Tissue Specificity Analysis Research Articles

Gene expression during the germination of coffee seed

Abstract: Germination of the coffee (Coffea arabica L.) seed is the result of events that occur simultaneously in the embryo and endosperm. To understand the molecular mechanisms responsible for these events, we undertook a transcriptome analysis of embryo, micropylar and lateral endosperms from 10-day-imbibed seeds. The sequencing yielded contigs coding for 16,813 proteins. From those, 14,005 (~ 83%) were highly similar to at least one protein sequence in the nr database. 162 genes were significantly expressed in the embryo, 36 in the micropylar endosperm and 72 in the lateral endosperm. The tissue specificity analysis of the significantly expressed genes showed that the embryo had the highest proportion of specific genes (113/162, ~70%), while 11 were expressed in the micropylar and lateral endosperms. In the embryo, genes were mainly associated with abiotic stress, cell growth, and intercellular communication. In the micropylar and lateral endosperms, they were associated with abiotic stress and cell wall degradation. The accuracy of RNA-seq data was confirmed by RT-qPCR. This work adds new information about the molecular mechanism involved in coffee seed germination.

Bioinformatic Identification of Candidate miRNA Biomarkers for Traumatic Brain Injury

The identification of peripheral blood biomarkers which are associated with traumatic brain injury (TBI) could lead to the development of molecular diagnostics which could be used to aid acute detection in scenarios where neuroimaging in unavailable, or to remotely track injury progression and recovery. It is becoming increasingly evident that brain tissue exhibits patterns of micro‐RNA (miRNA) expression which are distinct from those of other tissues. During TBI, the blood brain barrier becomes disrupted and molecules from damaged neural tissues are released into peripheral circulation. Thus, the detection of brain specific miRNAs in peripheral blood could serve as a surrogate marker of TBI. In this investigation, we aimed to systematically identify brain‐enriched miRNAs, and then test their potential utility for use as TBI biomarkers. First, we obtained publically‐available expression data for over 1,300 miRNAs generated from 31 different human post‐mortem tissues. Tissue specificity index (Tau) was calculated for each miRNA, and they were subsequently ranked in terms of their degree of brain‐specific expression. The abundances of the top five ranked miRNAs were then measured in serum samples collected from 10 TBI patients and 10 healthy controls using qPCR, and evaluated for their ability to discriminate between groups using k‐nearest neighbors (k‐NN). The top five miRNAs identified in our tissue specificity analysis consisted of miR‐137, miR‐219a‐5p, miR‐128‐3p, miR‐124‐3p, and miR‐138‐5p, which exhibited from 31 to 74‐fold higher expression levels in brain relative to other tissues. Three out of the five candidate miRNAs exhibited significantly higher abundance in serum samples from TBI patients relative to control patients in qPCR analysis, and their coordinate expression levels were able to discriminate between groups with 90% sensitivity and 80% specificity. The five candidate miRNAs identified in our analysis have potential utility for use as TBI biomarkers, and such a possibility warrants further investigation.Support or Funding InformationWork was supported by Case Western Reserve University FPB School of Nursing start‐up funds issued to Grant C. O'Connell.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Molecular characterization, activity analysis and transcriptional detection of chitinases encoded in the genome of Spodoptera exigua

Abstract Insect chitinases are necessary hydrolytic enzymes for chitin degradation, insect molting and metamorphosis. In this study, five chitinases encoded in the genome of Spodoptera exigua (SeCHIT7, SeCHIT11, SeCHIT12, SeCHIT13 and SeCHIT14) were identified by reverse transcription PCR. Phylogenetic analysis indicated that SeCHIT7 belonged to Group III, and SeCHIT11/12/13/14 belonged to Group VIII. Real-time quantitative PCR analyses showed that SeCHIT7 had an extensive transcription at the fourth- and fifth-instar larval and pupal stages, while SeCHIT11 had the highest transcription level at the egg stage, and the transcription of SeCHIT12 increased by over 1000 times from pre-pupal to pupal stage. Tissue-specific analyses showed that these three SeCHITs (SeCHIT7, SeCHIT11 and SeCHIT12) were mainly transcribed in the midgut and fat body. Chitinase activity assays suggested that the activity had a lower level at the egg stage and peaked at the pupal stage. The activities were increased by 9.4 times from egg to first-instar larval stage. Tissue specificity analysis showed that the highest activity was observed in the fat body, followed by hemolymph and cuticle of the pre-pupal stage. Overall, these results provided valid information for further research on the biological function and regulation of chitinases in S. exigua.

CpG oligodeoxynucleotide-specific duck TLR21 mediates activation of NF-κB signaling pathway and plays an important role in the host defence of DPV infection

TLR21 can recognize unmethylated cytosine-phosphate-guanine oligodeoxynucleotides (CpG-ODN) and activates NF-κB immune signaling pathway. However, the function of TLR21 in duck remains largely unclear. Here, the complete duck TLR21 (duTLR21) cDNA was cloned from Cherry Valley duck for the first time, and its immune response was preliminarily studied. Tissue specificity analysis showed duTLR21 was higher expressed in the peripheral blood, spleen, bursa of Fabricius and cecum. The expression of duTLR21 was significantly upregulated after stimulation with CpG-ODN or duck plague virus (DPV), but not Tembusu virus (TMUV), LPS or Poly (I:C). In addition, the transfection of DEF with duTLR21 stimulated by CpG-ODN activated NF-κB, through this signal pathway, the transcription of IL-1β, IL-6 and IFN-α were promoted, whereas knockdown of duTLR21 impaired the transcription of these genes. Furthermore, the overexpression of duTLR21 inhibited the replication of the DPV and the knockdown of duTLR21 by shRNA significantly promoted DPV replication in vitro. Altogether, these results indicate that duTLR21 can be activated by CpG-ODN, which mediates activation of NF-κB signaling pathway, and plays an important role in the host defence of DPV infection.

Microarray expression profile analysis of long non-coding RNAs in thoracic aortic aneurysm

Thoracic aortic aneurysm (TAA) is a highly lethal vascular disease. Long non-coding RNAs (lncRNAs) are newly discovered as a regulator of protein genes and play critical roles in cardiovascular physio-pathological processes. However, there were a few studies looking at lncRNAs in TAA. In this study, we profiled differential expression of lncRNAs between TAA (TAA group, N = 6) and normal thoracic aorta (control group, n = 6) by third-generation lncRNA microarray. We identified 1352 up-regulated and 1624 down-regulated lncRNAs with differential expression (log fold-change > 2.0, p < 0.01). Through nearby protein-coding gene associated with extracellular matrix (ECM) metabolism and vascular smooth muscle cell (VSMC) apoptosis, 12 up-regulated and 9 down-regulated lncRNAs were selected for further analysis. By calculating phastCons score of base, we identified 8 candidate lncRNAs (4 up-regulated and 4 down-regulated) with high conservation across species. By tissue specificity analysis, we found that 5 lncRNAs (HIF1A-AS1, RP11-465L10.10, LOC100506472, CTD-2184D3.5 and RP-399O19.5) were highly expressed in aortic tissues, suggested that they may be closely associated with TAA. Among them, 2 lncRNAs (RP11-465L10.10 and CTD-2184D3.5) with higher specificity in aorta (p < 0.01) were analyzed by bioinformatics. Further catRAPID analysis revealed a strong RNA-protein interaction between RP11-465L10.10 and myeloid zinc finger gene 1 (MZF1), a transcription factor of MMP9. However, no intense RNA-protein interactions were observed between CTD-2184D3.5 and transcript factors of MAPK6. In conclusions, our study showed differential expression profiles of lncRNAs in TAA and revealed the interaction between certain lncRNAs and coding genes. These data provides insights into new biomarker and therapeutic targets for TAA.

Molecular cloning and functional characterization of duck mitochondrial antiviral-signaling protein (MAVS)

Mitochondrial antiviral-signaling protein (MAVS), also called IPS-1/VISA/Cardif, is an important molecule involved in host defense and triggers a signal for producing type I IFN. Currently the function of MAVS in ducks (duMAVS) remains largely unclear while significant progress has been made in mammals. In this study, the full-length duMAVS cDNA was cloned from duck embryo fibroblasts (DEFs) for the first time. Tissue specificity analysis showed duMAVS was universally expressed in all detected tissues. DEFs transfected with duMAVS were able to induce interferon-β (IFN-β) expression through activating interferon regulatory factor 1 (IRF1) and nuclear factor kappa B (NF-κB). Both the CARD-like domain and transmembrane domain were required for duMAVS signaling via deletion mutant analysis. In addition, poly(I:C)- or Sendai virus (SeV)-induced IFN-β expression in DEFs were significantly decreased by knock-down of duMAVS with siRNA. Altogether, these results indicate that MAVS is a critical immunoregulator in duck innate immune system.

Serum cancer biomarker discovery through analysis of gene expression data sets across multiple tumor and normal tissues

The development of convenient serum bioassays for cancer screening, diagnosis, prognosis, and monitoring of treatment is one of top priorities in cancer research community. Although numerous biomarker candidates have been generated by applying high-throughput technologies such as transcriptomics, proteomics, and metabolomics, few of them have been successfully validated in the clinic. Better strategies to mine omics data for successful biomarker discovery are needed. Using a data set of 22,794 tumor and normal samples across 23 tissues, we systematically analyzed current problems and challenges of serum biomarker discovery from gene expression data. We first performed tissue specificity analysis to identify genes that are both tissue-specific and up-regulated in tumors compared to controls, but identified few novel candidates. Then, we designed a novel computation method, the multiple normal tissues corrected differential analysis (MNTDA), to identify genes that are expected to be significantly up-regulated even after their expressions in other normal tissues are considered, and, in a simulation study, showed that the multiple normal tissues corrected differential analysis outperformed the single tissue differential analysis combined with tissue specificity analysis. By applying the multiple normal tissues corrected differential analysis, we identified some genes as novel biomarker candidates. However, the number of potential candidates was disappointingly small, exemplifying the difficulty of finding serum cancer biomarkers. We discussed a few important points that should be considered during biomarker discovery from omics data.

Integrated proteomic profiling of cell line conditioned media and pancreatic juice for the identification of pancreatic cancer biomarkers.

Pancreatic cancer is one of the leading causes of cancer-related deaths, for which serological biomarkers are urgently needed. Most discovery-phase studies focus on the use of one biological source for analysis. The present study details the combined mining of pancreatic cancer-related cell line conditioned media and pancreatic juice for identification of putative diagnostic leads. Using strong cation exchange chromatography, followed by LC-MS/MS on an LTQ-Orbitrap mass spectrometer, we extensively characterized the proteomes of conditioned media from six pancreatic cancer cell lines (BxPc3, MIA-PaCa2, PANC1, CAPAN1, CFPAC1, and SU.86.86), the normal human pancreatic ductal epithelial cell line HPDE, and two pools of six pancreatic juice samples from ductal adenocarcinoma patients. All samples were analyzed in triplicate. Between 1261 and 2171 proteins were identified with two or more peptides in each of the cell lines, and an average of 521 proteins were identified in the pancreatic juice pools. In total, 3479 nonredundant proteins were identified with high confidence, of which ∼ 40% were extracellular or cell membrane-bound based on Genome Ontology classifications. Three strategies were employed for identification of candidate biomarkers: (1) examination of differential protein expression between the cancer and normal cell lines using label-free protein quantification, (2) integrative analysis, focusing on the overlap of proteins among the multiple biological fluids, and (3) tissue specificity analysis through mining of publically available databases. Preliminary verification of anterior gradient homolog 2, syncollin, olfactomedin-4, polymeric immunoglobulin receptor, and collagen alpha-1(VI) chain in plasma samples from pancreatic cancer patients and healthy controls using ELISA, showed a significant increase (p < 0.01) of these proteins in plasma from pancreatic cancer patients. The combination of these five proteins showed an improved area under the receiver operating characteristic curve to CA19.9 alone. Further validation of these proteins is warranted, as is the investigation of the remaining group of candidates.

A male genital tract-specific carbohydrate epitope on human CD52: implications for immunocontraception.

The identification of unique sperm surface epitopes that are not expressed or exposed in the female reproductive tract is a key element in the development of antibody-based contraceptives. Western blotting and immunohistochemistry were performed to define the tissue distribution of the S19 epitope, which has been proposed as a target for immunocontraception. S19 is an IgG1 murine monoclonal antibody (mAb) directed to an N-linked carbohydrate epitope on a 15-25 kDa glycoprotein, sperm agglutination antigen-1 (SAGA-1), containing a peptide core identical to that of the lymphocytic surface protein CD52. In this study, the S19 epitope was shown to be absent from human lymphocytes, demonstrating a distinction between this epitope and the CAMPATH epitope that is recognized by an antibody against the terminal tripeptide and GPI-anchor of CD52. Further tissue specificity analysis identified the S19 epitope in the epithelium of the human epididymis and vas deferens, as well as on both epididymal and ejaculated spermatozoa. In contrast, the S19 epitope was absent in the five human female reproductive tract and 18 other somatic tissues tested. These results support the use of the S19 epitope as a contraceptive immunogen and the suitability of the S19 mAb as an intravaginal contraceptive. To test the agglutinating activity of the S19 mAb in a formulation designed for vaginal use, S19 mAb were bound to the surface of Novasomes, a multilamellar liposome delivery vehicle. S19-Novasome formulations agglutinated human spermatozoa and were as effective as unbound S19 mAb, demonstrating the feasibility of spermistatic contraceptives targeted to the male reproductive tract specific carbohydrate epitope.

Antibodies to Hair Follicles in Alopecia Areata

Although alopecia areata is suspected to be an autoimmune disease, no direct evidence of an altered immune response to components of the hair follicle has been reported. We studied whether antibodies to normal human anagen scalp hair follicles are present in individuals with alopecia areata. Thirty-nine alopecia areata sera and 27 control sera were tested by Western immunoblotting for antibodies to 6 M urea-extractable proteins of normal anagen scalp hair follicles. At serum diluted 1:80, all alopecia areata subjects (100%), but only 44% of control individuals, had antibodies directed to one or more antigens of approximately 57, 52, 50, 47, or 44 kD. The incidence of antibodies to individual hair follicle antigens in alopecia areata was up to seven times more frequent than in control sera and their level up to 13 times greater and was statistically significant for all five antigens. Tissue specificity analysis indicated that these antigens were selectively expressed in hair follicles. These findings indicate that individuals with alopecia areata have abnormal antibodies directed to hair follicle antigens, and support the hypothesis that alopecia areata is an autoimmune disease.