Journal: Haematologica

Article Title: The process of somatic hypermutation increases polyreactivity for central nervous system antigens in primary central nervous system lymphoma

doi: 10.3324/haematol.2019.242701

Figure Lengend Snippet: Expression of SNRPC in primary lymphoma of the central nervous system (PCNSL). (A) Nearly all CD20+ (FITC) tumor cells of PCNSL show a nuclear expression of SNRPC (Cy3). (B) Intermingled with tumor cells of a PCNSL, single CD68+ (FITC) macrophages also express SNRPC (arrows, Cy3); additionally, there are also SNRPC-negative CD68+ macrophages (arrowheads). (C) Cortical NeuN+ (FITC) neurons express SNRPC (arrows, Cy3). (D) Strongly activated GFAP+ (FITC) reactive astrocytes express SNRPC (arrows, Cy3). (E) In the white matter, some Olig2+ (FITC) oligodendrocytes express SNRPC (arrows, Cy3). (F) CD34+ (FITC) endothelial cells of neocortical cerebral blood vessels do not express SNRPC (Cy3). (G) mRNA expression of SNRPC recognized on the ProtoArray in the normal central nervous system (CNS) and PCNSL. SNRPC is downregulated CNS and upregulated in PCNSL samples. The line represents the SNRPC tag on the U95Av2 microarray (Affymetrix) in individual samples of ten normal CNS tissues and 21 PCNSL published previously.15 SNRPC was significantly differentially expressed between CNS and PCNSL ( P <0.01, Student t -test). (A-F) Double immunofluorescence with rabbit anti-SNRPC (clone EPR16034, Abcam, Cy3) and mouse anti-CD20 (clone L26, DCS, FITC in A), mouse anti-CD68 (clone KP1, DCS, FITC in B), mouse anti-NeuN (clone A60, Millipore, FITC in C), mouse anti-GFAP (clone GA-5, Biogenex, FITC in D), mouse anti- Olig2 (clone Olig2/2400, Abcam, FITC in E), and mouse anti-CD34 (cloneQBend/10, Biogenex, FITC in F). Microphotographs were taken with an Axiophot (Zeiss, Jena, Germany) and Zen 2 software (Zeiss). Original magnification: x400 (objective: x40). Inserts: x750 (A-C), x1000 (D-F). Overlay of the microphotographs and adjustment for contrast and brightness were performed with Adobe Photoshop software version CC.

Article Snippet: Protein microarray slides (9374 human proteins, ProtoArray, Thermo Fisher Scientific) including internal controls were analyzed as described previously., ,

Techniques: Expressing, Microarray, Immunofluorescence, Software

Journal: Scientific Reports

Article Title: Identification of potential autoantigens in anti-CCP-positive and anti-CCP-negative rheumatoid arthritis using citrulline-specific protein arrays

doi: 10.1038/s41598-021-96675-z

Figure Lengend Snippet: Immunome slide design and protein categories on the protein microarray. ( A ) The Immunome protein microarray consists of four identical subarrays, each containing 1631 different proteins in addition to 11 control proteins (BCCP, BSA, Cy3BSA, IgA, IgG, IgM, and four different control probes for the BCCP tag acting as negative controls). The control proteins are located between the subarrays. ( B ) The proteins spotted on the microarray represent different protein groups, such as cancer-associated antigens, transcription factors, kinases, and signaling proteins. The number of proteins in each category is shown. BCCP biotin carboxyl carrier protein, BSA bovine serum albumin.

Article Snippet: Plasma from anti-CCP-positive or anti-CCP-negative RA patients was incubated with Immunome protein microarray slides containing native proteins or proteins citrullinated by PAD2 or PAD4.

Techniques: Microarray

Journal: Biomolecules

Article Title: Recent Progress in Development and Application of DNA, Protein, Peptide, Glycan, Antibody, and Aptamer Microarrays

doi: 10.3390/biom13040602

Figure Lengend Snippet: Different microarray printing strategies: ( A ) Fabrication of PDA patterned CYTOP (perfluoropolymer) glass slides via photolithography approach (reprinted with permission from ), ( B ) a representative of various steps involved in inkjet printing process (taken from https://gesim-bioinstruments-microfluidics.com/microarray-printer/ ; Accessed on 12 December 2022), and ( C ) GLAD method used to fabricate silver nanorods on glass slides (reprinted with permission from ).

Article Snippet: In various studies, commercially available microarrays, such as the Explorer Antibody Microarray (ASB6000, Full Moon Biosystems, Sunnyvale, CA, USA) with 656 antibodies [ ], VaxArray Coronavirus SeroAssay kit (#VXCV-5100, InDevR, Boulder, CO, USA) [ ], Human Cytokine Antibody Microarray slides (AAH-CYT-G4000 kit, RayBiotech, Peachtree Corners, GA, USA) [ ], Biotin-labeled Human antibody microarray (RayBio Biotech, Guangzhou, China) [ ], Protein Microarray Slides (Grace Bio-Labs, Bend, OR, USA) [ ], HuProt human proteome microarrays (CDI laboratories, Mayaguez, PR, USA) [ ], SBC Mouse ceRNA microarray V1.0 (Shanghai Biotechnology Corporation, Shanghai, China) [ ], and Agilent Whole Mouse 44 K ver.

Techniques: Microarray

Journal: Biomolecules

Article Title: Recent Progress in Development and Application of DNA, Protein, Peptide, Glycan, Antibody, and Aptamer Microarrays

doi: 10.3390/biom13040602

Figure Lengend Snippet: Detection methods: ( A ) FRET based approach to detect target DNA. In presence of target DNA only, the existing complex of fluorescence dye (Cy3) and quencher (BHQ2) is disrupted and generates fluorescence signal (). ( B ) CL based enzymatic approach. This approach involves multiple steps: immunoreaction, HCR amplification, enzyme conjugation, and enzyme-CL (luminol p-iodophenol) substrate reaction (). ( C ) Colorimetric detection (use of streptavidin-coated gold nanoparticles) of isothermal DNA amplification. Interaction of biotin and streptavidin was explored (). ( D ) OIRD, a label-free detection method to study protein/antibody interactions with target molecules on a protein microarray chip ().

Article Snippet: In various studies, commercially available microarrays, such as the Explorer Antibody Microarray (ASB6000, Full Moon Biosystems, Sunnyvale, CA, USA) with 656 antibodies [ ], VaxArray Coronavirus SeroAssay kit (#VXCV-5100, InDevR, Boulder, CO, USA) [ ], Human Cytokine Antibody Microarray slides (AAH-CYT-G4000 kit, RayBiotech, Peachtree Corners, GA, USA) [ ], Biotin-labeled Human antibody microarray (RayBio Biotech, Guangzhou, China) [ ], Protein Microarray Slides (Grace Bio-Labs, Bend, OR, USA) [ ], HuProt human proteome microarrays (CDI laboratories, Mayaguez, PR, USA) [ ], SBC Mouse ceRNA microarray V1.0 (Shanghai Biotechnology Corporation, Shanghai, China) [ ], and Agilent Whole Mouse 44 K ver.

Techniques: Fluorescence, Amplification, Conjugation Assay, Microarray

Journal: Biomolecules

Article Title: Recent Progress in Development and Application of DNA, Protein, Peptide, Glycan, Antibody, and Aptamer Microarrays

doi: 10.3390/biom13040602

Figure Lengend Snippet: ( A ) A pictorial workflow presentation of hybrid tetrahedral DNA structured probe in conjugation with hybridization chain reaction (DTSP-HCR) concept used to distinguish single-base mismatches in DNA () and ( B ) an overview of acute tuberculosis (ATB) biomarker identification using a two-phase strategy (discovery phase and validation phase) (). Venn diagram and microarray chip visual analysis revealed the potential of 5 protein biomarkers to distinguish ATB and LTBI/HC. LTBI represents latent tuberculosis infection, and HC represents healthy control.

Article Snippet: In various studies, commercially available microarrays, such as the Explorer Antibody Microarray (ASB6000, Full Moon Biosystems, Sunnyvale, CA, USA) with 656 antibodies [ ], VaxArray Coronavirus SeroAssay kit (#VXCV-5100, InDevR, Boulder, CO, USA) [ ], Human Cytokine Antibody Microarray slides (AAH-CYT-G4000 kit, RayBiotech, Peachtree Corners, GA, USA) [ ], Biotin-labeled Human antibody microarray (RayBio Biotech, Guangzhou, China) [ ], Protein Microarray Slides (Grace Bio-Labs, Bend, OR, USA) [ ], HuProt human proteome microarrays (CDI laboratories, Mayaguez, PR, USA) [ ], SBC Mouse ceRNA microarray V1.0 (Shanghai Biotechnology Corporation, Shanghai, China) [ ], and Agilent Whole Mouse 44 K ver.

Techniques: Conjugation Assay, Hybridization, Biomarker Assay, Microarray, Infection

Journal: Biomolecules

Article Title: Recent Progress in Development and Application of DNA, Protein, Peptide, Glycan, Antibody, and Aptamer Microarrays

doi: 10.3390/biom13040602

Figure Lengend Snippet: A generic workflow of the application of glycan microarray chip to efficiently profile enzyme activities and determine enzyme inhibitor IC 50 values ().

Article Snippet: In various studies, commercially available microarrays, such as the Explorer Antibody Microarray (ASB6000, Full Moon Biosystems, Sunnyvale, CA, USA) with 656 antibodies [ ], VaxArray Coronavirus SeroAssay kit (#VXCV-5100, InDevR, Boulder, CO, USA) [ ], Human Cytokine Antibody Microarray slides (AAH-CYT-G4000 kit, RayBiotech, Peachtree Corners, GA, USA) [ ], Biotin-labeled Human antibody microarray (RayBio Biotech, Guangzhou, China) [ ], Protein Microarray Slides (Grace Bio-Labs, Bend, OR, USA) [ ], HuProt human proteome microarrays (CDI laboratories, Mayaguez, PR, USA) [ ], SBC Mouse ceRNA microarray V1.0 (Shanghai Biotechnology Corporation, Shanghai, China) [ ], and Agilent Whole Mouse 44 K ver.

Techniques: Microarray

Journal: Cancers

Article Title: Understanding the Interplay between COX-2 and hTERT in Colorectal Cancer Using a Multi-Omics Analysis

doi: 10.3390/cancers11101536

Figure Lengend Snippet: Representative tissue microarray (TMA) slides with COX-1, COX-2, and TERT-staining. ( A ) Negative COX-1 staining was detected in 4/10 (40%) CRC samples; ( B ) Low COX-1 staining was detected in 5/10 (50%) CRC samples; ( C ) Medium intensity COX-1 staining was detected in 1/10 (10%) CRC samples; ( D ) Negative COX-2 staining was detected in 7/12 CRC samples (58.3%); ( E ) Low COX-2 staining was detected in 4/12 CRC samples (33%); ( F ) Medium COX-2 staining was detected in 1/12 (8.3%) CRC samples; TERT staining was negative in all CRC samples (11/11, 100%) ( G – I ). All slides were retrieved from the Human Protein Atlas (HPA). Magnification, ×100.

Article Snippet: The following are available online at https://www.mdpi.com/2072-6694/11/10/1536/s1 , Figure S1: PTGS1 or PTGS2 expression levels did not differ with regard to tumor staging ( p > 0.05), but PTGES3 expression was lower, and TERT expression was higher in high stage tumors (III and IV), compared to low-stage tumors (I and II); Figure S2: Heatmap depicting the PTGS1, PTGS2 and TERT expression levels (log2(TPM + 0.001)) in the TCGA-COAD and GTEx projects; Figure S3: Transcript analysis of PTGS1, PTGS2 and TERT in colorectal cancer (TCGA-Colon/Rectum Adenocarcinoma) vs. the normal colon (GTEx Colon); Table S1: Gene expression (RNAseq), promoter methylation, copy number aberration and somatic mutations (SNPs and small INDELs) in the COX-2 and TERT genes, across the TCGA-COAD dataset samples; Table S2: Summary table integrating all the generated data in the present study (mRNA expression data from TCGA and GTEx studies on Colon Adenocarcinoma (RSEM TPM), as well as immunohistochemistry (IHC) data from the tissue microarray (TMA) slides of the Human Protein Atlas (HPA).

Techniques: Microarray, Staining

Journal: bioRxiv

Article Title: Regulation of heterotopic ossification through local inflammatory monocytes in a mouse model of aberrant wound healing

doi: 10.1101/871574

Figure Lengend Snippet: a-c Injury site homogenates harvested from burn/tenotomy mice on day 0, 3, and 7 post burn/tenotomy. a ) Monocyte/Macrophage associated factors. b ) Monocyte/Macrophage and neutrophil maturation factors. c ) Cytokines stimulated by monocyte factors. d ) TGF family members. e ) Stem cell maintaining factors. Levels of cytokine and chemokines in pg/ug of Total protein, data represented as the median with interquartile range. Changes in cytokines and chemokines across day 3 and day 7 vs day 0 were analyzed by an analysis of variance (ANOVA) with post-hoc Dunnett test (n=3 mice/time point) significance. Non-heteroscedastic data identified by Levene’s test for homogeneity of variances were alternatively analyzed by Welch statistic and post-hoc Dunnett T3. Degrees of freedom (df or df1) across samples = 2. F statistic and significant post-hoc p-values respectively: CXCL1: 30.359, p(D0 vs. D7)=0.036, CXCL2: 8.504, CCL2: 268.773, p(D0 vs. D3)=0.000, p(D0 vs. D7)=0.000, CCL3: 16.430, CCL4: 22.441, p(D0 vs. D3)=0.014, G-CSF: 12.579, GM-CSF: 4.988, IL-1b: 3.486, IL-6: 13.019, TNF-α: 38.435, p(D0 vs. D7)=0.019, TGF-β1: 9.156, TGF-β2: 11.376, TGF-β3: 7.362, CCL5: 0.825, CXCL5: 0.825, LIF: 25.368, p(D0 vs. D3)=0.001, p(D0 vs. D7)=0.002 *p<.05 **p<.01. f) t-SNE dimensionality reduction analysis of single cell sequencing from day 3 cells harvested at the extremity injury site revealed 14 distinct cell clusters (representative, performed in triplicate). g,h) Feature plots displaying the single cell gene expression of g) monocyte/macrophage cytokines and chemokines increased in the homogenates and h) their receptors.

Article Snippet: After re-blocking the slides, the slides were incubated with a mouse anti-human TGF-β1 antibody (Cat No. MAB240, R&D Systems) at a dilution of 1:50 overnight.

Techniques: Sequencing, Expressing

Journal: bioRxiv

Article Title: Regulation of heterotopic ossification through local inflammatory monocytes in a mouse model of aberrant wound healing

doi: 10.1101/871574

Figure Lengend Snippet: a) Left: GSEA analysis of microarray data collected from buffy coat of human burn injury patients at increased risk of HO compared to post-surgical control patients. Right: GSEA analysis of RNAseq performed of tendon injury site 3 weeks after burn/tenotomy in mice. b) Western blot of whole tissue protein collected from the injury site of C57BL/6J mice after burn/tenotomy at indicated time points. A western blot for p-SMAD2 and H3 was performed on the nuclear fraction and SMAD2 and alpha-Tubulin were performed on the cytosolic fraction. n=5 were pooled for each time point. c) Co-localization of F4/80+ and TGF-β1 at tendon injury site 1 week after burn/tenotomy. Scale bars correspond to 100um. d) Left: Co-localization of CD68+ and TGF-β1 in early human HO. Right: Co-localization of p-SMAD2 and PDGFRα in human HO.

Article Snippet: After re-blocking the slides, the slides were incubated with a mouse anti-human TGF-β1 antibody (Cat No. MAB240, R&D Systems) at a dilution of 1:50 overnight.

Techniques: Microarray, Western Blot

Journal: bioRxiv

Article Title: Regulation of heterotopic ossification through local inflammatory monocytes in a mouse model of aberrant wound healing

doi: 10.1101/871574

Figure Lengend Snippet: a) Representative Safranin O stain of tendon injury site 3 weeks after burn/tenotomy in p7N3 (CD47 agonist) treated and PBS control mice. n=3/group. b) MicroCT analysis of tenotomy site 9 weeks after burn/tenotomy in PBS and p7N3 (CD47 agonist) treated mice. Left: Representative 3D reconstruction. Right: Quantification of total HO, floating HO and proximal HO.n=7/group. Total HO: t=3.415, df=7.840, p=0.009; Floating HO: t=2.201, df=12, p=0.048; Proximal HO: t=2.686, df=8.549, p=0.026. c) Levels of TGF-β1, TGFβ2, and TGFβ3 in pg/ug total protein and represented as median with interquartile range from Top: homogenates from the extremity injury (TGF-β1: t=-0.635, df=4, p=0.560; TGF-β2: t=-0.643, df=4, p=0.555; TGF-β3: t=-1.272, df=2.186, p=0.322) and Bottom: plasma from PBS and p7N3 (CD47) peptide treated mice 3days after burn/tenotomy (TGF-β1: t=1.544, df=2.037, p=0.260; TGF-β2: t=2.747, df=4, p=0.052; TGF-β3: t=-1.492, df=4, p=0.210). n=3 mice per treatment group. d) qPCR analysis of M1 ( iNos ) and M2 ( Arg1 and Mrc1 ) macrophage markers and Tgfb1 in macrophages isolated from the extremity injury site of naive (day 0), burn/tenotomy day 3, burn/tenotomy day 3 treated with PBS, and burn/tenotomy day 3 treated with p7N3 (CD47) peptide. Day 0 vs. Day 3 – iNOS : t=2.020, df=2, p=0.181; Arg1 : t=-6.084, df=3, p=0.009; Mrc1 : t=0.703, df=4, p=0.521; Tgfb1 : t=0.253, df=4, p=0.812. PBS vs. CD47 – iNOS : t=-0.834, df=2.043, p=0.491; Arg1 : t=0.895, df=4, p=0.421; Mrc1 : t=1.176, df=4, p=0.305; Tgfb1 : t=1.186, df=4, p=0.301. e) Representative images of phagocytosis assay using macrophages isolated from the extremity injury at day 3 after burn/tenotomy in mice treated with PBS or p7N3 (CD47). PBS n=3, CD47 n=4 approximately 25 cells/mouse. f) Measurement of cellular circularity Circularity: t=6.119, df=55.537, p=0.000 and quantification of mean fluorescent intensity phagocytosed by each macrophage. MFI: t=-0.357, df=111, p=0.722.

Article Snippet: After re-blocking the slides, the slides were incubated with a mouse anti-human TGF-β1 antibody (Cat No. MAB240, R&D Systems) at a dilution of 1:50 overnight.

Techniques: Staining, Isolation, Phagocytosis Assay