A Bi-Functional Polymeric Coating for the Co-Imobilization of Proteins and Peptides on Microarray Substrates

The analytical performance of the microarray technique in screening the affinity and reactivity of several probes towards a specific target, is highly affected by the coupling chemistry adopted to bind probes to the surface. However, the surface functionality limits the biomolecules that can be attached to the surface to a single type of molecule (DNA, protein, or peptide), thus forcing the execution of separate analyses to compare the performance of different species in recognizing their targets. Here we introduce a new N, N-dimethylacrylamide-based polymeric coating, bearing simultaneously different functionalities (N-acryloyloxysuccinimide and azide groups) to allow an easy and straightforward method to co-immobilize proteins and oriented peptides on the same substrate. The bi-functional copolymer has been obtained by partial post polymerization modification of the functional groups (NAS) of a common precursor. A deep characterization of the copolymer was carried out by means of NMR to quantify the percentage of NAS that has been transformed into azido groups. The polymer was then used to coat surfaces onto which both native antibodies and alkyne modified peptides were immobilized, to perform the phenotype characterization of extracellular vesicles (EVs). Ultimately, this strategy represents a convenient method to reduce the number of analysis, thus possible systematic or random errors, besides offering a drastic shortage in time, reagents and costs.

A Bi-Functional Polymeric Coating for the Co-Imobilization of Proteins and Peptides on Microarray Substrates

The analytical performance of the microarray technique in screening the affinity and reactivity of several probes towards a specific target, is highly affected by the coupling chemistry adopted to bind probes to the surface. However, the surface functionality limits the biomolecules that can be attached to the surface to a single type of molecule (DNA, protein, or peptide), thus forcing the execution of separate analyses to compare the performance of different species in recognizing their targets. Here we introduce a new N, N-dimethylacrylamide-based polymeric coating, bearing simultaneously different functionalities (N-acryloyloxysuccinimide and azide groups) to allow an easy and straightforward method to co-immobilize proteins and oriented peptides on the same substrate. The bi-functional copolymer has been obtained by partial post polymerization modification of the functional groups (NAS) of a common precursor. A deep characterization of the copolymer was carried out by means of NMR to quantify the percentage of NAS that has been transformed into azido groups. The polymer was then used to coat surfaces onto which both native antibodies and alkyne modified peptides were immobilized, to perform the phenotype characterization of extracellular vesicles (EVs). Ultimately, this strategy represents a convenient method to reduce the number of analysis, thus possible systematic or random errors, besides offering a drastic shortage in time, reagents and costs.

On the Feature Selection of Microarray Data for Cancer Detection based on Random Forest Classifier

Cancer is a disease that can affect all organs of humans. Based on data from the World Health Organization (WHO) fact sheet in 2018, cancer deaths have reached 9.6 million. One known way to detect cancer that is with Microarray Technique, but the microarray data have large dimensions due to the number of features that are very much compared to the number of samples. Therefore, dimension reduction should be made to produce optimum accuracy. In this paper, we compare Minimum Redundancy Maximum Relevance (MRMR) and Least Absolute Shrinkage and Selection Operator (LASSO) to reduce dimension of microarray data. Moreover, by using Random Forest (RF) Classifier, the performance of classification (cancer detection) is compared. Based on simulation, it can be concluded that LASSO is better than MRMR because it can produce an evaluation of 100% in lung and ovarian cancer, 92% colon cancer, 93% prostate tumor and 83% central nervous system.

Construction of heparan sulfate microarray for investigating the binding of specific saccharide sequences to proteins

Heparan sulfate (HS) is a heterogeneous, extracellular glycan that interacts with proteins and other molecules affecting many biological processes. The specific binding motifs of HS interactions are of interest, but have not been extensively characterized. Glycan microarrays are valuable tools that can be used to probe the interactions between glycans and their ligands while relying on relatively small amounts of samples. Recently, chemoenzymatic synthesis of HS has been employed to produce specific HS structures that can otherwise be difficult to produce. In this study, a microarray of diverse chemoenzymatically synthesized HS structures was developed and HS interactions were characterized. Fluorescently labeled antithrombin III (AT) and fibroblast growth factor-2 (FGF2) were screened against 95 different HS structures under three different printing concentrations to confirm the utility of this microarray. Specific sulfation patterns were found to be important for binding to these proteins and results are consistent with previous specificity studies. Furthermore, the binding affinities (KD,surf) of AT and FGF2 to multiple HS structures were determined using a microarray technique and is consistent with previous reports. Lastly, the 95-compound HS microarray was used to determine the distinct binding profiles for interleukin 12 and platelet factor 4. This technique is ideal for rapid expansion and will be pivotal to the high-throughput characterization of biologically important structure/function relationships.

Salinomycin Modulates the Expression of mRNAs and miRNAs Related to Stemness in Endometrial Cancer

Background: Salinomycin, an ionophore antibiotic, has a strong anti-cancer effect, inducing the apoptosis of cancer cells and cancer stem cells. Objective: The aim of the study was to assess the influence of salinomycin on the expression profile of genes related to stemness and miRNA regulating their expression in endometrial cancer cells. Methods: Endometrial cancer cells of cell line Ishikawa were exposed to salinomycin at concentrations in the range of 0.1- 100 µM, with the aim of determining its pro-apoptotic potential and the concentration which would cause the death of 50% of the cells (Sulforhodamine B test). In the following stages, the cells were incubated with the drug at a concentration of 1µM for 12,24 and 48 hour periods and compared to the control. Determining the changes in the expression of the genes related to stemness and regulating their miRNA was done using the microarray technique and RTqPCR. ELISA assay was performed in order to determining the level of TGFβ2, COL14A1, CDH2, WNT5A in cell culture under salinomycin treatment in comparison to the control. Results: Salinomycin caused the apoptosis of cells. For the concentration of 0.1 µM, a decrease in the population of living cells by 11.9% was determined. For 1 µM, it was 49.8%, for 10 µM -69.4%, and for a concentration of 100 µM - 87.9%. The most noticeable changes in the expression caused by the addition of salinomycin into the culture were noted for mRNA: TGFβ2; WNT5A (up-regulated); COL14A1; CDH2 (down-regulated), as well as miRNA: hsa-miR-411 (up-regulated); hsamiR-200a; hsa-miR-33a; hsa-miR-199a; hsa-miR-371-5p; hsa-miR-374; hsa-miR-374b (down-regulated). Conclusion: It was confirmed that salinomycin has the influence for the stemness process. The most noticeable changes in the expression were noted for mRNA: TGFβ2; COL14A1; CDH2; WNT5A, as well as for miRNA: hsa-miR-200a; hsa-miR33a; hsa-miR-199a; hsa-miR-371-5p; hsa-miR-411; hsa-miR-374a; hsa-miR-374b.

Detection of some Meat Adulterants in a Raw Minced Meat Product Model using Frozen Tissue Microarray Technique

In recent years, interest in meat authenticity has been increased. Therefore, twelve groups of beef kofta product were formulated as soya bean, skin, skin emulsion and mechanically deboned poultry meat (MDPM); each of these substances was added with three different concentrations (40, 60 and 80%) in addition to control one. All group samples were subjected to two types of histological examination, conventional paraffin embedding method and frozen tissue microarray. The results of histological examination revealed that both techniques were accurate methods for identification of the separate components of the formulated product and considered extremely useful tools to judge its quality. Moreover, frozen tissue microarray was very helpful in obtaining the result in a short time and allows for examination of a large number of samples at the same time comparing to the conventional method. In addition, no hazard materials like formalin is used, also no ethyl alcohol or xylene is required in this technique. Therefore, microarray technique is reliable and practicable technique that could be used in food quality control laboratory to assess the quality and safety of meat products.

2126. Comparison of Time to Appropriate Antibiotic Between Using Microarray Assay and Mass Spectrometry Technique for Identification of Positive Blood Cultures

Background Microarray-based, multiplexed, automated molecular method is a rapid diagnosis of bloodstream infections by directly identify bacterial pathogens and antibiotic resistance by detection resistance genes from positive blood culture. Previous studies showed significantly reduce time to organism identification from positive blood culture and antibiotic resistance gene with 97.1% sensitivity and 100% specificity. This study aimed to evaluate time to appropriate antibiotic between using Microarray Assay and Mass Spectrometry technique for bacterial identification. Methods one hundred and forty-five patients with bloodstream infection in medical ward were enrolled between 1 June 2018 and 31 January 2019. There were 2 study periods (pre-intervention and post-intervention), using MALDI-TOF combined with the conventional microbiological method as the current standard diagnostic method in pre-intervention group (N = 70) and microarray technique was used add-on to post-intervention group (N = 75). Antibiotic therapy was adjusted by infectious disease team in both periods of study. Results There were significantly faster bacterial identification and detection of antibiotic resistance (39.34 hours vs. 5 hours, P = 0.0001) as well as time to adjust specific antibiotic therapy (75 hours vs. 27.65 hours, P = 0.0001) resulted in earlier appropriate antibiotic therapy (31 hours vs. 0 hours, P = 0.005) and decrease unnecessary of antibiotic adjustment (51.4% vs. 37.3%). However, all-cause mortality within 2 weeks was not significantly reduced (11.4% vs. 14.7%), no differences cost of antibiotic therapy and length of hospital stay (13 days vs. 17 days). Conclusion Microarray technique has rapid turnaround time to bacterial identification and detection of some resistant genes. A combination of this technique and clinical judgement encourage earlier appropriateness antibiotic therapy and may be helpful in antibiotic stewardship program. Disclosures All authors: No reported disclosures.

Upregulation of Programmed Death-1 Ligand 2 (PDCD1LG2) Genevia the Effect of the Early Growth Response 2 (Egr2) Gene Expression in T-Lymphocyte Detected using Microarray-Technique-Based evaluation

The focus, in the current work, was applied on evaluation of Egr2-gene-dependent effects on the expression of the programmed death-1 ligand 2 (PDCD1LG2) gene in 2 types of T-lymphocytes (MF2), one type with the Egr2 gene (23 test samples (TS)) and other type without this gene (23 control samples (CS)). CDNA labelled with different fluorescent dyes, Cy3 and Cy5 for CSs and TSs, was mixed together and hybridized to a solid chip in a microarray-based technique. The resulted (upregulated, downregulated, and no changed) genes were categorized according to their functions using Go minor software. The results identified upregulated, downregulated, and no changed genes in the TSs compared to those in the CSs. Mainly, the PDCD1LG2, programmed death-1 ligand 2 was found to be changed in its levels. This gene is needed for T-cell-based proliferation and IFNG-based production. T-cell proliferation is inhibited via the interaction with PDCD1 via blocking cell cycle continuity. The current study presents important information that could be used as a guide for future in vitro and in vivo studies involving the PDCD1LG2 gene in links to the Egr2 gene.