Crystallization of \u3ci\u3eChlorella\u3c/i\u3e deoxyuridine triphosphatase

Deoxyuridine triphosphatase (dUTPase) is a ubiquitous enzyme that has been widely studied owing to its function and evolutionary significance. The gene coding for the dUTPase from the Chlorella alga was codon-optimized and synthesized. The synthetic gene was expressed in Escherichia coli and recombinant core Chlorella dUTPase (chdUTPase) was purified. Crystallization of chdUTPase was performed by the repetitive hanging-drop vapor-diffusion method at 298 K with ammonium sulfate as the precipitant. In the presence of 2\u27-deoxyuridine-5\u27-[(α,β)-imido]triphosphate and magnesium, the enzyme produced die-shaped hexagonal R3 crystals with unit-cell parameters a = b = 66.9, c = 93.6 A, ƴ = 120°. X-ray diffraction data for chdUTPase were collected to 1.6 A resolution. The crystallization of chdUTPase with manganese resulted in very fragile clusters of needles

Effect of Extracellular Matrix (ECM) Protein Micropatterns on the Behavior of Human Neuroblastoma Cells

Recent advances in patterning techniques and emerging surface microtechnologies have allowed cell micropatterning to control spatial location of the cells on a surface as well as cell shape, attachment area, and number of contacting neighbor cells. These parameters play important roles in cell cellular behaviors. Cell micropatterning has thus become one of the most important strategies for biomedical applications, such as, tissue engineering, diagnostic immunoassays, lab-on-chip devices, bio-sensing, etc., and cell biology studies as well. For neuronal cells, there have been attempts to distribute neuronal cells on specific patterns to control cell-to-cell interaction. However, there have been very limited understanding on the effects of micropattern size and specific ECM proteins used for patterning on neuronal cell behavior. In this work, in-vitro neuronal cell patterning was performed using various ECM protein lane widths with or without neuronal biochemical factor to investigate neuronal cell response to controlled microenvironments. We have developed a technique to organize cells into pre-assigned boundaries while maintaining their original properties of growth, proliferation, and differentiation. Soft lithography and microcontact printing were used to pattern arrays of Collagen-I ECM lanes with 5, 10, 20, 30, and 40 µm width separated by 50 µm gap. PLL-g-PEG was flooded before culturing SH-SY5Y cells. Cells did not prefer small adhesive lanes, but attached on as small as 5µm ECM lanes if cell-repellent backfilling was utilized. On 5 and 10 µm lanes, cell and nuclear growth was constrained as compared to unpatterned control, and wider lanes. Cells showed near perfect orientation along the adhesive lanes for 5 and 10 µm width lanes. With increasing lane width, neuronal cell orientation was influenced adversely, e.g., increased deviation from the patterning direction. When stimulated by retinoic acid (RA), cells patterned on 5 and 10 µm lanes showed well-developed, long neurites along the protein pattern connecting neighboring cells. The neurite length was shorter on wider lanes. Our data may provide a new insight for neuronal tissue engineering on how to regenerate damaged neurons using more controlled physical-biochemical environments. Adviser: Jung Yul Li

IMPACT OF COMMUNITY FORESTRY ON BIODIVERSITY CONSERVATION IN NEPAL

Master'sMASTER OF SCIENCE (ENVIRONMENTAL MANAGEMENT) (MEM

Functional Maintenance of Differentiated Embryoid Bodies in Microfluidic Systems: A Platform for Personalized Medicine

Hormone replacement therapies have become important for treating diseases such as premature ovarian failure or menopausal complications. The clinical use of bioidentical hormones might significantly reduce some of the potential risks reportedly associated with the use of synthetic hormones. In the present study, we demonstrate the utility and advantage of a microfluidic chip culture system to enhance the development of personalized, on-demand, treatment modules using embryoid bodies (EBs). Functional EBs cultured on microfluidic chips represent a platform for personalized, patient-specific treatment cassettes that can be cryopreserved until required for treatment. We assessed the viability, differentiation, and functionality of EBs cultured and cryopreserved in this system. During extended microfluidic culture, estradiol, progesterone, testosterone, and anti-mullerian hormone levels were measured, and the expression of differentiated steroidogenic cells was confirmed by immunocytochemistry assay for the ovarian tissue markers anti-mullerian hormone receptor type II, follicle-stimulating hormone receptor, and inhibin p-A and the estrogen biosynthesis enzyme aromatase. Our studies showed that under microfluidic conditions, differentiated steroidogenic EBs continued to secrete estradiol and progesterone at physiologically relevant concentrations (30-120 pg/ml and 150-450 pg/ml, respectively) for up to 21 days. Collectively, we have demonstrated for the first time the feasibility of using a microfluidic chip system with continuous flow for the differentiation and extended culture of functional steroidogenic stem cell-derived EBs, the differentiation of EBs into cells expressing ovarian antigens in a microfluidic system, and the ability to cryopreserve this system with restoration of growth and functionality on thawing. These results present a platform for the development of a new therapeutic system for personalized medicine

Functional Maintenance of Differentiated Embryoid Bodies in Microfluidic Systems: A Platform for Personalized Medicine

Hormone replacement therapies have become important for treating diseases such as premature ovarian failure or menopausal complications. The clinical use of bioidentical hormones might significantly reduce some of the potential risks reportedly associated with the use of synthetic hormones. In the present study, we demonstrate the utility and advantage of a microfluidic chip culture system to enhance the development of personalized, on-demand, treatment modules using embryoid bodies (EBs). Functional EBs cultured on microfluidic chips represent a platform for personalized, patient-specific treatment cassettes that can be cryopreserved until required for treatment. We assessed the viability, differentiation, and functionality of EBs cultured and cryopreserved in this system. During extended microfluidic culture, estradiol, progesterone, testosterone, and anti-müllerian hormone levels were measured, and the expression of differentiated steroidogenic cells was confirmed by immunocytochemistry assay for the ovarian tissue markers anti-müllerian hormone receptor type II, follicle-stimulating hormone receptor, and inhibin β-A and the estrogen biosynthesis enzyme aromatase. Our studies showed that under microfluidic conditions, differentiated steroidogenic EBs continued to secrete estradiol and progesterone at physiologically relevant concentrations (30–120 pg/ml and 150–450 pg/ml, respectively) for up to 21 days. Collectively, we have demonstrated for the first time the feasibility of using a microfluidic chip system with continuous flow for the differentiation and extended culture of functional steroidogenic stem cell-derived EBs, the differentiation of EBs into cells expressing ovarian antigens in a microfluidic system, and the ability to cryopreserve this system with restoration of growth and functionality on thawing. These results present a platform for the development of a new therapeutic system for personalized medicine