The recently suggested intestinal cancer stem cell marker DCLK1 is an epigenetic biomarker for colorectal cancer

Recently, Dclk1 expression was identified to be an intestinal cancer stem cell specific biomarker in mouse models, implicating a potential role for targeting the DCLK1-postive cancer cells as a treatment for colorectal cancer. Using quantitative methylation specific PCR (qMSP) we here demonstrated that the DCLK1 promoter is hypermethylated in the vast majority of colorectal cancers (134/164; 82%), with no methylation in the normal mucosa samples (0/106). We further showed by Affymetrix exon arrays that DCLK1 is significantly downregulated in human colorectal cancer (n = 125) compared with normal colonic mucosa (n = 15), which was further confirmed by real-time RT-PCR of a subgroup of the samples. Additionally, a significant negative correlation was observed between methylation and DCLK1 expression in 74 cancer cell lines derived from 15 different tissues, and gene expression increased significantly after epigenetic drug treatment of initially methylated cancer cell lines. These findings underscore the potential of DCLK1 as a colorectal cancer biomarker for early detection, but may also have clinical implications regarding the previously proposed therapy toward DCLK1-positive cancer cells. This therapy would at best affect the cancer stem cell population, but will, based on the present results, not be efficient to treat the bulk of the tumor.     

Development of Ultra-High-Density Screening Tools for Microbial “Omics”

High-throughput genetic screens in model microbial organisms are a primary means of interrogating biological systems. In numerous cases, such screens have identified the genes that underlie a particular phenotype or a set of gene-gene, gene-environment or protein-protein interactions, which are then used to construct highly informative network maps for biological research. However, the potential test space of genes, proteins, or interactions is typically much larger than current screening systems can address. To push the limits of screening technology, we developed an ultra-high-density, 6144-colony arraying system and analysis toolbox. Using budding yeast as a benchmark, we find that these tools boost genetic screening throughput 4-fold and yield significant cost and time reductions at quality levels equal to or better than current methods. Thus, the new ultra-high-density screening tools enable researchers to significantly increase the size and scope of their genetic screens.     

Evaluating the effectiveness of road mitigation measures

The last 20 years have seen a dramatic increase in efforts to mitigate the negative effects of roads and traffic on wildlife, including fencing to prevent wildlife-vehicle collisions and wildlife crossing structures to facilitate landscape connectivity. While not necessarily explicitly articulated, the fundamental drivers behind road mitigation are human safety, animal welfare, and/or wildlife conservation. Concomitant with the increased effort to mitigate has been a focus on evaluating road mitigation. So far, research has mainly focussed on assessing the use of wildlife crossing structures, demonstrating that a broad range of species use them. However, this research has done little to address the question of the effectiveness of crossing structures, because use of a wildlife crossing structure does not necessarily equate to its effectiveness. The paucity of studies directly examining the effectiveness of crossing structures is exacerbated by the fact that such studies are often poorly designed, which limits the level of inference that can be made. Without well performed evaluations of the effectiveness of road mitigation measures, we may endanger the viability of wildlife populations and inefficiently use financial resources by installing structures that are not as effective as we think they are. In this paper we outline the essential elements of a good experimental design for such assessments and prioritize the parameters to be measured. The framework we propose will facilitate collaboration between road agencies and scientists to undertake research programs that fully evaluate effectiveness of road mitigation measures. We discuss the added value of road mitigation evaluations for policy makers and transportation agencies and provide recommendations on how to incorporate such evaluations in road planning practices.     

Complete characterization of the human IGF-I nucleotide sequence isolated from a newly constructed adult liver cDNA library

A full-size cDNA sequence coding for insulin-like growth factor I (IGF-I) was isolated from a human liver library. For the construction of this bank, a new method was developed which anneals dG-tailed cDNA with a synthetic adaptor 5'-AATTCCCCCCCCCCC-3' followed by ligation into the EcoRI site of a lambda immunity-insertion vector. Based on the sequence analysis of the complete IGF-I messenger we concluded that the protein is synthesized as a precursor containing a signal peptide of 22 or 25 amino acid residues. In addition, the sequence extended in the 3'-direction and showed the presence of multiple polyadenylation sites in the IGF-I message.     

Lipolytic enzymes LipA and LipB from Bacillus subtilis differ in regulation of gene expression, biochemical properties, and three-dimensional structure

Bacillus subtilis secretes the lipolytic enzymes LipA and LipB. We show here that they are differentially expressed depending on the composition of the growth medium: LipA is produced in rich and in minimal medium, whereas LipB is present only in rich medium. A comparison of biochemical characteristics revealed that LipB is thermostable at pH 11 but becomes thermolabile at pH 5. However, construction of a variant carrying the substitution A76G in the conserved lipase pentapeptide reversed these effects. The atomic coordinates from the LipA crystal structure were used to build a three-dimensional structural model of LipB, which revealed that 43 out of 45 residues different from LipA are surface-located allowing to rationalize the differences observed in the substrate preferences of the two enzymes.     

The plantibody approach: expression of antibody genes in plants to modulate plant metabolism or to obtain pathogen resistance

Immunomodulation is a molecular technique that allows the interference with cellular metabolism or pathogen infectivity by the ectopic expression of genes encoding antibodies or antibody fragments. In recent years, several reports have proven the value of this tool in plant research for modulation of phytohormone activity and for blocking plant-pathogen infection. Efficient application of the plantibody approach requires different levels of investigation. First of all, methods have to be available to clone efficiently the genes coding for antibodies or antibody fragments that bind the target antigen. Secondly, conditions to obtain high accumulation of antigen-binding antibodies and antibody fragments in plants are being investigated and optimized. Thirdly, different strategies are being evaluated to interfere with the function of the target molecule, thus enabling immunomodulation of metabolism or pathogen infectivity. In the near future, optimized antibody gene isolation and expression, especially in reducing subcellular environments, such as the cytosol and nucleus, should turn immunomodulation into a powerful and attractive tool for gene inactivation, complementary to the classical antisense and co-suppression approaches.     

Serum trace element levels in HIV-infected subjects

The absolute number of T4 cells and the serum concentrations of Ca, Cu, Fe, K, Mg, P, Se, and Zn were determined in 59 Walter-Reed staged, HIV-infected men, compared to healthy controls, serum levels of Ca, Cu, and Fe were significantly higher, those of P and Se significantly lower in the HIV-infected subjects. In the HIV-infected cases, but not in the controls, the concentrations of Se and Zn, of Ca with Cu and Fe, and of Fe with P, were directly correlated. In the controls, the correlation between the levels of K and Mg was direct, and inverse between those of Zn and P. Trace element levels did not significantly correlate with WR-stage. However, the absolute number of T4 cells was directly correlated with the serum Mg concentration.     

Screening of the Philadelphia variant of galactokinase in racially unmixed black Africans: first results.

In previous reports, it was emphasized that the gene GALKA of galactokinase was the predominant allele in white populations and that another allele, GALKP, which reduces red blood cell activity (RBC GALK), was common in black people. In a group of black Americans living in Philadelphia, the frequency of GALKA was found to be very close to values expected from independent estimation of white admixture. The authors have suggested that the ancestors of these blacks might have been virtually all GALKP homozygous. We have looked for carriers of GALKP genotypes among 73 black Africans; only 33 probands were shown to have a low RBC GALK. To detect white admixture, immunoglobulin allotypes Km and Gm were investigated in 50 individuals of the sample; 15 GALKP carriers with low RBC GALK and 30 of 35 individuals with normal RBC GALK shared Gm phenotypes exclusive to blacks. Our work demonstrates for the first time the polymorphism of GALK in black Africans in the absence of white admixture.