the infectious disease and vaccine development community.

Abstract 898: Single-cell multiplex proteomics reveals synergistic activity of antigen and MyD88/CD40 stimulatory signals on promoting polyfunctional chimeric antigen receptor T cells

Abstract Introduction: GoCAR is a chimeric antigen receptor (CAR) platform that utilizes a rimiducid-dependent activation switch (inducible MyD88/CD40; iMC) to enhance CAR-T cell proliferation and to stimulate the host immune system. To understand how these unique CAR signaling molecules compare to conventional costimulatory domains (e.g., CD28 and 4-1BB), we employed a detection and bioinformatics platform to assess the functional attributes of CD19-specific CAR-T cells by simultaneous detection of 32 proteins secreted from single CAR-T cells upon various stimulation conditions. Methods: T cells were transduced with retrovirus encoding CD19-specific CARs with conventional costimulatory molecules (CD19.ζ, CD19.28.ζ or CD19.BB.ζ) or with GoCAR, a vector encoding iMC and a CD19-specific CAR (iMC-CD19.ζ). Control or modified T cells were enriched by anti-CD4 or anti-CD8 microbeads and stimulated with CD19 or ΔNGFR-expressing K562 cells with or without 10 nM rimiducid. After 24-hour, CD4 and CD8 T cells were collected and loaded into an IsoCode chip containing ~12,000 microchambers. Each chamber (~1.2 nL) was pre-patterned with a complete copy of a 32-plex antibody array. Cells on-chip were imaged, and single-cell cytokine signals were captured with a microarray scanner. The T cell polyfunctional profile (2+ cytokines per cell) of single T cells was evaluated across 5 functional groups: Effector: Granzyme B, TNFα, IFNγ, MIP1α, Perforin, TNFβ; Stimulatory: GM-CSF, IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, IL-15, IL-21; Chemoattractive: CCL11, IP-10, MIP-1β, RANTES; Regulatory: IL-4, IL-10, IL-13, IL-22, sCD137, sCD40L, TGFβ1; Inflammatory: IL-6, IL-17A, IL-17F, MCP-1, MCP-4, IL-1β. Results: Single-cell multiplex proteomic analysis demonstrated the synergistic effects of CD19-specific and rimiducid-induced stimulation on polyfunctional upregulation in both CD4 and CD8 GoCAR-T cells across donors. In the “off” state (i.e., no rimiducid), CD19-specific GoCAR-T showed a reduced polyfunctional strength index (PSI) compared to CD28 and 4-1BB CAR-T cells, which was predominated by antitumor-associated proteins. In contrast, activation of iMC dramatically increased GoCAR-T PSI, which was characterized by elevated “stimulatory” (GM-CSF, IL-2, IL-5, IL-8, IL-9), “chemotactic” (MIP-1b) and “regulatory” (IL-4, sCD137) molecules compared to conventional CAR constructs. Polyfunctional heatmaps revealed that GoCAR-T cells co-secreted combinatorial proteins in response to CD19-expressing target cells in combination with iMC-based costimulation. Further, both CD4+ and CD8+ GoCAR-T cells acquired the ability to produce IL-2 as part of their polyfunctional profile, which was only observed with iMC costimulation. Conclusions: Single-cell multiplexed proteomic profiling reveals that polyfunctionality can be regulated by small molecule activation of an inducible costimulatory switch, producing distinct “on/off” states. iMC signaling also enhances production of important stimulatory and chemoattractive molecules which may improve CAR-T cell activity and provoke a host anti-tumor immune response. Thus, the GoCAR-T platform may potentially be used to optimize CAR-T activity through rimiducid administration to maximize efficacy while mitigating CAR-T-related toxicities. Citation Format: Aaron Foster, Joanne Shaw, Matthew Collinson-Pautz, Aruna Mahendravada, Christine Gagliardi, Mariam Khalil, Patrick Paczkowski, Sean Mackay, Jing Zhou. Single-cell multiplex proteomics reveals synergistic activity of antigen and MyD88/CD40 stimulatory signals on promoting polyfunctional chimeric antigen receptor T cells [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 898.

Gene Expression Profiling in HeLa Cells After Proton and X-Ray Irradiation with Cisplatin

Our previous study demonstrated that cisplatin had more potent radiosensitizing effects and yielded a significantly higher percentage of apoptotic cell death when combined with proton beams than with X-ray irradiation. Although such results may be explained by differences in the DNA repair mechanisms, factors other than cell death may possibly be involved. In this study, we carried out the gene expression profiling in HeLa cells after proton and X-ray irradiation in combination with cisplatin. HeLa cells were provided by the RIKEN BioResource Center. We evaluated differences in the gene expression profile by cDNA microarray activated in response to proton and X-ray irradiation with or without cisplatin in HeLa. The cells were exposed to the medium containing 0 or 2.5 μmol/L cisplatin and were irradiated with single doses of 6 Gy with proton beams or X rays under the same biological conditions. One hour after irradiation, cisplatin was washed out from the medium. Each sample was cryopreserved at 2 h and 24 h after irradiation. Total RNA was further purified using a micro kit and amplified and labeled with Cyanine 3 using Agilent Low Input Quick Amp Labeling Kit (one-color) according to the manufacturer’s instructions. For each hybridization, 0.60 μg of Cyanine 3 labeled cRNA were fragmented, and hybridized at 65 °C for 17 h to an Agilent SurePrint G3 Human GE v3 8x60K Microarray. After washing, microarrays were scanned using an Agilent DNA microarray scanner. Intensity values of each scanned feature were quantified using feature extraction software. The Gene oncology (GO) analysis on microarray data was performed using a data analysis software. The data were then processed with Fisher’s exact test and multiple test correction to identify significant overrepresentation of GO annotations belonging to molecular functions. In addition, the pathway statistical analysis was performed on a pathway collection of WikiPathways database using PathVisio tool to determine pathways that contained the greatest change in expression. Gene expression profiling pathway analyses showed that the activation of different signaling and molecular networks was dependent on the radiation type, time, and presence/absence of cisplatin. In the 24-h samples irradiated with proton beams in combination with cisplatin, particularly, alterations in expression of many genes such as JAK-STAT cascade and MHC class I receptor activity were observed in GO analysis. In pathway analysis, there were significant differences in such as MAPK signaling and NRF2 between protons and X rays combined with cisplatin. In the microarray analysis, proton beam irradiation combined with cisplatin resulted in different gene expression alterations as compared with X rays and cisplatin. These differences suggested the possibility of leading to further development of irradiation methods and new therapeutic strategies using proton therapy.

Simultaneous detection of ofloxacin and lomefloxacin in milk by visualized microplate array

Analytical method for simultaneous detection of ofloxacin and lomefloxacin in milk by visualized microarray immunoassay without the need of time-consuming or complex pre-treatment steps was reported. A 96-well microplate was used as solid support, in which ofloxacin antigen and lomefloxacin antigen were immobilized, respectively. After immobilization, a mixture of standard solutions containing the analytes or samples and relevant antibodies were added to the array reaction areas, then added silver nanoparticles (AgNPs) labeled secondary antibody (goat anti-mouse IgG). Silver enhancement technique was applied to amplify the detection signals, which produced black image on array spots visible with naked eyes. The signals were detected with a microarray scanner; therefore the analyte residues could detect quantitatively by standard calibration curve. Ofloxacin and lomefloxacin dynamic range were all from 0.05 ng/mL to 12.8 ng/mL. The calibration curve of ofloxacin was calculated as: y = − 0.3446x + 0.4287, r = 0.9849. The calibration curve of lomefloxacin was calculated as: y = − 0.3055x + 0.4797, r = 0.9927. The spike recovery were within 80–130%, and RSD were less than 15%. The limits of detection (LOD) were estimated to be 0.24 ng/mL (ofloxacin) and 0.35 ng/mL (lomefloxacin) (n = 3, 3SD).

Glycan Profiling Analysis of Equine Amniotic Progenitor Mesenchymal Cells and Their Derived Extracellular Microvesicles.

Equine amniotic mesenchymal cells (eAMCs) are involved in many mechanisms in tissue regenerative processes. Their secreted vesicles are important effectors in a wide array of biological processes, and contribute to in vivo healing of equine tendon lesions and endometrial inflammation. Glycoconjugates are involved in cellular recognition and in the efficient uptake of extracellular vesicles (EVs) by recipient cells. In this study, we evaluated the surface glycosylation pattern of eAMCs and their EVs from the eAMCs released in conditioned medium. We used a microarray procedure in which eAMCs and eAMC-EVs were spotted on microarray slides, and incubated with a panel of 14 biotinylated lectins and Cy3-conjugated streptavidin. Signal intensity was detected using a microarray scanner. Both eAMC and eAMC-EV microarrays interacted with all the lectins, indicating the presence of N- and O-linked glycans. With respect to eAMCs, eAMC-EVs, were found to be (1) enriched in Galβ1,3GalNAc terminating O-glycans, α2,3-linked sialoglycans, and high-mannose N-glycans (Con A); (2) diminished in N-acetyllactosamine, GalNAc, Gal, GlcNAc, and fucose terminating glycans; and (3) unchanged in α2,6 linked sialoglycans content. These results suggest that eAMC-EVs emerge from a specific eAMC microdomain, and that the high simultaneous presence of Galβ1,3GalNAc, α2,3 sialic acid, and high-mannose N-linked glycans may constitute markers of the eAMC-EVs. The role of these sugars in equine regenerative medicine requires further investigation.

The autoimmune targets in IPEX are dominated by gut epithelial proteins

The autoimmune targets in IPEX are dominated by gut epithelial proteins

Low-cost microarray platform to detect antibiotic resistance genes

Versatile DNA-based approaches for the detection of pathogens and antibiotic resistance genes have been introduced to facilitate faster diagnostic test. This requires a notable amount of cost-intensive commercially labelled DNA oligonucleotides in high-throughput assays. Here, we introduce an alternative method that utilizes a terminal deoxynucleotidyl transferase reaction to significantly reduce expenses of oligonucleotides. Antibiotic resistance genes that are of high clinical relevance were used as relevant application. The alternative labelling was based on an elongation step in the presence of biotin-conjugated nucleotides. Fluorescence-labelled streptavidin was bound to biotin-conjugated nucleotides and detected via a standard high-throughput microarray scanner. The self-labelled detection oligonucleotides performed equally well regarding sensitivity and specificity compared to commercially labelled ones, but at only 10% of their cost.

Rapid selection of aptamers based on protein microarray.

We report a novel method for the efficient screening of aptamers from a complex ssDNA library based on a microarray chip, which was named Microarray-SELEX. In this method, the target protein (lactoferrin) and negative proteins (α-lactalbumin, β-lactoglobulin, bovine serum albumin, and casein) were each dotted and immobilized on a slide to form a protein microarray. Moreover, the library was added to this chip to interact with negative proteins, then with the target protein. The process of SELEX could be monitored on-line using a fluorescent microarray scanner and the whole process was performed in only six rounds. Finally, five aptamers (YFL-1, YFL-4, YFL-5, YFL-6 and YFL-7) were obtained, which showed good specificity towards lactoferrin in the presence of negative proteins. The equilibrium dissociation constants (Kd) of the aptamers were in the nanomolar range. Briefly, Microarray-SELEX is a rapid, easy, sensitive and efficient method for screening aptamers.

Role of Cyclic Adenosine Monophosphate in Myopic Scleral Remodeling in Guinea Pigs: A Microarray Analysis.

Myopia induction accompanies increased scleral cyclic adenosine phosphate (cAMP) levels and collagen degradation in mammalian models. We compared the scleral gene expression changes following monocular form deprivation (FD) with those induced by adenylate cyclase activation with forskolin (FSK) in guinea pigs. Guinea pigs were assigned to FD, FSK-treated, and age-matched (AM) control groups. FSK was injected monocularly into the inferior palpebral subconjunctiva daily for 4 days. After scleral RNA extraction, a gene microarray scanner and software were used to evaluate the gene expression patterns, followed by pathway analysis using Gene Ontology tools. Quantitative PCR (qPCR) was used to analyze the expression of 10 candidate genes in separate sets of form-deprived, vehicle-injected, and AM animals. FSK injections differentially regulated 13 collagen subtypes compared to AM and FD groups. FSK also downregulated Acta2 and Tgf-β2 compared to the AM eyes. Collagen subtypes and Acta2 underwent larger downregulation in the FSK group than during FD. FSK differentially regulated Rarb, Rxrg, Fzd5, Ctnnd2, Dkk2, and Dkk3, which have been linked to ocular growth. Only a few genes were differentially expressed between the FD and AM groups. There was 80% agreement in the direction of gene regulation between microarray and qPCR results. No significant differences were identified between vehicle-injected and AM eyes. Collagen, a major scleral extracellular matrix component, is degraded during myopia. Given that FSK and FD both promote myopia through increased collagen degradation, targeting cAMP signaling pathway genes could suppress myopia development.

The exploration of gene promoter methylation profiling in nasal polyp

Objective:To explore the gene promoter methylation profiles of nasal polyp, and to analysis the promoter methylation differences between the nasal polyp and the normal nasal mucosa.Method:Total DNA of the nasal polyp tissues and normal nasal mucosa were extracted. After immunoprecipitation and whole genome amplification, the DNA was labeled with Cy3/5 and hybridized in NimbleGen hybridization chamber. For array hybridization, Roche Nimblegen CpG Promoter array was used. The slides were scanned using the Axon GenePix 4000B microarray scanner. The different genes were analyzed through pathway and verified by Real-time PCR.Result:3010 genes were found to have promoter hypermethylation in normal nasal mucosa or nasal polyp.2,62%(79/3010) of the genes had promoter hypermethylation in all the nasal polyps, which were negative in normal nasal mucosa.10.66%(321/3010) of the genes had promoter hypermethylation in normal nasal mucosa, which were negative in all the nasal polyps. Three pathways were found in the promoter hypermethylation of the nasal polyps. Fourteen pathways were found in the negative hypermethylation of the nasal polyps.Conclusion:Genes promoter methylation plays an important role in the development of nasal polyps, and the gene promoter methylation profiling may yield new some clues on the mechanism of nasal polyps.

Primary and Stem Cell Microarrays: Application as Miniaturized Biotesting Systems.

The deposition of living cells on microarray surfaces can be used to create physiologically relevant architecture in vitro. Such living cell microarrays enable the reconstruction of biological processes outside the body in a miniaturized format and have many advantages over traditional cell culture. The present protocol offers an option for the preparation and analysis of living primary and stem cell-based microarrays utilizing the standard microarray equipment (contact-free piezoelectric nanoprinter, microarray scanner), as well as microscopy. To produce living cell microarrays, we applied two kinds of mesenchymal stem cells (MSCs) isolated from umbilical cord and adipose tissue, as well as human umbilical vein endothelial cells (HUVECs) as model cells. We used live imaging microscopy for the online monitoring of cell spots in total size, staining of viable cells with Calcein acetoxymethyl ester (Calcein-AM) and treatment of MSCs with differentiation media to analyze the proliferation, viability, and differentiation potential of printed cells. This way, the general applicability of the established living cell-based microarray production was demonstrated.