The Role of MicroRNA-200 in Progression of Human Colorectal and Breast Cancer

The role of the epithelial-mesenchymal transition (EMT) in cancer has been studied extensively in vitro, but involvement of the EMT in tumorigenesis in vivo is largely unknown. We investigated the potential of microRNAs as clinical markers and analyzed participation of the EMT-associated microRNA-200–ZEB–E-cadherin pathway in cancer progression. Expression of the microRNA-200 family was quantified by real-time RT-PCR analysis of fresh-frozen and microdissected formalin-fixed paraffin-embedded primary colorectal tumors, normal colon mucosa, and matched liver metastases. MicroRNA expression was validated by in situ hybridization and after in vitro culture of the malignant cells. To assess EMT as a predictive marker, factors considered relevant in colorectal cancer were investigated in 98 primary breast tumors from a treatment-randomized study. Associations between the studied EMT-markers were found in primary breast tumors and in colorectal liver metastases. MicroRNA-200 expression in epithelial cells was lower in malignant mucosa than in normal mucosa, and was also decreased in metastatic compared to non-metastatic colorectal cancer. Low microRNA-200 expression in colorectal liver metastases was associated with bad prognosis. In breast cancer, low levels of microRNA-200 were related to reduced survival and high expression of microRNA-200 was predictive of benefit from radiotheraphy. MicroRNA-200 was associated with ER positive status, and inversely correlated to HER2 and overactivation of the PI3K/AKT pathway, that was associated with high ZEB1 mRNA expression. Our findings suggest that the stability of microRNAs makes them suitable as clinical markers and that the EMT-related microRNA-200 – ZEB – E-cadherin signaling pathway is connected to established clinical characteristics and can give useful prognostic and treatment-predictive information in progressive breast and colorectal cancers.     

Single-Cell Dynamics Reveals Sustained Growth during Diauxic Shifts

Stochasticity in gene regulation has been characterized extensively, but how it affects cellular growth and fitness is less clear. We study the growth of E. coli cells as they shift from glucose to lactose metabolism, which is characterized by an obligatory growth arrest in bulk experiments that is termed the lag phase. Here, we follow the growth dynamics of individual cells at minute-resolution using a single-cell assay in a microfluidic device during this shift, while also monitoring lac expression. Mirroring the bulk results, the majority of cells displays a growth arrest upon glucose exhaustion, and resume when triggered by stochastic lac expression events. However, a significant fraction of cells maintains a high rate of elongation and displays no detectable growth lag during the shift. This ability to suppress the growth lag should provide important selective advantages when nutrients are scarce. Trajectories of individual cells display a highly non-linear relation between lac expression and growth, with only a fraction of fully induced levels being sufficient for achieving near maximal growth. A stochastic molecular model together with measured dependencies between nutrient concentration, lac expression level, and growth accurately reproduces the observed switching distributions. The results show that a growth arrest is not obligatory in the classic diauxic shift, and underscore that regulatory stochasticity ought to be considered in terms of its impact on growth and survival.     

Integrating biological pathways and genomic profiles with ChiBE 2

Background

Dynamic visual exploration of detailed pathway information can help researchers digest and interpret complex mechanisms and genomic datasets.

Results

ChiBE is a free, open-source software tool for visualizing, querying, and analyzing human biological pathways in BioPAX format. The recently released version 2 can search for neighborhoods, paths between molecules, and common regulators/targets of molecules, on large integrated cellular networks in the Pathway Commons database as well as in local BioPAX models. Resulting networks can be automatically laid out for visualization using a graphically rich, process-centric notation. Profiling data from the cBioPortal for Cancer Genomics and expression data from the Gene Expression Omnibus can be overlaid on these networks.

Conclusions

ChiBE’s new capabilities are organized around a genomics-oriented workflow and offer a unique comprehensive pathway analysis solution for genomics researchers. The software is freely available at http://code.google.com/p/chibe.     

Influence of detergents on the activity of the ABC transporter LmrA

The ABC transporter LmrA from Lactococcus lactis has been intensively studied and a role in multidrug resistance was proposed. Here, we performed a comprehensive detergent screen to analyze the impact of detergents for a successful solubilization, purification and retention of functional properties of this ABC transporter. Our screen revealed the preference of LmrA for zwitterionic detergents. In detergent solution, LmrA purified with FC-16 was highly active with respect to ATPase activity, which could be stimulated by a substrate (rhodamine 123) of LmrA. Both, high ATPase activity and substrate stimulation were not detected for LmrA solubilized in DDM. Interestingly, reconstituted LmrA showed an opposite behavior, with a high basal ATPase activity and stimulation by rhodamine 123 for a DDM-reconstituted, but only low ATPase activity and no substrate stimulation for a FC-16 reconstituted sample.     

Lowering bile acid pool size with a synthetic farnesoid X receptor (FXR) agonist induces obesity and diabetes through reduced energy expenditure

We evaluated the metabolic impact of farnesoid X receptor (FXR) activation by administering a synthetic FXR agonist (GW4064) to mice in which obesity was induced by a high fat diet. Administration of GW4064 accentuated body weight gain and glucose intolerance induced by the high fat diet and led to a pronounced worsening of the changes in liver and adipose tissue. Mechanistically, treatment with GW4064 decreased bile acid (BA) biosynthesis, BA pool size, and energy expenditure, whereas reconstitution of the BA pool in these GW4064-treated animals by BA administration dose-dependently reverted the metabolic abnormalities. Our data therefore suggest that activation of FXR with synthetic agonists is not useful for long term management of the metabolic syndrome, as it reduces the BA pool size and subsequently decreases energy expenditure, translating as weight gain and insulin resistance. In contrast, expansion of the BA pool size, which can be achieved by BA administration, could be an interesting strategy to manage the metabolic syndrome.     

In Vivo Disassembly and Reassembly of Protein Aggregates in Escherichia coli

Protein misfolding and aggregation are inevitable but detrimental cellular processes. Cells therefore possess protein quality control mechanisms based on chaperones and proteases that (re)fold or hydrolyze unfolded, misfolded, and aggregated proteins. Besides these conserved quality control mechanisms, the spatial organization of protein aggregates (PAs) inside the cell has been proposed as an important additional strategy to deal with their cytotoxicity. In the bacterium Escherichia coli, however, it remained unclear how this spatial organization is established and how this process of assembling PAs in the cell poles affects cellular physiology. In this report, high hydrostatic pressure was used to transiently reverse protein aggregation in living E. coli cells, allowing the subsequent (re)assembly of PAs to be studied in detail. This approach revealed PA assembly to be dependent on intracellular energy and metabolic activity, with the resulting PA structure being confined to the cell pole by nucleoid occlusion. Moreover, a correlation could be observed between the time needed for PA reassembly and the individual lag time of the cells, which might prevent symmetric segregation of cytotoxic PAs among siblings to occur and ensure rapid spatial clearance of molecular damage throughout the emerging population.     

Snapshots of pre-rRNA structural flexibility reveal eukaryotic 40S assembly dynamics at nucleotide resolution

Ribosome assembly in eukaryotes involves the activity of hundreds of assembly factors that direct the hierarchical assembly of ribosomal proteins and numerous ribosomal RNA folding steps. However, detailed insights into the function of assembly factors and ribosomal RNA folding events are lacking. To address this, we have developed ChemModSeq, a method that combines structure probing, high-throughput sequencing and statistical modeling, to quantitatively measure RNA structural rearrangements during the assembly of macromolecular complexes. By applying ChemModSeq to purified 40S assembly intermediates we obtained nucleotide-resolution maps of ribosomal RNA flexibility revealing structurally distinct assembly intermediates and mechanistic insights into assembly dynamics not readily observed in cryo-electron microscopy reconstructions. We show that RNA restructuring events coincide with the release of assembly factors and predict that completion of the head domain is required before the Rio1 kinase enters the assembly pathway. Collectively, our results suggest that 40S assembly factors regulate the timely incorporation of ribosomal proteins by delaying specific folding steps in the 3′ major domain of the 20S pre-ribosomal RNA.