BrainSpan human transcriptome database, across 16 anatomical brain regions spanning prenatal life through adulthood. We discovered modules of ASD candidate genes with biologically relevant temporal co-expression dynamics, which were enriched for functional ontologies related to synaptogenesis, apoptosis, and GABA-ergic neurons. Furthermore, we also constructed co-expression networks from the entire transcriptome and found that ASD candidate genes were enriched in modules related to mitochondrial function, protein translation, and ubiquitination. Hub genes central to these ASD-enriched modules were further identified, and their functions supported these ontological findings. Overall, our multi-dimensional co-expression analysis of ASD candidate genes in the normal developing human brain suggests the heterogeneous set of ASD candidates share transcriptional networks related to synapse formation and elimination, protein turnover, and mitochondrial function.Autism spectrum disorder (ASD) is a neurodevelopmental syndrome known to have a significant but complex genetic etiology. Hundreds of diverse genes have been implicated in ASD; yet understanding how many genes, each with disparate function, can all be linked to a single clinical phenotype remains unclear. We hypothesized that understanding functional relationships between autism candidate genes during normal human brain development may provide convergent mechanistic insight into the genetic heterogeneity of ASD. We analyzed the co-expression relationships of 455 genes previously implicated in autism using the The online version of this article (doi:10.1007/s12031-015-0641-3) contains supplementary material, which is available to authorized users. Autism spectrum disorder (ASD) is a neurodevelopmental syndrome characterized clinically by impairments in verbal and non-verbal communication, deficits in social interaction, and repetitive and/or restrictive patterns of behavior  to adulthood (40 years of age). This publicly available atlas presents a unique opportunity to understand the spatial and temporal specificity of ASD candidate genes.Transcriptome-based studies of the developing human brain have previously been limited in the sample size, number of brain structures analyzed, and developmental time points assessed, hampering the ability to evaluate the genetic contributors to neurodevelopmental disease comprehensively . They demonstrated that ASD risk genes were enriched in modules related to transcription and synaptic development, and furthermore that enriched in superficial cortical layers and glutamatergic projection neuron . Then, we created a transcriptome-wide co-expression network from all genes expressed in the brain, to discover significant “molecular interaction modules” and demonstrated that ASD candidate genes are enriched only in modules related to the processes of synaptogenesis, mitochondrial function, protein translation, and ubiquitination. Lastly, we identified hub genes within the ASD-enriched molecular interaction modules, whose functions supported our ontological results, and which may serve as additional ASD candidate genes. Our analysis of this multi-dimensional expression data suggests pathways previously independently implicated in autism are related to each other through shared neurodevelopmental transcriptional networks.Here, we present an analysis of the spatial-temporal co-expression of ASD candidate genes across the normal developing human brain using the ASD list. First, the correlation between each pair of ASD genes was calculated separately within each developmental stage based on the Spearman’s rank correlation between the two genes across all brain regions. For each gene pair, this resulted in a correlation value for each of the seven developmental stages, representing the brain-wide transcriptional similarity between the genes at each developmental stage . Finally, the correlation pattern for each module was summarized by averaging all the gene pair correlation patterns included in the respective module. It is worth noting that the patterns within the modules represent changes in co-expression across development .greater than 0.8. The surviving gene pairs clustered into three distinct modules. Two of these modules, the “green” module and the “blue” module, displayed distinct correlation patterns relative to pre- versus postnatal development. The green module  are summarized as shown in Fig. neuron projection, synapse, synaptic transmission, and behavior. The three modules were also enriched for disease terms including mental retardation and epilepsy. The green and blue modules were significantly enriched for neuron differentiation, cell morphogenesis, and learning/memory. The green module was specifically enriched in functional terms related to regulation of apoptosis and regulation of cell death, while the blue module was specifically enriched in terms related to ion channel, neurotransmitter receptor activity, and GABA receptor activity. Table To characterize these modules further, we used the gene ontology (GO) enrichment analysis tool DAVID 6.7 .None of the GO terms that were significantly enriched in the three ASD modules showed any significant enrichment in modules from ten randomly created sets , based on their expression profile across all samples . Genes were hierarchically clustered based on Spearman’s rank correlation and complete linkage between pairs of genes. The resulting network consisted of 32 modules of varying size (from 36 to 1386 genes), as shown in Fig. p values <0.001), as shown in Fig. The resulting transcriptome-wide co-expression modules were then assessed for enrichment of genes belonging to the ASD list using the hypergeometric probability test. Four modules—magenta, brown, orange, and purple—were significantly enriched for ASD candidate genes , above, which were also related to synaptogenesis. Neurological disease terms were also significant in the ASD-enriched modules: epilepsy (brown module), Parkinson’s (magenta and orange modules), Alzheimer’s (magenta and orange modules) and Huntington’s (magenta and orange modules).Then, these ASD-enriched modules were tested for enrichment of gene ontology terms, as shown in Fig. magenta, brown, and purple modules), as shown in Fig. Each module was also tested for enrichment of specific neural cell populations , as described earlier. Three out of the four ASD-enriched modules were enriched for neurons . This result is in line with the gene ontology enrichment for these modules  and a few are known to function as membrane ion channels . In addition, a number of hub genes function in apoptosis or myeloid/microglia cell processes . These hub gene functions largely recapitulate the ontologies of their respective networks, supporting our enrichment results and highlighting potential critical regulatory molecules of these networks.The ten most highly connected nodes (genes) within each graph were extracted and their putative functions determined by manual curation of the literature. Among these most highly connected hub genes, a number were of note. The most striking observation was that most of the highly connected hub genes in the magenta and brown modules are known to function in the processes of In order to gain insight into the molecular pathogenesis of ASD, we present a biologically driven computational approach to analyze a heterogeneous set of genes previously independently implicated in ASD, to understand if they may relate to each other through shared functional genomics mechanisms. The main goal of this work is to understand if ASD candidate genes relate to common cellular/molecular pathways when considered in the context of transcription during normal human brain development. Identifying such pathways has profound implications for understanding the pathophysiology of ASD, especially since the majority of ASD patients do not have an identifiable genetic mutation , while others are not as strongly associated, we have weighted all genes equally to avoid bias toward more severely affected patient cases. Future work could attempt to weigh genes differently within the co-expression networks to study different genetic subtypes of autism.We discovered subsets of ASD candidate gene modules that displayed biologically relevant co-expression dynamics, which were enriched for the processes of synaptogenesis, apoptosis, and GABA-ergic signaling. In addition, we assessed for functional genomic relationships between ASD candidate genes and the entire developing human brain transcriptome. This analysis revealed that ASD candidate genes are enriched within transcriptome-wide modules related to synaptogenesis, mitochondrial function, alternative splicing, protein translation, and ubiquitination. By identifying gene modules that have similar expression patterns in the brain (regardless of time period), we were able to infer that they are likely functioning in similar pathways. This allowed us to infer which cellular and molecular mechanisms are likely to be disrupted in autism. We also demonstrated the cell-type-specific enrichment of these modules being mostly neurons, further supporting the biological relevance of our computational approach, as the broad ASD phenotype is generally consider to ultimately result from neuronal/synaptic abnormalities Zoghbi . Althougp = 10−4), indicating that—based on their significantly strong co-expression across development—those ASD-associated genes are likely to be functionally related. We discovered three subsets of ASD-associated genes with distinct co-expression profiles around birth, even though the co-expression network for each developmental stage was calculated separately to avoid any bias toward pre/postnatal expression changes. All three of these modules were significantly enriched for the processes of synaptogenesis and behavior, in addition to the disease annotations of mental retardation and epilepsy. Two of the modules (the green and blue modules) were also significantly associated with cell morphogenesis, neuron differentiation, and learning. Moreover, the green module, which had highly correlated spatial expression at prenatal developmental stages with a dramatic loss of correlation at birth, was uniquely enriched for the process of apoptosis. Conversely, the blue module displayed an opposite co-expression trajectory—poor correlation in expression prior to birth, but strong co-expression beginning in infancy and increasing through adulthood—and was uniquely related to GABA-ergic signaling and ion channels. The distinct, biologically relevant expression patterns of these two modules around birth, a developmental period with the greatest shifts in gene expression , whereas autism symptoms are generally apparent by the age of two. Our results suggest that a heterogenous set of genes which were independently associated to ASD converge into few functional pathways late in normal development. However, our findings do not preclude the possibility that the pathways implicated by these modules are involved in ASD pathogenesis, as our analysis was on co-expression patterns, not absolute gene expression levels. It is possible that the genes in these modules are still expressed in early neurodevelopment but that they are most strongly co-expressed with other genes in the same module later in life. Consequently, disruption of the integrity of these genes  early in development is likely to disrupt the functions of those modules later in life.The functional ontologies of these networks are all pathways previously implicated ASD. Disrupted synaptogenesis has been one of the most replicated findings in ASD research  data generated from 579 tissue samples. These samples were collected from 41 developing and adult postmortem brains of neurologically unremarkable donors spanning early pre-natal development (8 postconception weeks (PCW)) to late adulthood (40 years of age).We downloaded the BrainSpan Atlas have missing data from certain brain regions. We excluded donors that had more than six regions missing. For donors with six or less missing regions, we imputed the data for the missing brain regions using a nearest neighbor approach. A full mathematical description of this is provided in the Some donor brains in the BrainSpan classification system   were discarded. The final ASD list consisted of 455 ASD susceptibility genes (Table A comprehensive yet high confidence list of common ASD susceptibility genes (herein named “ASD list”) was created by combining (taking the union) lists from three main ASD genes databases: AutDB . We used hierarchical clustering to cluster gene pairs using the Euclidean distance between the profiles and a complete linkage to merge clusters. Based on the heatmap of gene pair correlations across development, we cut the dendrogram to produce three clusters. The correlation pattern for each module was summarized by averaging all the gene pair correlation patterns included in the respective module.We constructed a transcriptome-wide co-expression from all genes expressed in the brain , based on the similarity of their expression profile across all samples (480 samples). We used hierarchical clustering to cluster gene pairs using Spearman’s rank correlation between the profiles and a complete linkage to merge clusters. We cut the dendrogam to produce 32 modules of varying size (from 36 to 1386 genes).p values were corrected for multiple testing using false discovery rate (FDR). All results reported are the −log10 of FDR-corrected p values, and only p values <0.001 were considered significant.Enrichment of transcriptome-wide molecular interaction modules for ASD candidate genes and cell-type-specific genes was assessed using the hypergeometric probability density function (hygepdf) in MATLAB R2011a . The resulting p value <0.01 are presented as significant.Gene list were assessed for shared biological pathways by testing for enrichment of gene ontology terms (GO) using DAVID Bioinformatics Resources 6.7 Fig. S2p = 10−4). Blue bars correspond to the 10,000 random gene sets analyzed. (PDF 92 kb)Distribution plot of the number of strongly correlated gene pairs per gene set. The distribution of the number of gene pairs remaining after applying the threshold  shows that that the number of strongly correlated gene pairs from the ASD list (dashed red line) is highly significant Average expression patterns of the 32 molecular interaction networks. Each graph shows the average expression pattern of all the genes within one of the 32 modules resulting from the transcriptome-wide co-expression network. Supplementary InformationFurther information regarding the details of our methods including: handling missing data, Spearman’s rank correlation, and random control networks. (PDF 159 kb)Table S1green, blue, and red modules. (XLS 60 kb)Modules of ASD candidate genes. A table listing member genes of the Table S2p value <0.01) GO terms in each of the ASD co-expression modules. (XLS 202 kb)GO enrichment analysis of ASD candidate genes. A table listing all the significantly enriched  are highlighted in green. (XLSX 402 kb)GO enrichment analysis of ten random gene sets. A table listing the GO enrichment analysis of the co-expression modules created by clustering ten random sets of 455 genes. Significantly enriched terms  as extracted from Table S6p value <0.01) GO terms in each of the transcriptome-wide molecular interaction modules. (XLS 1153 kb)GO enrichment analysis of transcriptome-wide molecular interaction modules. A table listing all the significantly enriched (Benjamini-adjusted Table S7ASD list. A table listing the 455 genes forming the list of ASD candidate genes in our study. (XLS 64 kb)


Human pluripotent stem cell (hPSC)-derived cardiomyocytes have attracted attention as an unlimited source of cells for cardiac therapies. One of the factors to surmount to achieve this is the production of hPSC-derived cardiomyocytes at a commercial or clinical scale with economically and technically feasible platforms. Given the limited proliferation capacity of differentiated cardiomyocytes and the difficulties in isolating and culturing committed cardiac progenitors, the strategy for cardiomyocyte production would be biphasic, involving hPSC expansion to generate adequate cell numbers followed by differentiation to cardiomyocytes for specific applications. This review summarizes and discusses up-to-date two-dimensional cell culture, cell-aggregate and microcarrier-based platforms for hPSC expansion. Microcarrier-based platforms are shown to be the most suitable for up-scaled production of hPSCs. Subsequently, different platforms for directing hPSC differentiation to cardiomyocytes are discussed. Monolayer differentiation can be straightforward and highly efficient and embryoid body-based approaches are also yielding reasonable cardiomyocyte efficiencies, whereas microcarrier-based approaches are in their infancy but can also generate high cardiomyocyte yields. The optimal target is to establish an integrated scalable process that combines hPSC expansion and cardiomyocyte differentiation into a one unit operation. This review discuss key issues such as platform selection, bioprocess parameters, medium development, downstream processing and parameters that meet current good manufacturing practice standards. Cardiovascular disease is the leading cause of death globally, accounting for 244.8 per 100,000 deaths in 2008 , indicatin vitro and have the ability to differentiate into all three germ layers, thus giving rise to all cell types of the human body [A potential cure for heart failure can be achieved through cardiovascular cell therapy, which aims to repopulate damaged myocardium with new contractile cells and restore the heart. Pluripotent stem cells have nearly unlimited self-renewal capability man body . Since tman body  and humaman body , stem ceman body -12. It wman body . Neverthman body . In caseman body , emphasiin vitro are also needed for cardiac safety pharmacology testing. Unforeseen cardiotoxicity is one of the most common causes of late-stage clinical attrition [in vitro cardiac models are limited as no current immortalized human cell lines accurately resemble functional cardiomyocytes of the heart for assessing preclinical cardiotoxic responses of drugs. Current cardiac models are typically animal models and in vitro assays, which lack cross-species translation due to differences in biological pathways and pharmacokinetic properties. Studies have already shown that hiPSC-derived cardiomyocytes will react to cardioactive drugs with expected responses, supporting the concept of large scale human cell-based predictive toxicology screens [Besides their significance in regenerative medicine, cardiomyocytes generated ttrition . Many drttrition . An exam screens . The use8 purified cells [A third application of hPSC-derived cardiomyocytes is drug discovery. Over the years, many animal models, particularly mouse, have been generated and widely used. Although these models have shed light on our understanding of the onset and progression of cardiac diseases, they do not always recapitulate the phenotype seen in patients. Several studies have demonstrated the usage of hiPSC-derived cardiomyocytes as models for human cardiac disease -21. Leveed cells , challenin vitro cardiomyocyte generation must be developed.The immense potential of hPSC-derived cardiomyocytes in regenerative medicine, cardiotoxicity pharmacology testing and drug discovery is indeed tantalizing. To effectively serve these applications, however, a reproducible, efficient and cost-effective platform for In this review, we examine the progress in development of large scale cardiomyocyte differentiation platforms from hPSCs. We describe the performance of existing reported systems and summarize the main obstacles for further research and optimization.The limited proliferating capacity of differentiated cardiomyocytes dictates the design of the production process. It is possible to isolate and cultivate cardiac progenitors as shown in several studies using fluorescence-activated cell sorting of the KDR+/PDGFRA- population  or genetPrior to establishment of a working cell bank and expansion method, it is important to select robust hPSC lines for growth ability and cardiomyocyte differentiation. Especially during the screening of hiPSC clones, it would be ideal to include parameters used in the later expansion as part of the selection criteria. For example, if microcarriers are chosen for hiPSC expansion, then suitable hiPSC clones should also be tested on microcarriers for cell growth and expression of pluripotent markers. In our group, we have observed variability in expansion capability of hPSCs as well as cardiomyocyte differentiation efficiency. Moreover, the mode of cell expansion  coating) can affect cardiomyocyte differentiation efficiency. Hence, it is important to verify these effects before large scale cardiomyocyte generation.As shown in Figure ®hESC SFM   coated plates in conditioned medium or commercial serum-free medium such as mTeSR™1  and StemPro6 cells/ml with 12.5- to 17.7-fold expansion using various hPSC lines [Cell aggregate culture has been developed as a scalable expansion platform for hPSCs, which utilizes the characteristic of hPSCs to form aggregates in suspension. hPSCs in single cell suspension usually undergo apoptosis or anoikis. In order to prevent these detrimental effects and establish a viable hPSC suspension culture, hPSCs are partially dissociated in order to form cell aggregates and/or ROCK-Myosin signaling pathway inhibitors (Y27632 or Blebbistatin) are added to improve survival of dissociated hPSCs. Despite significant cell loss during the initial cell aggregate formation (28 to 76%), several groups have obtained cell concentrations of 1 to 2 × 10SC lines ,58-60. SSC lines .6 cells/ml with 20-fold expansion in spinner flasks in serum-free medium. The microcarrier-expanded cells exhibited the capacity to differentiate in situ to neural progenitors [The development of the microcarrier platform for hPSC expansion has attracted interest for generating cardiomyocytes in a scalable manner. hPSCs cultured on microcarriers exhibited higher cell density and expansion than two-dimensional tissue or aggregate cultures, while maintaining pluripotency and karyotypic stability . A recengenitors , endodergenitors , and cargenitors ,65. Howegenitors . Comparegenitors ,65,67,68genitors . ROCK ingenitors .6 cells/ml in 11 days with a seeding density of 1.5 × 105 cells/ml (15-fold expansion). A comprehensive review of the bioprocess parameters for hPSC microcarrier culture was recently published [Scaling up of hPSC microcarrier cultures has been demonstrated by several groups using spinner flasks. Serra and colleagues  carried ublished . It is iublished . Leung aublished  reportedublished  and endoublished . Howeverublished ,74 exhibin vivo cardiogenesis, which depends on a series of complex molecular signaling pathways [Cardiomyocyte differentiation is based on the recapitulation of pathways ,76. The pathways . MoreoveCurrent cardiac differentiation protocols use either small molecule inhibitors or growth factors to induce the signals for cardiac differentiation. In both cases, the signaling leads to a cascade of three sequential stages: mesodermal induction, cardiac progenitor and cardiomyocyte generation and maintenance  Figure . MesoderCardiac progenitor induction is achieved by removal of the TGF-β pathway activators and addition of the growth factors, fibroblast growth factor-2 and/or vascular endothelial growth factor, which activate the ERK signaling pathway , or by sThe final stage of cardiomyocyte production (day 8 and later) focuses on maturation of cells to cardiomyocytes and their maintenance. Differentiated cardiomyocytes can be maintained in simple serum-free medium, which can minimize outgrowth of fibroblasts and maintain cardiomyocyte purity. Prolonged cultivation of cardiomyocytes has been shown to increase the amount of mature ventricular phenotype .After having established a scalable hPSC expansion method, a suitable cardiac differentiation process should be developed. The variety of protocols used by research groups indicates the complexity of the differentiation process and hence the difficulty of applying universally efficient differentiation protocols to the different hPSC lines and culture conditions Table . To dateIn the two-dimensional tissue culture differentiation platform, hPSCs expanded in monolayer can be directly differentiated to cardiomyocytes by a simple change of hPSC growth medium to cardiac differentiation medium. The first efficient directed differentiation protocol was reported by co-culturing hESCs with mouse endoderm-like cells (END-2), which generated 85% cells exhibiting ventricular-like action potentials . DiffereIn the EB differentiation platform, cells from two-dimensional cell cultures are usually dissociated into single cells followed by re-aggregation in order to generate homogeneous EB cultures with uniform sizes that are further differentiated to cardiomyocytes Figure e. In genSignificant progress has been made to improve EB-based cardiomyocyte differentiation efficiency with the identification of growth factors and pathways associated with cardiac differentiation Table . Most ofEB differentiation protocols are more complicated than those used in two-dimensional tissue cultures due to their three-dimensional shape and the effects of the EB microenvironment . Specifiin situ to cardiomyocytes as EBs [in situ differentiation is more scalable than monolayer expansion.Furthermore, hPSCs can be expanded as cell aggregates in suspension without microcarriers as discussed previously. Two studies showed that these aggregate cultures expanded in a spinner flask can be subsequently differentiated s as EBs ,58. Matss as EBs  reportedMicrocarriers can be applied in two ways for cardiomyocyte differentiation. Firstly, they can be used to assist EB formation, stabilization and prevention of agglomeration. Lecina and colleagues  used fiv6 cells/ml (40.4-fold cell expansion) with high cardiomyocyte purity of 67% cTnT and yields of 268 cardiomyocytes per hPSC seeded  for their potential to be coupled with various differentiation platforms. These platforms can be applied using a variety of media, culture methods and conditions. In the following section we discuss critical criteria that should be considered in selection of conditions for designing a scalable cardiomyocyte production system.The selection of cardiomyocyte production platform is dictated by the quantity of cardiomyocytes required for a particular application. Three methods of hPSC expansion and seven options for cardiomyocyte differentiation are available for cardiomyocyte production Table . The ini-1) showed lower lactate production and shorter lag phase than semi-continuous culture (50% medium exchange per day) [hPSC expansion in suspension with automated online process monitoring and controls has been reported . Serra aper day) . The perMaintaining a homogeneous suspension culture is critical in process monitoring and controls, but can be challenging considering the size variations of cell aggregate and cell-microcarrier aggregate cultures. Stirring can also induce shear stress responses. Recently, we have observed that agitation-induced shear stress in spinner microcarrier cultures reduced cardiomyocyte differentiation efficiency. Applying agitation during the first 3 days of cardiomyocyte differentiation (for both microcarrier- and EB-based methods) suppressed cardiomyocyte differentiation. This inhibition could be alleviated when intermittent agitation was employed. The literature reports that shear stress affects the TGF-β pathway, which is vital in cardiomyocyte differentiation ,86.Medium development is critical in the development of hPSC expansion and differentiation processes. In most papers, hPSCs were expanded in conditioned medium on Matrigel-coated plates or mitotically inactivated feeder cells Table . Medium Studies on development and optimization of downstream processing of hPSC-derived cardiomyocytes are very limited. The process of harvesting cardiomyocytes from monolayer, EB or microcarrier cultures has been demonstrated for small scale research purposes. Monolayer cultures, EBs or cell-microcarrier aggregates are treated with dissociation enzymes followed by passing through a sieve to remove undissociated cell aggregates and microcarriers . These pAnother aspect of downstream processing is the purification of cardiomyocytes from mixed cell populations ,89. ConsLastly, the purified cardiomyocytes can be either cryopreserved or applied directly for tissue engineering. Progress has been made showing the functionality of cryopreserved and dissociated cardiomyocytes. A study by Xu and colleagues  showed tProduction of cardiomyocytes at clinical commercial scales that follows the current good manufacturing practice (cGMP) standard will be challenging as most of the current processes are carried out for research purposes without any consideration for cGMP requirements. A defined xeno-free integrated process of expansion, differentiation and downstream purification within a closed system with appropriate monitoring and control systems would be the most suitable for cGMP manufacturing. To characterize the cardiomyocytes produced, quality control assays . Beyond these issues, several others have to be considered in future applications. For example, cardiomyocytes produced according to the current methods are immature in their marker expression and electrical and mechanical functionality . This poCardiovascular regeneration edited by Ronald Li. Other articles in the series can be found online at http://stemcellres.com/series/cardiovascularThis article is part of a thematic series on BMP: Bone morphogenetic protein; cGMP: current good manufacturing practice; cTnT: cardiac troponin T; EB: Embryoid body; ECM: Extracellular matrix; hESC: human embryonic stem cell; hiPSC: human induced pluripotent stem cell; hPSC: human pluripotent stem cell; MLC2: Myosin light chain 2; RT-PCR: Reverse transcriptase-polymerase chain reaction; TGF: Transforming growth factor.The authors declare that they have no competing interests.


Pups were then raised in normoxia until adulthood. Perinatal iNO administration completely restored acetylcholine-induced relaxation, as well as endothelial nitric oxide synthase protein content, in isolated pulmonary arteries of adult mice born in hypoxia. Right ventricular hypertrophy observed in old mice born in hypoxia compared to controls was also prevented by perinatal iNO treatment. Therefore, simultaneous administration of iNO during perinatal hypoxic exposure seems able to prevent adverse effects of perinatal hypoxia on the adult pulmonary circulation. Adverse events in utero are associated with the occurrence of chronic diseases in adulthood. We previously demonstrated in mice that perinatal hypoxia resulted in altered pulmonary circulation in adulthood, with a decreased endothelium-dependent relaxation of pulmonary arteries, associated with long-term alterations in the nitric oxide (NO)/cyclic GMP pathway. The present study investigated whether inhaled NO (iNO) administered simultaneously to perinatal hypoxia could have potential beneficial effects on the adult pulmonary circulation. Indeed, iNO is the therapy of choice in humans presenting neonatal pulmonary hypertension. Long-term effects of neonatal iNO therapy on adult pulmonary circulation have not yet been investigated. Pregnant mice were placed in hypoxia (13% O Numerous epidemiological studies have associated adverse events occurring in utero, like limitation of nutrients or oxygen supply, with an increased risk to develop chronic diseases in adulthood, including coronary artery disease, systemic hypertension, stroke, and noninsulin-dependent diabetes mellitus . TherefoThe fetal pulmonary circulation is characterized by low perfusion and high vascular resistances, which need to rapidly fall at birth to allow pulmonary blood flow to increase nearly 10-fold , 15. PulAdult pulmonary hypertension is also a severe disease of various origins, affecting life expectancy and quality of life , whose t2) 5 days before delivery and left with their litter for 5 days after birth. Pups were then bred in normoxia until adulthood when studied. We first demonstrated that the relaxation induced by the endothelium-dependent agent acetylcholine (ACh) in the adult main pulmonary artery (PA) was completely mediated by endothelial nitric oxide synthase (eNOS) -1-propanesulfonate). After a 45 min incubation on ice, the homogenates were centrifuged for 10 min at 3000 ×g at 4°C. Five μL of each supernatant was diluted in Laemmli buffer and heated for 5 min at 95°C before loading on a 7.5% polyacrylamide gel. Proteins were fractioned by SDS-PAGE (35 min at 200 V) and transferred to PVDF membrane  during 2 h at 100V. Blots were blocked overnight at 4°C in Tris-buffered saline plus 0.05% Tween 20 (TBS-T) containing 5% nonfat dry milk. All washing steps were performed using TBS-T. Membranes were immunoblotted for 1 h at room temperature using specific antibodies targeted against eNOS , or β-actin (Sigma), diluted 1 : 200 (for eNOS), or 1 : 250 (for β-actin) in TBS-T containing 5% nonfat dry milk. Blots were then incubated with ECL horseradish peroxidase-linked anti-mouse antibody (for eNOS detection) or anti-rabbit antibody (for β-actin) , diluted 1 : 10,000 in TBS-T. Finally, specific proteins were detected by chemiluminescence using the SuperSignal West Pico Chemiluminescent Substrate (Pierce) and exposition to X-ray film. Expression of eNOS was quantified using the UN-SCAN-IT gel software  and normalized to β-actin content. Relative protein content in homogenates of mice born in hypoxia was reported to the content measured in controls.Specific protein content in pulmonary arteries was investigated by western blotting analysis as previously described . Briefly2CO3 [Unless otherwise specified, all drugs were purchased from Sigma . Indomethacin was prepared in equal molar Na2CO3 . The othn represents the number of animals per group. Unless otherwise mentioned, the influence of perinatal exposure to hypoxia and/or iNO on the studied parameters was analyzed using the Mann-Whitney test. Two-way ANOVA was performed to compare the dose-response curves in isolated vessel tension studies. The difference was considered statistically significant when P < 0.05.Statistical analyses were performed using InStat 3.0 or Prism 4.0 . Data are expressed as mean ± SEM, and P = 0.0677, using the Mann-Whitney test), as well as between mice born in hypoxia plus iNO and controls .In young females (5-6-month-old mice), perinatal hypoxia did not influence the studied parameters, as previously published . SimultaIn old females (12–15-month-old mice), anatomical data were similar between mice exposed to perinatal hypoxia and controls, except for the RV/(LV + S) ratio, which was significantly higher in mice born in hypoxia than in controls. Old females submitted to simultaneous exposure to perinatal hypoxia and iNO displayed similar anatomical data to controls. However, RV weight and the RV/(LV + S) ratio were significantly reduced compared to mice exposed to perinatal hypoxia alone.Pharmacological response to cumulative doses of the endothelium-dependent relaxing agent acetylcholine (ACh) was tested in isolated PA preconstricted with phenylephrine . We prevWe previously showed that the eNOS protein relative content was significantly reduced in the main PA of adult females born in hypoxia compared to controls  Figure . In contWe previously demonstrated, in a murine model of perinatal hypoxia, that a transient exposure to hypoxia during a critical period of development induced long-term adverse effects on the adult pulmonary circulation , 27, 29.In the present study, we investigated in the same experimental model whether iNO administration during the whole exposure to perinatal hypoxia could have beneficial effects on the adult pulmonary circulation. Potential protective effects of iNO were tested by measurement of several parameters which were found to be altered in adult females exposed to perinatal hypoxia .In our previous report , we demoVascular reactivity studies showed that although ACh-induced relaxation was significantly reduced in isolated PA from adult females exposed to perinatal hypoxia compared to controls, simultaneous administration of iNO during perinatal hypoxic exposure completely restored the vasorelaxant response to ACh.Similarly, perinatal exposure to hypoxia with concomitant iNO treatment allowed a total recovery in eNOS protein content in the main PA of adult females, whereas eNOS protein expression was shown to be reduced by almost 40% in adult females born in hypoxia compared to controls .Anatomical data recorded in young adult mice (5-6 months) were similar between females born in hypoxic conditions and controls. Although previous hemodynamic studies showed a higher right ventricular pressure in 5-month-old mice exposed to perinatal hypoxia compared to controls , stronglTaken together our results suggest that concomitant administration of iNO during the whole exposure to perinatal hypoxia exerts a protective effect on the pulmonary circulation. Such results argue in favor of a potential long-term beneficial effect of neonatal iNO therapy. It will be nevertheless necessary to investigate whether neonatal  treatment would confer the same protection and whether this protective effect could also be observed on additional parameters, like right ventricular pressure, sensitivity to acute hypoxia, and molecular components of the NO/cGMP pathway, such as muscarinic receptors and phosphodiesterases, which were found to be altered in adult females exposed to perinatal hypoxia .Beneficial effects of iNO were also shown in other experimental models. For example, a 10-day treatment with iNO (10 ppm) during recovery from a 10-day exposure to neonatal hypoxia restored lung structure in eNOS-deficient mice, by stimulation of alveolar and vascular growth . SimilarThe mechanisms implicated in the observed protective effects of iNO treatment during the whole exposure to perinatal hypoxia remain to be elucidated. It will be first necessary to distinguish whether protective effects are mainly due to prenatal or postnatal treatment with iNO.Prenatal treatment with iNO probably mainly acts through reduction of pulmonary vascular resistances in pregnant mice. Indeed, closed-chest hemodynamic studies showed that acute exposure to iNO in hypoxic conditions was able to reverse hypoxia-induced increase in right ventricular pressure in anaesthetized adult mice (unpublished data). Due to the short half-life of gaseous NO, maternal exposure to iNO during hypoxia may not directly influence the fetal circulation, although there was recent evidence that extrapulmonary effects of iNO could be mediated, in addition to diffusion, by conversion of iNO into S-nitrosothiols further exported into the circulation by the type L amino acid transporter . HoweverIn our murine model of perinatal hypoxia, body weight measured in 5-day-old pups was decreased by about 10% after perinatal hypoxia compared to normoxia. Body weight in pups exposed to perinatal hypoxia plus iNO was also significantly reduced compared to controls and did not significantly differ from pups with perinatal hypoxia alone. This suggests that maternal exposure to iNO did not prevent growth restriction resulting from perinatal hypoxia. Therefore neonatal exposure to iNO during perinatal hypoxia probably contributes largely to the protective effects of iNO against adverse effects of perinatal hypoxia on the adult pulmonary circulation.Beneficial effects of neonatal iNO therapy could be explained by direct effect on the pulmonary circulation of pups exposed to hypoxia, thanks to the vasorelaxant properties of iNO allowing the counteraction of pulmonary vasoconstriction induced by hypoxia. Moreover, if prenatal exposure to hypoxia induced altered eNOS protein expression, exogenous NO could also act by compensation for endogenous NO production deficiency, allowing recovery of NO-mediated relaxation in pups, thus preventing further alterations in pulmonary vascular tone regulation. Such protective effects could be due to prevention of epigenetic modifications in pulmonary vessels leading to long-term alterations in the regulation of pulmonary circulation. In this line, it would be particularly of interest to investigate whether iNO could prevent the imbalance in the NO/cGMP pathway in adult pulmonary vasculature following perinatal hypoxia, with particular attention to muscarinic receptors and phosphodiesterases. Such a result would suggest that an appropriate intervention to counteract adverse effects of a hypoxic insult at a critical period of development could prevent long-term adverse effects of hypoxia on adult pulmonary circulation.It remains therefore to test whether a neonatal iNO treatment, introduced from birth to the end of the 10-day perinatal exposure to hypoxia, will be able to exert similar protective effects against adverse effects of perinatal hypoxia on the adult pulmonary circulation in our murine experimental model.It will be also necessary to confirm such observations in humans. Indeed, iNO therapy was introduced about 15 years ago to treat neonates with PPHN. However there is no data on the long-term effects of iNO on the regulation of adult pulmonary circulation in humans. For example, it was shown that young adults having recovered from PPHN before introduction of the iNO therapy  displayed exaggerated pulmonary vasoconstrictive response following reexposure to hypoxia, namely, during high-altitude exposure [In terms of basic research, our data may contribute to a better understanding of the regulatory mechanisms of the pulmonary circulation in the adult following a perinatal hypoxic exposure with or without iNO therapy. In terms of clinical implications, our studies may help to devise novel therapeutic strategies to prevent and treat adult pulmonary vascular disorders secondary to perinatal hypoxic events. More generally, such results should stimulate researchers to further investigate cellular and molecular mechanisms implicated in fetal programming of adult diseases in order to design novel targeted early therapeutic interventions to prevent long-term adverse effects of perinatal insults.It should be noted that most results of the present study are related to the main PA. The overall reactivity differs between the large conduit PA and the more distal resistant pulmonary vessels. The total pulmonary vascular resistance depends on the combination of the reactivity of all these vessels, among which large pulmonary arteries have also been shown to play a role , 40. HowMoreover, our experimental model was complex, because it combined pre- and postnatal stimuli. In our previous studies, the timing of exposure to hypoxia was chosen to cover the period of lung vasculogenesis, during which the functional units of gas exchange develop . FurtherRegarding the timing of iNO treatment, we aimed in the present study to show whether iNO administration during the whole exposure to hypoxia could reverse adverse effects of perinatal hypoxia on adult pulmonary circulation. Further investigation of potential beneficial effects of separate prenatal or postnatal treatment would be useful to determine the therapeutic window in which iNO administration could provide the greater protective effect. Such information could help to devise novel targeted therapeutic interventions. Particular attention will be paid to the postnatal treatment, which is closer to the current clinical practice.However, despite several limitations, the present paper points to beneficial effects of perinatal iNO administration against some adverse effects of perinatal hypoxia on the adult pulmonary circulation. Further investigations will of course be necessary to better understand the involved mechanisms. However, although several studies displayed short-term protective effects of iNO administration, our data are the first, to our knowledge, to demonstrate long-lasting beneficial effects of perinatal iNO therapy on the pulmonary circulation throughout the life.In the present report, we demonstrated that although a 10-day exposure to hypoxia in the perinatal period resulted in alterations of the adult pulmonary circulation, simultaneous administration of iNO during the whole exposure to perinatal hypoxia completely restored ACh-induced relaxation, as well as eNOS protein content, in isolated PA of adult females. Right ventricular hypertrophy observed in old mice born in hypoxia compared to controls was also prevented by perinatal iNO treatment. Therefore, concomitant administration of iNO during perinatal hypoxic exposure seems able to prevent adverse effects of perinatal hypoxia on the adult pulmonary circulation.


On the other hand phylogenetic analyses indicate lateral evolution of GS-ATase family and an early duplication event responsible for AMPylation and deAMPylation activity of GS-ATase. Our analysis also reveals molecular basis of substrate specificity of DrrA proteins.AMPylation is a novel post-translational modification (PTM) involving covalent attachment of an AMP moiety to threonine/tyrosine side chains of a protein. AMPylating enzymes belonging to three different families, namely Fic/Doc, GS-ATase and DrrA have been experimentally characterized. Involvement of these novel enzymes in a myriad of biological processes makes them interesting candidates for genome-wide search. We have used SVM and HMM to develop a computational protocol for identification of AMPylation domains and their classification into various functional subfamilies catalyzing AMPylation, deAMPylation, phosphorylation and phosphocholine transfer. Our analysis has not only identified novel PTM catalyzing enzymes among unannotated proteins, but has also revealed how this novel enzyme family has evolved to generate functional diversity by subtle changes in sequence/structures of the proteins. Phylogenetic analysis of Fic/Doc has revealed three new isofunctional subfamilies, thus adding to their functional divergence. Also, frequent occurrence of Fic/Doc proteins on highly mobile and unstable genomic islands indicated their evolution  AMPylation or adenylylation is a post-translational modification (PTM) involving the covalent attachment of AMP moiety onto the side chains of threonine/tyrosine residues of proteins12325689111215Legionella pneumophila effector protein, DrrALegionella’s survival in lysosomal vacuolesWhile catalytic activity of Fic domains is a more recent finding, the enzymatic ability of GS-ATase was discovered almost four decades ago217E.coli phage P1. These proteins contain a degenerate Fic motif, HPFx(D/E)GN(G/K)R, with the His being essential for its catalytic function615Pseudomonas syringae shares structural similarity with Fic/Doc family of proteins. AvrB causes hypersensitive death of plant cells by targeting host immune resistance protein Rin410Fic domain proteins do not share any sequence or structural similarity to GS-ATase but shares sequence similarity with another protein family known as Doc (Death on curing). PFAMa priori identification of AMPylation domains and their classification into various subfamilies. Even though AMPylation is involved in diverse biological processes, only a handful of AMPylating enzymes have been experimentally characterized. Due to high sequence divergence within each family of AMPylating enzymes all members cannot be identified by a simple BLAST search or profile based tools like PSI-BLAST. They are also limited in their ability to distinguish non-AMPylating from the AMPylating members. Therefore, in this work we have attempted to use machine learning approach like SVM and profile based approach like HMM to develop and benchmark a computational protocol for identification and classification of AMPylation domains. Using this computational approach we have identified large number of AMPylation domains belonging to various subfamilies in all three kingdoms of life. Phylogenetic analysis has also identified putative isofunctional subfamilies expanding the functional diversity of Fic domains. Analysis of phylogeny and synteny of these AMPylation domains suggested evolution of AMPylating enzymes, specifically Fic domains by extensive horizontal gene transfer. This was confirmed by the presence of many Fic proteins on highly unstable Genomic Islands. In addition, we have also attempted to decipher sequence determinants of the substrate specificity of DrrA type AMPylation domains towards a variety of Rab proteins.It is necessary to understand how these enzyme families have evolved across various organisms to generate functional diversity by subtle changes in sequence and structures of the proteins. Evolutionary analysis of functional diversity across organisms requires in silico identification of AMPylation domains and their classification into various subfamilies like Fic, Doc and AvrB. Even though iterative profile based searches increases the repertoire of Fic/Doc proteins compared to pair-wise BLAST search, it is often found that such methods cannot distinguish between the subfamilies. We wanted to investigate if machine learning based SVM classifiers and sensitive profile based HMMs can be used for identification and classification of AMPylation domains.Extensive sequence diversity coupled with functional divergence of Fic/Doc family poses a major challenge for Machine learning approaches like SVM (Support Vector Machines)25Hidden Markov Models (HMM), a fast and sensitive profile based method has been widely used for classification and identification of different protein domains2728Since the HMM method was found to be superior to SVM method in our benchmarking study for identification and classification of Fic, Doc and AvrB families, for analysis of AnkX, AR, AT and DrrA family members only HMM profiles were developed. Hence, only the developed HMMs for various subfamilies were used to identify AMPylating domains from nr database . SimilarLegionella pneumophila is a multi-domain protein containing Fic domain, a CMP-binding domain and several ankyrin repeats . As can be seen this sequence stretch HPFLEGNGRATR corresponding to the residues 96-107 in 2F6S adopts a helix-turn-helix (α4-turn-α5) structure which harbors most the active site residues  was that all groups are polyphyletic i.e. species of bacteria, archaea and eukaryotes are mixed in all the branches . Hence, E. coli APEC O1 showed many GEIs, one of them containing the Fic protein  showed evidence of HGT in literature (Many pathogenic Fic proteins are known to be transferred to their hosts using type IV or type III secretion systems and are encoded by Pathogenicity Island (PAI) 6103343 protein . GC contr genome . The aliterature . This futerature  did not et al. have proposed that, these conserved indels could be the reason for functional divergence of AR and AT domainsGS-ATase is a bifunctional enzyme with mutually antagonistic enzymatic activities residing in its two domains which share significant sequence similarityTo gain insight into the underlying determinants of functional divergence in GS-ATase family phylogenetic analysis was performed. Instead of complete sequences of GS-ATase, AT and AR domains, as classified by the HMMs, were taken separately and a phylogenetic tree was built . Colors Legionella pneumophila (Homo sapiens) has an overall positive charge in AMPylated Rab proteins, whereas Rab proteins that cannot be AMPylated by DrrA have a negatively charged or neutral amino acids in this sequence stretch. In the three dimensional structure of Rab proteins, the sequence stretch 53-58 is present on the surface and is in vicinity of Tyr77 which is AMPylated. As can be seen from DrrA is the latest addition to the repertoire of AMPylating enzymes. Though it shares the structural fold of GS-ATase, the sequences do not show significant sequence similarityumophila . Its tarumophila . To undeumophila . Except umophila . In factumophila  revealedin silico analysis involving BLAST, PSI-BLAST, profile HMM and SVMs has been carried out for identifying new Fido and GS-ATase domains from among the unannotated proteins in genomes of various organisms and distinguishing between various functional subfamilies. Systematic benchmarking of different computational protocols revealed that HMMs was distinctly superior to all types of SVM classifiers for identification and classification of various AMPylation subfamilies. Apart from identification of Fic, Doc, AvrB, GS-ATase AT and AR domains, the subfamily specific HMM profiles developed in this work can successfully distinguish AnkX type phosphocholinating domains from Fic type AMPylation domains. The HMM based computational protocol for identification and classification of AMPylating enzymes has also been made available online at http://www.nii.ac.in/novptmenzy.html.AMPylation of proteins has been known since last four decades5Using the HMM based computational protocol we have identified Fido and GS-ATase superfamily of enzymatic domains from various organisms and classified them into functional subfamilies. Evolutionarily conserved sequence determinants combined with systematic phylogenetic analysis were used to illuminate the functional diversity of Fic/Doc family and understand how protein sequence and function has coevolved. Three putative sub-families that might add to the increasing functional diversity of Fic/Doc family were identified. We expect more biological functions to be unearthed as more number of Fic sequences is experimentally characterized. In a curious evolutionary twist the sequences of bacterial Fic/Doc family was not only interweaved among themselves but also with eukaryotic and archaeal Fic/Doc proteins. The lack of an archaeo-eukaryotic branch also suggested that members of Fic/Doc family might not have been present in last universal common ancestor (LUCA) but have evolved in bacteria and subsequently spread to archaea and eukaryotes via horizontal gene transfer (HGT). We hypothesize that the reason for extensive HGT observed in Fic domains is because it is encoded by highly mobile and unstable GEIs. The evolution of GS-AT and GS-AR domains was studied through an explicitly phylogenetic approach tracing back the duplication and evolution of AT and AR domains. Phylogenetic and genome neighborhood analysis suggested that barring few taxonomical classes GS-ATase have probably evolved through lateral transfer. Phylogenetic analysis of DrrA substrates helped in identification of sequence stretches which are determinants of the substrate specificity of DrrA type AMPylation domains towards a variety of Rab proteins.-3. Overlapping hits were obtained in case of BLAST searches for Fic and Doc, because of significant sequence similarity between these two sub-families. In such cases, the obtained hits were classified as Fic or Doc based on their annotation. As AvrB does not share sequence similarity with Fic/Doc family members, no overlapping hits were obtained. Similarly, overlapping hits were obtained for GS-AT and GS-AR domains. In such cases GS-AT and GS-AR domains were classified based on presence of N169 in AR domain and G697 in AT domainftp://ftp.ncbi.nih.gov/blast/documents/blastclust.html) from the NCBI-BLAST packageThe sequences of all experimentally characterized AMPylating domains belonging to Fic/Doc, AvrB, GS-AT, GS-AR families were compiled based on literature search. This set also included the domains for which crystal structures were available in PDB. They consisted of 12, 1 and 6 sequences from Fic/Doc, AvrB and GS-ATase family respectively. A dataset of protein sequences for each family was compiled using sequence based searches like pair-wise BLASTp 48multiclass which is based on structural SVMsin silico identification and correct classification of putative AMPylating domains. SVMmulticlass allows classification of multiple classes of data by optimization of the models by varying various parameters like type of kernels  and trade-off value (C). For this study kernel was fixed to polynomial whereas C value was varied from 0.001 to 1. The SVMs were trained using features like amino acid composition, dipeptide composition, tripeptide composition and tetrapeptide composition. In addition SVMs were also trained using combination of multiple features, for example amino acid and tripeptide composition together, or amino acid, dipeptide and tetra peptide composition etc. Since the features were composition of Kmers in the sequences of AMPylation domains, the different feature vectors essentially consisted of one dimensional arrays of size 20k and the ith element of the array is fraction of k-mer of type i.SVMK. Thus the size of the feature vector corresponding to K = 1 i.e. amino acid composition is 20, while size of feature vectors for di-, tri- and tetra-peptide composition are 400, 8000 and 160000 respectively. Similarly these feature vectors were combined suitably to obtain feature vectors corresponding to multiple k-mer composition. SVM models for each family of AMPylation domains were developed by using sequences belonging to the given family as positive dataset and sequences belonging to the other families as negative dataset.where i = 1, 20http://www.nii.ac.in/novptmenzy.html.HMMs are statistical models that capture the consensus information from a set of related protein sequences at various sequence position. In order to develop profile HMM models for classification of AMPylation domains HMMER3multiclass was optimized based on the results of the 5-fold cross validation tests. Sensitivity (SN), specificity (SP), accuracy (ACC), Mathew’s Correlation Coefficient (MCC), F1 and normalized percentage better than random (S) were used as statistical measures to evaluate the performance of cross validation test. For Fic/Doc family both SVM and HMM were developed and performances of both the methods in identification and classification of Fic, Doc and AvrB were compared using the test dataset. Since the performance of HMM method was found to be distinctly superior to that of SVM, for analysis of AnkX, AR, AT and DrrA family members only HMM profiles were developed. Similarly for subsequent phylogenetic analysis of Fic, Doc, AR, AT and DrrA families sequences were identified from various genomes by using HHM profiles only.The performance of all the SVMs as well as HMMs was evaluated using fivefold cross validation methods. For 5-fold cross validation the total data set was randomly divided into five equal sized sets. One of these five datasets was used as test set while the remaining four sets were used to train the SVM models or derive the profile HMMs. This process was repeated five times such that each of the five parts was used for training as well as testing. The trade-off parameter (C) for the SVMThe Fic domain of AnkX shares a high sequence similarity with other Fic domains. Hence, HMMs based on its Fic domain could not be used to distinguish AnkX type Fic domains. Since AnkX proteins also contain a CMP binding domain  and a unique insert within the Fic domain (110-180) , these sEnterobacteria phage P1 was the representative structure from Doc class (colored red in Helicobacter pylori (PDB ID: 2F6S) was used to visualize active site of the Fic class  were built using Quick Tree tool of Phylip packageGenomic neighborhoods of AMPylation domains identified in different organisms by our profile based search were analyzed if the fully/partially sequenced genomes were available. Such genomic neighborhood analysis could be carried out for AMPylation domains from 970 organisms. Pfam domain information resource was utilized to find out domain annotations for five upstream and five downstream neighbors of AMPylation domain containing genes. Pfam domains which were found in the neighborhood of AMPylation domains at least 40 times were further analyzed to find out literature based evidence about these neighboring domains having evolutionary history of Horizontal Gene Transfer (HGT). Cytoscape58In order to analyze the transposons and integrases in the genomic neighborhood of AMPylation domains, neighboring genes annotated as transposon, transposase or insertion sequences were categorized under a broad heading of transposons and genes annotated as integrases were counted separately. In addition Genomic Island (GEI) predictions were carried out by using the IslandViewer4363http://bioinformatics.mpibpc.mpg.de/rab/)http://nbcr-222.ucsd.edu/pdb2pqr_2.0.0/)67Since substrate specificity of DrrA towards a number of Rab proteins were known, phylogeny of known DrrA substrates were also analyzed. Protein sequences of Rab 1a, 1b, 35, 8a, 13/8c, 3a, 37/26b, 27a, 5a, 22a, 31, 7a, 9a, 23, 32/32a, 38/32b, 4b, 11a, 14 and 6a were downloaded from Rab database .


Fanconi Anemia (FA) is a rare disorder with incidence of 1in 350,000 births. It is characterized by progressive bone marrow failure leading to death of many patients in their childhood while development of cancer at later stages of life in some. The treatment of FA is still a medical challenge. Current treatments of FA include androgen administration, hematopoietic growth factors administration and hematopoietic stem cell transplantation (HSCT). Clinical gene therapy trials are still ongoing. The partial success of current therapies has renewed interest in the search for new treatments. Generation of patient-specific induced pluripotent stem (iPS) has shown promising results for cell and gene based therapy. Small molecule interventions have been observed to delay tumor onset in FA. Tumors deficient in FA pathway can be treated by profiling of DNA repair pathway through synthetic lethality mechanism. Targeting toll-like receptor 8 (TLR8) dependent TNFα overexpression is yet another upcoming therapeutic approach to treat FA patients. In conclusion, in the present scenario of treatments available for FA, a proper algorithm of treatment decisions must be followed for better management of FA patients and to ensure their increased survival. Innovative therapeutic approaches that can prevent both anemia and cancer should be developed for more effective treatment of FA.The online version of this article (doi:10.1186/1877-6566-6-1) contains supplementary material, which is available to authorized users. Fanconi anemia (FA) is an autosomal recessive disorder (OMIM 227650). It is characterized by bone marrow failure (aplastic anemia), developmental delay, physical abnormalities such as short stature; microcephaly; abnormal skin pigmentation; malformations of skeletal system, limbs, eyes, ears, kidneys and urinary tract, heart, genitalia, gastrointestinal system and central nervous system; and increased incidence of solid tumors and leukemias . To dateFA proteins are also known to interact with other proteins such as FANCA and FANCC with α spectrin II; FANCC with cytochrome P-450 reductase and with glutathione S-transferase, Hsp70, STAT1; and FANCG with cytochrome P-450 2E1 . InteracPresently the available treatments for FA are androgen administration, administration of hematopoietic growth factors and bone marrow transplantation. Clinical Gene therapy trials still have a long way to go. Small molecule intervention, controlling TNFα overproduction and profiling of DNA damage repair pathway are the new emerging therapies under investigation. This review highlights the current treatments available for FA and discusses new and emerging therapeutic strategies that can be exploited for the treatment of FA in future.Androgens are male hormones  that stimulate production of red blood cell count, platelets, and white blood cells in some FA patients. Oxymetholone, a 17-a-alkylated androgen is the most common androgen used in the treatment of FA patients. Use of androgens has many pitfalls. Androgens are not a permanent cure, but only prolong the life of patients. Patients response to androgens only if the treatment is started at early stage. Though 70% of the patients respond to androgens, the response is slow, drug dependent and incomplete. Other limitations include several side effects such as masculinisation, acne, growth spurt and premature closing of epiphysis and risk to liver tumours . Thus, iHematopoietic growth factors (HGFs) bind with high affinity to specific receptors expressed on the surface of the target cells and stimulate primitive hematopoietic stem and progenitor cells as well as activate some mature cells.-/- mouse model showed that the short term use of G-CSF either alone or in combination with erythropoietin increased the peripheral blood count and delays the mitomycin C (MMC) induced bone marrow failure, but its long term administration leads to increased sensitivity to MMC and bone marrow hypoplasia. A study demonstrated that FANCC binds to STAT1 and facilitates its activation by gamma interferon and hematopoietic growth factors  interleukin (IL)-3 and granulocyte-macrophage colony-stimulating factor (rhGM-CSF), granulocyte colony-stimulating factor (rhG-CSF), have been used in human trials with FA patients . Adminis factors .Due to the limited success of the preliminary results, clinical trials of HGFs administration have not been carried out so far. Moreover, cytokine use is not recommended for patients with a clonal cytogenetic abnormality. Further studies are therefore needed to search novel cytokines and to establish the long-term efficacy of HGFs and their impact on progression of FA to acute myeloid leukaemia (AML) or myelodysplasia.Bone marrow transplantation (BMT) or HSCT is the only curative therapy for marrow failure in FA patients. Early attempts with BMT for the treatment of marrow failure had a limited success as excessive regimen-related toxicity, graft failure, and severe acute graft-versus-host disease (GVHD) leads to high mortality rate . There iIt was also demonstrated that in case of unrelated donor BMT, in which GVHD is the major limitation, the combined therapy of fludarabine-based preparatory regimens and T-cell depletion reduces the risk of GVHD and increases the overall survival . BecauseThough the HSCT has been successful in increasing the overall survival rate of patients but unfortunately occurrence of cancer has been observed in long-term survivors. In a study of a retrospective cohort of 145 North American FA patients, the crude rate for solid tumors prior to BMT, AML, or death was 0.7% per year, whereas the rate was 1.99% after BMT . In 2005The lack of HLA matched donor due to small families is the limitation in HSCT. However, preimplantation genetic diagnosis (PGD) facilities by umblical cord blood as an alternative source of hematopoietic stem cells provide a novel option in such case . The useThus, despite recent advances in the BMT that have lead to an increase in the overall survival rate of FA patients, there is still a need to establish new therapies for proper and cost effective treatment of FA.FANCA has shown that ex-vivo gene transfer is feasible as a treatment for the hematopoietic defects observed in FA  has shown a great therapeutic potential to obtain disease corrected patient specific cells . This obSmall molecule intervention, targeting TLR8 pathway to suppress TNFα production and profiling DNA damage repair pathways are new exciting emerging therapies under research.-/- mice  that can damage the DNA and therefore cause mutation and cancer , treatme-/- mice . SimilarBy High-Throughput cell screening, potential small molecule inhibitors of FA/BRCA pathway such as wortmannin, H-9, and alsterpaulone and curcumin were identified  in which-/- mice  and acute myeloid leukaemia (AML), suggesting that the FA proteins take part in DNA damage repair. Profiling of DNA repair pathway through synthetic lethality mechanism can be applicable as therapeutic approach for FA pathway deficient tumours. There are various DNA repair pathways; base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination (HR), nonhomologous end-joining (NHEJ), and translesion DNA synthesis (TLS) in the cell that plays a role when DNA damage occurs, depending upon its type. All FA proteins interact in a common pathway involving in homologous recombination. Loss of one DNA repair pathway may result in hyper-dependence on a second, compensatory DNA repair pathway. For example, cancer cells with a mutated FA pathway may be hyperdependent on the BER, NHEJ or TLS pathway . TherefoIn future, identification of the new partners of the FA pathway as well as other DNA damage repair pathways, understanding the crosstalk between these pathways and the mechanism of their regulation will help in generating new small inhibitor molecules that can act as drug target in devising a promising therapeutic strategy for targeting DNA damage repair pathways.To conclude, since the FA clinical trials are undergoing and research for a better therapeutic approach is underway, so in the present scenario, a proper algorithm based on the available established treatments and recent scientific advances should be devised for the treatment of the FA. If the clinical diagnosis has been done on the basis of physical abnormalities, bone marrow failure, haematopoiesis and organ abnormalities or cancer, first step should be to perform the chromosome breakage test. If the test is positive, molecular analysis through FANCD2 western blot can be done to confirm the diagnosis and to classify the patients according to FA/BRCA pathway. The patient should then be tested for complementation analysis and mutation testing. Relatives also need to be checked for chromosome fragility and mutation analysis. Histocompatibility antigen (HLA) typing should be done for stem cell transplantation. If HLA matched and non FA donor is available in the family, HSCT can be the first line of action. However, as the risk of graft failure increases in transfusions, transplantation should be performed before the patient is transfused. If HLA matched donor cannot be found then search for unrelated HLA matched donor should be started. Until the donor is found, androgens and growth factors will be good holding treatment to delay haematopoietic stem cell depletion. If the search for unrelated donor is still not successful, then in vitro fertilization, PGD and HSCT using umbilical cord blood collection may be a choice. However, it should be noted that HSCT treatment using HLA matched or unrelated donor has shown only marginal difference in the survival rate of the patient. Three year overall survival after HSCT in recipients of FA-negative HLA matched sibling grafts was 69-93% whereas using alternative donor grafts survival was 52-88%, . Finally


The incidence of foodborne diseases has increased over the years and resulted in major public health problem globally. Foodborne pathogens can be found in various foods and it is important to detect foodborne pathogens to provide safe food supply and to prevent foodborne diseases. The conventional methods used to detect foodborne pathogen are time consuming and laborious. Hence, a variety of methods have been developed for rapid detection of foodborne pathogens as it is required in many food analyses. Rapid detection methods can be categorized into nucleic acid-based, biosensor-based and immunological-based methods. This review emphasizes on the principles and application of recent rapid methods for the detection of foodborne bacterial pathogens. Detection methods included are simple polymerase chain reaction (PCR), multiplex PCR, real-time PCR, nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP) and oligonucleotide DNA microarray which classified as nucleic acid-based methods; optical, electrochemical and mass-based biosensors which classified as biosensor-based methods; enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay which classified as immunological-based methods. In general, rapid detection methods are generally time-efficient, sensitive, specific and labor-saving. The developments of rapid detection methods are vital in prevention and treatment of foodborne diseases. Foodborne diseases have become a major public health problem worldwide due to the significantly increased incidence of foodborne diseases over the last 20 years   which is complementary to the target sequence . PCR was invented about 30 years ago and it allows the detection of a single bacterial pathogen that present in food by detecting a specific target DNA sequence , 16S rDNA, invasion plasmid antigen H (ipaH), listeriolysin O (hlyA) and intimin (eaeA) gene, respectively. Ryu et al.  genetic analysis system allows high-throughput and detection of multiple pathogens in a single reaction. The analytical procedure involves primer design, PCR amplification and capillary electrophoretic separation of PCR products instead of agarose gel electrophoresis. The GeXP multiplex PCR amplification involves the use of chimeric primers, universal primers and capillary electrophoretic separation of PCR products instead of agarose gel electrophoresis. The chimeric primers contain a gene-specific sequence with a universal tag at the 5′end and they are used to produce amplicons with universal tags. Then, the universal primers which contain the same sequence as the universal tags used in the chimeric primers will drive the remaining PCR reactions. The forward universal primer is covalently labeled with a fluorescent dye at the 5′ end and it is used for the detection during capillary electrophoresis -binding fluorescent dye  was first reported by Liming and Bhagwat  in raw milk. Approximately 1 × 103 CFU/mL Staphylococcus aureus was detected by SYBR green qPCR whereas 1 × 104 CFU/mL Staphylococcus aureus was detected by TaqMan qPCR in raw milk , intimin (eae) and wzx genes. Taqman probe with different combinations of fluorophores were used to detect the product of multiplex qPCR with detection limit around 50 CFU per PCR. Additionally, a simultaneous detection of Vibrio parahaemolyticus, Vibrio cholerae, and Vibrio vulnificus by multiplex qPCR using primers that target vmrA, zot and vuuA genes respectively was developed by Kim et al. , AnDiaTec® Salmonella real time PCR Kit (AnDia Tec), Probelia™ Salmonella sp. (Sanofi Diagnostics Pasteur), and TaqMan™ Salmonella detection kit (PE Applied Biosystems)  and other commercial qPCR kit such as ADIAFOOD® Salmonella (AES chemunex), BIOTECON foodproof Salmonella Detection Kit (Biotecon Diagnostics), BioControl Assurance GDS® TM Salmonella (BioControl), Genedisc® Shiga Toxic E. colii and Salmonella spp. , BioRad iQ-Check Salmonella 2 (Biorad) and MicroSeq® Salmonella spp. Detection Kit (Applied Biosystems). A total of 49 Salmonella strains were included in this study and correctly identified by all the evaluated systems.The examples of commercial qPCR kits for the detection of n et al.  evaluatet et al.  evaluateListeria monocytogenes are BAX® Listeria monocytogenes Detection System , TaqMan® Listeria monocytogenes Detection Kit (Applied Biosystems), ADIAFOOD rapid pathogen detection system for Listeria monocytogenes (AES Chemunex), Probelia® Listeria monocytogenes PCR system (Biorad), LightCycler® Listeria monocytogenes Detection Kit (Roche/Biotecon) and GeneVision® Rapid Pathogen Detection System for Listeria monocytogenes  method 11290–1 for the detection of L. monocytogenes in salmon samples. The results indicated that Probelia® Listeria monocytogenes PCR system is as good as the ISO method and the detection limit was approximately 20 CFU/mL broth culture for pure culture of L. monocytogenes.The commercial qPCR kits available for the detection of the USDA  as a scrn et al.  conducteEscherichia coli and Campylobacter. For instance, Escherichia coli O157 BAX® PCR , ADIAFOOD rapid pathogen detection system for E. coli O157 and E. coli O157:H7 (AES Chemunex) and ADIAFOOD Campylobacter JCL qPCR commercial kit (AES Chemunex) for the detection of C. jejuni, C. coli and C. lari   biosensor due to their sensitivity. SPR employs reflectance spectroscopy for the pathogen detection  for the detection of pathogens  are immobilized on this crystal. Once the target antigens bind to the antibodies immobilized on the crystal, this will cause a measurable change in the vibrational frequency of the crystal which correlates with the added mass on the crystal surface. There are two major types of mass-based biosensors which are the bulk acoustic wave resonators (BAW) or quartz crystal microbalance (QCM) and surface acoustic wave resonators (SAW)  of pathogenic Vibrio parahaemolyticus. The detection limit of this assay was 103 cells of pathogenic Vibrio parahaemolyticus. Commercial ELISA test kit such as BIOLINE Salmonella ELISA Test is also available for the detection of Salmonella in food products. The detection limit of this test kit was 1 CFU/25 g sample with minimum four of the 20 food matrixes tested  and Assurance EIA (BioControl) are available for the detection of foodborne pathogens  is a commercial ELISA kit that allows automation and high-throughput testing Fung, . The Assle Fung, . Assurane et al. . Besidese et al.  performea et al.  performeELISA offers a sensitive and accurate detection of foodborne pathogens. However, the operation of ELISA requires specialized equipment and trained personnel  for Listeria, Salmonella, and Escherichia coli and DuPont™ Lateral Flow System  for Listeria  for ia Fung, . Lateralia Fung, . More exRapid methods provide various advantages for the detection of foodborne pathogens, however, they also have several limitations as summarized in Table Conventional methods for the detection of foodborne pathogens which based on culturing the microorganisms are selective, but they can be time-consuming and laborious. Hence, various rapid detection methods have been developed in order to overcome the limitations of conventional detection methods. Rapid methods are important for the rapid detection of foodborne pathogens in food products to prevent outbreaks of foodborne diseases and the spread of foodborne pathogens. Rapid detection methods are generally more sensitive, specific, time-efficient, labor-saving, and reliable than conventional methods.Nucleic acid-based methods such as PCR, mPCR, qPCR, and DNA microarray have high sensitivity and they are widely used for the detection of foodborne pathogens, but these methods require trained personnel and specialized instruments. Alternative nucleic acid-based methods such as NASBA and LAMP are available for the detection of foodborne pathogens and their toxins. NASBA and LAMP are relatively sensitive, specific and cost efficient. They do not require thermocycling system therefore they are useful especially in low resource settings. Furthermore, numerous biosensors-based methods have recently emerged and employed in the field of foodborne pathogen detection due to their rapidness and cost effectiveness. Biosensors-based methods are easy to operate and they do not require trained personnel, furthermore the techniques can be used for the detection of foodborne pathogens without sample pre-enrichment. However, improvement in food matrixes detection is still needed for these methods for on-site detection. Immunological-based methods such as ELISA and lateral flow immunoassay are also used for the detection of foodborne bacterial pathogens and their toxins. Immunological methods work best in the absence of interfering molecules in the samples such as non-targeted cells, DNA or proteins. Combination of several rapid methods for the detection of a particular foodborne pathogen is also possible as the use of only one detection method may not be sufficient to confirm the detected pathogen. Further studies on the effect of different combinations of rapid methods for foodborne pathogen detection are required in order to develop the most effective and accurate detection method.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 clinical outcomes of patients with NSCLC who progressed after first-line treatments remain poor. The purpose of this study was to assess the advantage of antiangiogenic therapy plus standard treatment versus standard treatment alone for this population of patients.We conducted a rigorous search using electronic databases for eligible studies reporting antiangiogenic therapy combined with standard second-line chemotherapy versus standard second-line treatment for patient who progressed after front-line treatment. Pooled risk ratio and 95% confidence intervals were calculated using proper statistical method. Predefined subgroup analyses were conducted to identify the potential proper patients.Thirteen phase II/III RCTs which involved a total of 8358 participants were included. Overall, there was significant improvement in OS , PFS , ORR  and DCR  in the group with antiangiogenic therapy plus standard treatment versus the group with standard treatment alone. Subgroup analysis showed that OS benefit was presented only in patients treated with docetaxel plus antiangiogenic agents  and patients with non-squamous NSCLC .This study revealed that the addition of antiangiogenic agents to the standard treatments could provide clinical benefit to NSCLC patients who failed their first-line therapy. Furthermore, proper selection of the combined standard cytotoxic agent, as well as the patient population by tumor histology, is warranted for future studies and clinical application of antiangiogenic therapy. Although several targeted therapies against driver mutations have been recently developed and led to extraordinary clinical benefit for NSCLC patients, more than half of the patients without known driver mutations still lack chance for targeted therapies . The firCurrently the recommended second-line or third-line treatments for NSCLC patients include single-agent docetaxel, erlotinib, pemetrexed or gemcitabine , 4–6. ClAngiogenesis plays an important role in cancer development. Several agents with antiangiogenic effect have been developed, including small-molecule multiple receptor tyrosine kinase inhibitors , and monoclonal antibodies . Previous studies were conducted to test the hypothesis that combining standard therapies and antiangiogenic agents might confer additional clinical benefit in advanced NSCLC patients. Eastern Cooperative Oncology Group 4599 study demonstrated that the addition of antiangiogenic agent (bevacizumab) to the standard chemotherapy could improve OS of NSCLC patients treated in the first-line setting . AdditioThe role of antiangiogenic therapy has been well recognized in first-line treatment for NSCLC patients. Two meta-analysis indicated significant improvement of ORR, PFS, and OS for the combination of antiangiogenic agent (bevacizumab) and chemotherapy compared with chemotherapy alone , 10. SevIn October 2014, all relevant articles were retrieved by searching through PubMed, Embase and the Central Registry of Controlled Trials of the Cochrane Library, as well as the ASCO and ESMO databases. Search strategety were the combination of “non-small-cell lung cancer” with any of the following: ‘‘angiogenesis inhibitors” or ‘‘sorafenib”, “sunitinib”, ‘‘bevacizumab”, ‘‘vandetanib”, ‘‘aflibercept”, ‘‘nintedanib”, ‘‘pazopanib”,”ramcirumab” or ‘‘axitinib”. Recent reviews and references of the included studies and were checked manually as a supplement. No language restriction was applied.Studies that met the following criteria were included: (1) Adult (≥18 years) patients with histologically or cytologically confirmed stage IIIB/IV NSCLC ; (2) Phase II or III RCTs that evaluate the efficacy of angiogenesis inhibitors plus a present standard single agent chemotherapy  as salvage cure for patients progressing after first-line treatment; (3) The control group must be the corresponding cytotoxic agent; (4) At least one endpoints  was reported. Trials were excluded if they fail to meet the including criteria. In cases of duplicate trials, the most complete reports were included.Angiogenesis inhibitors were defined as agent blocking angiogenic pathways mediated by vascular endothelial growth factor receptor (VEGFR). Oral small-molecule TKIs or monoclonal antibodies were classified as two types of angiogenesis inhibitors.http://www.cochrane.de). Researcher evaluated the quality of each eligible study according to the JADAD score [The data collection and assessment of methodological quality followed the QUORUM and the Cochrane Collaboration guidelines  and 95% confidence intervals (CI) for objective response rates (ORR) and disease control rates (DCR), median value, hazard ratio (HR) of overall survival (OS) and progression-free survival (PFS), were extracted by two investigators independently. Discrepancies were discussed by the third investigators to reach consensus. We tried to obtain additional unpublished data by contacting the primary authors. Meta-analyses was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statements as shown in The primary outcome was set as OS. Second outcomes included PFS, ORR and DCR. The extracted data of OS, PFS, ORR and DCR were pooled. Further exploration was conducted by subgroup analysis of survival outcomes according to the histological type (selective population for squamous carcinoma and non-squamous carcinoma), the second-line chemotherapy agents  and the classification of angiogenesis inhibitors .2). A random-effects model was employed in case of the existence of potential heterogeneity (I2≥50%); otherwise, the fixed-effect model was applied. Pooled hazard ratios (HRs) for survival outcomes (PFS and OS) and pooled risk ratio (RRs) for dichotomous data  with 95% CI were calculated using the proper algorithm. All calculations and assessment of the risk of bias were performed by Review Manager . Graphical funnel plots were generated to visually inspect for publication bias. P<0.05 was considered statistically significant for all analysis.We defined the experimental arm as angiogenesis inhibitors-containing group. The control arm was standard second-line single agent chemotherapy. Heterogeneity across studies was assessed with a forest plot and the inconsistency statistic .Angiogenensis inhibitor combined with pemetrexed or erlotinib slightly improved OS, however, the difference was not significant compared with chemotherapy alone.With respect to angiogenesis inhibitors, monoclonal antibody was only numerically superior to VEGF-TKI in decreasing the risk of death .Meanwhile, the distinguished combination of antiangiogenetic TKI and EGFR-TKI slightly decreased the risk of death, however, the difference was not statistically significant .All of the included studies were included for PFS and ORR analysis. However, the pooled result of DCR was based on 12 studies as one study  did not However, subgroup analysis showed that combination with angiogenesis inhibitor failed to bring additional efficacy to pemetrexed . For most studies included in this meta-analyses, low risk of bias existed for all key domains, including sequence generation, allocation concealment, blinding of participants or outcome assessment, incomplete outcome data, selective outcome reporting and other sources of bias. No high risk of bias was detected among the thirteen RCTs as shown in As shown in To our knowledge, this is the first meta-analysis to assess the role of antiangiogenesis combined with chemotherapy for the NSCLC patients in second-line setting. Data from our meta-analysis indicated that the addition of antiangiogenic agents to standard treatments could provide extra benefit for advanced NSCLC patients in terms of OS, PFS, ORR and DCR in the whole population. Further subgroup analysis implied that the patients with non-squamous NSCLC might be the potential target population, and docetaxel might be the best option for the combination treatment strategy.To date, the clinical outcome of NSCLC patients who failed from first-line treatment remains poor. Effective salvage therapies for this population are urgently needed. Considering the biological rationale for targeting angiogenesis, the combination of antiangiogenic therapy and standard treatment could be a reasonable option. However, outcomes of clinical trials evaluating this combination strategy were inconsistent.The benefit of this strategy has been questioned by some oncologists. According to our study, the combination of antiangiogenic treatment with standard therapy could increase anti-tumor efficacy, and improve overall survival in NSCLC patients versus standard therapy alone. Thus there is still a clinical rationale for targeting angiogenesis in patients with advanced NSCLC progressed after first-line treatment.Although improved overall survival was noted in the whole population receiving combination therapy, the subgroup assessment suggested that the OS improvement occurred only in patients treated with docetaxel plus antiangiogenic agents. The addition of antiangiogenic compounds to pemetrexed or erlotinib failed to show OS advantage. This finding was consistent with previous studies in the first-line setting , 27. PreToxicities are considered a vitally important outcomes for cancer treatment, especially in second-line setting. According to the included RCTs, combination regimen was associated with more severe side effects than standard second-line treatment alone but generally mild or moderate in severity and mostly manageable. However, given the heterogeneity of toxicity profile among various antiangiogenic and cytotoxic agents, we only conducted the analysis of toxicity-related death between the combination group and the control group. The pooled result indicated that the addition of antiangiogenic agents to standard second-line regimens slightly increased the risk of death caused by treatment toxicities  as shown in Due to the highly heterogeneous nature of NSCLC, it is possible that specific subgroups of NSCLC patients are more likely to benefit from antiangiogenic agents. Previous studies suggested that NSCLC tumor histology influences the response to both chemotherapy and targeted therapy –32. Our Our meta-analysis has several limitations. Firstly, this study may suffer from clinical heterogeneity due to the involvement of various standard treatment regimens and antiangiogenic agents. Secondly, our study is based on data abstracted from publications instead of individual patient data, which could offer more useful information. Finally, for certain subgroup analysis, publication bias existed due to unclear reasons. Publication status may be one of the contributing factors as ongoing studies were ineligible for inclusion.In conclusion, our study revealed that adding antiangiogenic agents to standard treatments could provide clinical benefits to NSCLC patient who failed their first-line therapy. Furthermore, proper selection of the standard treatment regimens and patients population by tumor histology is substantial for future studies and clinical application of antiangiogenic therapy.S1 Checklist(PDF)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.