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1.  Chicken chorioallantoic membrane as a reliable model to evaluate osteosarcoma—an experimental approach using SaOS2 cell line 
Osteosarcoma is the most common primary tumor that affects usually children. Due to its cellular complex and osteoid formation it is very difficult to understand the mechanism behind the progressiveness of osteosarcoma. Various animal models are available to study the issue but they are time consuming and costly. We aimed to understand the progressiveness and invasiveness of osteosarcoma induced by SaOS2 cells using chicken chorioallantoic membrane. CAM is a well-established model which allows in vivo studies of tumor induced angiogenesis and the testing of anti angiogenic molecules. However only a few reports showed the tumor forming ability of SaOS2 cells on CAM.
Angiogenic ability of SaOS2 cells on CAM was validated by various methods. Angiogenic ability was scored by direct visualization and scanning microscopic analysis. The sprouting ability and growth of the vessel was measured by Angioquant software under different cellular volume. The invasiveness was analyzed by histological staining. Involvement of angiogenic factors at differential stage of progressiveness was confirmed by the molecular and protein level expression analysis.
SaOS2 cells induces sprouting angiogenesis on CAM and shows its aggressiveness by rupturing the ectodermal layer of the CAM. Growth and development of osteosarcoma depends mainly on the activation of VEGF165, MMP2 and MMP9. CAM able to reproduce angiogenic response against the stimulation of SaOS2 cells exactly as in other animal models without inflammatory reactions.
CAM is an excellent alternative in vivo model for studying the aggressiveness and tumor progression of osteosarcoma using various angiogenic techniques in an easily, faster and affordable way. We further provided insight about the involvement of various angiogenic growth factors on the development of osteosarcoma which will enable to find the suitable therapeutic molecule for the treatment of osteosarcoma. CAM model could provide a wide space using modern techniques like micro array or in situ hybridization to have a better understanding about the progression and invasiveness of osteosarcoma cells to develop suitable therapeutic molecules.
PMCID: PMC4479062  PMID: 26109911
Osteosarcoma; SaOS2 cells; Angiogenesis; CAM- chicken chorioallantoic membrane
2.  The diversity of fungal genome 
The genome size of an organism varies from species to species. The C-value paradox enigma is a very complex puzzle with regards to vast diversity in genome sizes in eukaryotes. Here we reported the detailed genomic information of 172 fungal species among different fungal genomes and found that fungal genomes are very diverse in nature. In fungi, the diversity of genomes varies from 8.97 Mb to 177.57 Mb. The average genome sizes of Ascomycota and Basidiomycota fungi are 36.91 and 46.48 Mb respectively. But higher genome size is observed in Oomycota (74.85 Mb) species, a lineage of fungus-like eukaryotic microorganisms. The average coding genes of Oomycota species are almost doubled than that of Acomycota and Basidiomycota fungus.
PMCID: PMC4392786  PMID: 25866485
Ascomycota; Basidiomycota; Chytridiomycota; Monoblepharidomycota; Neocallimastigomycota; Blastocladiomycota; Glomeromycota; Entomophthoromycota; Stramenopiles and micorsporidia
3.  Evaluation of Epic® label-free technology to quantify functional recombinant hemagglutinin 
Alternative methods are being sought to measure the potency of influenza vaccines. Label-free technologies that do not require the use of hemagglutinin (HA)-specific antisera are particularly attractive as the preparation of antiserum delays availability of potency reagents. The objective of these experiments was to evaluate the use of a Corning Epic® label-free method to quantify functional influenza hemagglutinin in rHA preparations. The method was optimized to quantify recombinant HA (rHA) of B/Brisbane/60/2008 (B/BR/08). Fetuin was immobilized onto plates and the change in wavelength of refracted light measured using an Enspire (Perkin Elmer) instrument.
The change in wavelength measured in response to addition of rHA of B/BR/08 was proportional to its concentration and was optimal in the presence of native rHA conformations. However, the assay was strain-dependent and did not correlate with HAU measured using turkey red blood cells.
The Corning Epic® label-free method is suitable for quantifying the native forms of rHA for B/BR/08 and A/Brisbane/59/2007 (H1N1) and A/Hangxhou/3/2013 (H7N9). This method is a useful tool for research purposes but further investigation is needed to identify suitable glycoproteins to use as ligands that allow quantification of HAs from a broader range of virus strains.
Electronic supplementary material
The online version of this article (doi:10.1186/s12575-015-0019-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4359790  PMID: 25774096
Influenza; Hemagglutinin; Label-free technology; Potency; Fetuin; Corning; Epic; Enspire; Recombinant
4.  Recombinase-mediated cassette exchange (RMCE) system for functional genomics studies in Mycoplasma mycoides 
We have previously established technologies enabling us to engineer the Mycoplasma mycoides genome while cloned in the yeast Saccharomyces cerevisiae, followed by genome transplantation into Mycoplasma capricolum recipient cells to produce M. mycoides with an altered genome. To expand the toolbox for genomic modifications, we designed a strategy based on the Cre/loxP-based Recombinase-Mediated Cassette Exchange (RMCE) system for functional genomics analyses.
In this paper, we demonstrated replacement of an approximately 100 kb DNA segment of the M. mycoides genome with a synthetic DNA counterpart in two orientations. The function of the altered genomes was then validated by genome transplantation and phenotypic characterization of the transplanted cells.
This method offers an easy and efficient way to manipulate the M. mycoides genome and will be a valuable tool for functional genomic studies, such as genome organization and minimization.
Electronic supplementary material
The online version of this article (doi:10.1186/s12575-015-0016-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4359775  PMID: 25774095
5.  cDNA synthesis for BCR-ABL1 detection at the MMR level: the importance of using the appropriate kit 
The synthesis of complementary DNA (cDNA) for use in the detection of BCR-ABL1 at the Major Molecular Response (MMR) level is a well-established method used by clinical laboratories world-wide. However, the quality of cDNA provides sensitivity challenges and consequently affects the detection of Minimal Residual Disease (MRD).
Herein, we evaluated six commercially available kits for the synthesis of cDNA according to amplification success rate, linearity and ABL1 copy number. Based on our results, the Invitrogen SuperScript® III Reverse Transcriptase kit performed better, among the ones used in this study, for the cDNA synthesis, followed by the First Strand cDNA Synthesis Kit for RT-PCR (AMV), available from Roche Applied Sciences.
Accurate and sensitive testing for the detection of abnormal transcripts, allows the correct stratification and treatment of patients. Hence, the use of a suitable kit for the cDNA synthesis is of great importance. This study provides a comprehensive point of reference for clinical laboratories in an attempt to optimize BCR-ABL1 detection. We propose that the Invitrogen SuperScript® III Reverse Transcriptase kit is the most suitable, among the ones used in this study, for the cDNA synthesis to be used for the detection of BCR-ABL1 at the MMR level in a CML MRD assay.
Electronic supplementary material
The online version of this article (doi:10.1186/s12575-015-0014-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4321704  PMID: 25667568
BCR-ABL; cDNA synthesis; CML; MMR; MRD
6.  Evaluation of method performance for oxidative stress biomarkers in urine and biological variations in urine of patients with type 2 diabetes mellitus and diabetic nephropathy 
Oxidative stress biomarkers such as superoxide dismutase (CuZnSOD), catalase (CAT) and malondialdehyde (MDA) play an important role in the pathogenesis or progression of numerous diseases. Data regarding the biological variation and analytical quality specifications (imprecision, bias and total error) for judging the acceptability of method performance for oxidative stress biomarkers in urine are conspicuously lacking in the literature. Such data are important in setting analytical quality specifications, assessing the utility of population reference intervals (index of individuality) and assessing the significance of changes in serial results from an individual (reference change value; RCV).
Materials and methods
20 patients with type 2 diabetes mellitus (T2DM), 20 patients with diabetic nephropathy (DN) and 14 healthy individuals as control were involved in this study. Timed first morning urine samples were taken from patients and healthy groups on the zero, 1st, 3rd, 5th, 7th, 15th and 30th days. Index of individuality and reference change value were calculated from within-subject and between-subject variations. Methods of oxidative stress biomarkers in human blood were adopted in human urine and markers were measured as spectrophotometrically. Also, analytical quality specifications for evaluation of the method performance were established for oxidative stress biomarkers in urine.
Within-subject variations of oxidative stress biomarkers were significantly higher in patients with DN and T2DM compared to healthy subjects. MDA showed low individuality, and within-subject variances of MDA were larger than between-subject variances in all groups. However, CAT and CuZnSOD showed strong individuality, but within-subject variances of them were smaller than between-subject variances in all groups. RCVs of all analytes in diabetic patients were relatively higher, because of high within-subject variation, resulting in a higher RCV. Also, the described methodology achieves these goals, with analytical CVs of < 3.5% for all analytes. Goals for bias and total error were 6.0-7.9% and 12.5-23.3%, respectively.
RCVs concept for predicting the clinical status in diabetic patients represents an optimization of laboratory reporting and could be a valuable tool for clinical decision. Furthermore, for oxidative stress biomarkers’ measurements in urine, the desirable imprecision goals based on biological variation are obtainable by current methodologies.
PMCID: PMC4313470  PMID: 25649751
Biological variation; DN; Oxidative stress biomarkers; Reference change value; T2DM
7.  Comparison of Vibratome and Compresstome sectioning of fresh primate lymphoid and genital tissues for in situ MHC-tetramer and immunofluorescence staining 
For decades, the Vibratome served as a standard laboratory resource for sectioning fresh and fixed tissues. In skilled hands, high quality and consistent fresh unfixed tissue sections can be produced using a Vibratome but the sectioning procedure is extremely time consuming. In this study, we conducted a systematic comparison between the Vibratome and a new approach to section fresh unfixed tissues using a Compresstome. We used a Vibratome and a Compresstome to cut fresh unfixed lymphoid and genital non-human primate tissues then used in situ tetramer staining to label virus-specific CD8 T cells and immunofluorescent counter-staining to label B and T cells. We compared the Vibratome and Compresstome in five different sectioning parameters: speed of cutting, chilling capability, specimen stabilization, size of section, and section/staining quality.
Overall, the Compresstome and Vibratome both produced high quality sections from unfixed spleen, lymph node, vagina, cervix, and uterus, and subsequent immunofluorescent staining was equivalent. The Compresstome however, offered distinct advantages; producing sections approximately 5 times faster than the Vibratome, cutting tissue sections more easily, and allowing production of larger sections.
A Compresstome can be used to generate fresh unfixed primate lymph node, spleen, vagina, cervix and uterus sections, and is superior to a Vibratome in cutting these fresh tissues.
PMCID: PMC4318225  PMID: 25657614
Compresstome; Vibratome; Unfixed fresh tissue sectioning; Vagina; Cervix; Uterus; Spleen; Lymph node; Immunohistochemistry; in situ tetramer staining
8.  Model of Cation Transportation Mediated by High-Affinity Potassium Transporters (HKTs) in Higher Plants 
Trk/Ktr/HKT transporters probably were evolved from simple K+ channels KcsA. HKT transporters, which mediate Na+-uniport or Na+/K+-symport, maintain K+/Na+ homeostasis and increase salinity tolerance, can be classified into three subfamilies in higher plants. In this review, we systematically analyzed the characteristics of amino acids sequences and physiological functions of HKT transporters in higher plant. Furthermore, we depicted the hypothetical models of cations selection and transportation mediated by HKT transporters according to the highly conserved structure for the goal of better understanding the cations transportation processes.
PMCID: PMC4334588  PMID: 25698907
HKT transporters; Cation transport; K+/Na+ homeostasis; Na+-uniport; Na+/K+-symport
9.  A novel model of appendicitis and appendectomy to investigate inflammatory bowel disease pathogenesis and remediation 
The appendix contains copious lymphoid tissue and is constantly exposed to gut flora. Appendicitis and appendectomy (AA) has been shown to prevent or significantly ameliorate ulcerative colitis. In our novel murine AA model, the only existing experimental model of AA, the appendiceal pathology closely resembles that of human appendicitis; and AA offers an age-, bacteria- and antigen-dependent protection against colitis. Appendicitis and appendectomy performed in the most proximal colon curbs T helper 17 cell activity, curtails autophagy, modulates interferon activity-associated molecules, and suppresses endothelin vasoactivity-mediated immunopathology/vascular remodelling in the most distal colon. These AA-induced changes contribute to the limitation/amelioration of colitis pathology. Investigating strategies to manipulate and modulate different aspects of these pathways (using monoclonal antibodies, combinatorial peptides, and small molecules) would offer novel insight into inflammatory bowel disease pathogenesis, and will augment the development of new therapeutic options to manage recalcitrant colitis.
PMCID: PMC4082674  PMID: 24999306
Appendicitis; Appendectomy; Inflammatory bowel disease; Colitis; Autophagy; Antigen-processing
10.  A convenient, optimized pipeline for isolation, fluorescence microscopy and molecular analysis of live single cells 
Heterogeneity within cell populations is relevant to the onset and progression of disease, as well as development and maintenance of homeostasis. Analysis and understanding of the roles of heterogeneity in biological systems require methods and technologies that are capable of single cell resolution. Single cell gene expression analysis by RT-qPCR is an established technique for identifying transcriptomic heterogeneity in cellular populations, but it generally requires specialized equipment or tedious manipulations for cell isolation.
We describe the optimization of a simple, inexpensive and rapid pipeline which includes isolation and culture of live single cells as well as fluorescence microscopy and gene expression analysis of the same single cells by RT-qPCR. We characterize the efficiency of single cell isolation and demonstrate our method by identifying single GFP-expressing cells from a mixed population of GFP-positive and negative cells by correlating fluorescence microscopy and RT-qPCR.
Single cell gene expression analysis by RT-qPCR is a convenient means for investigating cellular heterogeneity, but is most useful when correlating observations with additional measurements. We demonstrate a convenient and simple pipeline for multiplexing single cell RT-qPCR with fluorescence microscopy which is adaptable to other molecular analyses.
PMCID: PMC4022543  PMID: 24834016
Single cell; RT-qPCR; Gene expression analysis; Fluorescence microscopy
11.  A novel recombinant baculovirus overexpressing a Bacillus thuringiensis Cry1Ab toxin enhances insecticidal activity 
Baculoviruses have been genetically modified to express foreign genes under powerful promoters in order to accelerate their speed of killing. In this study a truncated form of cry1Ab gene derived from Bacillus thuringinsis (Bt) subsp. aegypti isolate Bt7 was engineered into the genome of the baculovirus Autographa californica multiple nuclearpolyhedrosis wild type virus, in place of the polyhedrin gene by using homologous recombination in Spodoptera frugiperda (Sf) cells between a transfer vector carrying the Bt gene and the wild type virus linearized DNA. Recombinant wild type virus containing the cry1Ab gene was detected as blue occlusion-negative plaques in monolayers of Sf cells grown in the presence of X-Gal. In Sf cells infected with plaque-purified recombinant virus, the cry1Ab gene was expressed to yield a protein of approximately 82-kDa, as determined by immunoblot analysis. The toxicity of the recombinant virus expressing the insecticidal crystal protein (ICP) was compared to that of the wild-type virus. Infected-cell extract was toxic to cotton leaf worm Spodoptera littoralis second instar larvae and the estimated LC50 was 1.7 μg/ml for the recombinant virus compared with that of wild-type virus which was 10 μg/ml.
PMCID: PMC4001361  PMID: 24735532
Bacillus thuringiensis; Toxin; Baculovirus; Cry 1Ab; LC50
12.  Real-time vascular mechanosensation through ex vivo artery perfusion 
Cell-based perfusion studies have provided great insight into fluid-sensing mechanisms, such as primary cilia in the renal and vascular systems. However, the intrinsic limitations of in vitro cell culture, such as the inability to reflect cellular organization within tissues, has distanced observed paradigms from possible clinical developments. Here we describe a protocol that applies ex vivo artery perfusion and calcium imaging to observe real-time cellular responses to fluid-shear stress.
Through our ex vivo artery perfusion method, we were able to simulate physiological flow and initiate distinct fluid shear stress mechanosensory responses, as well as induced acetylcholine responses in mouse aortic tissue. The observed calcium profiles confirm results found through previous in vitro cell culture experiments. The overall procedure, including dissection, sample preparation and perfusion, takes around 3 hours to complete.
Through our unique method, we are able to induce laminar flow within intact mouse aortic tissue and illicit subsequent cellular responses. This method of ex vivo artery perfusion provides the opportunity to bridge the novel findings of in vitro studies with subsequent physiological models of fluid-shear stress mechanosensation in vascular tissues.
PMCID: PMC4392510  PMID: 24685068
13.  Detection and quantification of extracellular microRNAs in murine biofluids 
MicroRNAs (miRNAs) are short RNA molecules which regulate gene expression in eukaryotic cells, and are abundant and stable in biofluids such as blood serum and plasma. As such, there has been heightened interest in the utility of extracellular miRNAs as minimally invasive biomarkers for diagnosis and monitoring of a wide range of human pathologies. However, quantification of extracellular miRNAs is subject to a number of specific challenges, including the relatively low RNA content of biofluids, the possibility of contamination with serum proteins (including RNases and PCR inhibitors), hemolysis, platelet contamination/activation, a lack of well-established reference miRNAs and the biochemical properties of miRNAs themselves. Protocols for the detection and quantification of miRNAs in biofluids are therefore of high interest.
The following protocol was validated by quantifying miRNA abundance in C57 (wild-type) and dystrophin-deficient (mdx) mice. Important differences in miRNA abundance were observed depending on whether blood was taken from the jugular or tail vein. Furthermore, efficiency of miRNA recovery was reduced when sample volumes greater than 50 μl were used.
Here we describe robust and novel procedures to harvest murine serum/plasma, extract biofluid RNA, amplify specific miRNAs by RT-qPCR and analyze the resulting data, enabling the determination of relative and absolute miRNA abundance in extracellular biofluids with high accuracy, specificity and sensitivity.
PMCID: PMC3995583  PMID: 24629058
Extracellular microRNA; miRNA; Biofluid; RT-qPCR; Serum; Plasma
14.  Generation of a monoclonal antibody against the glycosylphosphatidylinositol-linked protein Rae-1 using genetically engineered tumor cells 
Although genetically engineered cells have been used to generate monoclonal antibodies (mAbs) against numerous proteins, no study has used them to generate mAbs against glycosylphosphatidylinositol (GPI)-anchored proteins. The GPI-linked protein Rae-1, an NKG2D ligand member, is responsible for interacting with immune surveillance cells. However, very few high-quality mAbs against Rae-1 are available for use in multiple analyses, including Western blotting, immunohistochemistry, and flow cytometry. The lack of high-quality mAbs limits the in-depth analysis of Rae-1 fate, such as shedding and internalization, in murine models. Moreover, currently available screening approaches for identifying high-quality mAbs are excessively time-consuming and costly.
We used Rae-1–overexpressing CT26 tumor cells to generate 60 hybridomas that secreted mAbs against Rae-1. We also developed a streamlined screening strategy for selecting the best anti–Rae-1 mAb for use in flow cytometry assay, enzyme-linked immunosorbent assay, Western blotting, and immunostaining.
Our cell line–based immunization approach can yield mAbs against GPI-anchored proteins, and our streamlined screening strategy can be used to select the ideal hybridoma for producing such mAbs.
PMCID: PMC3916315  PMID: 24495546
GPI-anchored protein Rae-1; Monoclonal antibody; Hybridomas; Streamlined screening strategy
15.  Reviewer acknowledgement 2013 
Contributing reviewers
A peer-reviewed journal would not survive without the generous time and insightful comments of the reviewers, whose efforts often go unrecognized. Biological Procedures Online has been blessed by the support of highly-qualified peer reviewers, and the Editor-in-Chief, Shulin Li, and staff of the journal would like to show their appreciation by thanking the following for their invaluable assistance with review of manuscripts for the journal in Volume 15 (2013).
PMCID: PMC3896796  PMID: 24444053
16.  Improved reduced representation bisulfite sequencing for epigenomic profiling of clinical samples 
DNA methylation plays crucial roles in epigenetic gene regulation in normal development and disease pathogenesis. Efficient and accurate quantification of DNA methylation at single base resolution can greatly advance the knowledge of disease mechanisms and be used to identify potential biomarkers. We developed an improved pipeline based on reduced representation bisulfite sequencing (RRBS) for cost-effective genome-wide quantification of DNA methylation at single base resolution. A selection of two restriction enzymes (TaqαI and MspI) enables a more unbiased coverage of genomic regions of different CpG densities. We further developed a highly automated software package to analyze bisulfite sequencing results from the Solexa GAIIx system.
With two sequencing lanes, we were able to quantify ~1.8 million individual CpG sites at a minimum sequencing depth of 10. Overall, about 76.7% of CpG islands, 54.9% of CpG island shores and 52.2% of core promoters in the human genome were covered with at least 3 CpG sites per region.
With this new pipeline, it is now possible to perform whole-genome DNA methylation analysis at single base resolution for a large number of samples for understanding how DNA methylation and its changes are involved in development, differentiation, and disease pathogenesis.
PMCID: PMC3895702  PMID: 24406024
DNA methylation; Epigenetics; Bisulfite sequencing; Clinical sequencing
17.  Fast automated yeast cell counting algorithm using bright-field and fluorescence microscopic images 
The faithful determination of the concentration and viability of yeast cells is important for biological research as well as industry. To this end, it is important to develop an automated cell counting algorithm that can provide not only fast but also accurate and precise measurement of yeast cells.
With the proposed method, we measured the precision of yeast cell measurements by using 0%, 25%, 50%, 75% and 100% viability samples. As a result, the actual viability measured with the proposed yeast cell counting algorithm is significantly correlated to the theoretical viability (R2 = 0.9991). Furthermore, we evaluated the performance of our algorithm in various computing platforms. The results showed that the proposed algorithm could be feasible to use with low-end computing platforms without loss of its performance.
Our yeast cell counting algorithm can rapidly provide the total number and the viability of yeast cells with exceptional accuracy and precision. Therefore, we believe that our method can become beneficial for a wide variety of academic field and industries such as biotechnology, pharmaceutical and alcohol production.
PMCID: PMC3829669  PMID: 24215650
Fast automated counting; Quantitative measurement; Yeast counting; Dual fluorescence
18.  Exploration of two methods for quantitative Mitomycin C measurement in tumor tissue in vitro and in vivo 
Two methods of quantifying Mitomycin C in tumor tissue are explored. A method of ultraviolet-visible absorption microscopy is developed and applied to measure the concentration of Mitomycin C in preserved mouse tumor tissue, as well as in gelatin samples. Concentrations as low as 60 μM can be resolved using this technique in samples that do not strongly scatter light. A novel method for monitoring the Mitomycin C concentrations inside a tumor is developed, based on microdialysis and ultraviolet-visible spectroscopy. A pump is used to perfuse a microdialysis probe with Ringer’s solution, which is fed to a flow cell to determine intratumor concentrations in real time to within a few μM. The success and limitations of these techniques are identified, and suggestions are made as to further development. To the authors’ knowledge these are the first attempts made to quantify Mitomycin C concentrations in tumor tissue.
PMCID: PMC3831870  PMID: 24206643
19.  Solution casting of chitosan membranes for in vitro evaluation of bioactivity 
Considerable research is focusing on the surface modification of titanium implants for the treatment of orthopaedic tissue injuries to increase the success of orthopaedic fixations. Chitosan is one of the natural materials under investigation based on several favourable properties. Numerous techniques have been described for the preparation of chitosan membranes, including solution casting methods for the investigation of bioactivity before applying coatings onto potential titanium implants. Solution casting enables the easy in-house evaluation of chitosan membranes and allows for the selection of promising chitosan materials.
We present a method for the standardized and easily applied preparation of chitosan membranes by solution casting. This protocol is suitable for chitosan materials spanning a wide degree of deacetylation, being derived from different chitin sources and chitosan derivatives with novel properties. We detail the preparation and quality control methods in order to prepare membranes with favourable bioactivity, sustaining cell attachment and proliferation for extended culture periods.
The possibilities associated with the use of chitosan in tissue engineering applications are far from being exhausted and numerous challenges remain prior to successful translation into the clinics. Based on our experience, we have developed simple in-house methods for quality control of homogeneous membrane casting and early prediction of successful experimental outcome.
PMCID: PMC4175485  PMID: 24192423
Chitosan; Membranes; Characterization; Titanium; Chitosan derivatives; MC3T3-E1; Fibronectin adsorption; Crosslinking
20.  Expression, purification and molecular analysis of the human ZNF706 protein 
The ZNF706 gene encodes a protein that belongs to the zinc finger family of proteins and was found to be highly expressed in laryngeal cancer, making the structure and function of ZNF706 worthy of investigation. In this study, we expressed and purified recombinant human ZNF706 that was suitable for structural analysis in Escherichia coli BL21(DH3).
ZNF706 mRNA was extracted from a larynx tissue sample, and cDNA was ligated into a cloning vector using the TOPO method. ZNF706 protein was expressed according to the E. coli expression system procedures and was purified using a nickel-affinity column. The structural qualities of recombinant ZNF706 and quantification alpha, beta sheet, and other structures were obtained by spectroscopy of circular dichroism. ZNF706's structural modeling showed that it is composed of α-helices (28.3%), β-strands (19.4%), and turns (20.9%), in agreement with the spectral data from the dichroism analysis.
We used circular dichroism and molecular modeling to examine the structure of ZNF706. The results suggest that human recombinant ZNF706 keeps its secondary structures and is appropriate for functional and structural studies. The method of expressing ZNF706 protein used in this study can be used to direct various functional and structural studies that will contribute to the understanding of its function as well as its relationship with other biological molecules and its putative role in carcinogenesis.
PMCID: PMC3848911  PMID: 24060497
Circular dichroism; Cloning; HSPC038; Molecular modeling; Protein expression; ZNF706 protein
21.  Simultaneous splicing of multiple DNA fragments in one PCR reaction 
Rapid and simultaneous splicing of multiple DNA fragments is frequently required in many recombinant DNA projects. However, former overlap extension PCRs, the most common methods for splicing DNA fragments, are not really simultaneous fusing of multiple DNA fragments.
We performed an optimized method which allowed simultaneous splicing of multiple DNA fragments in one PCR reaction. Shorter outermost primers were prior mixed with other PCR components at the same time. A sequential thermo cycling program was adopted for overlap extension reaction and amplification of spliced DNA. Annealing temperature was relatively higher in the overlap extension reaction stage than in the fused DNA amplification. Finally we successfully harvested target PCR products deriving from fusion of two to seven DNA fragments after 5–10 cycles for overlap extension reaction and then 30 cycles for fused DNA amplification.
Our method provides more rapid, economical and handy approach to accurately splice multiple DNA fragments. We believe that our simultaneous splicing overlap extension PCR can be used to fuse more than seven DNA fragments as long as the DNA polymerase can match.
PMCID: PMC3847634  PMID: 24015676
Simultaneous splicing; Multiple DNA fragments; Overlap extension PCR
22.  Quantification of bone changes in a collagen-induced arthritis mouse model by reconstructed three dimensional micro-CT 
Inflammatory arthritis is a chronic disease, resulting in synovitis and subchondral and bone area destruction, which can severely affect a patient’s quality of life. The most common form of inflammatory arthritis is rheumatoid arthritis (RA) in which many of the disease mechanisms are not well understood. The collagen-induced arthritis (CIA) mouse model is similar to RA as it exhibits joint space narrowing and bone erosion as well as involves inflammatory factors and cellular players that have been implicated in RA pathogenesis. Quantitative data for disease progression in RA models is difficult to obtain as serum blood markers may not always reflect disease state and physical disease indexes are subjective. Thus, it is important to develop tools to objectively assess disease progression in CIA.
Micro-CT (Computed Tomography) is a relatively mature technology that has been used to track a variety of anatomical changes in small animals. In this study, micro-CT scans of several joints of control and CIA mice were acquired at 0, 4, 7, and 9 weeks after the immunization with collagen type II. Each micro-CT scan was analyzed by applying a segmentation algorithm to individual slices in each image set to provide 3-dimensional representations of specific bones including the humerus, femur, and tibia. From these representations, the volume and mean density of these bones were measured and compared. This analysis showed that both the volume and the density of each measured bone of the CIA mice were significantly smaller than those of the controls at week 7.
This study demonstrates that micro-CT can be used to quantify bone changes in the CIA mouse model as an alternative to disease index assessments. In conclusion, micro-CT could be useful as a non-invasive method to monitor the efficacy of new treatments for RA tested in small animals.
PMCID: PMC3723793  PMID: 23855709
Rheumatoid arthritis; Micro-CT; Computed tomography; Imaging; Collagen induced arthritis; Bone density; Bone volume; Disease index
23.  An evaluation of the reliability of muscle fiber cross-sectional area and fiber number measurements in rat skeletal muscle 
The reliability of estimating muscle fiber cross-sectional area (measure of muscle fiber size) and fiber number from only a subset of fibers in rat hindlimb muscle cross-sections has not been systematically evaluated. This study examined the variability in mean estimates of fiber cross-sectional area as a function of the number of fibers measured, and tested whether counting a subset of fibers in a cross-section could predict total fiber number in middle-aged rats.
Soleus and extensor digitorum longus (EDL) muscle cross-sections from 23-month-old, male Fisher 344 x Brown Norway rats were stained for myofibrillar ATPase activity to identify muscle fiber type (either type I [slow-twitch] or II [fast-twitch]) and laminin to facilitate fiber cross-sectional measurements. We outlined the circumference of 1000 to 1600 single muscle fibers for measurement of fiber cross-sectional area within muscle sections. Mean type I fiber cross-sectional area was based on soleus muscle sections which were predominantly composed of type I muscle fibers. Mean type II fiber cross-sectional area was based on EDL muscle sections which were predominantly composed of type II muscle fibers. A bootstrapping resampling technique demonstrated that variability in sampling distribution of mean type I and II fiber cross-sectional areas decreased and gradually stabilized as the number of fibers measured increased with large declines in variability occurring at numbers below 150 fibers. Coefficients of variation for bootstrapped mean type I fiber cross-sectional areas were lower than for type II. In the same muscle sections, total fiber number was compared to fiber numbers within 1, 2, 3, and 4 fixed field areas (10x magnification; 1000 x 1500 pixels in size/field) on the cross-section. Fiber numbers from 3 to 4 fields (approximating 15 to 20% of the cross-section) provided a reasonably predictive value of total fiber number (r=0.57-0.59, P=0.003).
These data describe a pattern of improved precision in estimating mean fiber cross-sectional area as sample size of fibers measured increases to at least 150 in this rat model. Counting 15-20% of the fibers in cross-sections provides a reasonably reliable estimate of the total fiber number.
PMCID: PMC3599694  PMID: 23497012
Skeletal muscle; Muscle fiber cross-sectional area; Fiber number; Myofibrillar ATPase activity
24.  Fragmentation of DNA affects the accuracy of the DNA quantitation by the commonly used methods 
Specific applications and modern technologies, like non-invasive prenatal testing, non-invasive cancer diagnostic and next generation sequencing, are currently in the focus of researchers worldwide. These have common characteristics in use of highly fragmented DNA molecules for analysis. Hence, for the performance of molecular methods, DNA concentration is a crucial parameter; we compared the influence of different levels of DNA fragmentation on the accuracy of DNA concentration measurements.
In our comparison, the performance of the currently most commonly used methods for DNA concentration measurement (spectrophotometric, fluorometric and qPCR based) were tested on artificially fragmented DNA samples. In our comparison, unfragmented and three specifically fragmented DNA samples were used.
According to our results, the level of fragmentation did not influence the accuracy of spectrophotometric measurements of DNA concentration, while other methods, fluorometric as well as qPCR-based, were significantly influenced and a decrease in measured concentration was observed with more intensive DNA fragmentation.
Our study has confirmed that the level of fragmentation of DNA has significant impact on accuracy of DNA concentration measurement with two of three mostly used methods (PicoGreen and qPCR). Only spectrophotometric measurement was not influenced by the level of fragmentation, but sensitivity of this method was lowest among the three tested. Therefore if it is possible the DNA quantification should be performed with use of equally fragmented control DNA.
PMCID: PMC3576356  PMID: 23406353
DNA fragmentation; DNA quantitation; Spectrophotometry; PicoGreen; qPCR
25.  A do-it-yourself protocol for simple transcription activator-like effector assembly 
TALEs (transcription activator-like effectors) are powerful molecules that have broad applications in genetic and epigenetic manipulations. The simple design of TALEs, coupled with high binding predictability and specificity, is bringing genome engineering power to the standard molecular laboratory. Currently, however, custom TALE assembly is either costly or limited to few research centers, due to complicated assembly protocols, long set-up time and specific training requirements.
We streamlined a Golden Gate-based method for custom TALE assembly. First, by providing ready-made, quality-controlled monomers, we eliminated the procedures for error-prone and time-consuming set-up. Second, we optimized the protocol toward a fast, two-day assembly of custom TALEs, based on four thermocycling reactions. Third, we increased the versatility for diverse downstream applications by providing series of vector sets to generate both TALENs (TALE nucleases) and TALE-TFs (TALE-transcription factors) under the control of different promoters. Finally, we validated our system by assembling a number of TALENs and TALE-TFs with DNA sequencing confirmation. We further demonstrated that an assembled TALE-TF was able to transactivate a luciferase reporter gene and a TALEN pair was able to cut its target.
We established and validated a do-it-yourself system that enables individual researchers to assemble TALENs and TALE-TFs within 2 days. The simplified TALE assembly combined with multiple choices of vectors will facilitate the broad use of TALE technology.
PMCID: PMC3554550  PMID: 23316790
TALEN; TALE-TF; Golden Gate; Transcription-activator-like effector

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