Influence of cholesterol on cancer progression and therapy

Cholesterol is a fundamental molecule necessary for the maintenance of cell structure and is vital to various normal biological functions. It is a key factor in lifestyle-related diseases including obesity, diabetes, cardiovascular disease, and cancer. Owing to its altered serum chemistry status under pathological states, it is now being investigated to unravel the mechanism by which it triggers various health complications. Numerous clinical studies in cancer patients indicate an alteration in blood cholesterol level (either decreased or increased) in comparison to normal healthy individuals. This article elaborates on our understanding as to how cholesterol is being hijacked in the malignancy for the development, survival, stemness, progression, and metastasis of cancerous cells. Also, it provides a glimpse of how cholesterol derived entities, alters the signaling pathway towards their advantage. Moreover, deregulation of the cholesterol metabolism pathway has been often reported to hamper various treatment strategies in different cancer. In this context, attempts have been made to bring forth its relevance in being targeted, in pre-clinical and clinical studies for various treatment modalities. Thus, understanding the role of cholesterol and deciphering associated molecular mechanisms in cancer progression and therapy are of relevance towards improvement in the management of various cancers.     

High‐throughput sequencing and graph‐based cluster analysis facilitate microsatellite development from a highly complex genome

Despite recent advances in high‐throughput sequencing, difficulties are often encountered when developing microsatellites for species with large and complex genomes. This probably reflects the close association in many species of microsatellites with cryptic repetitive elements. We therefore developed a novel approach for isolating polymorphic microsatellites from the club‐legged grasshopper (Gomphocerus sibiricus), an emerging quantitative genetic and behavioral model system. Whole genome shotgun Illumina MiSeq sequencing was used to generate over three million 300 bp paired‐end reads, of which 67.75% were grouped into 40,548 clusters within RepeatExplorer. Annotations of the top 468 clusters, which represent 60.5% of the reads, revealed homology to satellite DNA and a variety of transposable elements. Evaluating 96 primer pairs in eight wild‐caught individuals, we found that primers mined from singleton reads were six times more likely to amplify a single polymorphic microsatellite locus than primers mined from clusters. Our study provides experimental evidence in support of the notion that microsatellites associated with repetitive elements are less likely to successfully amplify. It also reveals how advances in high‐throughput sequencing and graph‐based repetitive DNA analysis can be leveraged to isolate polymorphic microsatellites from complex genomes.     

High-resolution cDNA microarray CGH mapping of genomic imbalances in osteosarcoma using formalin-fixed paraffin-embedded tissue

Formalin-fixed paraffin embedded (FFPE) tumor tissue provides an opportunity to perform retrospective genomic studies of tumors in which chromosomal imbalances are strongly associated with oncogenesis. The application of comparative genomic hybridization (CGH) has led to the rapid accumulation of cytogenetic information on osteosarcoma (OS); however, the limited resolving power of metaphase CGH does not permit precise mapping of imbalances. Array CGH allows quantitative detection and more precise delineation of copy number aberrations in tumors. Unfortunately the high cost and lower density of BACs on available commercial arrays has limited the ability to comprehensively profile copy number changes in tumors such as OS that are recurrently subject to genomic imbalance. In this study a cDNA/EST microarray including 18,980 human cDNAs (which represent all 22 pairs of autosomal chromosomes and chromosome X) was used for CGH analysis of eight OS FFPE. Chromosomes 1, 12, 17, and X harbored the most imbalances. Gain/amplification of X was observed in 4/8 OS, and in keeping with other recent genomic analyses of OS, gain/amplification of 17p11.2 was often accompanied by a distal deletion in the region of the p53 gene. Gain/amplification of the X chromosome was verified using interphase FISH carried out on a subset of OS FFPE sections and OS tissue arrays.     

Use of whole genome amplification and comparative genomic hybridisation to detect chromosomal copy number alterations in cell line material and tumour tissue

We have established that whole genome amplification (WGA), in conjunction with genomic DNA array comparative genomic hybridisation (gaCGH) allows for the identification of genome-wide copy number abnormalities (CNAs) in DNA extracted from both cell line and patient material. To determine the fidelity and reproducibility of WGA to detect copy number imbalances using gaCGH, well characterized cell line genomic DNA was analysed. The gaCGH data obtained from non-amplified DNA and amplified DNA for the neuroblastoma cell line NUB7 and a paediatric medulloblastoma patient was almost identical. In addition, laser capture microdissection (LCM) of prostate tumour cells and subsequent WGA allowed for the detection of a number of CNAs that may not have been identified if DNA had been extracted in bulk from heterogeneous tissue. The results presented here demonstrate the use of WGA for generating sufficient DNA for gaCGH analysis without the introduction of significant sequence representation bias. The combination of amplification and gaCGH using DNA extracted from archival patient material has the potential for permitting the studying of DNA from small cancerous or pre-cancerous foci, which may help to identify potential genomic markers for early diagnosis.     

Comparison of the emulsion characteristics of Rhodococcus erythropolis and Escherichia coli SOXC-5 cells expressing biodesulfurization genes

Biodesulfurization of fuel oils is a two-phase (oil/water) process which may offer an interesting alternative to conventional hydrodesulfurization due to the mild operating conditions and reaction specificity afforded by the biocatalyst. For biodesulfurization to realize commercial success, a variety of process considerations must be addressed including reaction rate, emulsion formation and breakage, biocatalyst recovery, and both gas and liquid mass transport. This study evaluates emulsion formation and breakage using two biocatalysts with differing hydrophobic characteristics. A Gram-positive (Rhodococcus erythropolis) biocatalyst, expressing the complete 4S desulfurization pathway, and a Gram-negative biocatalyst (Escherichia coli), expressing only the gene for conversion of dibenzothiophene (DBT) to DBT sulfone, are compared relative to their ability to convert DBT and the ease of phase separation as well as biocatalyst recovery following desulfurization.     

Alterations of H19 imprinting and IGF2 replication timing are infrequent in Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is an overgrowth disorder resulting from dysregulation of multiple imprinted genes through a variety of distinct mechanisms. A frequent alteration in BWS involves changes in the imprinting status of the coordinately regulated IGF2 and H19 genes on 11p15. Patients have been categorized according to alterations in the imprinted expression, allele-specific methylation, and regional replication timing of these genes. In this work, IGF2/H19 expression, H19 DNA methylation, and IGF2 regional replication timing were studied in nine karyotypically normal BWS fibroblasts and two BWS patients with maternally inherited 11p15 chromosomal rearrangements. Informative patients (9/9) maintained normal monoallelic H19 expression/methylation, despite biallelic IGF2 expression in 6/9. Replication timing studies revealed no changes in the pattern of asynchronous replication timing for both a patient with biallelic IGF2 expression and a patient carrying an 11p15 inversion. In contrast, a patient with a chromosome 11;22 translocation and normal H19 expression/methylation exhibited partial loss of asynchrony and a shift toward earlier replication times. These results indicate that in BWS, (1) H19 imprinting alterations are less frequent than previously estimated, (2) IGF2 imprinting and H19 imprinting are not necessarily coordinated, and (3) alterations in regional replication timing are generally not correlated with either chromosomal rearrangements or the imprinting status of IGF2 and H19.     

Differential dynamics of RAS isoforms in GDP‐ and GTP‐bound states

RAS subfamily proteins regulates cell growth promoting signaling processes by cycling between active (GTP‐bound) and inactive (GDP‐bound) states. Different RAS isoforms, though structurally similar, exhibit functional specificity and are associated with different types of cancers and developmental disorders. Understanding the dynamical differences between the isoforms is crucial for the design of inhibitors that can selectively target a particular malfunctioning isoform. In this study, we provide a comprehensive comparison of the dynamics of all the three RAS isoforms (HRAS, KRAS, and NRAS) using extensive molecular dynamics simulations in both the GDP‐ (total of 3.06 μs) and GTP‐bound (total of 2.4 μs) states. We observed significant differences in the dynamics of the isoforms, which rather interestingly, varied depending on the type of the nucleotide bound and the simulation temperature. Both SwitchI (Residues 25–40) and SwitchII (Residues 59–75) differ significantly in their flexibility in the three isoforms. Furthermore, Principal Component Analysis showed that there are differences in the conformational space sampled by the GTP‐bound RAS isoforms. We also identified a previously unreported pocket, which opens transiently during MD simulations, and can be targeted to regulate nucleotide exchange reaction or possibly interfere with membrane localization. Further, we present the first simulation study showing GDP destabilization in the wild‐type RAS protein. The destabilization of GDP/GTP occurred only in 1/50 simulations, emphasizing the need of guanine nucleotide exchange factors (GEFs) to accelerate such an extremely unfavorable process. This observation along with the other results presented in this article further support our previously hypothesized mechanism of GEF‐assisted nucleotide exchange. Proteins 2015; 83:1091–1106. © 2015 Wiley Periodicals, Inc.