Escherichia coli (STEC)  served as a proof of concept. Genomic DNA was isolated from single colonies and sequencing was conducted on the Illumina MiSeq instrument with raw data sampling from the instrument following 4.5 hrs of sequencing. Modeling experiments indicated that datasets comprised of 21-nt reads representing approximately 4-fold coverage of the genome were sufficient to avoid significant gaps in sequence data. A novel bioinformatic pipeline was used to identify the presence of specific marker genes based on mapping of the short reads to reference sequence libraries, along with the detection of dispersed conserved genomic markers as a quality control metric to assure the validity of the analysis. STEC virulence markers were correctly identified in all isolates tested, and single colonies were identified within 9 hrs. This method has the potential to produce high-resolution characterization of STEC isolates, and whole-genome sequence data generated following the GeneSippr analysis could be used for isolate identification in place of lengthy biochemical characterization and typing methodologies. Significant advantages of this procedure include ease of adaptation to the detection of any gene marker of interest, as well as to the identification of other foodborne pathogens for which genomic markers have been defined.The timely identification and characterization of foodborne bacteria for risk assessment purposes is a key operation in outbreak investigations. Current methods require several days and/or provide low-resolution characterization. Here we describe a whole-genome-sequencing (WGS) approach (GeneSippr) enabling same-day identification of colony isolates recovered from investigative food samples. The identification of colonies of priority Shiga-toxigenic  E. coli (STEC) of public health concern), phenotypic methods are entirely impractical as a means of identification. Ultimately, these techniques are limited in terms of the type of information  that can be garnered from an isolate to underscore risk management decisions.Traditional techniques for the detection of pathogenic bacteria in foods rely on a multi-step process involving pre-enrichment in a selective broth, followed by plating to obtain colony isolates, which are then purified and subjected to a battery of biochemical and serological tests to confirm their identity. The process of definitively identifying bacterial colonies on primary isolation plates can take up to one week to complete because of the requirement for growth and expression of phenotypic characteristics specific to the organism. In some cases  [AggR or AaiC) it is necessary to re-develop and validate the PCR primers and conditions.STEC infections can result in serious medical conditions including bloody diarrhea, hemolytic-uremic syndrome (HUS), kidney failure, microangiopathic hemolytic anemia, and can occasionally be fatal. There are no biochemical features by which most so-called priority STEC strains can be differentiated from commensal Leading-edge genomic technologies open new possibilities for comprehensive analyses of microbial isolates recovered from food samples. Next-generation sequencing (NGS) technologies can now render a bacterial genome much faster and at a significantly lower cost than previously possible. The value of rapid benchtop sequencing in the investigation of foodborne disease outbreaks is becoming increasingly accepted –12. ImplWe set out to determine if NGS could be adapted to same-day identification and characterization of bacterial isolates recovered from foods while allowing the sequencing reaction to continue uninterrupted until completion. NGS data so generated could be analyzed almost immediately to support timely risk management actions without impacting the quality of whole-genome sequencing (WGS) data generated following the analysis. Here we present GeneSippr, a novel genomic tool for the analysis of single colony isolates based on the sampling (or “sipping”) of raw data during the early stages of the WGSprocess to determine the presence of pre-defined gene markers, and evaluate a set of quality metrics designed to validate the analysis.ad hoc basis. For example, in the course of a food safety investigation information may come to light which would prompt further investigation of genomic markers  to inform a risk management decision. Conventional tools such as PCR do not allow such ad hoc determinations within the time course of a food safety investigation because of the need to optimize and validate each new primer added to a reaction system. The priority STEC constitute a striking example of how genomic technologies can be used to discern the presence of gene markers pinpointing a family of pathogens otherwise not readily amenable to identification by classic means. For demonstration purposes, we focussed on the adaptation of GeneSippr to the identification of priority STEC in an approach modeling the previously described EHEC-7 CHAS method [This new approach may be regarded as an identification system providing ultimate multiplexing capacity in terms of the number of genomic markers which can be interrogated in a single procedure. Such an algorithmic approach has the inherent flexibility to enable determination of the presence of any genomic marker of interest on an S method  Fig 1)..ad hoc bin silico are referred to as e-probes. The EHEC-7 e-probes used in this study correspond to the amplicon-specific oligonucleotide capture probes currently used in the detection of priority STEC at the Canadian Food Inspection Agency (E. coli multilocus sequence typing (MLST) schemes. An e-probe created in the same manner from a housekeeping gene from Campylobacter jejuni, aspA [Two categories of gene targets were used in this study: 1) EHEC-7 targets, and 2) genomically dispersed conserved sequence (GDCS) quality control targets. The unique, diagnostic sequences used to detect these gene targets n Agency  [4, 13, ni, aspA , and theni, aspA  were incEscherichia coli O157:H7 Sakai  [D, using eq D corresponds to the expression defined in parentheses i.e., the number of reads, Nr, generated during a sequencing run times the length, Lr, of said reads divided by the size of the genome sequenced, Sg . Dadj, the adjusted depth of coverage, is obtained by adjusting D as a function of the ratio of the read length tested over the maximum read length studied (50 nt), and normalizing the number of reads to 0.5, 1, 1.5 and 2 million (M) by dividing 1M by the number of reads produced with a theoretical fold coverage of 10  previously performed in our laboratory were trimmed to 21 nt using FASTX Trimmer (v. 0.0.13.1) from the FASTX-Toolkit [The ART software  was used_002695) . Dataset-Toolkit .https://github.com/OLC-LOC-Bioinformatics/geneSippr. The percentage of sequence identity, corresponding to the proportion of e-probe bases mapped by the simulated reads, was determined using in-house Perl and R scripts and averaged for all probes . E. coli O157 was the only serotype used in the modeling, as the genome of E. coli O157:H7 Sakai  [Synthetic reads from each dataset were mapped to the e-probes using the Sequence Mapping and Alignment Tool (SMALT) (v. 0.7.4)  with a s_002695)  was usedE. coli strains with defined serological characteristics and virulence gene profiles were used to evaluate the performance of the GeneSippr  as previously described [A variety of eneSippr . The STEescribed .For the GeneSippr analysis, isolates  were culIn the experiments to determine the performance of real-time analysis of WGS data, base call (BCL) files were copied from the sequencing instrument following 37 cycles of sequencing. Files were then de-multiplexed and converted to FASTQ files using Illumina’s BCL2FASTQ conversion software (v. 1.8.3).E. coli O157:H7 Sakai closed genome  [Simulated and trimmed 21-nt reads, and reads derived from the GeneSippr analysis were mapped to the de novo whole-genome sequence assemblies were generated using SPAdes v. 3.1.1 [www.genomicepidemiology.org) [Following completion of the sequencing run, sequencing errors in reads were corrected using Quake (version 0.3 with a k-mer size of 15) , and de v. 3.1.1 . Detectiogy.org) . Web-tooThe main goal of this project was to determine whether a WGS approach to STEC colony identification and characterization could be completed within a typical food microbiology laboratory working day, which is the time frame for the EHEC-7 CHAS method currently used in food testing laboratories at the Canadian Food Inspection Agency . TypicalE. coli O157:H7 Sakai (EC20040078), were mapped to the e-probe sequences to determine the impact of read length, number of reads, depth of coverage, and mapping k-mer size on the identification of target sequences. The mean percent identity (MPI), which corresponds to the average proportion of e-probe bases covered by the reads, was used as a measure of identification. As the reads were generated from this reference genome, perfect coverage of a given e-probe would be expected to generate 100% identity, and any lower identity figure would indicate lack of coverage of the bases within that target. An average MPI ≥ 90% was arbitrarily chosen to serve as the threshold for accurate target detection. The EHEC-7 CHAS and 15 genomically dispersed, conserved sequences (GDCS) sequences were consistently detected using reads as short as 21 nt from the datasets containing 1 M reads or more  to 21 nt, and mapped the individual datasets to the same reference genome. The proportion of the genome covered by the synthetic reads was comparable (within 5 percent) to the coverage obtained from reads derived from laboratory data, indicating that the simulations accurately represented experimental conditions. We also combined the trimmed reads from the two best datasets to evaluate the benefit of sequencing redundancy and achieved a proportion of genome covered of 98.5% at 7-fold depth of coverage, indicating that sequencing duplicates during an urgent outbreak investigation can provide greater assurance of whole genome representation.To determine whether a sequencing run consisting of 21-nt reads would adequately cover the entire genome without leaving significant gaps, synthetic reads were mapped to the single ~5.5 Mb contig of the closed coverage . In ordeE. coli genome) was also included to test for non-specific detection of genetic markers , significantly delaying sequencing data processing. To reduce the time required to complete the index determination process, paired-end reads were generated asymmetrically, with 21-nt reads of the first strand followed by 281- or 581-nt reads of the second strand. To ensure adequate coverage of each isolate with 21-nt reads, a maximum of 8 isolates were multiplexed in each test run. Following the creation of FASTQ files from the primary output base-call files, 21-nt reads were retrieved and subsequently mapped to the e-probe sequences, while the sequencing run continued to completion. The outputs of this analysis included 1) determination of presence or absence of the 10 EHEC-7 genomic markers (identification of priority STEC isolate), and 2) a quality metric indicating the validity of the foregoing determination through analysis of the GDCS markers. This quality metric operates on the premise that detection of all 15 GDCS markers at the time of sampling the sequence data during its generation indicates that information covering the entire genome was available to support positive and negative assessments of the EHEC-7 targets . A set o markers . IsolateThe performance of GeneSippr with respect to the identification of the target EHEC-7 gene markers in STEC strains having different genotypes  was veriE. coli strains plus two Enterobacter cloacae isolates with an average yield of 85.6 ng/colony (Genomic DNA (gDNA) was purified from single colonies from a panel of 19 priority STEC strains , and an assortment of seven other g/colony . In genestx and eae markers in a small set of non-priority STEC strains . None of the EHEC-7 markers were identified in two strains each of generic E. coli and E. cloacae. These results demonstrate the reliability of GeneSippr in identifying the presence of the EHEC-7 markers in genomic DNA extracts from single colony isolates. Each of the 15 GDCS markers was identified by mapping to their respective e-probes in all of the E. coli strains. The ability to detect all of the GDCS markers in the E. coli strains underscores their suitability as indicators of comprehensive genome sampling for this organism. Only five of these markers  were detected in the E. cloacae strains despite adequate depth of coverage (5.7- and 8.2-fold) indicating that, as expected, not all of these sequences are present in non-target organisms. In any event, failure to detect all 15 GDCS markers in a given sample preparation would be indicative of either inadequate genome sampling or lack of sequence homology with the GDCS e-probes, flagging samples for further bioinformatic analysis. Lower coverage of 0.6–1.1 fold was observed for three strains in one run, due to the low numbers of clusters generated in the run for these strains. In these samples, GDCS could not be detected at the early time point, and, following the start of sequencing from the second strand, data from the instrument was manually retrieved after every 10 cycles and the GeneSippr procedure was repeated until GDCS were detected at cycle 41 (OLC-684), cycle 125 (OLC-683) and cycle 175 (OLC-715), after which the strains were correctly identified. The negative markers were not detected in any of the strains tested at any level of coverage.The gene markers corresponding to the EHEC-7 e-probes were correctly identified for each priority STEC strain as the GeneSippr results were in complete agreement with the EHEC-7 CHAS determinations performed separately on the strains . In addiE. coli EC20040078 strain from the Sakai outbreak [A potential risk of the GeneSippr analysis is that significant gaps in sequencing coverage could encompass target genes, resulting in false negatives, even if all GDCS markers could be identified. To evaluate the presence of gaps in the sequence, the 21-nt reads generated by two sequencing runs generated from a culture of the de novo. Raw data and assemblies have been deposited at DDBJ/EMBL/GenBank under BioProject PRJNA273275. Accession numbers are listed in E. coli genomes ranged between 4.1 and 106.9 .The timely generation and analysis of WGS information for isolates will not only be extremely valuable for their definitive identification, but also presents opportunities for risk profiling through the determination of other potentially relevant factors, such as virulence, antimicrobial resistance and typing markers. There may be instances where it is necessary to achieve more precise categorization of the risk attending a laboratory test result obtained in the context of a foodborne illness outbreak investigation. Furthermore, the definition of a pathogen or group of pathogens may change to reflect public health trends, for example, the STEC priority O serogroup designation may need to be adapted as new serogroups or strains emerge as significant public health risks –32. Indead hoc determinations within the time course of a food safety investigation because of the need to optimize and validate each new primer added to a reaction system. Unlike wet lab techniques such as PCR, GeneSippr can be readily adapted at the bioinformatic level by designating a suite of appropriate e-probes for the diagnostic and quality control marker features. New markers can be “validated” in silico using extensive public and in-house WGS databases of target and non-target isolates. Such databases are continuously growing and being refined, providing an unprecedented wealth of contemporary genomic information with which to verify the suitability of diagnostic sequences, far exceeding the more traditional “wet lab” approaches in which PCR reagents are evaluated using limited bacterial culture collections. Likewise, the adaptation of GeneSippr to the detection of other pathogenic bacteria can be readily achieved provided that suitable e-probe sequences are available.In the GeneSippr approach, the number of markers that can be assessed is not limited by technical aspects associated with laboratory methods. GeneSippr can be configured as a highly multiplexed detection system, where new markers are added as the need arises. Conventional tools such as PCR do not allow such E. coli was approximately $175 CDN per isolate. Increasing the number of multiplexed strains will reduce this cost. While this may appear to be more expensive than current PCR-based STEC detection methods [With the low level of multiplexing used in this study (8 isolates per sequencing run), the cost of sequencing a ~5 Mb organism such as  methods , it is iCurrent molecular methods for the identification of pathogenic bacteria generally determine the presence or absence of key virulence genes  using techniques such as PCR, which target short conserved sequences. Such an approach is limited in that it cannot determine the presence of a full-length functional gene, nor can it identify sequence variants with enhanced virulence. While the current version of GeneSippr does not target full length genes, reference mapping of the data derived in this procedure demonstrates that comprehensive coverage (>95%) of the genome is achieved at the early stages of the sequencing run. In future iterations of the method, data from positive isolates could be more fully analyzed to determine if virulence genes are likely to be functional, and to identify variants of virulence genes conferring higher risk –37. It sSalmonella enterica serovar Enteritidis, WGS approaches have been shown to be advantageous since current typing methods (e.g. PFGE) sometimes lack the resolution required for detection of outbreaks, traceback and determination of transmission routes [Comprehensive WGS data derived from the GeneSippr procedure conducted in front-line testing labs could also be used for high-resolution “DNA fingerprinting” of pathogens. Firstly, this would ensure that the strain analyzed can be unambiguously distinguished from in-house control strains, and secondly, data could subsequently be shared with public health stakeholders for molecular source tracking. Currently, PulseNet, the network of laboratories involved in molecular subtyping for outbreak detection, uses PFGE methods to type STEC isolates for source attribution purposes . Howevern routes –7. For sn routes , 6. In aE. coli strains within a time frame that is in line with current methods  was used to randomly generate triplicate datasets of simulated 21-nt reads at 2-, 2.5-, 5- and 7,5-fold coverage (108 datasets). The reads from individual datasets were then mapped to the target sequences using nine k-mer sizes ranging from 5 to 20 nt, and the mean percentage of sequence identity (MPI) was calculated for each dataset. The average and standard deviation of the three MPI values obtained for each dataset at different k-mer sizes are shown. An MPI above 90% was used as the threshold for accurate identification.The genome of (TIF)Click here for additional data file.S2 FigE. coli Sakai reference genome (EC20040078) and gaps of all sizes (bp) were counted using a custom script. Frequency of each gap size is indicated. No gaps larger than 102 bases were observed.The 21-nt reads sampled in real time from the sequencing run that provided the worst genome coverage were mapped against the (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Tablede novo. Quality metrics and accession numbers for raw and assembled data are provided.To demonstrate quality of WGS data generated by the GeneSippr procedure, following completion of the sequencing run, reads were assembled (XLSX)Click here for additional data file.S4 Tablewww.genomicepidemiology.org. Full length gene identification confirms probe-based gene identification determined using GeneSippr and with the EHEC-7 laboratory method.WGS assemblies generated from GeneSippr data were queried using the VirulenceFinder tool available at (XLSX)Click here for additional data file.


Arabidopsis thaliana after exposure to 10 nm Ag NPs. Our results show that Macerozyme R-10 treatment can release Ag NPs from Arabidopsis plants without changing the size of Ag NPs. The characteristics of Ag NPs obtained by SP-ICP-MS in both roots and shoots are in agreement with our transmission electron micrographs, demonstrating that the combination of an enzymatic digestion procedure with SP-ICP-MS is a powerful technique for quantitative determination of NPs in plant tissues. Our data reveal that Ag NPs tend to accumulate predominantly in the apoplast of root tissues whereby a minor portion is transported to shoot tissues. Furthermore, the fact that the measured size distribution of Ag NPs in plant tissue is centered at around 20.70 nm, which is larger than the initial 12.84 nm NP diameter, strongly implies that many internalized Ag NPs do not exist as intact individual particles anymore but are aggregated and/or biotransformed in the plant instead.Plants act as a crucial interface between humans and their environment. The wide use of nanoparticles (NPs) has raised great concerns about their potential impacts on crop health and food safety, leading to an emerging research theme about the interaction between plants and NPs. However, up to this day even the basic issues concerning the eventual fate and characteristics of NPs after internalization are not clearly delineated due to the lack of a well-established technique for the quantitative analysis of NPs in plant tissues. We endeavored to combine a quantitative approach for NP analysis in plant tissues with TEM to localize the NPs. After using an enzymatic digestion to release the NPs from plant matrices, single particle-inductively coupled plasma-mass spectrometry (SP-ICP-MS) is employed to determine the size distribution of silver nanoparticles (Ag NPs) in tissues of the model plant  The increasing use of nano-enabled products in our society inevitably leads to more release of engineered nanoparticles (NPs) into the environment. In the agriculture sector for instance, silver nanoparticles (Ag NPs) are often incorporated into nano-agrochemicals, nano-biosensors, and used for nano-agri-food production . AccumulThe primary challenge to study nanomaterials in plants lies in the detection of internalized NPs within plant tissue matrices and the measurement of their specific characteristics, such as particle size, number, size distribution, and concentration. Several techniques have been explored for the detection of NPs, including Raman spectroscopy, X-Ray absorption spectroscopy, and transmission electron microscopy (TEM) , but unfSingle particle-inductively coupled plasma-mass spectrometry (SP-ICP-MS) was based on the well-established ICP-MS technique and has proven to be a powerful tool to directly quantify single particle size, concentration, and size distribution . Dan et Single particle-inductively coupled plasma-mass spectrometry has been applied in the analysis of water, food matrices, materials, biological tissues, body fluids for trace metals, and toxic elements. Among all those tests it is the biological tissues that challenge the accuracy of SP-ICP-MS measurements most. The complex matrices require the use of a strong acid extraction procedure to release the NPs from the matrix, a procedure that could affect these NPs chemically. This problem could be solved by using a macerating enzyme digestion method . Dan et Arabidopsis thaliana (Arabidopsis) exposed to 10 nm Ag NPs. The characteristics of the internalized Ag NPs were determined with SP-ICP-MS. Localization of the NPs in plant tissues at subcellular level was examined by conventional TEM. Our goal is to establish a reliable method to determine Ag NPs uptake and accumulation in Arabidopsis tissues. Moreover, we aim to depict a deposition pattern of Ag NPs in plant tissues and to examine possible translocation of Ag NPs toward the aerial part of the plant.We have used the same macerating enzyme for the release of Ag NPs with the model plant species Arabidopsis thaliana Columbia (Col) wildtype seeds were surface-sterilized with 75% ethanol and 15% clorox followed by thrice washing using deionized water. After washing, 30 sterilized seeds were plated on 1/2 MS  medium supplemented with 1% sucrose and stratified at 4°C for 2 days in the dark. Seeds were germinated and grown in a nearly vertical position at 22°C with a 12 h/12 h light/dark regime and a light intensity of 120 μmol m-2 s-1 for 2 weeks before the seedlings were being subjected to NPs exposure.Arabidopsis seedlings to 1/2 MS media containing 0.02 mg/L Ag NPs which was mixed with the medium before solidification. After growing for 2 weeks, whole plants were sacrificed and separated into shoot and root tissues. These tissues were washed thrice with deionized water before homogenization by a hand-held tissue homogenizer in 2 mM citrate buffer, with the pH adjusted optimally in the range of 3.5–7.0 for Macerozyme R-10 in accordance with the manufacturer’s instructions. After homogenization, samples were treated with 5% Macerozyme R-10 and shaken in a 37°C incubator for 24 h. After digestion, the samples were settled and diluted with ultrapure water for SP-ICP-MS analysis.Silver (Ag) NPs (Cat #730785) with an average particle size of 10 nm were purchased from Sigma–Aldrich (USA) and stabilized in sodium citrate. The NPs were sonicated for 30 min at 37 kHz using an ultrasonic cleaner to aid homogenous suspension and reduction of aggregation. Dilutions were prepared using deionized water and subsequently filtered with 0.2 μm sterilized filter before application. The NP treatment was performed by transferring 2-week-old Rhizopus sp. containing cellulase, hemicellulase, and pectinase which enables it to digest plant tissues and liberate the internalized NPs. In this study, Macerozyme R-10 was first investigated for its effect on Ag NPs such as dissolution and/or aggregation. This was performed as described by Macerozyme R-10  is a complex macerating enzyme from 107Ag. The 30 nm Au NP standard was used for particle calibration to measure the particle size of Ag NPs in the samples and to determine the transport efficiency. This standard was also used at different concentrations to derive the detection limit. The detection limit was defined as the minimum detectable size of a single NP  transmission electron microscope at an accelerating voltage of 80 kV.For the image editing and composition of the figure we used the open source programs GNU Image Manipulation Program (GIMP) and Inkscape.Figure 1 shows the number of Ag counts in different sizes for Ag NPs that were subjected to Macerozyme R-10 and the non-treated control sample. The most frequent  size detected had a measured diameter of approximately 12.84 nm, slightly larger than the 10 nm NP size the manufacturer had described. This slight increase in size could be explained by the innate size of the Ag NP tested, because there was no significant difference in particle size distribution between enzyme-treated and non-treated: the number of counted NPs that were detected at 12.84 nm diameter did not differ significantly . These results demonstrated that application of Macerozyme R-10 causes no measurable effect (dissolution or aggregation) on the size distribution of Ag NPs as determined by SP-ICP-MS, which classifies it as a suitable enzyme for extraction of Ag NPs from plant tissues in this study.To investigate the effect of Macerozyme R-10 on Ag NPs during plant tissue digestion, Ag NPs were treated with Macerozyme R-10 and analyzed using SP-ICP-MS. Based on the sizes of Ag NPs detected, the particle size distribution histogram was generated. Figure 2A shows the raw data from the enzymatic digested roots (Replicate 1) that were previously dosed with 0.02 mg/L 10 nm Ag NPs for 2 weeks. Typical NP pulse signals were observed. Figure 2B shows the resulting particle size distribution of released Ag NPs as processed from the data in Figure 2A. Two replicates yielded highly similar results, displaying a peak at around 20.70 nm . As this observed NP size was larger than the initial dosed 12.84 nm, NPs may have been modified in the plant tissues.To determine the outcome of exposure to Ag NPs, Ag NPs treated plants were digested with Macerozyme R-10 prior to SP-ICP-MS analysis. Figure 3A) of Arabidopsis plants which grew on 1/2 MS medium containing 0.02 mg/L 10 nm Ag NPs. Compared to the root , fewer pulse signals  were detected in the shoot, indicating only a minor presence of Ag NPs in Arabidopsis shoot tissues. A nearly constant background signal at a level of circa 2 counts was observed in Syngistix . These continuous background signals indicate the presence of dissolved Ag in the solution  was processed from the data in Figure 3A. Similarly, particle size distribution in shoots also centered at 20.70 nm. Note that the particle size distribution in shoots showed less uniformity than in roots, as reflected by the large difference between the two replicates. The difference in counts between the root and shoot is striking, as root tissues had a five times higher reading frequency than shoot tissues . This experiment not only establishes the uptake and accumulation of Ag NPs by Arabidopsis root, but also strongly suggests that a translocation of Ag NPs occurs toward the plant shoot. Furthermore, the majority of the peaks for both histograms appeared in between 12.84 and 28.56 nm. The Syngistix software tabulated the most frequent size detected for both histograms as 19–20 nm and the mean size detected as 26–27 nm, which was approximately 1.52- and 2.06-fold larger than the size of an individual Ag NP. These results indicate that 10 nm Ag NPs hardly exist as intact individual Ag NPs in plant tissues.In the same experiment, SP-ICP-MS also detected Ag NPs signals in shoot tissues . Root-fed Ag NPs shown as individual particles and aggregated clumps were observed all over the cell wall, middle lamella plasma, intercellular space, and in the vacuole, demonstrating massive accumulation and internalization of Ag NPs in Arabidopsis root . The aggregated clumps could often be found in the middle lamella and the cell wall. We could not observe these universally scattered dark particles in the untreated control root cells whilst we did visualize the same kind of background dots there . Furthermore, in the cell wall and its middle lamella we observed only few black spots compared to the NP-fed roots where NPs were obviously retained across the cell wall, probably during the internalization process . We conclude that both the cell wall and the middle lamella were the primary accumulation sites for NPs.To establish whether the shootward Ag NPs translocation occurs as suggested by SP-ICP-MS analysis, we examined both root and leaf tissues using TEM to directly visualize the localization of Ag NPs at subcellular level. Micrographs revealed that electron-dense particles were predominantly present in the root as shown by numerous free NPs and some aggregated clumps in root cells . Ag NPs seemed to be prone to forming aggregates, resulting in varied size of clusters . In the absence of NPs, dark specks could be observed at several places in the leaf cell micrographs . These specks appeared to have a lighter density though, unlike the stodgy, much darker and rather compact particles that are present in NP-treated plants . The difference between the specks in untreated control plants and the dense particles in NP-treated plants are especially telling when examined under larger magnification . Therefore, it is believed that only the latter, darker particles represent the NP agglomerates. It is worth noting that, compared to the vast presence of Ag NPs in many root cells, Ag NPs could only be detected in some of the leaf cells, thereby establishing that silver NPs translocation from root to leaf did occur in vivo, albeit at a small scale only. Our TEM analysis agreed well with the SP-ICP-MS results that had demonstrated that the NPs that had accumulated in leaf tissues showed large variation in size and relatively few readings compared to NPs in root tissues.In the leaf of the same plant, deposition of Ag NPs was also observed around the cell wall and in the vacuole , in the sense that enzymatic treatment caused neither dissolution nor aggregation of Ag NPs in our experiment. Macerozyme R-10 treatment can release Ag NPs from Arabidopsis plants, which makes it suitable for the subsequent SP-ICP-MS analysis.Application of Macerozyme R-10 does not change the size of Ag NPs in SP-ICP-MS analysis  from the NP response in single particle mode in Syngistix, an accurate NP mass determination can be achieved, given the relatively low particle number concentrations used  represent individual particle events and the particle mass is determined by the intensity of the ICP-MS response. A continuous response, on the other hand, indicates the presence of dissolved metal in the solution . A propeons used .Figures 4C,D) suggests the intercellular passage of NPs through the cell wall. The existence of such a NP translocation pathway was reported earlier by Arabidopsis roots.The micrographs of the root sections clearly show that the cell wall and the middle lamella act as primary accumulation sites for NPs. It is at this location that we find the highest number of NPs accumulated. Although we are not sure whether subsequent symplastic transport occurs, the abundance of NPs inside the cell  and at the cell junction area where the cell wall connects to more than one cell  strongly indicates the predominant accumulation of Ag NPs in the root with only a minor translocation of Ag NPs toward the shoot. This is in line with our TEM analysis that showed more Ag NPs in root tissue than in shoot tissue . Micrographs reveal that NPs are widespread as monodisperse particles in many root cells, whereas variously sized NP aggregates were observed in a small portion of the leaf cells only, each aggregate displaying a distinct mass distribution and shape . During the preparation of the specimen for TEM, artifacts like precipitates can occur, as can be observed in the controls . For this reason, it is not possible to accurately pinpoint for an individual particle whether it is indeed a silver NP that has accumulated in the tissues. On the other hand, we did notice specific differences in location and accumulation patterns as well as a striking density difference between NPs and precipitates . Together with the SP-ICP-MS quantification data for Arabidopsis root and shoot, our results strongly indicate the presence of Ag NPs within these plant tissues.Earlier observations indicated that soil-grown n shoots . The facArabidopsis in this study.Other NPs such as titanium dioxide NPs and Au NPs have also been found to be transported toward the shoot area . It is iArabidopsis and tomato. Thus, similar types of NPs may behave differently in the process of internalization for different plants since the uptake is likely species-dependent, probably due to different properties of the cell wall and cytoplasm.From the SP-ICP-MS analysis, it can be concluded that the most frequently detected diameter of Ag NPs is 19–20 nm and the average diameter of Ag NPs detected is 26–27 nm, which is approximately 1.52 and 2.06 times larger than the initial diameter (12.84 nm) of monodisperse Ag NPs. These results strongly point toward possible transformations and/or aggregation of Ag NPs during or after their internalization. Whether they are modified in the cytoplasm itself or on their way before or during the entering of the cell is currently unknown. Interestingly, Arabidopsis. We found that in Ag NP treated plants the NPs accumulate predominantly at the middle lamella and cell walls in root tissue and that some Ag NPs can be translocated toward the leaves. The true nature of inter- and intracellular NP transport is not known yet nor do we know which transformations happen to the Ag NPs in plant tissues. The next challenge will be to bring light into the uptake and transport pathways and to unravel their underlying mechanisms.Using SP-ICP-MS in combination with TEM, we succeeded in combining a quantitative characterization of Ag NPs with visual NP localization in plant tissues, which enabled us to clarify where, and to what extent, NPs would be translocated in ZC initiated the project. DB did the TEM and data analysis. ZGO performed SP-ICP-MS. DB, ZGO, and ZC wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The Women's Health Initiative (WHI) Estrogen-Alone Trial randomized postmenopausal women, 50 to 79 years of age, with prior hysterectomy, to conjugated equine estrogens (CEE) or placebo with a 5.9-year median duration of CEE use. In 2013, the WHI published outcomes for additional extended follow-up. Reported here for the first time is an analysis of the number needed to treat with CEE rather than placebo for younger women (50–59 years) to prevent an adverse long-term outcome. For every 76 women randomized to CEE at 50–59 years, one less myocardial infarction occurred during the 13-year cumulative long-term follow-up. For every 37 women randomized to CEE at 50–59 years, one less woman experienced a global index endpoint  during the 13-year follow-up. Younger women (50–59 years), compared to older women, had more favorable cumulative long-term outcomes for MI and global index. Though a subgroup analysis is not an adequate basis for making primary prevention guideline recommendations, the WHI Estrogen-Alone Trial outcomes strongly suggest that a similar course of estrogen initiated at 50–59 years in postmenopausal women with prior hysterectomy results in significant long-term health benefit. The Women's Health Initiative (WHI) hormone trials are randomized, double blind, predominantly primary prevention trials, with long-term follow-up evaluating hormone therapy in postmenopausal women. The WHI Estrogen-Alone Trial randomized 10,739 postmenopausal women with prior hysterectomy to either 0.625 mg of conjugated equine estrogens (CEE) daily or placebo [However, outcomes resulting from postmenopausal hormone therapy trials may be affected by multiple factors, including the specific estrogen or progestogen agent used, duration of therapy, and characteristics of the group of women treated –8. The iThe current report makes the case in which outcomes clearly differ by age at time of randomization to CEE in the WHI Estrogen-Alone Trial and that cumulative long-term outcomes for women 50–59 years of age show a net benefit with CEE. In contrast, the WHI trial authors did not take the position in which the evidence from the WHI Estrogen-Alone Trial showed an overall cumulative long-term benefit for younger women randomized to estrogen . OutcomeP values and confidence intervals for hazard ratios in this paper are as provided by the WHI investigators in prior reports.For the analysis, in this paper, the number needed to treat (NNT) was assessed over the entire 13-year follow-up, including both the intervention and the postintervention phase, in order to help the clinician assess the overall impact of the effect of randomization to CEE in the WHI Estrogen-Alone Trial. The calculations were performed using SAS software (version 9.3) with an add-on module as per Bender , 11. CalAt 40 clinical centers in the United States, the WHI Estrogen-Alone Trial enrolled 10,739 women 50–79 years of age with prior hysterectomy. Subgroup analysis by age was prespecified in the trial protocol [The intervention phase lasted for a median duration of 7.2 years before the trial was stopped because of an elevated stroke rate in the CEE group  . The medThe majority of WHI Estrogen-Alone Trial participants were randomized to CEE or placebo at a median age older than the typical age for starting postmenopausal therapy in clinical practice. The age for initiating CEE or placebo in the trial is shown in The only statistically significant outcome for all participants (50–79 years) in the WHI Estrogen-Alone Trial for the 13-year cumulative long-term follow-up was a reduction in invasive breast cancer with CEE  [In the WHI Estrogen-Alone Trial, for 7.2-year median intervention phase, for all participants (50–79 years), there was no difference with CEE compared to placebo for CHD (coronary heart disease) defined as nonfatal MI or coronary death, the primary trial endpoint  . There wP = .02), total mortality (P = .04), colorectal cancer (P = .02), and global index (P = .02) for the intervention phase and myocardial infarction (P = .007) and global index (P = .01) for cumulative long-term follow-up [Multiple trial outcomes in the WHI Estrogen-Alone Trial were more favorable in younger compared to older participants randomized to CEE. Hazard ratios by decade of age of participant for primary and secondary trial outcomes with a significant trend by age are shown in ollow-up .P for interaction by age was not statistically significant [For the intervention phase (7.2 years), for women 50–59 years of age in the WHI Estrogen-Alone Trial, there was no statistically significant difference in CEE versus placebo for CHD  [For adverse outcomes in the intervention phase, for CEE compared to placebo, in women 50–59 years of age, there was an increase in DVT  which is consistent with the increase in DVT for all participants (50–79 years) , 21. P value (P = .77) for trend by age does not suggest that the hazard ratio for the younger age group (50–59 years) can reliably be considered different from the statistically significant elevated hazard ratio for stroke present in the entire group of women 50–79 years of age.)For women 50–59 years of age at trial entry, stroke rate for the intervention phase was similar for CEE and placebo groups  [A post hoc classification of stroke outcomes as ischemic or hemorrhagic showed a HR of 1.09  for women 50–59 years of age randomized to CEE for ischemic strokes . For womWith cumulative long-term follow-up of 13 years in the WHI Estrogen-Alone Trial, there were a number of outcomes with a reduced hazard ratio for CEE versus placebo in women 50–59 years of age at trial entry [Annualized incidence rates allow direct comparison of the magnitude of the difference between CEE and placebo groups, while hazard ratios provide information on the relative difference in outcome, but not the absolute differences in outcome. In The number of women needed to be randomized to CEE to prevent one woman from developing an adverse outcome during the cumulative 13-year follow-up in the WHI Estrogen-Alone Trial, for women 50–59 years of age, was calculated as noted in the Methods Section. This number-needed-to-treat analysis provides a measure of the summation effect of randomization to CEE versus placebo for the entire 13-year cumulative follow-up period. For every 76  women randomized to CEE rather than placebo at 50–59 years of age, there was one less woman having a myocardial infarction (MI) . For eveA number-needed-to-treat analysis for MI and global index for the intervention phase for women 50–59 years of age did not show a statistically significant difference in outcomes for the CEE versus placebo group.In the WHI Estrogen-Alone Trial with cumulative long-term follow-up, CEE use for a 5.9-year median duration compared to placebo resulted in a statistically significant reduction in invasive breast cancer for women 50–79 years of age  . Breast Outcome data stratified by years since menopause , 23 has There is a high degree of biologic plausibility that a woman's age and duration of time since menopause at initiation of hormone replacement therapy may affect clinical outcome . The timThe WHI Estrogen-Alone Trial results indicate that long-term follow-up is required to fully assess the effects of estrogen in postmenopausal women. For all participants (50–79 years) in the WHI Estrogen-Alone Trial, a reduction in invasive breast cancer only became statistically significant with approximately an additional 5 years of follow-up after the completion of the CEE intervention phase , 12. SimThe single published Kaplan-Meier estimate for women 50–59 years of age (known to the author) from the WHI Estrogen-Alone Trial is for the outcome of CHD for the intervention phase of the trial . The KapThe only published Kaplan-Meier estimate extending at least 10 years for hormone replacement therapy for women less than 60 years of age in a primary prevention trial is from the Danish osteoporosis prevention study (DOPS) . DivergiIf Kaplan-Meier cumulative hazard estimates extending through 10 years had been published for the WHI Estrogen-Alone Trial for CHD, death, MI, or global index endpoints for women 50–59 years of age, similar diverging outcome curves to the DOPS data would be shown. Long-term follow-up of cardiovascular, cancer, and mortality outcomes through 10 years, including at least 5 years of follow-up after completion of hormone therapy, is advisable to adequately assess the effects of hormone replacement therapy.Of note, in DOPS, where 81% of the women received both estrogen and progestin, participants had predominantly favorable outcomes , while wThe long duration of the follow-up of the WHI Estrogen-Alone Trial may have influenced outcomes. The 13-year median cumulative follow-up included the intervention phase and the 6.6-year median duration postintervention phase of the trial. The WHI trial study's authors reported on that time duration for their most recent comprehensive article on outcomes [The initial adverse effect of CEE on stroke and DVT documented during the intervention phase that diminished in the postintervention phase may have been simply the waning of the adverse effect that occurred after CEE was discontinued. The similarity of the relative risks in the intervention and postintervention phase for myocardial infarction, CHD, death, invasive breast cancer, and global index suggests a possible perseverance of a beneficial effect of CEE that lasted beyond cessation of the medication. If vascular beneficial effects occurred during the intervention phase, the ramification of these effects would tend to continue to manifest beyond the point in time when CEE was discontinued. The mechanism of the persistent effects of hormone therapy after cessation in regard to invasive breast cancer is beyond the scope of this paper.The unblinding occurred when the trial was stopped because of an increased risk of stroke in the CEE group . This woAge was prespecified in the WHI trial protocol as a subgroup for analysis. A limited number of subgroup analyses in the WHI hormone trial reports were thought to be important enough to warrant stratifying primary outcomes in the presentation of the initial WHI trial results , 2. In tP for interaction . This tP for interaction). Four primary or secondary endpoints in the intervention phase and two in the cumulative long-term follow-up had a significant trend by age at the .05 significance level. For the 20 primary and secondary trial endpoints [P for interaction would be expected by chance alone in the intervention phase and one by chance alone for cumulative long-term follow-up at the .05 level of significance.Multiple primary and secondary endpoints underwent evaluation for trend by age (ndpoints  in the WThe number-needed-to-treat analysis includes the extended follow-up phase which is unblinded and with incomplete participant follow-up. These same limitations are present for all WHI reported annualized incidence percentages and rates for postintervention and cumulative long-term follow-up , 33, 34.Nominal 95% confidence intervals are reported without adjustment for multiple outcomes, sequential repeated data analysis, multiple subgroup analyses, and extension of trial follow-up with incomplete participation. This is true for this report and for the majority of the published WHI trial outcome results , 32–34. Women 50–59 years of age at time of randomization to CEE or placebo in the WHI Estrogen-Alone Trial were similar in median age to women initiating hormone replacement therapy in clinical practice. In the intervention phase, for women 50–59 years of age with CEE, there was an increased risk of DVT, gall bladder disease, and stroke, while the reduction in MI, invasive breast cancer, and global index of events was not statistically significant. With cumulative 13-year long-term follow-up, women 50–59 years of age with CEE showed a reduction in MI, as well as a reduced global index of events. The increased risk of stroke and DVT in the intervention phase for women 50–79 years of age, which did not show significant trends with age, declined with cessation of CEE. Long-term follow-up including at least 5 years of follow-up after completion of hormone therapy is necessary to optimally evaluate effects of hormone replacement therapy on cardiovascular, cancer, and mortality outcomes. Though a subgroup analysis does not provide an adequate basis for making guideline recommendations for primary prevention, the preponderance of evidence in the WHI Estrogen-Alone Trial strongly suggests an overall benefit with CEE with cumulative long-term follow-up in women 50–59 years of age. These potential benefits only apply to women with prior hysterectomy and for duration of CEE use similar to what was used in the trial. The WHI Estrogen-Alone Trial data does not provide information on longer durations of use and strongly suggests that initiation of hormone therapy at significantly later ages is harmful.


Traditionally, resin composite restorations are claimed by reviews of the dental literature as being superior to glass-ionomer fillings in terms of restoration failures in posterior permanent teeth. The aim of this systematic review is to answer the clinical question, whether conventional high-viscosity glass-ionomer restorations, in patients with single and/or multi-surface cavities in posterior permanent teeth, have indeed a higher failure rate than direct hybrid resin composite restorations.versus resin composite restorations for direct head-to-head comparison were found. Three trials related to high-viscosity glass-ionomers versus amalgam and three trials related to resin composite versus amalgam restorations could be included for adjusted indirect comparison, only.Eight databases were searched until December 02, 2013. Trials were assessed for bias risks, in-between datasets heterogeneity and statistical sample size power. Effects sizes were computed and statistically compared. A total of 55 citations were identified through systematic literature search. From these, 46 were excluded. No trials related to high-viscosity glass-ionomers The available evidence suggests no difference in the failure rates between both types of restoration beyond the play of chance, is limited by lack of head-to-head comparisons and an insufficient number of trials, as well as by high bias and in-between-dataset heterogeneity risk. The current clinical evidence needs to be regarded as too poor in order to justify superiority claims regarding the failure rates of both restoration types. Sufficiently large-sized, parallel-group, randomised control trials with high internal validity are needed, in order to justify any clinically meaningful judgment to this topic. Fet al. (2004) found a survival rate for HVGIC  similar to that of amalgam but showed significantly lower survival rates for “low-viscosity” GICs  than for amalgam [Over the last twenty years, the term ‘high-viscosity’ or ‘high-viscous’ C-GICs (HVGIC) has emerged within the scientific dental literature. HVGICs appear distinct from other (low) viscosity C-GICs  in comparison of their survival rate to that of conventional amalgam restorations. The results of a meta-analysis by Frencken  amalgam . Subsequ amalgam , 10. In  amalgam , 10. Despite many published reviews of the dental literature -7, no syThe protocol of this systematic review has been registered with the International Prospective Register of Systematic Reviews (PROSPERO / Nr: CRD42013006487) and was published in an open access journal .1S1/Section . All databases were searched until 02.12.2013, the cut-off date. Citations were eligible for possible inclusion if in line with the following inclusion criteria:Electronic international databases: PubMed/Medline and CENTRAL (Cochrane Library); databases of open access journals: Directory of Open Access Journals (DOAJ) and Biomed Central; regional databases: IndMed and Sabinet, as well as databases for grey-literature: OpenSIGLE and GoogleScholar were searched by both authors independently. The details of the search strategies, including the strings of search terms (containing MeSH and text search terms) together with Boolean operators, per databases are presented in Additional file Full report of a prospective clinical control trialClearly reported parallel group design with dichotomous outcome measureOutcome measure: restoration failure due to fracture, wear, and secondary caries and/or retention loss Intervention in load bearing cavities of permanent posterior teethFollow-up period: 1 year or longer with longest comparable result per studyversus amalgamResin composite versus amalgamHVGIC versus resin compositeHVGIC Trials comparing:Trial participants comprised all patients of any age, gender or place of origin with suitable tooth cavities in posterior permanent teeth. Articles were further excluded according to the criteria: Tunnel restoration or preventive resin restoration  as intervention typeCermet or low-viscosity glass-ionomer cements as test interventionChemically curing composite resin or non-hybrid resin composite as control interventionLow-copper amalgam alloys as control intervention Resin composite and/or amalgam restorations placed with GIC liner or basen = number of failures; N = total number of evaluated units, per intervention group) reportedNo computable data  were included. For possible network-meta analysis or indirect treatment comparison (ITC), only datasets that matched in regard to restored cavity type and follow-up period, as well as with low statistical heterogeneity  scanned titles and abstracts of identified citations from data sources in duplication. Articles with suitable titles but lacking listed abstracts were retrieved in full copy. All included articles were judged separately by the authors: for possible exclusion, with reason, or for acceptance, in line with the exclusion criteria. Disagreements between authors were resolved through discussion and consensus. 2S1/Section . Verbatim quotes relevant to selection-, performance- and detection bias risk were extracted from all accepted trial reports. Assessment of the risk of selection bias and detection/performance bias was based on these, using criteria , shown iIn order to assess attrition bias risk, worst- and best-case scenarios were assumed. These were calculated in cases were the number of lost trial participants per intervention group was reported in the trial reports. The results were then compared to the intervention outcomes computed for participants available to follow-up. On this basis conclusions concerning attrition bias risk were drawn: high risk of attrition bias was assumed if the computed outcomes between worst- and best-case scenario and the intervention outcomes computed for participants available to follow-up differed significantly. Where the number of lost trial participants per intervention group was not reported, a high risk of attrition bias was assumed by default. The worst-case scenario was calculated by adding the number of lost-to-follow-up participants in the test group to the failures of that group and adding the number of lost-to-follow-up participants in the control group to the successes of that group. The best-case scenario was calculated by adding the number of lost-to-follow-up participants in the test group to the successes of that group and adding the number of lost-to-follow-up participants in the control group to the failures of that group. Intervention outcomes were computed for assessment of attrition bias as Relative risks (RR) with 95% Confidence intervals (CI). The two reviewers conducted the assessment separately. Disagreements were resolved through discussion and consensus. metaRegister of Controlled Trials (mRCT); South African National Clinical Trials Register; WHO Clinical Trials; IADR abstracts; International Poster Journal of Dentistry and Oral Medicine. Comparison of the number of identified unpublished with the number of published (accepted) trials was used to assess the possible extent of potential publication bias risk.In order to assess the risk of publication bias, the following trial registers and electronic sources were searched for abstracts and/or titles of trials in line with trial selection criteria as per sections: Australian New Zealand Clinical Trials Registry; Clinical Trials US; EU Clinical Trials Register; Graphical and statistical assessments of publication bias risk were planned if the number of extracted datasets exceeded ten.n) from the total number of evaluated units (N) for dichotomous data. Restoration failures were defined as: fractured restorations; secondary caries on restoration margins; partial or total loss of restoration retention; excessive wear of the restoration material. Only primary outcomes with either the patient or the tooth as unit of analysis were accepted. For each extracted dataset of n / N for the test- and control groups, the Odds ratio (OR) with 95% Confidence intervals (CI) and significance levels  were computed, using the statistical software programme Rev Man 4.2. The 5% significance level was used. In addition, each accepted dataset was assessed as to whether it contained the minimum sample size needed in order to have >80% power for detecting an effect size difference of 10% between directly compared interventions. . The effect size difference was chosen in keeping with past considerations for sample size calculation [Acceptable outcome measures were the number of restoration failures  and their upper confidence values were used in order to interpret the test results: 0-40% = might not be important; 30-60% = may represent moderate heterogeneity; 50-90% = may represent substantial heterogeneity; 75-100% = considerable heterogeneity [The ogeneity .2I > 40% and Cochrane’s Q-test/p-value < 0.10. However, datasets in compliance with these set test criteria were not assumed to be free of in-between heterogeneity, particularly due to the weak statistical power of both tests when the number of datasets are low [Across different trials, only datasets that showed lack of statistical in-between heterogeneity were considered eligible for indirect comparison. The cut-off point for statistical heterogeneity was set at  are low , but werDirect comparisons were considered to be comparisons between intervention groups within a controlled clinical trial setting . The sysversus amalgam trials with the clinical results of the resin composite versus amalgam trials through use of Indirect Treatment Comparison (ITC) or, if possible, network meta-analysis [It has been reported that only a few clinical trials that have directly compared HVGIC with resin composite restorations have yet been conducted . As it wanalysis , 20.et al. (1997) was applied [For ITC, the statistical methodology given by Bucher  applied , 22. The applied . All ind1) provides information on the number of articles identified. From the generated citations, 55 clinical trials were provisionally included. Of these, three could not be traced in full [. Details of the assessment results are presented in Table 1.Assessment of selection- and performance-/detection bias risk was based on verbatim quotes extracted from all six accepted trials  , 30. Non2 and indicate high bias risk for all except two datasets (CA01 and CA04) from two accepted trials [Only datasets that were judged sufficiently homogeneous in line with the trial inclusion criteria were quantitatively assessed for attrition bias risk. The results are presented in Table d trials . 8S2/Section ) identified six further studies that were presented from 2005 – 2013 at dental congresses. Of these, five studies appeared to remain unpublished to date; while one study was only partially published in the format of a case report in a regional dental magazine [S3).As the number of accepted datasets was < 10, no statistical and graphical assessment of publication bias risk was conducted. The search results from international and regional clinical trial registers and presented congress abstracts  [From the six accepted clinical trials, 21 datasets were extracted. In addition, the review authors computed three further datasets from the information given in one trial report . Each of the nine pairs was investigated for statistical in-between-datasets heterogeneity . Accordingly, five pairs were excluded and four pairs accepted for ITC (Table 4). All four pairs of accepted datasets appeared to be statistically homogeneous  and comprised:Of the total 24 datasets nine datasets from three trials comparing composite resin Two pairs of datasets, CA01/GA02 , 31 and One pair of datasets, CA02/GA07 , 30, regOne pair of datasets, CA04/GA17 , 29, reg7S2/Section . The type of information presented in the trial report about trial characteristics varied greatly and thus made further in-depth comparison within each dataset pair impossible. However, from the comparison tables it can be discerned that patients within the dataset pairs CA01/GA02 [7S2/Section ). No meta-analysis was conducted, due to lack of sufficient homogeneous data.The characteristics of the datasets in the four pairs are listed in Additional file A01/GA02 , 31 and A01/GA02 , 31 diff6S2/Section ).Assessment of all accepted datasets concerning the minimum sample size needed in order to have >80% power for detecting an effect size difference of 10% between directly compared interventions showed that the sample size of the datasets included in the two ITCs concerning Class II restorations after 24 months were too small. However, the sample sizes in all datasets for the ITCs concerning Class I restorations after 60 months and Class I/II restorations after 24 months appeared sufficient  [p > 0.05) than results from direct comparisons in RCTs. However, adjusted indirect comparison partially preserves the rigor of RCTs by considering the direct comparisons of both treatments of interest with the same controls [Notwithstanding the lack of trials directly comparing HVGIC . (1997) . However. (1997) . Accordicontrols . Therefo3) and were sufficiently homogeneous in terms of outcome measure; control intervention; cavity type restored; assessment method and length of follow-up period, it was not possible to eliminate the risks of heterogeneity between the available datasets, completely. One further potential limitation of indirect comparisons is related to the high risks of heterogeneity between trial characteristics of the compared groups. Although, in this systematic review great care was taken to only admit datasets for comparison that showed small risk of statistical heterogeneity . Most trial reports could not be accepted because they investigated older generations of restorative materials, such as chemically curing composite or low copper amalgam, that differed from those in current use and would have introduced further levels of clinical in-between-trial heterogeneity to the latter accepted RCTs. The number of excluded studies, particularly for resin composite versus amalgam restorations were excluded . Characteristics, such as higher risk for cavities and poorer oral hygiene and dietary behaviour in patients with at least two cavities, as opposed to patients with only one cavity in trials with parallel-group design, would have introduced clinical in-between-trial heterogeneity that needed to be avoided during ITC analysis. Such rather strict approach in trial selection according to aspects of lowest bias risk and highest achievable precision was needed, owing to the paucity of direct comparison/RCT in the current literature and in order to derive the most reliable and valid results through the more problematic adjusted indirect comparison method, instead.Care was further taken not to include trials with split-mouth study design. Thus three trials comparing HVGIC versus resin composite restorations were identified through the systematic literature search [et al. (2013) included composite restorations with HVGIC base, only, that would have introduced clinical HVGIC characteristics into the composite treatment group [et al. (2013) was only partially reported in a dental magazine at search cut-off date and, thus was unsuitable for further review [Two trials investigating direct comparison of HVGIC e search , 32. Hownt group . The triAll of the accepted trials appear limited by risk of either selection- and/or detection-/performance bias. 1) [Only two trials reported on the use of an adequate randomisation method , 30. Non1) . None ofFrom the onset, in all trials successful blinding or masking appeared not to have been possible, owing to the obvious differences in clinical appearance between the compared materials. For that reason, the allocation to either treatment group was visible to patients and operators. Only two trials reported adequate methods of masking/blinding of the trial evaluators , 30. How2), the risk for attrition bias may be regarded as high in all compared datasets, but particularly for the comparison of Class I tooth restorations after a 60-month period (DS CA02 versus GA07) [On the basis of the results of the quantitative risk assessment (Table us GA07) , 30. N  < 10) it was not possible to establish information concerning publication bias risk through graphical and statistical methods. However, the lack of listed trial protocols and relevant conference abstracts of unpublished trials suggests that the risk of publication bias to the reviewed topic may be low. Owing to the low number of available datasets (4). The relatively large width of the confidence intervals may confirm previous observations [6S2/Section ).Based on the conducted ITC analysis, the 95% Confidence intervals of all four Odds ratios (ORs) included 1.00, thus indicating no statistically significant difference between HVGIC and resin composite restorations (Table rvations  that adjThe available evidence suggests no difference in the failure rates between both types restoration beyond the play of chance, is limited by lack of head-to-head comparisons, an insufficient number of trials, as well as by high bias and in-between-dataset heterogeneity risk. Therefore the current evidence requires corroboration by large-sized, parallel-group, randomised control trials that compare HVGIC with resin composite restorations. Such future trials should avoid attrition bias risk through high loss-to-follow-up; include an investigation of performance/detection bias influence into its methodology, and test for selection bias risk, using the Berger-Exner test in order to quantitatively ascertain the level of adherence to the allocated random sequence throughout the trial . Superiority claims regarding the failure rates of direct hybrid resin composite restorations above that of conventional high-viscosity glass-ionomer restorations, in patients with single and/or multi-surface cavities in posterior permanent teeth, cannot be justified by the current poor clinical evidence to this topic. Direct head-to-head comparisons through clinical sufficiently large-sized parallel group randomised control trials with high internal validity are needed in order to justify any clinically meaningful judgement.


While there is an increasing pool of literature documenting the benefits of near-peer tutoring programme, little is known about the benefits for junior and senior peer tutors. Knowledge of the peer tutors’ perceived benefits at different levels of seniority will aid in the development of a near-peer tutoring programme that will better fulfil both curricula and personal aspirations of near-peer tutors. We, therefore, investigated the perceived benefits of participation in a near-peer tutoring programme for junior as well as senior near-peer tutors.Pre- and post-participation questionnaires were distributed to near-peer tutors after their clinical skills teaching sessions with Phase I undergraduate medical students. The Peer Tutor Assessment Instrument questionnaires were distributed to the 1) students, and to the 2) near-peer tutors (junior and senior) after each teaching and learning session for self-evaluation.p=0.03). As a whole, the near-peer tutors were more motivated  to participate in future teaching sessions but did not expect that having teaching experiences would make teaching as their major career path in the future . The senior near-peer tutors were evaluated significantly higher by the students (p=0.0001). Students’ evaluations of near-peer tutors on the domain of critical analysis was higher than self-evaluations (p=0.003).The senior near-peer tutors felt that their participation in the programme had enhanced their skills (Generally, the near-peer tutors perceived that they have benefited most in their skills enhancement and these near-peer tutors were scored highly by the students. However, senior near-peer tutors do not perceive that the programme has a lasting impact on their choice of career path. The emphasis of the General Medical Council (GMC) of the United Kingdom on the need for training medical students with teaching and learning skills encourages future doctors to become better teachers. The medical graduates must be able to ‘demonstrate appropriate teaching skills’, and as future physicians, they would need to understand the principles of medical education, be able to apply the various methods of teaching and learning, and be responsible to teach their colleagues . The iroMany studies reported that near-peer tutoring is effective, cost efficient, and is a popular educational intervention tool where students themselves are involved in imparting educational principles and knowledge. However, the capability of students to teach each other and junior students successfully relied heavily on the training of these students as tutors –10. The Literature on the effect of near-peer tutoring on the near-peer tutors themselves suggested that the programme could increase the development of self-esteem, with near-peers becoming more effective communicators and having a better understanding of learning and teaching principles –19. WhilThis study was conducted over a course of 8 months (September 2013 to May 2014) at the Clinical Skills and Simulation Centre, International Medical University (IMU), Kuala Lumpur, Malaysia.All the Year 1 and 2, Phase I, undergraduate medical students who were taught by the near-peer tutors were recruited for this study. The study population is made up of 35 junior and 16 senior near-peer tutors. For the junior near-peer tutors, a minimum of 19 students would need to be recruited as participants from the pool of near-peer tutors in the programme to achieve a 95% confidence level with an allowable error margin of ±7%. For the senior near-peer tutors, a minimum total of 15 students would need to be recruited as participants to achieve a 95% confidence level with an allowable error margin of ±0.1%.All the post-Year 3 medical students who have sat and passed their first Professional Examination were included in this study and categorised as the junior near-peer tutors. All medical students who have sat and passed their final year, newly graduated doctors waiting for their housemanship  at government hospitals, were included in this study and categorised as senior near-peer tutors. Participation was voluntary. All the junior and senior near-peers who gave informed consent were recruited for the study, and all those who did not give informed written consent were excluded.All post-Semester 5 and post-Semester 10 medical students were invited to apply for the near-peer tutoring programme. The potential near-peer tutors were invited to attend a compulsory 1 day training workshop where they were taught on adult learning and clinical skills learning. After the workshop, the potential near-peer tutors were assessed on their clinical skills teaching. They were to teach two junior medical students on a selected clinical skills, and their teaching skills were evaluated by the clinical skills lecturers. The acceptance rate of these medical students for the near-peer tutoring programme after clinical skills lecturers’ evaluation was about 80%.The near-peer tutors (junior and senior) were given pre-participation questionnaires to fill prior to their teaching experiences at the clinical skills and simulation centre. The near-peer tutors were given four clinical skills sessions each, and each teaching session lasted 2 hours. The skills taught were either physical examination skills or procedural skills. At the end of the fourth session, the near-peer tutors were given post-participation questionnaires to fill.The pre- and post-participation questionnaires (Peer Tutors Own Assessment) used in the assessment of the near-peer tutors were the adapted version of questionnaires developed by Buckley and Zamora . The queAll the Year 1 and 2, Phase I, undergraduate medical students who were taught by the near-peer tutors were given the Peer Tutor Assessment Instrument questionnaire to fill. The questionnaires were distributed to the medical students at the Clinical Skills and Simulation Centre at the end of every class taught by the near-peer tutors. The adapted version of the Peer Tutor Assessment Instrument  developeThe purpose of the study was explained to the students and consents were sought before the distribution of the hard copies of the questionnaires. The students were given 15 min each to complete the questionnaire.The study protocol was approved by the Joint Ethics Committee of the International Medical University in compliance with the Helsinki Declaration (Project Number: IMU 281/2013).t-test. The questionnaire completed by the students which assessed their acceptability of the teaching by the near-peer tutors (Peer Tutor Assessment Instrument) were evaluated using the independent t-test. In this study, a P-value of less than 0.05 was considered as statistically significant.Statistical Package for Social Sciences (SPSS) version 17.0 was used to analyse the data collected. The completed pre- and post-teaching questionnaires (Peer Tutors Own Assessment) by the near-peer tutors were evaluated using the independent as well as the paired n=34). Nineteen junior and 15 senior near-peer tutors were recruited for the study. The overall mean age ±SD of the participants was 21±2.75 years. An equal number of male and female participants were recruited (50%). Majority of the near-peer tutors did not have any previous teaching experiences (76.5%).n=19), as analysed from the post-participation scores, indicated that the near-peer tutor programme had the most impact on their motivation to do more teaching sessions in the future and to help their junior peers (5.53±0.48). The senior near-peer tutors (n=15) initially reported lesser expectation on the skills enhancement (5.07±0.80) and have shown less motivation (5.27±0.58) compared with their junior counterparts prior to joining the near-peer tutoring programme. However, in the post-participation questionnaires, the senior near-peer tutors were more inclined to report that the participation in this programme had enhanced their skills the most (p=0.03). Prior to participation, the senior near-peer tutors expected that participation in this programme were more likely to help to enhance their curriculum vitae (CV) and indicated that teaching would be a major component in their career path (4.93±1.02) when compared with the responses from the junior near-peer tutors (4.39±1.07). Post-participation, the senior near-peer tutors reported a lesser degree of agreement than previously stated: that the programme had helped to boost their career prospects (4.67±0.75).The male near-peer tutors in the pre-participation questionnaire responded that they were more likely to expect this programme to benefit them through skills enhancement (5.31±5.79) and to boost their career prospects (4.90±0.90) compared with the female near-peer tutors.n=1,053) of Phase I medical students (Year 1 to Year 3) participated and gave their evaluations on their near-peer tutors’ teaching sessions (n=34) using the Peer Tutor Assessment Instrument. Of all the near-peer tutors recruited for the study , only 13 senior near-peer tutors responded and completed the Peer Tutor Assessment Instrument after they had completed their teaching sessions. Therefore, the students’ evaluation of the two senior near-peer tutors who did not complete the self-evaluations were removed. The Phase I medical students’ (Year 1 to Year 3) respondents’ data (n=985) was analysed for this study.p=0.003) was seen in the scores given by the students compared with the near-peer tutors’ (junior and senior) self-evaluations on critical analysis.The senior near-peer tutors were evaluated significantly higher in all the domains assessed  when compared with their junior counterparts. There were no significant differences in the comparison of the self-evaluation scores of the senior and the junior near-peer tutors in all the domains assessed. Both groups scored lowest for the domain on critical analysis although the senior near-peers scored themselves slightly higher than the junior near-peers. Significant difference . Previous reports have identified that participation in a teaching programme such as this was perceived by near-peer tutors to be beneficial in the revision of their knowledge and provided them with an opportunity to reflect on their shortcomings, especially with regards to their knowledge depth. Additionally, participation in the programme helped to substantially provide them with a deeper level of understanding concerning the content of the knowledge taught , 26. AnoAlthough in general the near-peer tutors did see the importance of teaching and were more motivated to take up future teaching sessions, the senior near-peers did not feel that the programme had any effect on their choice of career paths or have any impact on the enhancement of their CV. It is therefore important to create more awareness of this career option by encouraging medical students to interact with more clinical teachers, introduce more formal teaching posts, and to expose them to this career path –29.The near-peer tutors’ scores on the pre- and post-participation questionnaires were on the higher end of the Likert scale with an average mean of five out of six points. The voluntary nature of the recruitment of near-peer tutors for this programme would have drawn in near-peer tutors that were highly motivated to teach when they first participated. Similarly, Wadoodi and Crosby did highlight in their article that voluntary recruitment processes rather than selecting those who are academically sound would draw and create opportunities for participation from potential near-peer tutors who could be academically weak but truly motivated to improve their clinical skills . There wAlthough the students scored their near-peer tutors very highly on the critical thinking skills, the near-peer tutors did not score themselves as high. This finding suggests that either some of the near-peer tutors have set possibly higher standards for themselves or some did not perform the teaching steps related to the domain of critical thinking. Although not reported in relation to near-peer teaching, there are contradicting reports from the literature on students’ ability to critically think. These reports suggest that in higher education, critical thinking skills either improve as the students increase in seniority –34 or thn=985) in the evaluation of their near-peer tutors and the consistency of high scores across the various near-peer tutoring sessions also suggested that students benefited from learning clinical skills from their near-peer tutors. Likewise, the high self-evaluation scores of their own teaching reflected that the near-peer tutors also have much confidence in the delivery of their teaching sessions. In fact, in some studies, it had been shown that near-peer tutors benefited more academically and cognitively when compared with their students (In the overall results, the students evaluated their near-peer tutors highly, indicating that students accept and recognise the teaching skills of the near-peer tutors. The large number of students’ response (students , 14, 38.students –41. Thisstudents , 43, in students .The limitations of this study are the relatively small sample sizes of near-peer tutors and the subjectivity of scorings of the questionnaires given due to the probable differences in the interpretation of the test items by the near-peer tutors as well as the students. Therefore, the results should be interpreted within these limitations. However, the results do imply that the near-peer tutoring programmes benefited the near-peer tutors equally regardless of their seniority but had not changed their perceptions of incorporating teaching as a long-term career consideration in the future. Efforts should be taken to optimise the exposure to teaching in medical schools to create awareness and interest to this career path. Although the near-peer tutors reported that the near-peer tutoring programme have enhanced their skills and the students’ responses stated likewise, there is a need to prove the effectiveness of this programme where their actual performance may need to be formally evaluated by faculty members and further analysed.Both groups of near-peer tutors equally benefited from the near-peer tutoring programme regardless of their seniority. Near-peer tutoring enhanced the skills domain of the near-peer tutor but formal analyses of their performances are needed to prove its effectiveness. The teaching skills of the near-peer tutors were evaluated highly by the students; however, the senior near-peer tutors were rated significantly higher. Although near-peer tutors did see the importance of teaching, the programme did not have any influence on their choice of future career paths for the senior near-peer tutors. Future studies on junior doctors’ perceptions of teaching would be useful to determine if there is a need to nurture the passion for teaching at an early stage in medical school.


The aim of the present study was to evaluate the clinical therapeutic effect of the combined application of alginate and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on the healing of refractory chronic skin ulcers. A single center, three arm, randomized study was performed at Jinan Central Hospital . A total of 60 patients with refractory chronic skin ulcers, which persisted for >1 month, were enrolled and randomly assigned into one of the following three groups: alginate dressing/rhGM-CSF group (group A), rhGM-CSF only group (group B) and conventional (vaseline dressing) group (group C). The wound area rate was measured, granulation and color were observed and pain was evaluated. The data were summarized and statistical analysis was performed. The results demonstrated that group A exhibited a significantly faster wound healing rate and lower pain score compared with the other groups (P<0.01). In conclusion, the combined application of alginate dressing and rhGM-CSF for the treatment of refractory chronic skin ulcers demonstrated significant advantages. It promoted the growth of granulation tissue, accelerated re-epithelialization and also effectively reduced wound pain, and thus improved the quality of life for the patient. This suggests that the combined application of alginate and rhGM-CSF may be an effective therapeutic strategy for the clinical treatment of refractory chronic skin ulcers. RecoIn order to achieve optimal results, rhGM-CSF must be continuously present in the wound at a certain concentration. In the present study, the combined effect of alginate and rhGM-CSF on the treatment of refractory chronic skin ulcers was investigated. It was hypothesized that the combination of alginate and rhGM-CSF significantly enhances wound healing compared with the treatment of rhGM-CSF alone.2, were selected for the present study. Patients with the following criteria were excluded from the present study: i) fasting plasma glucose levels >10.0 mmol/l, even following strict control; ii) patients with severe cardiac dysfunction (≥level III), severe renal dysfunction (≥level II), as well as severe diseases, including cancer, tuberculosis, chronic atrophic gastritis and systemic lupus erythematosus.A single center, three-arm, randomized clinical study was performed. Patients with refractory chronic skin ulcers were enrolled between October 2009 and March 2012 from Jinan Central Hospital . The present study was approved by the Ethics Committee of the Jinan Central Hospital and written informed consent was obtained from every patient. Patients with bedsores, varicose ulcers and diabetic foot ulcers, which persisted for >1 month following conventional and anti-inflammatory treatments, and with a wound area >10 cmPatients were randomly assigned to one of the following three treatment groups: group A, alginate dressing plus rhGM-CSF; group B, rhGM-CSF only; and group C, conventional treatment group using a vaseline gauze.Following cleaning the wounds with saline and drying them with sterile cotton, rhGM-CSF paste  containing 100 μg rhGM-CSF/10 g, covered by alginate dressing  was applied over the wound area for patients in group A. In group B, rhGM-CSF paste covered by a vaseline gauze was applied to the wounds, whilst in group C only a vaseline gauze was applied. The primary dressings were covered with cotton gauzes and bandages and the dressings were changed daily for the first 7 days and every other day thereafter.Visual observations were made on the wound color and the growth of granulation. The physicians who performed the evaluation were blinded to the treatment of the patients. The evaluation criteria were determined and scored on a four-point scale: 4 points, rosy colored wound surface and well-developed granulation tissue; 3 points, pink wound surface with moderate granulation tissue growth; 2 points, dark red wound surface with light granulation tissue growth; and 1 point, pale wound surface without granulation. The average scores for all the individuals in each group were calculated and expressed as the group healing score (GHS).The wound area was measured and recorded each time the dressing was changed. Healing was assessed as the percentage area that was healed [(pre-treatment area - post-treatment area)/pre-treatment area]. The ease with which the dressing was removed was also assessed, whilst the patient scored the comfort of the procedure. The dressing changes and treatment assessments were performed by unbiased nurses.Pain was assessed using the visual analogue scale (VAS) method. A 10 cm linear score was used, where 0 indicated painless and 10 indicated intolerable pain. Patients marked on the line based on their pain intensity.The data were analyzed using SPSS statistical software . Differences in the outcome variables were analyzed on an intention-to-treat basis. Differences in wound healing time between the groups were examined using the Chi-square test. P<0.05 was considered to indicate a statistically significant difference.2, with an average size of 17.2±8.0 cm2. The duration of the wound was 1–3.5 months, with an average time of 1.8±2.1 months prior to the start of the study. Patients were randomly assigned into one of the three treatment groups shown in A total of 60 patients were enrolled in the present study, including 35 males and 25 females, aged between 20 and 75 years with an average age of 50.6. In total, 25 patients had pressure sores, 15 had varicose ulcers and 20 had diabetic foot ulcers. The ulcer area ranged between 11 and 35 cmPre-treatment assessment was performed on the first day and was used as baseline for each patient. Observations were routinely performed each time the dressing was changed by physicians who were blinded to the treatment of the patients, and the data are summarized in Representative cases from each group are shown in Wound pain was assessed using the VAS method on a 10 cm scale. Group A showed significantly reduced pain compared with the other groups , and theet al (In the present study, the treatment of refractory chronic skin ulcers using a combination therapy of rhGM-CSF with sodium alginate was investigated. rhGM-CSF has been previously demonstrated to be a mitotic-promoting reagent capable of promoting wound healing. hGM-CSF was first used by da Costa et al  to treatet al , post-suet al  and hereet al , as wellet al ,20.+/Ca2+ ions with the exudate, forming a gel over the wound (Alginate is used for the treatment of wounds as it absorbs the exudate from the wound and exchanges Nahe wound . Throughhe wound . Besideshe wound . The alghe wound . Therefohe wound ,26, evenhe wound . The datSeveral previous studies have been performed using rhGM-CSF with intradermal or subcutaneous injection around the wound ,27–31. HIn the present study, patients with pressure sores, varicose ulcers and diabetic foot ulcers were enrolled, and each patient was randomly assigned to each group. However, statistical analysis to assess the differences based on the type of wound was not performed due to the limited number of cases. The results from the present study would have been more conclusive if patients with only one medical condition were selected. In addition, due to certain technical reasons the wound depth was not measured and instead the wound area was used as the sole standard for wound healing evaluation. Thus, further studies are required to validate the results from the present study.In conclusion, the present study demonstrated that alginate-rhGM-CSF dressing for refractory chronic skin ulcers promoted the growth of granulation tissue, accelerated re-epithelialization, whilst also effectively reducing wound pain. These results suggest that the combination of the two may be used for the routine treatment of refractory chronic skin ulcers.