Evidence of efficient stop codon readthrough in four mammalian genes.

Stop codon readthrough is used extensively by viruses to expand their gene expression. Until recent discoveries in Drosophila, only a very limited number of readthrough cases in chromosomal genes had been reported. Analysis of conserved protein coding signatures that extend beyond annotated stop codons identified potential stop codon readthrough of four mammalian genes. Here we use a modified targeted bioinformatic approach to identify a further three mammalian readthrough candidates. All seven genes were tested experimentally using reporter constructs transfected into HEK-293T cells. Four displayed efficient stop codon readthrough, and these have UGA immediately followed by CUAG. Comparative genomic analysis revealed that in the four readthrough candidates containing UGA-CUAG, this motif is conserved not only in mammals but throughout vertebrates with the first six of the seven nucleotides being universally conserved. The importance of the CUAG motif was confirmed using a systematic mutagenesis approach. One gene, OPRL1, encoding an opiate receptor, displayed extremely efficient levels of readthrough (∼31%) in HEK-293T cells. Signals both 5' and 3' of the OPRL1 stop codon contribute to this high level of readthrough. The sequence UGA-CUA alone can support 1.5% readthrough, underlying its importance.

Quantitative detection and identification of Naegleria spp. in various environmental water samples using real-time quantitative PCR assay.

Naegleria spp. is a free-living amoeba that can be found in various aquatic environments. There are some Naegleria spp. that can cause fatal infections in animals and humans, and the most important source of infection is through direct water contact. In this study, a real-time quantitative PCR was developed to detect and quantify the Naegleria spp. in various environmental water samples. The water samples were taken from rivershed, water treatment plants, and thermal spring recreation areas. The total detection rate was 4.0% (7/176) for Naegleria spp. The percentages of samples containing Naegleria spp. from river water, raw drinking water, and thermal spring water were 0% (0/100), 10.7% (3/28) and 8.3% (4/48), respectively. The concentration of Naegleria spp. in detected positive raw drinking water and thermal spring water samples was in the range of 3.9-12.6 and 1.1-24.2 cells/L, respectively. The identified species included Naegleria australiensis, Naegleria lovaniensis, and Naegleria spitzbergeniensis. The presence of Naegleria spp. in various aquatic environments is considered a potential public health threat.

Stimulation of -1 programmed ribosomal frameshifting by a metabolite-responsive RNA pseudoknot.

Specific recognition of metabolites by functional RNA motifs within mRNAs has emerged as a crucial regulatory strategy for feedback control of biochemical reactions. Such riboswitches have been demonstrated to regulate different gene expression processes, including transcriptional termination and translational initiation in prokaryotic cells, as well as splicing in eukaryotic cells. The regulatory process is usually mediated by modulating the accessibility of specific sequence information of the expression platforms via metabolite-induced RNA conformational rearrangement. In eukaryotic systems, viral and the more limited number of cellular decoding -1 programmed ribosomal frameshifting (PRF) are commonly promoted by a 3' mRNA pseudoknot. In addition, such -1 PRF is generally constitutive rather than being regulatory, and usually results in a fixed ratio of products. We report here an RNA pseudoknot capable of stimulating -1 PRF whose efficiency can be tuned in response to the concentration of S-adenosylhomocysteine (SAH), and the improvement of its frameshifting efficiency by RNA engineering. In addition to providing an alternative approach for small-molecule regulation of gene expression in eukaryotic cells, such a metabolite-responsive pseudoknot suggests a plausible mechanism for metabolite-driven translational regulation of gene expression in eukaryotic systems.

An intermolecular RNA triplex provides insight into structural determinants for the pseudoknot stimulator of -1 ribosomal frameshifting.

An efficient -1 programmed ribosomal frameshifting (PRF) signal requires an RNA slippery sequence and a downstream RNA stimulator, and the hairpin-type pseudoknot is the most common stimulator. However, a pseudoknot is not sufficient to promote -1 PRF. hTPK-DU177, a pseudoknot derived from human telomerase RNA, shares structural similarities with several -1 PRF pseudoknots and is used to dissect the roles of distinct structural features in the stimulator of -1 PRF. Structure-based mutagenesis on hTPK-DU177 reveals that the -1 PRF efficiency of this stimulator can be modulated by sequential removal of base-triple interactions surrounding the helical junction. Further analysis of the junction-flanking base triples indicates that specific stem-loop interactions and their relative positions to the helical junction play crucial roles for the -1 PRF activity of this pseudoknot. Intriguingly, a bimolecular pseudoknot approach based on hTPK-DU177 reveals that continuing triplex structure spanning the helical junction, lacking one of the loop-closure features embedded in pseudoknot topology, can stimulate -1 PRF. Therefore, the triplex structure is an essential determinant for the DU177 pseudoknot to stimulate -1 PRF. Furthermore, it suggests that -1 PRF, induced by an in-trans RNA via specific base-triple interactions with messenger RNAs, can be a plausible regulatory function for non-coding RNAs.

Triplex structures in an RNA pseudoknot enhance mechanical stability and increase efficiency of -1 ribosomal frameshifting.

Many viruses use programmed -1 ribosomal frameshifting to express defined ratios of structural and enzymatic proteins. Pseudoknot structures in messenger RNAs stimulate frameshifting in upstream slippery sequences. The detailed molecular determinants of pseudoknot mechanical stability and frameshifting efficiency are not well understood. Here we use single-molecule unfolding studies by optical tweezers, and frameshifting assays to elucidate how mechanical stability of a pseudoknot and its frameshifting efficiency are regulated by tertiary stem-loop interactions. Mechanical unfolding of a model pseudoknot and mutants designed to dissect specific interactions reveals that mechanical stability depends strongly on triplex structures formed by stem-loop interactions. Combining single-molecule and mutational studies facilitates the identification of pseudoknot folding intermediates. Average unfolding forces of the pseudoknot and mutants ranging from 50 to 22 picoNewtons correlated with frameshifting efficiencies ranging from 53% to 0%. Formation of major-groove and minor-groove triplex structures enhances pseudoknot stem stability and torsional resistance, and may thereby stimulate frameshifting. Better understanding of the molecular determinants of frameshifting efficiency may facilitate the development of anti-virus therapeutics targeting frameshifting.

Seasonal Distribution and Genotyping of Antibiotic Resistant Strains of ListeriaInnocua Isolated from A River Basin Categorized by ERIC-PCR.

Listeria innocua retains many conserved homologous domains with Listeria monocytogenes, which is a food-borne and water-borne diarrhea-causing bacterium. Studies of antimicrobial resistance in L. innocua showed that this microbe is more prone to acquire resistance than other bacteria in the genus Listeria. However, little is known about the seasonal population distribution and antimicrobial resistance patterns of L. innocua in natural water environments. The aims of the study were: (1) to investigate the occurrence of L. innocua isolates in a subtropical watershed and reconstruct the population structure and (2) to analyze the antibacterial resistance patterns of the identified L. innocua isolates according to ERIC type. A total of 288 water samples was collected from the Puzi River basin (23°28' N, 120°13' E) between March 2014 and March 2015, and 36 L. innocua isolates were recovered from 15 positive water samples. With regard to seasonal variation, L. innocua was only detected in the spring and summer. Eighteen enterobacterial repetitive intergenic consensus (ERIC)-PCR types were identified, and two genogroups with four subgroups were reconstructed in a minimum spanning tree. Isolates from different sampling areas that were located near each other were genetically different. All L. innocua isolates (including 41.7% of the multidrug-resistant (MDR) isolates) were resistant to oxacillin and showed high minimum inhibitory concentrations of tetracycline. These findings demonstrate the seasonal variations and differing geographical distributions of L. innocua in this subtropical water environment, as well as the existence of strong population structures and MDR and antimicrobial resistance patterns. Phylogenetic analysis based on ERIC-type showed that the Cluster A isolates were resistant to more antibiotics, and two types, ERIC8 and ERIC15 were multidrug resistant. The more commonly detected types, such as ERIC1 and ERIC12, were also more likely to be resistant to two or more antibiotics. Close monitoring of drug resistance in environmental L. innocua is warranted due to its potential for transferring antimicrobial resistance determinants to pathogenic Listeria.

In vitro activities of various piperacillin and sulbactam combinations against bacterial pathogens isolated from Intensive Care Units in Taiwan: SMART 2004 programme data.

We investigated the in vitro activity of various piperacillin and sulbactam combinations against Gram-negative bacterial isolates from Intensive Care Units (ICUs) in Taiwan. Antimicrobial susceptibility testing of 1030 bacterial isolates recovered from ICUs of nine major teaching hospitals was performed using the agar dilution method. Sulbactam was added to piperacillin either at a fixed sulbactam concentration of 4 mg/L and 8 mg/L or at a piperacillin:sulbactam ratio of 2:1 and 4:1. Piperacillin/sulbactam at a ratio of 2:1 or a fixed 8 mg/L concentration of sulbactam had better activities against Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis and Serratia marcescens than other piperacillin/sulbactam formulations. For Pseudomonas aeruginosa, piperacillin/sulbactam (2:1 or 4:1 ratios) had MIC(90) values (minimum inhibitory concentration for 90% of the organisms) of 64 mg/L (>90% susceptibility) compared with 64 mg/L for cefoperazone/sulbactam (68% susceptibility) and 128 mg/L for piperacillin/tazobactam (82% susceptibility). For Acinetobacter baumannii, both piperacillin/sulbactam (either 2:1 ratio or a fixed 8 mg/L sulbactam) and cefoperazone/sulbactam were the most potent agents. Adding sulbactam to piperacillin resulted in increased susceptibility rates among piperacillin-resistant P. aeruginosa (53-57% in either 2:1 or 4:1 ratios) and A. baumannii (38-46% in either 2:1 ratio or a fixed 8 mg/L concentration of sulbactam) isolates. Results of susceptibility tests with piperacillin/sulbactam are dependent on the method used. Piperacillin/sulbactam combinations possessed better in vitro activities than piperacillin alone or piperacillin/tazobactam against P. aeruginosa and A. baumannii.

Empirical testing of modified Salmonella MLST in aquatic environmental samples by in silico analysis.

Multilocus sequence typing (MLST) is an approach for prediction of Salmonella servoar and eBRUST groups (eBGs) based on seven typing scheme of housekeeping genes. Up to date, >220.000 allelic profiles and 65,973 Salmonella strains have been established in the MLST database. Several studies have modified MLST method with fewer targeted housekeeping genes for the purpose of economy and efficiency. Nevertheless, no study has conducted systematically to evaluate the correlation between the numbers of housekeeping genes targeted and the accuracy of prediction rate. In this study, we aimed to tackle this problem by extracting data from the MLST database as a whole using the software RStudio. Our results indicated that as the numbers of genes in MLST scheme increased, the accuracy of the eBGs prediction rate increased and reached 100% when the gene numbers are greater than or equal to 5. To examine the applicability of the approach, 395 environmental water samples were subjected to this study. A set of 52 Salmonella enterica isolates was initially used to develop MLST targeting seven housekeeping genes. A total of 29 sequence types, including 11 new sequence types were found among the 52 sequenced isolates that differentiated into 19 serotypes. Moreover, two novel sequence types did not belong to current classification. Our results show that the outcome in the three-gene sequence typing (aroC, hisD, and purE) was as accurate as in the seven-gene sequence typing for prediction of environmental Salmonella isolates. Our data suggested that this five-gene and reduced gene-number sequence-typing scheme can serve as an alternative modified MLST when effectiveness and financial management were the concerns.

Efficient large-scale preparation and purification of short single-stranded RNA oligonucleotides.

Sequence-specific RNA recognition by RNA-binding proteins plays a crucial role in the post-translational regulation of gene expression. Biophysical and biochemical studies help to unravel the principles of sequence-specific RNA recognition, but the methods used require large amounts of single-stranded RNA (ssRNA). Here we present a fast and robust method for large-scale preparation and purification of short ssRNA oligonucleotides for biochemical, biophysical, and structural studies. We designed an efficiently folding, self-cleaving hammerhead (HH) ribozyme to prepare ssRNA oligonucleotides. Hammerhead ribozyme RNAs self-cleave with over 95% efficiency during in vitro transcription as a function of magnesium concentration to produce high yields of the desired ssRNA products. The resulting ssRNAs can be purified from crude transcription reactions by denaturing anion-exchange chromatography and then desalted by weak anion-exchange chromatography using volatile ammonium bicarbonate buffer solutions. The ssRNA oligonucleotides produced this way are homogenous, as judged by mass spectrometry (MS), and are suitable for biochemical and biophysical studies. Moreover, for high-resolution NMR structure determination of RNA-protein complexes, our protocol enables efficient preparation of ssRNA oligonucleotides with various isotope-labeling schemes which are not commercially available.

Assessment of Legionella pneumophila in recreational spring water with quantitative PCR (Taqman) assay.

Legionella spp. are common in various natural and man-made aquatic environments. Recreational hot spring is frequently reported as an infection hotspot because of various factors such as temperature and humidity. Although polymerase chain reaction (PCR) had been used for detecting Legionella, several inhibitors such as humic substances, calcium, and melanin in the recreational spring water may interfere with the reaction thus resulting in risk underestimation. The purpose of this study was to compare the efficiencies of conventional and Taqman quantitative PCR (qPCR) on detecting Legionella pneumophila in spring facilities and in receiving water. In the results, Taqman PCR had much better efficiency on specifying the pathogen in both river and spring samples. L. pneumophila was detected in all of the 27 river water samples and 45 of the 48 hot spring water samples. The estimated L. pneumophela concentrations ranged between 1.0 × 10(2) and 3.3 × 10(5) cells/l in river water and 72.1-5.7 × 10(6) cells/l in hot spring water. Total coliforms and turbidity were significantly correlated with concentrations of L. pneumophila in positive water samples. Significant difference was also found in water temperature between the presence/absence of L. pneumophila. Our results suggest that conventional PCR may be not enough for detecting L. pneumophila particularly in the aquatic environments full of reaction inhibitors.

Application of molecular biological techniques to analyze Salmonella seasonal distribution in stream water.

Salmonella is a leading cause of waterborne diseases. Salmonella can survive for a long time in aquatic environments, and its persistence in the environment is of great concern to public health. Nonetheless, the presence and diversity of Salmonella in the aquatic environments in most areas remain relatively unknown. In this study, we examined three analytical processes for an optimum Salmonella detection method, and the optimized method was used to evaluate seasonal variations of Salmonella in aquatic environments. In addition, Salmonella strains were isolated by selective culture medium to identify the serotypes by biochemical testing and serological assay, and to identify the genotypes by pulsed-field gel electrophoresis based on the genetic patterns. A total of 136 water samples were collected in the study area in 9 months. Forty-one (30.1%) samples were found to contain Salmonella-specific invA gene, and most (24/41) of the detections occurred in summer. The serovars of Salmonella enterica were identified, including Bareilly, Isangi, Newport, Paratyphi B var. Java, Potsdam and Typhimurium.

Regulation of programmed ribosomal frameshifting by co-translational refolding RNA hairpins.

RNA structures are unwound for decoding. In the process, they can pause the elongating ribosome for regulation. An example is the stimulation of -1 programmed ribosomal frameshifting, leading to 3' direction slippage of the reading-frame during elongation, by specific pseudoknot stimulators downstream of the frameshifting site. By investigating a recently identified regulatory element upstream of the SARS coronavirus (SARS-CoV) -1 frameshifting site, it is shown that a minimal functional element with hairpin forming potential is sufficient to down-regulate-1 frameshifting activity. Mutagenesis to disrupt or restore base pairs in the potential hairpin stem reveals that base-pair formation is required for-1 frameshifting attenuation in vitro and in 293T cells. The attenuation efficiency of a hairpin is determined by its stability and proximity to the frameshifting site; however, it is insensitive to E site sequence variation. Additionally, using a dual luciferase assay, it can be shown that a hairpin stimulated +1 frameshifting when placed upstream of a +1 shifty site in yeast. The investigations indicate that the hairpin is indeed a cis-acting programmed reading-frame switch modulator. This result provides insight into mechanisms governing-1 frameshifting stimulation and attenuation. Since the upstream hairpin is unwound (by a marching ribosome) before the downstream stimulator, this study's findings suggest a new mode of translational regulation that is mediated by the reformed stem of a ribosomal unwound RNA hairpin during elongation.

Diversity and seasonal impact of Acanthamoeba species in a subtropical rivershed.

This study evaluated the presence of Acanthamoeba species in the Puzih River watershed, which features typical subtropical monsoon climate and is located just above the Tropic of Cancer in Taiwan. The relationship between the seasonal and geographical distributions of Acanthamoeba species in this rivershed was also investigated. Acanthamoeba species were detected in water samples using the amoebal enrichment culture method and confirmed by PCR. A total of 136 water samples were included in this study, 16 (11.7%) of which contained Acanthamoeba species. Samples with the highest percentage of Acanthamoeba (32.4%) were obtained during the summer season, mainly from upstream areas. The identified species in the four seasons included Acanthamoeba palestinensis (T2), Acanthamoeba sp. IS2/T4 (T4), Acanthamoeba lenticulata (T5), Acanthamoeba hatchetti (T11), Acanthamoeba healyi (T12), and Acanthamoeba jacobsi (T15). The most frequently identified Acanthamoeba genotype was T4 (68.7%). Acanthamoeba genotype T4 is responsible for Acanthamoeba keratitis and should be considered for associated human health risk potential in the rivershed.

Nationwide surveillance of antimicrobial resistance among non-fermentative Gram-negative bacteria in Intensive Care Units in Taiwan: SMART programme data 2005.

A nationwide surveillance of the antimicrobial susceptibilities of glucose non-fermentative Gram-negative bacteria isolates was conducted from 1 September 2005 to 30 November 2005 in Taiwan. A total of 456 isolates were recovered from patients hospitalised in the Intensive Care Units (ICUs) of ten major teaching hospitals. Rates of resistant pathogens, such as ciprofloxacin-resistant Pseudomonas aeruginosa (19%) and imipenem-resistant Acinetobacter baumannii (25%), were higher than those reported in 2000 (8% and 22%, respectively). Increased rates of isolates with resistant phenotypes correlated with prolonged length of ICU stay (48h to 7 days) for ceftazidime-non-susceptible P. aeruginosa (20.0% and 29.7%, respectively), imipenem-non-susceptible P. aeruginosa (4.0% and 13.5%, respectively) and imipenem-non-susceptible A. baumannii (15.4% and 29.8%, respectively), but not for ciprofloxacin-resistant P. aeruginosa. Alarming rates of emergence of extensively drug-resistant (XDR) A. baumannii (15%) and XDR P. aeruginosa (1.8%) were found, particularly among those isolates that were not susceptible to tigecycline and colistin. Interhospital dissemination of some clones of XDR A. baumannii in different ICUs was also noted. This study illustrates the crucial nature of continuous nationwide surveillance of resistant pathogens and implementation of effective strategies for ICU infection control and antibiotic restriction.

cDNA cloning, biological and immunological characterization of the alkaline serine protease major allergen from Penicillium chrysogenum.

BACKGROUND: Penicillium chrysogenum (Penicillium notatum) is a prevalent airborne Penicillium species. A 34-kD major IgE-reacting component from P. chrysogenum has been identified as an alkaline serine protease (Pen ch 13, also known as Pen n 13 before) by immunoblot and N-terminal amino acid sequence analysis. METHODS: In the present study, Pen ch 13 was further characterized in terms of cDNA cloning, protein purification, enzymatic activity, histamine release and IgE cross-reactivity with alkaline serine protease allergens from two other prevalent fungal species--P. citrinum (Pen c 13) and Aspergillus flavus (Asp fl 13). RESULTS: A 1,478-bp cDNA (Pen ch 13) that encodes a 398-amino-acid alkaline serine protease from P. chrysogenum was isolated. This fungal protease has pre- and pro-enzyme sequences. The previously determined N-terminal amino acid sequence of the P. chrysogenum 34-kD major allergen is identical to that of residues 116-125 of the cDNA. Starting from Ala116, the deduced amino acid sequence (283 residues) of the mature alkaline serine protease has a calculated molecular mass of 28.105 kD with two cysteines and two putative N-glycosylation sites. It has 83 and 49% sequence identity with the alkaline serine proteases from P. citrinum and A. fumigatus, respectively. The recombinant Pen ch 13 was recovered from inclusion bodies and isolated under denaturing condition. This recombinant protein reacted with IgE antibodies in serum from an asthmatic patient and with monoclonal antibodies (PCM8, PCM10, PCM39) that reacted with the 34-kD component from P. chrysogenum. The N-terminal amino acid sequence of the purified native Pen ch 13 is identical to that determined previously for the 34-kD major allergen in crude P. chrysogenum extracts. The purified native Pen ch 13 has proteolytic activity with casein as the substrate at pH 8.0. This enzymatic activity was inhibited by phenylmethylsulfonyl fluoride or diethylpyrocarbonate. Pen ch 13 was also able to degrade gelatin and collagen but not elastin. Basophils from 5 asthmatic patients released histamine (12-73%) when exposed to the purified Pen ch 13. In ELISA (enzyme-linked immunosorbent assay) experiments, IgE for Pen ch 13 was able to compete with purified Pen ch 13, Pen c 13 or Asp fl 13 in a dose-related manner. CONCLUSIONS: These results demonstrated that the 34-kD major allergen of P. chrysogenum is an alkaline serine protease. These results also indicated that atopic patients primarily sensitized by either of these prevalent fungal species may develop allergic symptoms by exposure to other environmental fungi due to cross-reacting IgE antibodies against this protease.