ABSTRACT
Objective To clone the full-length of cDNA protein marker cyanase (IP4) of Ophiocordyceps sinensis and predict the antigenic sites. Methods The information of protein marker of O. sinensis was obtained by proteomics technology. The transcriptome database of O. sinensis and mycelium constructed in our lab were used to analyze IP4 unigenes and appropriate primers designed to amplify IP4, which was then cloned and sequenced. The conserved sequence of IP4, homology comparison, and the antigenic site were analyzed by DNAMAN6.0 and DNAStar software. Results Two alternative splice variants of IP4 were discovered from the transcriptome data and belonged to the invariant alternative splicing. The protein marker of O. sinensis was identified as cyanate hydratase, 465 bp, encoding 154 aa. The analysis of the conserved and functional regions showed that catalytic site and binding site mainly lied in C-terminal of IP4. Analysis of DNAMAN 6.0 showed that species 1-39 aa and 55-81 aa were of higher species specificity, and DNAStar results showed that the epitope region of IP4 protein is distributed from 25 to 90 aa. Conclusion The optimal antigen region of IP4 lied in the region 25-39 and 55-81 aa, which lays a foundation for the subsequent large-scale preparation of IP4 protein and eutilizing ELISA to identify authenticity of O. sinensis.
ABSTRACT
Semen stain identification is one of the crucial tasks for collection of criminal evidence by forensic techniques. Substances such as DNA and RNA contained in semen stains can serve as a source of personalized evidence targeting the suspect. Therefore, semen stain identification is vital to inferring the case attributes and the facts of the crime. The conventional methods of forensic stain identification focus on the detection of specific-function protein and/or high-content protein, such as alkaline phosphatase and PSA. Although the specificity of such protein markers is relatively high, these methods yield a limited rate of success for several factors, including poor stability, low sensitivity of the target protein, and possible subjectivity of the performer. In order to overcome these limitations, new technologies such as Raman spectroscopy, mass spectrometry for protein markers, sperm-specific aptamer, mRNA, microRNA, and DNA methylation assays have been studied and recommended by many investigators. These new technologies are paving a new ground for personalized trace analysis and even for detection of over-timed specimens.
Subject(s)
Humans , Male , DNA , DNA Methylation , Forensic Medicine , Methods , MicroRNAs , RNA, Messenger , Semen Analysis , Methods , Sensitivity and Specificity , SpermatozoaABSTRACT
Background: Spermatogonial stem cells (SSCs) are classified as a unique adult stem cells that have capability to propagate, differentiate, and transmit genetic information to the next generation. Studies on human SSCs may help resolve male infertility problems, especially in azoospermia patients. Therefore, this study aims to propagate SSCs in-vitro with a presence of growth factor and detect SSC-specific protein cell surface markers. Methods: The sample was derived from non-obstructive azoospermic (NOA) patient. The disassociation of SSCs was done using trypsin. Specific cultures in serum-free media with added basic fibroblast growth factor (bFGF) were developed to support self-renewal division. This undifferentiated protocol was performed for 49 days. Cells were analysed on days 1, 7, 14, 21, and 49. Results: Human SSCs began to aggregate and form colonies after 14 to 21 days in specific culture. Then, the cells were successful expanded and remained stable for a duration of 49 days. Four specifics markers were identified using immunofluorescence in SSCs on day 49: ITGα6, ITGβ1, CD9, and GFRα1. Conclusion: This approach of using in vitro culture with additional growth factor is able to propagate SSCs from non-obstructive azoospermia patient via detection of protein cell surface markers.
ABSTRACT
A stem cell interacts with the neighboring cells in its environment. To maintain a living organism's metabolism, either cell-cell or cell-environment interactions may be significant. Usually, these cells communicate with each other through biological signaling by interactive behaviors of primary proteins or complementary chemicals. The signaling intermediates offer the stem cell's functionality on its metabolism. With the rapid advent of omics technologies, various specific markers by which stem cells cooperate with their surroundings have been discovered and established. In this article, we review several stem cell markers used to communicate with either cancer or immune cells in the human body.
Subject(s)
Human Body , Metabolism , Stem CellsABSTRACT
Both total proteome and differential proteins were effectively extracted, separated and selected by a combined approach of both differential centrifugation and 2D-PAGE in liver of Paralichtys olivaceus( POL) under the stress of cadmium chloride as an artificial pollution source. Approximately 800 spots for extraction of whole protein separated with 2D-PAGE were obtained by direct lysis in the POL. In addition, approximately 11 differential proteins in POL were also obtained under the stress of cadmium chloride. The differential centrifugal were used to prepare three sedimentation and a plasmolysis proteins, called POL component Ⅰ, POL component Ⅱ, POL component Ⅲ, and POL component Ⅳ(plasmolysis protein), respectively. Total protein spots for each gel were calculated to have 380, 550, 500, and 850, respectively, approximately 2280 spots in sum, while total spots are much higher than those by direct lysis approach. Using the comparison method, approximately 54 differential proteins in POL were obtained by a combined technology of both differential and two dimensional polyacylaminde gel electrophoresis(2D-PAGE) methods under the stress of cadmium chloride. In addition, these differential proteins can be further identified by peptide mass fingerprint(PMF). Here, these combined techniques can be effectively used to extract, separate and identify the whole proteins and the differential proteins including protein markers in the biological tissue.