Here, we present PGAweb, a user-friendly, web-based tool for bacterial pan-genome analysis, which is composed of two main pan-genome analysis modules, PGAP and PGAP-X. PGAweb provides key interactive and customizable functions that include orthologous clustering, pan-genome profiling, sequence variation and evolution analysis, and functional classification. PGAweb presents features of genomic structural dynamics and sequence diversity with different visualization methods that are helpful for intuitively understanding the dynamics and evolution of bacterial genomes. PGAweb has an intuitive interface with one-click setting of parameters and is freely available at  The advancement of sequencing technology and assembly tools has facilitated fast acquisition of genome sequences of various species over the past two decades. To make bacterial pan-genome analysis convenient and efficient, several tools, online databases, and web servers have been developed over the past two decades . PGAP Z, GET_HOMIn order to perform bacterial pan-genome analysis more efficiently, we have developed a user-friendly online tool, PGAweb, based on our previously published software, PGAP , PanGP , and PGAFigure 1). Compared with PGAP, PGAP-X introduces a novel algorithm for orthologous cluster identification, which can distinguish paralogs based on conserved genomic location from a genome alignment. PGAP-X also supports a visualization function for genome-wide sequence alignment, genome structure variation, and orthologous mapping based on sequence conservation. Users are able to choose one of the modules according to their research objectives.PGAweb consists of two core modules, PGAP and PGAP-X. PGAP supports five main analytic functions including orthologous clustering, pan-genome profiling, sequence variation analysis, species phylogeny, and gene functional classification. PGAP-X performs four comparative genomics assignments: genome-wide sequence alignment, orthologous clustering, pan-genome profiling, and sequence variation analysis  and GeneFamily (GF), are provided for orthologous clustering. In the MP method, first, orthologs between each pair of strains are identified by InParanoid ; MP is tf), and variation number (n) in 1 kb regions, are used to filter out high-substitution regions. Regions with the following traits will be identified as high-substitution regions: (a) no fewer than n substitution sites in the region; (b) variation frequency in the region is more than f; (c) the interval between any two substitution sites is less than 1/f. The high-substitution regions are marked on the genome structure. For pairwise variation analysis, a reference genome and a query genome should be selected.In this module, a new in-house algorithm is used for orthologous gene clustering . Whole-g1 as the front-end program. It adopts HTML5 and CSS3 protocols and uses D3.js2 and Echarts3 for result interaction. The server back-end adopts an Express Framework4 for Node.js MongoDB5 is used for the storage of related information. Docker6 is used as a container for packaging codes and software, which improves compatibility, portability, and safety. This website is open to all users, and login is not required.The web server is compatible with many platforms, including Safari, Firefox, Opera, Chrome, and Internet Explorer, by using Bootstrap FrameworkFigure 2. The first step is module selection for pan-genome analysis according to data type and research objectives. Two different pan-genome analysis modules are integrated in PGAweb. The PGAP module supports both draft and complete genomes and provides five main results, including orthologous clustering, pan-genome profiling, sequence variation analysis, species phylogeny, and gene functional classification. Most results generated by the PGAP module are text files. Detailed information can be found in these text files for in-depth pan-genome analysis. The PGAP-X module only supports analysis of complete genomes. The PGAP-X module’s capabilities include genome alignment, orthologous gene identification, genome variation detection, and pan-genome profile analysis. PGAP-X provides an in-house algorithm that can distinguish paralogous genes from orthologous genes based on conserved genomic location. The visualization function for genome structure conservation and gene distribution is integrated in the PGAP-X module.The general workflow of PGAweb is shown in Figure 2). First, specific input data for pan-genome analysis should be selected and uploaded. The PGAP module requires three types of files for each strain: nucleotide and protein sequences as well as annotations. Four input formats are selectable: (1) NCBI new data format: .cds_from_genomic.fna.gz, protein.faa.gz, and .feature_table.txt.gz extensions. These files can be downloaded from ftp://ftp.ncbi.nlm.nih.gov/genomes/all/. (2) NCBI old data format: .ffn, .faa, and .ptt extensions. These files can be downloaded at ftp://ftp.ncbi.nlm.nih.gov/genomes/archive/old_genbank/. (3) PGAP raw data format: .nuc, .pep, and .function extensions. These data may come from the user’s own laboratory..nuc files store nucleotide sequences in FASTA format; .pep files store corresponding protein sequences in FASTA format; .function files store annotations with three columns including gene name, COG classification, and function description. (4) NCBI GenBank (full) format: .gb extension. In addition, each protein sequence should have its corresponding nucleotide sequence and annotation information, while the length of the nucleotide sequence minus 3 (excluded stop codon) must be equal to three times the corresponding protein length. All uploaded files are auto-transformed to corresponding PGAP raw data format and criterion checking is performed. The PGAP-X module needs a complete genome sequence and annotation information for each strain. The input files should have .fna and .ptt extensions. More detailed description is available at http://pgaweb.vlcc.cn/doc. The second procedure is the selection of the orthologous gene identification method and pan-genome analysis functions. In the PGAP module, either the GF method or the MP method can be chosen for orthologous gene identification. The GF method is fast, but less accurate compared with the MP method. The GF method is suitable choice for handling more than 50 genomes with consideration of calculation time. In the PGAP-X module, there is only one method for orthologous gene identification. After selection of gene identification method, users need to select the desired functions for pan-genome analysis. Homologous gene clustering for the PGAP module and genome alignment for the PGAP-X module is required. Third, parameters for selected functions need to be set. Finally, users may provide e-mail addresses for obtaining notification of finished results and click the submit button for running PGAweb.This step contains four procedures . For the pan-genome profile, the relationships between the numbers of genomes and pan-genome size or core gene clusters are calculated. Whether a pan-genome is open or closed can be easily inferred from visual results. Pan-genome profiles provide indirect indications of lifestyle and opportunity for multiple mechanisms of DNA exchange with the global microbial gene pool of the specific species. Multiple algorithms for bacterial evolutionary analysis are provided to improve the accuracy of phylogenetic inference, especially for closely related species. All phylogenetic trees calculated by PHYLIP are output as .tree files, and the user can use visualization software to view the phylogenetic trees. Files with .outfile extension give users a simple view of the phylogenetic trees. For sequence variation analysis, information on positions with variation, amino acid types, nucleotide types, and variation types  are presented in tabular file 3.CDS.variation.txt. All statistical results of functional classification of core genes, dispensable genes, and strain-specific genes are dynamically displayed and compared. This result is useful for speculation of strain-specific physiological function and metabolic mechanism. Moreover, the actual gene lists  used in the functional classification can be downloaded for in-depth analyzes.Four parts of the pan-genome analysis results are showed in the PGAP-X output. Complete genome or pseudo-chromosome sequences are aligned in PGAP-X. In visualizing the alignment result, colored blocks are used to display homologous DNA fragments across strains, which highlight conserved regions, strain-specific blocks, and recombination events including insertions, inversions, and deletions on a large scale. The orthologous gene distribution in each conserved block is displayed in different colors that represent degrees of conservation for each gene. Genomic diversity of the bacterial population and differences in gene content are well depicted, and even horizontal gene transfer (HGT) or homologous recombination events are easily identified. Meanwhile, multiple-genome substitution and pairwise substitution regions are analyzed in PGAP-X. The high-substitution regions are marked on the genome structure with blue bars. For pairwise substitution region analysis, a reference strain must be selected; all sites of variation among paired genomes can be detected and reported in output text files but only high-substitution regions of selected strains are displayed. The influence of selective pressure on each genomic region or gene can be intuitively showed. Some highly variable regions may be involved in pathogenicity islands (PAIs) or related to strain adaptability. This may be used for SNP-based barcode design and gene selection for multilocus sequence typing (MLST). For the pan-genome profile, the result is the same as shown in the PGAP module.Streptococcus suis. The two modules, PGAP and PGAP-X, were used with default parameters (GF method was selected to identify orthologous genes in PGAP). The PGAweb results are described as follows.To evaluate the performance of PGAweb, we carried out a case study for S. suis genomes . The phylogenetic results give us a clear idea of the phylogenetic relationship among different serotypes of the 13 S. suis strains. Taking one of the phylogenetic trees (UPGMA tree based on the presence or absence of orthologous genes) as an example , the 13 strains are clearly grouped into two subgroups and all the serotype 2 strains are tightly grouped in the same clade. Serotype 1, 3, 7, and 9 strains could be assigned to a common clade. Serotype 14 and 1/2 strains were more closely related to the serotype 2 strains than to the other four serotypes. This result is consistent with earlier findings by Figure 3D), the 89K PAI exists in three serotype 2 strains isolated in China , while the other two Chinese serotype 2 strains (NC_017617 and NC_017622) harbored on different branches of the phylogenetic tree  do not contain this PAI. This result confirms an earlier observation that the 89K PAI is prevalent and specific for Chinese serotype 2 virulent strains . This suggests that these genes located in a cluster might have been introduced through HGT or homologous recombination with other closely related species . However, stand-alone tools always require basic programming skills for installation and operation. We developed PGAweb for in-depth inference and visualization in bacterial pan-genome analysis. PGAweb has a user-friendly, web-based interface, one-click input data submission, and smooth and efficient data analysis. No installation, command lines, or informatics skills are necessary for using PGAweb, making it very efficient for users, especially experimental scientists. PGAweb provides various interactive and customizable functions including orthologous clustering, pan-genome profiling, sequence variation, evolutionary analysis, and functional classification. Different visualization methods are used for enhanced genomic structural dynamics and sequence diversity display. These are helpful for intuitively understanding the dynamics and evolution of bacterial genomes. According to the pan-genome concept, three genomic data-sets is the minimum number for pan-genome analysis. In the case of technical and resource limitations, PGAweb is only suitable for performing analyzes on the pan-genome of a moderate number of strains (no more than 50 strains). The key issue for large scale pan-genome analysis is the input data uploading with limited network speed. We estimate that the input data size will be exceed 500 Mb for 50 strains pan-genome analysis, which will cost users a very long time for input data uploading. Furthermore, many factors may influence the runtime of PGAweb, such as the number input data sets, genome sizes, selected method, etc. The running time grows exponentially with the increase in number of input data sets due to the computational complexity . The current version of PGAweb still has some shortcomings, which only supports the pan-genome analysis of bacterial and archaeal genomes so far. With the development of sequencing technology, the pan-genome concept has also been widely utilized to perform comparative genomic analysis in fungi (Since the term of microbial pan-genome was introduced in 2005, several stand-alone and web-based tools have been developed for pan-genome analyses (in fungi  and planin fungi . In a fuHere, we present a fast and freely available online server, PGAweb, with integration of our previously published software, PGAP, PanGP, and PGAP-X. In brief, PGAweb has a user-friendly web interface, one-click input data submission, and smooth and efficient data analysis, and provides not only diverse methods for identification of orthologous genes but also powerful tools to display genome dynamics and structure characteristics. Case study indicates that PGAweb can intuitively reflect the characteristics of bacterial pan-genomes such as an open or closed pan-genome. Orthologous clustering, gene distribution based on conservation, genome structure conservation, and genetic variation, as well as their visualization, are all helpful for biologists to study bacterial genome dynamics to identify pathogenic and drug-resistant genes and their sequence contexts.JX, ZJ, and JAW conceived and designed the web server. XC performed the server front-end and back-end. YAZ carried out the case studies. YOZ, CS, MY, JNW, QL, BZ, and MC contributed to the web server testing. YAZ, ZZ, and JX wrote the paper. JY polished the language and gave many constructive suggestions.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.


In the present study we have used the wing-spot assay in Drosophila melanogaster to determine the genotoxic activity of Cu-NPs and Ni-NPs, and these data have been compared with those obtained with their microparticle forms (MPs). Additionally, a complete physical characterization of NPs using transmission electronic microscopy (TEM), dynamic light scattering (DLS), and laser Doppler velocimetry (LDV) techniques was also performed. Results obtained with Cu-NPs and Cu-MPs indicate that both failed to induce an increase in the frequency of mutant spots formation in the wings of the adults, suggesting a lack of genotoxicity in somatic cells of D. melanogaster. However, when Ni-NPs and Ni-MPs were evaluated, a significant increase of small single spots and total mutant spots was observed only for Ni-NPs (P<0.05) at the highest dose assessed. Thus, the genotoxicity of Ni-NPs seem to be related to their nanoscale size, because no genotoxic effects have been reported with their microparticles and ions. This study is the first assessing the in vivo genotoxic potential of Cu-NPs and Ni-NPs in the Drosophila model.Copper and nickel nanoparticles  are used in a variety of industrial applications, such as semiconductors, catalysts, sensors, and antimicrobial agents. Although studies on its potential genotoxicity already exist, few of them report Given their particular physical-chemical features and low cost of fabrication, copper and nickel nanoparticles  are being widely used in different industrial applications. On the one hand, Cu-NPs show high thermal and electrical conductivity and are being used in the manufacture of lubricants, polymers, plastics, metallic coatings, and electronic devices. Additionally, they have shown antimicrobial properties, and therefore Cu-NPs have been used as potential antibacterial and/or antifungal agents . On the The extremely small size of NPs allows reaching easily the nucleus of live cells through penetration via nuclear pore or during mitosis and interacting directly with DNA organized in chromatin or chromosomes causing genetic damage . However in vitro studies that suggest that Cu-NPs induced cell death and viability loss in neurons, suggesting a potential neurotoxicity and neurodegeneration mechanism associated [From the available literature, we found somesociated , 5. in vivo studies carried out with mice showed that Cu-NPs can induce several toxicological effects and injuries on kidney, liver, and spleen, but microparticles or bulk forms of copper do not [Instead,r do not . The abor do not . Recentlr do not . in vitro studies both metallic nickel fine and nanoparticles can induce high carcinogenic potential in mouse epidermal JB6 cells [ in vitro and in vivo genotoxic and mutagenic studies for both metallic nickel and nickel-based NPs, recent studies indicate that Ni-NPs had higher cytotoxic and genotoxic effects than Ni microparticles in in vitro and in in vivo studies under the same treatment doses using human cells and rat models [ in vitro and in Drosophila melanogaster model [Ni-NPs are undoubtedly the less studied among other metallic NPs. However, there is also some evidence that Ni-NPs may induce oxidative stress and apoptosis in human cells . In addiB6 cells . Althougt models . It shouer model . in vivo mutagenic and recombinogenic activity associated with the exposure of Cu-NPs and Ni-NPs measured by the wing-spot assay in Drosophila melanogaster was assessed. It is important to note that in recent years the fruit fly D. melanogaster has played a relevant role in detecting in vivo genotoxic and mutagenic action of several kinds of metal and metal-oxides NPs, offering several biological and genetic advantages to study acute and chronic effects and the underlying mechanisms of NPs action [In the present study, thes action . Drosophila strains were used for the wing-spot test: the multiple wing hairs strain with the genetic constitution y; mwhj; the flare-3 strain with the genetic constitution flr3/ln (3LR) TM3, Bds. The multiple wing hairs marker  is a completely recessive homozygous viable mutation, which is kept in homozygous condition. It produces multiple trichomes per cell instead of the normally unique trichome in the wing cells. The flare-3 marker  is a recessive mutation that affects the shape of wing hairs, producing malformed wing hairs that have a flare shape. Given their zygotic lethality, flare alleles have to be kept in stocks over balancer chromosomes carrying multiple inversions and a dominant marker that is a lethal homozygous . More detailed information on genetic markers and descriptions of the phenotypes are given by Lindsley and Zimm [ Drosophila  at a temperature of 25 ± 1°C and a relative humidity of ~60%.The following mutantand Zimm . Both stμm, CAS 7440-50-8, Ref. 102703) and nickel microparticles  were obtained from Merck Company .Copper nanoparticles  and nickel nanoparticles  were purchased from Sigma-Aldrich . Copper microparticles , dynamic light scattering (DLS), and laser Doppler velocimetry (LDV). TEM were carried out with a JEOLJEM- 2011 instrument to determine the size of NPs in dry form. DLS and LDV were performed with a Malvern Zetasizer Nano-ZS zen3600 instrument to measure the hydrodynamic diameter and zeta potential in aqueous suspension, respectively. For TEM analyses, metal-NPs were measured at a concentration of 2.56 mg/mL. For DLS and LDV techniques, NPs samples were measured at a concentration of 10 To particle preparation, we followed the methods of Carmona et al.  where va D. melanogaster strains were selected according to previous toxicity and viability studies carried out (data not shown). On the one hand, a range of doses from 0.1 to 10 mM was established for both particle sizes of copper, and within these dose range Cu-NPs showed higher toxicity than Cu-MPs. Hence, while for Cu-MPs a suitable larval viability (>70%) was reached at 10 mM, Cu-NPs could not be evaluated at concentration higher than 5 mM.The different concentrations of nano- and microparticles of Cu and Ni used in the genotoxicity experiments withOn the other hand, a range of doses from 1 to 10 mM was selected for Ni particles, and within this range a suitable viability was obtained (>70%). In general, both Ni-NPs and Ni-MPs showed similar toxicity levels; therefore both particles were assayed in the same concentrations. The main criterion to choose the final concentrations was the number of emerging larvae and adults after treatments were high enough to perform genotoxic experiments with the wing-spot test . multiple wing hairs (mwh) and flare-3 (flr3), in the wing blade of adult flies [mwh or flr3 phenotype) in wing slides preparations. This assay can detect a wide range of mutational events such as point mutations, deletions, certain types of chromosome aberrations (nondisjunction), and somatic recombination [The wing-spot test was used as a short test system based on the loss of heterozygosity (LOH) in normal genes and the corresponding expression of recessive markers, calledlt flies . Thus, tbination . flr3 strain were mated to mwh males as previously described [ Drosophila instant medium  prepared with 10 mL of various nontoxic concentrations of Cu-NPs , Cu-MPs, Ni-NPs, and Ni-MPs . Larvae were fed in this medium until pupation. The surviving adults were collected and stored in 70% ethanol. Afterwards, their wings were removed with fine tweezers and mounted in Faure's solution on microscope slides. The wings were scored at 400 times magnification for the presence of small single spots, large single spots, and twin spots. Small single flr3 spots also were scored in the wing samples, but these were included in the total mutant spots, as has been usually expressed in previous works [Virgin females of theescribed . Eggs frus works . In eachus works .α = β = 0.05 [ m (multiplication factor) times greater than in the control series. Since small single spots and total spots have a comparatively high spontaneous frequency, m was fixed at a value of 2 (testing for a doubling of the spontaneous frequency). For large single spots and twin spots, which have a low spontaneous frequency, m = 5 was used. The frequency of clone formation was calculated, without size correction, by dividing the number of mwh clones per wing by 24,400, which is the approximate number of cells inspected in one wing [The conditional binomial test was applied to assess differences between the frequencies of each type of spot in treated and concurrent negative control with significant levels β = 0.05 . The mulβ = 0.05 . The treone wing .In order to confirm the physical characteristics of metallic NPs given by the supplier TEM, DLS and LDV techniques were performed to assess diameter, shape, hydrodynamic size, and stability of Cu-NPs and Ni-NPs, respectively.TEM was used to characterize dry size of metal-NPs. On the one hand, Cu-NPs displayed spherical shapes and showed low levels of agglomeration . The sizOn the other hand, Ni-NPs exhibited mainly oval shapes and their NPs showed a certain level of agglomeration . The siz D. melanogaster larvae [Hydrodynamic characterization of metal-NPs was summarized in r larvae . Drosophila.The data of Cu-NPs and Cu-MPs from the wing-spot test was summarized in  Drosophila was chosen to evaluate genotoxicity of copper and nickel nanomaterials. This in vivo short-term test using a eukaryotic organism is a comprehensive genotoxic assay as diverse mutational events can be measured, such as point mutations, deletions, and certain types of chromosome aberrations. Besides, the wing-spot assay also distinguishes mitotic recombination in proliferative somatic cells, being the quantitation of the recombinogenic activity of a compound of primary importance for genotoxicity screening due to the strong relationship with carcinogenic process [In present study, the wing-spot test of process , 28. Thu process . Drosophila.In general, there are few genotoxic and mutagenic studies about Cu-NPs available in the literature, which are possibly difficult to be compared with the present study in in vitro studies indicate that Cu-NPs can induce cell death, viability loss, dopamine depletion, alteration of dopaminergic gene system expression, and oxidative stress in neurons of rats, suggesting a potential neurotoxicity and neurodegeneration mechanism associated with the acute exposition of Cu-NPs with average diameter between 40 and 90 nm [Danio rerio), Cu-NPs with average size of 80 nm have showed that they were acutely toxic to zebrafish at low concentration (1.5 mg/L), while nontoxic concentration (100 μg/L) can induce gill injury, affect biochemical markers of gill function, and alter stress and metal responsive gene expression in their gills [However,nd 90 nm . In addind 90 nm . In zebrir gills . Thus, t in vitro. Finally, Cu-NPs also induced DNA strand breaks measured by comet assay and oxidative DNA damage associated [ D. melanogaster. But these contrasts can be given by the use of different system assays (in vitro versus in vivo tests) and organisms models  for genotoxic screening. Thus, further comprehensive studies including different and combination of bioassays for toxic and genotoxic assessment are requested to clarify genetic effects of Cu-NPs; especially in vivo studies are needed to identify mechanisms of genotoxicity in whole organisms.Until now, only a single study had been published about genotoxicity of Cu-NPs. In this study, Cu-NPs were found to be mutagenic in the Ames test  and also found toxic for bacteria cells (cytotoxicity) in dose-dependent manner. Also, Cu-NPs induced significant increase in number of binucleated cells with micronuclei in monkey kidney cells at the highest concentration evaluated, suggesting chromosome damage Drosophila. Thus, the absence of genotoxicity found in this study could be related to the change of physical properties of Cu-NPs losing biological reactivity  and the capacity of generate DNA damage by direct or indirect mechanisms in somatic cells of D. melanogaster. However, the absence of positive results with Cu-NPs in the wing-spot test could be related also to their poor solubility of this nanomaterial in water media, because previously SMART studies indicated positive results with a more soluble form of copper: CuO-NPs [On the other hand, Cu-NPs showed high hydrodynamic size 261 nm) and low Z potential, indicating agglomeration and low stability of particles in aqueous medium, which represent the real conditions of NPs exposure in experiments with SMART test of1 nm and  CuO-NPs . in vitro and in vivo studies. Evidences from DNA fragmentation analysis using the comet assay showed that the Ni-NPs with average diameter of 52 nm cause primary DNA damage in a dose- and time-dependent manner. These Ni-NPs were also found to induce oxidative stress evidenced by the generation of reactive oxygen species (ROS) and depletion of glutathione (GSH), suggesting the capability of the Ni-NPs to induce oxidative stress resulting in genotoxicity for human skin cells (A431) [Although few data about genotoxic effects of single Ni-NPs are available in the literature, the results obtained in this research are in agreement with recent studies carried out ins A431) . Other n . Other  in vivo studies carried with female Wistar rats assessing genotoxicity with micronucleus, chromosome aberrations, and the comet assays indicate that the NiO-NPs administered through the oral route were capable of inducing chromosome alterations and DNA breaks at the high-dose level and these effects were more prominent at 24 h sampling time [On the other hand,ing time . in vitro [Besides the genotoxic effects, it should be noted that Ni-NPs have shown to induce effects on tumour promoter or suppressor gene expressions as well as on cell transformation in mouse epidermis cells (JB6 cells), suggesting that metallic Ni-NPs can be carcinogeneticin vitro . Thus, t in vivo wing-spot test of Drosophila. Treatments with high concentrations of Ni-NPs induce a significant increase on the frequency of single and total of spots analysed; however, Ni-MPs were negative to induce mutant spots. Due to the fact that Ni-NPs mainly induce single mutant and low number of twin spots, the genotoxicity observed for Ni-NPs can be attributed mainly for mutational events , not including recombinogenic activity. Considering that previous studies with ionic and soluble forms of nickel  have shown negative results in the Drosophila wing-spot test [ Drosophila. In addition, recent studies with NiO-NPs also indicate genotoxic and mutagenic in Drosophila with this kind of metallic nanoparticles supporting the genotoxic effects found in the present study [ Drosophila, previous studies have demonstrated that Ni-NPs can generate intracellular oxidative stress damaging DNA in cells, suggesting that this mechanism of action is responsible for their genotoxicity [Our results report for the first time data on the evaluation of Ni-NPs genotoxicity with thepot test , the gennt study . Althougtoxicity , 12, 34. D. melanogaster. However, when Ni-NPs were evaluated, a significant increase of small single spots and total mutant spots was observed at the highest dose assessed. Thus, the genotoxicity of Ni-NPs seems to be related to their nanoscale size, because no genotoxic effects have been reported with their microparticles and ion forms. This study was the first assessing the in vivo genotoxic potential of Cu-NPs and Ni-NPs in the Drosophila model.Cu-NPs failed to induce an increase of mutant spots in the wings of fruit flies adults, suggesting a lack of genotoxicity in somatic cells of


Current international guidelines recommend the use of a daily topical exit-site antimicrobial to prevent peritoneal dialysis (PD)-related infections. Although nonantibiotic-based therapies are appealing because they may limit antimicrobial resistance, no controlled trials have been conducted to compare topical antimicrobial agents with usual exit-site care for the prevention of PD-related infections among the Thai PD population. We propose a controlled three-arm trial to examine the efficacy and safety of a daily chlorhexidine gluconate-impregnated patch versus mupirocin ointment versus usual exit-site care with normal saline for the prevention of PD-related infections.Staphylococcus aureus colonization, (iii) healthcare costs, and (iv) skin reactions and adverse events. We plan to conduct a cost-utility analysis alongside the trial from the perspectives of patients and society. A Markov simulation model will be used to estimate the total cost and health outcome in terms of quality-adjusted life years (QALYs) over a 20-year time horizon. An incremental cost-effectiveness ratio in Thai Baht and U.S. dollars per QALYs gained will be illustrated. A series of probabilistic sensitivity analyses will be conducted to assess the robustness of the cost-utility analysis findings.This study is a randomized, double-blind, multicenter, active-controlled, clinical trial. Adult patients aged 18 years or older who have end-stage kidney disease and are undergoing PD will be enrolled at three PD Centers in Thailand. A total of 354 PD patients will be randomly assigned to either the 2% chlorhexidine gluconate-impregnated patch, mupirocin ointment, or usual exit-site care with normal saline dressing according to a computer-generated random allocation sequence. Participants will be followed until discontinuation of PD or completion of 24 months. The primary study outcomes are time to first PD-related infection (exit-site/tunnel infection or peritonitis) event and the overall difference in PD-related infection rates between study arms. Secondary study outcomes will include (i) the rate of infection-related catheter removal and PD technique failure, (ii) rate of nasal and exit-site The results from this study will provide new clinical and cost-effectiveness evidence to support the best strategy for the prevention of PD-related infections among the Thai PD population.NCT02547103. Registered on September 11, 2015.ClinicalTrials.gov,  Peritoneal dialysis (PD) is a well-established treatment modality of home renal replacement therapy (RRT) for end-stage kidney disease (ESKD) patients and has been available in Thailand for more than three decades. Although technical innovations and improvements in PD practice have significantly decreased PD-associated adverse outcomes, PD-related infections, including PD-related peritonitis and exit-site/tunnel infection, are still the most common complications –3. From Staphylococcus aureus. This strategy has been proven to be effective by several existing observational studies, randomized controlled trials (RCTs), and meta-analyses and is recommended by the International Society for Peritoneal Dialysis (ISPD) guidelines  only), Grade II , Grade III , and Grade IV (blisters and ulceration [skin breakdown]).No clinical trial insurance will be provided, and participants will not receive financial compensation for any trial-related injury. However, participants will receive full access to outpatient and in-hospital standard of care.A clinical monitor will visit the study sites every 2 weeks to check the progress of the study. Important points to be checked include whether the investigator has conducted the study as per protocol, how many participants were screened and enrolled, and if all eligible participants signed the informed consent form. Completeness of case report form and other essential documents, as well as records of any drop-outs or adverse events, will be checked for correctness and consistency with the source documents in a timely manner.Sample size calculation was based on information from the Thai Renal Outcomes Research-Peritoneal Dialysis database collected between 2006 and 2016, with an estimated rate of 0.31 episodes per patient-year and 0.39 episodes per patient-year for exit-site infection and peritonitis, respectively (composite rate of PD-related infections of 0.70 episodes per patient-year) [A paper-based case report form will be composed before the study commences. Each variable is carefully coded for auditing and statistical analysis. The participants’ general information will be recorded in the case report form by the responsible investigator, whereas participant-reported information will be documented in the case report form by participants.We will adopt a double data entry and double check approach to data management. All steps involved in the approach to data management will be independently conducted by two data administrators from the Pharmacoepidemiology and Statistics Research Center. If any inconsistency is identified in the data entry or logic consistence check, the investigators will be contacted for further information and clarification. To protect privacy, participants’ identification information  will not be used with the data management software.The DSMB will review the analyses in terms of efficacy and safety. While the trial is ongoing, DSMB members have access to original data but are blinded to participant allocation. The principle role of the DSMB is to provide a written recommendation in a timely manner to the investigator to discontinue a trial following discussion and assessment of efficacy and safety data.P-value less than 0.05 will be considered statistically significant.Baseline characteristics will be summarized as number (percentage) or mean ± standard deviation, or medians with interquartile range as suitable. Differences between treatment groups will be compared using Fisher’s exact test and analysis of covariance (ANCOVA) or Kruskal-Wallis test for categorical and continuous variables, respectively. All analyses will be conducted on both a per-protocol and intention-to-treat basis. All analyses will be performed using Stata software version 14.0  and Microsoft Excel version 2016. They will be two-tailed, and a For a primary analysis, Cox proportional hazards regression and Poisson regression analyses will be performed to assess the effectiveness of the chlorhexidine gluconate-impregnated patch and mupirocin ointment compared to usual exit-site care with normal saline for the first episode of PD-related infection and longitudinal rates, respectively. Secondary analysis using statistical methods as described above will then be analyzed to evaluate the effect on the prevention of PD-related infections between chlorhexidine gluconate-impregnated patch and mupirocin ointment. The treatment effects will be estimated as hazard ratios or incidence rate ratios with corresponding 95% confidence interval. Kaplan-Meier survival curves will be constructed for visual presentation of time-to-event comparisons.S. aureus colonization status , PD modality , PD vintage, and serum albumin (< 3.5 vs ≥3.5 mg/dL).Several a priori subgroup analyses with respect to the minimization variables for both primary and secondary outcomes will be investigated. For instance, the lists of variables include age (<65 vs. ≥65 years), gender, history of diabetes, history of PD-related infections (peritonitis or exit-site/tunnel infection), S. aureus colonization status, PD modality, PD vintage, and serum albumin); (ii) restricting the analysis to only PD-related infection episodes with positive cultures to account for misclassification bias; (iii) using the proportional sub-distribution hazards analyses by Fine and Gray method to address the completing risk outcomes, including mode switching to long-term hemodialysis, kidney transplantation, conservative treatment, or death; and (iv) using the multiple imputation method to evaluate the effect estimates when missing data are indicated.To address robustness of the treatment effects, further sensitivity analyses of the primary analysis will be performed by (i) adjusting for the several baseline covariates that seem to affect the outcomes  Regulations and 2006 Amended Regulations. We will conduct the investigation in accordance with the key principles of ethical conduct in research and the Declaration of Helsinki. The sponsors will not be involved with study design, or data assembly and analysis.Participants will be included in the study only if they provide written informed consent. Participant consent will be obtained by trained research assistants . No other gifts or payments will be offered.Participants may withdraw from the trial at any time for any reason. Furthermore, participants who meet any of the following conditions will be allowed to withdraw from the trial: (i) refusing to follow the study protocol; (ii) using other IMPs; (iii) pregnancy during the study period; (iv) switching mode to long-term hemodialysis, kidney transplantation, or conservative treatment; and (v) developing serious adverse drug reactions or suspected unexpected serious adverse reactions. According to the intention-to-treat approach, data collected prior to study withdrawal will be considered for participants who discontinue or deviate from the assigned intervention protocols.Participants and the public had no role in the trial design, recruitment, conduct, or monitoring. Our findings will be published in peer-reviewed journals and disseminated through scientific and professional meetings. Written lay summary results will be available to the public. At the end of the trial, the principal investigators will review and finalize the study report and data set. This report will be shared with the Health Systems Research Institute of Thailand. The investigators commit to reporting data as endorsed by the Consolidated Standards or Reporting Trials guidelines: Extension for Reporting of Multi-Arm Parallel-Group Randomized Trials , 36 and PD utilization and the number of PD centers are rising dramatically in Thailand owing to the “PD First” policy—the national health policy under universal coverage scheme , 39. GloS. aureus [S. aureus with no specified indications or time of use [Generally, the Centers for Disease Control and Prevention recommends short-term use of topical mupirocin alone in patients with . aureus . Furthere of use . The ISPe of use , 7. Accoe of use . For inse of use . Likewise of use .Although the ISPD guidelines recommend the use of daily topical exit-site antimicrobial to prevent PD-related infections (Level 1B), prophylactic antimicrobial agents for routine exit-site care were not always administered, with use varying worldwide from 6% to 96%—including in Thailand (73%) , 46. BecAt present, no direct head-to head comparison studies of prophylactic strategies for the prevention of PD-related infections have been conducted in Thailand. Taken together, stronger evidence in terms of clinical and cost-effectiveness outcomes is needed to support the best strategy for prevention of PD-related infections among the Thai PD population. As such, this is the first well-controlled trial to compare the safety, effectiveness, and cost-effectiveness of chlorhexidine gluconate-impregnated patch, mupirocin ointment, and usual exit-site care with normal saline dressing, for the prevention of PD-related infection. Given the “PD First” policy in Thailand, most patients are treated with continuous ambulatory peritoneal dialysis (CAPD), which may limit the generalizability of study findings to other PD modalities.In summary, the COSMO-PD trial will determine whether exit-site care with chlorhexidine gluconate-impregnated patch or mupirocin ointment decreases the risk of PD-related infections compared to standard exit-site care with normal saline dressing. Novel strategies are needed to prevent PD-related infection and alleviate the healthcare costs, while improving long-term survival in PD patients. The findings of this trial could provide the new clinical and cost-effectiveness evidence of the strategy for the prevention of PD-related infections.Trial protocol version 2–2016, dated May 9, 2016, was used to prepare this manuscript. The COSMO-PD trial is currently in the participant enrollment phase. A total of 288 eligible participants have been randomized as of July 2019. We anticipate that enrollment will be completed in October 2019.Additional file 1. SPIRIT 2013 Checklist: Recommended items to address in a trial protocol and related documents.Additional file 2: Appendix 1. Investigational medicinal products. Appendix 2. Participants’ information sheet and informed consent form (in Thai).


In patients with esophageal cancer, differentiation between lymph node metastasis and lymphadenopathies from sarcoidosis or sarcoid-like reactions of lymph nodes is clinically important. Herein, we report two esophageal cancer cases with lymph node involvement of sarcoid-like reaction or sarcoidosis.18F]-fluorodeoxyglucose positron emission tomography–computed tomography (FDG-PET/CT) was performed before and after chemo(radio)therapy. After the treatment, FDG uptake was not detected in the primary tumor, but it was slightly reduced in the hilar and mediastinal lymph nodes in both cases. These non-identical responses to chemo(radio)therapy suggest the presence of sarcoid-like reaction of lymph nodes associated with squamous cell carcinoma of the esophagus. Curative surgical resection was performed as treatment.One patient received chemotherapy and the other chemoradiotherapy as initial treatments. In both cases, [These FDG-PET/CT findings may be helpful to distinguish between metastasis and sarcoidosis-associated lymphadenopathy in esophageal cancer. Therefolignancy . Both palignancy . Therefolignancy .In esophageal cancer patients with suspected lymphadenopathies from sarcoidosis or sarcoid-like reactions, monitoring the response to chemotherapy via FDG-PET/CT may be helpful to diagnose whether the lymphadenopathies are benign or metastatic. However, this method may not be applicable when the esophageal cancer does not respond to chemotherapy/chemoradiotherapy. Nevertheless, 84% of patients with esophageal squamous cell carcinoma demonstrate good responses to neoadjuvant chemotherapy on FDG-PET/CT.The previous case monitored by Bekki et al. was a sarcoid-like reaction. The data of our first case support the suggestions of Bekki et al., and we showed that bilateral FDG accumulation in the hilar lymph nodes was only slightly changed after 15 months. In our second case, we have newly reported that FDG-PET/CT has diagnostic utility in the differential diagnosis of lymphadenopathy, even in patients with sarcoidosis. Therefore, monitoring responses by FDG-PET/CT is useful both for lesions associated with a sarcoid-like reaction and those from systemic sarcoidosis, although the incidence of esophageal cancer complicated by sarcoidosis is rare. Analyses in a large series of esophageal cancer patients with benign and malignant lymphadenopathy are needed to assess the differential diagnostic ability of FDG-PET/CT monitoring.


Objectives: This meta-analysis aims to study the effects of atomoxetine and methylphenidate on heart rate (HR), systolic blood pressure (SBP), and a number of adverse cardiac events on patients receiving treatment for attention-deficit hyperactive disorder (ADHD) in comparison to placebo and between atomoxetine and methylphenidate. Methods: We searched the following databases: PubMed, EMBASE, and ScienceDirect. Meta-analysis was performed on studies that examined the relationships between methylphenidate or atomoxetine and HR, SBP, as well as a number of adverse cardiac events. These studies were either placebo-controlled or comparison studies between methylphenidate and atomoxetine. Meta-regression identified patient- and treatment-related factors that may contribute to heterogeneity. Results: Twenty-two studies were included and the total number of participants was 46,107. Children/adolescents and adults treated with methylphenidate had more significant increases in post- vs. pre-treatment HR (p < 0.001) and SBP (p < 0.001) than those treated by placebo. Children and adolescents treated with atomoxetine had more significant increases post- vs. pre-treatment HR (p = 0.025) and SBP (p < 0.001) than those treated with methylphenidate. Meta-regression revealed mean age of participants, mean dose, and duration of atomoxetine and methylphenidate as significant moderators that explained heterogeneity. There were no differences in the number of adverse cardiac events between participants with methylphenidate treatment and placebo or atomoxetine. Conclusions: Children/adolescents and adults treated with methylphenidate resulted in significant increases in post- vs. pre-treatment HR and SBP as compared to placebo. Similarly, children and adolescents treated with atomoxetine had significant increases in post- vs. pre-treatment HR and SBP than those treated with methylphenidate. These findings have potential implications for continuous monitoring of HR and SBP throughout the course of treatment although the risk for adverse cardiac events were insignificant. Attention deficit hyperactive disorder (ADHD) is a psychiatric disorder characterized by inattention, hyperactivity, and impulsivity. ADHD is common in children and adolescents . In the The Multimodal Treatment Study of Children and Adolescents found that medications with or without behavioural treatment is the most effective for core ADHD symptoms . Recent There is substantial concern from clinicians, patients, parents, and the public about the cardiovascular safety of ADHD medications . The conA 2012 systematic review reported mixed findings on the association between prescription of stimulants and adverse cardiovascular outcomes . A 2014 For cardiovascular adverse effects, a nationwide self-controlled case series study was conducted in Korea. This study found that the relative risk of myocardial infarction and arrhythmia was increased after the start of methylphenidate treatment for ADHD in children and young people but not for hypertension, ischaemic stroke, and heart failure . The KorFurther, direct comparisons of the prevalence of cardiovascular adverse effects between methylphenidate and atomoxetine have not been performed to date. It is possible that certain risk factors are associated with higher risk of cardiovascular adverse effects. Having information on these risk factors will be helpful for healthcare professionals to identify patients who are more likely to develop cardiovascular adverse effects and apply necessary precautions. There has been a growing interest of cardiovascular safety of methylphenidate and atomoxetine in treating with patients suffering from ADHD. Additionally, examination of the effect of methylphenidate and atomoxetine on adults has yet to be completed, and studies that include adult patients with ADHD are generally small in this area of research. Hence, a meta-analysis is urgently needed to provide a better estimate of effect. The main objective of this meta-analysis was to conduct a head-to-head comparison of post- vs. pre-treatment HR, SBP, and the prevalence of cardiovascular adverse effects between patients taking methylphenidate and placebo or atomoxetine. This meta-analysis focused on SBP because SBP has been a better predictor of cardiovascular risk . The secThis meta-analysis adhered to a priori designed protocol. We searched the following databases: PubMed, EMBASE, and Science Direct. Our search started from inception of databases to 31 May 2016 for relevant articles. Our search terms were “methylphenidate” with varying combinations of other search terms: “atomoxetine”, “cardiovascular”, “heart rate”, “blood pressure”, “cardiovascular diseases”, “cerebrovascular accident”, “myocardial infarction”, “electrocardiography”, “vasculopathy”, “sudden cardiac”, and “death”. All fields were checked under the database search and suggested articles by databases were considered.We included all trials published in the English language that compared methylphenidate to either a placebo or atomoxetine in terms of its cardiovascular safety in the context of treatment for ADHD. The trials included in the data analyses met the following inclusion criteria: (i) compare methylphenidate and either placebo or atomoxetine in ADHD treatment; (ii) include outcome data on HR, SBP, or number of cardiovascular adverse events during treatment; and (iii) no concomitant administration of other psychotropic medication during the trial.We screened all citations and abstracts from the search strategy and identified articles for full-text extraction. Two investigators (ELC and SLZ) performed the literature search, screening, and data extraction independently. Disagreements at any phase of the review process were resolved by discussion. If a consensus was not reached, a third independent rate (RCH) determined eligibility. Studies that met the following exclusion criteria were excluded: (i) without a placebo control arm; (ii) methylphenidate or atomoxetine not used in the trial arm; (iii) nonhuman studies; (iv) inadequate sample size (less than 5); (v) review articles; and (vi) non-English articles and English abstracts that could not provide adequate information to calculate effect size. All data were independently extracted and organized into a standard electronic data extraction form. All publications were reviewed as full texts.The primary outcomes were the proportion of patients who experienced changes in cardiovascular parameters  after methylphenidate or atomoxetine treatment. Secondary outcomes include the occurrence of adverse cardiac outcomes, namely cerebrovascular accidents, myocardial infarction, sudden cardiac death, and significant electrocardiogram changes.The quality of each trial was independently assessed according to the standard Jadad scoring system . The assp value of <0.05 was taken as significant. Between-study heterogeneity was assessed with the I2 statistic [2 values of 25% were considered low, 50% moderate, and 75% high [p values were reported in the meta-regression analysis. Egger’s test was performed to assess for presence of publication bias. In the event that publication bias was detected, the classic fail-safe test was performed to establish the potential number of missing studies [This meta-analysis was reported according to the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guidelines . All statatistic . As a gu75% high . For mod75% high . The reg studies .Out of the 1075 potentially relevant articles identified in our initial searches, a total of 22 articles were included in our analysis. Studies were excluded at each stage of screening for inclusion and exclusion criteria see . Out of p < 0.001). Adults treated with methylphenidate had a more significant increase in post- vs. pre-treatment HR than those treated by placebo . Subgroup analysis showed no significant difference between children/adolescents and adults in post- vs. pre-treatment HR . A significant level of between-study heterogeneity was found . When we undertook meta-regression to explore the impact of our a priori sources of heterogeneity (p < 0.001), proportion of male gender , duration of treatment , and mean dose of methylphenidate . There was no publication bias .ogeneity , we founp = 0.025). A significant level of between-study heterogeneity was found . When we undertook meta-regression to explore the impact of our a priori sources of heterogeneity (see p < 0.0001), proportion of male gender , mean dose of methylphenidate , and mean dose of atomoxetine . There was no publication bias .eity see , we founp < 0.001). Adults treated with methylphenidate had a more significant increase in post- vs. pre-treatment SBP than those treated by placebo . Subgroup analysis showed no significant difference between children/adolescents and adults in post- vs. pre-treatment SBP . A significant level of between-study heterogeneity was found . When we undertook meta-regression to explore the impact of our a priori sources of heterogeneity (see p < 0.0001) and mean dose of methylphenidate . There was publication bias . The number of missing studies required to nullify results is 673 studies.eity see , we founp < 0.001). No between-study heterogeneity was found . Meta-regression was not performed because there was no between-study heterogeneity and because of the small number of studies that provided information on moderators. There was no publication bias .p = 0.18).p = 0.64).This meta-analysis found that children/adolescents and adults treated with methylphenidate resulted in significant increases in post- vs. pre-treatment HR and SBP as compared to placebo. Similarly, children and adolescents treated with atomoxetine had significant increases in post- vs. pre-treatment HR and SBP than those treated with methylphenidate. This meta-analysis is the first to perform a subgroup analysis that showed that children/adolescents and adults had similar risks in causing an increase in post- vs. pre-treatment HR and SBP after taking methylphenidate. We noticed a high level of heterogeneity that might be attributed to the differences in mean age of participants, mean doses, and duration of methylphenidate and atomoxetine treatment. In adults, there was no significant difference in the number of adverse cardiac events between participants treated with methylphenidate and placebo. In children/adolescents, there was no significant difference in the number of adverse cardiac events between participants treated with methylphenidate and atomoxetine.This meta-analysis found no statistically significant difference in the number of adverse cardiac events in adults receiving methylphenidate as compared to placebo. Our findings helped to address the mixed findings on the association between stimulant use and adverse cardiovascular outcomes reported by a systematic review in 2012 . We furtThis meta-analysis has confirmed that there were significant increases in post- vs. pre-treatment HR and SBP in children/adolescents and adults receiving methylphenidate or atomoxetine as compared to placebo, which is consistent with recent meta-analysis that reported similar findings in children and adolescents . We furtOur findings challenged the conclusion from a previous review which stated that methylphenidate and atomoxetine caused small and nonsignificant increases in mean HR and BP in children and adolescents . The afoMethylphenidate acts by inducing release of noradrenaline and dopamine into synaptic clefts and thus stimulating postsynaptic receptors  and stimFor atomoxetine, Wakamatsu et al. (2009) suggested that atomoxetine increased BP and HR without affecting the plasma adrenaline concentration but the exact mechanism remains unknown . MichelsThis meta-analysis can potentially inform both clinicians and patients about the cardiovascular side effects associated with atomoxetine and methylphenidate to make an informed decision about its potential risks to increase HR and SBP. Our findings support the recommendations proposed by the Medicines and Healthcare products and Regulatory Agency (UK) . It is iWe believe this is the first meta-analysis to study the effects of methylphenidate and atomoxetine on HR, SBP, and cardiac adverse events across all age groups including children/adolescents and adults. In this meta-analysis, 16 out of 22 studies (72.7%) achieved the Jadad score ≥3 that indicated good quality. Furthermore, 18 out of the 22 studies (81.8%) included are randomised controlled trials. In contrast, nearly 80% of studies included in a recent meta-analysis are open-label studies without a placebo group . We perfThere are a few limitations to our study. First, this meta-analysis mainly focused on SBP but not diastolic blood pressure (DBP). SBP has been a better predictor of risk than DBP . MoreoveOur meta-analysis demonstrated that children/adolescents and adults treated with methylphenidate resulted in significant increases in post- vs. pre-treatment HR and SBP as compared to placebo. Similarly, children and adolescents treated with atomoxetine had significant increases in post- vs. pre-treatment HR and SBP than those treated with methylphenidate. The results of this study can be applied by mental health professionals and paediatricians on patients who receive atomoxetine or methylphenidate treatment. Throughout the course of treatment, HR and SBP should be adequately monitored. Further research is required to elucidate how long-term atomoxetine treatment causes increases in HR and SBP. Patients and caregivers can be reassured that there was no significant difference in the number of adverse cardiac events in adults treated with methylphenidate and placebo as well as in children/adolescents treated with methylphenidate and atomoxetine.


Objectives: To evaluate clinicopathological and perioperative factors associated with the risk of focal and non-focal positive surgical margins (PSMs) after robot-assisted radical prostatectomy (RARP).Patients and methods: The study was retrospective and excluded patients who were under androgen-deprivation therapy or had prior treatments. The population included: negative SM cases (control group), focal and non-focal PSM cases (study groups). PSMs were classified as focal when the linear extent of cancer invasion was ≤1 mm and non-focal when >1 mm. The independent association of factors with the risk of focal and non-focal PSMs was assessed by multinomial logistic regression.Results: In all, 732 patients underwent RARP, from January 2013 to December 2017. An extended pelvic lymph node dissection was performed in 342 cases (46.7%). In all, 192 cases (26.3%) had PSMs, which were focal in 133 (18.2%) and non-focal in 59 (8.1%). Independent factors associated with the risk of focal PSMs were body mass index , percentage of biopsy positive cores , pathological extracapsular extension , and seminal vesicle invasion . High surgeon volume was a protective factor in having focal PSM . Independent predictors of non-focal PSMs were BPC , pT3a , and pT3b .Conclusions: In high-volume centres features related to host, tumour and surgeon volume are factors that predict the risk of focal and non-focal PSMs after RARP.Abbreviations: AJCC: American joint committee on cancer; AS: active surveillance; ASA: American society of anesthesiologists; BCR: biochemical recurrence; BMI: body mass index; BPC: percentage of biopsy positive cores; ePLND: extended lymph node dissection; H&E: haematoxylin and eosin; IQR, interquartile range; ISUP: international society of urologic pathology; LNI: lymph node invasion; LOS: length of hospital stay; mpMRI: multiparametric MRI; (c)(p)N:   nodal stage; OR: odds ratio; OT: operating time; PSA-DT: PSA-doubling time; (P)SM: (positive) surgical margin; (NS)(RA)RP: (nerve-sparing) (robot-assisted) radical prostatectomy; RT: radiation therapy; (c)(p)T:   tumour stage Prostate cancer is the most common non-cutaneous malignancy and the second leading cause of cancer-related deaths in men . The disThe unfavourable outcome of detecting PSMs after RP depends on both tumour biology and surgeon experience –10. In hThe study was retrospective, had Institutional Review Board approval, and included the period from January 2013 to December 2017. Each patient provided informed-signed consent for use of data. All patients who were under androgen blockade and/or had prior treatments were excluded.2). The serum levels of PSA (ng/mL) were determined by radioimmunoassay. Prostate biopsies had the following features: (i) at least 12–14 cores; (ii) number of positive cores; (iii) measurement of prostate volume (mL); (iv) tumour classified by biopsy grade groups according to the 2014 International Society of Urologic Pathology (ISUP) system [Preoperative patient data included age and body mass index  system . In each) system . TumoursExtended lymph node dissection (ePLND) was performed when the risk of lymph node invasion (LNI) was >5% . In low-RARP was performed using the da Vinci® Robot System  via a transperitoneal approach, with antegrade prostatic dissection . Nerve-sThe high-volume surgeon (W.A.) performed two-thirds of the procedures. Intraoperatively, operating time  and blood loss (mL) were measured. Preoperatively, patients’ surgical risk was evaluated using the American Anesthesiologists Score (ASA) system . PostopeSpecimens were processed according to the Stanford protocol by dedicated pathologists . ProstatSMs were considered positive when cancer invaded the inked surface of the specimen. When the linear extension of cancer involvement on the inked surface was ≤1 mm the PSM was classified as focal, otherwise it was coded as non-focal . AccordiThe patient population was divided into three groups including: negative SM cases , as well as focal and non-focal PSM patients (study groups). The associations of focal and non-focal PSMs with clinicopathological and perioperative factors were evaluated. Clinical variables assessed included: age, BMI, PSA level, prostate volume, BPC, biopsy grade group, cT stage, cN stage, and D’Amico’s class risk. The pathological factors assessed included: prostate weight, pathological grade group, pT, and pN. Perioperative factors assessed included: OT, blood loss, ePLND, number of dissected nodes, NSRP, surgeon volume, ASA score, LOS, Clavien–Dindo score, and readmission.Summary statistics and distributions of factors amongst the three groups  were assessed. Data on continuous variables were reported as medians with their respective interquartile ranges (IQRs). Data on categorical variables were presented as frequencies with relative percentages. Associations of factors amongst the three groups were analysed by the Kruskal–Wallis test for continuous variables, and by the Pearson’s chi-squared test or Fisher’s exact test, as appropriate for categorical ones. Significant factors were entered into the multivariate model. The multinomial logistic regression model  evaluated the associations between factors and the risk of focal and non-focal PSMs when compared to negative SM cases (control group). Clinical, pathological and perioperative factors were evaluated separately. An overall multivariate model including independent factors of each set  was also computed.P < 0.05 considered to indicate statistical significance.The software used for the analysis was the IBM Statistical Package for the Social Sciences (SPSS®), version 20 . All tests were two-sided with We evaluated 732 patients who underwent RARP . The medOverall , 192 patP = 0.009) and BPC  in the clinical model, by extracapsular extension  in the pathological model; by ePLND  and high surgeon volume  in the perioperative model (P = 0.006), BPC  and high surgeon volume . In the overall model, BMI , BPC , pT3a , pT3b  and high surgeon volume  were associated with the risk of focal PSMs  in the clinical model; by extracapsular extension  and seminal vesicle invasion  in the pathological model; and by no factor in the perioperative model (P = 0.044), extracapsular extension  and seminal vesicle invasion  were associated with the risk of non-focal PSMs . The length of the PSM is also an increasingly negative prognostic factor –10. AlthIt is important to identify predictive factors of PSMs in clinical practice, in order to define the risk, to plan the surgery, and to anticipate the potential need for adjuvant treatments after surgery. Therefore, patents who are at risk of PSM need appropriate counselling before and after RP due to the risk of needing adjuvant or salvage treatments . DetectiOur present study showed that other factors were able to differentiate the linear extent of PSM beyond BPC and pT stage (r = 0.37), which was measured by CT [High BMI and high surgeon volume were both independent factors that were associated with a reduced risk of focal PSMs. The influence of BMI during RARP is unclear. We previously found that BMI is associated with major postoperative complications after RARP . In the ed by CT . In the ed by CT  demonstrA systematic review of the literature investigated the subject of surgeon volume and oncological outcomes . The revOur present study has some limitations. First, the retrospective nature of the present study is a limitation in itself. Second, prostate volumes and biopsies performed elsewhere were not re-evaluated; however, their features had good standard quality to support their analysis. Third, in some cases the pathologists did not report the location, but just evaluated the linear extension of the PSM. Fourth, we did not provide a threshold for defining high- and low-volume surgeons due to the presence of a large difference between the single high- and four low-volume surgeons and the homogenous experience of the low-volume surgeons. For these reasons, we compared the cohort of the patients who were operated on by the single high-volume surgeon with the cohort of the patients who were operated on by the other four low-volume surgeons. However, beyond these limits, our present study has much strength as it includes a large contemporary cohort of patients and our dedicated pathologists assessed all specimens.In high-volume centres features related to host, tumour and surgeon volume are important factors that are associated with the risk of focal and non-focal PSMs after RARP. Particularly, high-volume surgeons have lower focal PSMs than low-volume surgeons. These issues should be discussed when counselling patients.


Hair cortisol concentration (HCC) is an increasingly used measure of systemic cortisol concentration. However, determinants of HCC in children and adolescents are unclear because few prospective studies have been conducted to date.We followed 725 children in Project Viva, a pre-birth cohort study of mothers and children, who provided hair samples at mid-childhood (median age: 7.7 years) or early adolescence (median age: 12.9 years). We examined associations of various factors measured from pregnancy to mid-childhood with HCC in mid-childhood and early adolescence, as well as change in HCC between these time points (ΔHCC).2, 95% CI: 0.01, 0.29) were positively associated with HCC. Child anthropometric measures and biomarker concentrations were not associated with HCC.There were 426 children with HCC measurements in both mid-childhood and early adolescence, 173 children with measures only in mid-childhood, and 126 with measures only in early adolescence. HCC was lower in mid-childhood  than early adolescence . In multivariable-adjusted regression models, female sex  and birth weight-for-gestational age z-score  were associated with lower mid-childhood HCC, while prenatal smoking was associated with higher mid-childhood HCC . In early adolescence, child age  female sex , and maternal pre-pregnancy body mass index (β = 0.15 per 5-kg/mMaternal pre-pregnancy BMI, maternal prenatal smoking, and low birth weight were associated with higher mid-childhood and adolescent HCC. However, few postnatal characteristics were associated with HCC. Hair cortisol concentration (HCC) is a relatively new measure of systemic cortisol concentration and hypothalamic-pituitary-adrenal (HPA) axis activity , 2. In eAlthough HCC is increasingly being used instead of other measures of cortisol secretion in epidemiologic investigations, few studies have examined HCC determinants in children and adolescents. Identifying HCC determinants in these populations is important to generate hypotheses about relationships between early life exposures and HPA axis activity as well as to adjust for confounders of associations of HCC with health outcomes. While some studies in children have observed positive associations of male sex and adiposity with HCC and inverse associations of household income and parental education with HCC , the curTo address these gaps, we explored prospective associations of various sociodemographic, environmental, behavioral, and cardiometabolic exposures with HCC in mid-childhood and early adolescence in Project Viva, a prospective pre-birth cohort of mothers and children. We specifically examined characteristics that are predictive of obesity and poor cardiometabolic health in children and adolescents because we hypothesized that these factors would also be the most important determinants of HCC in this population.Project Viva is an ongoing prospective cohort that recruited women carrying a singleton pregnancy during their initial obstetric care visit at Atrius Harvard Vanguard Medical Associates between 1999 and 2002 in eastern Massachusetts [White children who provided hair samples at the mid-childhood (n = 639) or early adolescent in-person visits (n = 567) were eligible for the present study. We excluded non-white children because of differences in hair texture and hair growth rate by race/ethnicity , which mWe collected hair samples measuring 3cm in length from the posterior vertex region of the scalp of participants at the mid-childhood and early adolescent visits. Hair strands were cut as close as possible to the scalp, tied to identify the scalp end, and stored in a paper envelope away from light. For measurement of hair cortisol concentrations, lab personnel first washed the hair strands in isopropanol, and subsequently extracted cortisol using liquid chromatography tandem mass spectrometry . We did Mothers reported their age, educational attainment, household income, pre-pregnancy weight, height, prenatal smoking (i.e. whether they smoked during pregnancy), and father’s weight and height via questionnaire and interview at recruitment. We calculated gestational weight gain by subtracting pre-pregnancy weight from the last recorded weight in clinical records in the four weeks prior to delivery. We then categorized this weight gain as excessive or not based on BMI category and Institute of Medicine guidelines .We extracted data on infant birth date and sex from hospital medical records. We calculated gestational age by subtracting the date of the last menstrual period from the date of delivery. If gestational age according to second-trimester ultrasound differed from that according to the last menstrual period by more than ten days, we used the ultrasound result to determine gestational age. We collected infant birth weight from hospital medical records, and calculated birth weight-for-sex-and-gestational age z-scores using national reference data . We usedAt the mid-childhood visit, we collected an 8-hour fasting blood sample. All samples were immediately refrigerated, processed within 24 hours, and stored at –80°C until time of analysis. These samples were used to measure glucose and insulin , adiponectin, high-density lipoprotein (HDL), triglycerides, C-reactive protein (CRP), interleukin-6 (IL-6), and leptin. Systolic blood pressure (SBP) measurements were taken five times using biannually-calibrated automated oscillometric monitors , from which we calculated mean SBP. HOMA-IR, HDL, SBP, triglycerides, and waist circumference were used to create a metabolic risk z-score, as described previously .At the mid-childhood visit, mothers reported children’s dietary behaviors on a PrimeScreen that assessed intake of 18 food groups , which we used to calculate Youth Healthy Eating Index scores as described previously . MothersWe performed all analyses using SAS version 9.4 . We used multivariable linear regression to estimate associations of various sociodemographic, environmental, behavioral, and anthropometric characteristics of children and their parents with mid-childhood and early adolescent HCC. We selected characteristics that we hypothesized would be associated with HCC (i.e. variables likely to be associated with stress and/or adiposity). Our primary analyses examined associations of these variables with mid-childhood HCC and early adolescent HCC separately. In secondary analyses, we explored associations between these variables with change in HCC from mid-childhood to early adolescence (ΔHCC). We log-transformed HCC values in all analyses, which greatly improved normality upon visual inspection. We also log-transformed HOMA-IR, CRP, IL-6, and leptin values, which improved normality.For all analyses, we first built models that included only demographic characteristics of children and their parents as covariates (Model 1). We next built models that examined prenatal (Model 2), infant (Model 3), and early-childhood (Model 4) characteristics. Models 1–4 were examined in relationship to mid-childhood HCC and early adolescent HCC separately. We then examined associations of mid-childhood lifestyle characteristics, anthropometric measures, and biomarkers  with early adolescent HCC only. Each model was adjusted for hypothesized confounders of the variables and HCC measure of interest. We did not include multiple anthropometric or biomarker measures in the same model because these measures are often highly correlated and including them in the same model can change the interpretation of the main variable of interest.2 values across imputed datasets for each model in our primary analyses. We reinterpreted our results after controlling for the false discovery rate (FDR) at q = 0.10 using the linear step up method of Benjamini and Hochberg [We also examined cross-sectional associations of mid-childhood characteristics with mid-childhood HCC, as well as of early adolescent anthropometric measures with early adolescent HCC, but these were considered exploratory because the exposures and outcomes occurred at the same time. We used multiple imputation to impute values of missing data for all exposures and covariates in all analyses. We did this by imputing 50 values for each missing observation and then combining the multivariable modeling estimates using PROC MIANALYZE. We calculated the range of RHochberg .For all analyses, we examined associations in males and females combined as well as in strata of sex, since we expected more females than males to have experienced onset of puberty at the time of early adolescent hair collection, which could influence cortisol secretion . We testTable 1). The median HCC was 0.98   in mid-childhood and 2.21   in early adolescence. The median ΔHCC was 0.78 .Compared to White children who participated in Project Viva but did not provide a hair sample at either visit, those who provided at least one hair sample (and were thus eligible for the current study), were more likely to be female, have a mother who graduated from college, and have been breastfed for one year or longer. Included participants were also less likely to have a mother who smoked during pregnancy or to be exposed to second-hand smoke in mid-childhood (Table 2), females had lower log-transformed HCC than males . We also found that maternal prenatal smoking , and lower birth weight-for-sex-and-gestational age z-score  were associated with higher HCC. Higher BMI z-score, waist circumference, and height in early childhood were associated with higher HCC in mid-childhood, but all CIs included the null. When controlling for the FDR at q = 0.10, we rejected null hypotheses for associations of sex and birth weight-for-sex-and-gestational age z-score. R2 values in the imputed datasets for the most complex model (Model 4) ranged from 0.05 to 0.06. We did not observe heterogeneity by sex for any associations with mid-childhood HCC (S1 Table). In cross-sectional analyses in mid-childhood, we did not observe associations of any measures of anthropometry, physical activity, diet quality, or secondhand smoke exposure with HCC, except for minor elevations in HCC associated with BMI z-score and waist-height ratio, though all CIs included the null (.In mid-childhood (Table 2), log HCC was higher in older children  and in females , opposite to the association we observed in mid-childhood. Higher maternal pre-pregnancy BMI was associated with higher adolescent HCC . We did not observe associations of any other early-childhood or mid-childhood factors with early adolescent HCC, though having a chronic illness was associated with marginally higher HCC . When controlling for the FDR at q = 0.10, we rejected null hypotheses for associations of age and sex. R2 values in the imputed datasets for the most complex model (Model 5) ranged from 0.09 to 0.11. We observed heterogeneity by sex for associations of annual household income , and maternal education  with early adolescent HCC (S1 Table). We also did not observe associations between early adolescent anthropometric measures and early adolescent HCC (S3 Table).In early adolescence (Table 3). However, ΔHCC was lower in children whose mothers smoked during pregnancy . Other parent and child characteristics were not associated with ΔHCC from mid-childhood to early adolescence. We observed heterogeneity between males and females for the association of household income with ΔHCC  (S4 Table).We observed that ΔHCC from mid-childhood to early adolescence was greater in children who were older in mid-childhood , in females , and in those with a chronic illness   with HCC. Indeed, some studies have observed the expected inverse associations between parental education and HCC in young children , 43, wheThe models we built explained a small amount of the variability in HCC, though the variables explained more variability in early adolescent HCC than mid-childhood HCC. The limited ability of these characteristics to account for the variability of HCC in children has been observed previously  and may We conducted many analyses in this study, implying that some of the associations we observed may be due to chance. This is supported by the fact that prenatal smoking and maternal pre-pregnancy BMI were not associated with HCC after controlling for the FDR at q = 0.10. However, our primary aim was to generate hypotheses regarding possible determinants of HCC in children and to identify potential confounders in future analyses of HCC and health outcomes. We therefore still consider prenatal smoking and maternal pre-pregnancy BMI possible determinants of HCC and believe that they should be considered as potential confounders in analyses that involve HCC. Future studies confirming and investigating the precise biological mechanisms underlying these associations are warranted.Strengths of this study include its prospective nature, long follow-up, collection of hair samples over two time points, which allowed us to examine associations in both mid-childhood and early adolescence (as well change in HCC between these time periods), and detailed collection of prenatal, environmental, lifestyle, and anthropometric information, which allowed us to examine relationships with HCC that have not been previously reported, as well as adjust for many likely confounders of these associations. However, our study has limitations as well. First, because of differences in hair growth and texture between racial and ethnic groups , we analIn summary, this study supports associations of maternal prenatal smoking, pre-pregnancy BMI, and low birth weight with higher childhood and adolescent HCC, but we overall observed few other associations for childhood socioeconomic, environmental, lifestyle, and anthropometric characteristics. Further studies are necessary to confirm the relationship of prenatal, parental, and childhood factors with HCC. Nevertheless, age, sex, birth weight-for-gestational-age z-score, maternal prenatal smoking, pre-pregnancy BMI, household income, and parental education may be determinants of HCC, and should be accounted for in analyses of HCC with childhood and adolescent outcomes.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Proper ciliary basal body positioning within a cell is key for cilia functioning. Centriole and basal body positioning depends on signaling pathways such as the planar cell polarity pathway (PCP) governed by Frizzled (Fz-PCP). There have been described two PCP pathways controlled by different protein complexes, the Frizzled-PCP and the Fat-PCP pathway. Centriole planar polarization in non-dividing cells is a dynamic process that depends on the Fz-PCP pathway to properly occur during development from flies to humans. However, the function of the Ft-PCP pathway in centrioles polarization is elusive. Here, we present a descriptive initial analysis of centrioles polarization in Fat-PCP loss of function (LOF) conditions. We found that Fat (Ft) and Dachsous (Ds) LOF showed a marked centrioles polarization defect similar to what we have previously reported in Fz-PCP alterations. Altogether, our data suggest that centriole planar polarization in Drosophila wings depends on both Ft-PCP and Fz-PCP pathways. Further analyses in single and double mutant conditions will be required to address the functional connection between PCP and centriole polarization in flies. Cilium is an organelle projected at the cell surface with several crucial functions during development and in adult organs. Cells use their cilia to communicate with the environment or to physically interact with the surrounding media . An examStructurally, cilia consist of membrane surrounding a cytoskeletal structure known as the axoneme, which is anchored to the basal bodies. In ciliopathies, cilium miss-functioning is usually associated to defective assembly or placement in a group of cells . Cilia aFat4 knock-out mice, hair cells are mis-orientated throughout the cochlea when looking at stereocilia  pathway, a conserved pathway from Drosophila to humans. PCP is established and maintained by two signaling cascades, the Frizzled PCP (Fz-PCP) and the Fat PCP (Ft-PCP) pathways. Fz-PCP pathway is governed by two protein complexes, the Frizzled/Disheveled/Flamingo/Diego (Fz/Dsh/Fmi/Dgo) complex and Vang/Prickle/Flamingo (Vang/Pk/Fmi) complex . The Ft-reocilia , howeverWe have recently found that centriole positioning was also controlled by the Fz-PCP pathway in Drosophila epithelial cells where cilia are absent ,b. In Drdecapentaplegic (dpp) wing compartment, a stripe between L3 and L4. In our experimental conditions, we found that only two lines (Ft and Ds) produced robust PCP phenotypes , Dachsous (Ds), Dachs and Four jointed (Fj). All culture and cross of fly lines were performed on standard medium and maintained at indicated temperatures (25 or 29°C). Following fly lines were used in this study: Ft-IR (9396/GD and 108863/KK VDRC stocks), Ds-IR (36219/DG and 4316/GD VDRC stocks), Dachs-IR , Fj-IR (6774/GD VDRC stock). GAL4/UAS system was used to direct UAS-RNAi constructs to enotypes . These penotypes . None ofenotypes  and out enotypes , only 1 enotypes .For adult trichomes analyses, wings were removed, washed in PBS 0.1% Triton X-100 (PBS-t) and mounted on a slide in 80% glycerol in PBS. Adult wings were imaged on a BX51 direct microscope (Olympus). Images acquisition was performed with a camera  and CellD software (Olympus).Fly pupae at white stage were collected and cultured at 29°C for 25 or 28.5 h. Wings were dissected in PBS-t and fixed for 1 h with 4% paraformaldehyde (PFA). Pupae were washed in PBS-t three times for 5 min and blocked in PBS-t with 2% bovine serum albumin (BSA) for 45 min. Dissected pupae were incubated overnight with primary antibody at room temperature in PBS-t-BSA. After incubation, samples were washed five times in PBS-t and incubated in fluorescent phalloidin and fluorescent secondary antibodies for 90 min, both diluted in PBS-t-BSA. Five additional washes were performed in PBS-t, and pupal wings were detached from the pupal cage and mounted on slides with medium for fluorescence . To stain the cellular membrane was used anti-Fmi (from DSHB). Secondary antibody conjugated with the fluorophore Alexa-405 (Invitrogen) and Alexa 594-phalloidin (Invitrogen) were used at 1:200.To orientate pupal wings, the distal part was always pointed to the right side. Images were acquired using FV 1000 confocal microscope (Olympus). After acquisition, images were processed using Fiji, to generate the cell borders mask , and Adobe Photoshop CC 2015 to create the color-coded mask based on the phenotype.We used density maps in order to visualize relative centriole positioning from a set of cells. Briefly, we modeled each cell to a regular hexagon with its area being the pixel numbers of the cell, and then we normalized all the modeled hexagon cells to the same size. Afterward, we normalized the position of the centriole relative to the centroid of its cell to the model cell. We have found that every cell in the Drosophila pupal wing has two centrioles but in many cases these two centrioles are too close to be separated, many times they are sitting one on top of the other. Our method compute each centriole individually. Finally, we represented the centriole density in the set of cells, so each pixel in the density map represents the probability of finding a centriole in that set of cells.Average Basal Body Position (ABP) for each sample was calculated following Each centriole was assigned to a specific Quartile (Q) depending on their relative position to the anterior-posterior axis of the cell that crosses the cell centroid . If the t-test (GraphPad Prism) for Q and ABP methods.Data were analyzed using We have previously described that centrioles polarization is a dynamic process that occurred during hair formation in Drosophila pupal wings. In wild type (WT) conditions, centrioles move from centered positions at the most apical part of pupal wing epithelial cells and as the actin-based hair is forming centrioles move off center toward the base of the hairs . To analFat4 knock-out mice have been described to have dilated tubules and cysts formation in their kidneys. This phenotype is connected to randomization of spindle orientation affecting oriented cell division, which is important for tubule elongation and single lumen formation , two Ds homologs (Ds1 and Ds2), and one Fj ortholog (Fjx1) . Fat4 knormation . In addiormation .pk in developing wings has recently been reported to reverse PCP orientation, resulting in actin hair formation being moved to the proximal cellular vertex without affecting Vang or Dsh localization (Moreover, over-expression of the Sple isoform of lization . We havelization ,b. In adlization . As a whlization . Hence, SG-J performed all of the experiments. A-CR designed and performed the data analysis. JC-G designed the experiments, analyzed data, and 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.