DESIGN: A case-control study.
METHODS: This study received ethical approval (NMRR Research ID 23957) and informed consent was obtained from all participants. It involved 20 participants with 20 samples of pterygium and 20 samples of normal conjunctiva that were obtained from the same eye of each participant. All the participants underwent history taking, slit lamp examination, and pterygium excision surgery. Both samples underwent immunohistochemistry procedure. Pretreatment procedure was conducted using heat-induced epitope retrieval with PT link, subsequently followed by EnVision FLEX staining procedure and incubation with anti‒IL-17 antibody and anti‒IL-23 antibody. Slides were examined in high-power fields (400x) for both samples in 3 different fields. Total positive stained cell counts in all 3 fields with IL-17 and IL-23 between pterygium and normal conjunctiva were analyzed by using Wilcoxon signed rank test.
RESULTS: IL-17 positive cell counts for normal conjunctiva showed mean 196.10 ± 80.487 but for pterygium was 331.10 ± 108.416. As for IL-23, the mean for positive cell counts for normal conjunctiva was 62.10 ± 33.462 and IL-23 positive cell counts for pterygium showed mean 102.95 ± 41.378. Both IL-17 and IL-23 were significantly increased in pterygium compared with normal conjunctiva (P < 0.001).
CONCLUSIONS: Both IL-17 and IL-23 were found to be significantly higher in the pterygium group than in the normal conjunctiva group with P < 0.001 by Wilcoxon signed rank test.
METHODS: In this study, new binders derived from shark VNAR domains library were investigated. Following immunization of a wobbegong shark (Orectolobus ornatus) with three recombinant malaria biomarker proteins (PfHRP2, PfpLDH and Pvaldolase), a single domain antibody (sdAb) library was constructed from splenocytes. Target-specific VNAR phage were isolated by panning. One specific clone was selected for expression in Escherichia coli expression system, and study of binding reactivity undertaken.
RESULTS: The primary VNAR domain library possessed a titre of 1.16 × 106 pfu/mL. DNA sequence analysis showed 82.5% of isolated fragments appearing to contain an in-frame sequence. After multiple rounds of biopanning, a highly dominant clone specific to PfHRP2 was identified and selected for protein production in an E. coli expression system. Biological characterization showed the recombinant protein expressed in periplasmic has better detection sensitivity than that of cytoplasmic proteins. Assays of binding activity indicated that its reactivity was inferior to the positive control mAb C1-13.
CONCLUSIONS: Target-specific bacteriophage VNARs were successfully isolated after a series of immunization, demonstrating that phage display technology is a useful tool for selection of antigen binders. Generation of new binding reagents such as VNAR antibodies that specifically recognize the malaria biomarkers represents an appealing approach to improve the performance of RDTs.
MATERIAL/METHODS: Fifty female mice, aged 4-6 weeks, were used in this study. Animals were superovulated, cohabitated overnight, and sacrificed. Fallopian tubes were excised and flushed. Embryos at the 2-cell stage were collected and cultured to obtain 4- and 8-cell stages before being cryopreserved using vitrification and slow freezing. Fixed embryos were stained with fluorescence-labelled antibodies against actin and tubulin, as well as DAPI for staining the nucleus. Labelled embryos were scanned using CLSM and images were analyzed with Q-Win software V3.
RESULTS: The fluorescence intensity of both vitrified and slow-frozen embryos was significantly lower for tubulin, actin, and nucleus as compared to non-cryopreserved embryos (p<0.001). Intensities of tubulin, actin, and nucleus in each stage were also decreased in vitrified and slow-frozen groups as compared to non-cryopreserved embryos.
CONCLUSIONS: Cryopreservation of mouse embryos by slow freezing had a more detrimental effect on the actin, tubulin, and nucleus structure of the embryos compared to vitrification. Vitrification is therefore superior to slow freezing in terms of embryonic cryotolerance.
METHODS: We therefore conducted a cluster analysis of the Autism Diagnostic Interview-Revised (ADI-R) scores from 85 individuals with ASD to predict subgroups and subsequently identified dysregulated genes by reanalyzing the transcriptome profiles of individuals with ASD and unaffected individuals. Proteome profiling of lymphoblastoid cell lines from these individuals was performed via 2D-gel electrophoresis, and then mass spectrometry. Disrupted proteins were identified and compared to the dysregulated transcripts and reported dysregulated proteins from previous proteome studies. Biological functions were predicted using the Ingenuity Pathway Analysis (IPA) program. Selected proteins were also analyzed by Western blotting.
RESULTS: The cluster analysis of ADI-R data revealed four ASD subgroups, including ASD with severe language impairment, and transcriptome profiling identified dysregulated genes in each subgroup. Screening via proteome analysis revealed 82 altered proteins in the ASD subgroup with severe language impairment. Eighteen of these proteins were further identified by nano-LC-MS/MS. Among these proteins, fourteen were predicted by IPA to be associated with neurological functions and inflammation. Among these proteins, diazepam-binding inhibitor (DBI) protein was confirmed by Western blot analysis to be expressed at significantly decreased levels in the ASD subgroup with severe language impairment, and the DBI expression levels were correlated with the scores of several ADI-R items.
CONCLUSIONS: By subgrouping individuals with ASD based on clinical phenotypes, and then performing an integrated transcriptome-proteome analysis, we identified DBI as a novel candidate protein for ASD with severe language impairment. The mechanisms of this protein and its potential use as an ASD biomarker warrant further study.