Preparation of Mouse Monoclonal Antibody for RB1CC1 and Its Clinical Application

RB1-inducible coiled-coil 1 (RB1CC1; also known as FIP200) plays important roles in several biological pathways such as cell proliferation and autophagy. Evaluation of RB1CC1 expression can provide useful clinical information on various cancers and neurodegenerative diseases. In order to realize the clinical applications, it is necessary to establish a stable supply of antibody and reproducible procedures for the laboratory examinations. In the present study, we have generated mouse monoclonal antibodies for RB1CC1, and four kinds of antibodies (N1-8, N1-216, N3-2, and N3-42) were found to be optimal for clinical applications such as ELISA and immunoblots and work as well as the pre-existing polyclonal antibodies. N1-8 monoclonal antibody provided the best recognition of RB1CC1 in the clinico-pathological examination of formalin-fixed paraffin-embedded tissues. These monoclonal antibodies will help to generate new opportunities in scientific examinations in biology and clinical medicine

Feeder Cells Support the Culture of Induced Pluripotent Stem Cells Even after Chemical Fixation

Chemically fixed mouse embryonic fibroblasts (MEFs), instead of live feeder cells, were applied to the maintenance of mouse induced pluripotent stem (miPS) cells. Formaldehyde and glutaraldehyde were used for chemical fixation. The chemically fixed MEF feeders maintained the pluripotency of miPS cells, as well as their undifferentiated state. Furthermore, the chemically fixed MEF feeders were reused several times without affecting their functions. These results indicate that chemical fixation can be applied to modify biological feeders chemically, without losing their original functions. Chemically fixed MEF feeders will be applicable to other stem cell cultures as a reusable extracellular matrix candidate that can be preserved on a long-term basis

Screening for Microsatellite Instability Identifies Frequent 3′-Untranslated Region Mutation of the RB1-Inducible Coiled-Coil 1 Gene in Colon Tumors

BACKGROUND: Coding region microsatellite instability (MSI) results in loss of gene products and promotion of microsatellite-unstable (MSI-H) carcinogenesis. Recent studies have indicated that MSI within 3'-untranslated regions (3'UTRs) may post-transcriptionally dysregulate gene products. Within this context, we conducted a broad mutational survey of 42 short 3'UTR microsatellites (MSs) in 45 MSI-H colorectal tumors and their corresponding normal colonic mucosae. METHODOLOGY/PRINCIPAL FINDINGS: In order to estimate the overall susceptibility of MSs to MSI in MSI-H tumors, the observed MSI frequency of each MS was correlated with its length, interspecies sequence conservation level, and distance from some genetic elements (i.e., stop codon, polyA signal, and microRNA binding sites). All MSs were stable in normal colonic mucosae. The MSI frequency at each MS in MSI-H tumors was independent of sequence conservation level and distance from other genetic elements. In contrast, MS length correlated significantly with MSI frequency in MSI-H tumors (r=0.86, p=7.2x10(-13)). 3'UTR MSs demonstrated MSI frequencies in MSI-H tumors higher than the 99% upper limit predicted by MS length for RB1-inducible coiled-coil 1(RB1CC1, mutation frequency 68.4%), NUAK family SNF1-like kinase 1(NUAK1, 31.0%), and Rtf1, Paf1/RNA polymerase II complex component, homolog (RTF1, 25.0%). An in silico prediction of RNA structure alterations was conducted for these MSI events to gauge their likelihood of affecting post-transcriptional regulation. RB1CC1 mutant was predicted to lose a microRNA-accessible loop structure at a putative binding site for the tumor-suppressive microRNA, miR-138. In contrast, the predicted 3'UTR structural change was minimal for NUAK1- and RTF1 mutants. Notably, real-time quantitative RT-PCR analysis revealed significant RB1CC1 mRNA overexpression vs. normal colonic mucosae in MSI-H cancers manifesting RB1CC1 3'UTR MSI (9.0-fold; p = 3.6x10(-4)). CONCLUSIONS: This mutational survey of well-characterized short 3'UTR MSs confirms that MSI incidence in MSI-H colorectal tumors correlates with MS length, but not with sequence conservation level or distance from other genetic elements. This study also identifies RB1CC1 as a novel target of frequent mutation and aberrant upregulation in MSI-H colorectal tumors. The predicted loss of a microRNA-accessible structure in mutant RB1CC1 RNA fits the hypothesis that 3'UTR MSI involves in aberrant RB1CC1 posttranscriptional upregulation. Further direct assessments are indicated to investigate this possibility.Bogdan C. Paun, Yulan Cheng, Barbara A. Leggett, Joanne Young, Stephen J. Meltzer, Yuriko Mor

Soft Substrates Promote Homogeneous Self-Renewal of Embryonic Stem Cells via Downregulating Cell-Matrix Tractions

Maintaining undifferentiated mouse embryonic stem cell (mESC) culture has been a major challenge as mESCs cultured in Leukemia Inhibitory Factor (LIF) conditions exhibit spontaneous differentiation, fluctuating expression of pluripotency genes, and genes of specialized cells. Here we show that, in sharp contrast to the mESCs seeded on the conventional rigid substrates, the mESCs cultured on the soft substrates that match the intrinsic stiffness of the mESCs and in the absence of exogenous LIF for 5 days, surprisingly still generated homogeneous undifferentiated colonies, maintained high levels of Oct3/4, Nanog, and Alkaline Phosphatase (AP) activities, and formed embryoid bodies and teratomas efficiently. A different line of mESCs, cultured on the soft substrates without exogenous LIF, maintained the capacity of generating homogeneous undifferentiated colonies with relatively high levels of Oct3/4 and AP activities, up to at least 15 passages, suggesting that this soft substrate approach applies to long term culture of different mESC lines. mESC colonies on these soft substrates without LIF generated low cell-matrix tractions and low stiffness. Both tractions and stiffness of the colonies increased with substrate stiffness, accompanied by downregulation of Oct3/4 expression. Our findings demonstrate that mESC self-renewal and pluripotency can be maintained homogeneously on soft substrates via the biophysical mechanism of facilitating generation of low cell-matrix tractions

Predicting stress-strain relationships in stratified rock mass by using machine learning techniques

The paper suggests a method based on machine learning techniques to predict the stress-strain relationships in the stratified composite rock mass. To construct the input output dataset for the learning stage, published experimental data for different stratified composite rock specimens have been used. The parameters of the covariance function have been optimized maximizing the log of the marginal likelihood for the experimental results. In the inference stage the obtained parameters are used to statistically predict the stress-strain state of the same rock mass for new stresses not used in the learning stage. The predictions are compared with the actual experimental data aimed to evaluate the validity and applicability of the suggested method. The results demonstrate that by conducting a limited number of experiments and applying the proposed machine learning techniques, it is possible to predict stress-strain relationships for stratified composite rock masses for different combinations of parameters. Application of the proposed method can significantly decrease the number of experiments required for constructing reliable mechanical models for rocks. The uncertainty of the predictions will be high in the case of very limited number of experiments; however it will be significantly reduced once new experimental results are provided to the proposed Bayesian predictive model.N.S. Melkoumia

Workplace experiences of international academic staff in South Australian universities

Nina Maadad and Noune Melkoumia

Effect of different stress path regimes on borehole instability in poorly cemented granular formations

Abstract not availableS.S. Hashemi, N. Melkoumia

A strain energy criterion based on grain dislodgment at borehole wall in poorly cemented sands

Abstract not availableS. S. Hashemi, N. Melkoumia

A case study on blending raw ores at open pit stockyard using decision support system for designing effective technological schemes for open pit

This research aims to develop a decision support system that will enable to identify the best way of ore blending at open pit stockyards. To demonstrate the need for such modeling, the potential for its application and to validate it a decision support system has been developed and discussed for a real-life ore blending process at mineral and iron ore deposits in the Northern Kazakhstan.L. Statsenko, N. Melkoumia