LAMC2, ANXA2, ADAM9, and APLP2 greatly influenced its decisions. An explanation approach for the RF model was successfully constructed. Moreover, protein expression of LAMC2, ANXA2, ADAM9, and APLP2 was found correlated and significantly higher in PC patients in independent cohorts. Survival analysis revealed that patients with high expression of ADAM9 (Hazard ratio (HR)OS = 2.2, p-value < 0.001), ANXA2 , and LAMC2  exhibited poorer survival rates. In conclusion, we successfully explore hidden biological insights from large-scale omics data and suggest that LAMC2, ANXA2, ADAM9, and APLP2 are robust biomarkers for early diagnosis, prognosis, and management for PC.Substantial alterations at the multi-omics level of pancreatic cancer (PC) impede the possibility to diagnose and treat patients in early stages. Herein, we conducted an integrative omics-based translational analysis, utilizing next-generation sequencing, transcriptome meta-analysis, and immunohistochemistry, combined with statistical learning, to validate multiplex biomarker candidates for the diagnosis, prognosis, and management of PC. Experiment-based validation was conducted and supportive evidence for the essentiality of the candidates in PC were found at gene expression or protein level by practical biochemical methods. Remarkably, the random forests (RF) model exhibited an excellent diagnostic performance and  Pancreatic cancer (PC) is the seventh leading cause of cancer-related death worldwide, with less than seven percent of patients surviving after five years from the initial diagnosis . Early dOnly a small fraction of an enormous number of cancer biomarkers has been successfully implemented in the clinical practice. Cancer is characterized by tumor heterogeneity and a variety of molecular biosignatures; this calls for a more effective strategy of simultaneously combining a set of biomarkers . A studyRecent large-scale gene expression data studies have been conducted to shed light on molecular characterization and to provide a system-level view of human cancers, with a minor focus on PC ,16. RemaAGR2, GAPDH, LAMC2, MMP11, and TAGLN2), 12 membrane , and six “dual”  protein-coding genes were selected for further analysis  was experimentally identified and validated by multiple techniques, such as microarray, quantitative proteomics, and antibody-based methods. According to the results, each gene was overexpressed with a change greater than two-fold at least once in PC or PC cell lines at the mRNA or protein level. For instance, SERPINB5 was more than 250 times overexpressed in the PC tissue compared to normal tissue (log FC = 8.35). LAMC2, CDH3, and ANXA2 exhibited the altered expression difference with log FC of 3.91, 2.72, and 1.16, respectively. Collectively, the experimental and in silico analyses largely supported our 23 selected candidates as reliable biomarkers for differentiating PC tissue from normal tissue. We further conducted an NGS gene expression analysis on a cohort containing six PDAC samples and six controls for experiment-based validation. Despite the heterogeneity and other confounding factors, the NGS result was promising, showing 17 out of 23 consistently overexpressed genes with a cut-off of two-fold change (log FC = 1.00), and 14 out of 23 genes using log FC = 1.50. Remarkably, AGR2, LAMC2, TAGLN2, MSLN, SERPINB5, CLDN18, EZR, FXYD3, GPRC5A, ITGA2, MST1R, NQO1, and SLC2A1 were upregulated in pancreatic neoplasms compared to normal tissue, while CDH3, EPHA2, GPRC5A, and SLC2A1 were upregulated in PC compared to pancreatic neoplasms, providing clues that they may be involved in PC initiation and development. Genetic alterations occur commonly in patients with curable pancreatic pre-malignant lesions, making the biomarker discovery at early stages indispensable . The altThe list of techniques for gene expression, identification, and validation, cES, and gene expression in premalignant pancreatic lesions are summarized in ADAM9, ANXA2, ITGA2, or MET, had both poorer OS and DFS rates than those with low gene expression. ADAM9, ANXA2, ITGA2, and MET expression. Of note, the HR was greater than 2 for both analyses, except for ITGA2 that had an HR of 1.9 for the DFS analysis. LAMC2 exhibited a significantly poor DFS . A multivariate Cox regression analysis of the TCGA cohort was adopted to evaluate the impact of the 23 genes in patient survival. The analysis revealed that LAMC2, ADAM9, ANXA2, CDH3, SERPINB5, EPHA2, GPRC5A, ITGA2, ITGB6, and MET were likely associated with the worse outcome of PC with FDR < 0.05. The detailed information regarding Cox regression survival analysis of these genes is described in The Kaplan-Meier (KM) survival analysis and the multivariate Cox regression analysis were conducted based on the available data from TCGA pancreatic cancer cohort. The KM survival analysis revealed that PC patients with high expression levels of 17 of the candidate genes exhibited a poorer overall survival (OS) or disease-free survival (DFS). Particularly, patients overexpressing CDH3 and LAMC2, were significantly higher in PDAC compared to normal tissue , ADAM9 (cut-off threshold = 6.74), LAMC2 (cut-off threshold = 6.07), AGR2 (cut-off threshold = 9.68), MMP11 (cut-off threshold = 6.58), APLP2 (cut-off threshold = 8.31), and SERPINB5 (cut-off threshold = 4.46) was an indication of PC in this investigation. The visualization of the above explanations of the four representative cases is given in Local Interpretable Model-Agnostic Explanations (LIME) algorithm was applied to explain the “decision rules” of our black-box model. The first case was classified into the normal group, with a probability of 0.96 based on the gene expression rules: LAMC2, ADAM9, and ANXA2 at the protein level using tissue microarray samples of 86 patients and normal controls. They were chosen due to their roles in the diagnostic model, prognostic impact, and profound validated evidence. Typical immunostaining is shown in p-value < 0.001). LAMC2, ADAM9, and ANXA2 exhibited moderate correlations with their protein levels according to Spearman’s pairwise correlation . The protein expression of APLP2 was also investigated in a cohort comprising 64 PC and 64 adjacent normal tissues , making them valuable for further investigation. The Pearson’s correlation analysis of gene expression between LAMC2–ADAM9 , ANXA2–LAMC2 , and ANXA2–ADAM9  revealed strong correlations among them  was employed to clarify the involvement of our candidates in the biological processes leading to PC according to the current ten hallmarks of cancer. Surprisingly, 18 of the 23 genes were implicated in at least five cancer hallmarks, suggesting that they may be highly associated with PC carcinogenesis and progression. Finally, a pathway enrichment analysis was performed based on the 397 upregulated genes with a cES greater than 1.5, derived from the meta-analysis of five datasets, to provide solid evidence on the PC related biological processes. As a result, various KEGG-annotated pathways were enriched in the PC tissue compared to normal tissue, including prominent cancer-related pathways, such as cell cycle and intrinsic molecular processes of PC and others, such as prostate cancer and chronic myeloid leukemia. The results indicate both unique and overlapping features of PC and other cancers, which would be helpful for further studies. In the protein–protein interaction network of 397 upregulated genes, strong connections among the nodes were observed (score > 0.900). There were two major clusters formed by pathways in cancer and cell cycles in the network . PathwayADAM9, ANXA2, MSLN, SERPINB5, EZR, GPRC5A, ITGA2, MET, and SLC2A1). The Pearson’s correlation coefficient between each candidate and the miRNA was also recorded. Finally, we input our genes in curated resources combined by the drug-gene interaction database (DGIdb) to examine if they coded for a drug-metabolizing enzyme or a drug transporter , and then determined possible drug-gene interactions. As an example of the results, ADAM9 was positively correlated with hsa-miR-126-3p  with strong evidence from the reporter assay, western blot, and qPCR. According to DrugBank, the drug Ilomastat was detected to inhibit the cell-surface protein ADAM9. The detailed information regarding miRNA–gene interaction and drug–gene interaction is presented in Increasing evidence suggests that aberrantly expressed miRNAs are attractive targets for preventing PC initiation, progression, metastasis, and chemoresistance. Given their enormous potential as therapeutic tools for cancer management, we checked whether the transcripts of our 18 membrane protein-coding genes were the targets of miRNAs based on experimentally validated evidence. It turned out that nine candidates exhibited a strong connection with miRNAs ; and miR-155 and miR-206 (ANXA2). In PC, miR-126 regulated ADAM9 by targeting the epithelial-mesenchymal transition, the initial step of the metastatic cascade [ANXA2 and KRAS were also direct downstream targets of miR-206 that modulated cancer cell growth, metastasis, and lymphangiogenesis in PDAC [ADAM9 and ANXA2, with high level of evidence of clinical validity, among other candidates, as drug candidates in PC management with regard to their associated miRNAs.It was expected that miRNA would diminish drug resistance of tumors through various regulatory mechanisms ,39. In o cascade . ANXA2 a in PDAC . In addi in PDAC . FurtherThe current study set out to introduce an integrative data mining model for translational research to facilitate the discovery and validation of new biomarkers. In particular, a comprehensive literature and experimental data mining approach to examine potential roles of overexpressed genes as biomarkers for diagnosis and prognosis of pancreatic cancer were conducted. Thus, as a limitation, down-regulated genes were neglected in all of our analyses, which might result in the missing of valuable candidates for further validation. In addition, a follow-up study integrating four proposed candidates and statistical learning is needed to examine their actual clinical validity.The Institutional Review Board (IRB) of Seoul National University reviewed and exempted our protocol for a full ethical approval application when using human gene expression data from the public database without personal information, survival data, and commercial tissue micro-array (SNU 16-11-014 and SNU 17-05-043). The IRB of Inha University approved the protocol of the NGS gene expression analysis (INHAUH 2016-08-008-001). Every patient signed an informed consent before any procedures were conducted. The experiments followed the Ethical Principles of the World Medical Association Declaration of Helsinki.ADAM9 antibody was purchased from Invitrogen , Annexin A2 (ANXA2) antibody was purchased from Cell Signaling , and LAMC2 and APLP2 antibodies were purchased from Sigma . Tissue samples were obtained from six PC patients, and six paired controls  using standard protocols of the Inha University Hospital. All samples were formalin-fixed paraffin-embedded and stored at 4 °C.The Affymetrix-based datasets GSE16515, GSE28735, GSE15471, GSE18670, and GSE41368, were included in the gene expression meta-analysis. After that, we divided the datasets into two groups with similar sample sizes: set A including, GSE16515 and GSE28735 for variable selection purposes; and set B, including GSE15471, GSE18670, and GSE41368 for statistical learning-based validation of the proposed set of novel biomarkers. Dataset GSE19650 was also included for the examination of the multistep PC development.Tissue microarrays containing PC tissues, adjacent normal tissues, and normal tissues (HPan-A150CS-02 and PA1001b) were purchased from US Biomax Inc. .g. The pellet was washed with 1 mL of ethanol and air dried. Next, 150 µL protease K digestion buffer containing 500 µg/mL protease K was added, and the mixture was incubated for 3 h. 1 mL of Trizol was added to the sample and incubated at room temperature for at least 5 min to dissociate nucleoprotein complexes. Subsequently, 0.2 mL chloroform was added, and the mixture was vigorously vortexed for 15 s and centrifuged at 12,000× g for 10 min at 4 °C. The aqueous phase was transferred to a fresh tube. Total RNA was precipitated by mixing with an equal volume of isopropyl alcohol at −20 °C for at least 1 h, followed by 10 min of centrifugation at 12,000× g at 4 °C. Finally, RNA pellet was washed with 70% ethanol, briefly air-dried, and resuspended in RNase-free water.Formalin-fixed paraffin-embedded tissue was cut using a razor blade (20 µm), transferred to a 1.5 mL tube, and filled with mineral oil. The tube was incubated at 70 °C for 3 min to melt the paraffin. The sample was then centrifuged for 1 min at 16,000× Homo sapiens. The reference genome sequence of Homo sapiens and the annotation data were downloaded from the UCSC website (http://genome.uscs.edu). The transcript counts at the gene level were calculated [To construct cDNA libraries, 1 µg of total RNA was used with TruSeq RNA library kit . The protocol included polyA-selected RNA extraction, RNA fragmentation, random hexamer-primed reverse transcription, and 100-nt paired-end sequencing by Illumina HiSeq2500 (Illumina). The libraries were quantified using qPCR and qualified using an Agilent Technologies 2100 Bioanalyzer . To estimate the expression level, the RNA-seq reads were mapped to the genome of lculated . Data noWe normalized raw gene expression data using a robust multi-array analysis implemented in the Bioconductor affy package . DatabasThe procedures of IHC were carried out as described previously . Two expWe treated the overexpressed genes coding for secretory and membrane proteins separately because of their distinct clinical roles. Only those coding for secretory proteins and not upregulated in pancreatitis from the compendium were selected for a further classification. The principal component analysis (PCA) was incorporated to facilitate data visualization and to detect potential outliers prior to the class assignment analysis using FactoMineR version 1.35 and factoextra version 1.0.4 . A heatmClassification and Regression Training (caret) package version 6.0.77 was used for data splitting and supervised classification analysis . Set B wPearson’s correlation analysis and Spearman’s rank correlation analysis were conducted for gene expression level and protein expression score, respectively.The overall survival and disease-free survival were investigated using the Kaplan-Meier method with the log-rank test. We set the high and low gene expression level groups by the median value. The overall survival plot and disease-free survival plot were obtained with the hazard ratios (HR) and the 95% confidence interval information. The whole process was implemented using the web-based tool GEPIA . AdditioKyoto Encyclopedia of Genes, Genomes (KEGG) pathway enrichment, and OmicsNet and STRING protein-protein interaction analyses and visualizations were conducted using the significantly overexpressed genes. The Database of Human Pancreatic Cancer (HPCDb) and the Pancreatic Expression Database (PED) were used as resources for literature data mining ,54. A puTo examine the connection between our membrane protein-coding genes with miRNA, we employed the miRTarBase . The miRp-value of 0.05 was used as the cut-off for significance in statistical tests. False discovery rate (FDR) (Benjamini-Hochberg method), when applicable , with a threshold of 0.05, was utilized for all multiple hypothesis tests.R statistic 3.4.2 was used to implement the analysis except otherwise stated. A NGS data generated and analyzed during the current study are available in the Gene Expression Omnibus (GEO) with the assession code of GSE119224. Other datasets analyzed during the current study are available in the GEO . All data generated or analyzed during this study are included in this published article and its LAMC2, ANXA2, ADAM9, and ALPL2 for innovating PC diagnosis, prognosis, and management. Future studies are expected to take a step forward and concentrate on validating biomarkers by integrating multi-omics data in an epidemiological and clinical context-dependent manner.Recent advances in high-throughput multi–omic technologies along with advanced bioinformatics and machine learning techniques have provided a new paradigm for the discovery of human disease biomarkers ,46,58,59


ALMS1, is critical for the development of specific treatments and may uncover pathways relevant to a range of other disorders including common forms of obesity and type 2 diabetes. Interest in ALMS1 is heightened by the recent discovery of its involvement in neonatal cardiomyocyte cell cycle arrest, a process with potential relevance to regenerative medicine. ALMS1 encodes a ~ 0.5 megadalton protein that localises to the base of centrioles. Some studies have suggested a role for this protein in maintaining centriole-nucleated sensory organelles termed primary cilia, and AS is now considered to belong to the growing class of human genetic disorders linked to ciliary dysfunction (ciliopathies). However, mechanistic details are lacking, and recent studies have implicated ALMS1 in several processes including endosomal trafficking, actin organisation, maintenance of centrosome cohesion and transcription. In line with a more complex picture, multiple isoforms of the protein likely exist and non-centrosomal sites of localisation have been reported. This review outlines the evidence for both ciliary and extra-ciliary functions of ALMS1.Alström syndrome (AS) is characterised by metabolic deficits, retinal dystrophy, sensorineural hearing loss, dilated cardiomyopathy and multi-organ fibrosis. Elucidating the function of the mutated gene,  Alström syndrome (AS) is a rare, recessively inherited disorder that affects numerous organ systems , 2. CardUnderstanding the molecular basis of AS is critical for the development of specific therapies. It may also provide new insights into the molecular pathways that go awry in common forms of conditions such as obesity and diabetes, which represent major public health concerns , 9.ALMS1, a widely expressed 23-exon gene located on chromosome 2p13 [The prevalence of AS is estimated at 1–9 cases per million individuals, with approximately 950 reported cases . It is come 2p13 , 12. Oveome 2p13 . Almost ome 2p13 , commensome 2p13 .ALMS1 also cause mitogenic cardiomyopathy, an ultra-rare form of DCM characterised by delayed cell cycle arrest of neonatal cardiomyocytes [Intriguingly, biallelic mutations in myocytes , 16. Thimyocytes . Thus, mmyocytes , 16. Mecmyocytes .The ALMS1 protein is a component of the centrosome , 19, a mCentrioles duplicate once per cell cycle, beginning at the G1/S transition, and the two resulting centrosomes go on to form the poles of the mitotic spindle  Fig. 2)2). In inCentrioles are vital for the assembly of cilia, hair-like projections that are either motile, as in multiciliated epithelial cells (MCCs), or non-motile. Most quiescent/terminally differentiated (G0) human cells possess a single non-motile (primary) cilium, nucleated by the mother centriole of the centrosome  Fig. . The corNotably, the light-sensing outer segment (OS) of vertebrate photoreceptors is a highly specialised form of primary cilium . The OS Interest in primary cilia has widened dramatically in recent years due to accumulating evidence of their importance for normal development, tissue homeostasis and energy balance –41. TheyWhile cilia-mediated signalling can influence the cell cycle, ciliation status is in turn controlled by cell cycle kinases . MoreoveDozens of genetic disorders have now been linked to cilia dysfunction . ClinicaThe phenotype of AS resembles that of the genetically heterogeneous ciliopathy Bardet-Biedl syndrome (BBS), which includes childhood obesity, retinal dystrophy, SNHL and susceptibility to type 2 diabetes , 60. TheTub gene, characterised by obesity, insulin resistance, retinal degeneration and SNHL [AS also bears similarity to the phenotype of mice with a mutation in the and SNHL , 69. Theand SNHL , 71, andand SNHL .While ALMS1 is known to localise to centrioles/basal bodies , 56, 57,ALMS1 gene regulation and the interaction network of the encoded protein are highlighted.This review focuses on the quest to understand AS at the molecular and cell biological levels. In addition to results from mouse model- and cell-based functional studies, recent insights into ALMS1 is a large (> 4000 residue) protein that lacks known catalytic domains , 12. It Orthologues of ALMS1 are readily detectable in chordates, although most of the N-terminal half of the protein, which mainly comprises the TRD, appears to be specific to mammals .ALMS1 as one of ~ 800 genes required for resistance to ionising radiation in U2OS cells [Experimentally defined sites of post-translational modification in ALMS1 include a cluster of ubiquitination sites around the third predicted coiled-coil domain  and a DNOS cells . Other cOS cells  but the ALMS1 is widely expressed [Alms1 mRNA declines during in vitro differentiation of 3T3-L1 preadipocytes [xpressed , 19, 93 ipocytes , 94, posipocytes . Conversipocytes  and in cipocytes , 96, 97.ipocytes , 99, posALMS1 promoter contains an evolutionarily conserved consensus binding site for ciliogenic RFX (regulatory factor X) transcription factors, three of which  have been implicated in activating ALMS1 transcription in response to serum-starvation [The arvation . Involvearvation , 101 (daarvation ). Vertebarvation , while Rarvation , 105. Hoarvation , 106.Xenopus differentiating MCCs, Alms1 transcription appears to be upregulated by a protein complex containing Multicilin and the DNA-binding transcription factor E2f4 [In tor E2f4 . Many oftor E2f4 .Numerous candidate ALMS1-interacting proteins have been reported. Members of the α-actinin family of actin-binding proteins were identified in yeast two-hybrid (Y2H) screens using the C-terminal region of murine Alms1 as bait, and co-immunoprecipitation data supported an interaction with α-actinin-4 (Actn4) in mouse kidney . ACTN4 aAn ongoing human interactome study based on affinity purification coupled with mass spectrometry (AP-MS) , 113 hasOther AP-MS studies have identified ALMS1 as a possible binding partner of the RNA polymerase II (RNAPII) subunit RPB1 ; the serβ-TrCP1 [thebiogrid.org [BioID has also identified ALMS1 as a potential interactor/substrate of the E3 ubiquitin ligase SCFβ-TrCP1 , and of β-TrCP1 , a proteβ-TrCP1 . Anotherβ-TrCP1 ). An updgrid.org .The notion that ALMS1 has a ciliary role probably first arose when BBS was linked to basal body/primary cilium dysfunction . As outlAlms1GT/GT mice) appear to form cilia normally, based on electron microscopy analysis of renal collecting ducts, photoreceptors and airway epithelium [Mice with a gene trap in intron 13  is often mis-positioned and some stereociliary bundles display defects in shape and orientation [Alms1GT/GT mice, suggesting that Alms1 dysfunction renders this cell type susceptible to a stochastic event during development [In the developing organ of Corti in entation  -induced nonsense mutation in exon 10 (Alms1L2131X/L2131X mice) exhibit loss of renal primary cilia by 6 months of age [Alms1L2131X/L2131X mice, similar to Alms1GT/GT mice. Also of note, sperm flagella, which are structurally similar to cilia, are reduced in number and length compared to wild-type [Mice with an s of age . Notablys of age . Cilia-ls of age  but whetild-type .Alms1foz/foz mice) have also been reported to exhibit age-dependent loss of primary cilia, in this case on hypothalamic neurons [Alms1foz/foz neurons. However, on the reasonable assumption that cilia are lost, the detection of Sstr3 and Mhcr1 in remaining cilia [Bbs and tubby mutant mice [Mice with a truncating mutation in exon 8  –141 and ng obese . Thus, rng obese . Althougme obese , 93, preme cells .Alms1L2131X/L2131X and Alms1foz/foz mice. Cilia-related deficits in a third model (Alms1GT/GT mice) include mislocalisation of rhodopsin and mispositioning of OHC kinocilia. Whether differences in the occurrence/timing of cilia-loss seen in these studies reflect the nature of the mutations, the focus on different cell types, or other factors, remains uncertain.In summary, the initial formation of primary cilia appears normal in all three mouse models reported to date, consistent with their lack of obvious developmental phenotypes. However, age-dependent loss of primary cilia has been observed in The formation of primary cilia seems to be unaffected in AS patient fibroblasts in vitro , 76, 142Alms1L2131X/L2131X and Alms1GT/GT mice, but not Alms1-depleted IMCD3 cells, using an N-terminal antibody [Alms1L2131X/L2131X embryonic fibroblasts [ALMS1 would likely disrupt ciliogenesis (assuming lack of adaptive compensation), leading to severe developmental defects and possibly embryonic lethality.Notably, Alms1 immunofluorescence is clearly visible at centrosomes in antibody , 57. MorAlms1foz/foz neurons [On the other hand, it could be argued that additional data are needed to confirm that the catastrophic ciliary defects seen in siRNA-treated cells are due to depletion of ALMS1. It is notable that Alms1 immunostaining was not detected in  neurons , nor in  neurons , implyinThe specific localisation of ALMS1 to the proximal ends of centrioles  is unusuCEP250 lead to retinal dystrophy and SNHL [There is some evidence that mutations in human and SNHL –148, andand SNHL –152. It and SNHL , 154. CEand SNHL , 155–157and SNHL . Anotherand SNHL . CEP250 and SNHL –162. ThuPCNT mutations lead to severe insulin resistance, dyslipidemia and diabetes in the context of microcephalic osteodysplastic primordial dwarfism type 2 (MOPDII) [In addition to CEP250 and NEK2, centrosomal levels of the PCM protein PCNT are modestly reduced in ALMS1-depleted hTERT-RPE1 cells . Like AL(MOPDII) . Centros(MOPDII) , making It is not hard to imagine ALMS1 performing a structural role such as tethering other proteins to the base of centrioles. Alternatively, it could influence centrosome composition indirectly. Recent data indicate that pericentrosomal levels of centriolar satellites, key regulators of centrosome composition, are enhanced in ALMS1-depleted cells . HoweverImmunofluorescence analysis of filamentous actin in AS patient fibroblasts revealed the presence of non-uniform and stunted filaments, in addition to stress fibres similar to those of control cells . A separImpairments in the trafficking and recycling of endocytosed transferrin (Tf) have been noted in AS patient fibroblasts . The traHES5 was detected in ALMS1-depleted HEK293 cells  trafficking . Insulin control . Moreovepe cells . Phosphoy of Akt . The acty of Akt  may provy of Akt , 170. HoAlms1GT/GT mice showed no such differentiation defect in vitro [Earlier RNAi experiments using 3T3-L1 preadipocytes indicated that Alms1 is important for adipogenic differentiation , whereasin vitro . This diin vitro . Of notein vitro , 95, 171Alms1GT/GT mouse adipose tissue, Alms1 did not appear to be required for proximal insulin signalling in 3T3-L1 adipocytes [Similar to the later analysis of ipocytes . Howeveripocytes . This stipocytes , 172. Hoipocytes .As noted earlier, ALMS1 has been identified as a potential binding partner of the RNAPII subunit RPB1 . In the Alms1GT/GT mice hints at involvement in the transition from vesicular to intraciliary transport, a process controlled in part by the small GTPase Rab8 and in which ciliopathy proteins including CC2D2A and BBS proteins have been implicated [Perhaps not surprisingly given the complexity of the AS phenotype, ALMS1 appears to have multiple functions. The photoreceptor phenotype of plicated , 176. Otplicated , may helplicated . HoweverThe application of proximity labelling has raised the intriguing possibility that ALMS1 interacts with centriole assembly factors, similarly to the ALMS motif-containing protein CEP295/KIAA1731. Although centriole-loss has not been observed in ALMS1-deficient cells, a subtler defect, which could affect both ciliary and extraciliary functions of the centrosome, remains a possibility. Loss of the centrosome cohesion protein CEP250/C-Nap1 from ALMS1-depleted centrioles could be a manifestation of such a defect. Indications that ALMS1 has roles in transcription  and possALMS1 transcripts produced by cells, their functional capacities and potential relevance to phenotypic variability in AS.Immunofluorescence data placing ALMS1 at locations such as the nucleus and cleavage furrow support the idea that multiple isoforms of the protein exist. However, it is important to note that these localisation data have yet to be confirmed by, for example, parallel analysis of ALMS1-depleted cells. Further work is needed to explore the range of motile cilia could contribute to other clinical aspects of AS including respiratory problems, chronic otitis media in childhood and abnormal spinal curvature in adolescence [ALMS1 expression during MCC differentiation is intriguing.It remains plausible that primary cilium or basal body dysfunction contributes to many aspects of the AS phenotype including obesity, retinal dystrophy, hearing-loss, kidney dysfunction, neurological disturbances and fibrosis , 52, 78.lescence , 180. AlWhether a ciliary defect could impair cardiomyocyte cell cycle arrest, leading to infantile DCM/mitogenic cardiomyopathy, is uncertain. Signalling pathways linked to primary cilia in other contexts  are among the many factors thought to regulate this arrest , 181. HoClearly, much remains to be learned about this giant protein. The identification of bona fide interacting partners remains an important goal, as does, more straightforwardly, confirmation of extra-centrosomal sites of localisation. Structural data may reveal homologies not detectable at the sequence level, helping to reveal the functions of individual domains. The establishment of zebrafish models of AS , 173 wil


Finally, physicochemical properties of the designed peptides have been evaluated. A peptide library was provided composed of 31 peptides which were designed based on the MIG6 structure. The results indicated that, two peptides were able to inhibit EGFRL858R mutant selectively. This computational study could be helpful in designing novel inhibitory peptides to inhibit oncogenic EGFR mutants which do not respond to available EGFR TKIs.EGFR  plays the critical roles in the vital cell activities, proliferation, differentiation, migration and survival in response to polypeptide growth factor ligands. Aberrant activation of this receptor has been demonstrated in many human cancers, particularly in non-small cell lung carcinoma (NSCLC). L858R point mutation is the most common oncogenic mutation in EGFR tyrosine kinase domain in patients with EGFR-mutated NSCLC. A feedback inhibitor of EGFR is MIG6 molecule which binds peptide-substrate binding site of the receptor and leads to degradation of activated EGFR. In this  The human epidermal growth factor receptor (EGFR) family that contains four closely related receptors  plays pivotal roles in the regulation of cell proliferation, differentiation, migration, and survival in response to polypeptide growth factor ligands. Overexpression or mutations of EGFR has been demonstrated in tumor cell formation and proliferation in some of human cancers such as liver, breast, stomach, colorectal cancers and particularly in glioblastoma and non-small cell lung carcinoma (NSCLC) .Before activation, the kinase is in an autoinhibited state by a feedback inhibitor called MIG6 (an encoded cytosolic protein by mitogen-inducible gene6). EGFR signalling induces MIG6 expression through the RAS-MAP kinase pathway and also activates Src. Src primes MIG6 molecule to bind EGFR via phosphosrylation of Tyr 395, then MIG6 binds EGFR and is double phosphorylated on Tyr 394 that leads to inhibit EGFR.C-terminal region of MIG6 protein comprises 2 segments, segment 1 residues 336–364) and segment 2 (residues 365–412) which includes a principal phosphorylation site, tyrosine residues (Tyr 394 and Tyr 395). This region plays the main role in binding and inhibition of EGFR tyrosine kinase by blocking the formation of an active dimer receptor 36–364 an. At thisThe peptide-substrate binding site is a shallow and solvent exposed cleft which includes two main pockets, phosphoacceptor and priming recognition pockets. Phosphoacceptor pocket as a significant pocket contains a catalytic base Asp 837) and is responsible for the phosphorylation of the substrates on a tyrosine residue in P position of phosphorylation site. Priming recognition pocket includes Lys 879 and Ala 920 which is able to recognize the substrates that are primed by prior phosphorylation of a tyrosine residue in P+1 position 7 and is , 8, 9.EGFR is one of the most studied receptor tyrosine kinases in the cancer drug target field , 10, 11.M value of kinases for ATP is in micromolar range, then the higher affinities in nanomolar to picomolar ranges is required for these inhibitors to inhibit EGFR in vivo [Although many of these ATP-competitive TKIs are approved by FDA, there are some important disadvantages reported for these inhibitors. Since the ATP-binding site of EGFR is highly conserved among human EGFR family members, this leads to off-target binding and toxicity of these compounds. Additionally, these inhibitors have to compete with intracellular ATP concentration in milimolar range while the K in vivo . Finally in vivo , 14, 16.M value or lower in vitro, therefor it is not required the inhibitors to have a high affinity in vitro activity [In order to avoid these drawbacks, the attention has been recently focused on peptide-substrate binding site to discover inhibitors to inhibit EGFR tyrosine kinase via inhibition of protein-protein interaction. In contrast to the ATP-binding pocket, the peptide-substrate binding site is less conserved across the EGF receptor tyrosine kinase family members that lead to improving the selectivity. In addition, the kinase substrates usually exhibit binding affinity at their Kactivity , 12.Over the recent years, peptide based therapeutics have been considered as a promising and novel approach to treat diseases, particularly cancers. Several favorable characteristics of inhibitory peptides including ease of synthesis and modification, low toxicity, biocompatibility and the higher target selectivity and potency make them effective alternatives to small chemical drugs –20.Given that the peptide-substrate binding site is a shallow cleft with several residues covering a wide surface area of the site and is less conserved across the EGFR tyrosine kinase family members compared to ATP-binding site , 12. AddAmong somatic mutations in the EGFR kinase domain including, deletions in exon 19 (Ex19Del), insertions in exon 20 (Ex20Ins) and point mutations L858R, T790M, G719X and L861Q, L858R in exon 21 is the widespread oncogenic mutation in this domain in patients with EGFR-mutated NSCLC , 10, 13.Since only the receiver subunit of WT-EGFR asymmetric dimer is active and contributes to EGFR signaling, thus, one single MIG6 molecule should be sufficient to inhibit the ligand-stimulated WT receptor. By contrast, both kinases in EGFRL858R are presumably active and contribute to EGFR signaling, therefore, both active kinases need to be inhibited. Given that MIG6 is not capable of accessing to the asymmetric dimer interface of the activator subunit and is not expressed sufficiently to bind both subunits, inhibitory peptides with higher affinity than MIG6 segment 2 seems to be effective in inhibiting the EGFRL858R .Computer-aided drug design has been recently applied broadly as a useful tool in biochemical and pharmaceutical sciences. These techniques can significantly contribute to the drug discovery and development of lead compounds as well as reduction of the experimental time and cost which is necessary to devote in laboratories . Moleculin silico techniques to target the peptide-substrate binding pocket by inhibitory peptides considering the structure of MIG6 segment 2, in order to inhibit EGFRL858R. Finally, physicochemical properties of the designed peptides were investigated.In this study, we have employed Crystal structures of EGFRL858R and WT-EGFR in complex with Mig6-pYpY were obtained from Protein Data Bank (PDB ID: 4R3R with resolution 3.25 Å and 4ZJV with resolution 2.7 Å). The ligand and water molecules were removed from the complexes. EGFR structures were in asymmetric homodimer form, so a monomer was used for the study. Swiss Model server was used in order to fix the chain breaks in the structure .The crystal structures of EGFRL858R and WT-EGFR are composed of 323 residues and the peptide-substrate binding site of both receptors consists of residues Asp837, Arg841, Asn842, Asp855, Lys875, Val876, Pro877, Ile878, Lys879, Trp880, Ser885, Ala920 .A peptide library is provided comprising 31 peptides  which arhttp://vakser.compbio.ku.edu/resources/gramm/grammx) was used to perform peptides docking on the WT-EGFR and EGFRL858R [GRAMM-X protein-protein docking server (v.1.2.0) (GFRL858R . Ten obthttp://flexpepdock.furmanlab.cs.huji.ac.il/) was used to perform local refinement of the best-estimated conformation of a peptide in complex with receptor using flexible side chain [Rosetta FlexPepDock server  method was applied on the MD simulations . G_mmpbsPhysicochemical properties of the designed peptides were investigated using FAF-Drugs4  web servIn the present study, molecular docking and dynamics simulation techniques in combination with MM-PBSA method have been employed to investigate the binding affinity of peptides in complex with both EGFRs. The results were compared with the crystal structures of WT-EGFR and EGFRL858R in complex with MIG6-pYpY (PDB IDs: 4ZJV and 4R3R respectively) and docked EGFR-MIG6YY complexes.In order to investigate the binding modes and calculate the binding free energy, GRAMM-X  and RoseBased on the structural investigations on the crystal structures of EGFR-MIG6pYpY complexes, MIG6-pYpY peptide covered 6 residues including Arg 841, Lys 875, Val 876, Pro 877, Lys 879 and Ala 920 in EGFRL858R complex and also covered the mentioned residues (except Arg 841) in WT-EGFR complex. . The intSTHHYYP), 6 (STHHYYL), 26 (KHTHYYD) and 27 (KRTHYYD), while the binding affinity of both receptors for peptide 10 (HTHYYLP) was very close to each other. Among these peptides, Peptides 5 and 26 (compared to MIG6), showed higher interaction scores (-19.640 and -19.982) and covered the almost whole residues of peptide-substrate binding site in EGFRL858R, including Asp 142, Arg 146, Asn 147, Asp 160, Arg 163, Lys 180, Val 181, Pro 182, Lys 184, Trp 185, Ala 225  are depicted in Also, the RMSF graphs of EGFR-peptide complexes showed that the most flexible regions of WT-EGFR and EGFRL858R were related to the loop shape region located in N-lobe (residues 37–48), activation loop (residues 160–190) and residues 300–320 .Furthermore, the RMSF values of the peptides were depicted in In order to calculate the total binding free energy and per-residue energy contribution, we applied the G_MMPBSA package . The estBased on the MM-PBSA results, despite peptide 27 is the most potent EGFR tyrosine kinase inhibitor but it is not able to inhibit EGFRL858R selectively, while peptides 5, 10 and 26 showed higher binding affinity to EGFRL858R rather than WT-EGFR. On the other hand, peptide 6 has higher binding affinity to WT-EGFR rather than EGFRL858R. Peptides 5 and 26 with binding affinities -23.923 Kcal/mol and -25.098 Kcal/mol, respectively had higher binding affinity than MIG6  to EGFRL858R that was in agreement with the docking results. In addition, peptide 10 with binding affinity -44.791 Kcal/mol showed higher affinity than MIG6 and the rest of peptides to EGFRL858R.3D representation of peptide 10 in complex with EGFRL858R is illustrated in THYYLLP), 5 (STHHYYP), 6 (STHHYYL), 10 (HTHYYLP), 26 (KHTHYYD) and 27 (KRTHYYD) in complex with WT-EGFR and EGFRL858R were calculated using G_MMPBSA  acts as an activated EGFR tyrosine kinase inhibitor and inhibits it by blocking the asymmetric dimer association through its segment 1 and occupying the peptide-substrate binding site via segment 2. The Y394-Y395 phosphorylation site in segment 2 plays the critical role to detect and bind tightly to the active EGF receptor and inhibit it. An interesting property of MIG6 is its ability in inhibiting constitutively active receptor, EGFRL858R, that is much more potent than its ability in inhibiting the basal activity of the WT-EGFR [in silico study to design inhibitory peptides in order to inhibit EGFRL858R.Peptide-substrate binding site is a shallow cleft with solvent exposed residues, Asp 837, Arg 841, Asn 842, Asp 855, Lys 875, Val 876, Pro 877, Ile 878, Lys 879, Trp 880, Ser 885 and Ala 920 which is our target in this To this purpose, we first provided a peptide library comprising of designed peptides considering the MIG6 segment 2 structure which its YY motif is maintained in their sequences, then performed molecular docking and molecular dynamic simulation studies on both WT-EGFR and EGFRL858R. The results of this study were compared with crystal structure of EGFR-MIG6 pYpY and docked EGFR-MIG6YY complexes.STHHYYP) and 26 (KHTHYYD) could be considered as selective inhibitors for EGFRL858R, since these peptides had higher binding affinities (-19.640 and -19.982) to EGFRL858R rather than WT-EGFR  showed approximately similar binding affinities to both receptors (-20.212 and -21.908) and peptides 6 (STHHYYL) and 27 (KRTHYYD) covered nearly similar residues of peptide-substrate binding site as MIG6. These peptides were also considered to be investigated for the next studies because of their high binding affinities , in complex with EGFRL858R.  in complex with peptides are more stable than free EGFRs during the simulation .Comparison of RMSF plots for free EGFRs and EGFR-peptide complexes revealed the difference in local flexibility which is related to the peptide-substrate binding site (residues 180–185). The flexibility of this region is higher in free EGFRs than EGFR complexes .The RMSF plots of the peptides in complex with both receptors indicated that all peptides (except peptide 26) showed more stability in complex with WT-EGFR than in complex with EGFRL858R and the lowest fluctuation is observed in their 4 last residues. While there is no significant difference in flexibility of the peptides in complex with mutant EGFR .The binding free energy of EGFR-peptide complexes was calculated using MM-PBSA.The results indicated that peptides 5, 10 and 26, have significant binding affinity to EGFRL858R than WT-EGFR, compared to MIG6-YY . These dIn addition, per-residue energy contributions of peptides MIG6-YY, 5, 6, 10, 26 and 27 in complex with both receptors were calculated. According to the results, Ser 1 of peptide 5, His 1 and His 3 of peptide 10 and Lys 1 of peptide 26 strongly interacted with the peptide-substrate binding site of EGFRL858R . Also, pin silico prediction of the physicochemical properties of the designed peptides suggested that peptides 5, 6 and 10 are suitable peptides to inhibit EGFRL858R compared to MIG6s (S3 Table).The STHHYYP) and 10 (HTHYYLP) as potential inhibitory peptides could be considered as mutant-selective inhibitors and are able to inhibit EGFRL858R more potently than MIG6-YY. Peptide 5 occupied both pockets (phosphoacceptor and priming recognition pockets) of peptide-substrate binding site , the docked peptides MIG6-YY (B1), peptide 6 (C1), peptide 10 (D1) and peptide 27 (E1) with EGFRL858R. The crystal structure of WT EGFR-MIG6 pYpY complex (A2), the docked peptides MIG6-YY (B2), peptide 6 (C2), peptide 10 (D2) peptide 27 (E2), peptide 5 (F) and peptide 26 (G) with WT-EGFR. (A1) MIG6-pYpY in complex with the crystal structure of EGFRL858R covered only 6 residues of 12 key residues of peptide-substrate binding site, Arg 841, Lys 875, Val 876, Pro 877, Lys 879, Ala 920 and occupied the priming recognition pocket (Lys 879 and Ala 920). Docked peptides MIG6-YY, 6, 10 and 27  with EGFRL858R covered 7, 7, 8 and 6 residues of the binding site, respectively and all of them occupied both pockets, phosphoacceptor site (Asp 142) and priming recognition pocket (Lys 184 and Ala 225) except peptide 10) occupied only phosphoacceptor site(. (A2) MIG6-pYpY in complex with the crystal structure of WT-EGFR covered only 5 residues of 12 key residues of peptide-substrate binding site, Lys 875, Val 876, Pro 877, Lys 879, Ala 920 and occupied the priming recognition pocket (Lys 875 and Ala 920). Docked peptides MIG6-YY, 6, 10 and 27  with WT-EGFR covered 8, 7, 10 and 8 residues of the binding site, respectively. Docked peptides 5 and 26 (F and G) with WT-EGFR covered 8 and 6 residues of binding site, respectively.(PDF)Click here for additional data file.S2 FigAll-atom RMSDs of EGFRL858R (A) and WT-EGFR (B) in complex with peptides MIG6-pYpY, MIG6-YY, 5, 6, 10, 26 and 27. EGFRL858R complexes were more stable than WT-EGFR during the simulation.(JPG)Click here for additional data file.S3 FigThe most flexible regions of WT-EGFR and EGFRL858R were related to the loop shape region located in N-lobe (residues 37–48), activation loop (residues 160–190) and residues 300–320.(JPG)Click here for additional data file.S4 FigAll-atom RMSD of EGFRL858R in free form, and in complex with peptides MIG6-pYpY, MIG6-YY, 5, 6, 10, 26, 27 (A) and RMSD of WT-EGFR in free form, and in complex with MIG6-pYpY, MIG6-YY, 5, 6, 10, 26, 27 (B). EGFR complexes (particularly EGFRL858R) are more stable than free EGFRs during the simulation.(JPG)Click here for additional data file.S5 FigRMSF plot of EGFRL858R in free form and in complex with peptides MIG6-pYpY, MIG6-YY, 5, 6, 10, 26, 27 (A) and RMSF plots of WT-EGFR in free form and in complex with MIG6-pYpY, MIG6-YY, 5, 6, 10, 26, 27 (B). The flexibility of the peptide-substrate binding site comprising residues 180 to 185 is higher in free EGFRs than EGFR-peptide complexes.(JPG)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


APC (Adenomatous polyposis coli) in eight out of ten patients. In the group of normal tissue comparison with colorectal adenoma tissue, somatic mutations were also detected in Catenin Beta 1 (CTNNB1), Family With Sequence Similarity 123B (FAM123B), F-Box And WD Repeat Domain Containing 7 (FBXW7), Sex-Determining Region Y-Box 9 (SOX9), Low-Density Lipoprotein Receptor-Related Protein 5 (LRP5), Frizzled Class Receptor 10 (FZD10), and AT-Rich Interaction Domain 1A (ARID1A) genes, which are involved in the Wingless-related integration site (Wnt) signaling pathway. In the normal tissue comparison with colorectal adenocarcinoma tissue, Kirsten retrovirus-associated DNA sequences (KRAS), Tumor Protein 53 (TP53), and Ataxia-Telangiectasia Mutated (ATM) genes are found in the receptor tyrosine kinase-RAS (RTK–RAS) signaling pathway and p53 signaling pathway, respectively. These results suggest that APC and TP53 may act as a potential screening marker for colorectal adenoma and early-stage CRC. This preliminary study may help identify patients with adenoma and early-stage CRC and may aid in establishing prevention and surveillance strategies to reduce the incidence of CRC.Colorectal adenomas are precursor lesions of colorectal adenocarcinoma. The transition from adenoma to carcinoma in patients with colorectal cancer (CRC) has been associated with an accumulation of genetic aberrations. However, criteria that can screen adenoma progression to adenocarcinoma are still lacking. This present study is the first attempt to identify genetic aberrations, such as the somatic mutations, copy number variations (CNVs), and high-frequency mutated genes, found in Thai patients. In this study, we identified the genomic abnormality of two sample groups. In the first group, five cases matched normal-colorectal adenoma-colorectal adenocarcinoma. In the second group, six cases matched normal-colorectal adenomas. For both groups, whole-exome sequencing was performed. We compared the genetic aberration of the two sample groups. In both normal tissues compared with colorectal adenoma and colorectal adenocarcinoma analyses, somatic mutations were observed in the tumor suppressor gene  Colorectal cancer (CRC) is the third most common cancer worldwide, with increasing numbers of estimated new cases in both males and females . ApproxiThe development of CRC is a complex and heterogeneous process. The transformation of normal colon tissue to a CRC sequence is known to be caused by several genetic aberrations, such as mutations that inactivate tumor suppressor gene function, chromosomal instability, and DNA methylation alteration ,12,13,14Salmonella spp. and HIV [A recent attempt to establish a screening protocol in the Thai population during July 2009–June 2010 examined new potential CRC screening methods in 1404 healthy volunteers using a fecal occult blood test (FOBT) and fecal immunochemical tests (FITs). The obtained results were compared with the screening results by colonoscopy. The study suggested the integration of colonoscopy into the national screening approach for the detection of early-stage CRC . Althoug and HIV . A non-i and HIV ,26,27.In general, the treatment of colorectal cancer is chemotherapy, radiotherapy, adjuvant therapy, and surgery . The resThe aim of this study was to identify the genetic abnormalities in genes associated with a high susceptibility of CRC from matched normal, colorectal adenoma, and CRC samples of Thai CRC patients using exome sequencing analysis for potential application as a noninvasive screening marker, such as the amplification of specific genes from stool DNA followed by mutation detection for early screening of precancerous and early-stage CRC.Genes with somatic mutations are mutations that occur in cancer cells and not healthy cells , and repAPC genes [APC, KRAS, Fc Receptor Like 5 (FCRL5), obscurin, cytoskeletal calmodulin and titin-interacting RhoGEF (OBSCN), Retinitis Pigmentosa 1 Like 1 (RP1L1), Dynein Axonemal Heavy Chain 17 (DNAH17), Zinc Finger Protein 568 (ZNF568), and Calcium Voltage-Gated Channel Subunit Alpha1 C (CACNA1C) [APC, TP53, SOX9, KRAS, PIK3CA, TTN, FBXW7, and SMAD4 [APC, AMA3, OR6X1, NMBR, EFR3A, RBFOX1, CDH20, BIRC6, KRT84, SLC15A3, FTHL17, and GLCCI1. The matched normal-CRC revealed the genes with somatic mutation in APC, FBXW7, FLT4, GSK3A, ZFP64, NRXN3, TGM7, GRIK1, KIF25, DTL, GNAL, ATF2, OR51E2, CUX1, PPAP2C, CORO1A, OR13J1, KRTAP19-7, POU4F3, PPP1R3C, NARS2, NFATC2, FAM109A, FAM54A, TFR2, ZNF781, RRP8, ZFP36L2, KRT31, RYR1, KIAA1409, NRG1, PGM1, ALPK1, FAM181A, FCRL3, and SDK1 [APC, while p4, p13, and p14 show mutations in CTNNB1. p6 and p11 show mutations in FAM123B. In addition, CRC samples of p5, p10, and p14 reveal the mutation in APC. As for CRC samples of p4, p5, and p10, the mutations of the SOX9 gene were found. While CRC of p2 and p10 exhibit mutations in the TP53 gene  . Based ond SMAD4 . The gennd SMAD4 ,22 -receptor pathway. The somatic mutation genes in normal-CRC are enriched in the glutamatergic synapse, phospholipase D signaling pathway, protein digestion and absorption, taste transduction, glioma, ECM-receptor interaction and focal adhesion pathway . The focAPC, CTNNB1, IGF1, and KLF5  increase of both incidence and mortality, (2) increase in incidence but decrease in mortality, and (3) decrease of both incidence and mortality . The redAPC, CTNNB1, LSR, TOPORS, KLF5, IGF1 and SOX9 are related to Wnt signaling pathways, KRAS and BRAF are involved in RAS signaling pathways [ATM and TP53 of the P53 signaling pathways are also at the pivot point of the colorectal tumorigenesis [CALM2, SHISA4, RSPO2, and SYF2 contributed to cell proliferation [The progression of normal epithelial cells to CRC involves multiple gene mutations within several signaling pathways, such as the Wnt signaling pathway, MAPK signaling pathway, PI3K signaling pathway, TGFβ signaling pathway, and p53 signaling pathway . While Apathways ,58,59,60igenesis ,62. In aferation ,65,66,67APC, CTNNB1, LRP5, FBXW7, and ATM -Src family kinase (SFK) signaling , which aAPC is a tumor suppressor gene and has many functions that affect the progression of cancer cells, such as proliferation, migration, cytoskeletal maintenance, and chromosome instability [TP53 gene plays an important function in either cell proliferation or trigger senescence and apoptosis [APC, TP53, and KRAS as early driver genes has been demonstrated in several reports [APC, TP53, and KRAS have been identified in colon adenoma as well as in CRC [Wnt/B-catenin is an important signaling cascade involved in both colorectal development and carcinogenesis . The muttability ,84. The poptosis . Evidenc reports ,86. Mutas in CRC ,86. Our Previous studies showed the gain of chromosome 20 in CRC patients and demonstrated its use as a marker to detect early-stage CRC ,42. Our Here, we identified variants, somatic mutation genes, high-frequency mutated genes, gene list enrichment, and copy number variations (CNVs) following the study flow shown in The patients included in this study were in a CRC screening cohort that underwent colonoscopy at Chulabhorn hospital between July 2009 and June 2010. The present study was approved by the Human Research Ethical Committee of the Chulabhorn Research Institute. All formalin-fixed, paraffin-embedded specimens of colorectal adenoma and adenocarcinoma were obtained from the pathology laboratory unit. The specimens were reviewed by the pathologist to confirm the diagnosis and distinguish between low-grade dysplasia and high-grade dysplasia before performing microdissection.A total of 1500 participants were enrolled in the project . The exc® 2.0 Fluorometer , respectively. Exome sequencing was performed by Macrogen  using the SureSelect human all exon kit V4+UTR , and the exome library of 101 bases paired reads were sequenced using the Illumina HiSeq2000  at 100 reads coverage.Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissue using a QIAmp DNA Microkit . The DNA quality and quantity were determined using a NanoDrop ND-1000  and QubitTo identify point mutations and somatic mutations, the raw FASTQ files were trimmed by trimmomatic , then al2 of ratios >0.5 [CNV was identified in matched normal-colorectal adenoma and normal-CRC samples using “ExomeCNV”  in the Rios >0.5  with defThe analyses of enriched genes in the colorectal adenoma and adenocarcinoma were performed by the use of genes with somatic mutations in the matched normal-colorectal adenoma and matched normal-CRC. Enrichr was used to identify the enriched genes and the enrichment pathway ,96.The matched normal-colorectal adenoma and matched normal-CRC data were used to identify genes with driver mutations using the MutSigCV application with the default parameters .The raw data can be found at the Sequence Read Archive (SRA), accession number PRJNA494574.To identify markers for the early detection of CRC, we explored the genetic alterations in six cases with matched normal colorectal adenoma and five cases with matched normal colorectal adenoma CRC. We performed whole-exome sequencing and bioinformatic analysis focused on CNV, somatic mutations, and candidate genes with driver mutations. Our discoveries showed that the gene sets identified in both matched normal colorectal adenoma and matched normal colorectal adenoma and the CRC group are involved in the Wnt, p53, and RTK–RAS signaling pathways. It might be used as a precancerous and early-stage screening marker candidate for CRC.However, this finding should be validated in a large sample size. One limitation of the current study is its small sample size, and thus more samples in a larger analysis are required for future studies to validate our findings.


Atherosclerosis is the main pathological basis for the occurrence of most cardiovascular diseases, the leading global health threat, and a great burden for society. It has been well established that atherosclerosis is not only a metabolic disorder but also a chronic, sterile, and maladaptive inflammatory process encompassing both innate and adaptive immunity. Macrophages, the major immune cell population in atherosclerotic lesions, have been shown to play critical roles in all stages of atherosclerosis, including the initiation and progression of advanced atherosclerosis. Macrophages have emerged as a novel potential target for antiatherosclerosis therapy. In addition, the macrophage phenotype is greatly influenced by microenvironmental stimuli in the plaques and presents complex heterogeneity. This article reviews the functions of macrophages in different stages of atherosclerosis, as well as the phenotypes and functions of macrophage subsets. New treatment strategies based on macrophage-related inflammation are also discussed. Ac. Ac12]. γ) and TNF and pathogen-associated molecular complexes (PAMPs), including lipopolysaccharides and lipoproteins [α, and IL-1β, and Th1 recruitment-associated chemokines, such as CXCL-9, CXCL-10, and CXCL-11, and low levels of IL-10 [β and chemokines CCL17, CCL22, and CCL24 [β and PAMPs, and produce both proinflammatory cytokines IL-1, IL-6, and TNF-α and the anti-inflammatory cytokine IL-10; M2c macrophages are the most prominent anti-inflammatory subtype and are induced by IL10, TGFβ, and glucocorticoids and produce IL10, TGFβ and pentraxin 3 (PTX3); last, M2d macrophages are induced by TLR signals and characterized by angiogenic properties, playing a role both in plaque growth and tumor progression. Activation of the peroxisome proliferator-activated receptor γ (PPAR-γ) and signal transducer and activator of transcription 6 (STAT6) pathways is the main signal for M2 polarization [In the simplified dichotomy, immune-activated proinflammatory macrophages (M1) and immunomodulatory alternatively activated macrophages (M2) are the most classical classification, mirroring the two types of T helper cells (Th1 and Th2), and represent the extreme phenotypes of the complicated activation states . M1 macrproteins . Granuloproteins . M1 macrof IL-10 , 106. Hoof IL-10 . At thisof IL-10 . M2 macrof IL-10 . Activatnd CCL24 . In factnd CCL24 , 109. M2rization . M1 and rization . M1 macrrization , 113.γ), M(IL-4), and M(IL-10). Recently, Piccolo et al. induced macrophages by dual stimulation with IFN-γ and IL-4, which are the inducers for M1 and M2 phenotype macrophages, respectively, and found that costimulation with two opposite stimuli drove macrophages to an intermediate state that we could call M(IFN-γ-IL-4) and displayed both M1- and M2-type specific gene transcriptome signatures [β and cyclooxygenase 2, and display defective phagocytic and chemotactic capacities [in vitro by hemoglobin-haptoglobin complexes and present a CD206+ CD163+ phenotype. M(Hb) macrophages have increased activity of LXRα, which results in increased cholesterol efflux and reduced lipid accumulation, increased ferroportin expression, which leads to reduced intracellular iron accumulation, and increased secretion of anti-inflammatory factors such as IL-10. The Mhem phenotype is polarized by heme and is characterized by increased expression of cyclic AMPdependent transcription factor- (ATF-) 1 and heme oxygenase 1 (HO1) and suppressed oxidative stress or lipid accumulation, sharing similar properties with H(Hb) macrophages. Both M(Hb) and Mhem cells are hemorrhage-associated phenotypes that are generally resistant to transformation to foam cells, suppressing oxidative stress and potentially serving atheroprotective roles. CXC motif chemokine 4 (CXCL-4) chemokine induces M4 phenotype macrophages in human atherosclerotic plaques, which are CD163− and characterized by expression of MMP-7 and the calcium-binding protein S100A8 and presentation of proinflammatory and proatherogenic properties [Along with a deep understanding of the phenotypes and functions of lesional macrophages, it has been clearly proven that the M1-M2 dichotomy does not actually reflect the complicated subsets of macrophages in atherosclerosis  4, 12, , 12, 111gnatures . In addignatures . Mox macpacities . Rupturepacities , 17. M are negatively correlated with PCSK9 expression [With a deep understanding of the nature of atherosclerosis as chronic inflammation and the fact that macrophages are involved throughout the entire process of atherosclerosis, including lesion initiation, progression, advanced lesion necrosis, and plaque breakdown , strategpression , 132.in vitro and in mice models have explored an abundance of plausible antibodies or inhibitory molecules targeted on proatherosclerotic biomarkers, such as adhesion molecules, scavenger receptors, efferocytosis-related receptors, ER stress signaling, oxidants, and macrophage inflammation. However, most strategies are far from being translated into therapeutic drugs. Several clinical trials have been undergoing targeting critical cytokines and chemokines involved in macrophage inflammation, such as CCR2, CX3CR1, TNF-α, IL-1β, and IL-6 [NCT02659150), IL-1β inhibitor Canakinumab , and IL01327846  are all 01327846 , and pat01327846 . Treatmerthritis . Antioxirthritis . Antioxirthritis . Clinicain vitro studies to electively clear macrophage-related inflammation.Notably, macrophage-related proatherosclerotic inflammatory responses are not easily distinguished from host defense, so therapeutic measures are likely to cause increased susceptibility to infections. The local target-based delivery systems would possibly lessen this problem by improving the drug efficacy. Anti-inflammatory biomarker drugs such as antibodies and siRNA carried by nanoparticles (NPs)  or stentAntiatherosclerotic biomarkers or lipid modulation strategies are nonspecific measures that suppress the functions of macrophages and other cells in the plaque, such as SMCs and ECs. However, therapies directly and specifically targeting macrophages are scarce, and studies have thus far been preclinical work, possibly owing to the complicated phenotypical and functional heterogeneity of lesional macrophages.in vitro or in animal models, and further studies are needed for more novel drugs and clinical translation.Currently, novel drug delivery systems such as NPs, stents, liposomes, glucan shell microparticles, oligopeptide complexes, and monoclonal antibodies make it possible to selectively modify macrophages. Macrophage surface markers such as F4/80, CD11b, CD68, CD206, and scavenger receptors provide unique targets for all macrophages or different subsets . Coupledin vitro via SDF-1/CXCR4 signaling [γ, can promote monocytes to polarize to the M2 phenotype by modifying the expression of M2 markers, such as mannose receptor (MR) and CD163 [In addition to the removal of macrophages, influencing macrophage polarization to an anti-inflammatory phenotype, M2 macrophages, rather than the M1 phenotype, is another option . Any facignaling . Thiazolnd CD163 .Macrophages, the major immune cell population in arterial plaques, have been proven to play critical roles in the initiation and progression of atherosclerosis. Lesion-derived signals induce macrophages into complicated subsets with distinct gene expression profiles, phenotypes, and functions. Based on these results, several strategies are suggested, including blocking proinflammatory cytokines and chemokines, activating anti-inflammatory macrophages, depolarizing macrophages, and enhancing efferocytosis.Although a number of studies have confirmed the critical functions of macrophages in atherosclerosis, many important problems remain unsolved. For example, the origins of macrophages from different organs or systems differ greatly, yet little is known about the proportions of proliferating resident macrophages or macrophages derived from circulating monocytes. This has important implications for the effectiveness of targeted drug therapy. In addition, the exact reason for the relation between intermediate monocytes and prognosis in CVD patients also needs to be clarified, as well as the functions of classical and nonclassical monocytes. In addition, more advanced techniques such as mass cytometry and single cell sequencing are needed to fully and more accurately characterize macrophage subsets and exploit novel therapeutic targets. Finally, much research is still needed before translating preclinical strategies directly targeting macrophages into clinical practice, including specific macrophage-targeted drugs and other targets, such as genetic modification.


Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an extremely rare tumour, which usually affects elderly males and presents in the skin with frequent involvement of the bone-marrow, peripheral blood and lymph nodes. It has a dismal prognosis, with most patients dying within one year when treated by conventional chemotherapies. The diagnosis is challenging, since neoplastic cells can resemble lymphoblasts or small immunoblasts, and require the use of a large panel of antibodies, including those against CD4, CD56, CD123, CD303, TCL1, and TCF4. The morphologic and in part phenotypic ambiguity explains the uncertainties as to the histogenesis of the neoplasm that led to the use of various denominations. Recently, a series of molecular studies based on karyotyping, gene expression profiling, and next generation sequencing, have largely unveiled the pathobiology of the tumour and proposed the potentially beneficial use of new drugs. The latter include SL-401, anti-CD123 immunotherapies, venetoclax, BET-inhibitors, and demethylating agents. The epidemiologic, clinical, diagnostic, molecular, and therapeutic features of BPDCN are thoroughly revised in order to contribute to an up-to-date approach to this tumour that has remained an orphan disease for too long. Blastic plasmacytoid dendritic cell neoplasm  is regarded as an orphan tumour due to its rareness and usual clinical aggressiveness with poor response to conventional chemotherapies . It deriNormal pDCs–originally described by Lennert and Remmele -have beeTCF4, BCL11A and IRF8 have been regarded as the main transcription factors determining the pDC development. The E-box transcription factor TCF4 controls the differentiation to the pDC lineage and its maintenance [BCL11A, is essential for pDCs generation and, along with TCF4, is used to distinguish the gene expression profile of pDCs from myeloid dendritic cells that, on contrary, are negative for BCL11A and highly positive for the B-cell lymphoma 6 protein (BCL6) [IRF8, induces in the lymphoid-primed multipotent progenitors and in CDPs the early commitment towards DC lineage and its expression increases during this transition [IRF-8 regulates different hematopoietic lineages and, if mutated, may cause pDC cytopenia and global immunodeficiency [At the time being, it is widely accepted that normal pDCs can recognise either myeloid or lymphoid derivation. Both common dendritic cell progenitors (CDPs) and common lymphoid progenitor (CLPs) can differentiate into pDCs, with the same ability to produce type 1 interferons (IFN-I), although only pDCs of myeloid origin can process and present the antigen . CDPs anntenance . The B-cn (BCL6) . The IFNficiency .BCL11A and TCF4 [IRF8 may be found mutated or mis-spliced [As normal pDCs, BPDCN cells express and TCF4 , while I-spliced ,15. The -spliced . However-spliced  and the -spliced ,19,20,21The condition has been given several names, such as NK-cell lymphoma, CD4+ NK cell leukaemia or blastic NK leukaemia/lymphoma. All of them are obsolete and reflect the misinterpretation of the histogenesis due to the expression of the CD56 molecule, which is also observed in NK lymphocytes and derived tumours . The terBPDCN more often affects males  in the seventh or eighth decade of life, although it can occur at any age, including childhood. Its incidence is 0.000045% .There are no data concerning the etiology of BPDCN, except for a certain association with myelodysplastic syndromes (MDS) and MDS/myeloproliferative neoplasms (MPN), with special reference to chronic myelomonocytic leukaemia, a fact that is not surprising taking into consideration the co-occurrence of several gene mutations (see below) .The disease tends to involve multiple sites . More ofMYC rearrangement [In its most common form, BPDCN is characterized by a diffuse, monomorphous infiltrate of medium-sized blasts reminiscent of either lymphoblasts or myeloblasts . Nuclei angement . Angioinangement ,37. In tangement . In lympangement . On periangement .Neoplastic cells are negative for myeloperoxidase, α-naphthylbutyrate esterase, ND naphthol AS-D chloroacetate esterase.BPDCN cells express CD4, CD43, CD45RA, and CD56, along with the pDC associated antigens CD123 (IL3 α chain receptor), CD303, TCL1A, CD2AP, and SPIB, and the type I interferon– dependent molecule MX1 ,43,44,45BPDCN patients are affected by frequent chromosomal alterations: up to 75% of them present a complex karyotype . Conventional cytogenetics studies reported a prevalence of genomic losses on gains and recognized six recurrent deletions of the regions 5q21 or 5q34 (72%), 12p13 (64%), 13q13-21 (64%), 6q23-qter (50%), 15q (43%) and of the entire chromosome 9 (28%) ,68. AlthMYC translocations were reported in the 39% of BPDCN patients, in association with the above mentioned immunoblast-like morphology [MYC rearrangement: it defined a subgroup of patients with a more aggressive behavior [MLL1 gene, also recorded in 18% of ALLs [ETV6), a transcription factor disrupted in other hematological malignancies [By FISH analysis, rphology . T [Akin to cytogenetics and FISH, a-CGH evidenced frequent deletions on the chromosomes 4, 9 and 13. Lucioni et al. analyzed by a-CGH 21 BPDCNs and found that the most affected chromosomes were Chr 9 (71%), 13 (61%), 12 (57%), 5 (19%), 7 (19%), 14 (19%), and 15 (14%). The deletions outnumbered the amplifications and resulted in the loss of nd IKZF1 . While aficiency , their i outcome . Wiesnernd TP53) , possiblnd TP53) ,80.RB1 and LATS2 tumor suppressor genes; (4) higher expression of various pDC-related genes, such as the TLRs, TLR9 and TLR10 [The first study of BPDCN gene expression profiling (GEP) was conducted in 2007 by Dijkman et al. Since BPDCN skin lesions could easily be confused with cutaneous myelomonocytic leukemia (c-AML), Dijkman et al. performed a-CGH and GEP by array of 5 BPDCN skin biopsies and 6 c-AML cases. According to their study, BPDCN displayed: (1) a transcriptome profile and a molecular karyotype indeed distinct from c-AMLs; (2) recurrent deletions of 4q34, 9, and 13q12-q31 chromosomal regions; (3) lower expression of nd TLR10 . In 2014nd TLR10 .Ceroi et al. performed transcriptional profiling of 12 BPDCN cases by array and focused on a specific signature of downregulated genes involved in cholesterol homeostasis and responsible for its accumulation within the tumor cells. These sets of downregulated genes, if activated, stimulated the cholesterol efflux from neoplastic cells, inhibited the NF-kB pathway and arrested the BPDCN tumor cell survival .TET2 are diffused in the myeloid lineage [TET2 was mutated in more than half of patients and was mostly affected by deleterious mutations (frameshift or nonsense). At diagnosis, TET2 mutations (54%) were recurrently flanked by TP53 mutations (38%) leading to hypothesize a synergistic effect between the two genes [TET2 in BPDCN [TET2 and RHOA [TET2, thanks to the targeted sequencing approach, many other myeloid-associated genes have been investigated in BPDCN. Taylor et al. presented to the ASH Meeting the first study of targeted sequencing of 219 myeloid-related genes in seven BPDCN samples. The most frequently mutated gene was the splicing factor ZRSR2 (57%) ex aequo with TET2 (57%), followed by ASXL1, TP53 and IDH2, KRAS, ABL1, ARID1A, GNA13, U2AF1, SRSF2, and the transcription factor IRF8 associated with dendritic cell deficiency [NRAS, ATM, KRAS, MET, IDH2, KIT, RB1, APC, TP53, RET, VHL, BRAF, and MLH1 genes, and deletions of CDKN2A, RB1, PTEN, and TP53 genes, already found by a-CGH [TET2 (36%), ASXL1 (32%), NRAS, NPM1, and IKZF family 1/2/3 (20%). Overall, 50% and 20% of patients with mutations in genes encoding for epigenetic factors or belonging to the IKAROS family respectively experienced a significantly reduced overall survival [The chromosomal lesions of BPDCNs fully reflect their myeloid origin and the same could be expected at the DNA mutational level. Starting from the premise that the mutations of the epigenetic regulator gene  lineage , Jardin wo genes . Alayed in BPDCN . Ladikouand RHOA . Besidesficiency . Later, by a-CGH . Menezessurvival .BCL2 overexpression in tumors. Furthermore, by the BH3-proling of two BPDCN cell lines (CAL-1 and GEN2.2), six primary patient samples, and six patient-derived xenografts, the same authors demonstrated the BCL2 dependence of BPDCN elements as well as their sensitivity to the BCL2 inhibitor venetoclax. In the light of this finding, two patients were then treated with venetoclax and experienced significant disease responses [More recently, integrated “omics” approaches have been applied aiming to better understand the tumor biology. Montero et al. analyzed, by RNA-sequencing, 12 BPDCN samples and four pDCs from healthy donors by confirming esponses ,89.TCF4 as a master regulator of the BPDCN oncogenic program: its downregulation provoked the loss of the BPDCN-specific gene expression signature along with tumor cell death. Already described as relevant in normal pDC development, the TCF4 gene product was positively detected by immunohistochemistry in all the 28 BPDCN samples examined and proposed as a new reliable diagnostic marker (see above) and potential therapeutic target for bromodomain and extra-terminal domain inhibitors (BETis) [Ceribelli et al. first performed an RNA interference screening study of the CAL-1 BPDCN cell line and recognized the transcription factor  (BETis) .NR3C1, defined a high-risk group of patients. NR3C1 is often juxtaposed with lncRNA3q, a novel nuclear noncoding RNA involved in the regulation of leukemia stem cell programs and G1/S transition and aberrantly overexpressed in BPDCN malignant cells. BETis successfully turned-off the expression of lncRNA3q and inhibited tumor cells growth [Emadali et al. further substantiated the use of BETis in BPDCN. They examined 47 tumor samples and the CAL-1 cell line by various techniques  and found that the loss of the glucocorticoid receptor gene, s growth .MYB gene rearrangement were identified in all the children (100%) and in four out of the nine adults (44%) [Suzuki et al., used RNA sequencing technology to discover novel fusion genes in 14 BPDCNs corresponding to five children and nine adults; recurrent ts (44%) .ASXL1, TET2, SUZ12, ARID1A, PHF2, CHD8) and the functional enrichment analysis of the mutational data showed that of all the biological programs explored, the epigenetic was the most affected. At transcriptomic level, the patients displayed the significant enrichment of gene signatures related to epigenetic pathways, predicting response to hypomethylating agents. Accordingly, the use of 5’-azacytidine in combination with decitabine significantly inhibited disease progression and extended survival in a preclinical mouse model [Sapienza et al. analyzed BPDCNs by WES, RNA and Chromatin Immunoprecipitation (ChIP) sequencing approaches. Several epigenetic factor genes were found mutated  are considered more effective compared to standard therapies  ,92,93. Iotrexate ,95.Regarding the role of hematopoietic stem cell transplant in BPDCN therapy, there are several reports suggesting better results in terms of enduring remissions and relapse rates with allogeneic-stem cell transplantation  compared to auto-SCT. These studies demonstrated durable complete remissions with allo-SCT, with OS rates ranging from 40% at 10 years, to 58% at 3 years depending on the follow-up period ,97. In gEligible patients should be considered for allo-SCT consolidation in first CR whenever feasible. It should be noted, however, that these patients represent the minority of BPDCN patients, as the disease normally affects elderly patients, with a median age of 68 years .For elderly patients, lower intensity treatments can be explored. Lower intensity chemotherapy regimens demonstrated some efficacy in BPDCN, such as single agent pralatrexate, bendamustine, or gemcitabine/docetaxel combinations ,102,103.A recent study by our group strongly supports the use of hypomethylating agents, demonstrating a significant enrichment in epigenetic modifiers mutations in the setting of BPDCN . In lineGiven the unsatisfactory results of low-intensity treatments, and the toxicity of intensive therapies and allo-SCT consolidation, there is strong rationale for the use of novel targeted agents for the treatment of BPDCN.SL-401 is a novel recombinant protein including components of diphtheria toxin fused to interleukin-3. As mentioned in previous sections, CD123 is expressed on the surface of BPDCN cells. In a phase I study of SL-401 in BPDCN the overall response rate was 77% (with 55% CR) in the evaluable patient population (seven out of eleven patients were able to complete the planned treatment) . A phasePhase I trials are ongoing with other immunotherapies targeting CD123, such as bispecific antibodies, immunoconjugates, and chimeric antigen receptor (CAR)-T-cells . In factSeveral recently published reports have described the activity of venetoclax in the setting of BPDCN ,111,112.Although the criteria for the diagnosis of BPDCN are well-defined , the kno


Bariatric surgery is an efficient treatment for weight loss in obese patients and for resolving obesity comorbidities. However, the mechanisms behind these outcomes are unclear. Recent studies have indicated significant alterations in the transcriptome after surgery, specifically in the differential expression of microRNAs. In order to summarize the recent findings, we conducted a systematic summary of studies comparing microRNA expression levels before and after surgery. We identified 17 animal model and human studies from four databases  to be enrolled in this meta-analysis. From these studies, we identified 14 miRNAs which had the same direction of modulation of their expression after surgery in at least two studies . Pathway analysis for these miRNAs was done using database resources (DIANA-TarBase and KEGG pathway database) and their predicted target genes were discussed in relation with obesity and its comorbidities. Discrepancies in study design, such as miRNA source, bariatric surgery type, time of observation after surgery, and miRNA profiling methods, were also discussed. Bariatric surgery was first performed in 1963 to help obese patients lose excess weight permanently . For exa2), it is also recommended for less obese patients (BMI > 35 kg/m2) with obesity comorbidities due to these beneficial outcomes [In addition to weight loss, many bariatric surgery patients demonstrate improvement in comorbidities of obesity post-operation. This includes recovery from Type 2 Diabetes Mellitus (T2DM) and achieving long-term favorable levels of cardiovascular risk factors, such as high-density lipoprotein cholesterol and hypertension ,15,16. Boutcomes .In the case of T2DM, glucose control via surgical treatment was reported to be better than medical therapy ,20,21,22The mechanisms of these long-term beneficial effects after bariatric surgery is poorly understood. The acronym “BRAVE” is often used to describe RYGB physiological effects, which are to alter bile flow, restrict stomach size, alter anatomy/flow of nutrients, manipulate vagal, and modulate enteric gut and adipose hormones . HoweverEpigenetic changes due to surgery could give insight into these mechanisms. Epigenetic machinery, such as DNA methylation, histone modifications, and non-coding RNAs, can respond to external environmental cues by altering gene expression levels without changing DNA sequence. In recent years, there is an increasing interest in studying the relationship between epigenetic changes and bariatric surgery outcomes . Among tSeveral miRNA studies have reported short- and long-term miRNA profile changes after bariatric surgery in various tissues of animal models and humans ,43,44,45The databases for the literature search were chosen based on a recommendation of the optimal database combinations  and dataDuring the screening stage, the exclusion criteria were: (1) non-English publications, (2) abstracts-only publications, case reports, comments, or reviews, (3) no report or comparison of miRNA profiles before and after surgery or between bariatric surgery-operated animals and sham-operated animals, or (4) added another intervention post-surgery before miRNA assessment. Inclusion criteria were (1) animal and human studies, (2) any profiling method, (3) any bariatric surgery method, (4) any biological sample type, and (5) reported cut-off criteria for differentially expressed miRNAs. One full-text study was later excluded due to inconsistent reporting of the direction of miRNA expression.The items collected from the full text and Supplementary Information followed a recent methods paper for meta-analysis of miRNAs studies . The itehttp://www.microrna.gr/ miRPathv3) [Only miRNAs reported in at least two independent studies were retained for analysis. The selected miRNAs were grouped into three categories based on their consistency. The first group included miRNAs with consistent report of expression direction in two or more studies. The second group included miRNAs with some discrepancies in the direction, but two or more studies agreed on a direction. The third group included miRNAs with no consistent reports of expression direction. Pathway analysis was done only for the first two miRNA groups. Pathway analysis was done using DIANA miRPath v.3 to predict their target genes and KEGG pathways (RPathv3) .A total of 164 articles were retrieved from Pubmed, OVID, Scopus, and Web of Science. After screening and assessment, 17 studies were selected for the meta-analysis . These sn = 13) (n = 4) [n = 15). However, a recent study in Austria profiled 58 patients [Most reported studies profiled miRNA levels before and after bariatric surgery in human patients (n = 13)  [29,30,3 (n = 4) ,35,39,45 (n = 4) ,41,42,43 (n = 4) ,40,44. Mpatients  and a stpatients , while an = 7) [The studies isolated miRNAs from different tissues: blood (plasma and serum) (n = 7) ,36,40,45n = 7) ,37, monon = 7) , circulan = 7) , adiposen = 7) ,42,43, ln = 7) ,35,45, an = 7)  Table 2n = 7 [Tan = 5), miRNeasy kits (n = 5), TRIzol reagent (n = 3) or other kits. Most of the studies then used qPCR (n = 7) or microarrays (n = 5) as their main profiling method. RNA sequencing was used as the main analysis in one study [n = 4) used a screening step using high-throughput profiling methods, such as microarrays and RNA sequencing, in a subset of their patients, then qPCR as validation in the final sample. Human studies using microarrays used the Robust MultiArray Average (RMA) method for normalization (n = 3). Studies with cells and tissue samples normalized their data using small non-coding RNAs with RNU6 and RNU6B being most commonly used (n = 6). Studies with plasma and serum samples used a number of stable miRNAs, which were unique for each study. One plasma study used RNA spike-in levels for normalization [The studies also differ in the miRNA profiling strategies . For isone study . Other slization  and the lization .n = 13) (The surgery type most commonly assessed is RYGB (n = 13)  [29,30,3n = 9), while others looked at long-term response .Lastly, the studies differ in the duration of study and number of observations after bariatric surgery . One stuAccording to the selected studies, a total of 50 miRNA families and 205 unique miRNAs were significantly differentially expressed after surgery compared to baseline. Among these, 32 differentially expressed miRNAs were identified in at least two different studies. The 32 miRNAs can be grouped based on the consistency of findings and reasons for discrepancies .Group 1 includes 14 miRNAs that changed in the same direction of expression, regardless of sample type and time of observation . Group 2 includes six miRNAs with inconsistent findings, but at least two studies agreed on a direction of expression . For example, hsa-miR-125b-5p was found to be downregulated in two studies profiling miRNA from plasma samples, but was upregulated in an exosome study. Lastly, group 3 includes 12 miRNAs reported in two studies but with no agreement in direction .DIANA-miRPath was used to identify pathways regulated by miRNAs in Group 1 and 2. The first analysis was done with only Group 1, and a total of 74 KEGG pathways were significantly predicted to be regulated by these miRNAs. The miRNAs were predicted to target genes involved in cancer, cell cycle, fatty acid metabolism, signaling pathways, infectious diseases, and RNA processes in cells . The incThe benefits of bariatric surgery beyond weight loss, such as T2DM remission, have been reported extensively ,20,21,22We found that through February 2019, there were 17 studies on miRNA profiles of patients before and after bariatric surgery. Although a relatively small number, there is a sharp increase in publications in the last five years. The first study among them was published in 2012  and 15 sHowever, these recent miRNA studies were highly variable in study design. Studies on rats looked into a wide range of tissues and included tissues inaccessible in human studies, such as the hypothalamus and liver. Most human studies profiled easily accessible tissues, including circulating miRNA in plasma, serum, exosomes, and monocytes. Some studies had access to adipose tissue biopsies which were collected from patients a few years after surgery. In contrast, human blood samples were able to be collected earlier and at more time points. The earliest time point was 21 days after surgery  and one Despite the high variability between studies, there were 14 human and rat miRNAs with consistent direction of differential expression after surgery. In at least two studies, hsa-miR-93-5p, hsa-miR-106b-5p, hsa-let-7b-5p, hsa-let-7i-5p, hsa-miR-16-5p, hsa-miR-19b-3p, hsa-miR-92a-3p, hsa-miR-222-3p, hsa-miR-142-3p, hsa-miR-140-5p, hsa-miR-155-5p, and rno-miR-320-3p were reported to have lower expression levels, while hsa-miR-7-5p and hsa-miR-320c had increased expression levels after surgery. These miRNAs are predicted to be important in various cellular pathways, including those related to lipid metabolism, insulin signaling pathway, and cardiac function. The genes within these pathways are interesting targets for functional studies to understand the mechanisms behind weight loss and remission of obesity-related comorbidity after surgery.For instance, the most significant pathway is the “proteoglycans in cancer” (hsa05205) and the 13 human miRNAs were predicted to target 140 genes in this pathway. One of them is FZD7, which is one of the Frizzled (Fzd) transmembrane receptors for Wnt proteins . ReducedThese miRNAs were also predicted to target 30 genes in the fatty acid metabolism pathway. The upregulated hsa-miR-7-5p was predicted to target FASN, which is inversely correlated with parameters of glycemic status  and its Although limited in sample size and the number of miRNAs analyzed, studies on SG patients and animal models suggest different miRNA profiles compared to other surgery types. A study in rats compared rno-miR-200a-3p expression levels between DJB and SG . In thisHowever, it is interesting that although many studies used high-throughput methods, only 32 miRNAs were reported in at least two studies. This might be due to the differences in miRNA isolation, profiling, and normalization strategies between studies. For isolation methods, some studies showed that miRNeasy isolation kits produce higher RNA quantity and better quality compared to miRVana ,81. MaxiThe highly different profiling methods between studies could also be the source of this limited agreement in their findings. Comparative studies have found low correlation between different profiling methods when used to analyze the same samples . DiffereFinally, normalization is crucial for providing robust expression data, but there is no consensus regarding normalization methods for miRNA results . SeveralIn addition to these study design limitations, our analysis has not considered population and sex differences, as well as analyzing miRNA results by sample type due to the limited number of studies published so far. Some studies have demonstrated population-specific miRNA expression between populations ,88. For We have identified 14 miRNAs with consistently altered expression after bariatric surgery, regardless of sample type, surgery type, and time of observation after surgery. However, these findings should be taken with caution. These miRNAs were identified from 13 studies with highly variable study design and small sample sizes. A consensus in miRNA profiling methods is crucial for a better comparative study of profiling studies. Until then, a better analysis would be to compare findings of studies with similar strategies. Future studies should also aim to profile a larger number of participants and untargeted profiling of SAT samples. Additionally, more profiling studies in different populations and in males are needed to investigate the generalizability of miRNA modulation after surgery. Studies investigating SG patients are also needed as this surgery type is becoming the most commonly used technique in many countries. Finally, functional studies are needed to understand the role of these miRNAs in promoting weight-loss and remission of obesity-related comorbidities after bariatric surgery. This may lead to novel targets for non-surgical treatment of obesity and its comorbidities and provide novel biomarkers for predicting bariatric surgery outcomes.