title: Gene expression profiles of 3D gene-edited bioprinted cells

authors: Turnbull, Catherine

subjects: Bioprinting

abstract: Cancer is the biggest cause of death in New Zealand each year and places a large economic strain on the health system. Previously, two-dimensional (2D) cell models have provided insight into the development of cancer but now, three-dimensional (3D) models provided more accurate information on how cell-cell interactions in the body affect the efficacy of different drug therapies. This research aims to investigate the gene expression profiles of 3D gene-edited bioprinted lung cancer cells to understand how mutations and the vital trace element Selenium (Se) affects gene expression. The human A549 lung cancer cell line was bioprinted into a set dome shape made of sodium alginate, gelatine, and cell media. The cells were then cultured for 21 days before a six-hour drug exposure with 2μM and 10μM Methylselenic Acid (MSA).
RNA was also extracted from 18 bioprints and the spectrophotometer data suggested high quality with a total RNA concentration range of 9 – 671μg/μL, respectively. The RNA integrity was further examined using the RNA tape station. The solvent control (n=2) and the 2μM MSA treatment (n=3) from the second independent experiment resulted in intact ribosomal bands. The protein concentration was determined using a Bradford assay and ranged from 0.1 – 3.42mg/mL. The electrophoresed protein showed a molecular weight size range from 120kDa – 10kDa. The protein was then transferred onto a western blot membrane and detected by two primary antibodies; (1) HSP70 - correctly fold proteins that are under cellular stress and (2) GAPDH – a loading control reference antibody. The HSP70 protein expression was measured for the drug-treated bioprints for three bioprinting experiment. We hypothesised an upregulation in HSP70 expression based on previous studies. However, the data set could not be replicated to conclusively downregulation in response to 2 and 10μM MSA. Thus, further optimisation into drug accessibility, cell and protein loading concentration is required.
Gene-editing using lipofectamine and CRISPR-Cas9 was attempted using A549 cells but no conclusive evidence that a mutation had been introduced into the Cyclin Dependent Kinase 4 (CDK4) gene was observed (n=5). Three research optimisations have been recommended for gene-editing and establishing a stable gene-edited cell line for future use in 3D bioprinting and gene expression analysis.

text: Gene expression profiles of 3D gene-edited bioprinted cells Cancer is the biggest cause of death in New Zealand each year and places a large economic strain on the health system. Previously, two-dimensional (2D) cell models have provided insight into the development of cancer but now, three-dimensional (3D) models provided more accurate information on how cell-cell interactions in the body affect the efficacy of different drug therapies. This research aims to investigate the gene expression profiles of 3D gene-edited bioprinted lung cancer cells to understand how mutations and the vital trace element Selenium (Se) affects gene expression. The human A549 lung cancer cell line was bioprinted into a set dome shape made of sodium alginate, gelatine, and cell media. The cells were then cultured for 21 days before a six-hour drug exposure with 2μM and 10μM Methylselenic Acid (MSA).
RNA was also extracted from 18 bioprints and the spectrophotometer data suggested high quality with a total RNA concentration range of 9 – 671μg/μL, respectively. The RNA integrity was further examined using the RNA tape station. The solvent control (n=2) and the 2μM MSA treatment (n=3) from the second independent experiment resulted in intact ribosomal bands. The protein concentration was determined using a Bradford assay and ranged from 0.1 – 3.42mg/mL. The electrophoresed protein showed a molecular weight size range from 120kDa – 10kDa. The protein was then transferred onto a western blot membrane and detected by two primary antibodies; (1) HSP70 - correctly fold proteins that are under cellular stress and (2) GAPDH – a loading control reference antibody. The HSP70 protein expression was measured for the drug-treated bioprints for three bioprinting experiment. We hypothesised an upregulation in HSP70 expression based on previous studies. However, the data set could not be replicated to conclusively downregulation in response to 2 and 10μM MSA. Thus, further optimisation into drug accessibility, cell and protein loading concentration is required.
Gene-editing using lipofectamine and CRISPR-Cas9 was attempted using A549 cells but no conclusive evidence that a mutation had been introduced into the Cyclin Dependent Kinase 4 (CDK4) gene was observed (n=5). Three research optimisations have been recommended for gene-editing and establishing a stable gene-edited cell line for future use in 3D bioprinting and gene expression analysis.

year: 2023

title: Scheduling contractors' farm-to-farm crop harvesting operations

authors: Basnet, Chuda

subjects: harvesting

abstract: The harvesting of renewable resources from an operations scheduling viewpoint is introduced, and a harvesting scenario arising in the agricultural context involving a commercial contracting enterprise that travels from farm-to-farm harvesting crops is discussed. This paper is an extension of previous work by two of the authors from the one-farm to the multi-farm case. In both cases, the duration of each operation is dependent upon the combination of constrained resources allocated to it, equipment and worker allocation is restricted, and minimum or maximum time lags on the start and completion of operations may be imposed. The present case incorporates harvesting at more than one farm and thus the sequence in which the farms are visited and the inter-farm travel times must be taken into account. We report on a harvesting scheduling model and solution procedures designed specifically for large-scale versions of the multi-farm case. The computational times experienced in solving general instances of the model of small-to-medium practical size by a commercial integer programming package are encouraging. Greedy and tabu search heuristics, which are capable of solving problems of relatively large dimensions in reasonable computational time are also included. The authors believe that the model and the solution techniques developed represent a useful addition to the farm crop contractor's tool kit.

text: Scheduling contractors' farm-to-farm crop harvesting operations The harvesting of renewable resources from an operations scheduling viewpoint is introduced, and a harvesting scenario arising in the agricultural context involving a commercial contracting enterprise that travels from farm-to-farm harvesting crops is discussed. This paper is an extension of previous work by two of the authors from the one-farm to the multi-farm case. In both cases, the duration of each operation is dependent upon the combination of constrained resources allocated to it, equipment and worker allocation is restricted, and minimum or maximum time lags on the start and completion of operations may be imposed. The present case incorporates harvesting at more than one farm and thus the sequence in which the farms are visited and the inter-farm travel times must be taken into account. We report on a harvesting scheduling model and solution procedures designed specifically for large-scale versions of the multi-farm case. The computational times experienced in solving general instances of the model of small-to-medium practical size by a commercial integer programming package are encouraging. Greedy and tabu search heuristics, which are capable of solving problems of relatively large dimensions in reasonable computational time are also included. The authors believe that the model and the solution techniques developed represent a useful addition to the farm crop contractor's tool kit.

year: 2006

title: Curriculum integration as treaty praxis.

authors: Fraser, Deborah

subjects: curricula

abstract: The article discusses the significance of curriculum integration on the promotion of principles of the Treaty of Waitangi, an agreement signed in 1840 by the Maori and the Crown people in New Zealand. It explores the challenges linked with this curriculum theory design. It also highlights a case study which analyzes the impact of curriculum integration in education.

text: Curriculum integration as treaty praxis. The article discusses the significance of curriculum integration on the promotion of principles of the Treaty of Waitangi, an agreement signed in 1840 by the Maori and the Crown people in New Zealand. It explores the challenges linked with this curriculum theory design. It also highlights a case study which analyzes the impact of curriculum integration in education.

year: 2002