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Decellularization of tissues provides extracellular matrix (ECM) scaffolds for regeneration therapy and an experimental model to understand ECM and cellular interactions. However, decellularization often causes microstructure disintegration and reduction of physical strength, which greatly limits the use of this technique in soft organs or in applications that require maintenance of physical strength. Here, we present a new tissue decellularization procedure, namely CASPER (Clinically and Experimentally Applicable Acellular Tissue Scaffold Production for Tissue Engineering and Regenerative Medicine), which includes infusion and hydrogel polymerization steps prior to robust chemical decellularization treatments. Polymerized hydrogels serve to prevent excessive damage to the ECM while maintaining the sophisticated structures and biological activities of ECM components in various organs, including soft tissues such as brains and embryos. CASPERized tissues were successfully recellularized to stimulate a tissue-regeneration-like process after implantation without signs of pathological inflammation or fibrosis in vivo, suggesting that CASPERized tissues can be used for monitoring cell-ECM interactions and for surrogate organ transplantation.

Recent advances in automated cell counters enable us to count cells more easily with consistency. However, the wide use of the traditional vital dye trypan blue (TB) raises environmental and health concerns due to its potential teratogenic effects. To avoid this chemical hazard, it is of importance to introduce an alternative non-hazardous vital dye that is compatible with automated cell counters. Erythrosin B (EB) is a vital dye that is impermeable to biological membranes and is used as a food additive. Similarly to TB, EB stains only nonviable cells with disintegrated membranes. However, EB is less popular than TB and is seldom used with automated cell counters. We found that cell counting accuracy with EB was comparable to that with TB. EB was found to be an effective dye for accurate counting of cells with different viabilities across three different automated cell counters. In contrast to TB, EB was less toxic to cultured HL-60 cells during the cell counting process. These results indicate that replacing TB with EB for use with automated cell counters will significantly reduce the hazardous risk while producing comparable results.

The need for efficient high-throughput gene delivery system for mammalian cells is rapidly increasing with the growing request for functional genomics studies and drug discoveries in various physiologically relevant systems. However, plasmid-based gene delivery has limitations in transfection efficiency and available cell types. Viral vectors have great advantages over plasmid-based vectors, but construction of recombinant viruses remains to be a big hurdle for high-throughput applications. Here we demonstrate a rapid and simple high-throughput system for constructing recombinant adenoviruses which have been used as efficient gene delivery tools in mammalian systems in vitro and in vivo. By combining Gateway-based site-specific recombination with Terminal protein-coupled adenovirus vector, the adenovirus high-throughput system (AdHTS) generates multiple recombinant adenoviruses in 96-well plates simultaneously without the need for additional cloning or recombination in bacteria or mammalian cells. The AdHTS allows rapid and robust cloning and expression of genes in mammalian cells by removing shuttle vector construction, bacterial transformation, or selection and by minimizing effort in plaque isolation. By shortening the time required to convert whole cDNA library into desired viral vector constructs, the AdHTS would greatly facilitate functional genomics and proteomics studies in various mammalian systems.

Understanding the structural organization of organs and organisms at the cellular level is a fundamental challenge in biology. This task has been approached by reconstructing three-dimensional structure from images taken from serially sectioned tissues, which is not only labor-intensive and time-consuming but also error-prone. Recent advances in tissue clearing techniques allow visualization of cellular structures and neural networks inside of unsectioned whole tissues or the entire body. However, currently available protocols require long process times. Here, we present the rapid and highly reproducible ACT-PRESTO (active clarity technique-pressure related efficient and stable transfer of macromolecules into organs) method that clears tissues or the whole body within 1 day while preserving tissue architecture and protein-based signals derived from endogenous fluorescent proteins. Moreover, ACT-PRESTO is compatible with conventional immunolabeling methods and expedites antibody penetration into thick specimens by applying pressure. The speed and consistency of this method will allow high-content mapping and analysis of normal and pathological features in intact organs and bodies.

Understanding the structural organization of organs and organisms at the cellular level is a fundamental challenge in biology. This task has been approached by reconstructing three-dimensional structure from images taken from serially sectioned tissues, which is not only labor-intensive and time-consuming but also error-prone. Recent advances in tissue clearing techniques allow visualization of cellular structures and neural networks inside of unsectioned whole tissues or the entire body. However, currently available protocols require long process times. Here, we present the rapid and highly reproducible ACT-PRESTO (active clarity technique-pressure related efficient and stable transfer of macromolecules into organs) method that clears tissues or the whole body within 1 day while preserving tissue architecture and protein-based signals derived from endogenous fluorescent proteins. Moreover, ACT-PRESTO is compatible with conventional immunolabeling methods and expedites antibody penetration into thick specimens by applying pressure. The speed and consistency of this method will allow high-content mapping and analysis of normal and pathological features in intact organs and bodies.

Low transfection and expression of target genes has been a problem in gene delivery for clinical therapy. In this study, we present a method for enhancing the transfection efficiency of target genes by electroporation using carrier genes. To evaluate the transfection efficiency, we transfected HeLa cells with luciferase genes (pGL3-control) and then measured the luciferase activity. In our experiments, the prokaryotic expression vector pCR2.1 was used as a carrier gene in the electro-mediated gene delivery. The result shows that the luciferase gene can be effectively transferred into the cell membrane with the aid of carrier genes. In the presence of carrier genes, luciferase activities increased two- to three-fold compared with that in the absence of carrier genes. We also investigated the effect of the weight ratio of luciferase genes to carrier genes on transfection efficiency and found no significant relationship between them. Consequently, we believe that carrier genes are potentially beneficial for promoting transfection and expression of target genes in biological applications.

We developed a multi-channel electroporation microchip made of polydimethylsiloxane (PDMS) and glass for gene transfer in mammalian cells. This chip produces multiple electric field gradients in a single microchip by varying the lengths of the microchannels from 2 to 4 cm. Electric fields of 0.65, 0.57, 0.49, 0.41, and 0.33 kV/cm were simultaneously produced in a single chip when the voltage of 1.3 kV was applied. We transferred enhanced green fluorescent protein genes (pEGFP) into HEK-293 and CHO cells, which were cultured within the microchannels. The feasibility of our device was demonstrated because it was able to produce five different transfection rates and survival rates at different electric fields produced in a single microchip. This system is expected to optimize the experimental conditions in gene transfection research more easily and faster than conventional electroporation methods.

CLOCK and BMAL1 are bHLH-PAS-containing transcription factors that bind to E-box elements and are indispensable for expression of core circadian clock components such as the Per and Cry genes. A key step in expression is the heterodimerization of CLOCK and BMAL1 and their accumulation in the nucleus with an approximately 24-h periodicity. We show here that nucleocytoplasmic shuttling of BMAL1 is essential for transactivation and for degradation of the CLOCK/BMAL1 heterodimer. Using serial deletions and point mutants, we identified a functional nuclear localization signal and Crm1-dependent nuclear export signals in BMAL1. Transient-transfection experiments revealed that heterodimerization of CLOCK and BMAL1 accelerates their turnover, as well as E-box-dependent clock gene transcription. Moreover, in embryonic mouse fibroblasts, robust transcription of Per2 is tightly associated with massive degradation of the CLOCK/BMAL1 heterodimer. CRY proteins suppressed this process during the transcription-negative phase and led to nuclear accumulation of the CLOCK/BMAL1 heterodimer. Thus, these findings suggest that the decrease of BMAL1 abundance during the circadian cycle reflects robust transcriptional activation of clock genes rather than inhibition of BMAL1 synthesis.

Technologies to assess the molecular targets of biomolecules in living cells are lacking. We have developed a technology called magnetism-based interaction capture (MAGIC) that identifies molecular targets on the basis of induced movement of superparamagnetic nanoparticles inside living cells. Efficient intracellular uptake of superparamagnetic nanoparticles (coated with a small molecule of interest) was mediated by a transducible fusogenic peptide. These nanoprobes captured the small molecule's labeled target protein and were translocated in a direction specified by the magnetic field. Use of MAGIC in genome-wide expression screening identified multiple protein targets of a drug. MAGIC was also used to monitor signal-dependent modification and multiple interactions of proteins.

Anaplastic thyroid carcinomas are a highly aggressive and extremely lethal form of human cancer, but the biological characteristics related to their aggressive nature are not understood. Moreover, Gadd45 family proteins have been implicated in a variety of growth-regulatory mechanisms, including DNA replication and repair, G(2)/M checkpoint control, and apoptosis. In this study we found that Gadd45gamma RNA was present at significantly lower levels in anaplastic cancer cells, compared with normal primary cultured thyrocytes. In addition, the adenovirus-mediated reexpression of Gadd45gamma significantly inhibited the proliferation of anaplastic thyroid carcinoma cells, ARO, FRO, and NPA cells, which was attributed to apoptosis. Furthermore, the adenovirus-mediated delivery of Gadd45gamma gene in anaplastic thyroid cancer resulted in the inhibition of tumor growth in vivo. This in vitro and in vivo activity of the adenovirus-mediated transduction of CR6/Gadd45gamma, on anaplastic thyroid cancer cell growth suppression, was reminiscent of the effects of p53. This study demonstrates that the Gadd45gamma gene has potential use as a candidate gene for gene therapy in anaplastic thyroid cancer.

Background: 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. Results: 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 (R-2 = 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. Conclusions: 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.

The Gadd45γ (growth arrest and DNA damage-inducible) gene is activated transcriptionally by at least two kinds of agents: DNA damaging agent such as methyl methanesulfonate (MMS) and UV radiation, or cytokines such as interleukin (IL)-6, IL-2 and granulocyte colony-stimulating factor (G-CSF). To investigate the sequences and transcription factors involved in induction of Gadd45γ after treatment with IL-6, the human gene was cloned and sequenced. We found C/EBP (CCAAT/enhancer-binding protein) family proteins, major transcription factors in the IL-6 signal transduction pathway, could regulate the transcriptional activity of the Gadd45γ promoter. In addition, a noncanonical C/EBP-binding site within the Gadd45γ promoter where C/EBPβ and C/EBPδ could bind, was identified by electrophoretic mobility shift assay (EMSA) and reporter gene analysis. Furthermore, we found a coordinated expression profile between Gadd45γ mRNA and C/EBPs (β and δ) protein during the differentiation of M1 cells: the amount of Gadd45γ transcripts became maximal when both C/EBPβ and C/EBPδ levels were high, on day 1 of differentiation of M1 cells after treatment with IL-6. These findings suggest that mitotic growth arrest coupled to M1 cell differentiation is mediated by C/EBPs stimulation of growth arrest-associated genes such as Gadd45γ.

Gadd45 family genes encode nuclear acidic proteins composed of Gadd45, MyD118, and CR6. Sequence analysis showed that Gadd45 family proteins (Gadd45, MyD118, and CR6) contain LXXLL signature motifs considered necessary and sufficient for the binding of several coactivators to nuclear receptors. Interaction between Gadd45 or CR6 and RXR alpha was confirmed by a two-hybrid test in yeast. Results from a series of GST pulldown assays showed that these Gadd45 family proteins interact with several nuclear hormone receptors including RXR alpha, RAR alpha, ER alpha, PPAR alpha, PPAR beta, and PPAR gamma2 in vitro. Interaction between Gadd45 family proteins and nuclear hormone receptors resulted in modest activation of transactivating function of nuclear hormone receptors in reporter systems. When fused to DNA binding domain of GAL4, Gadd45 and CR6 activated the UAS-mediated transcription in mammalian cells. These results suggest that Gadd45 family proteins bind to nuclear hormone receptors and act as nuclear coactivators.

Electroporation is widely used to achieve gene transfection. A common problem in electroporation is that it has a lower viability than any other transfection method. In this study, we developed a novel electroporation device using a capillary tip and a pipette that was effective on a wide range of mammalian cells, including cell lines, primary cells, and stem cells. The capillary electroporation system considerably reduced cell death during electroporation because of its wire-type electrode, which has a small surface area. The experimental results also indicated that the cell viability was dependent on the change in pH induced by electrolysis during electroporation. Additionally, the use of a long and narrow capillary tube combined with simple pipetting shortened the overall time of the electroporation process by up to 15 min, even under different conditions with 24 samples. These results were supported by comparison with a conventional electroporation system. The transfection rate and the cell viability were enhanced by the use of the capillary system, which had a high transfection rate of more than 80% in general cell lines such as HeLa and COS-7, and more than 50% in hard-to-transfect cells such as stem or primary cells. The viability was approximately 70-80% in all cell types used in this study.

The Gadd45 family of proteins includes Gadd45alpha, MyD118/Gadd45beta, and CR6/OIG37/Gadd45gamma. These proteins play important roles in maintaining genomic stability and in regulating the cell cycle. This study reports the cloning of a novel protein called CR6-interacting factor 1 (CRIF1) which interacts with Gadd45alpha, MyD118/Gadd45beta, and CR6/OIG37/Gadd45gamma. CRIF1 binds specifically to the Gadd45 family proteins, as determined by an in vitro glutathione S-transferase pull-down assay and an in vivo mammalian cell two-hybrid assay along with coimmunoprecipitation assays. CRIF1 mRNA is highly expressed in the thyroid gland, heart, lymph nodes, trachea, and adrenal tissues. CRIF1 localizes exclusively to the nucleus and colocalizes with Gadd45gamma. Recombinant CRIF1 inhibits the histone H1 kinase activity of immunoprecipitated Cdc2-cyclin B1 and Cdk2-cyclin E, and the inhibitory effects were additive with Gadd45 proteins. Overexpression of CRIF1 increases the percentage of cells in G1, decreases the percentage of cells in S phase, and suppresses growth in NIH3T3 cells. The down-regulation of endogenous CRIF1 by the transfection of the small interfering RNA duplexes resulted in the inactivation of Rb by phosphorylation and decreased the G1 phase cell populations. Expression of CRIF1 is barely detectable in adrenal adenoma and papillary thyroid cancer and much lower than in adjacent normal tissue. The results presented here suggest that CRIF1 is a novel nuclear protein that interacts with Gadd45 and may play a role in negative regulation of cell cycle progression and cell growth.

Objective: The purpose of this study was to detect the expression of cyclin G1 in leiomyoma and to investigate the alteration of its expression compared with normal myometrial tissue that was obtained from the same patient. Study Design: With the use of Northern blot analysis, Western blot analysis, and immunohistochemistry, we analyzed the expression of cyclin G1 in 24 patients who underwent hysterectomies. Results: We found that messenger RNA levels of cyclin G1 were elevated in human leiomyomas compared with their adjacent normal myometrial tissues. Consistent with elevated messenger RNA levels, high levels of cyclin G1 protein expression were detected by immunoblot analysis in all leiomyoma samples. Immunohistochemistry revealed that cyclin G1 is located mainly in the nucleus in both normal myometrium and leiomyoma. However, higher levels of cyclin G1 were apparent in tumor regions compared with adjacent normal myometrial regions. In addition, we found the expression levels of other cyclins (A and E) and CDK2 were elevated in leiomyomas compared with normal myometrium. Because cyclin G1 is a transcriptional target of the p53 tumor suppressor, we examined the p53 status of all eight leiomyoma samples and found no p53 mutations. Conclusion: These results suggest that cyclin G1 is frequently overexpressed in uterine leiomyoma in a p53-independent manner and that this abnormality could be attributed to the severe proliferation of human uterine leiomyomas. (Am J Obstet Gynecol 2003;188:634-9.)