Initial development of a multistage cancer model based on Syrian hamster embryo (SHE) cell transformation studies

Department Head: V. A. Murphy.1999 Fall.Includes bibliographical references (pages 86-91).To better incorporate biologic information into quantitative cancer modeling, the two-stage MVK (Moolgavkar-Venzon-Knudson) model has been modified for use with SHE cell neoplastic progression. Conceptually, five phenotypic stages are included in this model: normal cells can either become senescent or mutate into immortal cells followed by anchorage-independent growth and tumorigenic stages. Cells in each stage have distinct division, death and mutation rates, and mutation is assumed to occur during cell division. Model development and related experiments were focused on studying the abilities of lead, arsenic, chromium, and a mixture of these three metals to induce progression of SHE cells from one phenotype to the next. Cell division and death rates were assessed using flow cytometric analysis for inclusion in the model. Cell division rates were measured using bromodeoxyuridine (BrdU) incorporation with propidium iodide staining, which allows for the calculation of potential doubling time, a measure of cell cycle time that takes growth fraction, but not cell loss, into account. Potential doubling times of normal SHE cells ranged from 12 to 59 hours, depending on the degree of confluence of cell cultures. Cell death was measured by a flow cytometry method based on propidium iodide staining specifically related to membrane damage. The mean cell death rate is approximately equal to 1 % of the average value of division rates. The individual metals and their mixture did not induce immortalization or further mutations of SHE cells in our laboratory following a 2-day exposure. However, the growth of SHE cells was inhibited by 5.4 μM of arsenic, with cells becoming senescent after only 16 population doublings; whereas, normal cells and cells exposed to lower arsenic concentrations lasted for at least 30 population doublings. The model developed in our laboratory successfully predicted the growth of normal cells. The cell senescence rates under the impact of arsenic exposure were also calculated. Mechanisms responsible for induction of cellular senescence in SHE cells exposed to arsenic may be involved in the apparent inability of arsenic to induce neoplasia in experimental animals

Mitochondrial targeting of human NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) and its association with early-onset hypertrophic cardiomyopathy and encephalopathy

<p>Abstract</p> <p>Background</p> <p>NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2), containing one iron sulfur cluster ([2Fe-2S] binuclear cluster N1a), is one of the core nuclear-encoded subunits existing in human mitochondrial complex I. Defects in this subunit have been associated with Parkinson's disease, Alzheimer's disease, Bipolar disorder, and Schizophrenia. The aim of this study is to examine the mitochondrial targeting of NDUFV2 and dissect the pathogenetic mechanism of one human deletion mutation present in patients with early-onset hypertrophic cardiomyopathy and encephalopathy.</p> <p>Methods</p> <p>A series of deletion and point-mutated constructs with the <it>c-myc </it>epitope tag were generated to identify the location and sequence features of mitochondrial targeting sequence for NDUFV2 in human cells using the confocal microscopy. In addition, various lengths of the NDUFV2 N-terminal and C-terminal fragments were fused with enhanced green fluorescent protein to investigate the minimal region required for correct mitochondrial import. Finally, a deletion construct that mimicked the IVS2+5_+8delGTAA mutation in <it>NDUFV2 </it>gene and would eventually produce a shortened NDUFV2 lacking 19-40 residues was generated to explore the connection between human gene mutation and disease.</p> <p>Results</p> <p>We identified that the cleavage site of NDUFV2 was located around amino acid 32 of the precursor protein, and the first 22 residues of NDUFV2 were enough to function as an efficient mitochondrial targeting sequence to carry the passenger protein into mitochondria. A site-directed mutagenesis study showed that none of the single-point mutations derived from basic, hydroxylated and hydrophobic residues in the NDUFV2 presequence had a significant effect on mitochondrial targeting, while increasing number of mutations in basic and hydrophobic residues gradually decreased the mitochondrial import efficacy of the protein. The deletion mutant mimicking the human early-onset hypertrophic cardiomyopathy and encephalopathy lacked 19-40 residues in NDUFV2 and exhibited a significant reduction in its mitochondrial targeting ability.</p> <p>Conclusions</p> <p>The mitochondrial targeting sequence of NDUFV2 is located at the N-terminus of the precursor protein. Maintaining a net positive charge and an amphiphilic structure with the overall balance and distribution of basic and hydrophobic amino acids in the N-terminus of NDUFV2 is important for mitochondrial targeting. The results of human disease cell model established that the impairment of mitochondrial localization of NDUFV2 as a mechanistic basis for early-onset hypertrophic cardiomyopathy and encephalopathy.</p

The Vertebrate Codex Gene Breaking Protein Trap Library For Genomic Discovery and Disease Modeling Applications

The zebrafish is a powerful model to explore the molecular genetics and expression of the vertebrate genome. The gene break transposon (GBT) is a unique insertional mutagen that reports the expression of the tagged member of the proteome while generating Cre-revertible genetic alleles. This 1000+ locus collection represents novel codex expression data from the illuminated mRFP protein trap, with 36% and 87% of the cloned lines showcasing to our knowledge the first described expression of these genes at day 2 and day 4 of development, respectively. Analyses of 183 molecularly characterized loci indicate a rich mix of genes involved in diverse cellular processes from cell signaling to DNA repair. The mutagenicity of the GBT cassette is very high as assessed using both forward and reverse genetic approaches. Sampling over 150 lines for visible phenotypes after 5dpf shows a similar rate of discovery of embryonic phenotypes as ENU and retroviral mutagenesis. Furthermore, five cloned insertions were in loci with previously described phenotypes; embryos homozygous for each of the corresponding GBT alleles displayed strong loss of function phenotypes comparable to published mutants using other mutagenesis strategies (ryr1b, fras1, tnnt2a, edar and hmcn1). Using molecular assessment after positional cloning, to date nearly all alleles cause at least a 99+% knockdown of the tagged gene. Interestingly, over 35% of the cloned loci represent 68 mutants in zebrafish orthologs of human disease loci, including nervous, cardiovascular, endocrine, digestive, musculoskeletal, immune and integument systems. The GBT protein trapping system enabled the construction of a comprehensive protein codex including novel expression annotation, identifying new functional roles of the vertebrate genome and generating a diverse collection of potential models of human disease

HIP-KNEE control for gait assistance with Powered Knee Orthosis

A Powered Knee Orthosis (PKO) was developed for the elderly and patients with disordered gait to regain normal walking. In order to enhance the PKO performance and reduce system complexity especially for people with muscle weakness in their knee joints, an algorithm named HIP-KNEE control is proposed. This algorithm is based on the analysis of kinematic gait model, and the desired knee joint angle (KNEE) is estimated from the measurements of hip joint angle (HIP). The relationship between HIP and KNEE is modeled as a polynomial, which can be easily implemented to an embedded controller for real-time control. This control method is suitable to subjects with good function in hip joint, and it can provide help in walking without special training. An Inertia Measurement Units (IMU) is used for obtaining HIP input, and integrated with a footswitch for checking the heel condition; the gait assistance performance can be further improved

Experimental studies on kinematics and kinetics of walking with an assistive knee brace

Assistive knee brace is a species of wearable lower extremity exoskeletons. Such assistive equipment can enhance people's strength and provide desired locomotion to have advantages over wheelchairs, which are commonly used for patients with mobility disorders. However, the integration between the assistive knee brace and the user is challenging as inaccurate alignments may adversely affect the biomechanics of the knee joint. The goal of this study is to evaluate the changes between normal walking and walking with an assistive knee brace in "off" mode. The assistive knee brace was developed by integrating a multifunctional actuator with a custom-made knee-ankle-foot orthosis in order to minimize excessive shifting and to improve alignment to the knee joint. Spatial and temporal gait parameters, joint kinematics and joint kinetics parameters were compared. In general, the observed results showed that most of the gait parameters were not affected when walking with the knee brace. The only significant differences were found in knee flexion and knee rotational motions. These results indicated that walking with the developed knee brace provided minimal hindrance to the user and assured that assistive torque can be applied to the knee joint

Gait analysis for designing a new assistive knee brace

Assistive knee brace is a species of wearable lower extremity exoskeletons. In this research, an assistive knee brace was developed by integrating a multifunctional actuator with a custom-made knee-ankle-foot orthosis. In the study, the location of the actuator is moved up to the lateral side of the hip, instead of knee joint. Waist belt and shoulder belt are appended on the knee brace. This paper aimed to improve the design of the assistive knee braces through gait analysis. By walking with the knee braces, the spatial and temporal gait parameters, joint kinematics and joint kinetics parameters were evaluated, and the changes from normal walking were compared as well. The experimental results showed that walking with the developed knee brace provided minimal hindrance to the wearer. © 2011 IEEE

Genetic Polymorphism of XRCC1 Arg399Gln Is Associated With Survival in Non–Small-Cell Lung Cancer Patients Treated With Gemcitabine/Platinum

IntroductionElevated DNA-repair capacity has been related to chemoresistance of platinum doublet chemotherapy in non–small-cell lung cancer (NSCLC). We evaluated whether single nucleotide polymorphisms of DN- repair genes excision repair cross-complementing group 1 (ERCC1), ERCC2, x-ray repair cross-complementing group 1 (XRCC1), XRCC3, and RRM1 associate with treatment outcome in NSCLC patients receiving gemcitabine plus platinum as their first-line chemotherapy.MethodsGenotyping for eight polymorphisms in five DNA-repair genes was performed with the GenomeLab nucleotide polymorphismstream Genotyping System in 62 advanced NSCLC patients in a training set and 45 patients in a validation set treated with gemcitabine/platinum.ResultsIn the training set, the wild-type genotype of XRCC1 Arg399Gln (G/G) was associated with decreased median overall survival (OS) (22 months, 95% confidence interval [CI], 10–34 months versus not reached, log-rank test, p = 0.005) than those carrying variant genotypes (G/A+A/A). In addition, there was a statistically significant longer median OS in patients carrying wild-type ERCC2 Asp312Asn genotype (G/G) (51 months, 95% CI, 19–82 months versus 10 months, log-rank test, p < 0.001) than those carrying heterozygous variant genotypes (G/A). In the multivariate Cox model, we found a significant effect of XRCC1 Arg399Gln (G/A+A/A versus G/G, hazard ratio [HR] 0.290; 95%CI, 0.12–0.705, p = 0.006) and ERCC2 Asp312Asn (G/A versus G/G, HR 14.04; 95% CI, 2.253–87.513, p = 0.005) polymorphisms on patients’ OS. In the validation set, only XRCC1 399ConclusionsGenetic polymorphism of XRCC1 Arg399Gln may be a candidate for contributing interindividual difference in the OS of gemcitabine/platinum-treated advanced NSCLC patients

Tol2 Gene Trap Integrations in the Zebrafish Amyloid Precursor Protein Genes appa and aplp2 Reveal Accumulation of Secreted APP at the Embryonic Veins

Background—The single spanning transmembrane amyloid precursor protein (APP) and its proteolytic product, amyloid-beta (Aβ) peptide, have been intensely studied due to their role in the pathogenesis of Alzheimer’s disease. However, the biological role of the secreted ectodomain of APP, which is also generated by proteolytic cleavage, is less well understood. Here, we report Tol2 red fluorescent protein (RFP) transposon gene trap integrations in the zebrafish amyloid precursor protein a (appa) and amyloid precursor-like protein 2 (aplp2) genes. The transposon integrations are predicted to disrupt the appa and aplp2 genes to primarily produce secreted ectodomains of the corresponding proteins that are fused to RFP. Results—Our results indicate the Appa-RFP and Aplp2 fusion proteins are likely secreted from the central nervous system and accumulate in the embryonic veins independent of blood flow. Conclusions—The zebrafish appa and aplp2 transposon insertion alleles will be useful for investigating the biological role of the secreted form of APP