To stay or to leave: Stem cells and progenitor cells navigating the S1P gradient

Most hematopoietic stem progenitor cells (HSPCs) reside in bone marrow (BM), but a small amount of HSPCs have been found to circulate between BM and tissues through blood and lymph. Several lines of evidence suggest that sphingosine-1-phosphate (S1P) gradient triggers HSPC egression to blood circulation after mobilization from BM stem cell niches. Stem cells also visit certain tissues. After a temporary 36 h short stay in local tissues, HSPCs go to lymph in response to S1P gradient between lymph and tissue and eventually enter the blood circulation. S1P also has a role in the guidance of the primitive HSPCs homing to BM in vivo, as S1P analogue FTY720 treatment can improve HSPC BM homing and engraftment. In stress conditions, various stem cells or progenitor cells can be attracted to local injured tissues and participate in local tissue cell differentiation and tissue rebuilding through modulation the expression level of S1P1, S1P2 or S1P3 receptors. Hence, S1P is important for stem cells circulation in blood system to accomplish its role in body surveillance and injury recovery.     

DNA isolation by a rapid method from human blood samples: Effects of MgCl2, EDTA,storage time,and temperature on DNA yield and quality

The isolation of DNA from whole blood by a modified rapid method (RM) was tested using various detergents and buffer conditions. Extraction of DNA with either NP-40 or Triton X-100 gave a high yield of undegraded DNA in less than an hour. The concentration of magnesium ion in the buffers was critical to obtaining intact, high molecular weight (HMW) DNA. Greater than 10 mM MgCl₂ led to degradation. Addition of EDTA to the buffer inhibits this degradation. Preparation of DNA from blood stored at room temperature or incubated at 37°C for 24 hr resulted in the same amount and quality of DNA as from samples frozen at −70°C. DNA from blood samples that had undergone more than four freeze-thaw cycles was found to be partially degraded. The modified RM can be applied to extract DNA from as little as 10 μl of blood (340 ng of DNA) and from dried blood samples. DNA samples remained intact and undegraded for longer times when DNA was dissolved in higher concentrations of EDTA. This work was supported by grants from the Indiana Department of Mental Health and PHS RO1 AG10297.     

Isolation of Fucoxanthin from the Rhizoid of Laminaria japonica Aresch

Fucoxanthin was extracted from the intact rhizoid of Laminariajaponica Aresch with dimethyl sulfoxide （DMSO）, and then recovered from the DMSO extract by partitioning into ethyl acetate and subsequent evaporation. Some isolation conditions such as solvent volume and extraction time were screened. The quantity and quality of the extracted fucoxanthin were determined by spectral analysis （absorption spectra and fluorescence emission spectra）. The results indicated that： （1） the average total content of fucoxanthin was 122.1μg in 1 g of fresh L. japonica rhizoid; （2） in comparison with the widely used organic solvent, acetone, DMSO was much more effective for the extraction of fucoxanthin; （3） both DMSO volume and extraction time influenced extraction efficiency such as the recovery rate and purity of fucoxanthin （1 g of fresh L. japonica rhizoid treated with 4 mL DMSO for 60 min, yielded 〉 88% of the total fucoxanthin with purity 0.63）; （4） when （NH4）2SO4 concentration was in the range of 0.5-1.0 mol/L, the pigments rapidly and entirely moved from DMSO into the ethyl acetate phase; （5） the ethyl acetate and DMSO were recycled using a rotary evaporator.     

Cellular factors derived from mesenchymal stem and progenitor cells with regeneration effects

Regeneration effects with cellular factors are considered essential in regenerative treatments. Cellular factors derived from multiple cells can be applied in such therapies. Various clinical trial phases are based on studies of mesenchymal stem and progenitor cells (MSPCs). Mesenchymal stem cells (MSCs) are pluripotent stem cells which have multi-directional differentiation potential. MSPCs may exhibit full stem cell functions and can be obtained from different tissues, such as adipose tissue, umbilical cord and bone marrow etc. MSPCs reside in the perivascular niche that is proximal to blood vessels, which allow MSPCs capable of exerting their potential of homing and migration across the endothelium barrier toward lesion sites for tissue repairing or regeneration. MSPCs can be stimulated to release various factors, including surface molecules, growth factors and inhibitory factors. MSPCs’ homing potential depends on the expressing of certain surface molecules. The growth and inhibitory factors contribute to tissue regeneration and immunomodulation effects. This review provides details of how cellular factors derived from MSPCs can be used for homing and repair mechanisms, and ultimately be applied to clinical settings.     

DNA Extraction Protocol for Biological Ingredient Analysis of Liuwei Dihuang Wan

Traditional Chinese medicine(TCM) preparations are widely used for healthcare and clinical practice. So far, the methods commonly used for quality evaluation of TCM preparations mainly focused on chemical ingredients. The biological ingredient analysis of TCM preparations is also important because TCM preparations usually contain both plant and animal ingredients,which often include some mis-identified herbal materials, adulterants or even some biological contaminants.For biological ingredient analysis, the efficiency of DNA extraction is an important factor which might affect the accuracy and reliability of identification. The component complexity in TCM preparations is high, and DNA might be destroyed or degraded in different degrees after a series of processing procedures. Therefore, it is necessary to establish an effective protocol for DNA extraction from TCM preparations. In this study, we chose a classical TCM preparation,Liuwei Dihuang Wan(LDW), as an example to develop a TCM-specific DNA extraction method.An optimized cetyl trimethyl ammonium bromide(CTAB) method(TCM-CTAB) and three commonlyused extraction kits were tested for extraction of DNA from LDW samples. Experimental results indicated that DNA with the highest purity and concentration was obtained by using TCM-CTAB. To further evaluate the different extraction methods, amplification of the second internal transcribed spacer(ITS2) and the chloroplast genome trnL intron was carried out.The results have shown that PCR amplification was successful only with template of DNA extracted by using TCM-CTAB. Moreover, we performed high-throughput 454 sequencing using DNA extracted by TCM-CTAB. Data analysis showed that 3–4 out of 6 prescribed species were detected from LDW samples, while up to 5 contaminating species were detected, suggesting     

Human adult pluripotency: Facts and questions

Cellular reprogramming and induced pluripotent stem cell(IPSC) technology demonstrated the plasticity of adult cell fate, opening a new era of cellular modelling and introducing a versatile therapeutic tool for regenerative medicine.While IPSCs are already involved in clinical trials for various regenerative purposes, critical questions concerning their medium-and long-term genetic and epigenetic stability still need to be answered. Pluripotent stem cells have been described in the last decades in various mammalian and human tissues(such as bone marrow, blood and adipose tissue). We briefly describe the characteristics of human-derived adult stem cells displaying in vitro and/or in vivo pluripotency while highlighting that the common denominators of their isolation or occurrence within tissue are represented by extreme cellular stress. Spontaneous cellular reprogramming as a survival mechanism favoured by senescence and cellular scarcity could represent an adaptative mechanism. Reprogrammed cells could initiate tissue regeneration or tumour formation dependent on the microenvironment characteristics. Systems biology approaches and lineage tracing within living tissues can be used to clarify the origin of adult pluripotent stem cells and their significance for regeneration and disease.     

Absent MicroRNAs in Different Tissues of Patients with Acquired Cardiomyopathy

MicroRNAs (miRNAs) can be found in a wide range of tissues and body fluids, and their specific signatures can be used to determine diseases or predict clinical courses. The miRNA profiles in biological samples (tissue, serum, peripheral blood mononuclear cells or other body fluids) differ significantly even in the same patient and therefore have their own specificity for the presented con-dition. Complex profiles of deregulated miRNAs are of high interest, whereas the importance of non-expressed miRNAs was ignored. Since miRNAs regulate gene expression rather negatively, absent miRNAs could indicate genes with unaltered expression that therefore are normally expressed in specific compartments or under specific disease situations. For the first time, non-detectable miRNAs in different tissues and body fluids from patients with different diseases (cardiomyopathies, Alzheimer’s disease, bladder cancer, and ocular cancer) were analyzed and com-pared in this study. miRNA expression data were generated by microarray or TaqMan PCR-based platforms. Lists of absent miRNAs of primarily cardiac patients (myocardium, blood cells, and serum) were clustered and analyzed for potentially involved pathways using two prediction platforms, i.e., miRNA enrichment analysis and annotation tool (miEAA) and DIANA miRPath. Extensive search in biomedical publication databases for the relevance of non-expressed miRNAs in predicted pathways revealed no evidence for their involvement in heart-related pathways as indicated by software tools, confirming proposed approach.     

Epigenetic reprogramming of embryos derived from sperm frozen at −20°C

Cryopreservation of spermatozoa is a strategy that has been used to conserve the sperm of animal species and animal strains that are valuable for biomedical research. A simple method for preserving spermatozoa after application of intracytoplasmic sperm injection (ICSI) is much needed. It has been shown previously that spermatozoa frozen at 20°C can activate oocytes and support full-term embryo development. However, epigenetic reprogramming could be affected by the environment and by the in vitro manipulation of gametes. Here, we investigated embryo epigenetic reprogramming including DNA methylation and histone modification, in embryos derived from sperm preserved at 20°C without cryoprotectants. The results showed that although both fertilization and embryo developmental competence were decreased, the dynamic epigenetic reprogramming of embryos derived from frozen sperm was similar to the reprogramming of embryos derived from fresh sperm. The results reported in this study indicate that sperm frozen without cryoprotectant is epigenetically safe for ICSI.     

A Chinese perspective on factors and molecular mechanisms that influence the curative effects of platelet transfusion

Platelet transfusion, as the primary treatment for severely low platelet count (thrombocytopenia) or disorders of platelet function, is used extensively in patients with blood system diseases or active hemorrhaging. In recent decades platelet related researches in China have steadily increased. In the clinical application of platelet transfusion, there are large differences between the treatment’s effects on different patients. Immune or nonimmune factors, such as platelet-specific antibodies, platelet gene analysis and matches, and auxiliary treatment, may affect the curative effect, and even result in ineffective platelet transfusion. Platelets used in clinical therapy include platelets made from whole blood and hemapheresis platelets, and the latter is the main application type used in China. Both preparation and preservation methods can affect the quality of platelets and influence the therapeutic effect. Researches on the molecular basis or the curative effect of platelet transfusion are increasing, such as the relationship between platelet-activating factors, tissue factors, thrombopoietin, membrane glycoprotein and platelet activation, and their influence on the curative effect of platelet transfusion, the inhibition of antiplatelet therapy in platelet activation, and platelet signal transduction pathways and their significance in the curative effect of platelet transfusion. This article aims to review the research progress on the curative effect of platelet transfusion and its molecular mechanisms.     

Studies of five microelement contents in human serum,hair, and fingernails correlated with aged hypertension and coronary heart disease

Context: Complementary alternative medicine therapies based on the use of cesium chloride preparations for the treatment of cancer and radiation poisoning, have generated therapeutic interest; but oral or intravenous administration of cesium chloride (CsCl) to cancer patients as an alternative mode of cancer therapy have not been approved by the U.S. Food and Drug Administration (FDA). Objective: Cesium (Cs) levels from human tissue were measured to determine exposure to an alternative medical treatment. Cesium levels are reported from two patients who were administered cesium chloride in conjunction with aloe vera as part of an alternative cancer treatment. Design: The samples were analyzed by graphite furnace atomic absorption spectrometry with Zeeman background correction. As a reference, Cs was also determined in brain, liver, kidney, and whole blood from control case materials retrieved from the National Tissue Repository of the Armed Forces Institute of Pathology. Results: High levels of cesium were found in brain, liver, kidney, bile, gastric content, and whole blood collected at autopsy as compared to reference levels. The administration of cesium chloride resulted in blood levels a factor of 1100 higher than normal. The highest Cs concentrations were found in the liver (1029 μg/g, dry wt), followed by the kidney (815 μg/g, dry wt) and brain (219 μg/g, dry wt). Conclusion: The high accumulation in the liver suggests that hepatotoxicity from Cs might be an initial presenting symptom in Cs-poisoning cases. This is the first report describing two cases with high Cs levels in human tissues. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army, Department of Defense, or the Armed Forces Institute of Pathology.     

Extraction of cellular DNA from human cells and tissues fixed in ethanol

DNA can be extracted from ethanol-fixed lymphoid cells and tissues. The fixation procedure is simple and rapid, and the DNA extraction itself is the same as that normally used for fresh tissue or cells. DNA extracted from ethanol-fixed material is indistinguishable from DNA extracted from fresh samples based on its purity, its ability to be digested with restriction endonucleases, and its ability to specifically hybridize to DNA probes. The capability to extract DNA from ethanol-fixed cells and tissues eliminates the need for stringent handling and storage requirements of fresh or frozen specimens.     

Excavation of a buried treasure--DNA, mRNA, miRNA and protein analysis in formalin fixed, paraffin embedded tissues

Fresh or frozen tissue samples will always be the best tissue source for the analysis of nucleic acids and proteins from tissues. However, their long-term storage is expensive and laborious. Much interest has therefore been focused on the question whether the almost infinite resources of formalin fixed and paraffin embedded tissue samples in the archives of pathology and histology departments can be used for research on biomarkers and molecular mechanisms of disease. In recent years the methods and protocols for the extraction of DNA, mRNA, miRNA and proteins from formalin-fixed and paraffin-embedded tissue samples have improved enormously. Especially, the possibilities of analysing DNA and miRNA in FFPE have reached a level that allows their application as a first line approach in the search for biomarkers. In contrast, many questions remain in terms of quantification of mRNA and protein expression levels in formalin-fixed and paraffin-embedded tissue samples. This review gives an overview on current potentials and limitations of the quantification of DNA, miRNA, mRNA and the proteome in FFPE tissue samples. The chemical events during formalin fixation and paraffin embedding and alternatives to formalin fixation are described. In addition, methods and general problems of DNA, miRNA, mRNA and protein extraction and the current knowledge on the feasibility and accuracy of quantitative gene expression analysis in FFPE tissues is summarized.     

Use of dextran and post-primary antibody fixation in immunoperoxidase staining of fresh frozen tissue. Detection of immunoglobulin associated with squamous carcinomas of the head and neck

Use of unfixed fresh frozen tissue sections for immunocytochemical studies reduces the possibility of denaturation of antigenic determinants compared to formalin fixation and paraffin embedding procedures. However, tissue and cellular morphology can be extensively altered in the numerous application and washing steps with frozen tissue sections. We tested a number of buffer solutions and showed that the use of dextran-containing buffers and fixation by glutaraldehyde after primary antibody application preserves tissue morphology. The procedures described here are also applicable to ascertaining the presence of Fc receptors of leukocytes in sections of carcinoma tissues. The buffered dextran washes and post-primary antibody fixation method was used to demonstrate the presence of immunoglobulin associated with squamous carcinoma cells. The immunoglobulin was not removed by washing of tissue sections at 37 degrees C but could be removed by low or high pH buffer washes, suggesting that the immunoglobulin is bound in a specific manner.     

Tissue proteomics using chemical immobilization and mass spectrometry

Proteomics analysis is important for characterizing tissues to gain biological and pathological insights, which could lead to the identification of disease-associated proteins for disease diagnostics or targeted therapy. However, tissues are commonly embedded in optimal cutting temperature medium (OCT) or are formalin-fixed and paraffin-embedded (FFPE) in order to maintain tissue morphology for histology evaluation. Although several tissue proteomic analyses have been performed on FFPE tissues using advanced mass spectrometry (MS) technologies, high-throughput proteomic analysis of OCT-embedded tissues has been difficult due to the interference of OCT in the MS analysis. In addition, molecules other than proteins present in tissues further complicate tissue proteomic analysis. Here, we report the development of a method using chemical immobilization of proteins for peptide extraction (CIPPE). In this method, proteins are chemically immobilized onto a solid support; interferences from tissues and OCT embedding are removed by extensive washing of proteins conjugated on the solid support. Peptides are then released from the solid phase by proteolysis, enabling MS analysis. This method was first validated by eliminating OCT interference from a standard protein, human serum albumin, where all of the unique peaks contributed by OCT contamination were eradicated. Finally, this method was applied for the proteomic analysis of frozen and OCT-embedded tissues using iTRAQ (isobaric tag for relative and absolute quantitation) labeling and two-dimensional liquid chromatography tandem mass spectrometry. The data showed reproducible extraction and quantitation of 10,284 proteins from 3996 protein groups and a minimal impact of OCT embedding on the analysis of the global proteome of the stored tissue samples.     

2‐D PAGE and MS analysis of proteins from formalin‐fixed,paraffin‐embedded tissues

In the past decade, encouraging results have been obtained in extraction and analysis of proteins from formalin‐fixed, paraffin‐embedded (FFPE) tissues. However, 2‐D PAGE protein maps with satisfactory proteomic information and comparability to fresh tissues have never been described to date. In the present study, we report 2‐D PAGE separation and MS identification of full‐length proteins extracted from FFPE skeletal muscle tissue. The 2‐D protein profiles obtained from FFPE tissues could be matched to those achieved from frozen tissues replicates. Up to 250 spots were clearly detected in 2‐D maps of proteins from FFPE tissue following standard mass‐compatible silver staining. Protein spots from both FFPE and frozen tissue 2‐D gels were excised, subjected to in situ hydrolysis, and identified by MS analysis. Matched spots produced matched protein identifications. Moreover, 2‐D protein maps from FFPE tissues were successfully subjected to Western immunoblotting, producing comparable results to fresh‐frozen tissues. In conclusion, this study provides evidence that, when adequately extracted, full‐length proteins from FFPE tissues might be suitable to 2‐D PAGE‐MS analysis, allowing differential proteomic studies on the vast existing archives of healthy and pathological‐fixed tissues.     

Noncryogenic Preservation of Mammalian Tissues for DNA Extraction: An Assessment of Storage Methods

Reliable field methods for the storage of tissues to be used for DNA extraction and amplification are critical to many studies employing molecular techniques. Protection from DNA degradation was compared among three commonly used methods of noncryogenic storage of tissues over a time scale of 2 years. All three methods prevented DNA degradation during storage for at least 6 months. DMSO (dimethyl sulfoxide)-salt solution provided the best protection from DNA degradation of tissues stored for up to 2 years. High molecular weight DNA was recovered from lysis buffer in which tissue was stored for 2 years, however, moderate amounts of degraded DNA was also present. High molecular weight DNA was recovered from tissues stored in ethanol for 2 years, however, the yield was relatively small compared to the other two noncryogenic storage techniques. Much of the DNA degradation in ethanol preserved tissues appeared to occur during the extraction procedure and can be reduced by soaking the tissue in lysis buffer for a few hours prior to beginning the extraction. The yield of PCR products was greatest from DNA extracted from DMSO-salt solution preserved tissues, whereas DNA from tissues stored in either lysis buffer or ethanol produced lower yields.     

Comparison of DNA isolation using salting-out procedure and automated isolation (MagNA system)

Isolation of genomic DNA is a key step in genetic analysis. The aim of the study was to evaluate the suitability of isolation of DNA from peripheral blood with manual salting-out procedure and automated MagNA system under specific conditions. The impact of storage conditions, type of material (whole blood or blood cells), and method used for DNA extraction were evaluated in terms of DNA yield, its purity, and integrity. Fresh material, and material stored at 2–8°C for 1–4 weeks and frozen at ?80°C were tested. For fresh samples, salting-out method gives higher yield than MagNA, irrespectively, on material used. Neither the yield of salting-out method nor its purity decreases during the storage of the samples in the fridge (2–8°C) during 4 weeks. Concerning MagNA, storage of blood cells in the fridge decreases the yield of DNA as well as its purity. For frozen samples, for whole blood, MagNA gives better results while for blood cells, salting-out method seems to be better. For fresh samples, salting-out method is the preferred one, and both whole blood and blood cells can be used. For frozen samples, the preferred method depends on the material.     

Use of DNA from dry leaves for PCR and RAPD analysis

Fresh or frozen tissue is usually used as a source of DNA for PCR and RAPD analysis. We have found that leaves can be allowed to dry at room temperature before extraction of DNA. Heating the leaves or microwave drying resulted in poor recovery of DNA. Storage of fresh leaves in paper envelopes in the laboratory was the most successful approach. This allowed the tissue to dry out over a period of several days and DNA could be extracted at any time, providing a convenient method for the collection and analysis of field material. DNA from leaves stored for four months at room temperature was suitable for PCR analysis.     

Using Unfixed,Frozen Tissues to Study Natural Mucin Distribution

Mucins are complex and heavily glycosylated O-linked glycoproteins, which contain more than 70% carbohydrate by weight^1-3. Secreted mucins, produced by goblet cells and the gastric mucosa, provide the scaffold for a micrometers-thick mucus layer that lines the epithelia of the gut and respiratory tract^3,4. In addition to mucins, mucus layers also contain antimicrobial peptides, cytokines, and immunoglobulins^5-9. The mucus layer is an important part of host innate immunity, and forms the first line of defense against invading microorganisms^8,10-12. As such, the mucus is subject to numerous interactions with microbes, both pathogens and symbionts, and secreted mucins form an important interface for these interactions. The study of such biological interactions usually involves histological methods for tissue collection and staining. The two most commonly used histological methods for tissue collection and preservation in the clinic and in research laboratories are: formalin fixation followed by paraffin embedding, and tissue freezing, followed by embedding in cryo-protectant media.Paraffin-embedded tissue samples produce sections with optimal qualities for histological visualization including clarity and well-defined morphology. However, during the paraffin embedding process a number of epitopes become altered and in order to study these epitopes, tissue sections have to be further processed with one of many epitope retrieval methods¹³. Secreted mucins and lipids are extracted from the tissue during the paraffin-embedding clearing step, which requires prolong incubation with organic solvents (xylene or Citrisolv). Therefore this approach is sub-optimal for studies focusing on the nature and distribution of mucins and mucus in vivo.In contrast, freezing tissues in Optimal Cutting Temperature (OCT) embedding medium avoids dehydration and clearing of the sample, and maintains the sample hydration. This allows for better preservation of the hydrated mucus layer, and thus permits the study of the numerous roles of mucins in epithelial biology. As this method requires minimal processing of the tissue, the tissue is preserved in a more natural state. Therefore frozen tissues sections do not require any additional processing prior to staining and can be readily analyzed using immunohistochemistry methods.We demonstrate the preservation of micrometers-thick secreted mucus layer in frozen colon samples. This layer is drastically reduced when the same tissues are embedded in paraffin. We also demonstrate immunofluorescence staining of glycan epitopes presented on mucins using plant lectins. The advantage of this approach is that it does not require the use of special fixatives and allows utilizing frozen tissues that may already be preserved in the laboratory.     

Assessment of the 2-D Gel-Based Proteomics Application of Clinically Archived Formalin-Fixed Paraffin Embedded Tissues

Hospital tissue repositories possess a vast and valuable supply of disease samples with matched retrospective clinical information. Detection and characterization of disease biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues will greatly aid the understanding of the diseases mechanisms and help in the development of diagnostic and prognostic markers. In this study, the possibility of using full-length proteins extracted from clinically archived FFPE tissues in two-dimensional (2-D) gel-based proteomics was evaluated. The evaluation was done based on two types of tumor tissues (breast and prostate) and two extraction protocols. The comparison of the 2-D patterns of FFPE extracts obtained by two extraction protocols with the matching frozen tissue extracts showed that only 7–10 % of proteins from frozen tissues can be matched to proteins from FFPE tissues. Most of the spots in the 2-D FFPE’s maps had pl 4–6, while the percentages of proteins with pl above 6 were 3–5 times lower in comparison to the fresh/frozen tissue. Despite the three-fold lower number of the detected spots in FFPE maps compared to matched fresh/frozen maps, 67–78 % of protein spots in FFPE could not be matched to the corresponding spots in the fresh/frozen tissue maps indicating irreversible protein modifications. In conclusion, the inability to completely reverse the cross-linked complexes and overcome protein fragmentation with the present day FFPE extraction methods stands in the way of effective use of these samples in 2-D gel based proteomics studies.