Cigarette smoking is the leading cause of lung cancer worldwide. In this study, we evaluated the serum autoantibody (AAb) repertoires of non-small cell lung cancer (NSCLC) patients and smokers (SM), leading to the identification of overactivated pathways and hubs involved in the pathogenesis of NSCLC. Surface- and solution-phase biopanning were performed on immunoglobulin G purified from the sera of NSCLC and SM groups. In total, 20 NSCLC- and 12 SM-specific peptides were detected, which were used to generate NSCLC and SM protein datasets. NSCLC- and SM-related proteins were visualized using STRING and Gephi, and their modules were analyzed using Enrichr. By integrating the overrepresented pathways such as pathways in cancer, epithelial growth factor receptor, c-Met, interleukin-4 (IL-4) and IL-6 signaling pathways, along with a set of proteins (e.g. phospholipase D (PLD), IL-4 receptor, IL-17 receptor, laminins, collagens, and mucins) into the PLD pathway and inflammatory cytokines network as the most critical events in both groups, two super networks were made to elucidate new aspects of NSCLC pathogenesis and to determine the influence of cigarette smoking on tumour formation. Taken together, assessment of the AAb repertoires using a systems biology approach can delineate the hidden events involved in various disorders. Approximately 85% of all new cases of lung cancer are classified as non-small cell lung cancer (NSCLC), including adenocarcinoma, squamous cell carcinoma and large-cell carcinoma2. Despite an overall increase in the rate of survival for most cancers, 5-year survival for lung cancer has not improved significantly1. On the other hand, smoking is a primary risk factor for lung cancer, and nearly 14–25% of lifetime smokers (SM) are expected to develop lung cancer2. Accordingly, the carcinogenic effects of tobacco smoke through deregulation of various cellular pathways and DNA adduct formation have been extensively studied in the past decades3.Lung cancer is recognized as a fatal malignancy, accounting for more than a quarter of all cancer deaths in the United States and 1.6 million deaths every year worldwide5. Imaging methods are usually applied in the diagnosis of lung cancer. However, the high costs of these methods, radiation exposure, and false positive or negative results, restrict their applications as screening tests4. Moreover, resistance to conventional chemotherapeutics, such as platinum agents, antitubulars, topoisomerase II inhibitors, and antimetabolites, is quite common among NSCLC patients who eventually develop resistance to chemotherapeutics5.The low success rate of lung cancer treatment originates from delayed diagnosis, which reduces the chance of total tumour excision via surgery and acquired resistance to available medication regimens6. Since AAb production occurs during the early stages of disease, it may serve as a functional tool for NSCLC diagnosis7. Therefore, detection of AAbs in NSCLC patients has been the subject of a few investigations to determine the AAb repertoires and identify AAbs against particular proteins14. Nevertheless, for comparison of the AAb repertoires and identification of common autoantigens, limited research has been performed on the sera of NSCLC patients and SM.Autoantibody (AAb) production, as a reaction of the immune system to tumour antigens, is an inevitable phenomenon in cancers19, a few studies have focused on smoking with a systems biology approach; for instance, expression-based network analysis and assessment of protein-protein interactions have revealed changes in human blood20, human buccal mucosa21, and mouse lung samples22 due to cigarette exposure. For exhibiting the direct correlation between smoking and lung cancer, Ying Liu et al. analysed three protein expression datasets of lung adenocarcinoma and divided them into two subcategories of SM and non-SM. The results revealed Ras signalling pathway (SP) and proteoglycans in cancer as the key pathways in lung cancer development in SM23. Furthermore, Wang et al. constructed a graphical model to assess gross network alterations during transition from normal to cancer tissue in SM. The findings demonstrated a gain of epithelial growth factor receptor (EGFR) and platelet-derived growth factor receptor A (PDGFRA) modules in addition to a loss of interleukin-6 (IL-6) module in lung cancer24.In contrast to studies assessing lung cancer via bioinformatics toolsIn the present study, we carried out biopanning with a phage-displayed random peptide library on immunoglobulin G (IgG) purified from the sera of NSCLC patients and SM to detect peptides based on their AAbs. NSCLC- and SM-specific proteins, predicted from the detected peptides, were investigated with systems biology tools to demonstrate their involvement as hubs in significant pathways driving lung cancer pathogenesis. Furthermore, they were used to construct two super networks for clarifying multidimensional contribution of smoking to NSCLC.U1-20; SL, FLL21-30) and 15 SM  clones were selected, which showed the highest intensities in comparison to the controls  containing sequences with three consecutive histidine residues, and one SM-specific peptide (FSL25) due to its similarity to another peptide sequence in this group, were omitted. Notably, among NSCLC-specific clones, FLU6, FLU9, and FLU15 (-CRNVHHKNC-), FLL22 and FLL29 (-CTRHWPHHC-), in addition to FLL26 and FLL27 (-CQSLHGANC-), showed similar sequences  to clones FLL26 and FLL27  and solution-phase (SL) biopanning, all the input and output phages were screened via polyclonal phage enzyme-linked immunosorbent assay (ELISA). The highest signals were observed in the third round of SU and SL biopanning (data not shown). Based on monoclonal phage ELISA on random clones obtained from the third round of biopanning, 30 NSCLC  followed by an assessment in the UniProt database and review of the literature, did not present major proteins; accordingly, these peptides were deleted from the final peptide lists. However, blasting the remaining peptide sequences led to the prediction of 531 NSCLC and 346 SM proteins with 50 common proteins (Supplementary dataset). Figure U19, FLL26, FLL27, and FLL30 accounted for the lowest number of NSCLC-related proteins (<10), while peptides FLU5 and FLU10 represented the highest number of NSCLC-related proteins (>40). Moreover, peptides FSU1, FSU15 and FSU17 were individually related to more than 40 proteins whereas a group of SM-specific peptides  accounted for less than 10 proteins in the SM protein dataset. Markedly, retinoic acid-induced protein 1 (RAI1), dermatan-sulfate epimerase (DSE), and protein kinase C delta (PRKCD) were proteins predicted from more than one peptide in both NSCLC and SM groups.Evaluation of the selected peptides in the MimoDB demonstrated that 21 NSCLC- and 13 SM-specific peptides, were true target binders. Blasting peptides FLp-value < 0.05). APC-stimulated guanine nucleotide exchange factor 2 (ASEF2)/Adenomatous polyposis coli (APC) complex (2/2), Early elongation complex with hyperphosphorylated RNA polymerase II C-terminal domain (Pol II CTD) (4/4), Positive transcription elongation factor (P-TEFb) complex (2/3), and Tankyrin 1-tankyrin 2-telomeric repeat binding factor (TRF1) complex (2/3) were the most significant complexes in the SM group . Histone deacetylase 2 (HDAC2) and lysine-specific demethylase 1 (LSD1) complexes were common in the datasets. Table p-value of each complex.Gene set enrichment by Consensus PathDB (CPDB) revealed multiple physically associated protein complexes, with at least two members in the NSCLC or SM protein dataset. Proline-rich tyrosine kinase 2 (PYK2)/Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2) complex , ADP-ribosylation factor 6 (ARF6)/Guanosine triphosphate (GTP)/Nucleoside diphosphate kinase A (NME1)/T-lymphoma invasion and metastasis-inducing protein 1 (Tiam1) complex (2/3), proto-oncogene tyrosine-protein kinase Src (SRC)-PRKCD-CUB domain-containing protein 1 (CDCP1) complex (2/3), and Janus kinase 1 (JAK1): Interleukin-13 receptor subunit alpha-1 (IL-13RA-1): Interleukin-4 receptor alpha (IL-4RA): Tyrosine kinase 2 (TYK2) complex (2/4) were among the most significant complexes in the NSCLC group , differentiation , and migration  as the top three processes associated with the highest proteins count , carcinogenesis , metastasis , abnormality , neoplastic transformation , malignant transformation , and neoplasm invasion , as the most important diseases linked to the NSCLC group  was a major event, involving 19% of SM proteins. Other events in this group included metastasis , abnormality , congenital malformation , neoplastic transformation , neurodegeneration , malignant transformation , and neurodegenerative disease . Although carcinogenesis, metastasis, and abnormality owned similar shares in both groups, cancer state and neurodegenerative disease were major disorders which were specifically associated with the NSCLC and SM groups, respectively. The percentage indicates the ratio of involved proteins to the total number of proteins in each dataset.Diseases analysis demonstrated cancer  SP, phosphoinositide 3-kinase-Akt (PI3K-Akt) SP, JAK-signal transducer and activator of transcription (JAK-STAT) SP, gonadotropin-releasing hormone (GnRH) SP, chemokine SP, natural killer cell (NKC) mediated cytotoxicity, pathways in cancer, signalling of hepatocyte growth factor receptor (HGFR), EGF/EGFR SP, IL-4 SP, IL-6 SP, type II interferon signalling (IFNγ), mitogen-activated protein kinase (MAPK) SP, signalling pathways regulating pluripotency of stem cells, ataxia telangiectasia mutated (ATM) SP, Fanconi anaemia pathway, nicotine addiction, gamma-aminobutyric acid (GABAergic) synapse, and Ras SP Fig. . SimilarAccording to features such as betweenness centrality and degree implemented in Gephi, several NSCLC- and SM-specific hubs were identified Table . Proteinp-value: 0.014), epithelial cancers , lung diseases , and carcinoma , as well as several malignancies and disorders related to cancer. Evaluation of SM-specific hubs revealed confirmative results such as precancerous conditions , substance-related disorders , chromosome aberrations , and stress , in addition to adenocarcinoma and neoplasms.Assessment of NSCLC-specific hubs in the WebGestalt database demonstrated disorders such as lung neoplasms . In contrast, they showed poor binding to the sera of age-matched healthy subjects , promote various pathways leading to PLD activation33. Another remarkable receptor in this network, EGFR, stimulates PLD via multiple routes containing proteins, some of which were found in this study, as well , and MAPK SPs, are induced through phosphatidic acid (PA) produced by the enzymatic activity of PLD36. Components of ECM  induce the synthesis of PA by activation of PLD which stimulates matrix metalloproteinases (MMPs) and enhances the invasive capacity of tumor cells37. Among pathways connected to the PLD SP, pathways in cancer was one of the critical events observed in both NSCLC and SM groups and was linked to other significant pathways such as microRNAs in cancer, DDR, and small cell lung cancer. A set of proteins, including LPAR4, LPAR6, collagen alpha-3(IV) chain-like (COL4A3), PLCB2, and granulocyte-macrophage colony-stimulating factor receptor subunit alpha (CSF2RA) from the NSCLC protein dataset as well as APC, LAMA1, LAMA5, and PLD1 from the SM protein dataset, were involved in pathways in cancer. All NSCLC- and SM-specific proteins playing roles in the above-mentioned SPs are presented in Fig. The PLD SP was undoubtedly the most significant NSCLC pathway. Remarkably, PLD was found among SM-specific proteins. Furthermore, phospholipid metabolic process and phospholipid binding were overrepresented GO terms identified through NSCLC-related module analysis. According to the literature review and evaluation of pathways involved in tumour formation and development, the PLD SP took centre stage in the pathogenesis of NSCLC in this study. Curation of the PLD SP derived from the KEGG database with the information attained through data-mining and Pathway Studio® led to the formation of a super network constructed with several NSCLC  and SM  hubs. Among the mentioned hubs, RPTOR, a SM-specific hub, was also found in the NSCLC protein dataset. As exhibited in Fig. 38, IL-1740 and cigarette smoking41. There is a two-way interaction between IL-6 and HGF, resulting in increased invasiveness of a lung cancer cell line42 and an autocrine loop between IL-6 and TGF-β1 which its dysregulation causes an array of inflammatory disorders43. As shown in Fig. 45. Production of LIF, an IL-6 family cytokine, by synovial fibroblasts is also induced by IL-1739. Another interleukin, IL-4, can increase the transcription of MUC4 (via JAK/STAT) and MUC16 (through JAK/STAT and NCOA3)47. Furthermore, IL-4 triggers M2 macrophages to produce much more collagen a3 (VI)48. Expression of TJP1 is also decreased by IL-4, and its distribution at the interface of adjacent cells is disrupted by IL-17 receptor C (IL-17RC)50. The major cytokine in this network, TGF-β, contributes to the modification of tumour microenvironment via MUC4 upregulation in an interdependent manner with retinoic acid and interferon type II (IFNγ)51. TGF-β increases TJP1 expression52 and inhibits collagens degradation through activation of TGFBI53. Locally-secreted cytokines not only modulate the structure of tumour microenvironment, but also disrupt the protective function of the immune system. TGF-β signalling plays critical roles in the inhibition of tumour-specific CD8 T cell- and NK cell-mediated cytotoxicity through regulatory T cells55. In addition, IL-4, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) increase the population of myeloid-derived suppressor cells (MDSCs) which interrupt the identification of tumour antigens by T cells through induction of nitric oxide synthase and consequent nitration of T cell receptor56. The final link is related to the TNF family which seems to be involved in tumour suppression57. However, the binding of TNFSF15 to its receptor, TNFRSF25, amplifies the expression of IL-4, IL-6, GM-CSF, and IL-1759, which may explain the overactivity of these tumour-promoting cytokines in our network.The second super network was constructed to describe how cytokines alter the tumour microenvironment (e.g. ECM proteins) and how tumour recruits them to fabricate the immune system Fig. . This ne61. According to the literature, AAbs exist in the plasma of patients several months to years before the emergence of any clinical signs, reflecting the functional immunoreactivity against tumour antigens62. AAbs against tumour-specific antigens generated due to antigen mutation, overexpression, or altered structure, have been recruited to discover cancer biomarkers63. Aside from the important role of AAbs in the discovery of tumour antigens for the development of T cell-based vaccines, they can act as therapeutic antibodies by either blocking tumour antigens or recruiting the immune system63. Various studies have assessed cancer-specific antibodies in different types of malignancies63. Similarly, several studies have examined NSCLC patients and have identified a panel of AAbs binding to rho-associated protein kinase 1, protein kinase C beta, complement factor H, c-myc, cyclin A, B1 and D1, cyclin-dependent kinase 2, MUC1, Dickkopf-related protein 1, p53, NY-ESO-1, and annexins (I and II)14. Furthermore, Yao, et al. introduced four combined AAb biomarkers identifying nucleolar and coiled-body phosphoprotein 1, metastasis-associated lung adenocarcinoma transcription 1, hyaluronan mediated motility receptor, and spermine oxidase for early detection of NSCLC7.A combination of events such as cigarette smoking, nicotine addiction, delayed diagnosis, and resistance development during chemotherapy, has led to major concerns about lung cancer. Therefore, it can be useful to compare the AAb repertoires of NSCLC patients with SM to discover proteins involved in lung cancer pathogenesis65, PI3K-Akt66, PLD36, Ras67, EGF/EGFR71, c-Met74, LPARs75, IL-476 and IL-679 SPs which play significant roles in tumorigenesis and cancer development. Remarkably, we found tumour suppressor pathways such as Fanconi anaemia pathway80 and ATM SP81 in the NSCLC group, which might be activated as feedbacks to the overactivated pathways in cancer cells.In the present study, classification of the predicted proteins into significant cellular processes revealed some overlapped areas as well as dissimilarities in the cellular behaviour of NSCLC and SM. Network enrichment analysis of NSCLC- and SM-related modules resulted in the identification of remarkable pathways which play key roles in cancer initiation, progression, or suppression. NSCLC-specific pathways included MAPK82 SPs as well as negative regulation of Wnt SP83 in the SM group, show activation of cellular defensive mechanisms against the carcinogenic compounds of cigarette smoke. Moreover, we found TGF-β and TNF SPs in this group, known as “double-edged swords”, which can promote or suppress cancers85. As the results demonstrate, carcinogenic events are strongly significant in SM and may accelerate the emergence of disorders such as cancer.Considerably, analysis of SM-related modules revealed multiple tumour-promoting pathways such as PI3K-Akt, PLD, and EGF/EGFR SPs which were commonly found in the NSCLC group. Conversely, activation of tumour suppressor pathways, including DDR and FoxO86. Moreover, a significant correlation has been reported between CFTR downregulation and lung cancer progression and metastasis87. In contrast to studies reporting CFTR suppression in epithelial cells due to exposure to cigarette smoke, Li, et al. showed CFTR upregulation through nicotine-derived nitrosamine ketone (NNK), as one of the most toxic tobacco-specific carcinogens in cigarette smoke88. CFTR as an ABC transporter promotes protection against tobacco-specific carcinogens in cigarette smoke88. On the other hand, AAbs can lead to the development of lung cancer by blocking CFTR.Among NSCLC-specific hubs, there are four proteins which can be considered as the key players involved in lung cancer development. The first hub is CFTR which its dysregulation has been reported in a group of cancers such as breast, gastric, prostate, lung, and colorectal cancers (CRC)30. It has been reported that PTK2B is upregulated in NSCLC. In fact, its higher expression and greater activity lead to the progression of NSCLC through modulating extracellular signal-regulated kinase 1/2 (ERK1/2)89. Furthermore, wound-induced activation of PTK2B stimulates EGFR activation in the epithelial cells of lung lesions90. PTK2B was also detected as a sunitinib-specific target in NSCLC patients with kirsten rat sarcoma viral oncogene (KRAS) mutations91. Likewise, prescription of crizotinib and merelotinib in NSCLC patients underscores the importance of PTK2B in the pathogenesis of lung cancer. KRAS-activated PRKCD promotes growth, invasion, and migration of cancer cells, through MAPK signalling in NSCLC cell lines (and mouse model), whereas PRKCD activation in cells without active KRAS leads to tumour suppression92. In this regard, the PRKCD inhibitor, rottlerin, can effectively potentiate chemotherapy-induced apoptosis, particularly if it is used concomitantly with trastuzumab93. Liu, et al. found that CHEK1 overexpression is associated with poor overall survival in NSCLC patients, and inhibition of CHEK1 by miR-195 suppresses tumour cell growth, migration, and invasion94. In this regard, Xiaojie, et al. revealed that miR-195 synergizes with microtubule-targeting agents (eribulin and paclitaxel) via CHEK1 regulation, leading to the inhibition of NSCLC cells growth95. In agreement with other studies, we confirmed the involvement of CFTR, PTK2B, PRKCD, and CHEK1 in NSCLC pathogenesis.The second hub, PTK2B, is a part of protein complexes found in this study. This hub plays major roles in cancer cells due to its connections to critical pathways such as PLD, EGFR, and c-Met SPs97. Mutations and upregulation of Notch1 have been reported in 10% and 30% of NSCLC patients, respectively98. Konishi, et al. showed that suppression of NOTCH signaling inhibits the growth of NSCLC99. Furthermore, NOTCH signaling can crosstalk with other SPs such as Wnt and TGF-β driving cancer development98. Therefore, targeting NOTCH signaling by therapeutic agents like antibodies which can inhibit delta-like protein 4 (Dll4) binding to NOTCH1, may be advantageous for NSCLC patients98. In this regard, a humanized anti-Dll4 antibody, demcizumab, is currently under evaluation in three Phase 1b studies on pancreatic cancer, CRC, and NSCLC98.In terms of major cancer-related proteins among SM-specific hubs, NOTCH1, ARFGEF1, PLD2, and RPTOR were strongly aligned with hallmarks of NSCLC. NOTCH1 contributes to cell proliferation, invasion, and chemoresistance. Its involvement in tumor progression and prognosis of NSCLC has been demonstrated in different studies100. Among these three proteins, PLDs have been garnering attention as potential contributors to cellular signalling of tumours101. Surprisingly, PLD represented an important intersection for other pathways in this study, and the first super network was constructed based on this enzyme. The existence of PLD isozymes (PLD1 and PLD2) in the SM protein dataset indicates the role of these junction boxes in the initiation of tumorigenesis. PLD is activated through a variety of signals such as neurotransmitters, hormones, and growth factors, transducing signals into pivotal cellular events, including proliferation, secretion, respiratory burst, and actin cytoskeletal reorganization102. As mentioned in the results section, PLD network involves proteins such as LPAR4, LPAR6, HCRTR1, and GRM5, as well as pathways like EGF/EGFR SP, which their roles have been proven in initiation and progression of several cancers. Phosphatidic acid, the product of PLD enzymatic activity, can be also converted to LPA and form a positive loop of LPA-LPARs30. LPA is involved in various cancer-related processes such as proliferation, growth, and survival75. Even though LPAR4 and LPAR6 are newly-discovered members of LPAR family, their contributions to hepatocellular carcinoma103 and thyroid cancer104 have been demonstrated, respectively. Although HCRTR1 stimulation with high doses of orexin causes apoptosis in colon and prostate cancers106, elevated serum orexin level has not been detected in cancer patients107. It seems that the normal serum level of orexin is not sufficient to induce apoptosis in cancer cells. Physiologically activated HCRTR1 may activate some pathways other than apoptosis, such as the PLD SP which contributes to tumour progression105. Therefore, pharmacologically activated HCRTR1 may be promising in cancer prevention or treatment.As illustrated in the results section, three interconnected proteins, ARFGEF1, PLD2, and RPTOR, were successively activated, stimulating the cancer driver mTOR SP. Intriguingly, mutations in ARFGEF1, an activator of ARF1, have been reported in lung cancer107. Noteworthy, GRM4 and 8 have been reported as susceptibility genes in NSCLC31. One of the critical pathways in both NSCLC and SM groups is the EGFR SP. More than half of lung tumours express receptors from EGFR family and one-fifth of lung tumours carry EGFR mutations, causing proliferation, migration, and metastasis71. Despite the preliminary response of all mutated lung tumours to tyrosine kinase inhibitors, they essentially acquire resistance68. Activation of mTOR, a master regulator of cell growth and proliferation, is one of the most important consequences of PLD activation. Accordingly, combination therapy of mTOR antagonists with standard regimens for lung cancer has enhanced treatment efficacy in early-stage trials108. Similarly, the MAPK SP, triggered by both PA and PLD, is associated with P53 degradation and MMPs induction, leading to cell survival and motility, respectively36. Overall, the PLD super network clarifies why treatment fails in this type of lung cancer and presents new insights into NSCLC chemotherapy. Moreover, this network underscores the pivotal function of PLDs in the occurrence of lung cancer in smokers.Hypoxia, as a common feature of tumour microenvironment, increases the transcription of glutamate receptors, promotes the activation of GRM1, 3, 4, and 5, and supports proliferation and survival of several types of tumours through MAPK and PI3K/Akt SPs56. Incidentally, the significance of IL-4 and IL-6 SPs in the present study may suggest inappropriate activation of this unfavourable arm of immunity. IL-4 SP promotes metastasis by changing the microenvironment of epithelial tumours76. It has been reported that serum IL-6 levels in NSCLC patients  are higher than healthy subjects78, and blockade of IL-6/STAT3 signalling results in the inhibition of lung tumour growth109. Besides, STAT3 is persistently activated in GPCR C5a-knockout (KO) mice, a spontaneous lung cancer developing model, and the use of an antibody against LIF leads to STAT3 inactivation in this model110. The IL-6 and c-Met loop causes cell invasion through MMP activation in A549 cells, as well72. Proteins such as IL-6 and c-Met, in cooperation with IL-17 and TGF-β, play various roles in the regulation of proteins such as mucins, collagens, and TJP1. It has been demonstrated that mucins such as MUC4 and MUC13 are associated with cancer development; the circulating level of MUC16 is even used to monitor patients with ovarian cancer113. Regarding collagens, their increased and decreased expressions are correlated with malignancy114; accordingly, collagen XXIII has been suggested as a biomarker for the diagnosis of NSCLC115. Another study showed that COL4A3 cleavage by MMP-9 produces tumstatin which is a circulating anti-angiogenic factor116. Hence, AAb against COL4A3 may worsen the state of cancer by preventing the generation of tumstatin. Additionally, COL27A1 is aberrantly expressed in idiopathic pulmonary fibrosis117, a condition associated with lung cancer118. Ni and colleagues found that the expression of TJP1 was decreased in NSCLC tissue in comparison to adjacent non-tumour tissue and its overexpression was correlated with better prognosis in NSCLC patients119. Considerably, increased MDSCs population as well as dysfunction of NKCs and CD8+ T cells highlight an inefficient immune system in NSCLC122. Notably, our network exhibits how IL-4, IL-6, GM-CSF, and TGF-β predispose these phenomena.Another super network constructed in this study is a complicated linkage between inflammatory cytokines and tumour microenvironment. Inflammation is a mixed blessing in cancer and the T helper 2 response deteriorates the state of cancer through suppression of cellular immunityIn conclusion, given the rapid emergence of lung cancer in smoker populations across the world, new aspects of NSCLC pathogenesis and effects of cigarette smoke should be addressed. Tailoring the AAb profiling with a systems biology approach provides an excellent image of the network of AAbs targeting proteins which may be key drivers of cancer-interacting SPs such as PLD, PI3K-Akt, MAPK, EGF/EGFR, c-Met, Ras, DDR, IL-4, IL-6, and TGF-β SPs in the NSCLC and SM groups. Based on the PLD SP and inflammatory cytokines network, two super networks were constructed with NSCLC- and SM-specific proteins such as PLD1/2, PRKCD, PTK2B, LPARs, RPTOR, NOTCH1, TJP1, laminins, mucins, and collagens as well as pathways found in this study. Focusing on these super networks will help to design novel therapeutic agents targeting proteins involved in the pathogenesis of lung cancer.123, Tehran, Iran, as well as Kurdistan Hospital in Sanandaj, Iran. The patients aged 22–77 years  were all confirmed for NSCLC via histopathological tests and were classified into different stages based on the tumour/node/metastasis scale (TNM) staging system. Furthermore, 57 subjects  were allocated to the SM group according to the complete blood cell count, blood level of C-reactive protein (≤6 mg/dL), erythrocyte sedimentation rate (≤32 mm/h), rheumatoid factor (negative), and chest X-ray imaging to evaluate their overall health. All the subjects in the SM group were current smokers with different smoking patterns, ranging from a few cigarettes per week to heavy smoking.For obtaining a repertoire of NSCLC AAbs, 67 blood samples of untreated newly-diagnosed adenocarcinoma and squamous cell carcinoma patients  were collected from different centres, including Baqiyatallah and Masih Daneshvari hospitals125 and was approved by the Ethics Committee of Baqiyatallah University of Medical Sciences, the Ethics Committee of Masih Daneshvari Hospital123, and the Ethics Committee of Kurdistan University of Medical Sciences. All participants provided written informed consent before enrollment. The characteristics of all three groups are presented in Table A control group of 92 healthy non-SM volunteers  were also analysed, based on the same criteria considered for the subjects in the SM group and were enrolled in the study. All healthy participants avoided anti-inflammatory drugs for three days prior to blood collection. The demographic information was collected from the subjects, and they were interviewed for potential risk factors for cancers and autoimmune diseases, including personal and family medical history. This study was done in accordance with the Helsinki DeclarationThe sera of each group  were mixed separately and IgG purification was performed using Melon™ Gel IgG Purification Kit  according to the manufacturer’s instructions. The purification accuracy was confirmed by reducing SDS-PAGE analysis.TM-C7C Phage Display Peptide Library Kit , SU and SL biopanning were carried out for three successive cycles on the purified IgG of NSCLC and SM groups. For SU biopanning, 96-well Nunc MaxiSorp flat-bottom plates were coated with IgG of HC, NSCLC and SM groups. In addition, for SL biopanning, Protein G Mag Sepharose Xtra  was employed to expose IgG, according to the manufacturer’s instructions. In both procedures, the phages of three inputs were subtracted on HC IgG before being transferred to the wells coated with IgG (NSCLC or SM IgG) or microtubes containing IgG (NSCLC or SM IgG) according to the instructions of Ph.D.TM-C7C Phage Display Peptide Library Kit and Protein G Mag Sepharose Xtra).Using Ph.D.TM-C7C kit) (see Supplementary information).Polyclonal phage ELISA was conducted on phages obtained from three rounds of SU and SL biopanning on NSCLC and SM IgG. The specificity of peptides displayed on 60 selected phages (30 clones from SU biopanning and 30 clones from SL biopanning) to NSCLC and SM IgG was individually assayed by monoclonal phage ELISA according to the manufacturer’s instructions  (http://immunet.cn/bdb/) to discard target-unrelated peptides126. Based on default parameters for the Blastp (score ≥18.5), the selected peptides were blasted against the Refseq_protein database for the Homo sapiens127.To extract the single-stranded phage DNAs, 20 and 10 clones obtained from SU and SL biopanning on NSCLC IgG, along with 15 and 10 clones obtained from SU and SL biopanning on SM IgG, were selected, respectively, according to the Ph.D.http://cpdb.molgen.mpg.de) to find significant protein complexes with at least two members in the NSCLC or SM protein dataset .NSCLC- and SM-specific proteins obtained through blasting (score ≥18.5) and preliminary analysis of the literature and UniProt database, were assessed in CPDB  was used to exhibit the top cellular processes and diseases in two datasets. For this purpose, the NSCLC and SM protein datasets were separately introduced to Pathway Studio® and the Summary Analysis function was used to present a quick analytical summary of the datasets.Pathway Studio® 11.2.5.9 (http://string-db.org)128 and the nodes (proteins) and edges (protein-protein interactions) were extracted and transferred to Gephi 0.9.1129. The combined scores (from STRING) were defined as the edge weights. Modularity function implemented in Gephi was used for clustering, and the nodes were coloured differentially based on their modules. To identify GO terms and pathways related to the top six NSCLC and SM modules, Metascape (http://metascape.org)130 and Enrichr web tools (http://amp.pharm.mssm.edu/Enrichr/)131 were recruited. Disease Association Analysis in the WebGestalt database 132 was performed for 25 NSCLC- and 26 SM-specific nodes (hubs) which showed the highest scores of betweenness centrality and degree. Target-based search were also implemented with DrugBank 5.0 (https://drugbank.ca)133 and Reaxys® (https://reaxys.com) to identify marketed drugs affecting NSCLC-specific hubs.For visualizing molecular interaction networks, the NSCLC and SM protein datasets were introduced to STRING 10.0  and NOTCH1 (R&D systems), as the representatives of NSCLC and SM groups, were selected for ELISA, respectively (see Supplementary information).p-value < 0.05) was determined by two-tailed Student’s t-test.The statistical analyses were carried out using GraphPad prism®. The data were stated as the mean ± SD and the statistical significance (Supplementary InformationSupplementary Dataset


The p62–Keap1–Nrf2 pathway plays a protective role in normal cells; however, recent studies indicate that this pathway induces tumorigenesis of pre-malignant cells, and promotes the growth and drug resistance of tumor cells via metabolic reprogramming mediated by Nrf2 activation. These findings suggest that impairment of autophagy is involved in the acquisition of malignancy and maintenance of tumors, and furthermore, that p62/SQSTM1 could be a potential target for chemotherapy in cancers that harbor excess p62.Autophagy and the Keap1–Nrf2 system are major cellular defense mechanisms against metabolic and oxidative stress. These two systems are linked  Autophagy is a bulk degradation process in which cytoplasmic components are sequestered in a double-membrane structure to form an autophagosome. The contents of the autophagosome are subsequently degraded after fusion with a lysosome Figure  1). Und. Und1). via direct interaction with microtubule-associated protein light chain 3 (LC3) on the autophagosome  acts as a hub protein in various cellular signaling pathways, including NF-κB, mechanistic target of rapamycin (mTOR), Caspase 8, and nuclear factor erythroid 2-related factor 2 (Nrf2) –7. p62 mhagosome . There ahagosome –12. Ubiqhagosome , while uhagosome .via characteristic domains, such as the zinc finger (ZZ) domain, TRAF6 binding (TB) domain, LIR, and Keap1-interacting region (KIR), which interacts with RIP kinase, TRAF6, Raptor, LC3, and Keap1, respectively   . Keap1 b (Keap1) –34, Keap (Keap1) . The relIn previous reports, we demonstrated that phosphorylation of p62 at serine 349 results in Nrf2 activation , which ap62 in an HCC cell line markedly abrogates tumor growth, whereas forced expression of a phosphorylation-mimic allele of p62, but not a phosphorylation defective mutant, resulted in recovery of the growth defect  has been found in hepatic adenoma in autophagy-deficient livers and in hepatitis C virus-positive human hepatocellular carcinoma (HCC) , 37. Knoh defect . These r pathway . Consistng liver , 37, kidng liver , 39, lunng liver , breast ng liver , 42, panng liver , prostatng liver , 45, heang liver , 47, ovang liver , 49, orang liver , 51, colng liver , 53, endng liver , skin  in normal cells. Recent studies demonstrate that Nrf2 increases the expression of multiple enzymes involved in the pentose phosphate pathway, purine nucleotide synthesis, as well as glutathione synthesis, and glutaminolysis in lung cancer, which activates the phosphatidylinositol 3-kinase-Akt pathway . In suppBy chemical screening, we have identified an inhibitor for the Keap1-phosphorylated p62 p-S349) protein–protein interaction—the acetonyl naphthalene derivative K67  (Figure 49 proteiIn recent years, there are many reports showing that p62–Keap1–Nrf2 pathway plays a protective role in oxidative and stress conditions. For example, ER stress-induced apoptosis is prevented by the activation of p62–Keap1–Nrf2 pathway . QuercetAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Lactobacillus gasseri AL3 and AL5 that allow an in-depth investigation of the genes involved in oxidative metabolism and the stress response.Adaptation to the aerobic environment has been investigated in heterofermentative lactobacilli, while data on how homofermentative lactobacilli adapt to oxygen are limited. We report here the draft genome sequences of the aerobic strains  Lactobacillus gasseri is abundantly present in the vaginal microbiota, is found in the human intestine, and is receiving particular attention due to its industrial applications  broth, and pellets were sent for genomic DNA extraction and sequencing on an Illumina HiSeq platform at the GATC Biotech AG Company (Germany). Sequencing led to 11,092,500 (AL3) and 12,136,300 (AL5) paired-end reads. Low-quality bases (Phred score <20) were trimmed, and reads shorter than 60 bp were discarded using the SolexaQA++ software (bd-I oxidase [CydABCD]), and oxidative stress response . A match was considered valid when showing more than 40% of identity over at least 90% of the query length. Functional annotation of all genes was carried out using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The presence of ribA and ribH genes was used as a Lactobacillus gasseri species-specific marker . A total of 1,945 and 1,998 genes were found in the AL3 and AL5 genomes, respectively. The AL3 draft genome contains 1,874 predicted coding sequences (CDSs), 77 pseudogenes, 61 tRNAs, and 71 rRNAs. The AL5 draft genome contains 1,932 CDSs, 100 pseudogenes, 56 tRNAs, and 66 rRNAs. Plasmids were not found by using PlasmidFinder 1.3  with genes encoding NOX of Lactobacillus brevis, GshA of Micrococcus luteus, SOD of Atopostipes suicloacalis, and Dps of Lactobacillus reuteri. Genes encoding GshB, GshF, CAT, and Man-CAT were not found.ications –5. Lactosoftware , and asssoftware . Genes wc marker . The AL3MTZT00000000 and MUJA00000000 for strains AL3 and AL5, respectively. The versions described in this paper are versions MTZT00000000 and MUJA01000000.This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession numbers


Evidence on biological activities of cooked black rice is limited. This study examined the effects of washing and cooking on the bioactive ingredients and biological activities of black rice.Cooked rice was prepared by washing 0–3 times followed by cooking in a rice cooker. The acidic methanol extracts of raw and cooked rice were used for the analyses.Raw black rice, both washed and unwashed, had higher contents of polyphenols, anthocyanins, and cyanidin-3-glucoside (C3G), but lower protocatechuic acid (PA), than did cooked samples. Similarly, raw rice extracts were higher in ferric-reducing antioxidant power (FRAP) activities than extracts of cooked samples. Nonetheless, extracts of raw and cooked rice showed similar inhibitory potencies on nitric oxide, tumor necrosis factor-α, and interleukin-6 productions in lipopolysaccharide-activated macrophages, whereas equivalent amounts of C3G and PA did not possess such inhibitory effects.Thermal cooking decreased total anthocyanin and C3G contents and the FRAP antioxidative capacity, but did not affect anti-inflammatory activities of black rice. Neither C3G nor PA contributed to the anti-inflammatory activity of black rice. Oryza sativa L. indica) is a cultivar widely consumed in Asian countries and is rich in anthocyanins, especially cyanidin-3-glucoside (C3G) and peonidin , a major degradation product of anthocyanins –12. Surhs-triazine, trifluoroacetic acid, potassium persulfate, sodium hydroxide, Folin–Ciocalteu's reagent, acetonitrile, and methanol were purchased from Sigma-Aldrich . PA was from Cayman . Dulbecco's modified Eagle medium (DMEM) and fetal bovine serum (FBS) were obtained from GIBCO . All chemicals and solvents used in the study were of reagent grade.C3G, gallic acid, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (TROLOX), dimethyl sulfoxide (DMSO), 2,4,6-tripyridyl-Black rice was purchased from a local market in Taipei, Taiwan. The rice was washed 0–3 times followed by immediate cooking with an electronic rice cooker for 25 min. Washing was performed by soaking the rice in cold water for 5 min, and discarding the water. Ground dry raw and cooked rice samples were then extracted with water, methanol, ethanol, acidic methanol , or acidic ethanol  for 24 h, and the respective extracts were obtained after centrifugation. All experiments were performed at least three times, and the results are expressed on a dry matter basis.TPCs in black rice extracts were determined spectrophotometrically at 755 nm after adding the Folin–Ciocalteu reagent , and the18 column  under a visible wavelength of 520 nm (Hitachi) using an authentic C3G standard. The mobile phase consisted of solvent A (4.5% formic acid in water) and solvent B (50% acetonitrile with 4.5% formic acid in water) with a gradient of solvent B of 10% (0 min), 25% (30 min), 33% (34 min), 90% (42 min), and 10% (45–50 min)  with a C–50 min) , 15. PA,To understand whether the TPCs or TACs were associated with antioxidant activities, the antioxidant power of the extracts was measured by a ferric-reducing antioxidant power (FRAP) assay using TROLOX as a standard .2 environment. Cells (1–5×105) were treated with lipopolysaccharide (LPS at 100 ng/ml) in the presence of different treatments for 16 h, and the medium and cells were collected for analysis.Murine RAW 264.7 macrophages were used to study the anti-inflammatory activities of black rice. Cells were obtained from the Culture Collection and Research Center , and were grown in DMEM supplemented with 10% FBS at 37°C under a 5% COH-tetrazolium (MTS) produced by live cells at 490-nm wavelength. The production of inflammatory mediators, including nitric oxide (NO), tumor necrosis factor (TNF)-α, and interleukin (IL)-6, secreted into the medium was determined. NO, as quantitated by nitrite, was determined by the Griess reaction, and TNF-α and IL-6 were analyzed by commercial enzyme-linked immunosorbent assay kits .Cell proliferation was evaluated by measuring the absorbance of the formazan product of [3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2t-test was used to compare means between the LPS and control groups. A one-way ANOVA followed by the least significance difference test was used to compare means between groups of LPS-treated macrophages. All statistical analyses were performed with the aid of SPSS software version 19 . Differences between groups were considered significant at p<0.05.Values are expressed as the mean±standard deviation (SD). A two-way analysis of variance (ANOVA) with repeated measures was carried out to compare effects of the cooking and washing processes. A Pearson correlation analysis was performed to express the relationships of total polyphenols, total anthocyanins, C3G, and PA with antioxidant activities. Student's Polyphenols, particularly anthocyanins, are major contributors to the dark-purple color of black rice, but the contents may vary with different cooking processes and extraction solvents, so TPCs and TACs in black rice extracts from different preparations were analyzed. Results showed that acidic methanol extracts had 285–730% higher TACs from raw black rice than other extracts, including water, ethanol, and acidic ethanol , so acidic methanol extracts were used in the following experiments. C3G and PA, as one of the major anthocyanins and anthocyanin degradation products in black rice , 17, resIn order to understand the antioxidative activities of the acidic methanol extracts of black rice, the FRAP antioxidant capacity activity was determined. Results showed that washing slightly decreased the FRAP antioxidant capacity, but cooked rice had a lower FRAP capacity than did raw samples . In addiTo understand the biological effects of black rice, LPS-stimulated RAW 264.7 cells were used to study the anti-inflammatory activities of extracts of different black rice preparations. Results indicated that extracts of black rice  showed no cytotoxicity (data not shown), but extracts from both raw and cooked rice showed similar inhibitory effects on the production of LPS-stimulated inflammatory mediators, including NO a, TNF-α In this study, we demonstrated that thermal cooking for 25 min with no presoaking significantly reduced the TPCs, TACs, and C3G levels in black rice, and such decreases were associated with decreased FRAP antioxidant activity, indicating that ordinarily consumed cooked black rice possessed lower polyphenol contents and antioxidative activities. On the contrary, washing up to three times did not have significant effects on the polyphenol contents, although it slightly decreased the FRAP antioxidant capacity. We observed that 1 g of dry raw and cooked black rice respectively contained 5.3–5.9 versus 3.1–4.4 mg polyphenols, 2.1–2.4 versus 1.0–1.2 mg anthocyanins, and 650–791 versus 238–296 µg C3G, which agreed well with results obtained by Sompong et al.  showed tPolyphenols and anthocyanins are labile to heat treatment and decreased anthocyanin contents after thermal treatment were observed in various polyphenol-rich foods, such as blueberry juice, pomegranate juice, and black rice , 20. ResAlthough cooked rice had lower phenolic compounds and antioxidative activity, it possessed similar anti-inflammatory activities as raw rice, as determined by assays of NO, TNF-α, and IL-6. Prolonged inflammation evoked by over-activated macrophages is positively associated with a variety of pathological conditions, including autoimmune diseases, cardiovascular diseases, and cancers. Therefore, compounds that decrease the overproduction of inflammatory mediators may protect the body from inflammation-associated diseases . In thisThermal cooking rather than washing decreased total polyphenol, anthocyanin, and C3G contents of black rice, and these contents were associated with decreased FRAP antioxidative activity. On the contrary, cooked rice possessed similar anti-inflammatory activities as raw rice, and neither C3G nor PA contributed to the anti-inflammatory activities of black rice. These results suggest that cooking processes do not attenuate the potential health-promoting effects of black rice.Click here for additional data file.


Pseudomonas palleroniana MAB3, a 1-aminocyclopropane-1-carboxylate deaminase-producing bacterium isolated from an environmental soil Amanita mushroom, is presented here. The genome of P. palleroniana MAB3 contains a single circular chromosome of 6.29 Mb and an average GC content of 60.5%.The near-complete genome sequence of  Ethylene plays an important role in the developmental processes of plants and basidiomycete mushrooms . BacteriPseudomonas palleroniana MAB3, an ACC deaminase-producing bacterium isolated from an environmental soil Amanita mushroom (fruit body), which confers significant beneficial effects on both mushroom and plant development.In this work, we report the genome sequence of de novo genome assembly was performed using SOAPdenovo version 2.04 , constructed based on a guided assembly against the genome sequence of P. palleroniana BS3265  using MAUVE version 2.4.0 progressive alignments  according to the manufacturer’s instructions. The DNA library was constructed using the Illumina TruSeq DNA Nano kit (automated) and was sequenced using the MiSeq platform and the MiSeq V3 reagent kit (2 × 300 bp), generating a total of 2,362,402 reads. The initial Pipeline .P. palleroniana MAB3 contains a single circular chromosome of 6.29 Mb in length with an average GC content of 60.5%. A total of 5,753 open reading frames were predicted, in which 5,679 correspond to protein-coding sequences (CDSs). A total of 74 RNA-related genes were also found.The genome of BlastKOALA analysis resulted in the functional annotation of 3,099 from a total of 5,677 CDSs (54.6%) in which environmental (974) and genetic (647) information processing functions were assigned for most of the annotated CDSs, followed by cellular processes (454) and amino acid (362), carbohydrate (283), cofactor and vitamin (209), energy (203), lipid (112), and nucleotide (103) metabolism. AntiSMASH analysis revealed the presence of several gene clusters involved in the production of nonribosomal peptides, siderophores, bacteriocins, and aryl-polyene. Additionally, genetic traits involved in plant and mushroom growth promotion, such as the ACC deaminase gene and indole-3-acetic acid and cytokinin biosynthesis genes, were found in the genome.P. palleroniana MAB3 will bring new insights into the genetics and molecular interactions between beneficial bacteria, mushrooms, and plants, which play a key role in the development of important agricultural and biotechnological applications.The genome sequence of P. palleroniana MAB3 has been deposited in GenBank under the accession no. CP025494.The near-complete genome sequence of


Arabidopsis USP gene (AtUSP) showed induction in response to abscisic acid (ABA) and various abiotic stresses viz. heat, dehydration, salt, osmotic, and cold stresses. Additionally, in silico analysis of AtUSP promoter identified several cis-elements responsive to phytohormones and abiotic stresses such as ABRE, ERE, DRE, and HSE, etc. To functionally validate the AtUSP promoter, the 1115 bp region of promoter was characterized under phytohormone and abiotic stress treatments. Deletion analysis of promoter was carried out by cloning the full length promoter (D0) and its three 5′ deletion derivatives, D1 (964 bp), D2 (660 bp), and D3 (503 bp) upstream of the β-glucuronidase (GUS) reporter gene, which were then stably transformed in Arabidopsis plants. The AtUSP promoter (D0) showed minimal activity under non-stress conditions which was enhanced in response to phytohormone treatments (ABA and ACC) and abiotic stresses such as dehydration, heat, cold, salt, and osmotic stresses. The seedlings harboring D1 and D2 deletion fragments showed constitutive GUS expression even under control condition with increased activity almost under all the treatments. However, D3 seedlings exhibited complete loss of activity under control condition with induction under ACC treatment, dehydration, heat, oxidative, salt, and osmotic stresses. Thus, present study clearly showed that AtUSP promoter is highly inducible by phytohormones and multiple abiotic stresses and it can be exploited as stress inducible promoter to generate multi-stress tolerant crops with minimal effects on their other important traits.Universal stress proteins (USPs) are known to be expressed in response to various abiotic stresses in a wide variety of organisms, such as bacteria, archaebacteria, protists, algae, fungi, plants, and animals. However, in plants, biological function of most of the USPs still remains obscure. In the present study,  During their life cycle, plants have to cope with various abiotic stresses, such as drought, salinity, high or low temperature, oxidative stress, etc. To counter these stresses, plants have evolved various stress tolerance and avoidance mechanisms. These mechanisms involve signal transduction cascades that respond to external stimuli by activating stress-responsive genes, which in turn bring about some morphological, physiological, and metabolic changes that help plants to survive these adverse conditions . In the Escherichia coli, six USP paralogs have been identified namely, UspA, UspC, UspD, UspE, UspF, and UspG  were first discovered in bacteria to be overexpressed in response to various stresses . Accumuland UspG . Loss ofand UspG . Broadlyn kinase . Kerk etrachiata . Howevercis-acting regulatory elements present in the promoters of the genes are a type of transcriptional gene regulatory elements that are required for the specific expression pattern of a gene. Till date, various stress-responsive cis-elements have been identified in the promoters of stress inducible genes that allow their stress specific expression. The promoters of multiple stress-responsive genes have a number of regulatory elements that respond to multiple stresses, such as ABRE (abscisic acid-responsive element), LTRE (low temperature-responsive element), MYC and DRE (dehydration-responsive element) are the potential targets for regulating stress inducible expression of transgenes in transgenic plants  expression which was lost in the subsequent deleted promoter fragments. This study showed that AtUSP promoter is multiple stress-responsive and can be used for guiding multiple stress-responsive expression of transgene in transgenic plants.In the present study, bidopsis . TherefoArabidopsis thaliana grown on ½ strength Murashige and Skoog  and transgenic koog MS;  medium sFor expression and promoter analysis, 2 week-old WT and transgenic seedlings were subjected to different abiotic stress and hormone treatments. ABA (100 μM), ACC , salt , osmotic  and oxidative (50 μM methyl viologen) treatments were given by placing the seedlings on filter paper saturated with respective chemical in the ½ MS solution for 8 h . DehydraAtUSP (At3g53990) and GUS genes (Supplementary File S1) were designed using Primer Express 3.0.1 software (Applied Biosystems). The β-actin was used as internal control and the amplification specificity was determined by melt curve analysis. The expression analysis was performed with three biological and three technical replicates and relative gene expression was analyzed using 2-ΔΔCT method as described previously  excluding the coding sequence. Based on the position of different stress-responsive elements identified through in silico analysis, three 5′ promoter deletions were made by sequentially deleting 151, 455, and 612 bp from the 5′region and were named as D1, D2, and D3, respectively. Same reverse primer with NcoI site was used to amplify D0, D1, D2, and D3 fragments with SalI site in the D0 and EcoRI site in the D1, D2, and D3 forward primers (Supplementary File S2). All these fragments were cloned into promoter-less vector pCAMBIA1391z fused with GUS gene and their schematic representation is depicted in Supplementary Figure S1.A 1115 bp region of AtUSP promoter sequence was analyzed using PLACE1 and PLANTCARE2 databases to identify various cis-regulatory elements present in the AtUSP promoter.The Arabidopsis thaliana (WT) plants grown at 22–23°C with 16/8h photoperiod and light intensity of 100 μmolm-2s-1 were used for Agrobacterium-mediated transformation using vacuum infiltration method. For this, the unopened buds were dipped in the Agrobacterium tumefaciens (GV3101 strain) suspension in YEP medium with 30% sucrose and 0.2% Silvet-L77 solution by inverting the pots and applying the vacuum of 400 mm Hg for 5 min. The transformed plants were kept in the horizontal position in the dark for 24 h and then grown until the T0 generation seeds were harvested. T0 generation seeds were screened on 1X MS medium (1% sucrose and 0.8% agar) with hygromycin selection (20 μg/ml). The true transformants that were hygromycin resistant and able to reach the four leaf stage were transferred to pots containing a 1:1:1 mixture of vermiculite:perlite:coco peat. These plants were grown to maturity until the T1 generation seeds were harvested. Similarly, T1 and T2 seeds were germinated and plants were grown to maturity to harvest T2 and T3 generation seeds, respectively. Genomic DNA of eight T3 transgenic lines of Arabidopsis transformed with empty vector pCAMBIA1391z and recombinant vectors with D0, D1, D2, and D3 promoter fragments were isolated and analyzed for the presence of respective promoter fragment using PCR amplification . The positive T3 transgenic Arabidopsis lines were used for functional characterization of AtUSP promoter and transgenic lines harboring empty vector pCAMBIA1391z were used as negative control.Four-week old Arabidopsis seedlings with or without treatment were kept in GUS staining buffer containing 1 mM 5-bromo-4-chloro-3-indolyl β-D-glucuronidase (HiMedia), 100 mM sodium phosphate (pH 7.5), 0.5 mM potassium ferricyanide and 0.5 mM potassium ferrocyanide, 10 mM ETDA and 0.1 % (v/v) Triton-X 100 and a vacuum of 400 mm of Hg was applied for 5 min. Seedlings were incubated at 37°C overnight and cleared with 70% ethanol in order to remove chlorophyll for clear visualization of blue color of GUS stain. The stained seedlings were then observed and photographed under Carl-Zeiss Stereo DiscoveryV12 with AxioVision software.Histochemical GUS assay was carried out using the method as described previously . Two weeD-glucuronide as substrate . Protein was extracted from stress treated seedlings frozen in liquid nitrogen by grinding in GUS extraction buffer [50 mM sodium phosphate (pH 7.0), 10 mM β-mercaptoethanol, 10 mM EDTA, 0.1% (w/V) N-lauroyl sarcosine , and 0.1% (v/v) Triton X-100). The tissue extract was centrifuged for 15 min at 4°C at 13000 rpm. The 10 μL of the protein extract was mixed with 390 μL of aliquoted GUS assay solution containing 22 mg MUG in 50 ml extraction buffer and incubated at 37°C for 1 h. The 100 μL of sample was taken from each tube at 0, 30, and 60 min incubation time, and 4.9 ml of 0.2 M Na2CO3 was added to stop the reaction. Fluorescence was measured with a fluorometer. Protein concentration was determined according to Bradford reagent using BSA standard  in response to phytohormone treatments (ABA and ACC) and abiotic stresses such as dehydration, salt, osmotic, oxidative, heat, and cold stress . The expression of AtUSP was highly induced under ABA treatment and all the abiotic stress conditions, except for the oxidative stress. The highest induction was observed after heat exposure (22.9-fold) followed by cold (17.7-fold), and dehydration (16.4-fold). Salt and osmotic stresses also led to induction of AtUSP transcript levels by 2.3- and 3.7-fold, respectively . ABA treatment resulted in 8.7-fold induction, while ACC treatment led to no significant change in the AtUSP expression. AtUSP gene expression was downregulated by 1.7-fold in response to oxidative stress.The expression level of AtUSP was found to be multiple abiotic stress-responsive, its 1115 bp region upstream to translation start site was selected to characterize its promoter. This region was amplified from the genomic DNA of WT Arabidopsis and cloned in pGEM-T easy vector. The promoter sequence was analyzed using PLACE and PLANTCARE databases that led to identification of different putative cis-acting regulatory elements present in the AtUSP promoter (Table 1). The sequence motifs for ABA (ABRELATERD1 and ABRERATCAL), ethylene (ERELEE4), and gibberellin (GARE2OSREP1) were present at -700, -701, -238, and -377 positions, respectively . Two motifs for salicylic acid (WBOXATNPR1) were present at positions -5 and -965. Five MYCCONSENSUSAT motifs at -153, -595, -678, -730, -1089, and one motif of each MYCATRD22 and MYBCONSENSUSAT were present at positions -678 and -662, respectively. Additionally, three motifs of MYBCORE elements known to be water stress-responsive were also present at -168, -659, and -662 positions. Five motifs of GT1GMSCAM4 responsible for pathogen and salt inducible expression were also found to be present in AtUSP promoter. It also contained two motifs of PREATPRODH known to be hypo-osmolarity responsive. AtUSP promoter had a number of cis-acting responsive elements for tissue specific expression such as CAATBOX1 and GATABOX present in several repeats . It also contained certain motifs required for organ-specific expression, for example, PROXBBNNAPA for seed-specific expression, CACTFTPPCA1 for mesophyll cell-specific expression, RAV1AAT for high expression in rosette leaves and roots, RHERPATEXPA7 for root-hair specific expression, ROOTMOTIFTAPOX1 for root-specific expression, POLLEN1LELAT52 for pollen-specific expression, and TAAAGSTKST1 for guard cell-specific expression.Since AtUSP translation start site was cloned in the promoterless vector pCAMBIA1391z at SalI/NcoI sites and named as D0 construct containing the full length AtUSP promoter. Then, three deleted fragments were amplified by sequentially deletion of 151, 455, and 612 bp from the 5′ side of the full length promoter . These fragments were then individually cloned into the promoterless vector pCAMBIA1391z at EcoRI/NcoI sites and were designated as D1 (964 bp), D2 (660 bp), and D3 (503 bp) constructs . The D0, D1, D2, and D3 constructs were then transformed into Arabidopsis (WT) by Agrobacterium-mediated transformation using vacuum infiltration method. True transformants were first screened on 1X MS agar plates containing hygromycin and then further checked for the presence of desired fragment by PCR amplification using forward primer of each promoter fragment and reverse primer from GUS gene.The 1115 bp region just upstream of 3 seedlings of D0, D1, D2, and D3 transgenic Arabidopsis lines under non-stress and stress conditions . Under control conditions, the GUS gene showed 12.4- and 6.4-fold induction in the D1 and D2 lines, respectively, while 1.4-fold reduction in D3 lines with respect to D0 lines . The D0 lines exhibited 2.5- and 4.5-fold GUS gene induction in response to ABA and ACC treatments, respectively. The D0 lines showed 7.2-, 6.6-, 5.0-, 4.0-, and 2.5-fold induction in the GUS gene expression under, dehydration, heat, cold, salt and osmotic stress, respectively . While in case of oxidative stress, GUS expression was unaltered in D0 lines . The D1 lines showed GUS gene induction only under cold (4.1-fold) stress with respect to D1 lines under non-stress conditions . The GUS expression was downregulated in response to ABA, ACC, oxidative and heat stress in the D1 lines . In case of D2 lines, the GUS expression was induced (1.9-fold) only under cold stress , while, GUS expression was downregulated in response to ABA, ACC, oxidative dehydration, salt and osmotic stress . The D3 seedlings showed induction in GUS expression in response to ACC treatment (2.7-fold), oxidative (1.6-fold), dehydration (3.1-fold), heat (2.8-fold), and salt stress (1.8-fold) with respect to D3 seedlings under non-stress conditions . The GUS expression was downregulated by 3.4-fold in the D3 seedlings only under ABA treatment .The GUS transcript level was measured using qRT-PCR in the 2 week old TAtUSP gene was found to be induced under multiple abiotic stresses, T3 generation of transgenic plants harboring AtUSP promoter and its three 5′ deleted fragments were analyzed for GUS induction under hormone treatments (such as ABA and ACC) and abiotic stresses . Under control conditions, strong GUS stain was detected in roots, while, a slight GUS staining was also detected in the region surrounding the mid-rib of the leaves, and hypocotyl of the D0 seedlings . In contrast, a strong GUS stain was observed in the entire leaf and root of the D1 and D2 seedlings under control condition. However, GUS stain was not detected in the D3 seedlings under control condition. Under ABA treatment, dark GUS stain was detected in the leaf tips, stomata, and roots of the D0 seedlings. However, in the D1 seedlings, GUS expression was found to be in the entire leaf, hypocotyls, and roots under ABA treatment. The GUS staining in D2 seedlings in response to ABA treatment was comparable to GUS stain found in untreated D2 seedlings which was evident by comparable GUS activities observed in the D2 seedlings under both the conditions . However, GUS staining was completely absent in D3 seedlings. ACC treatment resulted in slight GUS staining in the region surrounding mid-rib of leaves, hypocotyls, and roots in the D0 seedlings. Similar GUS staining pattern was observed in the leaves and roots of the D1 and D2 seedlings. ACC treated D3 seedlings also exhibited GUS staining in the leaves and roots. Dehydration stress also resulted in similar GUS staining pattern as that observed in case of ABA treatment. Strong GUS expression was observed in the leaf tip and stomata as in case of ABA treated D0 lines. The D1 and D2 seedlings also showed GUS expression following dehydration. However, in contrast to ABA treatment, D3 seedlings also exhibited GUS staining in response to dehydration, which was further confirmed by GUS activity assay with 1.9-fold increase in GUS activity observed under dehydration . In D0 and D1 seedlings, GUS stain was detected in the region surrounding the leaf tips, hypocotyl, stomata, and roots under heat stress. Similar GUS staining was detected in D1 seedlings except for roots as GUS stain was detected only in root apex. Comparatively, weaker expression was observed in D2 and D3 seedling under heat stress, where GUS stain was detected in the entire leaf, hypocotyls, and roots. With cold treatment, GUS stain was detected only at the leaf tip, hypocotyl, stomata, and roots in the D0 seedlings. The tissue specific cold induction in GUS expression was observed in D1 seedlings with stronger expression in the region surrounding the leaf tip, stomata, hypocotyl with weak GUS expression in the roots. There was complete loss of GUS staining in the D3 seedlings under cold stress. Oxidative stress resulted in the weak GUS expression at the leaf tips, stomata, and roots of D0 and D1 seedlings as compared to control conditions. Interestingly, significant GUS activity (2.8-fold) was detected in D3 seedlings when subjected to oxidative stress . Under salt stress, GUS stain can be detected in the region surrounding the leaf tip, mid-rib region, hypocotyl, and in the entire root of the D0 seedlings. Similar GUS stain was also detected in the D1 seedlings with lower staining in the roots. Strong GUS staining was also observed in the salt treated D2 and D3 seedlings. Osmotic stress also resulted in similar GUS expression pattern in D0 seedlings as observed under salt stress, except in roots where GUS staining was found only in the root tip. Slight induction in GUS expression was observed in the D1 and D2 seedlings, while, weaker induction was still observed in D3 seedlings .Since USP genes has been shown to be induced under various abiotic stresses, but their precise molecular function is still unknown  and its three 5′ deletion derivatives were generated and used to drive expression of GUS reporter gene by cloning them in a promoterless vector pCAMBIA1391z. These constructs were then transformed into WT A. thaliana by Agrobacterium tumefaciens-mediated transformation. The 1115 bp region of AtUSP promoter contained several cis-regulatory elements required for tissue specific, hormone, and stress-responsive expression. AtUSP promoter contained MYCCONSENSUSAT, MYCATRD22, MYBCORE, and MYBCONSENSUSAT elements which were reported to be also present in the promoter of dehydration-responsive RD22 that is known to play role in ABA, drought and cold signaling  promoter was strongly activated by ABA, ACC, dehydration, salt, osmotic, heat, and cold stresses. These results strengthen our qRT-PCR based gene expression analysis which also highlighted the multiple stress-responsive characteristic of AtUSP. Recently, AtUSP possess molecular chaperone like function which is regulated by ROS homeostasis. They also showed that AtUSP plays an essential role in heat and oxidative stress tolerance in Arabidopsis. ABA accumulation has been shown to act as trigger to mediate stress tolerance during dehydration, salt, and cold stresses . ABA induction of GUS activity was lost in the D2 and D3 seedlings with deletion of two ABRE motifs. ABA-independent signaling involves dehydration-responsive elements in promoter region that are bound by DRE binding transcription factors (DREBs/CBFs) to induce the expression of downstream genes. Several studies have shown that overexpression of DREB/CBF lead to drought, salinity, and freezing tolerance in crops (AtUSP promoter also contains several DRE elements that might be responsible for its multiple stress-responsive characteristic. D3 seedlings also showed weak induction with respect to non-stress conditions in response to dehydration which might be due to the fact that it still contains dehydration-responsive elements such as MYCCONSENSUSAT, LTRECOREATCOR15, DRECRTCOREAT, CBFHV, and DRE2COREZMRAB17 which have been shown to be part of ABA-independent signaling. Strong induction of GUS expression in D0 and D1 seedlings under osmotic stress in comparison to their respective controls can be attributed to presence of all the motifs of ABRE, MYB2CONSENSUSAT, MYCCONSENSUSAT, DRECRTCOREAT, and DRE2COREZMRAB17 which are directly or indirectly involved in osmotic stress response. Ethylene has been shown to play a positive role in mediating the plant salt tolerance (AtUSP gene under oxidative stress.Based on the multiple stress-responsive characteristic of ignaling , 2003. Signaling . ABA-resectively . GT1GMSCectively  were alselements . For D0 elements . The comelements . Under celements . The fulstresses . Therefostresses . AtUSP pin crops . AtUSP polerance . Similarolerance . Strongeolerance . Strong AUSP gene and its promoter was functionally characterized under phytohormone and abiotic stress treatments. The GUS expression and histochemical studies showed that AtUSP promoter can respond to phytohormones such as ABA, ethylene, and several abiotic stresses such as dehydration, salt, osmotic, heat, and cold stress. Therefore, the D0 promoter fragment can be considered as potential multiple stress inducible promoter to derive the expression of transgenes that may confer stress tolerance to the plants.The present study reveals the multiple stress-responsive characteristic of AS conceived and designed the experiments. MB and PG performed the experiments. MB and AS performed the data analysis. AS and SK secured the funds to support this research. MB wrote the manuscript. MB and AS revised and finalized the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Accurate, rapid, and early diagnosis of dengue virus (DENV) infections is essential for optimal clinical care. Here, we evaluated the efficacy of the quantitative real-time PCR (qRT-PCR)-LightMix dengue virus EC kit for DENV detection using samples from a dengue outbreak in Taiwan in 2015.Sera from patients with suspected DENV infection were analyzed and compared using the LightMix kit, a Dengue NS1 Ag + Ab Combo kit for detection of NS1 antigen and DENV-specific IgM and IgG antibodies, and an “in-house” qualitative DENV-specific RT-PCR assay.2 and 106 copies/reaction, and the minimum detection limits for DENV serotype 1 (DENV1) and DENV2, DENV3, and DENV4 were 1, 10, and 100 focus forming units (FFU)/mL, respectively. There was 88.9% concordance between the results obtained using the NS1 antigen combo kit and by LightMix analysis, and the diagnostic sensitivity and specificity of the two methods were 89.4 and 100%, and 84.7 and 100%, respectively. Notably, fatal cases were attributed to DENV2 infection, and 79.5% (27/34) of these cases occurred in patients ≥ 71 years of age. Among these older patients, 82.3% (14/17) were NS1/IgM/IgG (+/-/-), exhibiting VLs between 106–109 copies/mL, which was markedly higher than the rate observed in the other age groups.A total of 8,989, 8,954, and 1581 samples were subjected to NS1 antigen detection, IgM and IgG detection, and LightMix assays, respectively. The LightMix assay yielded a linear curve for viral loads (VL) between 10The LightMix assay was effective for early diagnosis of DENV infection. Our data indicate that high VLs during primary infection in elderly patients may be a positive predictor for severe illness, and may contribute to high mortality rates. The LightMix dengue virus EC qRT-PCR assay is effective for early diagnosis of DENV infection. High viral loads during primary infection in elderly patients may comprise a positive predictor for severe illness, and may contribute to high mortality rates. Flavivirus (family Flaviviridae) that is transmitted by mosquitos, causing endemic and epidemic outbreaks in tropical and subtropical areas. It is estimated that approximately 390 million people are infected with DENV globally each year, resulting in disease manifestation in roughly 96 million patients , DENV2-454009A (6 × 105 FFU/mL), DENV3-8700829 (4 × 105 FFU/mL), and DENV4-59201818 (2 × 105 FFU/mL) were kindly provided by the Center of Infectious Disease and Signal Research, NCKU.The viral reference strains DENV1-8700828 [4 × 101 to 106 copies/reaction), which were provided with LightMix dengue virus EC kit. Within-run samples were tested in triplicate during the same run, while positive values from another two runs were used for evaluating between-run precision, which represent the coefficient of variation (CV). In parallel, the analytical specificity of the assay was tested using clinical samples containing various viral species. Lastly, the diagnostic sensitivity and specificity of the assays were determined by comparing the results obtained from clinical specimens collected from suspected DENV-infected patients with those obtained by the qRT-PCR assay, in-house qualitative dengue RT-PCR, and NS1 antigen assays.The accuracy, precision, and analytical sensitivity of the LightMix dengue virus EC kit were evaluated using the reference strains listed above or triplicates of serially diluted plasmid DNA standards .Frequency distributions for analyses of viral load (VL) levels and the results of NS1 antigen, and IgM and IgG assays were compared by χth week of August and the 4th week of September , 11.2% , 6.9% , and 40.2%  tested positive for NS1, IgM, and IgG, and tested positive for DENV in the qRT-PCR assay, respectively. As shown in eptember .Of the 44 samples collected from patients with fatal cases  of dengue, 34 tested positive for DENV with the LightMix dengue EC kit, which targets the conserved region of DENV serotypes 1–4. Notably, each of these 34 samples, as well as 67 of the 85 samples obtained from patients diagnosed with severe cases , tested positive for DENV2 by our in-house dengue RT-PCR serotyping assay. Conversely, none of these samples was positive for DENV1, DENV3, or DENV4. The age of the patients associated with fatal disease ranged from 41 years to 91 years, but 42 (95.4%) of the patients were older than 60 years of age.1 to 106 copies/reaction) provided with the LightMix kit. While the CVs for the within-run precision when using 102 to 106 plasmid copies/reaction were 0.29%–7.24%, the CV when using 101 copies/reaction was markedly higher  of the LightMix dengue virus EC qRT-PCR assay for the four subtypes was 1 FFU/mL for DENV1 and DENV2, 10 FFU/mL for DENV3, and 100 FFU/mL for DENV4 , varicella zoster virus, herpes simplex virus, parvovirus B19, John Cunningham virus (JCV), BK virus, Epstein-Barr virus (EBV), or norovirus. Notably, each of the clinical samples tested negative for DENV, indicating that the presence of these 11 other viruses had no effect on the performance of the LightMix dengue virus EC qRT-PCR assay.A total of 199 clinical specimens with suspected dengue virus infection were subsequently evaluated simultaneously by both the LightMix dengue virus EC qRT-PCR assay and another in-house dengue-specific RT-PCR assay. The results obtained from each sample were then compared to a composite reference standard, defined as the detection of DENV in one or both assays. The diagnostic sensitivity of the LightMix dengue virus EC qRT-PCR and in-house RT-PCR assay were 99.4% (179/180) and 72.6% (130/180), respectively.6–109 copies/mL; P < 0.05) than the specimens in the other NS1/IgM/IgG categories. Furthermore, 71.8% (61/85) and 52.3% (23/44) of the samples in the NS1/IgM/IgG (+/-/-) category collected from patients with severe and fatal dengue contained detectable VLs, respectively, which was higher than the rate observed for other NS1/IgM/IgG categories . Furthermore, of the 289 (a + b) samples that were positive for IgM and/or IgG , 37.3%  contained detectable VLs. Meanwhile, VLs of <106 copies/mL were detected in 88.5% ) and 75% ) of the NS1-positive and NS1-negative categories, respectively. Of the 104 (j1 + j2) samples that tested positive for NS1 antigen but exhibited no detectable VL, the majority  also tested positive for IgM and/or IgG. In contrast, the majority of the samples that tested negative for NS1 antigen but had a detectable VL ; 83.3%) were negative for IgM and IgG. Of the 996 LightMix dengue virus EC qRT-PCR assay-negative samples, a statistically significant majority (79.8%) were negative for NS1, IgM, and IgG (P < 0.05). To investigate the detection efficiency of the NS1 and the LightMix dengue virus EC qRT-PCR assays for DENV during the acute phase of infection, the results obtained from each assay were compared. Notably, the concordance between the two assays was 88.9%  tested positive for DENV in the LightMix dengue virus EC qRT-PCR assay. Meanwhile, among these 585 positive samples, 417 (71.3%) were NS1-positive and IgM/IgG-negative (+/-/-), and contained detectable VLs, of which 274  exhibited significantly higher viral copy numbers ) and 57.1% ) of the NS1/IgM/IgG (+/-/-) specimens collected from patients with fatal and severe dengue aged ≥71 years, respectively. Although 10 samples from fatal cases and 17 samples from severe cases were qRT-PCR-negative for DENV, these patients were still diagnosed with dengue infection, as they were positive for either NS1 antigen or IgM/IgG (Among the 34 (a1 + a + b) LightMix dengue virus EC qRT-PCR assay-positive specimens collected from the 44 patients with fatal dengue, half  had VLs of 10opies/mL . Meanwhi IgM/IgG .Currently, detection of DENV-specific nucleic acids during the acute phase of infection is widely applied for dengue diagnosis. Indeed, many studies have evaluated the efficacy of real-time RT-PCR assays for the detection of DENV. The results of these previously published reports are summarized in 2 and 106 copies/reaction (R2 = 0.99). Notably, the assay was more sensitive for detection of DENV1 and DENV2 than for DENV3 and DENV4. Such differences in sensitivity for distinct viral serotypes have been reported in the other studies [A linear curve for the LightMix dengue virus EC qRT-PCR assay was obtained between 10 studies , 24. Ind6–109 copies/mL). Meanwhile, the highest percentage (88.5%) of specimens with medium VLs (<106 copies/mL) was observed in the NS1-positive and IgM and/or IgG-positive category. Notably, the highest ratios ) of antibody-positive and -negative specimens ) were observed in the NS1/VL (+/-) and NS1/VL (-/+) categories, respectively  reported >5 × 106 copies of DENV RNA in samples collected from two suspected dengue secondary infection patients that were IgG-positive but IgM-negative on the first and second day after the onset of illness [Therefore, we evaluated the diagnostic sensitivity of the assay to validate its use for detection of DENV in specimens derived from infected patients. For these analyses, we compared the results obtained by NS1 antigen, IgM, IgG, and LightMix dengue virus EC qRT-PCR assay analyses of samples collected from suspected DENV-infected patients during an outbreak in Taiwan in 2015. The NS1/IgM/IgG (+/-/-) category was associated with the highest proportion 274/417; 65.7%) of samples exhibiting high VLs  observed maximal sensitivity of the NS1 antigen and real time RT-PCR assays for DENV detection on the second and third day of illness, respectively . The groThere were limitations to our study. Notably, the NS1/IgM/IgG (+/-/-) category contained 20 VL-negative specimens , which mS1 Checklist(DOCX)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.


Re-challenging these resistant tumors in mice, we showed that second-line treatment with everolimus particularly affected vasculogenic mimicry and tumor cell differentiation compared to sorafenib and axitinib. Finally, our results highlighted the phenotypic and genotypic changes at the tumor cell and microenvironment levels during sunitinib response and progression and the subsequent improvement second-line therapies bring to the current renal cell carcinoma treatment paradigm.Angiogenesis is hallmark of clear cell renal cell carcinogenesis. Anti-angiogenic therapies have been successful in improving disease outcome; however, most patients treated with anti-angiogenic agents will eventually progress. In this study we report that clear cell renal cell carcinoma was associated with vasculogenic mimicry in both mice and human with tumor cells expressing endothelial markers in the vicinity of tumor vessels. We show that vasculogenic mimicry was efficiently targeted by sunitinib but eventually associated with tumor resistance and a more aggressive phenotype both  Several of these genes can also be considered as marker of tumor differentiation or contribute to epithelial-to-mesenchymal transition (EMT) such as CDH1, EDN1 or ERBB2 for instance. In fact, at the protein level, tumors of mice that progressed during sunitinib treatment expressed high levels of CD133 and vimentin compared to tumors of mice that responded to sunitinib , 1 mg/kg/day everolimus (n = 8), 2.5 mg/kg/day everolimus (n = 8), 5 mg/kg/day everolimus (n = 8), or 60 mg/kg/day sorafenib (n = 5). In the second set, mice received vehicle (n = 4), 5 mg/kg/day everolimus (n = 4), 10 mg/kg/day everolimus (n = 5), 20 mg/kg/day everolimus (n = 4), or 60 mg/kg/day axitinib (n = 5). No appreciable toxicity, defined by weight loss, unkempt appearance, mortality, and behavioral changes, was observed during treatment with everolimus, sorafenib, or axitinib. Median TTP with the highest dose of everolimus (20 mg/kg/day) was 13 days, which was significantly longer than the median TTP of 5 days observed in the control group (P < 0.001)   Figure . Similarn = 5 for each group), sorafenib (n = 5), axitinib (n = 5), and vehicle control (n = 8) revealed that the number of vessels was significantly decreased in all everolimus- and sorafenib-treated tumors compared to control tumors (P < 0.001)  . In axitinib-treated and control tumors, CA9 staining was mainly confined to the tumor edges, whereas in the everolimus- and sorafenib-treated tumors, staining was evenly distributed.CD31 staining of tumors from sunitinib-refractory CAKI-1-bearing mice treated with 5, 10, and 20 mg/kg/day everolimus  Figure ; everoli) Figure . Neither) Figure . EveroliImmunohistochemical analysis demonstrated that sunitinib-resistant tumors of mice treated with everolimus expressed CD31mm, but not CD31hs, indicating that vessels contained endothelial cells of murine origin only Figure . Conversp ≤ 0.05) in 3835  genes in the axitinib treatment, 5314  genes in the sorafenib treatment, and 1057  genes in the everolimus treated tumors relative to the placebo (Data File S2). Of these, 606 genes were similarly expressed in all three treatments compared to the control, while a number of genes were uniquely expressed in only one of the therapies , cytoskeletal regulation , angiogenesis (PDGFA), and miscellaneous . At the protein levels, tumors of mice treated with everolimus expressed low levels of CD133 and vimentin compared to tumors of control mice  displayed an atypical mixed phenotype with expression of human and mouse CD31 and human FvW (FvWhs). Co-staining of CD31 or CD10 with FvWhs revealed that these human endothelial markers were expressed by tumors cells and only in the tumor vessels' vicinity, which explained the atypical mixed phenotype of these tumor vessels. This finding is in line with results of a recent study, which demonstrated endothelial cell differentiation of glioblastoma stem-like cells and suggested that the ability of cancer stem-like cells to directly contribute to tumor vascularization through differentiation into endothelial cells may represent a new mechanism of angiogenesis /2. Tumor progression was defined as 3 consecutive increases in tumor volume and a tumor volume that was double the initial tumor volume. These progressive tumors were considered to have developed acquired resistance to sunitinib. Within each group, tumor volume was correlated with tumor weight, which was measured after mice were killed. Progressive tumors under first-line sunitinib treatment were randomly assigned to receive second-line treatment with everolimus, sorafenib, or axitinib. Everolimus was administered at 5 doses  by oral gavage (5 days per week for 3 weeks). The doses of everolimus used in this study were previously shown to be active in mouse xenografts. Sorafenib and axitinib, both dosed at 60 mg/kg/day, were used as reference treatments using the same administration protocol. Axitinib was shown to be active in a preclinical human colon cancer xenograft model at doses ranging from 30 to 100 mg/kg/day, and axitinib 30 mg/kg twice daily corresponded to an ED70 for inhibition of tumor growth and to the plasma IC50 for VEGFR-2 at 24 hours [3, mice were sacrificed. Body weight and tumor weight were recorded. Time to progression (TTP), defined as the time from start of drug administration to tumor progression, was analyzed for first-line sunitinib treatment and for all second-line treatments.All  France) . Assuminterature  and to e24 hours . Sorafen24 hours . After 32 atmosphere, and regularly checked for absence of Mycoplasma.The human RCC cell lines CAKI-1 (VHL+/+) and 786–0 (VHL−/−) were obtained from the American Type Culture Collection . CAKI-Suni and 786-Suni are variants of CAKI-1 and 786–0 exposed to sunitinib for more than 6 months. Cells were grown as monolayers in RPMI medium supplemented with 10% fetal calf serum , 2 mM glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37°C in a humidified 5% COTissues samples were obtained from tumors stored in the Beaujon and St Louis Hospital Tissues Bank. Paraffin-embedded tissues were obtained from patients who provided informed consents for biological analysis of tumor tissues according to French National Guidelines.® 555 Antibody labeling kit, Invitrogen, France) and Alexa 488  respectively. Double immunofluorescent staining was performed on a Discovery XT  by co-incubating tissue sections with the two bounded primary antibodies. Tissue sections were analyzed on a motorized Z-axis BX63 Olympus microscope . Each fluorescent immunostaining was captured through a UPlan Fl/40 × /0.75 objective with a digital camera DP71 using Olympus CellSens Dimension software , using specific wavelength for fluorophore excitation. Merged pictures were performed to assess the co-localization of CD10 and CD31.To characterize the mechanisms of acquired resistance to sunitinib, tumors from the control group, the sunitinib-responders group, and the sunitinib-resistant group were analyzed for necrosis, hypoxia, and angiogenesis. To characterize the effects of everolimus, sorafenib, and axitinib as second-line treatments, tumors from sunitinib-resistant mice treated with 5, 10, and 20 mg/kg everolimus, sorafenib 60 mg/kg, axitinib 60 mg/kg, and placebo were analyzed for necrosis, hypoxia, and angiogenesis. Immunohistochemistry was performed on OCT-embedded subcutaneous tumor stained with hematoxylin-eosin (HE) to evaluate necrosis, CA-IX  to evaluate hypoxia, and CD31 (anti-human and anti-murine antibodies) to evaluate angiogenesis, using an automated immunohistochemical stainer. The images were captured and analyzed with a Zeiss Observer Z1 microscope. Quantifications were performed using Histolab software . For the immunofluorescence assay, tumor slices were incubated with CD31, CD10, or von Willebrand factor (FvW) primary antibodies, followed by incubation with the secondary antibodies. Nuclei were stained with DAPI. Image analysis was performed using Zeiss Observer Z1 fluorescent microscope and AxioVision software. For the human samples, CD31/CD10 double immunofluorescence analyses were performed on 5-μm formalin-fixed paraffin-embedded tissue sections. Primary antibodies CD10  and CD31  were bound with Alexa 555 (APEX Alexa fluorTBP gene (an endogenous RNA control) and relative to a calibrator (1× sample), consisting of the cell line sample from our tested series that contained the smallest amount of target gene mRNA. For microarray experiments, isolated RNA from each tumor was amplified, and biotin-labeled using Illumina TotalPrep RNA Amplification Kit . 750 ng of biotinylated aRNA was then briefly heat-denatured and loaded onto expression arrays to hybridize overnight. Following hybridization, arrays were labeled with Cy3-streptavidin and imaged using the Illumina ISCAN. Intensity values were transferred to Agilent GeneSpring GX microarray analysis software and data was filtered based on the quality of each call. Statistical relevance was determined using ANOVA with a Benjamini Hochberg FDR multiple testing correction . Data were then limited by fold change analysis to statistically relevant data points demonstrating a 1.75-fold or greater alteration in expression of each gene when averaging the normalized values from each sample within an experimental group. Omics pathway analysis was performed with Metacore integrated software suite  and functional association networks were created using the String database (www.string-db.org).To evaluate and compare gene expression in the different tumor samples, RNA was extracted from 20 tumor slices from each first-line sunitinib treatment group  by RNABLE  standard protocol. Total RNA (1 μg) was reverse transcribed and the resulting cDNA were analyzed by quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) for expression of selected target genes related to sunitinib resistance. mRNA expression of CXCR4, CXCL12, ErbB3, ErbB4, PDGFRA, PDGFRB, CDH1, and vimentin was analyzed in parental and sunitinib-resistant CAKI- 1 and 786–0 cells. Quantitative real- time RT-PCR was conducted through use of the ABI Prism 7900 Sequence Detection System . Results were expressed as n-fold differences in target gene expression relative to the To evaluate and compare protein expression in the different tumor groups, total proteins were extracted from 25 to 30 tumor sections from each first-line sunitinib treatment group and analyzed for expression of CXCR4, vimentin, MET, ErbB3, AKT, p-AKT, p-ERK1/2, mTOR, p-S6, and actin by Western blot. Total proteins were also extracted from 25 to 30 tumor sections from each of the second-line treatment groups and analyzed for expression of p-S6, p-AKT, p-ERK1/2, vimentin, and actin by Western blot. Protein expression of p-Akt, p-ERK, Akt, PTEN, p27, and GAPDH in parental and sunitinib-resistant CAKI-1 and 786–0 cells and of p-S6, p-4EBP1, p-Akt, Akt, p-PKCα, p-ERK, vimentin, and GAPDH in cells exposed to 0.1 μM everolimus for 0, 1, 5, and 24 hours was assessed using Western Blot analysis.3 cells/well. After 48 hours of incubation with sunitinib and 24 hours of postincubation in drug-free medium, cells were incubated with 0.4 mg/mL MTT. After incubation, formazan precipitates were dissolved and absorbance was measured at 560 nm . Wells with untreated cells or with drug-containing medium without cells were used as positive and negative controls, respectively. Growth inhibition curves were plotted as the percentage of untreated control cells.Parental and sunitinib-resistant CAKI-1 and 786–0 cells were exposed to 1 μM, 5 μM, 10 μM, and 20 μM everolimus and assessed for inhibition of cellular proliferation after 24–, 48–, 72–, and 96-hour exposure, using the MTT assay . In brief, cells were seeded at a density of 2 × 10in vitro model of pseudotube formation by renal cancer cells was used to assess the in vitro angiogenesis in hypoxic and normoxic conditions. Briefly, renal cancer cells after 24 hour hypoxia (100 μM CoCl2) or normoxia incubation were plated on Matrigel-coated μ-Slides Angiogenesis  at concentration of 7 × 103 per well. Pseudotube formation after 4 hours was visualized using phase contrast microscopy. The architecture of the matrix-associated vascular channels in vitro, which was characterized by interconnected loops and networks, modelled the network patterns observed in cultures by endothelial cells. Number of meshes, junctions and segments was calculated using the “Angiogenesis Analyzer” software from Image J software developed by J. Carpentier  (Well-established  France) .t test was used to calculate the significance of differences between groups . Kaplan-Meier curves were constructed for survival analysis.All statistical analyses were performed using GraphPad Prism, Version 5.00, GraphPad Software. A nonlinear regression curve fit  was used to analyze dose response experiments. Two-tailed unpaired