Involvement of miRNAs in the Differentiation of Human Glioblastoma Multiforme Stem-Like Cells

Glioblastoma multiforme (GBM)-initiating cells (GICs) represent a tumor subpopulation with neural stem cell-like properties that is responsible for the development, progression and therapeutic resistance of human GBM. We have recently shown that blockade of NFκB pathway promotes terminal differentiation and senescence of GICs both in vitro and in vivo, indicating that induction of differentiation may be a potential therapeutic strategy for GBM. MicroRNAs have been implicated in the pathogenesis of GBM, but a high-throughput analysis of their role in GIC differentiation has not been reported. We have established human GIC cell lines that can be efficiently differentiated into cells expressing astrocytic and neuronal lineage markers. Using this in vitro system, a microarray-based high-throughput analysis to determine global expression changes of microRNAs during differentiation of GICs was performed. A number of changes in the levels of microRNAs were detected in differentiating GICs, including over-expression of hsa-miR-21, hsa-miR-29a, hsa-miR-29b, hsa-miR-221 and hsa-miR-222, and down-regulation of hsa-miR-93 and hsa-miR-106a. Functional studies showed that miR-21 over-expression in GICs induced comparable cell differentiation features and targeted SPRY1 mRNA, which encodes for a negative regulator of neural stem-cell differentiation. In addition, miR-221 and miR-222 inhibition in differentiated cells restored the expression of stem cell markers while reducing differentiation markers. Finally, miR-29a and miR-29b targeted MCL1 mRNA in GICs and increased apoptosis. Our study uncovers the microRNA dynamic expression changes occurring during differentiation of GICs, and identifies miR-21 and miR-221/222 as key regulators of this process.     

miR-192 Directly Binds and Regulates Dicer1 Expression in Neuroblastoma

Neuroblastoma (NB) arises from the embryonic neural crest and is the most common extracranial solid tumor in children under 5 years of age. Reduced expression of Dicer1 has recently been shown to be in correlation with poor prognosis in NB patients. This study aimed to investigate the mechanisms that could lead to the down-regulation of Dicer1 in neuroblastoma. We used computational prediction to identify potential miRs down-regulating Dicer1 in neuroblastoma. One of the miRs that were predicted to target Dicer1 was miR-192. We measured the levels of miR-192 in 43 primary tumors using real time PCR. Following the silencing of miR-192, the levels of dicer1 cell viability, cell proliferation and migration capability were analyzed. Multivariate analysis identified miR-192 as an independent prognostic marker for relapse in neuroblastoma patients (p=0.04). We were able to show through a dual luciferase assay and side-directed mutational analysis that miR-192 directly binds the 3'' UTR of Dicer1 on positions 1232-1238 and 2282-2288. An increase in cell viability, proliferation and migration rates were evident in NB cells transfected with miR-192-mimic. Yet, there was a significant decrease in proliferation when NB cells were transfected with an miR-192-inhibitor We suggest that miR-192 might be a key player in NB by regulating Dicer1 expression.     

Atypical Human Infections by Animal Trypanosomes

The two classical forms of human trypanosomoses are sleeping sickness due to Trypanosoma brucei gambiense or T. brucei rhodesiense, and Chagas disease due to T. cruzi. However, a number of atypical human infections caused by other T. species (or sub-species) have been reported, namely due to T. brucei brucei, T. vivax, T. congolense, T. evansi, T. lewisi, and T. lewisi-like. These cases are reviewed here. Some infections were transient in nature, while others required treatments that were successful in most cases, although two cases were fatal. A recent case of infection due to T. evansi was related to a lack of apolipoprotein L-I, but T. lewisi infections were not related to immunosuppression or specific human genetic profiles. Out of 19 patients, eight were confirmed between 1974 and 2010, thanks to improved molecular techniques. However, the number of cases of atypical human trypanosomoses might be underestimated. Thus, improvement, evaluation of new diagnostic tests, and field investigations are required for detection and confirmation of these atypical cases.

Key Learning Points

• The classical human trypanosomoses are human African trypanosomosis (HAT) or sleeping sickness, and Chagas disease, the Latin American human trypanosomosis.
• Atypical human infections caused by Trypanosoma species that normally are restricted to animals have been reported. These cases of atypical human trypanosomoses (a-HT) are mostly transient, but some require treatment and can be fatal.
• Only a few cases of a-HT have been fully confirmed, especially in Asia, leading to the hypothesis that the actual prevalence is probably underestimated.
• The detection of a case of a-HT should be based on observation of the parasite by direct microscopy. Evaluating/improving the diagnoses through serological and PCR assays would help in detecting and identifying atypical trypanosomosis infections in humans. These laboratory research and field activities are needed to evaluate the actual occurrence of atypical cases.

Top Five Papers

1. Verma A, Manchanda S, Kumar N, Sharma A, Goel M, et al. (2011) Trypanosoma lewisi or Trypanosoma lewisi-like infection in a 37-day-old infant. Am J Trop Med Hyg 85: 221–224.
2. Deborggraeve S, Koffi M, Jamonneau V, Bonsu FA, Queyson R, et al. (2008) Molecular analysis of archived blood slides reveals an atypical human Trypanosoma infection. Diagn Microbiol Infect Dis 61: 428–433.
3. Vanhollebeke B, Truc P, Poelvoorde P, Pays A, Joshi PP, et al. (2006) Human Trypanosoma evansi infection linked to a lack of apolipoprotein L-I. N Engl J Med 355: 2752–2756.
4. Joshi PP, Shegokar V, Powar S, Herder S, Katti R, et al. (2005) Human trypanosomiasis caused by Trypanosoma evansi in India: the first case report. Am J Trop Med Hyg 73: 491–495.
5. Howie S, Guy M, Fleming L, Bailey W, Noyes H, et al. (2006) A Gambian infant with fever and an unexpected blood film. PLoS Med 3: e355. doi:10.1371/journal.pmed.0030355.

     

A Subset of Mouse Colonic Goblet Cells Expresses the Bitter Taste Receptor Tas2r131

The concept that gut nutrient sensing involves taste receptors has been fueled by recent reports associating the expression of taste receptors and taste-associated signaling molecules in the gut and in gut-derived cell lines with physiological responses induced by known taste stimuli. However, for bitter taste receptors (Tas2rs), direct evidence for their functional role in gut physiology is scarce and their cellular expression pattern remained unknown. We therefore investigated Tas2r expression in mice. RT-PCR experiments assessed the presence of mRNA for Tas2rs and taste signaling molecules in the gut. A gene-targeted mouse strain was established to visualize and identify cell types expressing the bitter receptor Tas2r131. Messenger RNA for various Tas2rs and taste signaling molecules were detected by RT-PCR in the gut. Using our knock-in mouse strain we demonstrate that a subset of colonic goblet cells express Tas2r131. Cells that express this receptor are absent in the upper gut and do not correspond to enteroendocrine and brush cells. Expression in colonic goblet cells is consistent with a role of Tas2rs in defense mechanisms against potentially harmful xenobiotics.     

snoRNPs Regulate Telomerase Activity in Neuroblastoma and Are Associated with Poor Prognosis

Amplification of the MYCN oncogene is strongly associated with poor prognosis in neuroblastoma (NB). In addition to MYCN amplification, many studies have focused on identifying patients with a poor prognosis based on gene expression profiling. The majority of prognostic signatures today are comprised of large gene lists limiting their clinical application. In addition, although of prognostic significance,most of these signatures fail to identify cellular processes that can explain their relation to prognosis. Here, we determined prognostically predictive genes in a data set containing 251 NBs. Gene Ontology analysis was performed on significant genes with a positive hazard ratio to search for cellular processes associated with poor prognosis. An enrichment in ribonucleoproteins (RNPs) was found. Genes involved in the stabilization and formation of the central small nucleolar RNP (snoRNP) complex were scrutinized using a backward conditional Cox regression resulting in an snoRNP signature consisting of three genes: DKC1, NHP2, and GAR1. The snoRNP signature significantly and independently predicted prognosis when compared to the established clinical risk factors. Association of snoRNP protein expression and prognosis was confirmed using tissue microarrays. Knockdown of snoRNP expression in NB cell lines resulted in reduced telomerase activity and an increase in anaphase bridge frequency. In addition, in patient material, expression of the snoRNP complex was significantly associated with telomerase activity, occurrence of segmental aberrations, and expression-based measurements of chromosomal instability. Together, these results underscore the prognostic value of snoRNP complex expression in NB and suggest a role for snoRNPs in telomere maintenance and genomic stability.     

Forty Years in the Making: Understanding the Molecular Mechanism of Peptide Regulation in Bacterial Development

Signal transduction systems are influenced by positive and negative forces resulting in an output reflecting the sum of the opposing forces. The Rap family of regulatory protein modules control the output of two-component signal transduction systems through protein∶protein and protein∶peptide interactions. These modules and their peptide regulators are found in complex signaling pathways, including the bacterial developmental pathway to sporulation, competence, and protease secretion. Two articles published in the current issue of PLOS Biology reveal by means of crystallographic analyses how the Rap proteins of bacilli are regulated by their inhibitor Phr peptide and provide a mechanistic explanation for a genetic phenotype isolated decades earlier. The Rap-Phr module of bacterial regulators was the prototype of a family that now extends to other bacterial signaling proteins that involve the use of the tetratricopeptide repeat structural fold. The results invite speculation regarding the potential exploitation of this module as a molecular tool for applications in therapeutic design and biotechnology.Cell signaling by oligopeptides is a critical component of the biology of eukaryotic and prokaryotic cells. In microorganisms such as Gram-positive bacteria, small peptides have been found to regulate a variety of cellular functions, providing the bacteria with the ability to communicate and change behavior of the same or of other species in response to conditions and perturbations of the environment [1]. Studies in the spore-forming model organism Bacillus subtilis were among the first to identify pathways in which peptide signaling played a regulatory role.