RNase Protection Research Articles

Analysis of intron-containing pre-mRNA and spliced mRNA in maize chloroplasts by RT-PCR

Chloroplast RNA splicing is usually studied by complicated methods, such as Northern blot hybridization, RNAse protection, or primer extension assays. The use of a simpler RT-PCR technique frequently results in the underestimation of unspliced pre-mRNA levels. Five protein-coding genes from the maize plastome were studied to analyze which factors can lead to an apparent reduction in the pool of introncontaining transcripts compared to mature RNA. It was found that the accurate determination of the unspliced RNA level should take into account the DNase inactivation mode, the cDNA synthesis temperature, and the type of DNA polymerase. For one gene, the portion of unspliced RNAs decreased with increasing number of PCR cycles. It was found that intron-containing and intron-free strands of amplicons can form heteroduplexes after PCR. A simple and effective technique of investigating chloroplast spliced mRNA and unspliced pre-mRNA is suggested based on the obtained data.

Minimal-length Synthetic shRNAs Formulated with Lipid Nanoparticles are Potent Inhibitors of Hepatitis C Virus IRES-linked Gene Expression in Mice

We previously identified short synthetic shRNAs (sshRNAs) that target a conserved hepatitis C virus (HCV) sequence within the internal ribosome entry site (IRES) of HCV and potently inhibit HCV IRES-linked gene expression. To assess in vivo liver delivery and activity, the HCV-directed sshRNA SG220 was formulated into lipid nanoparticles (LNP) and injected i.v. into mice whose livers supported stable HCV IRES-luciferase expression from a liver-specific promoter. After a single injection, RNase protection assays for the sshRNA and 3H labeling of a lipid component of the nanoparticles showed efficient liver uptake of both components and long-lasting survival of a significant fraction of the sshRNA in the liver. In vivo imaging showed a dose-dependent inhibition of luciferase expression (>90% 1 day after injection of 2.5 mg/kg sshRNA) with t1/2 for recovery of about 3 weeks. These results demonstrate the ability of moderate levels of i.v.-injected, LNP-formulated sshRNAs to be taken up by liver hepatocytes at a level sufficient to substantially suppress gene expression. Suppression is rapid and durable, suggesting that sshRNAs may have promise as therapeutic agents for liver indications.

Chitosan Combined with Poly-L-arginine as Efficient, Safe, and Serum-Insensitive Vehicle with RNase Protection Ability for siRNA Delivery

Chitosan (CS) combined with poly-L-arginine (PLA) was formulated and evaluated for its performance to deliver siRNA to HeLa cells expressing enhanced green fluorescent protein (EGFP). Compared with the formulations using single polymer in which the polyplexes were completely formed at the weight ratio of >20 : 1 for CS/siRNA or 1 : 1 for PLA/siRNA, the combination of CS and PLA could reduce the amounts of the polymers required for the complete complexation with siRNA, thereby forming positively charged, nanosized polyplex at the weight ratio of CS/PLA/siRNA of 5 : 0.5: 1. In addition, while the transfection efficiency of CS/siRNA and PLA/siRNA was very low at physiological pH (7.4), CS/PLA/siRNA at the optimal weight ratio of 5 : 0.5 : 1 satisfactorily silenced the endogenous EGFP gene at pH 7.4 as well as at pH 6.4 without the deterrent effect from serum. The combined polymers could protect siRNA from RNase degradation over a period of at least 6 h. Furthermore, MTT assay results demonstrated that CS/PLA/siRNA complexes showed acceptably low cytotoxicity with 75% cell viability. Therefore, CS combined with PLA is easy to prepare, safe, and promising for use as an efficient siRNA delivery vehicle.

LQT2 Nonsense Mutations Generate Trafficking Deficient N-Terminally Truncated Channels by the Reinitiation of Translation

Long QT syndrome type 2 (LQT2) is caused by mutations in the human ether-a-go-go related gene (hERG). The reinitiation of translation has recently been reported as a novel pathogenic mechanism of LQT2 by which early PTC mutations generate N-terminally truncated channels with altered gating properties. We have previously shown that hERG transcripts containing the Q81X mutation were translated at the downstream M124 codon following premature termination and generated channels that exhibited increased deactivation kinetics and decreased resurgent outward current during action potential repolarization. We have now identified two additional LQT2 nonsense mutations C39X and C44X that escape nonsense mediated mRNA decay by the reinitiation of translation. Unlike the Q81X channels, which are expressed at the cell surface, C39X and C44X channel proteins are trafficking defective. We show that C39X and C44X channels undergo translation reinitiation at M60 which deletes the hERG N-terminus including the first 34 residues of the Per, Arnt, and Sim (PAS) domain. In contrast to the reinitiation of Q81X channels at M124, in which the PAS domain is nearly completely deleted, reinitiation at M60 disrupts the folding of this highly structured domain, precluding the efficient folding and trafficking of the mutant channels. The RNase protection assay, western blot analysis and electrophysiology were used to characterize the LQT2 mutants at the RNA, protein, and functional level. Our findings indicate that translation reinitiation may give rise to trafficking as well as functional defects of mutant hERG channels associated with LQT2 nonsense mutations.

Ultrasound-Targeted Microbubble Destruction to Deliver siRNA Cancer Therapy

Microbubble contrast agents can specifically deliver nucleic acids to target tissues when exposed to ultrasound treatment parameters that mediate microbubble destruction. In this study, we evaluated whether microbubbles and ultrasound-targeted microbubble destruction (UTMD) could be used to enhance delivery of EGF receptor (EGFR)-directed siRNA to murine squamous cell carcinomas. Custom-designed microbubbles efficiently bound siRNA and mediated RNAse protection. UTMD-mediated delivery of microbubbles loaded with EGFR-directed siRNA to murine squamous carcinoma cells in vitro reduced EGFR expression and EGF-dependent growth, relative to delivery of control siRNA. Similarly, serial UTMD-mediated delivery of EGFR siRNA to squamous cell carcinoma in vivo decreased EGFR expression and increased tumor doubling time, relative to controls receiving EGFR siRNA-loaded microbubbles but not ultrasound or control siRNA-loaded microbubbles and UTMD. Taken together, our results offer a preclinical proof-of-concept for customized microbubbles and UTMD to deliver gene-targeted siRNA for cancer therapy.

Stat6 cooperates with Sp1 in controlling breast cancer cell proliferation by modulating the expression of p21Cip1/WAF1 and p27Kip1

The signal transducer and activator of transcription 6 (Stat6), a member of the family of DNA-binding proteins, has been identified as a critical cell differentiation modulator in breast cancer cells. As of yet, the mechanisms underlying this function have remained largely unknown. To further elucidate the role of Stat6 in breast cancer development, we investigated the consequences of exogenous Stat6 expression. Proliferation assays and flow cytometry assays were conducted to evaluate the putative role of Stat6 on cell proliferation. To this end, we produced synchronized cells after a double thymidine block, as confirmed by FACS analysis. mRNA levels of Stat6 were measured by RNase protection analysis. To confirm the interaction among proteins, we employed GST pull-down assays and immunoprecipitation assays. Luciferase assays and ChIP assays were used to assess the transcriptional activity. Compared to control breast cancer cells, we found that exogenous Stat6 expression plays a critical role in controlling cell proliferation. Also in different breast tumor cell lines, endogenous Stat6 expression was found to be positively related to a lower proliferation rate. Interestingly, in human breast cancer cells Stat6 functions in G1/S cell cycle progression, and the growth-inhibitory effect of Stat6 was shown to be mediated by induction of the G1 cyclin-dependent kinase inhibitors p21(Cip1/WAF1) (p21) and p27(Kip1) (p27). Simultaneously, G1-related cyclin/cyclin-dependent kinase activities and pRB phosphorylation were markedly reduced, and cell cycle progression was blocked in the G1 phase. Stat6 knockdown resulted in enhanced cell proliferation and a decrease in p21 and p27 mRNA levels in the steroid-responsive and non-responsive T-47D and MDA-MB-231 cell lines, respectively. In addition, the stimulatory effect of Stat6 on p21 and p27 gene transcription was found to be associated with interaction of Stat6 with the transcription factor Sp1 at the proximal Sp1-binding sites in their respective promoters. Together, these results identify Stat6 as an important cell differentiation regulatory protein functioning, at least in part, by interacting with Sp1 to activate the p21 and p27 gene promoters in breast cancer cells.

Processing of snoRNAs as a new source of regulatory non‐coding RNAs

Recent experimental evidence suggests that most of the genome is transcribed into non-coding RNAs. The initial transcripts undergo further processing generating shorter, metabolically stable RNAs with diverse functions. Small nucleolar RNAs (snoRNAs) are non-coding RNAs that modify rRNAs, tRNAs, and snRNAs that were considered stable. We review evidence that snoRNAs undergo further processing. High-throughput sequencing and RNase protection experiments showed widespread expression of snoRNA fragments, known as snoRNA-derived RNAs (sdRNAs). Some sdRNAs resemble miRNAs, these can associate with argonaute proteins and influence translation. Other sdRNAs are longer, form complexes with hnRNPs and influence gene expression. C/D box snoRNA fragmentation patterns are conserved across multiple cell types, suggesting a processing event, rather than degradation. The loss of expression from genetic loci that generate canonical snoRNAs and processed snoRNAs results in diseases, such as Prader-Willi Syndrome, indicating possible physiological roles for processed snoRNAs. We propose that processed snoRNAs acquire new roles in gene expression and represent a new class of regulatory RNAs distinct from canonical snoRNAs.

DNA microarray analysis identified molecular pathways mediating the effects of supplementation of branched‐chain amino acids on CCl4‐induced cirrhosis in rats

This study addresses the effects of branched-chain amino acids (BCAA) on global gene expression in liver and skeletal muscle and the molecular mechanisms underlying the improvement in liver cirrhosis using DNA microarray analysis combined with RNase protection assay. Male Wistar rats administered carbon tetrachloride (CCl(4) ) repeatedly for 19 weeks as a decompensated cirrhosis model were thereafter given BCAA-enriched diet (AL) or normal diet (LC) for 5 weeks. The control-diet rats without CCl(4) administration were used as a normal control group. Gene expression in AL was reversed by twofold greater than in LC in the microarray were selected to elucidate the improvements in nutritional and metabolic disorders. Downregulation of fatty acid translocase (FAT)/Cd36, glutamine synthetase, and pyruvate dehydrogenase kinase isoenzyme 4 is believed to promote lower uptake of fatty acids, lower ammonia incorporation, and higher uptake of glucose, and thus to provide an energy source without using BCAA. Ultimately, the catabolism of BCAA and skeletal muscle protein would be slowed, maintaining BCAA concentrations in blood. We established, for the first time, the regulatory gene pathways of processes involved in hepatic fibrosis and energy metabolism (hypoalbuminemia, hyperammonemia, and carbohydrate catabolism, and their relationships) under BCAA supplementation.

Expression and promoter analysis of a highly restricted integrin alpha gene in vascular smooth muscle

Full genome annotation requires gene expression analysis and elucidation of promoter activity. Here, we analyzed the expression and promoter of a highly restricted integrin gene, Itga8. RNase protection and quantitative RT-PCR showed Itga8 to be expressed most abundantly in vascular smooth muscle cells (SMC). Transcription start site mapping of Itga8 revealed the immediate 5′ promoter region to be poorly conserved with orthologous sequences in the human genome. Further comparative sequence analysis showed a number of conserved non-coding sequence modules around the Itga8 gene. The immediate promoter region and an upstream conserved sequence module were each found to contain a CArG box, which is a binding site for serum response factor (SRF). Luciferase reporter assays revealed activity of several Itga8 promoter constructs with no apparent restricted activity to SMC types. Further, neither SRF nor its coactivator, Myocardin (MYOCD), was able to induce several distinct Itga8 promoter constructs. Transgenic mouse studies failed to reveal Itga8 promoter activity, indicating distal regulatory elements likely control this gene's in vivo expression profile. Interestingly, although the promoter was unresponsive to SRF/MYOCD, the endogenous Itga8 gene showed increases in expression upon ectopic MYOCD expression even though knockdown of SRF both in vitro and in vivo failed to demonstrate a corresponding change in Itga8. Collectively, these data demonstrate that Itga8 expression is CArG-SRF independent, but MYOCD dependent through an as yet unknown sequence module that is distal from the promoter region.

Isolation of Total RNA from Yeast Cell Cultures: Figure 1.

This article describes two procedures for isolating total RNA from yeast cell cultures. The first allows the convenient isolation of total RNA from early log-phase cultures (vegetative cells). RNA isolated in this way is intact and sufficiently pure for use in microarray experiments, primer extension, and RNase protection mapping. With additional treatment to remove contaminating genomic DNA, the preparation is suitable for reverse transcription-polymerase chain reaction (RT-PCR), quantitative PCR (qPCR), cDNA library construction, high-throughput sequencing of RNA, or other manipulations. However, compared to vegetative cells, the isolation of RNA from cells late in meiosis (asci and ascospores) requires additional effort. This is because a tough cell wall composed of heavily cross-linked polysaccharides and proteins is built around the four spores during meiosis and ascospore development. Therefore, an alternative protocol is presented for extracting RNA from cells late in meiosis. This alternative may also be preferable for cells from stationary cultures or from yeast strains and other fungal species isolated from the environment.

Inhibitory Effect of Epstein-Barr Virus Gene-Ebna1 on Human Tnfrp55 Gene Expression

Abstract The aim of the study was to assess the expression of TNFRp55 mRNA and to examine if the antisense inhibition of Epstein-Barr virus (EBV) encoded EBNA1 gene product alters the expression of gene encoding TNFRp55 in lymphoblastoid cell line (LCL). The experiment was performed on LCL derived from EBV infected human peripheral blood B lymphocytes. The lymphocytes were isolated and cultured. RNA was isolated and examined according to the RNase protection assay. The hybridisation was done with HCR-4 probe. RNA was quantified by densitometry and presented in extinction units. The level of expression was calculated with TotaILab software programme. The results of the study suggest that EBV gene, responsible for the synthesis of EBNA1 protein, has an inhibitory effect on human TNFRp55 gene expression in LCL.

Reversal of Liver Fibrosis in Chronic Murine Schistosomiasis mansoni by Safironil/Praziquantel

Purpose: To evaluate the safety, pharmacological effect and mechanism of action of an antifibrotic compound, safironil (SAF)/praziquantel (PZQ) combination on reversal of liver fibrogenesis in chronic murine Schistosomiasis mansoni. Methods: The antifibrotic effect of 0.5, 1 and 2 mg/ml of SAF was evaluated in vitro myofibroblast cell culture, using RNAse protection assay for collagen I mRNA expression and quantitative immunoblot for smooth muscle α-actin protein extract. Ninety Swiss albino mice were infected with 50 Schistosoma mansoni cercariae. SAF was provided in drinking water at a concentration of 1.5 mg/ml while praziquantel (PZQ) was given by gavage in a dose of 500 mg/kg. Mice, divided into five groups: infected non-treated; infected and PZQ-treated; infected PZQ- and SAF-treated; infected and SAF-treated; and control. After sacrificing the animals, the liver of each mouse was taken, weighed and used for histopathological examination, hydroxyproline assay and collagen determination. Results: SAF prevented myofibroblast activation at the pre-transcriptional level in a dose-dependent manner as monitored by collagen I mRNA levels (expression reduced by 40, 70 and 90 % at doses of 0.5, 1 and 2 mg/ml, respectively) or by smooth muscle α-actin (expression reduced by 70, 85 and 95 %, respectively). SAF decreased the production of collagen I by 60 % and laminin by 55 % but increased collagen III by 50 % relative to control. SAF had no effect on liver granuloma size and did not alter total hydroxyproline but altered the pattern of fibrosis by increasing collagen III and decreasing collagen I deposition. The most significant reduction in liver fibrosis was noticed in mice treated with SAF combined with PZQ. No toxic pharmacological effect was noticed during SAF treatment. Conclusion: When SAF was combined with PZQ, augmented reduction of liver fibrogenesis was achieved. The mechanism is probably through inhibition of new liver injury induced by parasite egg deposition and interruption of collagen type I synthesis with attenuation of pre-existent collagen. Keywords: Schistosomiasis mansoni, Mice, Liver fibrosis, Antifibrotic, Praziquantel

Molecular characterization of totiviruses in Xanthophyllomyces dendrorhous

BackgroundOccurrence of extrachromosomal dsRNA elements has been described in the red-yeast Xanthophyllomyces dendrorhous, with numbers and sizes that are highly variable among strains with different geographical origin. The studies concerning to the encapsidation in viral-like particles and dsRNA-curing have suggested that some dsRNAs are helper viruses, while others are satellite viruses. However, the nucleotide sequences and functions of these dsRNAs are still unknown. In this work, the nucleotide sequences of four dsRNAs of the strain UCD 67–385 of X. dendrorhous were determined, and their identities and genome structures are proposed. Based on this molecular data, the dsRNAs of different strains of X. dendrorhous were analyzed.ResultsThe complete sequences of L1, L2, S1 and S2 dsRNAs of X. dendrorhous UCD 67–385 were determined, finding two sequences for L1 dsRNA (L1A and L1B). Several ORFs were uncovered in both S1 and S2 dsRNAs, but no homologies were found for any of them when compared to the database. Instead, two ORFs were identified in each L1A, L1B and L2 dsRNAs, whose deduced amino acid sequences were homologous with a major capsid protein (5’-ORF) and a RNA-dependent RNA polymerase (3’-ORF) belonging to the Totiviridae family. The genome structures of these dsRNAs are characteristic of Totiviruses, with two overlapped ORFs (the 3’-ORF in the −1 frame with respect to the 5’-ORF), with a slippery site and a pseudoknot in the overlapped regions. These structures are essential for the synthesis of the viral polymerase as a fusion protein with the viral capsid protein through −1 ribosomal frameshifting. In the RNase protection analysis, all the dsRNAs in the four analyzed X. dendrorhous strains were protected from enzymatic digestion. The RT-PCR analysis revealed that, similar to strain UCD 67–385, the L1A and L1B dsRNAs coexist in the strains VKM Y-2059, UCD 67–202 and VKM Y-2786. Furthermore, determinations of the relative amounts of L1 dsRNAs using two-step RT-qPCR revealed a 40-fold increment of the ratio L1A/L1B in the S2 dsRNA-cured strain compared to its parental strain.ConclusionsThree totiviruses, named as XdV-L1A, XdV-L1B and XdV-L2, were identified in the strain UCD 67–385 of X. dendrorhous. The viruses XdV-L1A and XdV-L1B were also found in other three X. dendrorhous strains. Our results suggest that the smaller dsRNAs (named XdRm-S1 and XdRm-S2) of strain UCD 67–385 are satellite viruses, and particularly that XdRm-S2 is a satellite of XdV-L1A.

Targeting a pre-mRNA structure with bipartite antisense molecules modulates tau alternative splicing

Approximately 15% of human genetic diseases are estimated to involve dysregulation of alternative pre-mRNA splicing. Antisense molecules designed to alter these and other splicing events typically target continuous linear sequences of the message. Here, we show that a structural feature in a pre-mRNA can be targeted by bipartite antisense molecules designed to hybridize with the discontinuous elements that flank the structure and thereby alter splicing. We targeted a hairpin structure at the boundary between exon 10 and intron 10 of the pre-mRNA of tau. Mutations in this region that are associated with certain forms of frontotemporal dementia, destabilize the hairpin to cause increased inclusion of exon 10. Via electrophoretic mobility shift and RNase protection assays, we demonstrate that bipartite antisense molecules designed to simultaneously interact with the available sequences that immediately flank the tau pre-mRNA hairpin do indeed bind to this structured region. Moreover, these agents inhibit exon 10 splicing and reverse the effect of destabilizing disease-causing mutations, in both in vitro splicing assays and cell culture. This general bipartite antisense strategy could be employed to modulate other splicing events that are regulated by RNA secondary structure.

Autolytic activity and molecular characteristics of Staphylococcus haemolyticus strains with induced vancomycin resistance

The aim of this study was to investigate the molecular characteristics of induced vancomycin resistance in Staphylococcus haemolyticus. Autolytic properties and phenotypic characteristics of passage-selected vancomycin-resistant S. haemolyticus strains were examined. In addition, expression of autolysis-related genes (atl, lrgAB, sarA and lytS) was investigated using the RNase protection assay (RPA). The RPA results indicated that only the expression of the atl gene was significantly upregulated (2.5- to 6-fold increase) in vancomycin-intermediate and vancomycin-resistant strains. The vancomycin-resistant strains exhibited lower expression of murein hydrolase proteins and reduced autolytic activity compared with the parent strain. In addition, a reduced growth rate, cell wall thickening and higher survival rate in the presence of lysostaphin were observed in vancomycin-intermediate and vancomycin-resistant induced strains compared with the parent strain. In conclusion, altered autolytic properties, in particular upregulation of the atl gene, may contribute to vancomycin resistance in S. haemolyticus.

Detection of miRNA in Cell Cultures by Using Microchip Electrophoresis with a Fluorescence-Labeled Riboprobe

The analysis of a microRNA (miRNA), miR-222 isolated from the PC12 cell line, was performed by use of the ribonuclease (RNase) protection assay, cyanine 5 (Cy5)-labeled miR-222 riboprobe, and a Hitachi SV1210 microchip electrophoresis system, which can be used to evaluate the integrity of total RNA. The fluorescence intensity corresponding to the protected RNA fragment increased in a dose-dependent manner with respect to the complementary-strand RNA. More highly sensitive detection of miRNA by microchip electrophoresis than by conventional method using fluorescence-labeled riboprobe could be obtained in 180 s. An obvious increase in miR-222 expression induced by nerve growth factor in PC12 cells could be observed. These results clearly indicate the potential of microchip electrophoresis for the analysis of miRNA using RNase protection assay with a fluorescence-labeled riboprobe.

A greatly extended PPARGC1A genomic locus encodes several new brain-specific isoforms and influences Huntington disease age of onset†

PGC-1α has been implicated in the pathogenesis of neurodegenerative disorders. Several single-nucleotide polymorphisms (SNPs) located in two separate haplotype blocks of PPARGC1A have shown associations with Huntington's disease (HD) and Parkinson's disease, but causative SNPs have not been identified. One SNP (rs7665116) was located in a highly conserved 233 bp region of intron 2. To determine whether rs7665116 is located in an alternative exon, we performed 5'-RLM-RACE from exon 3 and discovered multiple new transcripts that initiated from a common novel promoter located 587 kb upstream of exon 2, but did not contain the conserved region harboring rs7665116. Using real-time polymerase chain reaction, RNase protection assays and northern blotting, we show that the majority of these transcripts are brain specific and are at least equally or perhaps more abundant than the reference sequence PPARGC1A transcripts in whole brain. Two main transcripts containing independent methionine start codons encode full-length brain-specific PGC-1α proteins that differ only at their N-termini (NTs) from PGC-1α, encoded by the reference sequence. Additional truncated isoforms containing these NTs that are similar to NT-PGC-1α exist. Other transcripts may encode potential dominant negative forms, as they are predicted to lack the second LXXLL motif that serves as an interaction site for several nuclear receptors. Furthermore, we show that the new promoter is active in neuronal cell lines and describe haplotypes encompassing this region that are associated with HD age of onset. The discovery of such a large PPARGC1A genomic locus and multiple isoforms in brain warrants further functional studies and may provide new tissue-specific targets for treating neurodegenerative diseases.

Selective Estrogen‐Receptor Modulators Genistein, Resveratrol, and Catechin fail to stimulate the Hepatic Expression of Estrogen‐Responsive Genes Encoding the Avian Apolipoprotein II and Vitellogenin

Apolipoprotein (apo) II and vitellogenin (VTG) I, II & III are yolk proteins expressed in the avian liver in response to estrogen during egg‐laying. Estrogenic stimulation of the hepatic expression of apo II and VTG is due to both transcriptional increase and mRNA stabilization. We had previously shown the estrogen‐responsive mRNA stabilizing factor, which stabilizes apo II mRNA to be stimulated by estrogen, and selective estrogen receptor modulators (SERMs) such as genistein, resveratrol, tamoxifen, Aroclor, chlordecone (Kepone) and hexachlorophene, but inhibited by clomiphene, toxaphene and bisphenol‐A (Ratnasabapathy et al. 53:1425–1434, 1997, Ratna and Oyemalu, C. J. Steroid Biochem. Mol Biol. 80:383–393, 2002, Ratna, W.N. & Simonelli, Life Sci. 70:1577–1589, 2002; Ratna, W.N. Life Sci 71:865–877, 2002). Reverse Transcription‐Polymerase Chain Reaction (RT‐PCR), Northern blot analysis, RNase protection assays on whole cell RNA and Western blot analysis of cytosolic extracts from livers of roosters treated with estrogen or SERMs such as genistein, resveratrol and catechin showed stimulation of transcription and expression of apo II and VTG I, II & III by estrogen but not the SERMs, demonstrating selective regulation of estrogen‐responsive genes by the latter. This work was supported by science research funds from Arnold and Marie Schwartz College of Pharmacy, Long Island University, NY.

Detection of human norovirus in cherry tomatoes, blueberries and vegetable salad by using a receptor-binding capture and magnetic sequestration (RBCMS) method

In this study, we developed a sensitive receptor-binding capture and magnetic sequestration (RBCMS) method capable of concentrating human norovirus (HuNoV) from various food samples within few hours. We found that distilled water was suitable for the elution of HuNoV from inoculated tomatoes and blueberries, and glycine buffer improved the elution of HuNoV from inoculated salad. A significant improvement in post-extraction RNA yield was achieved by sequentially heat-releasing and column-extracting over either technique alone. The viral recovery of the RBCMS method was significantly higher than both the same-day PEG method (90 min PEG precipitation) and the two-day PEG method (overnight PEG precipitation) with a recovery rate of 8.75%, 1.03% and 5.40%, respectively. The detection limit of HuNoV by RBCMS method was significantly improved to 0.056 RTU. The estimated minimal concentration powers (MCPs) were 6.11, 30.48, and 63.60-fold for the same-day PEG, two-day PEG, and RBCMS methods, respectively. RNase protection assay suggests that the viral genome was protected from RNase attack by remaining within the viral capsid. The signal detected by the RBCMS method might be more biologically relevant, as it requires both intact viral capsid to bind to HBGA receptors and the presence of viral genome to be amplified. Overall, the RBCMS method takes significantly less time than current PEG precipitation methods, recovers a higher yield of HuNoV from various food samples, and hence exhibits higher sensitivity.

Aclarubicin enhances tumor necrosis factor‐related apoptosis‐inducing ligand‐induced apoptosis through death receptor 5 upregulation

Anthracycline drugs are potent anti-tumor agents. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a death ligand with promising anti-cancer effects. However, some tumor types develop resistance to TRAIL. We examined the effect of aclarubicin (ACR), an anthracycline, in combination with TRAIL. The combination of TRAIL and ACR synergistically induced apoptosis in human acute lymphoblastic leukemia Jurkat cells and human lung cancer A549 cells. In contrast, another anthracycline, doxorubicin (DOX), only slightly sensitized Jurkat cells and A549 cells to TRAIL-induced apoptosis, with weaker enhancement of death receptor 5 (DR5) expression than ACR. The RNase protection assay, real time RT-PCR and western blot demonstrated that ACR upregulated the expression of a TRAIL receptor, DR5. Caspase inhibitors and dominant negative DR5 efficiently reduced the apoptotic response to the treatment with ACR and TRAIL, indicating that the combined effect depends on caspase activities and the interaction between TRAIL and its receptor. ACR but not DOX increased the activity of the DR5 gene promoter in Jurkat cells carrying a mutation in the p53 gene, suggesting that ACR upregulates DR5 expression through p53-independent transcription. These results suggest the combination of TRAIL and ACR to be a promising treatment for malignant tumors.