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1.  Isolation of T cells from mouse oral tissues 
Utilizing mouse models provides excellent immunological and experimental tools to study oral immune responses. However for functional assays, isolating T lymphocytes from the oral tissues has proved to be challenging due to the absence of reliable methods that yield viable cells with consistency. To study adaptive immune cell interactions in the oral mucosal tissues, it is necessary to isolate T cells with a good viability and study them at the single cell level.
We have established an improved method to isolate immune cells, including Tregs and Th17 cells from intra-epithelial niches and lamina propria of the tongue, gingival and palatal tissues in the oral mucosa of mice.
This new method of isolating immune cells from oral tissues will enable us to further our understanding of oral tissue immune cells and their role during oral infections and oral inflammation.
PMCID: PMC3984730  PMID: 24612879
Murine oral tissue; Leukocyte isolation and oral T cells; Treg; Th17; ILC
2.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  Increasing the library size in cDNA display by optimizing purification procedures 
The library size is critical for selection in evolutionary molecular engineering (directed evolution). Although cDNA display has become a promising in vitro display technology by overcoming the instability of mRNA display, it is hindered by low yields. In this study, we improved the yield of cDNA display molecules by carefully examining each step of the preparation process.
We found that steric hindrance of ribosomes binding to the mRNA-protein fusion molecules was interfering with biotin-streptavidin binding. Additionally, reducing buffer exchange by performing RNase digestion in the His-tag-binding buffer to release the cDNA display molecules improved their His-tag purification.
Our optimized conditions have improved the yield of cDNA display molecules by more than 10 times over currently used methods, making cDNA display more practically available in evolutionary molecular engineering.
PMCID: PMC3680162  PMID: 23697943
Directed evolution; In vitro protein selection; mRNA/cDNA display; Protein engineering; Puromycin
11.  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
12.  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
13.  Next generation sequencing in cancer research and clinical application 
The wide application of next-generation sequencing (NGS), mainly through whole genome, exome and transcriptome sequencing, provides a high-resolution and global view of the cancer genome. Coupled with powerful bioinformatics tools, NGS promises to revolutionize cancer research, diagnosis and therapy. In this paper, we review the recent advances in NGS-based cancer genomic research as well as clinical application, summarize the current integrative oncogenomic projects, resources and computational algorithms, and discuss the challenge and future directions in the research and clinical application of cancer genomic sequencing.
PMCID: PMC3599179  PMID: 23406336
Next generation sequencing; Cancer research; Clinical application
14.  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
15.  Determine the quality of human embryonic stem colonies with laser light scattering patterns 
With the prompt developments of regenerative medicine, the potential clinical applications of human embryonic stem cells have attracted intense attention. However, the labor-intensive and complex manual cell selection processes required during embryonic stem cell culturing have seriously limited large-scale production and broad applications. Thus, availability of a label-free, non-invasive platform to replace the current cumbersome manual selection has become a critical need.
A non-invasive, label-free, and time-efficient optical platform for determining the quality of human embryonic stem cell colonies was developed by analyzing the scattering signals from those stem cell colonies. Additionally, confocal microscopy revealed that the cell colony morphology and surface structures were correlated with the resulting characteristic light scattering patterns. Standard immunostaining assay (Oct-4) was also utilized to validate the quality-determination from this light scattering protocol. The platform developed here can therefore provide identification accuracy of up to 87% for colony determination.
Our study here demonstrated that light scattering patterns can serve as a feasible alternative approach to replace conventional manual selection for human embryonic stem cell cultures.
PMCID: PMC3560278  PMID: 23316759
Light-scattering; Human embryonic stem cell; Pluripotency; Label-free detection
16.  A new method of kidney biopsy using low dose CT-guidance with coaxial trocar and bard biopsy gun 
To explore a new method of kidney biopsy with coaxial trocar and bard biopsy gun under low dose computed tomography (CT)-guidance and evaluate its accuracy, safety, and efficacy.
Sixty patients underwent renal biopsy under CT-guidance. They were randomly divided into two groups: group I, low dose CT-guided (120 kV and 25 or 50 mAs) and group II, standard dose CT-guided (120 kV and 250 mAs). For group I, the coaxial trocar was accurately placed adjacent to the renal capsule of the lower pole, the needle core was removed, and samples were obtained with a bard biopsy gun. For group II, the coaxial trocar was not used. Total number of passes, mean biopsy diameter, mean glomeruli per specimen, mean operation time, mean scanning time, and mean radiation dose were noted. Dose-length product (DLP) was used to calculate the radiation doses. After 24 hours of the biopsy, ultrasound was repeated to identify any subcapsular hematoma.
Success rate of biopsy in group I was 100% while using low dose CT-guidance along with coaxial trocar renal. There was no statistic differences bewteen group I and II in the total number of passes, mean biopsy diameter, mean glomeruli per specimen and mean time of operation and CT scanning. The average DLP of group I was lower as compared to the value of group II (p <0.05).
Kidney biopsy using coaxial trocar and bard biopsy gun under low dose CT was an accurate, simple and safe method for diagnosis and treatment of kidney diseases. It can be used for repeat and multiple biopsies, particularly suitable for obese and renal atrophy patients in whom the kidneys are difficult to image.
PMCID: PMC3561167  PMID: 23294600
Kidney biopsy; Low dose CT scanning; Bard biopsy gun; Coaxial trocar
17.  Spontaneous arthritis and ankylosis in male DBA/1 mice: further evidence for a role of behavioral factors in “stress-induced arthritis” 
Ageing male DBA/1 mice spontaneously develop arthritis in the hind paws. We and others have demonstrated that this model shares striking features with human spondyloarthritis, in particular entheseal involvement, progressive ankylosis but also dactylitis. Here, we report on our recent experience with this model highlighting how changes in the animal facility affect the development of the disease.
Ageing male DBA/1 mice from different litters were caged together (6 mice per cage) at the age of 10 weeks. The mice were checked twice a week for clinical signs of arthritis. Disease severity was assessed in further detail post-mortem by scoring for histomorphological characteristics. DBA/1 mice spontaneously develop macroscopically detectable arthritis, presenting as joint swelling or toe stiffness. Standard settings with open cages lead to an almost 100% incidence by the age of 26 weeks. The introduction of larger cages and filter tops reducing exposure to other cages dramatically affected incidence. Other negative factors include excess bedding material reducing the impact of walking and running. Switching back to the original conditions resulted again in a high incidence, further optimized by sensory exposure to female mice. We also showed that the related DBA/2 strain is sensitive to the disease.
Changing environmental factors in the housing conditions of DBA/1 mice severely affects the spontaneous development of arthritis. This points out that the model is very sensitive to external stress and sensory factors that are likely affecting the behavior of the male mice and that the model needs to be optimized in different situations.
PMCID: PMC3537550  PMID: 23253472
Spondyloarthritis; Spontaneous arthritis; Behavioral factors; Ankylosis
18.  Modified protocol for in vivo imaging of wild-type mouse retina with customized miniature spectral domain optical coherence tomography (SD-OCT) device 
This protocol outlines and evaluates a modified scanning procedure for a customized spectral domain optical coherence tomography (SD-OCT) imaging apparatus within the wild-type C57Bl/6 mouse posterior segment. This modified protocol allows for the capture of a 50 degree field of view spanning 3 mm by 3 mm perimeter with the optic disc as the central point. By utilizing this scanning protocol a more reliable measurement of retinal thickness can be achieved outside the fluctuating region of the optic disc. This protocol, when applied to this high resolution device, enables non-invasive in vivo histological imaging and biometric assessment of the various layers of the rodent posterior segment within a 20 – 30 min procedural time-frame. This protocol could establish a standardized method for evaluating morphological changes, with this commercial SDOCT device, when assessing longitudinal disease pathophysiology and treatment response in mouse models for future vision science research.
PMCID: PMC3520836  PMID: 23057840
Spectral domain optical coherence tomography; Customized; Retinal imaging; Mouse
19.  A robust dual reporter system to visualize and quantify gene expression mediated by transcription activator-like effectors 
Transcription activator-like effectors (TALEs) are a class of naturally occurring transcription effectors that recognize specific DNA sequences and modulate gene expression. The modularity of TALEs DNA binding domain enables sequence-specific perturbation and offers broad applications in genetic and epigenetic studies. Although the efficient construction of TALEs has been established, robust functional tools to assess their functions remain lacking.
We established a dual reporter system that was specifically designed for real-time monitoring and quantifying gene expression mediated by TALEs. We validated both sensitivity and specificity of this dual-reporter system in mammalian cells, and demonstrated that this dual reporter system is robust and potentially amenable to high throughput (HTP) applications.
We have designed, constructed and validated a novel dual reporter system for assessing TALE mediated gene regulations. This system offers a robust and easy-to- use tool for real-time monitoring and quantifying gene expression in mammalian cells.
PMCID: PMC3509018  PMID: 22871120
Dual reporter; Gene editing; Transcription activator-like effector; Green fluorescent protein; Firefly luciferase
20.  Competitive DNA transfection formulation via electroporation for human adipose stem cells and mesenchymal stem cells 
Adipose stem cells have a strong potential for use in cell-based therapy, but the current nucleofection technique, which relies on unknown buffers, prevents their use.
We developed an optimal nucleofection formulation for human adipose stem cells by using a three-step method that we had developed previously. This method was designed to determine the optimal formulation for nucleofection that was capable of meeting or surpassing the established commercial buffer (Amaxa), in particular for murine adipose stem cells. By using this same buffer, we determined that the same formulation yields optimal transfection efficiency in human mesenchymal stem cells.
Our findings suggest that transfection efficiency in human stem cells can be boosted with proper formulation.
PMCID: PMC3388581  PMID: 22512891
Electroporation; Formulation; Stem cells; Transfection; Cell therapy
21.  Novel mouse mammary cell lines for in vivo bioluminescence imaging (BLI) of bone metastasis 
Tumor cell lines that can be tracked in vivo during tumorigenesis and metastasis provide vital tools for studying the specific cellular mechanisms that mediate these processes as well as investigating therapeutic targets to inhibit them. The goal of this study was to engineer imageable mouse mammary tumor cell lines with discrete propensities to metastasize to bone in vivo. Two novel luciferase expressing cell lines were developed and characterized for use in the study of breast cancer metastasis to bone in a syngeneic mouse model.
The 4 T1.2 luc3 and 66c14 luc2 cell lines were shown to have high levels of bioluminescence intensity in vitro and in vivo after orthotopic injection into mouse mammary fat pads. The 4 T1.2 luc3 cell line was found to closely model the sites of metastases seen in human patients including lung, liver, and bone. Specifically, 4 T1.2 luc3 cells demonstrated a high incidence of metastasis to spine, with an ex-vivo BLI intensity three orders of magnitude above the commercially available 4 T1 luc2 cells. 66c14 luc2 cells also demonstrated metastasis to spine, which was lower than that of 4 T1.2 luc3 cells but higher than 4 T1 luc2 cells, in addition to previously unreported metastases in the liver. High osteolytic activity of the 4 T1.2 luc3 cells in vivo in the bone microenvironment was also detected.
The engineered 4 T1.2 luc3 and 66c14 luc2 cell lines described in this study are valuable tools for studying the cellular events moderating the metastasis of breast tumor cells to bone.
PMCID: PMC3473320  PMID: 22510147
Breast cancer; Mammary cancer; Bone metastasis; in vivo imaging; 4 T1 cells; 4 T1.2 cells; Osteolysis; Syngeneic Balb/c model
22.  A reinvestigation of somatic hypermethylation at the PTEN CpG island in cancer cell lines 
PTEN is an important tumour suppressor gene that is mutated in Cowden syndrome as well as various sporadic cancers. CpG island hypermethylation is another route to tumour suppressor gene inactivation, however, the literature regarding PTEN hypermethylation in cancer is controversial. Furthermore, investigation of the methylation status of the PTEN CpG island is challenging due to sequence homology with the PTEN pseudogene, PTENP1. PTEN shares a CpG island promoter with another gene known as KLLN. Here we present a thorough reinvestigation of the methylation status of the PTEN CpG island in DNA from colorectal, breast, ovarian, glioma, lung and haematological cancer cell lines.
Using a range of bisulphite-based PCR assays we investigated 6 regions across the PTEN CpG island. We found that regions 1-4 were not methylated in cancer cell lines (0/36). By allelic bisulphite sequencing and pyrosequencing methylation was detected in regions 5 and 6 in colorectal, breast and haematological cancer cell lines. However, methylation detected in this region was associated with the PTENP1 promoter and not the PTEN CpG island.
We show that methylation of the PTEN CpG island is a rare event in cancer cell lines and that apparent methylation most likely originates from homologous regions of the PTENP1 pseudogene promoter. Future studies should utilize assays that reliably discriminate between PTEN and PTENP1 to avoid data misinterpretation.
PMCID: PMC3342897  PMID: 22490388
DNA methylation; Epigenetic; PTEN; KILLIN; PTENP1; Pseudogene; Cowden syndrome
23.  A microplate technique to simultaneously assay calcium accumulation in endoplasmic reticulum and SERCA release of inorganic phosphate 
Traditional analyses of calcium homeostasis have separately quantified either calcium accumulation or release mechanisms. To define the system as a whole, however, requires multiple experimental techniques to examine both accumulation and release. Here we describe a technique that couples the simultaneous quantification of radio-labeled calcium accumulation in endoplasmic reticulum (ER) microsomes with the release of inorganic phosphate (Pi) by the hydrolytic activity of sarco-endoplasmic reticulum calcium ATPase (SERCA) all in the convenience of a 96-well format.
PMCID: PMC3388579  PMID: 22472432
Calcium; SERCA activity; Microsomes; Inorganic phosphate; Malachite green
24.  An improved competitive inhibition enzymatic immunoassay method for tetrodotoxin quantification 
Quantifying tetrodotoxin (TTX) has been a challenge in both ecological and medical research due to the cost, time and training required of most quantification techniques. Here we present a modified Competitive Inhibition Enzymatic Immunoassay for the quantification of TTX, and to aid researchers in the optimization of this technique for widespread use with a high degree of accuracy and repeatability.
PMCID: PMC3337821  PMID: 22410273
Tetrodotoxin; CIEIA; HPLC
25.  Frequency shifting approach towards textual transcription of heartbeat sounds 
Auscultation is an approach for diagnosing many cardiovascular problems. Automatic analysis of heartbeat sounds and extraction of its audio features can assist physicians towards diagnosing diseases. Textual transcription allows recording a continuous heart sound stream using a text format which can be stored in very small memory in comparison with other audio formats. In addition, a text-based data allows applying indexing and searching techniques to access to the critical events. Hence, the transcribed heartbeat sounds provides useful information to monitor the behavior of a patient for the long duration of time. This paper proposes a frequency shifting method in order to improve the performance of the transcription. The main objective of this study is to transfer the heartbeat sounds to the music domain. The proposed technique is tested with 100 samples which were recorded from different heart diseases categories. The observed results show that, the proposed shifting method significantly improves the performance of the transcription.
PMCID: PMC3396354  PMID: 21970368

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