ABSTRACT
Basic research in life science and medicine has dug into single cell level in recent years. Single-cell analysis offers to understand life from diverse perspectives and is used to profile cell heterogeneity to investigate mechanism of diseases. Single cell technologies have also found applications in forensic medicine and clinical reproductive medicine, while the techniques are rapidly evolving and have become more and more sophisticated. In this article, we reviewed various single cell isolation techniques and their pros and cons, including manual cell picking, laser capture microdissection and microfluidics, as well as analysis methods for DNA, RNA and protein in single cell. In addition, we summarized major up-to-date single cell research achievements and their potential applications.
Subject(s)
Animals , Cell Separation , DNA , Laser Capture Microdissection , RNA , Single-Cell AnalysisABSTRACT
This study investigated the molecular markers of DS-1-47, a component of an implantation- promoting traditional Chinese medicine consisting of Astragalus mongholicus, Atractylodes macrocephala, Scutellaria baicalensis and Dipsacales, in an attempt to clarify the molecular mechanism and action targets of DS-1-47. Controlled ovarian stimulation (COS) method was used to establish the implantation dysfunction models of mice. Animals were divided into normal pregnant group, COS model group and DS-1-47 group. Laser capture microdissection-double dimensional electrophoresis-mass spectrum (LCM-DE-MS) was used to analyze the uterine protein molecules that were possibly involved in the promotion of implantation. Twenty-three proteins in DS-1-47 group were significantly changed as compared to those in COS model group, with 7 proteins down-regulated and 16 proteins up-regulated. Except for some constituent proteins, the down-regulated proteins included collagen α-1 (VI) chain, keratin 7, keratin 14, myosin regulatory light chain 12B, myosin light polypeptide 9, heat shock protein β-7, and C-U-editing enzyme APOBEC-2; the up-regulated proteins included apolipoprotein A-I, calcium regulated protein-3, proliferating cell nuclear antigen, L-xylulose reductase, and calcium binding protein. These 23 proteins that were regulated by DS-1-47 represented a broad diversity of molecule functions. The down-regulated proteins were associated with stress and immune response, and those up-regulated proteins were related to proliferation. It was suggested that these proteins were important in regulating the uterine environment for the blastocyst implantation. By identification of DS-1-47 markers, proteomic analysis coupled with functional assays is demonstrated to be a promising approach to better understand the molecular mechanism of traditional Chinese medicine.
Subject(s)
Animals , Female , Mice , Pregnancy , Drugs, Chinese Herbal , Pharmacology , Embryo Implantation , Ovulation Induction , Proteome , Genetics , Metabolism , Uterus , Metabolism , PhysiologyABSTRACT
An extensive guide on practicable and significant quantitative proteomic approaches in neuroscience research is important not only because of the existing overwhelming limitations but also for gaining valuable understanding into brain function and deciphering proteomics from the workbench to the bedside. Early methodologies to understand the functioning of biological systems are now improving with high-throughput technologies, which allow analysis of various samples concurrently, or of thousand of analytes in a particular sample. Quantitative proteomic approaches include both gel-based and non-gel-based methods that can be further divided into different labelling approaches. This review will emphasize the role of existing technologies, their advantages and disadvantages, as well as their applications in neuroscience. This review will also discuss advanced approaches for targeted proteomics using isotope-coded affinity tag (ICAT) coupled with laser capture microdissection (LCM) followed by liquid chromatography tandem mass spectrometric (LC-MS/MS) analysis. This technology can further be extended to single cell proteomics in other areas of biological sciences and can be combined with other ‘omics’ approaches to reveal the mechanism of a cellular alterations. This approach may lead to further investigation in basic biology, disease analysis and surveillance, as well as drug discovery. Although numerous challenges still exist, we are confident that this approach will increase the understanding of pathological mechanisms involved in neuroendocrinology, neuropsychiatric and neurodegenerative disorders by delivering protein biomarker signatures for brain dysfunction.
ABSTRACT
The aim of the present study was to explore the differentially expressed genes in the blood vessel endothelial cells (BVECs) between diffuse large B-cell lymphoma (DLBCL) and reac- tive lymph node hyperplasia (RLNH), and to perform an initial bioinformatics analysis on a novel gene, C20orf14, which is highly expressed in lymph node of lymphoma. The mRNA of the tissue from the BVECs of DLBCL and RLNH tissues was labeled with biotin respectively and hybridized with expression profile microarray, and the differentially expressed genes were obtained. Initial bio- informatics analysis was performed on a novel gene named C20orf14. Its gene structure, genomic lo- calization, the physical and chemical characteristics of the putative protein, subcellular localization, functional domain etc. were predicted, and the systematic evolution analysis was performed on the similar proteins among several species. By using expression profile microarray, many differentially expressed genes were uncovered. The efficient bioinformatics analysis have fundamentally identified that C20orfl4 was a nuclear protein, and may be involved in the post-transcription modification of mRNA. Therefore, microarray is an efficient and high throughout strategy for the detection of differ- entially expressed genes, and C20orf14 is thought to be a potential target for tumor metastasis re- searches by bioinformatics analysis.
ABSTRACT
La actividad del virus LCM fue informada en Argentina a comienzos de la década del 70 y sólo han sido aisladas cinco cepas a partir del roedor Mus domesticus y dos de humanos. El objetivo de este trabajo consistió en investigar características biológicas de las cepas argentinas de virus LCM para compararlas entre sí y respecto a las cepas históricas WE y Armstrong. En células L 929 se obtuvieron placas bajo agarosa tanto con las cepas humanas como con las cepas de ratón, pero en células Vero sólo se obtuvieron placas con las cepas humanas. No se observó ninguna característica morfométrica de las placas que distinguiera nítidamente a las cepas históricas de las cepas argentinas, ni se observaron diferencias que se relacionen con las especies de origen de las cepas. Las cepas históricas y las cepas argentinas no fueron letales para ratón recién nacido (rrn) generando una infección persistente, según se comprobó al inocular ratones recién nacidos (rrn) por vía intracerebral con cepas de virus LCM y detectarse virus en los cerebros cosechados a diferentes días post inoculación. La única excepción fue la cepa Cba An 13065 que resultó virulenta para rrn ya que con sólo 0.026 UFP se logró 1 DL50. Todas las cepas resultaron letales en ratón adulto (rad), siendo las cepas de ratón más virulentas que las cepas de humanos. Estos resultados permitieron evidenciar el diferente comportamiento en cultivos celulares de las cepas de ratón con respecto a las cepas humanas, e identificar marcadores de virulencia mediante la respuesta a la inoculación por vía intracerebral del rad y del rrn.
The activity of LCM virus was first reported in Argentina at the beginning of the seventies and only five strains have been isolated from rodents Mus domesticus and two from humans. The objective of this paper was to find differential biological characteristics of Argentine strains of LCM virus comparing them in relation to the historical strains WE and Armstrong. Regarding the results obtained in tissue culture, when L 929 cells were used, plaque forming units (PFU) were obtained with human and mouse strains, whilst on Vero cells only human strains developed PFU. Differentials characteristics of historical and Argentine strain's plates were not found, neither differences related to the strain's origin. Neither historical nor Argentine strains were lethal to new-born mice giving a persistent infection, that was demonstrated when we inoculated new-born mouse by intracranial route with different strains of LCM virus and virus was isolated from brains harvested at different days post inoculation. The only exception was Cba An 13065 strain that exhibited virulence in new-born mice, only with 0.026 PFU was obtained 1 DL50. All the strains resulted lethal to adult mice. The mouse strains were more virulent than human strains, being Cba An 13065 the most virulent. These results demonstrate a different behavior in tissue culture between human and mouse strains and allow the identification of virulence markers by intracranial inoculation into new-born or adult mice.
Subject(s)
Humans , Animals , Mice , Lymphocytic Choriomeningitis/virology , Lymphocytic choriomeningitis virus/pathogenicity , Rodentia/virology , Argentina , Biomarkers , Cell Line , Host-Pathogen Interactions , Immunocompromised Host , Lymphocytic Choriomeningitis/immunology , Lymphocytic choriomeningitis virus/isolation & purification , Rodentia/genetics , Species Specificity , VirulenceABSTRACT
PURPOSE: Serum level of soluble form CD30 (sCD30), a marker for T helper 2-type cytokine-producing T cells, is used as a marker of immunologic status of pre-transplant recipient that can predict graft rejection and graft survival. This study compared pre-transplant serum sCD30 levels with conventional pre-transplant immunologic parameter, such as panel- reactive antibodies (PRA) and lymphocyte cross matching (LCM). METHODS: Adult seventy two patients were enrolled this study. The blood for tests was sampled simultaneously. Measurement of serum sCD30 level was performed using enzyme-linked immunosorbent assay kit (Bender MedSystems, Co. CA, USA). We tested PRA using a commercial ELISA kit (Lambda Cell Tray Lymphocytotoxicity assay)(One Lambda Inc. CA, USA). We established LCM tests for T cells by Modified NIH (National institute center of health)/Johnson's Method/AHG (Anti human globulin), and for B cells by warm test. RESULTS: Mean score of sCD30 was 90.3+/-6.4 U/mL, ranged from 12.2 to 244.4 U/mL. There was no significant correlation between patient's age or sex and sCD30 level. The correlation between sCD30 and mode or duration of dialysis was not statistically significant clinical situation. The result of LCM didn't show significant correlation with sCD30 level (87.3+/-55.7 U/mL in LCM positive group versus 91.9+/-1.3 U/mL in LCM negative group, P=0.696). And sCD30 level equal to or more than 86 U/mL could not predict the positive result of LCM. The positive and negative predictive value of sCD30 to LCM was merely 27.8% and 58.3% (P=0.322). Also the correlation between sCD30 level and PRA was not significant (P=1.0). CONCLUCION: There was no significant correlation between serum sCD30 level and conventional immunologic parameter such as PRA or LCM. That means the pre-transplant monitoring of the sCD30 level can be used as an independent immunologic parameter.
Subject(s)
Adult , Humans , Antibodies , B-Lymphocytes , Dialysis , Enzyme-Linked Immunosorbent Assay , Graft Rejection , Graft Survival , Lymphocytes , T-LymphocytesABSTRACT
Objective To develop a method to analyze functional grouping cDNA microarray gene expression in microvessels and neurons from rat brain using laser capture microdissection(LCM). Methods Microvessels and neurons were captured using the PixCell ⅡLCM instrument. The total RNA was extracted from the LCM samples according to the manufacturer's protocol of RNAqueous-Micro kit. The total RNA were processed for one round of T7-based RNA amplification; cDNA probe were synthesized using gene-specific primer and labeled for cDNA microarray analysis. Results Amplified RNA from microvessels and neurons allowed us to measure 96 gene expression in functional grouping cDNA microarray. Conclusion LCM allowed us to harvest pure microvessels and neurons from their native tissue environment, combined with methods of T7 RNA amplification and gene-specific primer amplification so that we can analyze functional grouping cDNA profiling in LCM samples.