Natural habitats have the ability to protect coastal communities against the impacts of waves and storms, yet it is unclear how different habitats complement each other to reduce those impacts. Here, we investigate the individual and combined coastal protection services supplied by live corals on reefs, seagrass meadows, and mangrove forests during both non-storm and storm conditions, and under present and future sea-level conditions. Using idealized profiles of fringing and barrier reefs, we quantify the services supplied by these habitats using various metrics of inundation and erosion. We find that, together, live corals, seagrasses, and mangroves supply more protection services than any individual habitat or any combination of two habitats. Specifically, we find that, while mangroves are the most effective at protecting the coast under non-storm and storm conditions, live corals and seagrasses also moderate the impact of waves and storms, thereby further reducing the vulnerability of coastal regions. Also, in addition to structural differences, the amount of service supplied by habitats in our analysis is highly dependent on the geomorphic setting, habitat location and forcing conditions: live corals in the fringing reef profile supply more protection services than seagrasses; seagrasses in the barrier reef profile supply more protection services than live corals; and seagrasses, in our simulations, can even compensate for the long-term degradation of the barrier reef. Results of this study demonstrate the importance of taking integrated and place-based approaches when quantifying and managing for the coastal protection services supplied by ecosystems. Increasingly, coastal and marine ecosystems are perceived, promoted and used as alternatives to traditional coastal protection structures –4. The hThe typical approach of using a single habitat to only protect coastal regions against specific forcing conditions merely treats natural systems as alternatives to traditional mono-functional hard coastal structures, and it underutilizes the potential of all the habitats present on the entire seascape. Indeed, this approach overlooks the fact that natural systems can help protect coasts from a host of hazards that occur under different forcing conditions. For example, while coral reefs and wetlands can reduce the impacts of tsunamis, storm surge and storm waves ,18,19, tHere, using the Caribbean country of Belize as a characteristic setting , we quanTropical seascapes generally have 3 distinct habitat types: coral reefs, seagrass meadows and mangrove forests. The coral reef may be a barrier reef, a fringing reef or an atoll, and is covered by coral colonies. Seagrass meadows generally grow shoreward of the reef, and they cover as much of the seabed as light and water quality conditions allow. Mangrove forests occupy the nearshore intertidal and coastal regions, extending from mean lower low water (MLLW) to well landward of the mean higher high water (MHHW) mark.We quantify the difference in coastal protection services supplied by habitats living in two idealized tropical 1-D seascapes: one bounded offshore by a barrier reef and the other by a fringing reef Figs ; Table 1Thalassia testudinum) meadows :,61:Db innd b = 1 ,60.Df in Eq . Consequently, wave heights transmitted by the reefs and observed immediately shoreward of the reef flats are also identical in the two systems. Therefore, results presented in this section apply to both systems.We find that reefs are natural breakwaters , but theAs mean sea level increases, live reefs maintain their relative depth and coral cover, while dead reefs degrade further and do not grow. Consequently, live reefs provide the same amount of wave attenuation in present and future scenarios , Table 3Ht can be computed as Ht = A1 tanh [Ho / A2] . The presence and condition of corals on the reef, or the reef’s health, control the values of the maximum transmitted wave height, A1, and of the minimum offshore wave height filtered by the reef, A2.These results suggest that reefs act as dynamic low-pass filters for waves ,83 Fig , where tAnalysis of energy flux dissipation due to breaking and bottom friction indicates that, in the absence of corals, nearly all of the incoming wave energy is dissipated by breaking on the reef face and rim . In the 2, than what would have been observed if the reef were alive  to dissipate their energy via bottom friction. Also, in both profiles, dissipation occurs via bottom friction; waves barely break in the lagoons.We find similar results for the fringing reef profile. Under present day conditions, nearshore waves and bed shear stresses shoreward of a dead fringing reef are, at 0.23 m and 0.70 N/mIn both the barrier and fringing reef profiles, once waves pass the reef, they encounter and propagate through seagrass meadows that further attenuate the incoming waves, resulting in lower wave heights and bed stresses nearshore than the no live habitat and the coral only scenarios .2, which is below the threshold for sediment motion  shoreward of the mangrove forest, and to reduce mud bed scour in the mangrove forest. We also quantify the combined and individual importance of live corals and seagrasses at reducing the potential for sand erosion just offshore of the mangrove forest.We find that, in the absence of habitats, differences in the geomorphology of the two profiles yield significant differences in inundation and sediment loss. Because the barrier reef lagoon is so long, the hurricane generates a 1.2 m surge in the barrier reef profile compared to only a 0.5 m surge in the fringing reef profile. Also, after breaking over the reefs, hurricane waves re-generate to a maximum height of 2.1 m in the barrier reef lagoon compared to only 0.5 m in the fringing reef lagoon . TherefoExamining the role of the different habitats, we find that in both profiles mangroves alone systematically reduce inundation levels and mud bed scour volumes, while live corals and seagrasses play a relatively minor role Figs  Figs.3/m , under present day and future (+1 m) sea-level conditions, and during both non-storm and storm conditions. We find that together, these habitats substantially moderate incoming wave energy, inundation levels and loss of mud sediment ,18,51,85Another major finding is that the ability of different habitats to provide coastal protection services varies as a function of local geomorphology, or setting, and forcing conditions ,89. DuriOur results also illustrate the importance of taking into account the presence of live corals when computing the ecosystem service function of reefs ,42,83, sFinally, results presented herein also illustrate the importance of clearly identifying the metrics used to quantify protection services. Live corals in the two profiles supply the same level of protection if the service  is measured at the shoreward edge of the reef. However, if the service is measured nearshore, live corals in the fringing and barrier reef profiles do not provide the same amount of services. The same argument can be made for seagrass meadows far from the coast . This poIn conclusion, we demonstrate the importance of taking an integrated approach to assessing the protection services supplied by marine habitats when crafting conservation or restoration plans. The merits of such an integrated approach has been established for fisheries management ,94. Our S1 FigLeft: Complete wave record from WW3 grid point. Center: Distribution of wave height and wave period. Right: Wave rose of the complete record. Wave rose of non-storm record is similar.(EPS)Click here for additional data file.S2 Fig2>0.8). Right: scatter plot of frictional dissipation; solid line represents an approximation of the best fit (R2>0.8). Results presented for different combinations of coral presence and absence, under present and future conditions.Left: Average ratio of breaking and frictional dissipation over the reef profiles. Center: scatter plot of breaking dissipation; solid line represents an approximation of the best fit (R(EPS)Click here for additional data file.S3 FigBox plots of wave height (top subplots) and bed shear stress (bottom subplots) values computed as a function of the presence or absence of live coral or seagrasses. Circles represent mean values.(EPS)Click here for additional data file.S4 FigBox plots of wave height values computed in both profiles in the presence and absence of a portion or the whole meadow. The offshore meadow is the one closest to the reef. The nearshore meadow is the one closest to the shore. In the fringing reef, the seagrass meadow was split in two equidistant meadows. Circles represent mean values.(EPS)Click here for additional data file.S5 FigBox plots of wave height (top subplots) and bed scour (bottom subplots) values computed as a function of different combination of presence or absence of live coral, seagrasses and mangroves. Circles represent mean values.(EPS)Click here for additional data file.S6 Fig(EPS)Click here for additional data file.S7 FigSurge attenuation by mangroves is ignored.(EPS)Click here for additional data file.S8 FigSurge attenuation by mangroves is ignored.(EPS)Click here for additional data file.


There is an error in the affiliation of the last author. Pingyu Zhou is also affiliated with Shanghai Skin Disease Hospital, Clinical School of Anhui Medical University, Shanghai, People′s Republic of China.


Sulfolobus solfataricus. More than 40% of the targeted mRNA could be cleaved, as demonstrated by quantitative polymerase chain reaction. Cleavage of the mRNA was visualized by northern analyses and cleavage sites were mapped. In vitro, the same substrates were cleaved by the purified CRISPR-associated CMR complex from Sulfolobus solfataricus. The in vivo system was also re-programmed to knock down mRNA of a selected chromosomal gene  using an artificial miniCRISPR locus. With a single complementary spacer, ∼50% reduction of the targeted mRNA and of corresponding intracellular protein activity was achieved. Our results demonstrate in vivo cleavage of mRNA in a prokaryote mediated by small RNAs  and the re-programming of the system to silence specific genes of interest.The recently discovered clustered regularly interspaced short palindromic repeat (CRISPR)-mediated virus defense represents an adaptive immune system in many bacteria and archaea. Small CRISPR RNAs cause cleavage of complementary invading nucleic acids in conjunction with an associated protein or a protein complex. Here, we show CRISPR-mediated cleavage of mRNA from an invading virus in the hyperthermophilic archaeon  In vivo activity has been demonstrated in six model systems of bacteria and in three model organisms of archaea, proving the effective CRISPR-mediated immunity against DNA of invading viruses or plasmids by type I, type II and type IIIA CRISPR systems . For growth of the uracil auxotrophic mutant M18, uracil at a final concentration of 0.0125 mg/ml was added to the media. Electro-competent cells were prepared as previously described  to OD (600 nm) = 1. Cells were then inoculated into tryptone (BD Biosciences) 0.1% (w/v) media with a reduced sucrose concentration (0.04%) until OD (600) = 0.2–0.3. Expression of the protospacer constructs D63-7U, D63-HA and A53* (control) was induced through the addition of 20% (m/v) arabinose . After 4 h, samples of the cultures were taken for DNA and RNA extraction. In each experiment, at least three biological replicates were sampled and analyzed.S. solfataricus cells were performed with a basic phenol-chloroform extraction with TENS and TENST (mirVana™ kit (Ambion®), followed by DNAseI treatment (Promega™), following manufacturer’s instructions. RNA quality and purity were measured by NanoDrop  and agarose gel electrophoresis, respectively. Absence of DNA contamination in the RNA preparation was verified by polymerase chain reaction (PCR) amplification of 100 ng of DNAse I-treated RNA before further analysis. The cDNA reaction was carried out using 1 µg of RNA and M-MuLV Reverse Transcriptase  following the manufacturer’s instructions. The cDNA was then purified using columns  and was used after dilution to ∼ 20 ng/µl for real-time PCR quantification.DNA extractions from nd TENST  as lysisS. solfataricus strain P2 and P1) was placed into ORF 406 of the conjugative plasmid pNOB8  following the manufacturer’s recommendations , yielding pEntryA53* (with match to spacer A53 in strain P2 but no match in strain P1). The recombinant plasmid was further mutated using the Overlapping Extension-PCR (OE PCR) method  in the presence or absence of 100 µM ATP and pre-incubated at room temperature for 10 min before the addition of 500 nM 5′-32P-end-labeled synthetic target RNA to the reaction mix . The reaction was further incubated at 75°C for 10 min, then stopped with EDTA and analyzed by gel electrophoresis and phosphor imaging.eviously . In brieet al. sequence had an extra A at position 32 that was introduced by error. This difference has no effect on the cleavage activity observed.RNA oligonucleotides were chemically synthesized (Integrated DNA Technologies). The D63 crRNA guide sequence shown here differs by one base from that used previously . The oricrD63: 5′-AUUGAAAGUUUCGUAUAAGGACCAGAACGGCAAUACCCAAACUGU-3′D63-7U: 5′-CGAAUCAAUAGUUUGGGUAUUGUUGUUUUGGUUUUUAUAUGAAAGAGAGC-3′D63-HA: 5′-AUAGUUUGGGUAUUGUUGUUUUGGUUUUUAUAUGAAACUUUCAAUGAG-3′crBG-HA: 5′-AUUGAAAGAUGUUAUACAUAUGCCUACGGGAAAGUUCAAAGCUUAGG-3′BG-HA: 5′-CCUAAGCUUUGAACUUUCCCGUAGGCAUAUGUAUAACAUCAUUCAAG-3′The sequences are:S. solfataricus can recognize and trigger degradation of a protospacer located on viral DNA. This study also showed that the efficiency of plaque formation decreases with an increasing number of mismatches between spacer and protospacer .We used qPCR on reverse-transcribed total RNA to analyze the amount of cleaved mRNA with two specific primer pairs. The first PCR product Q1 in  covered Supplementary Table S1B).The amount of cleaved over uncleaved mRNA is shown in in vivo RNA cleavage mediated by the crRNA D63, northern blot analysis . Beside the uncut full-length mRNA that was the only band in the northern hybridization detected in the control, an additional smaller band of the size expected for mRNA cut in the protospacer site was detected for D63-7U and D63-HA transfectants, whose abundance was in accordance with that measured by qPCR (Supplementary Table S1A). The 3′ end of the mRNA (downstream of the protospacer) could not be visualized in northern analyses probably because of fast degradation in the cell. More accurate lengths of the cut mRNA species generated through RNA interference in the transformed cells were determined in RACE experiments. About two-thirds of all cut sites in D63-HA and D63-7U transfectants were mapped inside the protospacer regions of the mRNA or in close vicinity, i.e. 3 nt inside the region of the 8-nt handle region, with an accumulation of mRNA ending at AU and UU sites . Other mRNA ends (10 of 25 for D63-7U and 6 of 18 for D63-HA) were located toward the 5′end of the full mRNA, indicating more progressively degraded products (from 3′ to 5′ direction). No cuts were located by RACE beyond the protospacer toward the 3′ end of the full mRNA, strongly supporting the conclusion that initial cutting of the mRNA occurred inside the protospacer and then progressed toward the 5′ end.For confirmation of analysis B and rapin vitro assays were performed as described earlier . These data confirm our in vivo results and show that the CMR complex does not have an absolute requirement for a mismatch opposite the 5′-handle. Interestingly, in vitro cleavage of the D63-HA substrate was not stimulated by ATP, whereas cleavage of the other substrate was, which is presumably due to the effect of complete base pairing between the 5′-handle of the crRNA D63 and the D63-HA target .To investigate if CRISPR-mediated RNA interference can also be programmed to degrade mRNA of specific chromosomal genes, artificial miniCRISPR loci with and without a spacer targeting the mRNA of β-galactosidase  were constructed and expressed in  locus D  of the s locus D A and D.  locus D . On tranin vitro results with purified CMR complexes in two different archaea  and by grant [BB/K000314/1] from the Biotechnology and Biological Sciences Research Council (to M.F.W.). Funding for open access charge: Austrian Science Fund.Conflict of interest statement. None declared.


Self-rated health is a brief measure for general health. It is a comprehensive and sensitive index for prediction of health in future. Due to the high internet usage in medical students, the current study designed to evaluate the self-rated health (SRH) in relationship with internet addiction risk factors in medical students.This cross sectional study conducted on 254 students of Qom University of Medical Sciences 2014. Participants selected by two stage sampling method including stratified and simple random sampling. The Young’s questionnaire of internet addiction and SRH question used for data collection. Chi-square, t-test, and logistic regression used in data analysis.p=0.002). Using internet for Entertainment, using private Email and chat rooms were the most important predictors of affecting to internet addiction. Moreover, internet addiction is the most predictors of SRH and increased the odds of bad SRH.More than 79.9% of students reported their general health good and very good. The student’s mean score of general health was higher than the average. In addition, the prevalence of internet addiction was 28.7%. An inverse significant correlation observed between SRH and internet addiction score (r=-0.198, The good SRH of medical students was higher than general population but in health faculty’ students were lower than others. Due to the effect of internet addiction on SRH and increasing trend of internet use in medical students, as well as low age of participants, attention to psychological aspects and the job expectancy in future, can effective on increasing the good SRH. The rate of good SRH was 79.9% in medical students and was higher than general population. In addition, the mean score of general health was higher than the average of people.The prevalence of internet addiction was 28.7%. Using internet for Entertainment, using private Email and chat rooms were the most important predictors of affecting to internet addiction. Moreover, internet addiction is the most predictors of SRH and increased the odds of bad SRH.An inverse significant correlation exist between SRH and internet addiction score.It is predicted that poor SRH will increased by higher usage of internet and mobile services and social networks in future. Therefore, preparing cultural and scientific needs for students in leisure times is necessary.Self-rated health (SRH) has become as increasing common measure for public health monitoring and has been reported as predictor of mortality in previous studies -4. SRH iInternet addiction is a psychological disorder due to heavy and long usage of internet. An European study showed 68% of people 16-24 age used the Internet compared to 27% of people over age 54 and only 10% of people over 65 .Today, undergraduate university students contain a large portion of the younger population in Iran  and an iA cross sectional study conducted on 254 students in Qom Medical University those were in second term or higher. Subjects selected by two stage sampling method including stratified proportional sampling in the first stage and faculty considered as stratum. In the second stage, simple random sampling was used to all students have equal chance for selection.Data collection conducted by standard questionnaires including demographic characteristics, Young internet addiction questionnaire (YIAQ) and SRH question.This questionnaire has 20 items in Likert scale and the reliability and validity of YIAQ is measured and validated by several studies in Iran , 12, 13.The SRH measured by tree different questions with likert scale. 1) SRH-5; how would you rate your general health status? With reply alternatives: very good, quite good, neither good nor poor, quite poor, and poor; 2) SRH-7; how do you regard your health? With reply alternatives ranging from 1 to 7, where 1 = Very poor, and 7 = Excellent, could not be better; 3) SRH-age; how would you assess your general health status compared to that of others of your own age? With reply alternatives: Much better, slightly better, neither better nor worse, slightly worse and much worse. The reliability and validity of these questions are assessed in other studies , 17.Data entered in SPSS software and were analyzed by chi-square, t-test analysis of variance, and Spearman correlation coefficient tests. Rating health as very good to very poor seems a simple concept that is unlikely to contain surprises, but ratings of health as fair or poor are predictors of morbidity and mortality after adjusting for clinical health status . TherefoOverall, 254 medical students were studied with mean age of 21.7 ± 2.9 years which 84.3% (214 students) were female and 77.6% (197 students) were single. From total, 36.6% (93 students) resident in dormitory and 89.8% have not any health problem or disease history.More than 81.9% reported their self-health as good quietly (50%) or very good (31.9) and only 3.2% were poor quietly or very poor. Other students (14.9%) reported as moderate. In addition, 41.5% of students reported their health status as the same of other coeval while in 12% were worse than and in 46% were better than ones. The mean of three different SRH questions are depicted in Table p=0.002). Nevertheless, marital status, living in dormitory, educational level, conducting scientific research, and having Lap Top were not significant factors.The mean score of internet addiction was 35.22 ± 12.1 and subjects weekly used on average 10.5 ± 11.35 and 7.6 ± 8.8 hours by PC (private computer) and internet, respectively. Moreover, 71.3% were normal regarding to internet use. The prevalence of internet addiction was 28.7% including 27.2% mild internet addiction and 1.6% were severe addicted with frequent problems due to internet. According to Chi-square test . Also, using Email increase to three fold  and using internet for entertainment increase to 2.2 fold  the odds of affecting to internet addiction. Moreover, internet addiction was the most predictors of SRH so that increase the odds of bad SRH more than 3 fold .et al. study lower literature was related by poor SRH (In the current study, poor and moderate response to SRH was 18.1%. in another study conducted by Babones this rate was 8.3% . In a napoor SRH . In addipoor SRH , 24-26.The prevalence of internet addiction was 28.7% and this prevalence was higher than other studies conducted in Iran  and KoreAccording to our results using internet for entertainment, using from chat rooms and private Email are predictors of internet addiction. In addition, internet addiction is the most important predictors of SRH in medical students. Another study showed the effect of internet use on SRH . AccordiHowever, since these relationships accessed in a cross-sectional study the temporality of association is unclear. Therefore, authors suggested that prospective studies conducted in general population in different age groups based on psychological outcomes and common disease to find the effect on SRH as predictors of health.The rate of good SRH in Qom medical university was higher than general population but low in comparing to their age. Poorer SRH was observed in students of health faculty. Since the target population of our study was young, the poor response of SRH in medical students is considerable. It is predicted that this prevalence will increased by higher usage of internet and mobile services and social networks in future. Therefore, medical universities shall to prepare the cultural and scientific needs of students in leisure times. Therefore, increasing self-steam, insured, and clear future can remove fear and anxiety and help for increasing of student is SRH.


RNA sequencing (RNA-seq), a next-generation sequencing technique for transcriptome profiling, is being increasingly used, in part driven by the decreasing cost of sequencing. Nevertheless, the analysis of the massive amounts of data generated by large-scale RNA-seq remains a challenge. Multiple algorithms pertinent to basic analyses have been developed, and there is an increasing need to automate the use of these tools so as to obtain results in an efficient and user friendly manner. Increased automation and improved visualization of the results will help make the results and findings of the analyses readily available to experimental scientists.By combing the best open source tools developed for RNA-seq data analyses and the most advanced web 2.0 technologies, we have implemented QuickRNASeq, a pipeline for large-scale RNA-seq data analyses and visualization. The QuickRNASeq workflow consists of three main steps. In Step #1, each individual sample is processed, including mapping RNA-seq reads to a reference genome, counting the numbers of mapped reads, quality control of the aligned reads, and SNP (single nucleotide polymorphism) calling. Step #1 is computationally intensive, and can be processed in parallel. In Step #2, the results from individual samples are merged, and an integrated and interactive project report is generated. All analyses results in the report are accessible via a single HTML entry webpage. Step #3 is the data interpretation and presentation step. The rich visualization features implemented here allow end users to interactively explore the results of RNA-seq data analyses, and to gain more insights into RNA-seq datasets. In addition, we used a real world dataset to demonstrate the simplicity and efficiency of QuickRNASeq in RNA-seq data analyses and interactive visualizations. The seamless integration of automated capabilites with interactive visualizations in QuickRNASeq is not available in other published RNA-seq pipelines.The high degree of automation and interactivity in QuickRNASeq leads to a substantial reduction in the time and effort required prior to further downstream analyses and interpretation of the analyses findings. QuickRNASeq advances primary RNA-seq data analyses to the next level of automation, and is mature for public release and adoption. RNA sequencing (RNA-seq) has emerged as a powerful technology in transcriptome profiling –3. Our pShort reads generated by RNA-seq experiments must first be aligned, or mapped, to a reference genome or transcriptome assembly. The general objective of mapping or aligning a collection of sequence reads to a reference is to discover the true location (origin) of each read with respect to that reference. Although a large number of read mapping algorithms have been developed in recent years –10, the The second important step in most RNA-seq analyses is gene or isoform quantification. A common method to estimate gene or transcript abundance from RNA-seq data is to count the number of reads that map uniquely to each gene or transcript. RPKM (reads per kilobase per million reads) is widely used to represent the relative abundance of mRNAs for a gene or transcript. A number of algorithms have been developed to infer gene and isoform abundance , 18, incSelecting appropriate software packages and setting software-specific parameters. Making the right or best choice can be difficult because many similar tools are available. Setting software parameters is even harder if not impossible, because it often requires both an in-depth understanding of the algorithms and sufficient hands-on experience, which disadvantages researchers new to this field.Writing scripts to make different components work seamlessly in a pipeline. A variety of algorithms have been designed to perform different tasks, but they have been developed (and/or maintained) independently by different research groups and often use different programming languages. Moreover, those algorithms do not understand each other well, and the output(s) from one algorithm often cannot be used as input(s) for another algorithm. As a result, additional bridging scripts are necessary, which ideally requires a data analyst who is familiar with a number of programming languages, including Shell script, Perl, Python, Java, C/C++, and R.Integrating and summarizing analyses results from individual samples. In general, most algorithms are implemented to process an individual sample. Consequently, the results of primary RNA-seq data analyses have to be further processed, integrated, and summarized for reporting, presentation, and downstream analysis. Usually, data integration and summarization are tedious and not easy to execute efficiently.Identifying RNA-seq sample outliers. It is not uncommon that some samples have low quality and often substitute samples are not available, especially for RNA-seq of clinical specimen. RNA-seq is a complicated multistep process that involves sample collection/stabilization, RNA extraction, fragmentation, cDNA synthesis, adapter ligation, amplification, purification, and sequencing. Any mistake in this complex sequence of protocols can result in biased or even unusable data. Therefore, it is necessary to establish stringent RNA-seq data quality metrics to identify outliers that should be excluded from further downstream data analysis.Detecting sample swapping and mislabeling. For large-scale RNA-seq studies in which hundreds or even thousands of RNA samples are sequenced and analyzed, it is not unusual that some samples are mishandled and appear to be swapped or sequenced more than once. Such errors can become a serious problem for downstream data analyses and interpretation of results, especially for longitudinal sample analyses. It is difficult to identify such mistakes based only on RNA-seq QC metrics and/or gene expression profiles. To confirm whether samples are from the same subject, it is more reliable to compare genetic markers among samples, such as single nucleotide polymorphisms (SNPs).Sharing the results of RNA-seq data analyses with experimental scientists. Nearly all RNA-seq data analyses are performed using Linux clusters or workstations; however, analyses results in Linux are often inaccessible to most experimental scientists. RNA-seq data analyses typically generate a large number of files and large amounts of data that are difficult to comprehend or digest directly by experimental scientists. Therefore, easily accessible interfaces are needed that not only provide a quick and easy way for non-expert users to obtain high-level visualizations of the main RNA-seq analyses outputs , but also allow them to drill down further or export the results into additional analysis applications of their choice. To the best of our knowledge, very few RNA-seq related open source packages provide all these options.Although the time and cost for generating RNA-seq data are decreasing, the analysis of massive amounts of RNA-seq data still remains challenging. Numerous software packages and algorithms for basic data quality control (QC) and analyses have been developed, which has led to the need to apply these tools efficiently to obtain results within a reasonable timeframe, especially for large datasets. Based on our own experience with in-house analyses of multiple RNA-seq datasets of varying size using open source tools, the main challenges, gaps, and bottlenecks for large-scale RNA-seq data analyses can be summarized as follows:To address these challenges, we have implemented a new pipeline named QuickRNASeq to advance the automation and visualization of RNA-seq data analyses results, and have constantly improved and refined its implementation since its inception. QuickRNASeq significantly reduces data analysts’ hands-on time, which results in a substantial decrease in the time and effort needed for the primary analyses of RNA-seq data before proceeding to further downstream analysis and interpretation. Additionally, QuickRNASeq provides a dynamic data sharing and interactive visualization environment for end users. All the results are accessible from a web browser without the need to set up a web server and database. The rich visualization features implemented in QuickRNASeq enable non-expert end users to interact easily with the RNA-seq data analyses results, and to drill down into specific aspects to gain insights into often complex datasets simply through a point-and-click approach.QuickRNASeq is designed for simplicity and visual interactivity. A few important principles dictate its implementation. First, all components of the pipeline are freely available in the public domain. Second, it is easy to deploy and use. Third, all analyses results including RNA-seq QC metrics, sample correlations, and gene quantifications are accessible via a web browser and can be further explored interactively. An overview of QuickRNASeq Fig.  illustraIn addition to raw sequence reads in FASTQ format, the only other required inputs are a reference genome file in FASTA format and a corresponding gene annotation file in GTF (gene transfer format). QuickRNASeq can be applied to any species as long as its genome and gene annotations are available; for example, human, mouse, rat, and cynomolgus or rhesus monkeys. A gene annotation file can exist in many formats, but GTF has become the de facto standard; however, not all tools accept gene annotation files in GTF format as input. For example, RSeQC   accepts This step consists mainly of read mapping, counting, aligned read QC, and SNP calling, and the corresponding algorithms used to perform these tasks are STAR , 27, feaThe RSeQC  package Computational algorithms for RNA-seq analyses are continuously being improved, including STAR, featureCounts, RSeQC, and VarScan. Therefore, we designed our pipeline to be independent of its underlying software version and ensured that it can handle RNA-seq samples from a variety of species. To decouple the dependence of QuickRNASeq pipeline upon underlying computational algorithms and species, we introduced a plain text configuration file that can store project, species, and software-specific parameters. This configuration file also improves the reproducibility of RNA-seq data analyses and simplifies the command lines in QuickRNASeq. For the convenience of QuickRNASeq users, a configuration file template has been provided for easy customization.Merge mapping, counting summaries, and RSeQC metrics from individual samples.Generate a read counting table ready for downstream analysis of all annotated genes.Calculate a SNP (and gene expression) correlation matrix among samples.Perform correlation-based sample QC, calculation of MADScore (median absolute deviation score), and data normalization.Produce RNA-seq metrics and correlation plots ready for PowerPoint presentations.Generate a comprehensive HTML QC report for individual sample.Produce a dynamic and integrated QC metrics plot for individual samples.Generate a master HTML entry webpage for data analyses results.Step #2 aims mainly to merge results generated in Step #1 for each individual RNA-seq sample. Additionally, it runs many across-sample calculations, such as correlation-based QC and a SNP correlation matrix. As shown in Fig. star-fc-qc.sh and star-fc-qc.summary.sh). All the plots generated in Step #2 are ready for presentations, and the gene counting table can feed downstream differential analysis algorithms. The highly automated features in Step #2 make QuickRNASeq an efficient tool for typical standard RNA-seq analyses, and our pipeline substantially reduces the hands-on time  that data analysts have to spend on primary RNA-seq data analyses.Each individual task listed above is performed by a corresponding Bash, Perl, or R script, and a master script coordinates the execution of all these tasks. The main scripts and their functions are listed in Table We implemented a correlation-based QC to detect potential outliers in the RNA-seq data by calculating a MADScore for each sample. In general, an outlier appears to deviate markedly from other samples in a RNA-seq study, and thus its correlation with other samples will be relatively low. The MADScore is calculated as follows. For each sample, calculate the correlation difference, which is simply the difference between the average of all the pairwise correlations that involve the sample and the average of all the pairwise correlations that do not involve the sample. If a sample is an outlier, then the difference will be negative. Accordingly, there will be a vector of values (one for each sample). Then this vector of difference is converted to MADScores (robust Z-scores) by subtracting the medians and dividing it by median absolute deviations (MAD). A standard MADScore cutoff  is set to determine the outliers.Primary RNA-seq data analyses results are represented by a standard file folder structure, and an integrated report provides comprehensive QC metrics and a gene expression table. RNA-seq data analyses typically generate a variety of files and large amounts of data, and the master entry webpage generated in Step #2 makes data navigation and visualization more convenient. More importantly, the project report offers interactive visualizations of RNA-seq QC and gene expression levels, and provides analytical tools to gain insights into the data. All required JavaScript libraries have already been packaged into the QuickRNASeq project report; thus, deployment of the data into a web server is optional.JavaScript has become the hallmark of the web 2.0 technologies because it greatly enhances interactive visualizations. The availability of JavaScript-based open source visualization libraries has fueled the adoption of this technique. We implemented the interactive data visualization in QuickRNASeq using these libraries, including JQuery , D3  is new, while the other splice site is annotated (known). While the majority of junctions in Fig. complete_novel or partial_novel.Two strategies are used to determine the read duplication rate, as indicated in Fig. One of the most important objectives in many RNA-seq studies is to estimate gene expression levels under certain biological or disease conditions. With the help of the visualization tools shown in Fig. All samples can be processed in parallel in Step #1 of the QuickRNAseq pipeline  , a powerREADME.txt is provided along with the QuickRNASeq package, which explains step-by-step how to run QuickRNASeq. In addition, users can examine the configuration and sample annotation file under the test_run folder in the QuickRNASeq package. QuickRNASeq can be run without a sample annotation file, but it is strongly recommended that users provide meaningful annotations for all samples. A proper annotation file should be tab delimited, and QuickRNASeq requires that the first and second columns correspond to sample and subject identifiers, respectively. Sample names should start with a letter, and should not contain any white spaces.For gene quantifications, QuickRNAseq requires a complete genome sequence and well-annotated genes as inputs. The pipeline is not intended for the discovery of novel isoforms. QuickRNASeq is designed for use by bioinformaticians, experimental biologists, and geneticists in the fields of genome-scale analysis, functional genomics, and systems biology; however, downloading, installing, and running the QuickRNASeq pipeline in a Linux environment will require some basic computer-based expertise. A In QuickRNASeq, we selected FeatureCounts, a union exon based approach, for gene quantification. According to our own most recent research , union eThe current version of QuickRNASeq focuses on the automation of primary processing steps in RNA-seq data analyses, and these steps are in general biological question independent. We plan to expand QuickRNASeq to downstream analyses in the future, including differential analysis and pathway enrichment. Downstream analyses are usually driven by biological questions and experimental designs and thus different from project to project. How to automate such analyses in a user friendly manner remains a challenge for our practical implementation.While this paper was in preparation, Wagle et al.  publisheBy combing the best open source tool sets developed for RNA-seq data analyses and the most advanced web 2.0 technologies, we implemented the QuickRNASeq pipeline, which significantly reduces the efforts involved in primary RNA-seq data analyses and generates an integrated project report for data sharing and interactive visualization. The dynamic visualization features enable end users to explore and digest RNA-seq data analyses results intuitively and interactively, and to gain deep insights into RNA-seq datasets. The configuration file contains project, species, and software related parameters, and thus improves the reproducibly in RNA-seq data analyses. We have already applied QuickRNASeq to in-house large scale RNA-seq projects, and its current version is stable and mature for public release and adoption.Project name: QuickRNASeq pipelineProject home page:http://quickrnaseq.sourceforge.netOperating system: LinuxProgramming languages: Bash scripting, Perl, R, JavaScriptDependencies: R packages edgeR, reshape2 and ggplot2Other requirements: NoneLicense: GNU GPL version 3


O-dealkylase (PROD) activity. Analyses by high-performance liquid chromatography and liquid chromatography mass spectroscopy revealed that propofol was metabolized by phenobarbital-treated rat liver microsomes into 4-hydroxypropofol. In comparison, PROD activity and 4-hydroxy-propofol production from propofol metabolism were suppressed by orphenodrine, an inhibitor of CYP2B1/2, and a polyclonal antibody against rat CYP2B1/2 protein. Furthermore, exposure of rats to propofol did not affect the basal or phenobarbital-enhanced levels of hepatic CYP2B1/2 protein. Meanwhile, propofol decreased the dealkylation of pentoxyresorufin by phenobarbital-treated rat liver microsomes in a concentration-dependent manner. Taken together, this study shows that rat hepatic CYP2B1/2 plays a critical role in the ring-oxidative metabolism of propofol into 4-hydroxypropofol, and this anesthetic agent can inhibit CYP2B1/2 activity without affecting protein synthesis.Propofol, an intravenous anesthetic agent, is widely used for inducing and maintaining anesthesia during surgical procedures and for sedating intensive care unit patients. In the clinic, rapid elimination is one of the major advantages of propofol. Meanwhile, the biotransformation and drug interactions of propofol in rat livers are still little known. In this study, we evaluated the ring-oxidative metabolism of propofol in phenobarbital-treated rat livers and possible drug interactions. Administration of phenobarbital to male Wistar rats significantly increased levels of hepatic cytochrome P450 (CYP) 2B1/2 and microsomal pentoxyresorufin  Propofol  is an intravenous anaesthetic agent widely used to induce and maintain anaesthesia during surgical procedures , 2. Propb5, and a multiplicity of CYP isoforms. In human and canine livers, CYP2B isoforms are reported to participate in propofol metabolism  for 4-hydroxypropofol and at m/z 178.9 for propofol. Conditions for the electrospray mass spectra were as follows: a spray voltage of 4.5 kV; capillary voltage of 10 V; capillary temperature of 270°C; 80 units of sheath gas; and 20 units of auxiliary gas. Acquisition and processing of data from the mass spectrometer were performed using Xcalibur software revision 1.0 (ThermoQuest).The LC-MS analysis was carried out as described previously . The LC A chemical inhibition assay was analyzed following a previously described method . Orphenaμg protein) were preincubated with a rabbit pAb against rat CYP2B1/2 (Chemicon) for 30 min at room temperature. The reaction was initiated by adding propofol and an NADPH cofactor solution. After the reaction, samples were extracted with cyclohexane and analyzed by HPLC.An immunoinhibition assay was carried our according to a previous method . Rat liv t-test, and differences were considered statistically significant at P values of <0.05. Statistical analysis between groups over time was carried out using two-way analysis of variance (ANOVA).Statistically significant differences between the control and drug-treated groups were determined using Student's top panel, lane 1). Meanwhile, exposure of rats to phenobarbital obviously increased the amounts of CYP2B1/2 in liver microsomes (lane 2). Amounts of β-actin were immunodetected as the internal control (bottom panel). These immunorelated protein bands were quantified and analyzed . The MS analysis further showed that this compound had the same fragment distribution as that of propofol (right panel). In comparison, after propofol was metabolized by phenobarbital-treated liver microsomes, in addition to propofol, a major peak was observed at 22.7 min ( left panel). The MS analysis demonstrated that this compound was 4-hydroxypropofol (right panel).Following metabolism by rat liver microsomes, propofol and its metabolites were analyzed using LC-MS and HPLC Figures  and 3. RThe HPLC analysis showed that 4-hydroxypropofol appeared at 5.35 min . After bμM orphenadrin, respectively, caused significant 38%, 56%, 60%, 76%, 78%, 84%, and 87% reductions in PROD activity . Propofol alone did not affect hepatic CYP2B1/2 synthesis (lane 3). In comparison, treatment of rats with propofol did not influence phenobarbital-induced CYP2B1/2 expression (lane 4). Levels of β-actin were immunodetected as the internal control (bottom panel). These immunorelated protein bands were quantified and analyzed (μM did not affect PROD activity in liver microsomes from phenobarbital-treated rats (Drug interactions with propofol were evaluated by analyzing CYP2B1/2 synthesis and its specific PROD activity . Exposuranalyzed . Cotreatted rats . in vivo data to demonstrate the metabolic elimination of propofol in rat livers mainly due to the contribution of CYP2B1/2.CYP2B1/2 plays a critical role in the ring-oxidative metabolism of propofol in rat livers. This study showed that exposure of rats to phenobarbital increased CYP2B1/2 expression and its specific enzyme activities. In parallel, analyses by HPLC and LC-MS demonstrated the ring-oxidative metabolism of propofol into 4-hydroxypropofol by phenobarbital-treated microsomes. In comparison, when CYP2B1/2 activity was reduced by orphenadrine and its specific antibody, 4-hydroxypropofol production was concurrently inhibited. Thus, the results presented in this study show that rat hepatic CYP2B1/2 contributes to the ring-oxidative metabolism of propofol into 4-hydroxypropofol. Propofol can be used to induce anaesthesia during surgical procedures and sedate ICU patients . When apThe CYP2Bs responsible for propofol hydroxylation are species-specific. An immunoblot analysis revealed that levels of CYP2B1/2 in rat livers were greatly enhanced after exposure to phenobarbital. Previous studies disclosed that phenobarbital is a good stimulator for inducing CYP2Bs , 24. The4-Hydroxypropofol is a major metabolite in CYP2B1/2-involved monooxygenation of propofol in rat livers. The HPLC analysis revealed that after propofol was biotransformed by phenobarbital-treated rat liver microsomes, a major metabolite was observed in the profile. The major metabolite was more hydrophilic than the parent compound, propofol. Results of the LC-MS analyses further showed that this metabolite was a hydroxylated product. Court et al. reported that hydroxypropofol can automatically be oxidized into the relative quinine form . Accordi in vitro and in vivo studies [Propofol causes drug interactions with hepatic CYP2B1/2. After exposure to propofol, the basal and phenobarbital-enhanced levels of CYP2B1/2 in rat livers were not altered. Meanwhile, coincubation of propofol led to a significant reduction in CYP2B1/2-specific PROD activity. A previous study showed that when propofol was intravenously administered to rats, CYP2B-involved testosterone 16beta-hydroxylation and pentoxyresorufin depentylation in rat liver microsomes decreased . Adminis studies . In addi studies , 21. Tho studies , in rat  studies , 23. ThuIn the human liver, multiple CYP isoforms, including CYP2A6, -2B6, -2C8, -2C18, -2C19, and -1A2, are reported to participate in propofol metabolism –17. In cIn summary, the present study shows that administration of phenobarbital to rats increased CYP2B1/2 protein synthesis and enzyme activities. HPLC and LC-MS analyses disclosed that propofol was metabolized into 4-hydroxypropofol by phenobarbital-treated liver microsomes. A chemical inhibition assay demonstrated the simultaneous reduction in PROD activity and propofol hydroxylation. The role of CYP2B1/2 in the biotransformation of propofol into 4-hydroxypropofol was alternatively confirmed by an immunoinhibition assay. Exposure of rats to propofol did not influence basal or phenobarbital-augmented levels of CYP2B1/2 in liver microsomes. However, propofol exhibited competitive inhibition against PROD activity. Taken together, this study demonstrates the roles of CYP2B1/2 in the ring-oxidative metabolism of propofol into 4-hydroxypropofol in rat livers and drug interactions of this intravenous anaesthetic agent with this monooxygenase.


Our knowledge of the variety and abundances of RNA base modifications is rapidly increasing. Modified bases have critical roles in tRNAs, rRNAs, translation, splicing, RNA interference, and other RNA processes, and are now increasingly detected in all types of transcripts. Can new biological principles associated with this diversity of RNA modifications, particularly in mRNAs and long non-coding RNAs, be identified? This review will explore this question by focusing primarily on adenosine to inosine (A-to-I) RNA editing by the adenine deaminase acting on RNA (ADAR) enzymes that have been intensively studied for the past 20 years and have a wide range of effects. Over 100 million adenosine to inosine editing sites have been identified in the human transcriptome, mostly in embedded Alu sequences that form potentially innate immune-stimulating dsRNA hairpins in transcripts. Recent research has demonstrated that inosine in the epitranscriptome and ADAR1 protein establish innate immune tolerance for host dsRNA formed by endogenous sequences. Innate immune sensors that detect viral nucleic acids are among the readers of epitranscriptome RNA modifications, though this does preclude a wide range of other modification effects. Until now, identifications of modified bases in mRNAs have relied mainly on mass spectrometry or antibodies specific for the modified base, or on the detection of different responses of the modified base versus the normal base to some chemical modification  have received a lot of recent attention. m6A does not change base-pairing preferences of RNA and cannot recode open reading frames. It has been found in approximately 7,000 mRNAs with an enrichment around the stop codon and in the 3′UTR regions of transcripts . The. TheAdarc system –36 nor ic system  as, otheto birth . HoweverAdar1; P53 MEF cells reduces the level of transcription of ISGs, whereas catalytically inactive ADAR1 does this to an observable but considerably lower extent [Adar1 mutant phenotype appears to be mainly due to loss of inosine in RNA, with Mavs rescuing as it blocks the aberrantly activated signalling from both of the RLR dsRNA sensors. The effect of inosine-containing dsRNA is dominant; dsRNA oligonucleotide-containing inosine can bind to RIGI and MDA5, thus inhibiting the innate immune response [Restoring expression of either ADAR1 or an ADAR2 mutant that localizes to the cytoplasm in stably transfected r extent . Thus, tresponse .Adar1E861A catalytically inactive mutation in mice gives an embryonic phenotype that is similar to, but not as severe as, the complete null [Adar1E861A catalytically inactive mutant embryos die two days later (E14.5 versus E12.5) [Ifih1-/-), rescues the Adar1E861A mutant but not Adar1 null mutant embryos [Adar1E861A protein partially rescues the Adar1 null mutant phenotype in the whole embryo, presumably because it still binds the most critical immune-inducing RNAs. Enzymes that introduce epigenetic modifications in chromatin also show partial rescues by catalytically-inactive mutants [Part of the ADAR1 effect is due to an editing-independent function. A recent study demonstrated that knocking-in the ete null . The Adas E12.5) , and a d embryos . This im mutants ,50. Main mutants ,52. For While it is tempting to associate the ADAR1 editing-independent activity with Alu or SINE RNAs, this remains an entirely open question. ADAR proteins are not abundant, and many adenosines in Alu RNA hairpins are edited at efficiencies below 1%. The editing-independent ADAR1 effect may involve particular immune-inducing ADAR target RNAs present in both mice and humans. ADAR1 tightly binds many of the conserved pre-mRNA structures that are site-specifically edited by ADAR2; some such RNA structures are entirely in exons or UTR regions and might reach the cytoplasm to affect innate immune sensors. The inactive ADARs, the brain specific ADAR3, and the two testis-specific ADAR-related ADAD proteins present in the genome may also bind particular RNAs that are tissue-specific (for review ).ADAR1 [ADAR1 mutations catches a transient viral infection, reduced ADAR1 dosage or activity leads to unedited cellular RNAs in the cytoplasm binding to RLRs, stimulating an innate immune response (The importance of epitranscriptome base modification for innate immune regulation is well illustrated by Aicardi-Goutières syndrome (AGS), which is caused by mutations in ADAR1 . AGS is response . After 15C, m6A, m5U, pseudouridine, or 2′-O-methylated nucleotides dampen the innate immune response when transfected into mammalian dendritic cells, whereas RNA that is unmodified will stimulate it [Data from the Weissman group has demonstrated that in vitro transcribed RNA containing various modified nucleotides such as mulate it –56. The 6A also weakens base pairing in dsRNA even though it does not alter base pairing preferences [In the case of inosine effects on RLRs, the inosine–uracil (I-U) wobble base pair weakens dsRNA base pairing and several I-U base pairs together cause dsRNA melting . It is lferences . 2′-O-meN-cyclohexyl-N′-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulphonate) a chemical that forms a stable interaction with pseudouridine [Levels of base modifications often change in response to stress or virus infection. Pseudouridine has been found in mRNA in both yeast and mammalian cells in the past year by three groups –61, all ouridine . When thouridine .6A occurs in some viral RNA at a higher level than would be predicted from a random occurrence; for example, Rous sarcoma viral mRNA has seven m6A sites, whereas SV40 has more than ten [6A is present in RNA [Base modification is important in the conflict between virus and host because viruses can benefit from evading host innate immune responses if they can capture some modifications. Thus, mthan ten : it has t in RNA .Escherichia coli but not by tRNA from human cells in PBMCs [Helm and co-workers have demonstrated that stimulation of the innate immune receptor TLR7, by tRNA purified from in PBMCs , is due Kluyveromyces lactis cleaves tRNAsGlu/Gln/Lys from Saccharomyces cerevisiae on the 3′ side of an anticodon base that is modified. The γ-toxin recognizes the cleavage sites due to the 5-methoxycarbonylmethyl-2-thiouridine wobble base modification present on these tRNAs in S. cerevisiae but not in K. lactis [Conflicts between self and non-self nucleic acids are much older than the animal innate immune systems , and tRN. lactis –69.Some modified bases lead to particular patterns of misincorporation at the modified base position in standard RNA-Seq and indications of RNA modification events can be obtained from existing RNA sequence databases . New RNAAs Albert Einstein once said, “Fundamental ideas of science are essentially simple.” The use of host RNA modification to distinguish between host and parasite nucleic acids is reminiscent of the restriction and modification that occurs in bacterial DNA. With the plethora of modifications in RNA, it’s easy to envisage these would generate a unique bar code that would be species-specific, allowing intricate “self”-“non-self” discrimination.


It is widely believed that constructed response written questions test higher order cognitive processes in a manner that multiple-choice questions cannot, and consequently have higher validity.Written assessments fall into two classes: An extensive review of the literature suggests that in summative assessment neither premise is evidence-based. Well-structured open-ended and multiple-choice questions appear equivalent in their ability to assess higher cognitive functions, and performance in multiple-choice assessments may correlate more highly than the open-ended format with competence demonstrated in clinical practice following graduation. Studies of construct validity suggest that both formats measure essentially the same dimension, at least in mathematics, the physical sciences, biology and medicine. The persistence of the open-ended format in summative assessment may be due to the intuitive appeal of the belief that synthesising an answer to an open-ended question must be both more cognitively taxing and similar to actual experience than is selecting a correct response. I suggest that cognitive-constructivist learning theory would predict that a well-constructed context-rich multiple-choice item represents a complex problem-solving exercise which activates a sequence of cognitive processes which closely parallel those required in clinical practice, hence explaining the high validity of the multiple-choice format.The evidence does not support the proposition that the open-ended assessment format is superior to the multiple-choice format, at least in exit-level summative assessment, in terms of either its ability to test higher-order cognitive functioning or its validity. This is explicable using a theory of mental models, which might predict that the multiple-choice format will have higher validity, a statement for which some empiric support exists. Given the superior reliability and cost-effectiveness of the multiple-choice format consideration should be given to phasing out open-ended format questions in summative assessment. Whether the same applies to non-exit-level assessment and formative assessment is a question which remains to be answered; particularly in terms of the educational effect of testing, an area which deserves intensive study.The online version of this article (doi:10.1186/s12909-014-0249-2) contains supplementary material, which is available to authorized users. ContentIndeed, knowledge may be acquired and held at varying depths. Aspects of this are reflected in the cognitive levels of learning constituting Bloom's taxonomy of learning -14] , mini-essay questions, single-word and single-sentence questions and the modified essay question (MEQ). The selected-response or closed-ended format is typified by the multiple-choice question (MCQ) assessment, where candidates select the most appropriate answer from a list of options rather than generating an answer spontaneously. Many variants of the multiple-choice format have been used: current best practice recommends the use of one-best-answer , and extended matching item (EMI) formats . In. Inof lelearning ,22],. Ap. Apth ce-writing .Throughout its history, the multiple-choice format has had many detractors. Their principal arguments are that closed-ended questions do not stimulate or test complex constructive cognitive processes, and that if the ability to construct rather than choose a correct answer is not actively assessed, there is a potential that it will be neither taught nor learnt -41]..41].As Rotfield has stated: "Students proudly show off their high grades, from multiple-choice exams, as if their future careers will depend on knowing which choice to make instead of discerning which choices exist" . Self-evmodified essay question (MEQ). The format is of a clinical scenario followed by a series of sequential questions requiring short answers. This was expressly designed to bridge a perceived gap between multiple-choice and SAQ as it was believed that it would prove better at testing high-order cognitive skills than multiple-choice while allowing for more standardised marking than the standard open-ended question . Th. Th44]. question .et al. have stressed the necessity for interpreting reliability in terms of outcome, particularly in terms of pass/fail misclassification, and not merely in terms of numeric scores such as Cronbach’s alpha ; in; in39]; The revised Bloom's taxonomy of learning -12] is  is 12] icontext-rich and context-free questions. In clinical assessment, a context-rich question is typically presented as a case vignette. Information within the vignette is presented to candidates in its original raw format, and they must then analyse, interpret and evaluate this information in order to provide the answer. The stimulus reflects the question which the candidate must answer and is therefore relevant to the content of the question. An example of a final-year question in Internal Medicine is shown in the following example. Such a question requires analysis (What is the underlying problem?), application (How do I apply what I know to the treatment of this patient?) and evaluation , none of which can be answered without both knowledge and understanding. Thus 5 of Bloom’s 6 levels have been tested.Those who understand the principles underlying the setting of high-quality multiple-choice items have no difficulty in accepting that multiple-choice is capable of assessing high-order cognition ,13],,. Indeed,Example of a context-rich multiple-choice item in internal medicineA24-year-oldwoman is admitted to a local hospital with a short history of epistaxis. On examination she is found to have a temperature of 36.9°C. Sheis wasted, has significant generalised lymphadenopathy and mild oral candidiasis but no dysphagia. A diffuse skin rash is noticed, characterised by numerous small purple punctate lesions.A full blood count shows a haemoglobin value of 110 g/L, a white cell count of 3.8×109per litre and platelet count of 8.3×109per litre. Which therapeutic intervention is most urgently indicated in this patient?Antiretroviral therapyFluconazoleImipenemPrednisonePlatelet concentrate infusionNone of the options offered are obviously unreasonable or easily excluded by the candidate who attempts to shortcut the cognitive processes required in answering it by searching for clues in the options themselves. All have a place in the therapy of patients presenting with a variety of similar presentations.Answering this item requires:Analysis. In order to answer this item successfully, the candidate will have to recognise (1) that this patient is highly likely to be HIV-positive , (2) that the presentation is suggestive of immune thrombocytopenic purpura , (3) that other commonly-seen concomitant features such as severe bacterial infection and extensive esophageal candidiasis are excluded by a number of negative findings.Evaluation. Further, in order to answer this item successfully, the candidate will have to (1) consider the differential diagnosis for the principal components of the clinical vignette and, by process of evaluation, decide which are the most likely; (2) decide which of the diagnoses require treatment most urgently, (3) decide which form of therapy will be most appropriate for this.Knowledge, understanding and application. It is utterly impossible to “recognise” the correct answer to this item without having worked through this process of analysis and evaluation, and the knowledge required to answer it must clearly be informed by deep learning, understanding and application. Hence five of the six levels of Bloom’s taxonomy have been tested. Furthermore it would appear an eminently reasonable proposition that the candidate who correctly answers this question will indeed be able to manage such a patient in practice, hence implying structural validity.Though guessing has a 20% chance of providing the correct answer, this will be eliminated as a factor by assessing performance across multiple such items and applying negative marking to incorrect answers.stimulus format is more important than the response format. Scores on questions in open-ended format and multiple-choice format correlate highly (approaching 100%) for context-rich questions testing the same material. In contrast, low correlations are observed for different content using the same question format . Th. ThaskinIs the contention true? Essentially, this may be explored at two levels. The first is to correlate outcomes between the two formats. The second is to perform appropriate statistical analysis to determine whether these formats are indeed testing different dimensions or “factors”.Validity is an indicator of how closely the assessment actually measures the quality it purportedly sets out to test. It is self-evident that proficiency in many domains, including clinical practice, requires not only the ability to recall factual knowledge, but also the ability to generate and test hypotheses, integrate knowledge and apply it appropriately as required.Modern conceptualisations of validity posit a single type; namely construct validity -59]. Th. Th59]. et al. compared the two formats prospectively and showed a strong correlation between the two sets of scores ..et al. cIn general correlations between the two formats are higher when the questions in each format are specifically designed to be similar , and lower where the items in the two formats differ. However, the difference is not great: in a meta-analysis, Rodriguez found a correlation across 21 studies of 0.92 for stem-equivalent items and 0.85 across 35 studies for non-stem-equivalent items. The scores may not always be identical, but they are highly correlated ,65]..65].et al. compared a one factor model with a two factor model to examine the relationship of the open-ended and closed-ended formats and found that in general the single factor provided a better fit. This suggests that essentially the two formats are testing the same thing ,,,. In summd recall , thus suet al. reported that discrepancies in the outcome between open-ended and multiple-choice tests were largely due to the low reliability of the open-ended component and inappropriate testing strategies . In. Inet alstrategy . As a geIt is also important to be aware that open-ended assessments may bring confounding factors into play, for example testing language mastery or skills rather than the intended knowledge domain itself , and hanIn comparison with the multiple-choice format, is the open-ended format superior in predicting subsequent performance in the workplace? This has been assessed and the answer, surprisingly, is that it may be less predictive. Rabinowitz and Hojat  correlatet al. concluded: “Thus, while we are sympathetic to… the arguments… regarding the advantages of open-ended format, we have yet to see convincing psychometric evidence supporting them. We are awash in evidence of their drawbacks”, and further, “… We are forced to conclude that open-ended items provide this information in more time at greater cost than the multiple-choice items. This conclusion is surely discouraging to those who feel that open-ended items are more authentic and, hence, in some sense, more useful than multiple-choice items. It should be” , an, an93], essments . There iessments .et al. stress the need for reference beyond the existing medical education literature to relevant scientific disciplines, including cognitive psychology , as, as48], ychology . In the Third is the counter-intuitiveness of this finding. Indeed, the proposition that the open-ended format is more challenging than MCQ is intuitively appealing. Furthermore, there is the “generation effect”; experimental work has shown that spontaneous generation of information, as opposed to reading enhances recall ,19]. Al. Al19]. assessment for learning, and given very interesting new insights into the cognitive basis of memorisation, recall and reasoning, a field which is as yet largely unexplored, and may be expected to have a significant impact on the choice of assessment format. For diagnostic purposes, the open-ended format may hold value, since it is better able to expose the students intermediate thinking processes and therefore allow precise identification of learning difficulties , th, thassesiculties ,23], th, thassesiculties , and theiculties  are cleaiculties  and may iculties . Though iculties .et al. elaborated causal networks, knowledge encapsulation and illness scripts . Th. Th96].  scripts ,106]. C. Cet al. scripts ,108]; t; tet al. scripts . In comp scripts . As Normtuation” . The protuation” .an internal representation of a system that the learner brings to bear in a problem-solving situation . I. I111]. ituation ,104],..an interZhang has introduced the concept of a “distributed cognitive task”: a task requiring that information distributed across both the internal mind and the external environment is processed . If we eet al. , an, an21], et al. , an, anet alProblem-solving skills are poorly generalizable . This isI suggest that the problem with many assessments in the MEQ format is that they are essentially linear. By requiring the candidate to think one step at a time, the assessment effectively misses the crux of the problem-solving process, which is to look at and respond to a complex problem in its entirety, and not stepwise. The context-rich vignette-based multiple-choice item by contrast presents a complex problem which must be holistically assessed. Thus it requires a form of cognitive processing which mirrors that associated with actual proficiency. Hybrid formats such as key feature assessments in effect also break down the clinical reasoning process into a sequence of sequential steps; whether this is regarded as a drawback will depend on the relative importance ascribed to decision-making at critical points in the decision tree and global assessment of a problem viewed holistically. This is a critical area for future research in clinical reasoning.Educators who mistrust the multiple-choice format have tended to concentrate on the final, and cognitively the least important, step in this whole process: the selection of a particular option as the answer, while ignoring the complex cognitive processes which precede the selection. Indeed, in a good assessment, the candidate is not “selecting” an answer at all. They recognise the external representation of a problem, subject the internalised representation to high level cognitive processing, and then externalise the product as a solution , which (The multiple-choice format is by no means unlimited in its capacity to test higher-order thinking. The literature on problem-solving stresses the importance of highly-structured complex problems, characterised by unknown elements with no clear path to the solution and indeed a potential for there to be many solutions or even no solution at all . The staIn effect the choice would then lie between an assessment comprising one or two unstructured essay length answers with low objectivity and reliability, and a large number of highly reliable multiple choice items which will effectively test high-order problem-solving, but will stop short of a fully complex situation. Perhaps this is a restatement of the assertion that “measuring something that is not quite right accurately may yield far better measurement than measuring the right thing poorly” , the sitet al. —es—es100]—eTherefore, if candidates demonstrate evidence of extensive, deeply-learned knowledge, and the ability to solve complex problems, be it through the medium of multiple-choice assessment or any other form of assessment, then it is safe to assume that they will be able to apply this knowledge in practice. This accounts for the extensive correlation noted between multiple-choice performance, performance in open-ended assessments, and tests of subsequent performance in an authentic environment.et al. . As. Aset alThat the multiple-choice format demonstrates high validity is due in part to the observation that well-constructed, context-rich multiple-choice questions are fully capable of assessing higher orders of cognition, and that they call forth cognitive problem-solving processes which exactly mirror those required in practice. On a theoretical basis it is even conceivable that the multiple-choice format will show superior performance in assessing proficiency in contrast with some versions of the open-ended format; there is indeed empirical evidence to support this in practice ,92]. Pa. Pa92]. assessment for learning is important, such as in formative assessment and in summative assessment at early and intermediate stages of the medical programme given that conclusive evidence with respect to the learning effects of the two formats is as yet awaited.Though current evidence supports the notion that in summative assessment open-ended items may well be redundant, this conclusion should not be uncritically extrapolated to situations where The author was solely responsible the literature and writing the article.RJH is currently Dean and Head of the School of Clinical Medicine at the University of KwaZulu-Natal, Durban, South Africa. He studied at the University of Cape Town, specialising in Internal Medicine and subsequently hepatology, before moving to Durban as Professor of Medicine. He has a longstanding interest in medical education, and specifically in the cognitive aspects of clinical reasoning, an area in which he is currently supervising a number of research initiatives.


Tospovirus based on the homology of N gene sequence to those of tospoviruses. In this study, the complete sequence of the tripartite RNA genome of MVBaV was determined and analyzed. The L RNA has 8905 nucleotides (nt) and encodes the putative RNA-dependent RNA polymerase (RdRp) of 2877 aa amino acids (aa) in the viral complementary (vc) strand. The RdRp of MVBaV shares the highest aa sequence identity (85.9%) with that of Watermelon silver mottle virus (WSMoV), and contains conserved motifs shared with those of the species of the genus Tospovirus. The M RNA contains 4731 nt and codes in ambisense arrangement for the NSm protein of 309 aa in the sense strand and the Gn/Gc glycoprotein precursor (GP) of 1,124 aa in the vc strand. The NSm and GP of MVBaV share the highest aa sequence identities with those of Capsicum chlorosis virus (CaCV) and Groundnut bud necrosis virus (GBNV) . The S RNA is 3294 nt in length and contains two open reading frames (ORFs) in an ambisense coding strategy, encoding a 439-aa non-structural protein (NSs) and the 277-aa nucleocapsid protein (N), respectively. The NSs and N also share the highest aa sequence identity  with those of CaCV. Phylogenetic analysis of the RdRp, NSm, GP, NSs, and N proteins showed that MVBaV is most closely related to CaCV and GBNV and that these proteins cluster with those of the WSMoV serogroup, and that MVBaV seems to be a species bridging the two subgroups within the WSMoV serogroup of tospoviruses in evolutionary aspect, suggesting that MVBaV represents a distinct tospovirus. Analysis of S RNA sequence uncovered the highly conserved 5’-/3’-ends and the coding regions, and the variable region of IGR with divergent patterns among MVBaV isolates.Mulberry vein banding associated virus (MVBaV) that infects mulberry plants with typical vein banding symptoms had been identified as a tentative species of the genus  Morus spp.) is an economically important plant grown widely throughout Asia, for the cultivation of the silkworms (Bombyx mori Linn.) and for the sericulture industry. Diverse virus-like symptoms, including mosaic, vein banding, vein necrosis, chlorotic ringspots, and leaf deformation were frequently observed on mulberry. The viral diseases have been a major factor restricting yield and quality of mulberry. Two mulberry-infecting viruses, Mulberry latent virus (Genus Carlavirus) [Mulberry ringspot virus (Genus Nepovirus) [Tospovirus, temporarily named Mulberry vein banding virus (MuVBV), was found in China and identified to be a new species of Tospovirus based on the homology of N protein sequence to other Tospovirus species [The mulberry (lavirus)  and the povirus) , have be species .Tospovirus genus include foliar necrotic spots, necrotic stems, bronzing, wilting, and ring spots in leaves and fruits [Tospoviruses cause significant loss of yield and quality to vegetables, legumes, and ornamental crops worldwide and are transmitted by thrips in a circulative and propagative manner , 5. Typid fruits , 6. Tospd fruits . The 3' d fruits .The L RNA is with a negative polarity and encodes a putative RNA-dependent RNA polymerase (RdRp), which is also called L protein, in the viral complementary (vc) strand for virus replication . The othIn recent years, new tospoviruses such as Pepper necrotic spot virus (PNSV) , SoybeanWatermelon silver mottle virus (WSMoV), Tomato spotted wilt virus (TSWV), Groundnut yellow spot virus (GYSV), and Iris yellow spot virus (IYSV) [Impatiens necrotic spot virus (INSV) was classified together as a distinct serotype [Tospovirus [Tospovirus [Tospoviruses are classified into four major serogroups, designated by their serological relatedness to the type viruses s (IYSV) ,19. Impaserotype . BeNMV aspovirus , 20. As spovirus , 21–24.Tospovirus belonging to the WSMoV serogroup.In our previous survey, a new tospovirus tentatively named Mulberry vein banding virus (MuVBV) with highest N protein homology of 74.4% to Capsicum chlorosis virus (CaCV) was identified from mulberry with vein-banding symptom in Guangxi Province of China by RT-PCR and sequencing of a fragment of the S RNA of the viral genome . Since tThis work was supported by the government of Guangxi province and the sampling activities were conducted with the permission of the agricultural authority of local counties or authorities in charged. The mulberry orchards where the plant samples were collected were in public domain and in regular agricultural farm lands where no endangered or protected species were involved. The sampling locations of the isolates of MVBaV in this study and the authorities who issued the permission were listed in During 2011 to 2012, ten isolates of MVBaV designated XCSY-3, XCBY-1, XZDL-1, SL-3, HX-2, NN-5, NN-10, NN-16, YZ-3, YZ-4, were collected from mulberry plants showing characteristic symptoms including vein banding, mosaic, chlorotic ringspots, necrotic ringspots or vein necrosis on leaves in mulberry orchards around Guangxi province, China. Due to difficulty of sap transmission, the virus was maintained on mulberry by grafting to the virus-free mulberry seedlings. The leaf samples were stored at −80°C until total RNA was extracted. Ultrathin sections of diseased tissues were prepared as described  and examGroundnut bud necrosis virus (GBNV) for WSMoV serogroup, Groundnut ringspot virus (GRSV)/Tomato chlorotic spot virus (TCSV) for TSWV serotype, and antisera against TSWV for TSWV serogroup, IYSV for IYSV serogroup, and INSV for the distinct serotype, were purchased from Agdia Inc. . Indirect ELISA was performed as described Chen et al. (2010) with minor modifications [Mixed antisera of WSMoV/ications . Crude eTotal RNA was extracted from the infected mulberry leaves using RNAplant plus reagent  according to the manufacturer's instruction and the quality of the purified RNA was evaluated by gel electrophoresis on 0.8% (w/v) agarose gels. The 5′ and 3′-terminal sequences of the viral genomic RNA were determined by using the RACE cDNA Amplification Kit  coupling with Sanger sequencing. Primers were designed according to the available sequences obtained through viral small RNA deep sequencing. The RACE products were purified using DNA gel recovery kit  and cloned into the pCR2.1-TOPO vector  before sequencing.MVBaV-specific primer pairs were designed based on the sequence information obtained by 5′ and 3′ RACE, and used for the amplification of complete fragments of the MVBaV genome. The primers LRNAF1/LRNAR1 and LRNAF2/LRNAR2 were used for the PCR amplification of MVBaV L RNA, MRNAF/MRNAR for MVBaV M RNA, and SRNAF/SRMAR for S RNA, respectively. The reverse transcription reaction was carried out using the Reverse Transcriptase Kit (Thermo Fisher Scientific) and the PCR was carried out in a total reaction volume of 25 μl. After denaturizing at 95°C for 2 min, the PCR were performed with the following parameters: 35 cycles with each cycle at 94°C for 30 s, 55–58°C for 30 s (the temperature varied with the length of template fragments), 72°C for 45 s, and final extension at 72°C for 10 min. The PCR products were analyzed by electrophoresis on a 0.8% (w/v) agarose gel. The PCR products of interest were recovered from the gel and cloned into pCR2.1-TOPO vector plasmid. The primers used for RACE and amplifying the MVBaV Genome are shown in The cloned PCR products were sequenced with sequencer ABI 3700. At least three independent clones were sequenced for a target PCR product. The assembled full-length sequence data of MVBaV genome were deposited in the GenBank with accession numbers KM819698-KM819709.N- and O-linked glycosylation sites of the GP of MVBaV were predicted using the software programs SignalP 4.1, NetNGlyc 1.0, and NetOGlyc 3.1, respectively. For transmembrane domain prediction, the TMHMM Server 2.0 program was used. The sequences of Tospoviruses were down loaded from the GenBank and listed in Sequence analysis was carried out using programs within the software VECTOR NTI V11.0 (Thermo Fisher Scientific). Phylogenetic trees were constructed by the neighbor-joining method with 1000 bootstrap replications using a program included in MEGA 6.06 . The putThe symptoms of MVBaV-infected leaves varied widely, depending on the growing stage of mulberry and the environmental conditions. Initial symptoms of vein banding, mosaic, or chlorotic ringspots  were obsThe diseased samples reacted strongly with WSMoV/GBNV mixed antisera, but not with antisera to TSWV, INSV, IYSV, and GRSV/TCSVin ELISA assays (data not shown), indicating that MVBaV is serologically related to the members of the WSMoV serogroup.In our previous deep sequencing of small RNAs from MVBaV-infected samples, three incomplete RNA segments of MVBaV responding to the L, M, and S RNA had been assembled (our unpublished data). The complete genome sequence of the MVBaV isolate XCSY-3 was obtained by combining the deep sequencing data, RT-PCR and RACE data , Table 1Two overlapping PCR fragments generated by RT-PCR constitute the complete MVBaV L RNA of 8905 nt in length (GenBank accession number KM819698). There is a single open reading frame (ORF) in the viral complementary strand (vc-strand) that encodes a deduced RdRp (L protein) of 2877 aa with a molecular mass of 331.6 kDa. The 5' untranslatable regions (UTRs) and 3'-UTRs of the L RNA were 241 and 30 nt in length, respectively. The 17-nt termini of the L RNA formed a panhandle structure with a mismatch at the 11th nucleotide.The RdRp protein of MVBaV has a conserved region similar to those of other tospoviruses. The five conserved motifs, motif A (DXXKW), motif B (QGXXXXXSS), motif C (SDD), motif D (K), and motif E (EXXS), were all found within the RdRp of MVBaV. Furthermore, three motifs, Motif F1 (TDF), Motif F2 (KxQRTK) and Motif F3 (DREIY), which were found in the RdRp of CaCV, IYSV, TYRV, WBNV, GBNV, WSMoV, TZSV, and CCSV , were alThe complete nucleotide sequence of the MVBaV M RNA is 4731 nt in length and contains two ORFs encoding NSm and GP, in an ambisense coding strategy and separated by an IGR of 322 nts (GenBank accession number KM819699). The 5'- and 3'-UTRs of MVBaV M RNA contain 57 and 47 nts, respectively, forming a panhandle structure with 21 base pairs and 3 mismatches at the termini. The ORF on the viral strand codes for the NSm protein of 309 aa with a molecular mass of 34.3 kDa. The ORF on the vc-strand was 3,375 nt in length and codes for a 1,124 aa GP with a molecular mass of 128.1 kDa.The NSm protein, probably involved in cell-to-cell movement, belongs to the 30 K movement protein superfamily , 28. TheN-glycosylation sites  and five transmembrane domains  were predicted and two potential signal peptides with predicted cleavage sites at aa 24 (VYL-LN) and aa 436 (SIA-LQ) to yield the glycoproteins Gn (47.8 kDa) and Gc (77.7 kDa) were present, whereas Arg-Gly-Asp (RGD) motif was not found.In topology of MVBaV GP protein, four The complete nucleotide sequence of the MVBaV S RNA is 3294 nt in length (GenBank accession code KM819701). The S RNA contains two ORFs in an ambisense coding strategy separated by an AU-rich IGR of 1008 nt in length spun from coordinates 1386–2393 in the v-strand. The 65- and 67-nt untranslated regions in the 5'- and 3'-UTRs can form a panhandle structure of 22 bp with 2 mismatches. The ORF on the v-strand codes for a 439 aa NSs protein with a molecular mass of 49.1 kDa and the ORF on the vc-strand encodes a 277 aa nucleoprotein (N protein) of 30.69 kDa. Three highly conserved motifs, Walker A (GxxxxGKT), Walker B (DEXX), and YL, were present in the NSs proteins of some tospoviruese . Walker MVBaV RdRp shares the highest sequence homology to that of WSMoV (85.8%) and high sequence homology to GBNV, WBNV, and CaCV (83.9–84.9%). To the rest members of the WSMoV serogroup , the homology were 76.1–77.8%. Intriguingly, the RdRp homology between MVBaV and topoviruses of different serotypes was all less than 70%, with members of TSWV and the distinct serotypes being the lowest (42.3–45.2%) . An inspOf particular interest is the envelop glycoproteins (GPs). Although the aa homology between MVBaV and most members of WSMoV serogroup is greater than 70%, two members, TNRV and MYSV, share only 63.4% and 65.3% homology, compatible to the 62.9% of TYRV, a member belonged to IYSV serogroup.The phylogenetic trees constructed with RdRps, NSm, GnG, NSs, and N all clearly showed that MVBaV was a distict member of WSMoV serogroup, more closely related to a subgroup composed of CaCV, WBNV, WSMoV, and GBNV .To assess the genome sequence diversity of MVBaV, the S RNA of nine additional MVBaV isolates were cloned, sequenced, and compared with that of the type isolate XCSY-3. As shown in Tospovirus.The N protein sequence, in combination with biological characters such as the thrips vector species and host range, represents the main classification criteria for the establishment of a new tospovirus species. In term of molecular differentiation, N protein sequence identity of 90% has been set as the threshold to establish a new species of tospovirus. As of to date, nine definitive species and fourteen tentative species of tospoviruses have been recognized by the International Committee on Taxonomy of Viruses , 32. AmoTospovirus, particularly the WSMoV serogroup , establishing a positive correlation of MVBaV to the disease. Of important note, 32 out of 48 (66.7%) mulberry plants with viral disease-like symptoms were found to be infected with MVBaV in our preliminary survey, demonstrating the dominant nature of MVBaV in the field [S1 Table(DOC)Click here for additional data file.