In bioinformatics, exon skipping (ES) event prediction is an essential part of alternative splicing (AS) event analysis. Although many methods have been developed to predict ES events, a solution has yet to be found. In this study, given the limitations of machine learning algorithms with RNA-Seq data or genome sequences, a new feature, called RS (RNA-seq and sequence) features, was constructed. These features include RNA-Seq features derived from the RNA-Seq data and sequence features derived from genome sequences. We propose a novel Rotation Forest classifier to predict ES events with the RS features (RotaF-RSES). To validate the efficacy of RotaF-RSES, a dataset from two human tissues was used, and RotaF-RSES achieved an accuracy of 98.4%, a specificity of 99.2%, a sensitivity of 94.1%, and an area under the curve (AUC) of 98.6%. When compared to the other available methods, the results indicate that RotaF-RSES is efficient and can predict ES events with RS features. The complex and diverse process alternative splicing (AS) involves removing noncoding intronic sequences and remaining exons to generate mature mRNA . AS is gexon to read counts on the alternative exon, and predict and quantify alternative isoforms derived solely from exon expression levels. Because Solas only uses Nexon and ignores other features associated with ES events, the prediction result of this algorithm contains many false positive samples. Burge et al. [ni that reads counts supporting the inclusive exon and Nne that reads counts supporting the exclusive exon, without considering other features associated with ES events. Similar to Solas, the prediction results of PSI contain many false positive samples. To detect differential alternative splicing events from RNA-Seq data, Shen et al. [exon, Nni, Nne, Nup intron that reads counts on upstream introns, and Ndown intron that reads counts on downstream introns. The framework obtained a high real-time polymerase chain reaction (RT-PCR) validation rate of 86% for differential ES events. Katz et al. [ni and Nne, but also Nexon, Nup intron, and Ndown intron. To predict exon splicing, MISO used a Bayesian probabilistic model by calculating the different exon splicing conditions and retaining the posterior probability. Compared to the other methods, MISO results contain the least false positive samples, demonstrating that these new features accurately predict AS events. In addition, a comprehensive review of AS event prediction methods from RNA-Seq data was completed [Because experiment methods are costly, labor intensive, and have inherent biases and limited coverage, computational prediction of ES events is becoming increasingly popular. Some studies have demonstrated that two kinds of data can regulate the prediction of ES events: genome sequence information and RNA-Seq data. Many classical models have been constructed for genome sequence information. For instance, Sorek et al.  combinede et al.  developen et al.  construcz et al.  proposedompleted  and Fengompleted  listed sThe slow updating of genome sequences results in lower ES event prediction. For RNA-Seq data, some special RNA expression conditions lead to lower ES event prediction. In addition, the features extracted from single data may have some noise caused by the incompleteness of these data, and these noises may produce unexpected results. Therefore, a method that can reduce the data error and improve accuracy is urgently needed for ES event prediction.To solve the above-mentioned problems and find more features to describe the ES events, we were inspired by predicting protein complexes in protein-protein interaction networks through multiple information sources . We propRotaF-RSES is a novel method using a Rotation Forest algorithm to facilitate better analysis and ES event prediction with RNA-Seq data and genome sequence information. The method involves the following two main steps .Firstly, according to the known exon, we extracted the RS features from the RNA-Seq data and genome sequence. After that, these features were used to train the model based on the Rotation Forest.Secondly, the new exon was sent to the classifier to determine whether the exon is an ES event.Some studies have demonstrated that ES event prediction can be regulated through genome sequences or RNA-Seq data. Given their drawbacks mentioned above, we think that if genome sequence information is combined with the RNA-Seq data, better prediction results may be obtained compared to genome sequence or RNA-Seq data. To validate this idea and explore the effects of different data on ES event prediction, some experiments with different features were organized. Because the Random Forest algorithm  has beenThese experiments were constructed based on the initial features using Random Forest (RF-IFES), equilibrium features using Random Forest (RF-EFES), RNA-Seq features using Random Forest (RF-RFES), sequence features using Random Forest (RF-SFES), and RS features using Random Forest (RF-RSES), with their optimal parameters. The optimal parameters were the same as in the original paper , and 100From For the RF-SFES method, we analyzed the results. The test data contained 51 ES events and 255 non-ES events. The RF-SFES predicted 9 true ES events and 11 false ES events, 244 true non-ES events and 42 false non-ES events. The RF-SFES were initially disappointing, but then the results of RF-RSES were combined with RNA-Seq features and sequence features. From these results, RF-RSES had the best overall ability to predict the ES event compared to RNA-Seq data or genome sequence. This result validates our idea.Due to the effect of different random decision values, ROC provides a reliable performance comparison. Therefore, Although Random Forest was used to perform the predictions in the previous section and achieved good results, we wanted to investigate other machine learning algorithms with RS features. We carefully analyzed and compared other traditional machine learning methods including Random Tree, Naïve Bayes, Bayes Net , Naïve BIn this work, all classification algorithms were derived in the Waikato environment (WEKA) . The RanAs seen in We analyzed the experimental results of Naïve Bayes and SVM. The test data included 51 ES events and 255 non-ES events. Naïve Bayes predicted 45 true ES events and 141 false ES events, 114 true non-ES events and 6 false non-ES events. SVM predicted 1 true ES event, 255 true non-ES events, and 50 false non-ES events. To further illustrate the performance of different algorithms, According to the analysis in the previous section, RotaF-RSES was found to be more suitable for ES event prediction. However, we did not know if the initial features of Rotation Forest (RotaF-IFES), equilibrium features in Rotation Forest (RotaF-EFES), RNA-Seq features with Rotation Forest (RotaF-RFES), or the sequence features with Rotation Forest (RotaF-SFES) could obtain better results than RotaF-RSES. To validate this idea, some experiments were performed with different features of the Rotation Forest algorithm. In this experiment, Rotation Forest contained 21 seeds. To further illustrate the performance of different features of Rotation Forest, We first compared RotaF-RSES with the state-of-the-art method ESFinder (a Random Forest classifier to identify ES events from RNA-Seq data) . ESFindeFrom We investigated why the specificity and sensitivity of RotaF-RSES were higher than ESFinder, but the AUC of ESFinder was higher than RotaF-RSES. To answer the question, we checked their experimental results. The test data contained 51 ES events and 255 non-ES events. ESFinder predicted 46 true ES events and 7 false ES events, 248 true non-ES event and 5 false non-ES events. However, RotaF-RSES predicted 48 true ES events and 3 false ES events, 253 true non-ES event and 2 false non-ES events. As RotaF-RSES had many true non-ES events (253), the AUC of RotaF-RSES is was slightly smaller than that of the ESFinder.To further illustrate the performance of both ESFinder and our method, Next, we compared RotaF-RSES with MATS  , MISO 1, and SI We constructed independent test data that contained the existing ES events derived from the UCSC Alt events dataset, with a total of 83454 ES event instances. For the skeletal muscle and brain RNA-Seq data, 306 events were used as the test data, and 612 as the training data, so the remaining 82,536 events were used as the independent test data.To validate our method on the independent test data, we compared RotaF-RSES to ESFinder, MATS, MISO, and SI. The results are shown in The classic method to identify ES events involved features derived from genome sequences using a machine learning method ,25. WithIn this work, we observed some relationships between the RS, sequence, and RNA-Seq features. We analyzed the effect of different features on ES event prediction based on Random Forest, and found that the RS features obtained a sensitivity of 92.2%, and the sequence features and RNA-Seq features were the highest at 90.2%, whereas the accuracy, specificity, and AUC were similar. Experimental results showed that the predictive power of the RS features was higher than RNA-Seq features or the single sequence features alone.Some researchers have shown that different methods have different effects on identifying ES events based on the same features ,30. We aIn conclusion, we found that the predictive power of the RS features was higher than the RNA-Seq features or the single sequence features alone. Our experiments showed that RotaF-RSES is an efficient method for ES event prediction. In the future, we will analyze the reason that these sequence features achieve better results when binding RNA-Seq features.To compare our method to the existing state-of-the-art method, we used the same benchmark dataset as ESFinder . It was http://ailab.ahu.edu.cn:8087/RotaF-RSES/index.html.The genome sequences were collected from the UCSC  Genome Browser Home. Given the strand, the start and end positions of the exon, and the upstream and downstream introns, we accurately obtained sequence information. The RNA-Seq data were collected from human brain GSM325476) and skeletal muscle (GSM325479). The raw RNA-Seq were mapped by Tophat2 to the genome sequences, then sorted and stored in a BAM file . In addi5476 and The RS features were composed of RNA-Seq features derived from the RNA-Seq data and sequence features derived from genome sequences. These sequence features were composed of structure features and short motif features. The RNA-Seq features were composed of initial features and equilibrium features. A detailed description is given below.The sequence features were composed of structure features and short motif features. Here, structure features included the length of the upstream intron, exon, and downstream intron, which are important for ES event prediction. Generally, the length of the intron is much larger than the adjacent length of exon .The short motif features include single-tuple counts, computed separately for downstream introns, exons, and upstream introns, resulting in a total of 4 × 3 = 12 features. These short motif features have been previously shown to be helpful for ES event prediction ,34.exon, Nup intron, Ndown intron, Nni, Nne, and Ngene. exon, BNup intron, BNdown intron, BNni, BNne, and BNgene were obtained from human brain RNA-Seq data, and the other six features SNexon, SNup intron, SNdown intron, SNni, SNne, and SNgene were obtained from human skeletal muscle RNA-Seq data. These 12 features constitute initial features. The RNA-Seq features were composed of initial features and equilibrium features. For each RNA-Seq data, the following six basic features were extracted: Nexon, NORMup intron, NORMdown intron, NORMni, NORMne, and NORMgene. e is the length of alternative exon, Lr is the length of RNA-Seq read, o is the length of the anchor [num is the number of the total mapped read in the sample, and Lg is the length of gene. Le − Lr + 1 is the effective length of exon where reads are mapped; Lr + 1 − 2o is the effective length of upstream intron where reads are mapped; Le − Lr + 1 + 2 × (Lr + 1 − 2o) is the effective length of the inclusive isoform where reads are mapped; and Lg − Lr + 1 is the effective length of the gene where reads are mapped. Six normalized features were obtained through human brain RNA-Seq data: BNORMexon, BNORMup intron, BNORMdown intron, BNORMni, BNORMne, and BNORMgene. Another six normalized features were obtained through human skeletal muscle RNA-Seq data: SNORMexon, SNORMup intron, SNORMdown intron, SNORMni, SNORMne, and SNORMgene. The equilibrium features were composed of normalized features, P features, and divergence features. For each RNA-Seq data, the following six basic normalized features were extracted NORMe anchor , Tnum isni, defined by Equation (1), that results in two features from brain (BP) and human skeletal muscle (SP) RNA-Seq data. In addition, seven features, named divergence features, that indicate divergence of normalized read count features between skeletal muscle and brain RNA-Seq data were also used: Δexon, Δup intron, Δdown intron, Δni, Δne, Δgene, and Δp. These seven features are defined by Equations (2) through (8), respectively. In summary, Feature P simultaneously describes the percentage of NORMRotation Forest  is an en(1)We split D randomly into K subsets. The feature number of each subset was (2)Using PCA to obtain feature conversion, for example, subset Si and obtain M feature vector, and M’ feature vectors (non-zero) were selected to form a feature vector matrix (3)Step (2) is repeated and the result is inputted into a matrix R. We found these features and their initial position in S according to the feature vector in R. Each feature vector was rearranged according to the initial position to obtain a new R*, and a new sample (4)Multiple base classifiers were obtained using the above procedure. The final result was determined by the maximum class confidence.Given initial instances set The prediction of an ES event is a binary classification problem. In this experiment, accuracy, specificity, and sensitivity were chosen to measure the performance of classifiers:formance . A classThe area under a curve (AUC) for the ROC curve is also used. When the AUC value of a predictor is larger than the area of other ROC curves, the predictor is considered better than other predictors . BecauseIn this study, to reduce the error caused by RNA-Seq data or genome sequence on ES event prediction, we propose a novel method named RotaF-RSES, which uses Rotation Forest with RS features composed of RNA-Seq features and sequence features. We explored the effects of two kinds of data on ES event prediction. Five different feature sets derived from the above data were selected for the analysis. The results indicated that RS features are better than any individual dataset for ES event prediction. To investigate the ability of different machine learning algorithms with RS features, ten algorithms were used for comparison and analysis. The results showed that Rotation Forest had the best performance. In addition, we analyzed the impact of different feature sets with Rotation Forest. The above five features were also selected for analysis. The results reinforced our ideas. Finally, to confirm the ability of RotaF-RSES to predict ES events, four methods were used to compare the performance of RotaF-RSES. The results confirmed that RotaF-RSES is efficient and has a strong ability to predict ES events. RotaF-RSES could provide biologists more accurate results for ES event studies.


Deep-sequencing technology has been widely adopted in miRNA investigations. However, it is still a big mysterious why nearly all sequencing data contain miRNA sequences from exogenous species, called exo-miRNAs. In this study, we developed a novel platform, exo-miRExplorer, for mining and identifying exo-miRNAs from high-throughput small RNA sequencing experiments which originated from tissues and cell lines of multiple organisms. Thousands of exo-miRNAs are characterized with their expression abundance, the RNA families, original organisms and the sequencing platforms presented in exo-miRExplorer. Subsequently, we used exo-miRExplorer to perform further analysis. Comparative analysis of the exo-miRNAs between different sequencing datasets revealed significant correlation of exo-miRNAs between experiments in the same study. The plant-derived exo-miRNAs analysis provided robust evidence for non-diet source of exo-miRNAs. Virus-derived exo-miRNA analysis showed that pathogen RNAs could transfer to host cells and exist in deep-sequencing result at abundance level. In conclusion, exo-miRExplorer provides users with an integrative resource to facilitate detection and analysis of exo-miRNAs. exo-miRExplorer is available at the following URL:  MicroRNA (miRNA) is a class of small RNA with 18–24 nt length, and widespread distribution in animals, plants, and viruses  or deep sequencing technology could rapidly retrieve almost all RNA sequences in cells, which becomes a powerful method to investigate microRNAs. Interestingly, most, if not all, sequenced datasets contain small RNA fragments from the genome of exogenous species, called exo-miRNAs (exo-miRNAs) Jeang, . These fThese studies sparked keen debate and drew much attention from both academic and commercial community , mouse , chicken  and Ciona intestinalis (JGI v2.0) genome sequences were downloaded from the UCSC Bioinformatics website; Caenorhabditis remanei, Caenorhabditis elegans, and Caenorhabditis briggsae genome sequences were downloaded from WormBase  values , a strict filtering standard was used to get rid of all the metazoa miRNAs . In the “Expression” tool, only “non-metazoa” exo-miRNAs are shown in the expression page Figure . Next, wp-value < 0.0001) ; A similar result was observed between the comparation of “dataset 2” and “dataset 3”  , it has a high probability to be the pollution during the process of sequencing. On the other hand, if one exo-miRNA frequently presents in the datasets performed by different research groups (“inter-study”), it may be genuine functional exo-miRNA in the cell. Following this idea, we used the “compare” tool to perform the analysis. For “intra-study” analysis, two groups of small RNA sequencing datasets which were performed by the same laboratory we chosen  Figure . We next) Figure  (Stark e) Figure . When co) Figure , only 3.) Figure . The com) Figure . Combine) Figure , we can To determine whether the plant miRNAs exist in the samples of animal and other species, all samples collected in exo-miRExplorer were examined to find exo-miRNAs derived from plants. The results showed that plant-derived exo-miRNAs existed in all of the categories in exo-miRExplorer Figure . In humaT. brucei and prokaryote Escherichia coli. It should be noted that T. brucei is an early-branched single-cell animal. It has been believed that microRNA genes are absent from this ancient organism . This system is used to against invading phages and plasmids , and autism susceptibility candidate 2 (AUTS2)  Whether other miRNAs from the same species could also be observed in the sample? The ratio of miRNAs observed in other species to the total miRNAs in their original specie. If one species could release miRNA to another species, it should not release only one or two types of microRNAs. Although rice miRNAs are frequently observed in other species, most of them are miR-168a and miR-156a. Of 713 miRNAs recorded in rice, only 6.17% of them could be observed in other species. In comparison, 86.36% of recorded 44 EBV miRNAs could be observed in human samples. We consider that these three features can be used to distinguish the real exo-miRNAs from the potential contamination RNAs.In our experience, we consider that the most important and difficult thing is to distinguish the contamination exo-miRNAs from the actual infected exo-miRNAs. Here we provide three major features for users to recognize the actual exo-miRNAs from contamination: (1) The observed frequency. If one miRNA could be observed in multiple samples performed by independent groups, it should highly possible be the real exo-miRNA. exo-miRExplorer provide “supporting number” for users to inspect this feature. (2) The evolutionary distance between the species which contain the same type of exo-miRNAs. In our analysis, human and Considering the false positive in miRNA identification and high homology of miRNAs between different organisms, we cannot completely exclude the potential miRNAs in which some fragments are actually derived from endogenous RNAs, though strict filter standard has been used in our analysis. Other possibilities also exist of course. For instance, if genome-sequencing is incomplete, it will inevitably contain sequencing error, and if the RNAs go through some RNA process events like novel splicing, RNA editing, or gene fusion events, they can result in endogenous fragments exactly the same to exo-miRNAs. exo-miRExplorer will keep updating by improving the algorithms and integrating more sequencing datasets to filter false positive results. We also try to answer the questions regarding the mechanism of exo-miRNAs transfer or even communicate with the host molecules.LQ, JY, ZL, and LZ conceived and designed the study. LZ, KD performed the data analysis pipeline. AD, JW collected, classified, and handled the source data. LQ, JY, ZL, JW, AD, KD, and LZ wrote the paper. All authors read and approved the manuscript.This research is supported by the Ministry of Science and Technology of China; National Basic Research Program (No. 2011CB811300) from the National Basic Research program (“973” program) to LQ; and the National Natural Science Foundation of China ; Guangzhou science and technology plan projects (201504010022). This research is supported in part by the Guangdong Province Key Laboratory of Computational Science and the Guangdong Province Computational Science Innovative Research Team.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 interaction of influenza infection with the pathogenesis of acute heart failure (AHF) and the worsening of chronic heart failure (CHF) is rather complex. The deleterious effects of influenza infection on AHF/CHF can be attenuated by specific immunization. Our review aimed to summarize the efficacy, effectiveness, safety, and dosage of anti-influenza vaccination in HF. In this literature review, we searched MEDLINE and EMBASE from January 1st 1966 to December 31st, 2016, for studies examining the association between AHF/CHF, influenza infections, and anti-influenza immunizations. We used broad criteria to increase the sensitivity of the search. HF was a prerequisite for our search. The search fields used included “heart failure,” “vaccination,” “influenza,” “immunization” along with variants of these terms. No restrictions on the type of study design were applied. The most common clinical scenario is exacerbation of pre-existing CHF by influenza infection. Scarce evidence supports a potential positive association of influenza infection with AHF. Vaccinated patients with pre-existing CHF have reduced all-cause morbidity and mortality, but effects are not consistently documented. Immunization with higher antigen quantity may confer additional protection, but such aggressive approach has not been generally advocated. Further studies are needed to delineate the role of influenza infection on AHF/CHF pathogenesis and maintenance. Annual anti-influenza vaccination appears to be an effective measure for secondary prevention in HF. Better immunization strategies and more efficacious vaccines are urgently necessary. Influenza is a respiratory RNA virus with two types, A and B, causing epidemic human disease in a seasonal pattern . Based oIn the absence of a global anti-influenza vaccine conferring permanent immunity, influenza remains one of the leading causes of widespread viral diseases, with high morbidity and mortality. An estimated 5–10% of adults and 20–30% of children are infected by influenza worldwide, while 250,000–500,000 severely infected individuals are estimated to die annually .The association between influenza and cardiovascular morbidity and mortality is confounded by several factors. Firstly, significant under-testing and under-diagnosis occurs in affected patients. Point-of-care molecular testing is becoming gradually widely available with emphasis on specificity during the influenza season and on sensitivity outside it . SecondlThus, a wide-spectrum of influenza-related consequences can be seen in patients with chronic disease including cardiac patients ranging from uncomplicated recover within 1 week of illness without requiring any medical intervention to a severely ill patient requiring intensive care unit treatment. Statistical modeling methods and health system records have estimated a high annual burden of influenza-attributable hospitalizations and deaths resulting in a significant strain on national health systems and substantial economic loss .The aim of the present clinical review was to evaluate the literature about the efficacy, effectiveness, safety, and dosage of anti-influenza vaccination in HF patients. A search was conducted for English language publications in MEDLINE and EMBASE databases from January 1st 1966 to December 31st, 2016. The following broad search terms, including Medical Subject Headings (MeSH), were used: chronic heart failure (CHF), acute heart failure (AHF), acute decompensated heart failure (ADHF), influenza infection, vaccination, immunization, hospitalizations, mortality, death, and heart failure decompensation. Two investigators (NK and ST) performed the literature research. With the exception of case studies, all other study designs were considered (both randomized and non-randomized), whether prospective or retrospective. We excluded studies that did not report heart failure outcomes. The reference list of the included articles was checked to identify other relevant papers for inclusion.Numerous observational studies have shown an elevated risk for acute cardiovascular events  in general population, not only within the first few days of infection –13, but .It is still obscure whether influenza infection can be a primary cause of heart failure (HF). A wealth of information implicates the involvement of inflammatory activation in the development and progression of CHF . NeverthFig. .Fig. 1PInfection-induced ADHF may develop on the top of pre-existing HF, already diagnosed or not , 20. A sImmunization against seasonal influenza has risen during the last decades as a potential measure to reduce hospitalization rate , 28 and Regarding those controversies, the largest international scientific societies have considered the magnitude of data as robust evidence of annual anti-influenza vaccination for secondary, but not primary, prevention in patients with cardiovascular diseases, like those with CAD or HF , 39. As The advocacy of anti-influenza vaccination in HF is based on limited number of large, observational studies. There are additional data derived from population-based registries Table . MethodoTo address that question, a previous observational, population-based study examined the clinical impact of anti-influenza vaccination on patients with diagnosis of CAD and/or ischemic HF . There wA sub-analysis of the large-scale PARADIGM-HF trial was recently published . The maiIn terms of effectiveness, anti-influenza vaccination seems to reduce HF-related hospitalizations and all-cause mortality, especially during influenza periods, in patients with pre-existed HF.The vaccine effectiveness relies upon intact immune responses, which may be compromised in HF. Following influenza immunization most individuals develop both antibody titers and T-cell immune responses. The efficacy (laboratory measured) and consequently the effectiveness  of anti-influenza vaccine seem to decrease with increasing age and the presence of co-morbidities . EvidencA more aggressive approach of vaccination using high rather than standard-dose vaccine in elderly patients has been proposed . NotablyDespite the previously mentioned overwhelming evidence of the burden of influenza and the high effectiveness of anti-influenza vaccine, the fear of negative side effects has discouraged many people from getting vaccinated. Evidence deriving from the general population has failed to show an association between influenza vaccines and significant side effects , 57. GivInfluenza vaccines confer only short-lived, strain-specific immunity, and annual revaccination is required. Optimal timing is difficult to predict, since the influenza season starts at different times each year and influenza activity may vary among geographic regions. In clinical practice, the administration of anti-influenza vaccine in HF patients is wise to take place in pre-influenza seasons (prior to winter), with renewed formulation each time. Interestingly, a large retrospective study in male veterans admitted to hospitals due to HF underpinned the survival benefits of anti-influenza vaccination not only among patients vaccinated before, but also during hospitalization . TherebyThe majority of large studies have enrolled community-dwelling populations with mixed characteristics , where it is difficult to distinguish HF patients. Moreover, HF is mostly referred as the admission reason for patients with concomitant influenza infection, while only a limited number of studies have clearly defined cohorts with pre-existed HF. Hence, the future research should point towards populations with established HF. Details of pre- and post-infection clinical characteristics and medications records are necessary in order to retrieve the pure effect of anti-influenza vaccination on HF progression and precisely assess the incidence of HF decompensation. It would be also prudent to evaluate the vaccination effects along the classes of HF severity. Another important drawback of observational studies and meta-analyses is the difficulties to evaluate classify a death as influenza-related mortality. For this purpose, most researchers have presented results on all-cause mortality instead of specific-cause mortality. Therefore, a residual confounding in the estimates of vaccine effectiveness cannot be entirely excluded.According to prevailing recommendations, the anti-influenza vaccination should be encouraged in HF patients, who are at high mortality risk. Unfortunately, they often have suboptimum vaccine uptake. A significant proportion of elderly people with chronic HF remain annually unvaccinated against influenza , 44. A pLarge-scale and adequately powered studies targeting the effectiveness and safety of anti-influenza vaccination will assess beyond any doubt the cost-effectiveness of anti-influenza vaccination in HF patients (secondary prevention) or those at increased risk for HF development (primary prevention).Seasonal influenza infection, especially among elderly populations, is associated with high occurrence of AHF, increased cardiovascular morbidity and all-cause mortality. Based on data from the general population, annual anti-influenza vaccination seems to be an effective measure to prevent AHF episodes and in some degree to improve survival. Confining literature research in patients with pre-existed HF, there are limited data favoring the incorporation of anti-influenza vaccination as an adjunctive therapy, to reduce hospitalizations and all-cause mortality rates. Unambiguously, future studies will verify the required dose and the potential benefits and risks of annual anti-influenza vaccination in the context of primary prevention or standard care processes of established HF .


BMC Evolutionary Biology and BMC Genomics covered the proceeding of BGRS\SB-2016 conference and SBB-2015 School in Novosibirsk [https://bmcgenomics.biomedcentral.com/articles/supplements/volume-15-supplement-12).Current collection continues the series of BioMed Central special post-conference issues presenting the highlights from the set of conferences on bioinformatics and systems biology held in Novosibirsk and Moscow (Russia) in 2017. This thematic issue of BMC Evolutionary Biology issue cover the papers presented at Young Scientists School “Systems Biology and Bioinformatics - 2017” (SBB-2017) and Belyaev memorial conference “Belyaev Readings - 2017” (BR-2017). Previously published special issues of osibirsk  as well http://conf.bionet.nsc.ru/belyaev100/en).In it important to note that Year 2017 marks the 100th anniversary since birth of Full Member of the USSR Academy of Sciences, Professor Dmitry K. Belyaev (1917–1985), an outstanding scientist, evolutionist and geneticist. In view of this memorable date, the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences (ICG SB RAS) held international Belyaev Conference on Genetics and Evolution  geneticist Dmitry Konstantinovich Belyayev embarked on a journey to turn aggressive, wily silver fox into a domestic animal as friendly and tail-wagging as a dog. Belyaev was inspired by Darwin’s early observations on similarity of morphologic and physiologic changes observed in various domestic animals. Hence, he hypothesized that domestication is, in fact, a selection for disposition toward humans and, therefore, it should involve the changes in regulatory genes governing the behavior, the stress and the metabolism of sex hormones. As a result, a unique, world-famous tame fox population was generated. Belyaev demonstrated that the selection for human-friendly demeanor disrupts physiological and morphological systems of silver fox, thus, shifting the phenotype to that similar to the dogs and other now-tamed animals. With that, Belyaev opened up a totally new chapter in the theory of morphogenetic processes that explores interactions between two main factors in evolution: variability and selection. The results of a breathtaking experiment with thousands of animals led Belyaev to postulate a very special form of directional, destabilizing selection taking place in domestication. It destabilizes regulatory systems underlying ontogenesis and, as a consequence, sharply increases the rates at which new phenotypic forms emerge.http://icg.nsc.ru/belyaev100/en/monument/). The tamed fox gives the Professor a paw and wags the tail . The article by Prof. V. K. Shumny [In 2017, “Vavilov Journal of Selection and Breeding” published a series of memoirs publications about Prof. Belyaev , this research team made several adjustments to the phylogeny of these plant species, while also showing both intra- and interspecific divergence of their rRNA-encoding genes.Nadezhda L. Bolsheva and co-authors  presenteAlexey S. Rozanov et al.  describeElena V. Ignatieva  and collBMC Genetics, BMC Genomics, BMC Medical Genomics, BMC Plant Biol and BMC Neuroscience.Follow-on series of related works in the areas of classical and medical genomics, genetics, and plant biology discussed at “Belyaev conference – 2017” and other related meetings in Novosibirsk and Moscow will be published in Special Issues of


Objective: Telavancin is approved to treat complicated skin and skin structure infections, hospital-acquired, and ventilator-associated bacterial pneumonia caused by Staphylococcus aureus. A previous meta-analysis of randomized controlled trials suggested that it might be an alternative to vancomycin in cases of difficult-to-treat meticillin-resistant S. aureus infections. We did a meta-analysis including one new trial to access the efficacy and safety of telavancin.Methods: We searched PubMed, Cochrane Central Register of Controlled Trials, EMBASE and ClinicalTrials.gov up to December 30, 2015 to identify randomized controlled trials that assessed the clinical efficacy, eradication efficiency, adverse events and laboratory abnormalities of telavancin vs. other antibiotic agents for bacterial infection. Meta-analysis was performed using Review Manager 5.3.0.Results: Five studies (3790 participants) were included in the meta-analysis. There was no significant difference in treatment success with telavancin than with control antibiotic agents. The pooled pathogen eradication for the telavancin group was numerically higher than that for the control groups, but there was no significant difference. While all-cause mortalities and serious adverse events were comparable between telavancin and control antibiotic agents, adverse event-related withdrawals  were higher in telavancin group. The total number adverse events were more in the telavancin group than in the control groups, especially in the digestive system , nervous system  and urogenital system . Serum creatinine increase  and hypokalemia  occurred more frequently in telavancin group compared to control groups.Conclusion: Telavancin may be as effective as but no better than the comparison therapy for S. aureus infection. However, because of the high risk of adverse event-related withdrawals and potential nephrotoxicity, prudence with the clinical use of telavancin in infections is required. Staphylococcus aureus is one of the most common and virulent clinically encountered Gram-positive bacteria  with meticillin-resistant S. aureus (MRSA) as the predominant pathogen. Currently, glycopeptide antibiotics such as vancomycin and teicoplanin are the gold standard for the treatment of serious infections caused by Gram-positive bacteria, especially MRSA. The emergence and prevalence of multidrug-resistant Gram-positive pathogens underscores the urgent need for development of new antimicrobials.S. aureus and other Gram-positive bacteria, including methicillin resistant and vancomycin-intermediate strains of S. aureus . In the US, telavancin was approved for complicated skin and skin structure infections (cSSSI) in September 2009 and for hospital-acquired and ventilator-associated bacterial pneumonia caused by S. aureus in June 2013. In Europe, telavancin had been approved as second-line treatment for hospital-acquired pneumonia, including ventilator-associated pneumonia (VAP), known or suspected to be caused by MRSA when other alternatives are not suitable .So far, several studies have suggested that telavancin had comparable efficacy and higher MRSA eradication rate comparing with other antibiotics for the treatment of gram-positive bacteria  checklist.A computerized search in PubMed, Cochrane Central Register of Controlled Trials and EMBASE up to December 30, 2015 was conducted independently by two individuals (Zhu and Zhong) with the search terms “telavancin” or “TD-6424.” We also did a search in ClinicalTrials.gov up to December 30, 2015 to screen completed trials about telavancin but with no published results. Furthermore, the references of retrieved literatures were manually screened for more eligible studies.post-hoc analyses, bactericidal activity studies, pharmacokinetic, or pharmacodynamic studies.Studies that met the following criteria were considered as eligible for this meta-analysis (1) they were randomized controlled trials (RCTs); (2) studies compared the outcomes of telavancin and other antibiotic agents in infection treatment; (3) studies assessed the clinical efficacy, efficiency for eradication of pathogens, adverse events and laboratory abnormalities of both therapeutic regimens. Two independent reviewers searched the databases and screened all retrieved articles according to the inclusion criteria. Studies were excluded if they were animal studies, retrospective studies, Two of our authors (Zhang and He) independently extracted data for each eligible study. In cases of discrepancy, a third author (Chuan) was consulted. The following information was recorded for the included trials: study title, name of first author, year of publication, study design, type of infection, drug regimens, treatment duration, time to test of cure (TOC), number of patients, clinical and microbiological outcomes, and data on safety.The Intention-to-Treat (ITT) population included all randomized patients in the group to which they were randomly assigned, regardless of the treatment they actually received, and regardless of subsequent withdrawl from treatment or deviation from the protocol  and laboratory abnormalities. Eradication of pathogens was based on ME populations. Adverse effects and laboratory abnormalities were assessed in mITT populations.The statistical analyses were carried out in Review Manager (version 5.3.0) . Random effects model (REM) was chosen since the included studies involving different infections, different control regimens, different sample size, which introduced obvious heterogeneity across the trials. Odds ratio (OR), with 95% confidence interval (CI), was used for all primary and secondary outcomes by Mantel-Haenszel method. The publication bias was not assessed due to the small number of the included studies. Sensitivity analysis was performed to investigate the influence on the overall results by omitting a single trial at a time.The outcome of the search was shown in Figure S. aureus bacteremia and compared telavancin IV at 10 mg/kg/24 h with standard therapy  guidelines recommended 15–20 μg/mL of vancomycin for serious infections, such as pneumonia and severe skin and soft tissue infections , whereas the difference was not significant in our study . The results were the same between Polyzos's meta-analysis and our study when the data from trials involving HAP and uncomplicated S. aureus bacteremia were removed. Thus, these results suggested that telavancin showed higher eradication rate of MRSA for cSSSI but not for HAP and uncomplicated S. aureus bacteremia.In Polyzos and colleagues' meta-analysis of telavancin  were sponsored by Theravance Inc., a pharmaceutical company producing telavancin (brand name as VIBATIV). This could be considered a factor that might introduce bias in the assessment of outcomes.There were several limitations in our study. Only one new study assessing the treatment of telavancin for uncomplicated S. aureus infection, including MRSA infection. However, telavancin is associated with a higher frequency of adverse events than the comparators, especially in the digestive system, nervous system and urogenital system. Because of significant serum creatinine increase and consequent potential nephrotoxicity, prudence with the clinical use of telavancin in infections is required.In conclusion, telavancin has clinical efficacy and microbiological eradication rate similar to control antimicrobial regimens in Conceived and designed the experiments: JC, YuaZ, and HX. Performed the experiments: JC, YuaZ, and XH. Searched the databases: YuxZ, LZ. Study inclusion: YuxZ, LZ. Data collection: JC, YuaZ, and XH. Analyzed the data: JC, YuaZ, and XH. Wrote the paper: JC, YuaZ, and HX. Critical revision of the manuscript for important intellectual content: YuaZ, DY.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.


Cell-free transcription–translation is an expanding field in synthetic biology as a rapid prototyping platform for blueprinting the design of synthetic biological devices. Exemplar efforts include translation of prototype designs into medical test kits for on-site identification of viruses (Zika and Ebola), while gene circuit cascades can be tested, debugged and re-designed within rapid turnover times. Coupled with mathematical modelling, this discipline lends itself towards the precision engineering of new synthetic life. The next stages of cell-free look set to unlock new microbial hosts that remain slow to engineer and unsuited to rapid iterative design cycles. It is hoped that the development of such systems will provide new tools to aid the transition from cell-free prototype designs to functioning synthetic genetic circuits and engineered natural product pathways in living cells. Escherichia coli cell extracts to study coupled transcription–translation (TX–TL). Together with Har Khorana and Robert Holley, this resulted in a shared Novel Prize in Physiology or Medicine in 1968. On this theme, the efforts of Alfred Goldberg led to the unveiling of an ATP-dependent mechanism for protein degradation by ubiquitin in a mammalian cell-free system [Cell-free systems represent a historically important component of the founding of the field of biochemistry. Ever since the pioneering efforts of the Nobel laureate Eduard Buchner (Nobel Prize in Chemistry in 1907) and his discovery of fermentation in yeast cell extracts –4, whiche system .Today, with the rise of synthetic biology and the design and construction of synthetic life , cell-frCell-free coupled TX–TL utilises the core machinery of RNA polymerase holoenzyme, the translation apparatus  and energy regeneration enzymes to amplify a set of DNA instructions into the target protein(s) of choice . TherefoE. coli platforms, which are based on either a crude cell extract [purified recombinant elements (PURE) [The choice of a well-characterised cell-free system almost entirely resides with  extract –23 or a s (PURE) ,25. A vis (PURE) . Proteins (PURE) . In addis (PURE) .E. coli translation machinery including ribosomes, 22 tRNA synthetases, initiation factors, elongation, release and termination factors, which when combined with T7 RNA polymerase, tRNA, energy regeneration enzymes, substrates (amino acids and creatine phosphate) and synthetic DNA instructions, this reconstitutes the entire TX–TL network within a test tube. This rather remarkable engineering feat is commercially available as the PURExpress® kit (New England Biolabs). While the high cost of the system prohibits scaled-up applications, a variety of cell-free researchers utilise the PURExpress® system to study the dynamics and kinetics of TX–TL [First, in respect to the PURE system , this inof TX–TL ,28–32. Tof TX–TL , which rE. coli strains and user preference [In contrast, a crude cell extract provides an inexpensive route to protein synthesis. In addition, unlike the PURE system, reactions are scalable into high-volume fermentation conditions ,34. Howeeference .in vivo based approach can take several days to weeks. Thus, cell-free provides a prototyping approach  soil bacteria, it has long been appreciated that these hosts provide unique and well-characterised platform for the assembly of a rich repertoire of natural products [Streptomyces cell-free, the recently developed high-yielding Streptomyces lividans and Streptomyces venezuelae host platforms [Streptomyces cell-free could be utilised for incorporating non-natural or potentially toxic substrates into natural products, towards expanding the chemical space of biosynthesis. A proof of concept of how cell-free can be utilised to incorporate non-natural amino acids into protein backbones was demonstrated for GFP synthesis in E. coli cell-free [E. coli. Thus, with further developments, cell-free potentially provides a potentially fast route to prototype and engineer the application of novel chemistry to natural product biosynthesis [Viewed from a different perspective, synthetic biology has begun to examine the prospects of engineering non-model microbial hosts  that canrevisiae ,57, Streces spp. –61 and Blus spp. ,62. Suchproducts . For Strlatforms ,61 can pell-free . While tell-free , which hell-free . Howeverynthesis .The rise of cell-free systems from its historical links in foundational biochemistry has provided a platform to this expanding field in synthetic biology. Perhaps, the greatest challenge of cell-free studies is to establish and define the boundaries and limitations of mimicking cellular biology within cell-free systems. One understudied area is the impact of molecular crowding on enzyme velocities ,69 and s


In conventional fundus photography, trans-pupillary illumination delivers illuminating light to the interior of the eye through the peripheral area of the pupil, and only the central part of the pupil can be used for collecting imaging light. Therefore, the field of view of conventional fundus cameras is limited, and pupil dilation is required for evaluating the retinal periphery which is frequently affected by diabetic retinopathy (DR), retinopathy of prematurity (ROP), and other chorioretinal conditions. We report here a nonmydriatic wide field fundus camera employing trans-pars-planar illumination which delivers illuminating light through the pars plana, an area outside of the pupil. Trans-pars-planar illumination frees the entire pupil for imaging purpose only, and thus wide field fundus photography can be readily achieved with less pupil dilation. For proof-of-concept testing, using all off-the-shelf components a prototype instrument that can achieve 90° fundus view coverage in single-shot fundus images, without the need of pharmacologic pupil dilation was demonstrated. Traditional fundus cameras employ trans-pupillary illumination, i.e., a donut-shaped illumination pattern projected to the peripheral area of the pupil. After passing through the pupil, the light diverges and illuminates the posterior of the eye5. To illuminate the retina homogenously, the diameter and divergence of the illumination pattern on the pupil plane must be carefully adjusted, requiring careful design and sophisticated construction of the optical imaging system7. According to ISO 10940:20098, external-angle is commonly used to specify field of view (FOV) in traditional fundus cameras. However, eye-angle has been recently adopted to determine the FOV in wide-field fundus imagers, such as a Retcam , Optos , etc. In order to avoid unnecessary confusion, we provide both external- and eye-angle numbers in the following discussion. Traditional fundus cameras provide 30°–45° external-angle (45°–67.5° eye-angle) FOV9. Additional challenges with trans-pupillary illumination include glare caused by light reflection from the cornea and crystalline lens7, and the requirement of adequate pupil dilation for wide field examination. Pharmacologic pupil dilation may make patients suffer from light glare and focusing difficulty for hours and even days in some cases.Wide field fundus photography is desirable for screening, diagnosis, and treatment evaluation of diabetic retinopathy (DR)11. Trans-scleral illumination delivers the illumination light from the region outside of the pupil, and thus can increase the available FOV for fundus photography (see Methods: Trans-pars-planar illumination). Panoret-1000™ employed trans-scleral illumination to image the retina from the optic disc to the ora serrata in a single-shot image12. However, clinical deployment of trans-scleral illumination was not successful, and the product Panoret-1000™ is no longer commercially available. Clinical deployment of trans-scleral illumination failed due to several limiting factors. First, the employed contact-mode imaging was not favorable for patients. Direct contact of the illuminating and imaging parts to the eyeball might produce potential inflammation, contamination, and abrasion to the sclera and cornea. Second, it was difficult to operate the system to obtain good retinal images. In Panoret-1000™, the digital camera and light illuminator were apart from each other. To capture a retinal image, one hand was used to operate the camera, and the other hand was used to adjust the light illuminator. The simultaneous need of both hands for imaging operation made the device very difficult to use. Moreover, the image quality was highly dependent on the illumination location. Without one objective, automated method for quickly optimizing location of the illuminator, it may take too long to train a photographer, and the ratio of readable retinal images was poor.Trans-scleral illumination has been proposed as one alternative illumination method to achieve wide field fundus examination not requiring pharmacologic pupil dilation13. Using a fiber illuminator contacting the eyelid, the trans-palpebral illumination excluded the possibility of eyeball inflammation, contamination, and abrasion associated with trans-scleral illumination. Without the need for pharmacologic pupil dilation, we have demonstrated nonmydriatic wide field fundus photography using a smartphone-based prototype13. However, contact-free imaging is desirable for easy-to-use clinical deployments.We recently demonstrated trans-palpebral illumination for wide-angle fundus photography13 or sclera 11 trans-pars-planar illumination is totally contact-free to project illuminating light through the pars plana. Without the complications of contact optics and pupil dilation, the trans-pars-planar illumination promises a low-cost, easy-to-use, wide field fundus photography to advance routine examination of DR, ROP and other eye diseases that are known to produce morphological changes of the retinal periphery.In this article, contact-free trans-pars-planar illumination for wide field fundus photography is reported. Instead of using a light illuminator contacting the eyelid Using all off-the-shelf components, we constructed the prototype camera for proof-of-concept validation of trans-pars-planar illumination. Without the need of pharmacologic pupil dilation, a 60° external-angle (90° eye-angle) fundus view coverage was achieved in single-shot fundus images.As shown in Fig. All images in Fig. 14. In order to verify the potential feasibility of using the trans-pars-planar illumination based fundus camera for quantitative imaging, we explored automated classification of arteries and veins, quantitative analysis of blood vessel diameter and tortuosity, and arteriolar-to-venular diameter ratio (AVR). It is known that retinopathy can affect arteries and veins differently. For example, some studies have shown that in ROP the increase in arterial tortuosity is more significant than that of veins15, and in DR the diameter of arteries decrease and diameter of veins increase17. Therefore, separate analysis of arteries and veins can provide improved sensitivity for quantitative fundus image analysis and classification.Quantitative analysis of fundus images is essential for objective and automated classification of eye diseases18. As shown in Fig. 19. Figure Figure A clinical mydriatic fundus camera  was used for comparative imaging to evaluate the image quality of the trans-pars-planar illumination based nonmydriatic fundus photography. Figure 7, the imaging light is typically collected through the central pupil only. Therefore, the available FOV of conventional fundus camera is congenitally limited, and pupil dilation is frequently required for fundus examination of the retinal periphery. Sophisticated system design, with delicate optical devices, were mandatory to balance the pupil usages for illumination light delivery and imaging light collection, making traditional fundus cameras complex and expensive. By freeing the entire pupil for collecting imaging light only, the demonstrated trans-pars-planar illumination dramatically simplified the complexity of optical system required for wide filed fundus photography. Moreover, the trans-pars-planar illumination eliminates all contact parts in previously demonstrated trans-scleral12 and trans-palpebral13 illumination for wide filed fundus photography. Therefore, totally contact-free trans-pars-planar illumination promises next generation low-cost, ultra-wide field, nonmydriatic, snapshot fundus cameras, which will foster clinical deployment of wide filed fundus photography to enable better ROP management, early DR detection, and improved accuracy in predicting DR progression and diabetic macular edema (DME) development, etc.21.A contact-free trans-pars-planar illumination for snapshot, wide field fundus photography without pharmacologic pupil dilation was demonstrated. In conventional fundus cameras with trans-scleral illumination, both illumination path and imaging path share the pupil. Typically, the illuminating light is delivered through the periphery of the pupil. In order to minimize the effect of reflections from the cornea and crystalline lens on fundus image17, separate analysis of arteries and veins can provide improved sensitivity for quantitative fundus image analysis and classification. Comparative evaluation was conducted using a clinical mydriatic fundus camera to demonstrate the potential of using the nonmydriatic trans-pars-planar illumination for clinical application. Moreover, we have recently demonstrated the feasibility of using smartphone for trans-scleral illumination based fundus photography with single-shot view up to 152°13. Unique combination of the contact-free trans-pars-planar illumination, low-cost smartphone technology, quantitative image analysis, and widely available internet technology promises a low-cost, ultra-wide field, nonmydriatic fundus camera to enable affordable telemedicine to reduce health disparities in rural and underserved areas, where both experienced ophthalmologists and expensive devices are limited.Using fundus images acquired with the prototype camera, the feasibility of automated classification of arteries and veins, quantitative analysis of blood vessel diameter, tortuosity, and AVR were validated. Because different eye diseases can affect arteries and veins differentlyWhile trans-pars-planar illumination provides an easy solution for wide field fundus photography, the image quality is highly dependent on illumination delivery location. In other words, accurate identification of pars plana is important. In our current prototype, the illumination position was manually controlled through a mechanical translation stage. In principle, automatic identification of pars plana can be achieved by dynamic monitoring of overall brightness of individual digital images as shown in Fig. We are aware of different color efficiencies for trans-pars-planar illumination. It is known that light attenuation due to absorption and scattering is wavelength dependent. Because the transmission of long wavelength light is much higher than short wavelength light, we chose a LED that had strong short wavelength emission (yellow and green color) and weak long wavelength emission (red color)  for trans-pars-planar illumination. However, the fundus images were still red predominated. It is well established that details of retinal vasculatures are mainly reflected in short wavelength images. In order to produce retinal blood vessel enhanced images, we have demonstrated the feasibility of digitally normalizing intensity values of individual color channels Fig. . HoweverOne second exposure time was used for collecting the fundus image in Fig. In conclusion, contact-free trans-pars-planar illumination for nonmydriatic, snapshot, wide field fundus photography was demonstrated. Using all off-the-shelf components, a simple and low-cost prototype instrument was constructed to validate a 90° fundus view in single-shot images, without the need of pharmacologic pupil dilation. The fundus images provided sufficient quality to allow computer-aided classification of arteries and veins, and quantitative analysis of blood vessel diameter, tortuosity, and AVR. Further development of the trans-pars-planar illumination based fundus camera will foster clinical deployment and telemedicine of using wide filed fundus photography for better disease screening, diagnosis, and treatment management of DR, ROP, DME, and other chorioretinal conditions.23. The pars plana lacks muscle, blood vessels and pigmentation23, thus is more transparent than other area of the sclera, making it a good location for delivering light into the eye. Figure Figure 11. The trans-scleral illumination frees the entire pupil for imaging, and thus nonmydriatic wide field fundus imaging is possible. However, the contact mode illumination is not favorable, and thus it failed clinical deployment. We recently reported a prototype of trans-palpebral illumination fundus camera, which delivered illumination light through the eyelid, without direct contact to the sclera (Fig. Figure era Fig. . Howeverera Fig. . As showera Fig. . Since cera Fig. .Figure 525, as well as to reject scattering light from the sclera. A lens was positioned behind the camera viewfinder and a cross that was illuminated by an LED lamp was placed in front of the lens to serve as a fixation target, so that the testing subjects could fix their eyes by looking into the camera through the lenses and look at the cross. A single-shot fundus image could be easily acquired by pressing the camera shutter button.Figure In conventional fundus cameras, a beam splitter can be used to split light paths so that a fixation target could be implemented. However, a beam splitter wastes a fraction of the light from the retina. In our prototype system, there was no beam splitter required due to the single reflex feature of the camera. As shown in Fig. This study was approved by the Institutional Review Board of the University of Illinois at Chicago and was in compliance with the ethical standards stated in the Declaration of Helsinki. Images shown were captured from one healthy Asian female subject and one health Turkish male subject with informed consent. No discomfort or vision impairment was reported by the subjects after fundus photos being taken.26. The transmission of the sclera in visible wavelength is 10–30%27. To be conservative, 30% was used for calculation. According to the ISO 15007-2:2007 standard, a maximum of 10 J/cm2 weighted irradiance is allowed on the retina28 without photochemical hazard concern. The weighted irradiance was calculated using the photochemical hazard weighting function provided in the standard. For the proof-of-concept experiment, the weighted irradiance on the sclera was calculated to be 0.5 mW, the area of the arc-shaped light was 13 mm2. For the worst case estimation, we assumed all illumination light directly expose to the retinal area behind the illuminated sclera area. Therefore, the maximum allowed exposure time isPotential photochemical and thermal hazards of the retina were carefully evaluated. There is no retina present at the pars planar area. However, it is possible that the light passes through sclera and illuminates the retina. The thickness of the sclera is ~0.5 mm2. Thus the maximum allowed exposure time through the pupil is >30 minutes. For thermal hazard, the maximum weighted power intensity allowed on the sclera without thermal hazard concern is 700 mW/cm2 28. The calculated weighted power intensity was 230 mW/cm2, which was more than three times lower than the maximum limit. Therefore, there was no thermal hazard concern.If the illumination light accidently fell into the pupil, the illuminated area on retina was estimated to be >9 mm29. The segmentation algorithm was based on the traditional matched filtering method first developed in 198929. Briefly, 2D Gaussian kernels that matched blood vessel profiles were used. Kernels of 12 different orientations and 10 different sizes were used to cover all blood vessel directions and diameters. After subtracting their means, each kernel was used to convolve the green channel fundus image and a total of 120 convolved images were generated. Among the 120 convolved images, the maximum intensity projection was used as the final enhanced blood vessel image. Top-hat filtering was used to correct uneven illumination of enhanced blood vessel image, then global thresholding was applied to generate segmented blood vessel map (Fig. 18. The ODR image was then multiplied by the blood vessel map (Fig. 18, and diameter of artery was smaller than accompanying vein30. Details of blood vessel tortuosity and diameter quantification have been reported in previous publication14.To identify arteries and veins, the blood vessel map was first segmented using the green channel of the fundus image. The green channel Fig.  was usedmap Fig. . ODR imamap Fig. . Blood vThe datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.


Imatinib mesylate is an inhibitor of the tyrosine kinase Bcr–Abl and a first-line treatment for Philadelphia chromosome-positive chronic myeloid leukaemia (CML). Dermatological side effects include superficial oedema, pustular eruption, lichenoid reactions, erythroderma, and skin rash. Depigmentation of the skin and/or mucosa is uncommon, and hyperpigmentation is rare.We present the case of a 63-year-old Caucasian male with widespread hyperpigmentation of the hard palate associated with a 9-year history of imatinib therapy to treat CML. He did not complain of any symptoms. Clinical examination did not reveal any abnormal pigmentation of the skin or other region of the oral mucosa. He did not smoke cigarettes or drink alcohol. His medication regimen was a proton pump inhibitor, a beta-blocker, cardioaspirin, atorvastatin, and imatinib 400 mg/day. Histopathologically, melanin and haemosiderin deposits were evident in the lamina propria. The lesion persisted, with no clinical change, through several follow-ups. We reviewed the literature to explore the possible relationship between oral hyperpigmentation and long-term imatinib mesylate treatment.We diagnosed oral pigmentation associated with imatinib intake based on the medical history and clinical features of the pigmented macules. Oral pigmentation may have a variety of causes, and differential diagnosis requires nodal analysis. Clinicians should be aware of possible oral mucosal hyperpigmentation in patients taking imatinib mesylate. Such pigmentation is benign and no treatment is needed, but surveillance is advisable. Pigmentation of the oral mucosa associated with overproduction of melanin is relatively common and may involve any region of the oral cavity. The prevalence varies by geographical region and ethnicity. A cross-sectional study of 1275 Jordanian subjects found that 30.2% exhibited oral pigmentation . The aetImatinib mesylate , a tyrosine kinase inhibitor targeting the Bcr–Abl protein, is a first-line treatment for Philadelphia chromosome-positive CML . The derIn January 2016, a 63-year-old Caucasian male was referred to us for evaluation of painless grey–blue hyperpigmentation of the hard palate, noted by his dentist during a routine dental examination (Fig. Histopathological examination revealed a non-inflamed palatal mucosa with pigment-containing histiocytes in the mucous membrane Fig. . ImmunohAt the 6-month follow-up, neck ultrasonography did not reveal any swollen lymph node. We took close-up colour photographs of the lesion to confirm the absence of any morphological change. In May 2017, palatal hyperpigmentation was still evident, and the clinical appearance was unchanged, but he reported no symptoms.Diagnostic considerations when encountering oral melanosis should include physiological, pathological, and environmental variables. Physiological oral melanosis is usually localised to the gingival and buccal mucosa and is bilateral and symmetrical, brownish in colour, and clinically more common among dark-skinned populations . Oral meA history of occupational or environmental exposure to heavy metals and clinical signs of metal toxicity help to identify pigmentations of the oral mucosa. Heavy metals such as bismuth, lead, copper, arsenic, gold, copper, cobalt, chromium, silver, mercury, and magnesium can induce the development of a bluish-black line, the so-called Burton’s line, along the gingival margin, the thickness of which is proportional to the extent of gingival inflammation . In someOf the 15 cases published in the English language literature, eight reported the histopathological features, i.e. deposits of melanin and/or haemosiderin in the lamina propria. Of these, four described co-existing melanin and haemosiderin deposits. Our findings are consistent with those of the cited reports; both Fontana–Masson staining for melanin and Perl’s Prussian blue staining for haemosiderin were positive Table .Table 2SThe pathophysiological mechanism of mucocutaneous pigmentation induced by imatinib remains unclear. Imatinib targets the ATP-binding site of the Bcr–Abl tyrosine kinase and also inhibits the actions of other tyrosine kinases, including platelet-derived growth factor receptor-b, C-kit, and C-ABL . C-kit iThe diagnosis of imatinib-associated oral pigmentation requires a thorough history-taking and clinical examination of the melanotic maculae. Medical and dental practitioners should be aware of possible oral mucosal hyperpigmentation in patients taking imatinib mesylate. The hyperpigmented lesions are benign; no treatment is required. However, annual follow-up is advisable to monitor changes in morphology or colour over time.


N = 887) from a public fcMRI database was used to characterize adult lifespan connectivity dynamics within and between seven brain networks: the default mode, salience, dorsal attention, fronto-parietal control, auditory, visual and motor networks. The entire cohort was divided into young ; middle-aged ; and old  subgroups. Correlation matrices as well as a mixed model analysis of covariance indicated that within high-order cognitive networks a considerable connectivity decline is already evident by middle adulthood. In contrast, a motor network shows increased connectivity in middle adulthood and a subsequent decline. Additionally, alterations in inter-network interactions are noticeable primarily in the transition between young and middle adulthood. These results provide evidence that aging-related neural changes start early in adult life.As the world ages, it becomes urgent to unravel the mechanisms underlying brain aging and find ways of intervening with them. While for decades cognitive aging has been related to localized brain changes, growing attention is now being paid to alterations in distributed brain networks. Functional connectivity magnetic resonance imaging (fcMRI) has become a particularly useful tool to explore large-scale brain networks; yet, the temporal course of connectivity lifetime changes has not been established. Here, an extensive cross-sectional sample (21–85 years old,  Aging has long been suggested to be accompanied by cognitive decline, even in the absence of dementia or other neurological insults. This decline is more pronounced in cognitive abilities such as processing speed, working memory, and encoding new information into episodic memory, whereas other capacities, particularly semantic knowledge, and emotional processing, seem to remain relatively stable along the adult lifespan . The preAge-associated cognitive impairment has frequently been linked to structural modifications in both gray matter (GM) and white matter (WM) , though In addition to structural modifications, alterations in neurotransmitter function have been described in healthy aging, particularly reductions in the binding potential of monoaminergic transmitters . Age-relImportantly, several other neurobiological measures potentially related to cognitive aging are greatly modified by middle-age. For instance, deficits in adult dentate and subventricular zone neurogenesis, considered to play a role in aging, were reported as already established in middle-aged animals . The levThe timing of age-related structural modifications was reported to vary across brain regions with earlier deterioration in high-order association areas relative to primary sensory and motor cortices . AssociaThe present study aims to fill this gap and extend our view of intra- and inter-network functional reorganization during aging. The authors believe that a comprehensive outlook of the lifetime dynamics of brain functional connectomics is crucial for deciphering the mechanisms underlying brain aging, determining intervention targets and their optimal timing. Herein, an extensive fcMRI cohort of 887 healthy individuals between the ages of 21 and 85 was used to characterize functional connectivity changes across the adult lifespan in seven robust well-established ICNs: the default mode (DMN), salience (SN), dorsal attention (DAN), fronto-parietal control (FPCN), auditory (AN), visual (VN), and motor (MN) networks . Consist1 ; middle-aged ; and old . Seventeen research sites were included  were selected for further statistical analysis as detailed below.Intrinsic functional connectivity MRI data recorded at rest of 887 healthy individuals  were obtained from the online dataset of the International Neuroimaging Data-sharing Initiative (INDI), ‘1000 Functional Connectomes’ Project1 . The entTable 1. Functional images were preprocessed using FSL software  and SPM software  following conventional methods as previously described (Acquisition data are shown in escribed . PreprocTable 3). Correlation maps were produced by extracting the time course from each of the above seeds and computing the Pearson correlation coefficient (r) between this time course and the time course of each voxel across the whole brain. SPM8 software was used to compute statistical maps of each network across participants. Maps of a young age group  were used to identify peak coordinates of additional regions of interest (ROIs) representing each network. All regions were defined as 6 mm radius spheres around the peak coordinate (Table 3). For statistical tests, the Fischer’s r-to-z transformation (z(r) = 0.5 ln[(1 + r)/(1 -r)]) was applied. Using MATLAB version R2012b , node pair correlation matrices were computed for the three age groups: young (21–40 years), middle-aged (41–60 years), and old (61 years and above). Additionally, between age group difference matrices were calculated for the young vs. the middle-aged group and the middle-aged vs. the old group. These matrices exhibit the age group difference in connectivity strength [z(r) value] for each node pair. Correction for multiple comparisons was carried out using the false discovery rate (FDR) procedure (t-test implemented in SPM8. Three significant ROIs were chosen for each of the four contrasts to present z(r) values of ROI-to-network seed (LPCC or LMC) across participants within each age group. Regions were defined as 4 mm radius spheres around the peak coordinate. Box-and-whisker plots were drawn using MATLAB.Seed-based analysis was performed as previously described  to studyrocedure  implemenAs head motion is widely considered a significant confound in fcMRI  and in v2. Freesurfer uses intensity and continuity information from the entire three dimensional magnetic resonance volume in segmentation and deformation procedures to produce representations of cortical thickness  values of node pair correlations within each network were calculated for each participant. These connectivity scores were then analyzed as the dependent variable by a mixed model analysis of covariance (ANCOVA). This modeling method allowed us to examine the effect of a number of factors, and their interactions, while accounting for (1) the effects of covariates on connectivity; (2) systematic differences between test centers; and (3) the influence of individual subjects who each provided data on all networks. Specifically, network, age group, gender and all their factorial interactions served as fixed independent factors; FD, DVARS and cortical thickness served as covariates; test center, assumed to be a random sample from the population of all possible test centers, served as a random effect; and participant, presumed to be part of a random sample of all possible subjects, served as a random factor, necessarily nested in age group, gender and center (utilization of both fixed and random factors makes the model “mixed”). The factor ‘age group’ had three levels: young (21–40 years), middle-aged (41–60 years), and old (61 years and above), to fit our hypotheses. Gender was included as a factor following prior literature suggesting a gender effect on functional connectivity in general . This implies that variances associated with each center and each subject were different, and that there was no covariance between either centers or subjects.Inspection of ANCOVA diagnostic plots, particularly residual information, did not indicate violation of required normality assumptions, so no transformations were applied to the data. Although not a focus of our research, a full treatment of models under mixed modeling generally examines the patterns of relationships (covariance structures) between elements of random effects; when there is no p ≤ 0.05.The above described model does not provide tests of specific hypotheses of interest regarding the age-group-related connectivity changes. Yet, it does provide the basic information for constructing specific contrasts which are of direct relevance to our hypotheses. Therefore, specific, pre-planned, multiplicity-corrected contrasts were constructed to examine: (1) whether a significant difference emerged within each of the seven networks in the transition from young to middle-aged vs. middle-aged to old; (2) whether these changes in each high-order cognitive network (from young to middle-aged vs. middle-aged to old) were significantly different from equivalent changes in each primary sensory and motor network. The contrasts were multiplicity-corrected (for multiple tests) using the simulation method offered by SAS PROC MIXED. Statistical significance after multiplicity correction was at the level of Figure 1A). In contrast, the MN typically showed increased connectivity in the transition from young to middle-aged and decreased connectivity in the transition from middle-aged to old. Increased connectivity in the transition to middle-age was particularly noted in the connections between the supplementary motor area and the primary motor cortex bilaterally. Within the AN and VN both comparisons yielded significant decrements.To study connectivity dynamics across the adult lifespan, connectivity matrices were computed for each age group  for the following networks: DMN, SN, DAN, FPCN, AN, VN, and MN. Difference matrices were then calculated for each network between the young and middle-aged (Y-M) and the middle-aged and old (M-O) groups. These comparisons yielded similar results for all high-order cognitive networks : Y-M matrices mostly showed significant connectivity reductions, while M-O matrices revealed non-significant decreases, or a tendency for increased connectivity in some pairs, particularly within the DMN and DAN (Table 4). Network and age group showed a highly significant main effect (p < 0.0001) and a gender main effect was significant at p = 0.05. Network × age group and network × gender interactions both reached significance , but network × age group × gender interaction did not (p = 0.38). Least-squares means (LSM) and standard errors (SE) of the network × age group interaction are plotted in Figure 1B. These analyses confirmed the findings of the correlation matrices, showing an early decline in connectivity within high-order cognitive networks, predominantly in the DMN and SN. Pre-planned, multiplicity-corrected contrasts of the transition from young to middle-aged against middle-aged to old within each network revealed significant differences for the DMN and MN (Table 5). For the DMN, a significant decrement from young to middle-aged was followed by a minor increment from middle-aged to old. The MN showed a completely different pattern of lifelong connectivity changes: an increase from young to middle-age, followed by an equivalent decrease at old age. Contrasts of the transition between the young to middle-aged and middle-aged to old were compared between high-order cognitive networks and primary sensory and motor networks. Pre-planned, multiplicity-corrected comparisons showed a significant difference between the MN and all other high-order cognitive networks (Table 6).To account for potential sources of bias such as brain atrophy, head motion and varying acquisition parameters, a mixed-model ANCOVA was conducted on a subset of 318 participants of the entire cohort, after equalizing the number of participants in each age group , the right lateral temporal cortex (LTC), and the left frontal pole . Increased correlation with the LPCC was also observed, mainly within frontal regions bilaterally . These regions were found to be associated with the DMN anticorrelated network ; therefore, connectivity increments in this case represent reductions in the magnitude of anticorrelation with the LPCC. Within the MN, the comparison between young and middle-aged participants showed decreased connectivity in the latter group in an anterior region of the cerebellum, and increased anticorrelations with regions in the thalamus, basal ganglia and posterior cerebellum . Increased connectivity in middle-aged relative to young individuals was noticed mainly in cortical regions of the MN or border regions adjacent to the MN .To anatomically characterize the unique and opposed effects of age on connectivity within the DMN and MN, a two-sample Figure 3).Connectivity matrices calculated for the three age groups showed connectivity dynamics not only within networks as described above, but also between networks. Most of the inter-network connectivity changes occurred in the transition between the young and middle-aged groups and involved high-order cognitive networks. Early significant reduced connectivity was found between the DMN and MN, SN and AN, and DAN and VN. Early significant increased connectivity was found between the DAN and MN, DAN and AN, SN and VN, AN and MN, parts of the DMN and FPCN, and parts of the AN and VN. Another early change was a decline in anticorrelations: DMN-SN, DMN-DAN, and DAN-FPCN. Late changes were much less pronounced. Significant alterations were noticed in only 10 out of 752 node pairs, compared with 203 pairs in the transition from young to middle-age. Late changes included reduced connectivity between the DMN and FPCN and AN and MN, along with increased connectivity between the SN and FPCN, SN and VN, and AN and VN  decline in high-order cognitive networks is already evident by middle-age, as expressed by fcMRI measures. The time course of FC alterations within a motor network is characterized by an early increment followed by a decrement. Finally, most inter-network connectivity changes occur in the transition from young to middle adulthood, highlighting that several potentially meaningful FC changes onset early in the adult lifespan.Growing evidence now suggests that age-related cognitive decline is associated with FC alterations within large-scale brain networks . The DMNEven more remarkable is the fact that within high-order cognitive networks young to middle-age FC decline is more pronounced than the changes observed between middle-aged and older adults. While this finding conforms with earlier structural connectivity findings  as well In addition to temporal aspects of connectivity decline, spatial features should be considered. Our study supports the view that different brain systems are differentially affected by aging. Here, the MN showed a significantly different FC time course compared with high-order cognitive networks (early FC increment followed by a late decrement). Age-related changes in FC within the MN have been studied only rarely and results are conflicting with both decreases  and incrThe observed difference between the motor and high-order cognitive networks is in line with the “last in, first out” hypothesis. Moreover, differential vulnerability to aging is also noted within the same network. Interestingly, the vmPFC, LTC and frontal pole, here showing maximal connectivity reduction among DMN regions, were previously reported as being regions of advanced age-related atrophy and greater expansion during evolution . As for Age-related alterations in interrelations between networks represent another central finding of the current study. Most prominent alterations were increased correlations  and decreased anticorrelations  between networks in the transition from young to middle adulthood. Our observations strongly support accumulating evidence for enhanced communication between functional networks with aging . Increas2 reactivity . The institutional review board of Rambam healthcare campus approved the receipt and analysis of these data.The imaging data of the current study were obtained from 17 research sites through the online dataset of the International Neuroimaging Data-sharing Initiative (INDI). Each center’s ethics committee approved submission of de-identified data as detailed in the NITRC: ‘1000 Functional Connectomes’ Project site (TS-T, NB, AE, JA-P, and IK contributed to the conception and design of this work. TS-T, NB, and IK collected, analyzed, and interpreted the data. RP contributed to data analysis. ES designed and performed the statistical analyses. TS-T, NB, ES, JA-P, and IK contributed to in-depth discussions about methods and results. TS-T and IK wrote the paper. All authors revised and approved 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 reviewer NE and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


P = 0.06), progression-free survival , and overall survival  in patients with LA-HNSCC. Moreover, the addition of anti-EGFR agents increased the risk of skin toxicities  and mucositis . In conclusion, this meta-analysis indicates that the addition of an anti-EGFR agent to RT or CRT do not improve clinical outcomes compared with CRT in patients with LA-HNSCC. Except for patients with coexisting medical conditions or decreased performance status, concurrent CRT should remain the standard of care for patients with LA-HNSCC.There has been a debate over whether the addition of anti-epidermal growth factor receptor (EGFR) agents to the conventional treatments has beneficial effects in patients with head and neck squamous cell carcinoma (HNSCC). This meta-analysis was performed to investigate the role of anti-EGFR agents in patients with locoregionally advanced HNSCC (LA-HNSCC). A systematic search of the electronic databases was carried out. From eight randomized controlled trials, 2,263 patients were included in the meta-analysis. Compared with chemoradiotherapy (CRT), the addition of an EGFR inhibitor to radiotherapy (RT) or CRT did not improve locoregional control (hazard ratio (HR) = 1.19 [95% confidence interval (CI): 0.99–1.42],  Head and neck cancer (HNC) is the sixth most common cancer worldwide, with about 650,000 patients newly diagnosed annually , 2. DespIn the late 1990s, surgery followed by postoperative radiotherapy (RT) or RT alone was the standard therapeutic modality for locoregionally advanced HNSCC (LA-HNSCC). Since chemotherapeutic agents were identified to have additional effects when combined with RT, chemoradiotherapy (CRT) has become the standard treatment over the last decade for patients with LA-HNSCC who were not candidates for surgery. Clinical trials have demonstrated that concurrent CRT can improve overall survival (OS) compared with RT alone .The epidermal growth factor receptor (EGFR) is expressed in about 80% of patients with HNSCC . EGFR ovA recent phase III trial showed no synergistic effect when cetuximab was added to platinum-based CRT in patients with LA-HNSCC . HoweverFigure The characteristics of the eight included studies are summarized in Table Four studies provided the results of subgroup analysis according to p16 status , 13, 14.P = 0.06) compared with CRT    Figure  in patieWe also analyzed LRC, PFS, and OS in the subgroups according to the treatment modalities (CRT vs. RT + an anti-EGFR agent or CRT vs. CRT + an anti-EGFR agent). Regardless of the treatment type, EGFR inhibitors added to RT or CRT led to no significant improvement of clinical outcomes Figures –4.P = 0.18) and OS  in patients with p16-positive LA-HNSCC  and OS  outcomes. The fixed-effects model was selected because there was no significant heterogeneity across the studies in each analysis.From 4 studies , 13, 14,P < 0.00001)   Figure . Skin to) Figure .Visual inspection of the funnel plots for LRC, PFS, and OS showed symmetry, indicating there were no substantial publication biases Figure .In this meta-analysis, we evaluated the efficacy and safety of adding an anti-EGFR agent in patients with LA-HNSCC treated with RT or CRT. There was a couple of meta-analysis evaluating the role of cetuximab in LA-HNSCC , 18. Howet al. [The treatment of choice for inoperable LA-HNSCC is concurrent CRT. Considering the results from the phase III randomized trial by Bonner et al. , 19, howet al. , variousIn studies comparing an EGFR inhibitor plus RT with CRT, the major interest was whether an anti-EGFR agent could replace cisplatin, the standard partner of RT. In the subgroup analysis of the three studies –12, howeSubgroup analysis of the five studies that compared the combination of an EGFR inhibitor and CRT with CRT alone also confirmed that anti-EGFR agents had no benefits in patients with LA-HNSCC. The lack of additional benefit may be due to anti-EGFR agents and cisplatin having a similar mechanism of radio-sensitization  , 21. Theet al. reported that elevated basal antibody-dependent cell-mediated cytotoxicity (ADCC) and high EGFR expression predicted favorable outcome in patients with LA-HNSCC treated with cetuximab and RT [This meta-analysis indicates that anti-EGFR agents should not replace cisplatin or be added to CRT in patients with LA-HNSCC. However, there might be some patients who benefit from EGFR inhibitors in the treatment of LA-HNSCC –24. Receb and RT . To dateb and RT , 6, 25, b and RT . Prospecb and RT , 24. In Our study has several inherent limitations. First, this meta-analysis included a small number of studies. Second, there was a significant heterogeneity across the studies especially in the subgroup analyses. Third, patients had tumors located at various primary sites of the head and neck. Fourth, the studies adopted different treatment modalities and used various anti-EGFR agents. Fifth, different RT techniques among studies may have influenced treatment outcomes. Finally, this study included only papers published in English, which might have biased the results.In conclusion, this meta-analysis demonstrates that the addition of an anti-EGFR agent to conventional RT or CRT do not improve clinical outcomes compared with CRT in patients with LA-HNSCC. These results indicate that anti-EGFR agents should not replace cisplatin or be added to CRT routinely in patients with LA-HNSCC. Except for patients with coexisting medical conditions or decreased performance status, concurrent CRT should remain the standard of care for patients with LA-HNSCC.PubMed, MEDLINE, EMBASE, and the Cochrane Library databases (up to June 2017) were searched for articles with the following terms in their titles, abstracts, or keyword lists: “epidermal growth factor receptor inhibitor or EGFR inhibitor,” and “cetuximab or panitumumab or nimotuzumab or erlotinib or zalutumumab,” and “head and neck cancer or head and neck neoplasm.” All eligible studies were retrieved and their bibliographies were checked for other relevant publications.Eligible studies should meet the following inclusion criteria: prospective randomized controlled phase II or III trials conducted in patients with LA-HNSCC; randomization of patients to either CRT or an EGFR inhibitor plus RT/CRT; reporting HRs and 95% CIs for LRC, PFS, and/or OS; providing data for incidence of serious adverse events; articles written in English. Studies were also deemed eligible if HRs and their 95% CIs could be calculated from the available data.We excluded studies comparing an anti-EGFR agent plus RT with RT alone because RT alone is not an optimal treatment for LA-HNSCC.Data were carefully extracted from all eligible studies by two authors (BJK and JHJ). If these two authors did not agree, the principle investigator (JHK) was consulted to resolve the discrepancies.The following data were collected from each study: the first author's name; year of publication; trial phase; number of patients; treatment modalities; p16 status; LRC, PFS, and OS, including their HRs and 95% CIs; incidence of serious AEs of interest, including their Ors and 95% CIs.The methodological quality of the included studies was scored using the Jadad 5-item scale, taking into account randomization, double blinding, and withdrawals . The finThe statistical values used in the meta-analysis were obtained directly or indirectly from the original articles. If the HR and 95% CI were not provided, they were calculated as described , 28. TheI2 inconsistency test and the chi-square-based Cochran's Q statistic test, in which P < 0.1 and I2 > 50% were regarded as indicators of significant heterogeneity. In the absence of substantial heterogeneity, the fixed-effects model (Mantel-Haenszel method) was used to calculate the pooled HR and OR. When substantial heterogeneity was observed, the random-effects model (DerSimonian-Laird method) was utilized. The RevMan version 5.2 was used to combine the data. The plots show a summary estimate of the results from all combined studies. The size of each square represents the estimate from each study and reflects its statistical ‘weight.’ Results are presented as forest plots, with diamonds representing estimates of the pooled effect and the width of each diamond representing its precision. The line of no effect is number one for binary outcomes, which depicts statistical significance if not crossed by the diamond [P-values were two-sided and P < 0.05 was considered statistically significant.Heterogeneity across studies was estimated using the  diamond . Publica diamond . All rep