Animal Technology Institute Taiwan

Miaoli, Taiwan

Animal Technology Institute Taiwan

Miaoli, Taiwan
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Chang L.-C.,National Defense Medical Center | Yang C.-Y.,Animal Technology Institute Taiwan | Chua A.C.N.,National Defense Medical Center | Lin Y.-J.,Animal Technology Institute Taiwan | Lai S.-M.,National Ilan University
Molecular Pharmaceutics | Year: 2011

The current regimen of factor IX (FIX) injection is of an episodic format, which leads to limited efficacy. A sustained release dosage form is beneficial in terms of reducing the injection frequency and improving the therapeutic effectiveness. The aim of this study was to formulate a new microsphere form of a FIX-containing preparation to diminish these shortcomings. Using the water-in-oil-in-water (W/O/W) double emulsion technique, injectable long-acting FIX microspheres were prepared with transgenic recombinant human FIX (rhFIX) and poly(lactic-co-glycolic acid) (PLGA) polymer. The rhFIX microspheres prepared had diameters ranging between 25-350 μm and easily passed through a small-gauge-number needle for subcutaneous injection. In in vitro release testing, the microspheres had a sustained release profile featuring an initial burst and sustained release spanning a 5-day period. In in vivo pharmacodynamic testing, normalization of the bleeding of hemophilic mice was maintained for 5 days with microsphere injection as compared with 2 days with native rhFIX. Taken together, these results indicated that long-acting FIX microspheres were successfully prepared for potential use in hemophilic prophylaxis. © 2011 American Chemical Society.


Chang K.C.,Chung Yuan Christian University | Chang K.C.,Animal Technology Institute Taiwan | Su J.J.,National Taiwan University of Science and Technology | Cheng C.,Chung Yuan Christian University
Food Chemistry | Year: 2013

An online sampling and matrix reduction technique coupled liquid chromatography electrospray-iontrap mass spectrometry was developed for rapid analysis of maduramicin (MAD) residue in chicken meat. Multiple-reaction monitoring of mass spectrometry in positive ion mode was used to detect maduramicin. A post-column continuous infusion of internal standard (nigericin) with matrix-matched calibration method was utilised for quantification. The linear concentration range of the calibration curve was 0- 10.0 ng mL -1 (r2 = 0.999). The limit of detection (quantification) was 0.08 ng g-1 (0.28 ng g-1). The analytical accuracy of chicken meat samples for four spiked MAD concentrations (0.5, 1.0, 5.0, and 10.0 ng g-1) was 84-97% and their corresponding intra-day and inter-day precisions were 3.7-5.0% and 5.8-7.9%, respectively. The analysis time for one sample was 10 min. The application of the method for incurred chicken samples elucidates that MAD residue in chicken meat decreases during the withdrawal period. © 2013 Elsevier Ltd.


Patent
Stresspro Biomedicine Incorporation and Animal Technology Institute Taiwan | Date: 2010-02-17

An expression system for a recombinant target protein in a host cell, comprising a nucleic acid segment consisting essentially of a nucleotide sequence coding for a protein transduction domain of SEQ ID No: 1, a nucleotide sequence coding for a Hsp40-J domain, and a nucleotide sequence coding for a target protein, wherein the nucleic acid segment is operatively linked to host specific transcription and translation regulatory elements for expressing the recombinant target protein in the host cell is provided. The invention also provides a method for producing a recombinant target protein with an enhanced yield in the host cell.


Hsu P.-C.,National Tsing Hua University | Yang C.-Y.,Animal Technology Institute Taiwan | Lan C.-Y.,National Tsing Hua University
Eukaryotic Cell | Year: 2011

Candida albicans is an opportunistic fungal pathogen that exists as normal flora in healthy human bodies but causes life-threatening infections in immunocompromised patients. In addition to innate and adaptive immunities, hosts also resist microbial infections by developing a mechanism of "natural resistance" that maintains a low level of free iron to restrict the growth of invading pathogens. C. albicans must overcome this irondeprived environment to cause infections. There are three types of iron-responsive transcriptional regulators in fungi; Aft1/Aft2 activators in yeast, GATA-type repressors in many fungi, and HapX/Php4 in Schizosaccharomyces pombe and Aspergillus species. In this study, we characterized the iron-responsive regulator Hap43, which is the C. albicans homolog of HapX/Php4 and is repressed by the GATA-type repressor Sfu1 under iron-sufficient conditions. We provide evidence that Hap43 is essential for the growth of C. albicans under low-iron conditions and for C. albicans virulence in a mouse model of infection. Hap43 was not required for iron acquisition under low-iron conditions. Instead, it was responsible for repression of genes that encode irondependent proteins involved in mitochondrial respiration and iron-sulfur cluster assembly. We also demonstrated that Hap43 executes its function by becoming a transcriptional repressor and accumulating in the nucleus in response to iron deprivation. Finally, we found a connection between Hap43 and the global corepressor Tup1 in low-iron-induced flavinogenesis. Taken together, our data suggest a complex interplay among Hap43, Sfu1, and Tup1 to coordinately regulate iron acquisition, iron utilization, and other ironresponsive metabolic activities. © 2011, American Society for Microbiology. All Rights Reserved.


Tsai P.-W.,National Tsing Hua University | Yang C.-Y.,Animal Technology Institute Taiwan | Chang H.-T.,China Medical University at Taichung | Lan C.-Y.,National Tsing Hua University
PLoS ONE | Year: 2011

Candida albicans is the major fungal pathogen of humans. Its adhesion to host-cell surfaces is the first critical step during mucosal infection. Antimicrobial peptides play important roles in the first line of mucosal immunity against C. albicans infection. LL-37 is the only member of the human cathelicidin antimicrobial peptide family and is commonly expressed in various tissues, including epithelium. We previously showed that LL-37 significantly reduced C. albicans adhesion to plastic, oral epidermoid OECM-1 cells, and urinary bladders of female BALB/c mice. The inhibitory effect of LL-37 on cell adhesion occurred via the binding of LL-37 to cell-wall carbohydrates. Here we showed that formation of LL-37-cell-wall protein complexes potentially inhibits C. albicans adhesion to polystyrene. Using phage display and ELISA, we identified 10 peptide sequences that could bind LL-37. A BLAST search revealed that four sequences in the major C. albicans cell-wall β-1,3-exoglucanase, Xog1p, were highly similar to the consensus sequence derived from the 10 biopanned peptides. One Xog1p-derived peptide, Xog1p90-115, and recombinant Xog1p associated with LL-37, thereby reversing the inhibitory effect of LL-37 on C. albicans adhesion. LL-37 reduced Xog1p activity and thus interrupted cell-wall remodeling. Moreover, deletion of XOG1 or another β-1,3-exoglucanase-encoding gene EXG2 showed that only when XOG1 was deleted did cellular exoglucanase activity, cell adhesion and LL-37 binding decrease. Antibodies against Xog1p also decreased cell adhesion. These data reveal that Xog1p, originally identified from LL-37 binding, has a role in C. albicans adhesion to polystyrene and, by inference, attach to host cells via direct or indirect manners. Compounds that target Xog1p might find use as drugs that prevent C. albicans infection. Additionally, LL-37 could potentially be used to screen for other cell-wall components involved in fungal cell adhesion. © 2011 Tsai et al.


Tsai P.-W.,National Tsing Hua University | Yang C.-Y.,Animal Technology Institute Taiwan | Chang H.-T.,China Medical University at Taichung | Lan C.-Y.,National Tsing Hua University
PLoS ONE | Year: 2011

Candida albicans is the major fungal pathogen of humans. Fungal adhesion to host cells is the first step of mucosal infiltration. Antimicrobial peptides play important roles in the initial mucosal defense against C. albicans infection. LL-37 is the only member of the human cathelicidin family of antimicrobial peptides and is commonly expressed in various tissues and cells, including epithelial cells of both the oral cavity and urogenital tract. We found that, at sufficiently low concentrations that do not kill the fungus, LL-37 was still able to reduce C. albicans infectivity by inhibiting C. albicans adhesion to plastic surfaces, oral epidermoid OECM-1 cells, and urinary bladders of female BALB/c mice. Moreover, LL-37-treated C. albicans floating cells that did not adhere to the underlying substratum aggregated as a consequence of LL-37 bound to the cell surfaces. According to the results of a competition assay, the inhibitory effects of LL-37 on cell adhesion and aggregation were mediated by its preferential binding to mannan, the main component of the C. albicans cell wall, and partially by its ability to bind chitin or glucan, which underlie the mannan layer. Therefore, targeting of cell-wall carbohydrates by LL-37 provides a new strategy to prevent C. albicans infection, and LL-37 is a useful, new tool to screen for other C. albicans components involved in adhesion. © 2011 Tsai et al.


Sun Y.-L.,Animal Technology Institute Taiwan | Chang Y.-S.,Animal Technology Institute Taiwan | Lin Y.-S.,Animal Technology Institute Taiwan | Yen C.-H.,Animal Technology Institute Taiwan
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2012

Valuable pharmaceutical proteins produced from the mammary glands of transgenic livestock have potential use in the biomedical industry. In this study, recombinant human clotting factor IX (rhFIX) produced from transgenic sow milk for preclinical animal studies have been established. The transgenic sow milk was skimmed and treated with sodium phosphate buffer to remove abundant casein protein. Then, the γ-carboxylated rhFIX fraction was segregated through the Q Sepharose chromatography from uncarboxylated one. For safety issue, the process included virus inactivation by solvent/detergent (S/D) treatment. Subsequently, the S/D treated sample was loaded into the Heparin Sepharose column to recover the rhFIX fraction, which was then reapplied to the Heparin Sepharose column to enhance rhFIX purity and lower the ratio of activated form rhFIX (rhFIXa) easily. This was possible due to the higher affinity of the Heparin affinity sorbent for rhFIXa than for the rhFIX zymogen. Furthermore, an IgA removal column was used to eliminate porcine IgA in purified rhFIX. Finally, nanofiltration was performed for viral clearance. Consequently, a high-quality rhFIX product was produced (approximately 700. mg per batch). Other values for final rhFIX preparation were as follows: purity, >99%; average specific activity, 415.6 ± 57.7. IU/mL and total milk impurity, <0.5. ng/mg. This is the first report that described the whole process and stable production of bioactive rhFIX from transgenic sow milk. The overall manufacturing process presented here has the potential for industrial production of rhFIX for treatment of hemophilia B patients. © 2012 Elsevier B.V.


Lee K.-H.,Animal Technology Institute Taiwan
Methods in Molecular Biology | Year: 2014

The generation of a fertile embryonic stem cell (ESC)-derived or F0 (100 % coat color chimerism) mice is the final criterion in proving that the ESC is truly pluripotent. Many methods have been developed to produce chimeric mice. To date, the most popular methods for generating chimeric embryos is well sandwich aggregation between zona pellucida (ZP) removed (denuded) 2.5-day post-coitum (dpc) embryos and ESC clumps, or direct microinjection of ESCs into the cavity (blastocoel) of 3.5-dpc blastocysts. However, due to systemic limitations and the disadvantages of conventional microinjection, aggregation, and coculturing, two novel methods (vial coculturing and hypertonic microinjection) were developed in recent years at my laboratory. Coculturing 2.5-dpc denuded embryos with ESCs in 1.7-mL vials for ~3 h generates chimeras that have significantly high levels of chimerism (including 100 % coat color chimerism) and germline transmission. This method has significantly fewer instrumental and technological limitations than existing methods, and is an efficient, simple, inexpensive, and reproducible method for "mass production" of chimeric embryos. For laboratories without a microinjection system, this is the method of choice for generating chimeric embryos. Microinjecting ESCs into a subzonal space of 2.5-dpc embryos can generate germline-transmitted chimeras including 100 % coat color chimerism. However, this method is adopted rarely due to the very small and tight space between ZP and blastomeres. Using a laser pulse or Piezo-driven instrument/device to help introduce ESCs into the subzonal space of 2.5-dpc embryos demonstrates the superior efficiency in generating ESC-derived (F0) chimeras. Unfortunately, due to the need for an expensive instrument/device and extra fine skill, not many studies have used either method. Recently, ESCs injected into the large subzonal space of 2.5-dpc embryos in an injection medium containing 0.2-0.3 M sucrose very efficiently generated viable, healthy, and fertile chimeric mice with 100 % coat color chimerism. Both vial coculture and hypertonic microinjection methods are useful and effective alternatives for producing germline chimeric or F0 mice efficiently and reliably. Furthermore, both novel methods are also good for induced pluripotent stem cells (iPSCs) to generate chimeric embryos. © Springer Science+Business Media New York 2014.


Patent
Animal Technology Institute Taiwan | Date: 2012-07-20

The present invention provides a pharmaceutical preparation in powder-like form, comprising a therapeutically effective amount of a human Factor IX (hFIX) encapsulated by a lipophilic biodegradable polymer or copolymer to form a microsphere, whereby the pharmaceutical preparation provides a sustained release of hFIX and a prolonged biological activity.


Patent
Animal Technology Institute Taiwan | Date: 2011-08-10

The present invention provides a pharmaceutical preparation in powder-like form comprising a therapeutically effective amount of human Factor IX (hFIX) encapsulated by a lipophilic biodegradable polymer or copolymer to form a microsphere, whereby the pharmaceutical preparation provides a sustained release of hFIX and a prolonged biological activity.

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