Optimising the biocompatibility of 3D printed photopolymer constructs in vitro and in vivo.

3D printing is a rapid and accessible fabrication technology that engenders creative custom design solutions for cell scaffolds, perfusion systems and cell culture systems for tissue engineering. Critical to its success is the biocompatibility of the materials used, which should allow long-term tissue culture without affecting cell viability or inducing an inflammatory response for in vitro and in vivo applications. Polyjet 3D printers offer arguably the highest resolution with the fewest design constraints of any commercially available 3D printing systems. Although widely used for rapid-prototyping of medical devices and 3D anatomical modelling, polyjet printing has not been adopted by the tissue engineering field, largely due to the cytotoxicity of leachates from the printed parts. Biocompatibility in the context of cell culture is not commonly addressed for polyjet materials, as they tend to be optimised for their ability to fabricate complex structures. In order to study the potential issues surrounding the leaching of toxins, we prepared cell culture substrates using the commercially available MED610 photopolymer. The substrates were cleaned using either the manufacturer-specified 'biocompatible' washing procedures, or a novel protocol incorporating a sonication in isopropanol and water step. We then compared the effectiveness of these both in vitro and in vivo. Using primary mouse myoblast cultures, the manufacturer's protocol led to inconsistent and poorer cell viability when compared to the sonication protocol (p = 0.0002 at 48 h after indirect exposure). Subdermal implantation of MED610 into nude rats demonstrated a significant foreign body response with a greater number of giant cells (p = 0.0161) and foreign bodies (p = 0.0368) when compared to the sonication protocol, which was comparable to the control (sham) groups. These results present an improved, cytocompatible cleaning protocol of printable photopolymers to facilitate creative 3D-printed custom designs for cell culture systems for both in vitro and in vivo tissue engineering applications.

Nanoscale platinum printing on insulating substrates.

The deposition of noble metals on soft and/or flexible substrates is vital for several emerging applications including flexible electronics and the fabrication of soft bionic implants. In this paper, we describe a new strategy for the deposition of platinum electrodes on a range of materials, including insulators and flexible polymers. The strategy is enabled by two principle advances: (1) the introduction of a novel, low temperature strategy for reducing chloroplatinic acid to platinum using nitrogen plasma; (2) the development of a chloroplatinic acid based liquid ink formulation, utilizing ethylene glycol as both ink carrier and reducing agent, for versatile printing at nanoscale resolution using dip-pen nanolithography (DPN). The ink formulation has been printed and reduced upon Si, glass, ITO, Ge, PDMS, and Parylene C. The plasma treatment effects reduction of the precursor patterns in situ without subjecting the substrate to destructively high temperatures. Feature size is controlled via dwell time and degree of ink loading, and platinum features with 60 nm dimensions could be routinely achieved on Si. Reduction of the ink to platinum was confirmed by energy dispersive x-ray spectroscopy (EDS) elemental analysis and x-ray diffraction (XRD) measurements. Feature morphology was characterized by optical microscopy, SEM and AFM. The high electrochemical activity of individually printed Pt features was characterized using scanning electrochemical microscopy (SECM).

Standardization of PCR amplification for fragile X trinucleotide repeat measurements.

To provide the clinical diagnostics community with accurate protocols and measurements for the detection of genetic disorders, we have established a quantitative measurement program for trinucleotide repeats associated with human disease. In this study, we have focused on the triplet repeat associated with fragile X syndrome. Five cell lines obtained from the Coriell Cell Repository were analyzed after polymerase chain reaction (PCR) amplification and size separation. These cell lines were reported to contain CGG repeat elements (ranging from 29 to 110 repeats). Our initial measurements focused on measurement variability: (a) between slab-PAGE and capillary (CE) separation systems (b) interlane variability (slab-PAGE) (c) intergel variability, and (d) variability associated with amplification. Samples were run in triplicate for all measurements, and the analysis performed using Gene Scan analysis software. The repeat sizes were verified by DNA sequence analyzes. The standard deviations for interlane measurements in slab-gels ranged from 0.05 to 0.35. There was also little variation in size measurements performed on different gels and among PCR amplifications. The CGG repeat measurements performed by capillary electrophoresis were more precise, with standard deviations ranging from 0.02 to 0.29. The slab-PAGE and CE size measurements were in agreement except for the pre-mutation alleles, which yielded significantly smaller sizes by CE.

Prediction of DNA single-strand conformation polymorphism: analysis by capillary electrophoresis and computerized DNA modeling.

We have analyzed previously three representative p53 single-point mutations by capillary-electrophoresis single-strand conformation polymorphism (CE-SSCP). In the current study, we compared our CE-SSCP results with the potential secondary structures predicted by an RNA/DNA-folding algorithm with DNA energy rules, used in conjunction with a computer analysis workbench called STRUCTURELAB. Each of these mutations produces measurable shifts in CE migration times relative to wild type. Using computerized folding analysis, each of the mutations was found to have a conformational difference relative to wild type, which accounts for the observed differences in CE migration. Additional properties exhibited in the CE electropherograms were also explained using the computerized analysis. These include the appearance of secondary peaks and the temperature dependence of the electrophoretic patterns. The results yield insight into the mechanism of SSCP and how the conditions of this measurement, especially temperature, may be optimized to improve the sensitivity of the SSCP method. The results may also impact other diagnostic methods, which would benefit by a better understanding of DNA single-strand conformation polymorphisms to optimize conditions for enzymatic cleavage and DNA hybridization reactions.

Long PCR for VNTR analysis.

The Polymerase Chain Reaction (PCR) has revolutionized the analysis of DNA from a variety of sources. With its sensitivity and ability to amplify degraded DNAs and small quantities of samples, coupled with fast turn-around-time, PCR is often the analytical method of choice for DNA profiling in forensic laboratories. RFLP methods, while requiring larger amounts of high molecular weight DNA and needing approximately 6-8 weeks of analytical time, still provide a higher power of discrimination per locus than that achieved using the loci currently available for PCR. The combination of both RFLP and PCR would be advantageous for some applications. A new technique, Long PCR, allows for the effective amplification of long DNA targets from approximately 0.5 kb to > 20 kb of genomic DNA. Currently, several Long PCR systems are commercially available. Using a Taq/Pyrococcus DNA polymerase enzyme system and DNA isolated from bloodstains, we have successfully amplified 1-20 ng of Chelex-extracted DNA, an amount commonly used in Amp-FLP technology. The robustness of Long PCR in comparison to RFLP was also examined through the use of partially degraded blood samples. Long PCR was then used to amplify both D2S44 and D5S110 RFLP loci. Although all D2 and D5 alleles were detected, the larger alleles were amplified at significantly lower levels than the smaller alleles.

Detection of p53 gene mutations: analysis by single-strand conformation polymorphism and Cleavase fragment length polymorphism.

We have generated a collection of clones containing single point mutations within the exon 5-9 hot spot regions of the p53 gene by using polymerase chain reaction (PCR) to amplify select regions of the gene from characterized cell lines. These clones were then used to address the sensitivity of mutation detection using slab-gel single-strand conformation polymorphism (SSCP) and Cleavase fragment length polymorphism (CFLP) assay systems. Both methods exhibited high sensitivities for the detection of mutations in cloned p53 mutations in this study: 97% for CFLP and 94% for SSCP. In addition to resulting in higher sensitivity of mutation detection, CFLP has the capability to analyze longer fragments. In this study, CFLP identified five intronic mutations which were not investigated in the exon-specific SSCP assay. These results agree with those found elsewhere and demonstrate that CFLP scanning can have practical advantages when used for the identification of sequence alterations within the p53 gene.

Identification of known p53 point mutations by capillary electrophoresis using unique mobility profiles in a blinded study.

This study is part of an ongoing project at the National Institute of Standards and Technology (NIST) that generates a panel of DNA clones containing the most common mutations found in the human p53 tumor suppressor gene. This panel will be made available as a reference source for evaluation and testing for p53 mutations. Single strand conformation polymorphism (SSCP) analysis has found widespread acceptance as a tool for simply and rapidly screening for mutations, albeit with a detection rate that can be below 100%. We have begun to analyze mutations found in exon 7 of the p53 gene by SSCP using laser induced fluorescence capillary electrophoresis (LIF-CE). PCR fragments, containing single point mutations, were amplified from genomic DNA isolated from cell lines using primers labeled with two different fluorophores. This dual labeling approach allowed better traceability of mobility shifts as a function of the experimental conditions. While analyzing the clones H596, Colo320, Namalwa and wild type (reference samples) at different temperatures, ranging from 25 to 45 degrees C, it was observed that each mutation responded in a unique way to changes in temperature both in magnitude and direction of shifts relative to the wild type sample. In a blinded study, ten p53 exon 7 samples were matched automatically, using ABI PRISM Genotyper software, against the four reference samples. From these 10 samples, six were correctly identified as containing one of the reference mutations, two corresponded to wild type, and two were correctly identified as non-reference mutations. This approach should prove helpful in the rapid screening of target sequences that are known to bear a limited number of mutations.

Detection of tyrosinase mRNA in melanoma by reverse transcription-PCR and electrochemiluminescence.

Increased sensitivity and improved quantitation of analytical tests used in biotechnology and clinical chemistry are goals of many laboratories. We have used tyrosinase primers to specifically amplify by RT-PCR the tyrosinase mRNA expressed by the M12 melanoma cell line in a background of mRNA from breast cancer cells. An electrochemiluminescence detection procedure was used as a readout system for this study. Biotinylated post-PCR cDNA samples were hybridized to a tris(2,2'-bipyridine)ruthenium(II) (TBR) chelate-labeled oligonucleotide probe, and the hybrid was subsequently captured by streptavidin-coated Dynabeads. When either the QPCR System 5000 or the Origen 1 Analyzer System were used, the luminescence emitted by the TBR-chelate of the captured specific post-PCR product was assessed. Tyrosinase-specific mRNA isolated from approximately 1-10 melanoma cells in a background of 10(7) cells could be detected. We improved the sensitivity and logistics of the assay through the use of rTth for reverse transcription and amplification. Tyrosinase mRNA was detected in blood from 7 of 16 melanoma patients, whereas none of the 5 healthy donor bloods were positive (P = 0.01; Wilcoxon test).

Development of standard reference materials for diagnosis of p53 mutations: analysis by slab gel single strand conformation polymorphism.

We have amplified by polymerase chain reaction (PCR) a 2.0 kbp region of the p53 gene containing exons 5--9 from seven cell lines reported in the literature to contain the majority of mutations reported for this gene. Sequence analysis of these products show that all seven cell lines contain mutations within the mutational hot spots of the p53 gene. Six of the seven clones have single base substitutions and the seventh has a single base deletion. We have analyzed the seven p53 single point mutations by single strand conformation polymorphism (SSCP) analysis using fluorescence slab gel electrophoresis (SG-SSCP). Fluorescent-labeled PCR primers were used for amplification of specific exons for mutation detection. SG-SSCP was conducted using Model 373 and Model 377 DNA sequencers with GeneScan Software (Perkin Elmer, Applied Biosystem Division). Nine different gel systems were first tested for their ability to resolve the p53 mutations using the Model 373 instrument. Two gel systems were capable of resolving all of the mutations that were screened. Optimal results were obtained with 12% w/v acrylamide 50:1 plus 10% v/v glycerol. This gel system was used to evaluate the effect of temperature on the ability to resolve the mutations. The separation with respect to wild type varied for each mutation examined. Subambient temperature (20 degrees C) was preferable overall for discrimination of these mutations as a group. We intend to use this system to examine a much larger panel of p53 mutation standards that are now under development.

Detection of p53 point mutations by single strand conformation polymorphism: analysis by capillary electrophoresis.

We have analyzed five p53 single point mutations by single strand conformation polymorphism using capillary electrophoresis (CE-SSCP) and have compared these measurements to measurements obtained by slab gel electrophoresis (SG-SSCP). PCR primers were used for amplification of specific exons for mutation detection. 5' Primers were labeled with FAM (5-carboxyfluorescein) and 3' primers were labeled with JOE (2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein). CE-SSCP was performed using the Perkin Elmer ABI PRISM 310 Genetic Analyzer with GeneScan Software and the Beckman P/ACE 5510 CE equipped for laser-induced fluorescence detection. Although the shifts in migration times for the p53 mutations relative to the corresponding wild-type strands could be successfully detected by either SG or CE analysis, the individual electrophoresis run times were about tenfold faster and more automated with capillary electrophoresis. The CE-SSCP measurements were performed at temperatures ranging from 10 to 60 degrees C on a prototype instrument. For mutations measured at ambient temperature (25 degrees C), characteristic shifts in direction and magnitude were observed in the migration times of both strands of all mutations relative to the wild type. This demonstrated the ability of CE at ambient temperature to resolve these mutations. However, the magnitude and direction of shifts in migration time varied with temperature in a discrete pattern for each mutation and resulted in a temperature-specific profile for each mutation. This demonstrated that extended temperature control will be an important advantage in resolving single point mutations by CE-SSCP. In addition, by using CE, discrete intra-strand isoforms could be easily observed at different temperatures. The combination of mutation-specific temperature profiling and analysis of isoforms by CE-SSCP should be of help to the diagnostic community in the detection of genetic mutations.

Detection of p53 mutations in B cell non-Hodgkin's lymphoma cell lines.

The p53 tumor suppressor gene is frequently mutated within its evolutionarily conserved regions in a number of human cancers. Previous reports demonstrated mutations of this gene in both Burkitt's lymphoma and B cell chronic lymphocytic leukemia. However, dissimilar results were obtained in non-Hodgkin's lymphoma (NHL). In one study, no mutation was detected in 43 NHL tissues. A second study reported p53 mutations in eight (all with advanced stage disease) out of 48 tissues obtained from Japanese NHL patients. Using both immunoblotting and radio-immunoprecipitation, we detected mutant p53 proteins in nine out of 10 B cell lines established from NHL tissues. The mutations were confirmed by reverse transcription polymerase chain reaction-mediated single-strand conformational polymorphism (RT-PCR-SSCP) analysis in eight cell lines. The high frequency of p53 mutation in NHL B cell lines and the relatively low frequency of p53 mutations in fresh lymphoma tissue suggests that p53 gene alteration may play a role in lymphomagenesis and/or disease progression in a subset of B cell lymphomas and that the p53 mutation conveys a proliferative advantage on lymphoma cells that permits their in vitro growth.

Cloning and characterization of human and murine homologues of the Drosophila polo serine-threonine kinase.

We have cloned both human and murine complementary DNAs that are homologous to the Drosophila serine/threonine polo kinase and the recently cloned murine polo related kinase (PLK). Both the human and murine clones are about 2.1 kilobases with open reading frames of 1.8 kilobases, encoding proteins of 603 amino acids with a predicted size of 66 kilodaltons and an apparent size of 67 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. During embryonic development of the mouse, the mRNA was expressed in all tissues examined, whereas in adult tissues, expression was limited to thymus and ovaries. All cell lines examined also expressed mRNAs of similar size. Microinjection of in vitro transcribed sense mRNA into serum-starved murine NIH3T3 cells induced tritiated thymidine incorporation, whereas microinjection of antisense RNA into growing NIH3T3 cells blocked tritiated thymidine incorporation. When PC12 rat cells were induced to differentiate with nerve growth factor, gene expression of PLK was greatly reduced. Together, these results suggest that PLK expression is restricted to, and is perhaps required by, proliferating cells.

Detection of c-mos proto-oncogene expression in human cells.

Although the human c-mos proto-oncogene has been characterized for more than a decade, very little is known about its protein product and its expression in somatic cells. We generated three human c-mos-specific antisera and report here the detection of c-mos protein in a human neuroblastoma cell line, SK-N-BE2 (BE2). Both Western (immuno-) blot and immunoprecipitation analyses detected a p37 as the major form and p40 and p35 as minor forms of the c-mos protein. Using Northern blot analysis, 3.5- and 1.7-kb c-mos messages were detected. Using a highly sensitive method that combines reverse transcription and the polymerase chain reaction (RT-PCR), c-mos RNA was detected in all the human samples examined. With Western blot analysis, we further showed that c-mos proteins are expressed in cervical carcinoma-derived cell lines. This ubiquitous expression of low levels of c-mos suggests a fundamental role for the c-mos proto-oncogene.

Antigen binding and idiotype analysis of antibodies obtained after electroporation of heavy and light chain genes encoding phosphocholine-specific antibodies: a model for T15-idiotype dominance.

Antibodies bearing the T15 idiotype dominate the murine primary immune response to phosphocholine (PC). Analysis of antigen binding of antibodies derived from V1:DFL16.1:JH1 (VH1) germline and N region-derived variant heavy (H) chains and kappa 22, kappa 24, and kappa 8 light (L) chains demonstrates that the T15H:kappa 22L (T15) antibody binds PC at least 20-40 times better than other antibodies derived from alternate germline forms of the VH1 H chain and kappa 22, kappa 24, or kappa 8 L chains. To achieve affinities in the same range as the T15 antibody, kappa 24 and kappa 8 L chain-containing antibodies must have H chains derived from variant N region or somatically mutated VH1 genes. Single amino acid differences at the VD junction of the various germline and N region variant VH1 H chains dictate the L chain that can associate with the H chain to produce a PC-specific antibody. Several H:L combinations give rise to T15 or M167 idiotype-positive antibodies that lack specificity for PC, and single amino acid substitutions or insertions at the VH1:D junction result in the loss of T15 or M167 idiotopes. Based on these observations, our data support a molecular model involving both preferential gene rearrangement and antigen-driven B cell selection to explain T15 idiotype dominance in the immune response to PC. In the absence of N region diversification, large numbers of neonatal B cells bearing the T15H:kappa 22L surface immunoglobulin M (sIgM) receptors would be selected and expanded by autologous or environmental PC antigen into the long-lived peripheral B cell pool.

Phorbol ester-induced, cell-cycle-specific, growth inhibition of human B-lymphoma cell lines.

The activation, growth, and differentiation of three B-cell- and one non-B-cell-derived human lymphoma cell lines were examined after treatment with protein kinase C-activating phorbol esters. Treatment with these agents resulted in early activation events similar to those observed in normal B cells. However, in contrast to their growth-promoting effect on normal human B lymphocytes, exposure to these phorbol esters induced profound growth inhibition of the three B-cell-derived lymphoma lines. Maximal inhibition was achieved within 24 hours of culture initiation and could be reversed if the phorbol ester was removed after 12, but not 20, hours in culture. Cell-cycle analysis of phorbol ester-treated lymphoma cells revealed a G1/S block in one line, whereas cells from the other two lines accumulated in G2/M. These data demonstrate that protein kinase C-binding phorbol esters can interrupt the cell cycle in two places in actively dividing human B-lymphoma cells. These findings may prove valuable with regard to potential therapy of human malignant lymphomas.

The long terminal repeat sequences of a novel human endogenous retrovirus.

The complete nucleotide sequence of both the 5' and 3' long terminal repeats (LTR's) has been determined for a human endogenous retroviral genome. These sequences are 593 and 590 nucleotides long and have diverged from one another by 8.8 percent. The LTR's resemble those of functional mammalian type C retroviruses in length and in the presence and location of eukaryotic promoter sequences. The 5' LTR is followed by a presumptive primer binding site unlike that of any known mammalian type C retrovirus, exhibiting 17 out of 18 nucleotides complementary to arginine transfer RNA rather than proline transfer RNA.