Regression of murine lung tumors by the let-7 microRNA.

MicroRNAs (miRNAs) have recently emerged as an important new class of cellular regulators that control various cellular processes and are implicated in human diseases, including cancer. Here, we show that loss of let-7 function enhances lung tumor formation in vivo, strongly supporting the hypothesis that let-7 is a tumor suppressor. Moreover, we report that exogenous delivery of let-7 to established tumors in mouse models of non-small-cell lung cancer (NSCLC) significantly reduces the tumor burden. These results demonstrate the therapeutic potential of let-7 in NSCLC and point to miRNA replacement therapy as a promising approach in cancer treatment.

Comparison of arsenic concentrations in simultaneously-collected groundwater and aquifer particles from Bangladesh, India, Vietnam, and Nepal.

One of the reasons the processes resulting in As release to groundwater in southern Asia remain poorly understood is the high degree of spatial variability of physical and chemical properties in shallow aquifers. In an attempt to overcome this difficulty, a simple device that collects groundwater and sediment as a slurry from precisely the same interval was developed in Bangladesh. Recently published results from Bangladesh and India relying on the needle-sampler are augmented here with new data from 37 intervals of grey aquifer material of likely Holocene age in Vietnam and Nepal. A total of 145 samples of filtered groundwater ranging in depth from 3 to 36 m that were analyzed for As (1-1000 mug/L), Fe (0.01-40 mg/L), Mn (0.2-4 mg/L) and S (0.04-14 mg/L) are compared. The P-extractable (0.01-36 mg/kg) and HCl-extractable As (0.04-36 mg/kg) content of the particulate phase was determined in the same suite of samples, in addition to Fe(II)/Fe ratios (0.2-1.0) in the acid-leachable fraction of the particulate phase. Needle-sampler data from Bangladesh indicated a relationship between dissolved As in groundwater and P-extractable As in the particulate phase that was interpreted as an indication of adsorptive equilibrium, under sufficiently reducing conditions, across 3 orders of magnitude in concentrations according to a distribution coefficient of 4 mL/g. The more recent observations from India, Vietnam and Nepal show groundwater As concentrations that are often an order of magnitude lower at a given level of P-extractable As compared to Bangladesh, even if only the subset of particularly reducing intervals characterized by leachable Fe(II)/Fe >0.5 and dissolved Fe >0.2 mg/L are considered. Without attempting to explain why As appears to be particularly mobile in reducing aquifers of Bangladesh compared to the other regions, the consequences of increasing the distribution coefficient for As between the particulate and dissolved phase to 40 mL/g for the flushing of shallow aquifers of their initial As content are explored.

MicroRNAs as potential cancer therapeutics.

MicroRNAs (miRNAs) have been shown to have an important role in various cellular processes, such as apoptosis, differentiation and development. Recent studies have shown that miRNAs are mis-expressed in human cancers where they can exert their effect as oncogenes or tumor suppressors. Here, we review the potential for using miRNAs as biomarkers for diagnosis, prognosis and cancer therapies.

Genome-wide profiling identifies epithelial cell genes associated with asthma and with treatment response to corticosteroids.

Airway inflammation and epithelial remodeling are two key features of asthma. IL-13 and other cytokines produced during T helper type 2 cell-driven allergic inflammation contribute to airway epithelial goblet cell metaplasia and may alter epithelial-mesenchymal signaling, leading to increased subepithelial fibrosis or hyperplasia of smooth muscle. The beneficial effects of corticosteroids in asthma could relate to their ability to directly or indirectly decrease epithelial cell activation by inflammatory cells and cytokines. To identify markers of epithelial cell dysfunction and the effects of corticosteroids on epithelial cells in asthma, we studied airway epithelial cells collected from asthmatic subjects enrolled in a randomized controlled trial of inhaled corticosteroids, from healthy subjects and from smokers (disease control). By using gene expression microarrays, we found that chloride channel, calcium-activated, family member 1 (CLCA1), periostin, and serine peptidase inhibitor, clade B (ovalbumin), member 2 (serpinB2) were up-regulated in asthma but not in smokers. Corticosteroid treatment down-regulated expression of these three genes and markedly up-regulated expression of FK506-binding protein 51 (FKBP51). Whereas high baseline expression of CLCA1, periostin, and serpinB2 was associated with a good clinical response to corticosteroids, high expression of FKBP51 was associated with a poor response. By using airway epithelial cells in culture, we found that IL-13 increased expression of CLCA1, periostin, and serpinB2, an effect that was suppressed by corticosteroids. Corticosteroids also induced expression of FKBP51. Taken together, our findings show that airway epithelial cells in asthma have a distinct activation profile and identify direct and cell-autonomous effects of corticosteroid treatment on airway epithelial cells that relate to treatment responses and can now be the focus of specific mechanistic studies.

RNase P-mediated inhibition of cytomegalovirus protease expression and viral DNA encapsidation by oligonucleotide external guide sequences.

External guide sequences (EGSs) are oligonucleotides that consist of a sequence complementary to a target mRNA and recruit intracellular RNase P for specific degradation of the target RNA. In this study, DNA-based EGS molecules were chemically synthesized to target the mRNA coding for the protease of human cytomegalovirus (HCMV). The EGS molecules efficiently directed human RNase P to cleave the target mRNA sequence in vitro. When EGSs were exogenously administered into HCMV-infected human foreskin fibroblasts, a reduction of about 80-90% in the expression level of the protease and a reduction of about 300-fold in HCMV growth were observed in the cells that were treated with a functional EGS, but not in cells that were not treated with the EGS or with a "disabled" EGS carrying nucleotide mutations that precluded RNase P recognition. Moreover, packaging of the viral DNA genome into the capsid was blocked in the cells treated with the functional EGS. These results indicate that HCMV protease is essential for viral DNA encapsidation. Moreover, our study provides direct evidence that exogenous administration of a DNA-based EGS can be used as a therapeutic approach for inhibiting gene expression and replication of a human virus.

Effective inhibition of herpes simplex virus 1 gene expression and growth by engineered RNase P ribozyme.

Using an in vitro selection procedure, we have previously isolated ribonuclease P (RNase P) ribozyme variants that efficiently cleave an mRNA sequence in vitro. In this study, an M1GS RNA variant was used to target the mRNA encoding human herpes simplex virus 1 (HSV-1) major transcription activator ICP4. The variant is about 15 times more efficient in cleaving the ICP4 mRNA sequence in vitro than the ribozyme derived from the wild type RNase P ribozyme. Moreover, the variant is also more effective in inhibiting viral ICP4 expression and growth in HSV-1-infected cells than the wild type ribozyme. A reduction of approximately 90% in the expression level of ICP4 and a reduction of 4000-fold in viral growth were observed in cells that expressed the variant. In contrast, a reduction of <10% in the ICP4 expression and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. These results provide direct evidence that RNase P ribozyme variants can be highly effective in inhibiting HSV-1 gene expression and growth and furthermore, demonstrate the feasibility of developing highly effective RNase P ribozyme variants for anti-HSV applications by using in vitro selection procedures.

Treatment of bacillary dysentery in Vietnamese children: two doses of ofloxacin versus 5-days nalidixic acid.

Nalidixic acid (NA: 55 mg/kg daily for 5 days) is the recommended treatment for uncomplicated bacillary dysentery in areas where multidrug-resistant Shigella are prevalent. An open randomized comparison of this NA regimen with 2 doses of ofloxacin (total 15 mg/kg) was conducted in 1995/96 in 135 Vietnamese children with fever and bloody diarrhoea. Sixty-six children with a bacterial pathogen isolated were eligible for analysis. Of the 63 Shigella isolates, 39 (62%) were resistant to multiple antibiotics. Resolution times for fever and diarrhoea were similar in the 2 groups, but excretion time of stool pathogen was significantly longer in the NA recipients [median (range) days 1 (1-9) vs 1 (1-2), P = 0.001]. There were 9 (25%) treatment failures in the NA regimen and 3 (10%) in the ofloxacin group; P = 0.1. Two patients had NA-resistant Shigella flexneri. One of these isolates was selected during NA treatment. From a clinical and public health standpoint a 2-dose regimen of ofloxacin is preferable to nalidixic acid in the treatment of bacillary dysentery.

A ribozyme derived from the catalytic subunit of RNase P from Escherichia coli is highly effective in inhibiting replication of herpes simplex virus 1.

A sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the mRNA encoding human herpes simplex virus 1 (HSV-1) major transcription activator, ICP4. A reduction of more than 80% in the expression level of ICP4 and a reduction of about 1000-fold in viral growth were observed in cells that stably expressed the ribozyme. In contrast, a reduction of less than 10 % in ICP4 expression and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Thus, M1GS ribozyme is highly effective in inhibiting HSV-1 growth and can be used as a general gene-targeting agent for anti-HSV applications.

Effective inhibition of human cytomegalovirus gene expression and replication by a ribozyme derived from the catalytic RNA subunit of RNase P from Escherichia coli.

A sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the overlapping exon 3 region of the mRNAs encoding the major transcription regulatory proteins IE1 and IE2 of human cytomegalovirus. A reduction of more than 80% in the expression levels of IE1 and IE2 and a reduction of about 150-fold in viral growth were observed in human cells that stably expressed the ribozyme. In contrast, a reduction of less than 10% in the IE1/IE2 expression and viral growth was observed in cells that either did not express the ribozyme or produced a "disabled" ribozyme that carried mutations that abolished its catalytic activity. Examination of the expression of several other viral early and late genes in the cells that expressed the M1GS ribozyme further revealed an overall reduction of at least 80% in their expression. These results are consistent with the notion that the antiviral effects in these cells are due to the fact that the ribozyme specifically inhibits the expression of IE1 and IE2 and, consequently, abolishes the expression of viral early and late genes as well as viral growth. Our study is the first, to our knowledge, to use M1GS ribozyme for inhibiting human cytomegalovirus replication and demonstrates the utility of this ribozyme for antiviral applications.

RNase P ribozymes selected in vitro to cleave a viral mRNA effectively inhibit its expression in cell culture.

An in vitro selection procedure was used to select RNase P ribozyme variants that efficiently cleaved the sequence of the mRNA encoding thymidine kinase of herpes simplex virus 1. Of the 45 selected variants sequenced, 25 ribozymes carried a common mutation at nucleotides 224 and 225 of RNase P catalytic RNA from Escherichia coli (G(224)G(225) --> AA). These selected ribozymes exhibited at least 10 times higher cleavage efficiency (k(cat)/K(m)) than that derived from the wild type ribozyme. Our results suggest that the mutated A(224)A(225) are in close proximity to the substrate and enhance substrate binding of the ribozyme. When these ribozyme variants were expressed in herpes simplex virus 1-infected cells, the levels of thymidine kinase mRNA and protein were reduced by 95-99%. Our study provides the first direct evidence that RNase P ribozyme variants isolated by the selection procedure can be used for the construction of gene-targeting ribozymes that are highly effective in tissue culture. These results demonstrate the potential for using RNase P ribozymes as gene-targeting agents against any mRNA sequences, and using the selection procedure as a general approach for the engineering of RNase P ribozymes.

Nuclease footprint analyses of the interactions between RNase P ribozyme and a model mRNA substrate.

RNase P ribozyme cleaves an RNA helix substrate which resembles the acceptor stem and T-stem structures of its natural tRNA substrate. By linking the ribozyme covalently to a sequence (guide sequence) complementary to a target RNA, the catalytic RNA can be converted into a sequence-specific ribozyme, M1GS RNA. We have previously shown that M1GS RNA can efficiently cleave the mRNA sequence encoding thymidine kinase (TK) of herpes simplex virus 1. In this study, a footprint procedure using different nucleases was carried out to map the regions of a M1GS ribozyme that potentially interact with the TK mRNA substrate. The ribozyme regions that are protected from nuclease degradation in the presence of the TK mRNA substrate include those that interact with the acceptor stem and T-stem, the 3' terminal CCA sequence and the cleavage site of a tRNA substrate. However, some of the protected regions (e.g. P13 and P14) are unique and not among those protected in the presence of a tRNA substrate. Identification of the regions that interact with a mRNA substrate will allow us to study how M1GS RNA recognizes a mRNA substrate and facilitate the development of mRNA-cleaving ribozymes for gene-targeting applications.