Unsupervised computational approaches were used for disease deconstruction. We found that hip and knee osteoarthritis are not identical in respect to the inflammatory processes that take place in the synovial membrane. Instead, we report that principally CD14+ macrophages are expanded fourfold in the synovial membrane of patients with knee OA compared to hip OA, with a trend to higher expression in CD8+ T cells, while CD4+ T cells, B cells, and NK cells were found at comparable quantities. Upon isolation and culture of cells from synovial membrane, isolates from hip OA released higher concentrations of Eotaxin (CCL11), G-CSF, GM-CSF, INF-γ, IP-10 (CXCL10), TNF-α, MIP-1α (CCL3), MIP-1β (CCL4), IL-4, IL-10, IL-17, and lower concentrations of stem cell factor (SCF), thereby highlighting the difference in the nature of hip and knee osteoarthritis. Taken together, this study establishes hip and knee OA as immunologically distinct types of OA, and creates a resource of the cytokine expression landscape and mononuclear cell infiltration pattern of patients with hip and knee osteoarthritis.Osteoarthritis (OA) is a progressive joint disease driven by a blend of inflammatory and biomechanical processes. Studies using human samples to understand inflammatory mechanisms in OA frequently recruit OA patients with different affected joints, even though recent evidence indicates that OA is a heterogeneous disease which only culminates in a common end point. Differences in age of onset and the dynamics of disease progression suggest that different joints may represent different disease entities, thereby diluting the discovery potential in a combined analysis. We hypothesized that different OA joints may also differ in immunopathology within the synovium. To investigate this hypothesis, we profiled the immune cell contribution (flow cytometry) and cytokine release profiles (ELISA) in purified synovial membrane mononuclear cells from 50 patients undergoing either hip ( Osteoarthritis (OA) is a heterogeneous disease, and with a prevalence of symptomatic OA of 10% in men and 18% in women aged over 60, represents the most frequent cause of disability in older adults according to the World Health Organization . As a re+ T helper cells are the two principal cell types in osteoarthritic synovial membrane, while additional effector cells contributing to joint damage are synovial fibroblasts and osteoclasts [OA was traditionally viewed as a non-inflammatory joint disease with ageing, with mechanical stress being considered the major contributors to cartilage breakdown and bone remodeling. This still being relevant, evidence for an inflammatory component to OA pathogenesis is significantly increasing ,4,5. Theeoclasts . Infiltreoclasts . In the eoclasts . IL-1β aeoclasts . IL-17 iIn contrast to traditional belief, osteoarthritis is now believed to be a heterogeneous disease, where diverse disease courses culminate in a common end point ,7. Up toUltimately, defining the pathophysiology of OA subtypes and characterizing common but also different pathways could prove helpful in understanding disease pathophysiology and developing specialized osteoarthritis disease-modifying interventions.50 patients with OA (34 patients with hip OA and 16 patients with knee OA) were included in this study. Due to methodological setup, one part of the study population was utilized for flow cytometry analysis and the other part for multiplex analysis of cytokine expression A. The st5 events were assessed and analyzed with a MACS-Quant flow cytometer . Data analysis was performed using FlowJo version 9.8.1 . A representative gating strategy is shown in + T cells, CD8+ T cells, CD14+ macrophages, CD19+ B cells, and CD16+CD56+ natural killer (NK) cells. The cut-off for all cell surface markers was defined based on isotype controls.Prior to surgery, peripheral blood samples were obtained and collected in EDTA-containing tubes. Synovial membrane (SM) samples were harvested from the resected capsule of the hip and the suprapatellar pouch of the knee during surgery and prepared as previously described . SM sampSynovial membrane samples were harvested from hip and knee OA joints as described above. SM samples were rinsed twice with phosphate buffered saline (PBS), minced finely with sterilized scissors, and taken into culture for 24 h. The supernatants were harvested and stored at −80 °C. The Pro-Human Cytokine Multiplex Assays  was used to analyze the cytokines in synovial fluid samples. The 27-plex analyses for Eotaxin (CCL11), FGF-Basic, G-CSF, GM-CSF, interferon (INF)-γ, IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8 IL-9, IL-10, IL-12-p70, IL-13, IL-15, IL-17, IP-10, monocyte chemotactic protein 1 , macrophage inflammatory protein-1α , MIP-1β (CCL4), PDGF–bb, regulated upon activation normal T cell expressed and activated , TNF-α, and vascular endothelial growth factor (VEGF). The 21-plex contains CCL27, GRO-α, HGF, IFN-α2, IL-1α, IL-2Rα, IL-3, IL-12p40, IL-16, IL-18, leukemia inhibitory factor (LIF), MIF, MCP-3 (CCL-7), M-CSF, MIG (CXCL9), β-NGF, stem cell factor (SCF), SCGF-β, SDF1α (CXCL-12), TNF-β, and TRAIL. Multiplex assays were carried out according to the manufacturers’ instructions and run on the Luminex 200 platform. Bio-Plex Manager version 5.0 was used for data processing. Cytokine and chemokine concentrations were calculated by reference to the standard curve. The sensitivity of the multiplex kit was <5 pg/mL.n = 3 of 1012 measurements) were substituted by the highest measured value for the respective soluble mediator. Values below the detection threshold (n = 17 of 1012 measurements) were substituted by zero. Then, expression values were standardized between 0 and 1. Soluble mediators for which less than 25% of measurements were usable were excluded , retaining 44 cytokines for downstream analysis. From the resulting matrix, we calculated the Pearson correlation between all samples and the spearman correlation between all soluble mediators. In order to test if cytokines were differentially expressed between samples from hip and knee OA, we used the calculated mean and standard deviation for each cytokine.Following ELISA measurements, we used a two-step computational pipeline to test the association between different samples. First, to allow simultaneous analysis of different soluble mediators with concentration profiles that differ in several orders of magnitude, the expression value of each soluble mediator was transformed: Values above the detection threshold , SPSS version 22·0 , and R version 3.5.1.Demographic parameters between study groups were compared using the unpaired : Hip OA patients were significantly younger , while the average BMI was significantly higher in knee OA (hip: 26.0 ± 4.6 kg/m2 vs. knee: 29.7 ± 6.2 kg/m2p = 0.022). The laboratory parameters C-reactive protein (CRP) and leucocytes were within standard range, and thus did not suggest any signs of systemic inflammation at the time of surgery. Our first approach was to analyze peripheral blood of both patient groups for frequency of mononuclear cell subsets (p = 0.4431), CD14+ macrophages , CD8+ cytotoxic T cells , CD4+ T helper cells , CD19+ B cells , and CD16+CD56+ natural killer cells .The two study groups showed significant differences regarding the demographic parameters age at surgery and BMI : Hip OA  subsets A,B. TherNext, in the absence of systemic inflammatory differences, we proceeded to directly test the site of joint destruction: the synovial membrane.p = 0.0662). Only very few CD16+CD56+ NK cells were detected in the SM of both groups  were significantly increased in knee OA, and CD8+ cytotoxic T cells showed a trend for increase in knee OA   D, suggesIn the next step, we cultured unmanipulated synovial membrane samples and analyzed their cytokine release profile in the supernatant by multiplex ELISA analysis. Standardized cytokine expression values were used to calculate pairwise Pearson correlation coefficients between all individual samples. Hierarchical clustering of these sample to sample distances revealed a striking separation, predominantly driven by the joint involved in osteoarthritis A: Two ma(We further examined the pairwise Spearman correlation between individual soluble mediators. While both positive and negative correlations between soluble mediators were found, a cluster of strongly correlated cytokines consisting of CCL3, IL-17, CCL4, IL-9, CCL11, IL-4, and IFN-γ stood out B. In hipA. IntereB. Other As both the hip and knee OA group contained patients with radiographic Kellgren and Lawrence scores III and IV, we additionally examined the data separately for radiographic scores. In the cytokine release data, all samples from knee OA were scored as K&L III. In hip OA, 11 samples were from K&L III, and 4 samples had K&L IV. No cytokines were differentially expressed between K&L score III hip OA and K&L score III knee OA, suggesting that this asymmetry did not influence our initial results. Direct comparison of cytokine expression from K&L III in hip OA vs. K&L III in knee OA identified significantly higher production of IL-17, GM-CSF, CXCL10, CCL3, CCL4, PDGF-BB, and TNF in the Hip OA cohort, and higher expression of SCF, SCGF-beta, and IL-13 in the knee OA cohort . Of thesConsistent with differences in mononuclear cell infiltration into the joints, we found differences in the cytokine release profiles between hip and knee OA isolates. These differences in cytokine release appeared to be coordinated, as we identified highly correlated expression pairs, such as IFN-γ–IL-4, IFN-γ–IL-9 and CCL4–IL-17 that were positively correlated in both hip and knee OA. A. HoweveOsteoarthritis is increasingly recognized as a heterogeneous disease ,14. In dHowever, up to this point osteoarthritis of hip and knee have been viewed as a manifestation of the same disease in different joints, and many studies on OA pool samples from different joints for analysis. If OA from different joints had distinct cellular and molecular properties, then attempts to identify disease biomarkers or prognostic parameters could potentially miss discovery power in a pooled analysis . Therefo+ macrophages were the largest cell population in hip OA and knee OA, accompanied by CD4+ T cells, CD19+ B cells, CD8+ T cells, and CD16+CD56+ NK cells in descending order.Healthy synovial membrane is an acellular structure assembled by an intimal lining layer and a synovial sublining layer. Infiltration of immune cells, structural changes such as stromal edema, and proliferation of blood vessels accelerate the loss of the SM-lining structure . Especia+ and CD8+ T cells, and the intimal layer predominantly CD4+ T cells. Our study is in accordance with previous publications, which showed predominant CD4+ and CD8+ mononuclear cell infiltration in mid to severe OA [+ T cells are described to induce synovitis [Previous immunohistochemical studies of osteoarthritic joints have shown that T cells are detectable in synovial tissue at an early disease stage . Using tevere OA . These fevere OA . Especiaynovitis . Our stu+ macrophages appear to mediate structural progression and pain in knee OA by playing a central role in maintenance of inflammation [As first part of active immune response, activated resident macrophages represent the major cell population, followed by T-lymphocytes and plasma cells, which immigrate in the affected joints where they play a relevant role in initiation of OA and induce subsequently pathologic elevation of inflammatory cytokines ,15,21. Iammation . Our resTaken together, CD14+ macrophages and CD4+ T cells appear to mediate much of OA immunopathology. Thus, functional investigations of macrophage and T cells and their interaction within synovial tissues could be of utmost relevance for the understanding of pathological events in OA. In addition to immune cells, fibroblasts are critical effector cells in arthritis. Recent studies have described distinct subsets of fibroblasts based on different surface protein expression, transcriptome, and functional properties, including inflammatory cytokine release. ,25,26 InBesides the immune cell infiltration, an inflammatory cytokine environment is present in OA joints. We have previously shown that in end-stage OA, a shift towards a pro-inflammatory cytokine environment takes place . A numbeIn direct comparison between different joints, our study reveals that hip OA is affected by significantly higher cytokine levels in comparison to knee OA, with a marked shift towards a pro-inflammatory milieu including IFN-γ, TNF-α, and IL-17. IFN-γ is produced by a broad variety of cells like NK and T-cells, is an important activator of macrophages, and has an immunostimulatory and immunomodulatory character. The significantly higher presence of IL-17 in hip OA could suggest an important role in hip OA pathophysiology, as it is able to induce inflammatory cytokines such as TNF-α, IL1β, and IL-6 to amplify joint destruction ,34,35. TInterestingly, the two anti-inflammatory cytokines IL-4 and IL-10 showed a higher concentration in hip OA than knee OA. IL-4 and IL-10 stimulate proliferation and differentiation of T and B cells, and are associated with a chondroprotective effect. Both cytokines inhibit secretion of IL1β, TNF-α, MMPs and can thereby suppress inflammation of the synovial membrane and reduce variations of proteoglycans which maintain OA pathophysiology ,37.Growth factors like SCGF-β, G-CSF, HGF, and VEGF showed the highest absolute concentrations in both disease groups. First, they could play an important role in progression of OA by promoting abnormal osteoblast expression from the subchondral bone plate and inducing subchondral bone sclerosis . Second,Regarding MIG–CXCL9, other studies suggest a higher concentration in OA by elevating expression of pro-inflammatory cytokines and suppression of chondrocyte multiplication . IP-10 (Besides the above-mentioned differences in cytokine release patterns between both OA groups, a broad variety of pro-inflammatory mediators such as LIF, CCL27, IL-1a, and IL-12-p70 was present in the affected joints without a significant difference between groups.In summary, were able to show significantly higher levels of many inflammatory mediators in isolates from hip OA which potentially accelerate disease progression. Thus, different affected joints in OA may differ in inflammatory pathophysiology.+ T cell infiltration in knee OA could also be affected by the higher age of this group. Therefore, confounding was excluded by use of a multivariable linear regression model.In our study, hip OA and knee OA displayed significant differences regarding BMI and age at surgery, and these differences have implications for data interpretation: Obesity is one of the strongest risk factors of OA, with a higher correlation especially in knee OA. Beside the increased loading stress in joints, obesity leads to metabolic abnormalities and systemic inflammation . Thus, tThe detected demographic heterogeneity between hip and knee OA with respect to age at time of surgical intervention and BMI is additional evidence for differences in disease pathophysiology between OA joints. This idea is further supported by clinical observations: Patients with manifested hip OA suffer more severe symptoms at an earlier age and require total joint arthroplasty after a shorter disease course. In contrast, knee OA manifests at later age with less severe symptoms and a longer period until arthroplasty. Our study supports these differences in age at surgery and BMI between both disease groups, which are both higher in knee OA. Although we did not observe differences in K&L score, more severe radiographic changes were displayed in hip OA, which undergirds hip OA as a more severe and faster declining condition, as supported by previous studies . The resWe acknowledge that our study has limitations. By focusing on the comparison between hip and knee OA using tissue obtained during joint arthroplasty, this study did not include healthy controls. The inclusion of healthy synovial tissue may be possible in the future through the increasing availability of synovial tissue microbiopsies. Furthermore, by only including patients with primary OA, our study excluded secondary factors that also predispose joints to OA. Also, patients with polyarticular osteoarthritis were not included because of the vague definition in the literature and the potentially multimodal causes which lead to a generalized osteoarthritis . By cultTaken together, this study establishes hip and knee OA as distinct disease subsets with respect to the immune cell infiltration pattern in the synovium and cytokine release profiles. Exploration of immune cell infiltration and inflammatory pathways is highly relevant to understand disease pathophysiology. This study represents an early step in deciphering OA heterogeneity, and sets the stage for subsequent, functional studies on the role of inflammatory biomarkers and mononuclear cells in OA, which are required to assess disease progression. This and potentially other stratification strategies may inform clinical decision making in the future and pave the way for personalized therapies in osteoarthritis.


Nrf and CNC homology), because of their strong conservation with all the known CNC and NF-E2 p45 subunit-related factors Nrf1 and Nrf2. Further experimental evidence has also been provided, revealing that Nach1 and Nach2 from the marine bacteria exert distinctive functions, when compared with human Nrf1 and Nrf2, in the transcriptional regulation of antioxidant response element (ARE)-battery genes. Collectively, further insights into these Nach/CNC-bZIP subfamily transcription factors provide a novel better understanding of distinct biological functions of these factors expressed in distinct species from the marine bacteria to humans.Normal growth and development, as well as adaptive responses to various intracellular and environmental stresses, are tightly controlled by transcriptional networks. The evolutionarily conserved genomic sequences across species highlights the architecture of such certain regulatory elements. Among them, one of the most conserved transcription factors is the basic-region leucine zipper (bZIP) family. Herein, we have performed phylogenetic analysis of these bZIP proteins and found, to our surprise, that there exist a few homologous proteins of the family members Jun, Fos, ATF2, BATF, C/EBP and CNC (cap’n’collar) in either viruses or bacteria, albeit expansion and diversification of this bZIP superfamily have occurred in vertebrates from metazoan. Interestingly, a specific group of bZIP proteins is identified, designated Nach ( The transcriptional networks formed by distinct arrays of such dimerization of the bZIP superfamily are known to play vital roles in cell division, proliferation, differentiation, maintenance, and other life processes, particularly in multicellular organisms [d” positions, and mediates dimerization of bZIP proteins [Notably, one of the most conserved TFs is the basic-region leucine zipper (bZIP) superfamily. They are involved in the transcriptional regulation of differential subsets of target genes by forming homo- and hetero-dimers with their cognate partners before binding their specific rganisms . Conversrganisms ,9,10,11.rganisms . The basproteins ,14.Monosiga brevicollis) and protist (Capsaspora owczarzaki) [Currently, with the availability of whole genome sequences from distinct species, an ever- increasing number of bZIP proteins are identified as key players in defending against abiotic stresses in plants, including Arabidopsis , rice 1, apple , and maizarzaki) , in addizarzaki) ,20, fungzarzaki) , and plazarzaki) . For exazarzaki) . Consequzarzaki) . NonetheCaenorhabditis elegans protein Skn-1 [Drosophila melanogaster Cnc protein [Nrf1α cells. Taken altogether, the regulatory network demonstrates the importance of Nrf1α hub with the rest of bZIP genes.The CNC-bZIP subfamily contains a founding consensus CNC domain situated in front of its BRLZ domain, which is uniquely distinctive from other bZIP subfamilies. Among its previously assigned members are nuclear factor-erythroid 2 (NF-E2) p45 and related factors (Nrfs), along with two transcriptional repressors Bach1 and Bach2 in vertebrates, and the in Skn-1 , in addi protein . Here, wGallid herpesvirus 2 (Gh2), Cyprinid herpesvirus 1 (Ch1), Endozoi- comonas numazuensis (En), Endozoicomonas arenosclerae (or sp. ab112) (Ea/Es), Dictyostelium discoideum (Dd), Vitrella brassicaformis (Vb), Saccharomyces cerevisiae (Sc), Monosiga brevicollis (Mb), Capsaspora owczarzaki (Co), Trichoplax adhaerens (Ta), Amphimedon queenslandica (Aq), Nematostella vectensis (Nv), Caenorhabditis elegans (Ce), Drosophila melanogaster (Dm), Helobdella robusta (Hr), Octopus bimaculoides (Ob), Strongylocentrotus purpuratus (Sp), Danio rerio (Dr), Xenopus tropicalis (Xt), Gallus gallus (Gg), Anolis carolinensis (Ac), Mus musculus (Mm), and Homo sapiens (Hs). Across these species, a total of 441 of the bZIP proteins were identified, after removal of both incomplete and repeated sequences from the resulting searches from BLASTP and HMMER databases. For the Gallid herpesvirus 2, only one bZIP protein  was identified, with a BRLZ domain that has a high sequence identity of 60.82% with the BATF subfamily (Cyprinid herpesvirus 1 (with accession No. YP_007003813 in GenBank), with an 87.5% BRLZ sequence consistency with human Jun  up to 19 proteins. The more bZIP proteins were identified in vertebrates, e.g., the human (Homo sapiens) up to 53 bZIP proteins, six of which belong to the CNC-bZIP subfamily.To investigate the origin of the bZIP family members, some known bZIP sequences were used as queries for BLASTP  in the non-redundant protein sequences database and for the HMMER  search. As shown in ubfamily a. Anotheuman Jun b. More iproteins . In metaproteins a, for exa to g), in which the typical residues at two positions “a” and “d” are key in forming a hydrophobic interface and essential for the dimerization of related bZIP proteins. Within the LZ regions, almost all the “d” positions are highly conserved and occupied primarily by leucine (L) or other hydrophobic residues. The third “a” position asterisked is also highly conserved by asparagine (N), while glutamic (E) at the first to fourth ‘g’ positions are relatively conserved -homologous protein), Mm-CHOP, Dr-CHOP and Xt-CHOP, were collectively gathered into a branch Yap subfamily, by employing the well- supported bootstrap values. The remaining 430 BRLZ sequences were clustered into additional 16 branches, each of which has a potential individual difference from others, within their mutual relatively independent and interrelate connective evolutionary trajectories  in the Jun subgroup.Notably, r branch . The XBPMore interestingly, the BATF and Jun subfamilies (including other AP-1 family members Fos and ATF2) are inferable to be originated from the putative earliest primogenitor existing in the viruses a,b, wherDrosophila and Nematodes, respectively [Caenorhabditis elegans and Drosophila melanogaster, the original homologues with the CNC-bZIP proteins are, indeed, objectively present in the marine bacteria E. sp. ab112 (Nach1) and E. numazuensis (Nach2). Besides Nach1 and Nach2, other orthologues with CNC-bZIP were further searched in the multicellular organisms including Amphimedon queeslandica (Nach4/5), Trichoplax adhaerens (Nach3), Nematostella vectensis (Nach6), Octopus bimaculoides (Nach7), and Strongylocentrotus purpuratus (Nach8). Hence, these Nrf and CNC homologues 1 to 8, according to the evolutionary status from origin to advance of the distributed species (on the left panel of Danio rerio (zebrafish) has also given rise to 10 CNC-bZIP homologues, but only four of CNC-bZIP proteins exist in Gallus gallus (chicken) with a constructive loss of NF-E2 p45 and Nrf3 from within the genome.According to the current literature as far as we know, the CNC subfamily of bZIP transcription factors is composed of NF-E2 p45, Nrf1, Nrf2, Nrf3, and their transcriptional repressors Bach1 and Bach2 in vertebrates, in addition to both Cnc and Skn-1 proteins found in ectively . To furtDm_Cnc and Ob_Nach7. In contrast, a parallel clade comprised mostly of two subgroups of Nrf2 and p45 proteins. All these Nrf proteins were gathered together and also shared a last common progenitor with Sp_Nach8. The progenitor was also likely situated at a similar generation to another progenitor shared by between Bach1 and Bach2. These two progenitors were inferable to be originated from the hierarchical root comprising mostly Nach proteins. These putative ancestral Nach proteins retain a high evolutionary conservation with zebrafish Nrf1a, Nrf2b and Nrf3, in addition to Skn-1 of Nematodes   within their CNC domains  or the former 23rd position-specific serines . In addition to their 24th position-specific arginines in both subgroups, which are distinctive from any hydrophobic residues occupying at these same corresponding positions in all other CNC-bZIP subgroups. Overall, the motif is highly conserved by its sequence identity with equivalents existing among those advanced eukaryotes from octopus to humans, but in some lower lineages, it is a relatively less conservative across all their CNC domains. For example, glutamine (Q) is occupied specifically at the 13th position of the bacterial Nach1 and Nach2, but is replaced by proline (P) within almost all other CNC-bZIP proteins  membrane through the N-terminal homology box 1 (NHB1) peptides ,28, howeREBP-TM2 a, upper.REBP-TM2 a.bona fide TM domains are dictated by their constitutive core hydrophobic (h)-regions spanning across membranes, while distinct orientations of these segments within membranes are predominantly determined by the charge differences along and between its n-region and c-region flanking the core h-region. Therefore, we further aligned multiple sequences of putative TM domains in the CNC-NHB1 subgroup including Nach proteins , XBP1u, ATF6, OASIS, as well as SREBP-TM1 and -TM2 were wheeled by the HeliQuest tool, with their aliphatic indexes and hydropathicity estimated d. These The above membrane-bound bZIP and bHLH-ZIP transcription factors were, here, summarily classified into four different categories, which are represented by Nrf1, XBP1u, ATF6, and SREBP1 in order to provide a better understanding of distinct topovectorial processes of these TM-containing proteins integrally folded within and around the ER membranes e. FirstlIn order to gain an in-depth insight into the evolutionary diversity of human bZIP subfamilies during the nature selection, their neighbor-joining phylogenetic tree was further constructed with their full-length amino acid sequences a. The reNrf1α was knocked out by Talens-mediated gene editing in the liver cancer HepG2 cells [Bach2, MafK, MafF, Jun, FosB, Fra1, ATF3, ATF4, NRL, HLF, TEF, CREB5, CEBPE, E4BP4, BATF3, and CREM were up-regulated significantly  . In addition, BATF, MAF, and CREBH are possibly expressed at much lower levels, so that they were not detected by RNA-sequencing. Conversely, the stable tetracycline-inducible expression of Nrf1α in HEK293CNrf1α cells  b,d. By c1α cells c,d cause−/−Nrf1α cells, ATF3, ATF4, CHOP, MafF, and NRL were significantly up-regulated at their mRNA levels (p < 0.05), whereas Nrf1, ATF2, DBP, and JunD were markedly down-regulated (p < 0.05), but expression of ATF1 was unaltered (Nrf1α by tetracycline treatment of HEK293CNrf1α cells resulted in significant increases (p < 0.05) in the inducible expression of Nrf1, ATF1, ATF2, DBP, and CHOP , in addition to no obvious changes in the expression of ATF3, ATF4, JunD, and NRL. These collective results of all other genes, except for CHOP, are in accordance with the RNA-seq data.To ensure that the above RNA sequencing results are reliable, 10 of human bZIP proteins were randomly selected for further examination by real-time quantitative RT-PCR. The results revealed that in naltered e. These and CHOP f, but onFor an in-depth insight into functional domains of Nach1 and Nach2 required for the regulation of ARE-driven genes  , their cTo determine the biological function of Nach1 and Nach2, HepG2 cells were transfected with each of expression constructs for Nach or CNC-bZIP protein, of which C-terminal ends are tagged by the V5 peptides. Western blotting revealed that either Nach1 or Nach2 was expressed as a major protein of 65-kDa estimated on 10% PAGE gels d, which Next, to further identify a role of the putative DSGxSL degron within Nach1, its mutant 1  and Mut2 (in which the DSGLSL motif was mutated to DAGLAL) were subjected to co-expression with Nrf2 in ARE-driven reporter assays c,h. As aFurther pulse-chase experiments of HepG2 cells that had treated with cycloheximide  alone or in combination with the proteasome inhibitor MG132 revealed that Mut1 caused an obvious increase. When compared with wild-type Nach1 protein having a shorter half-life defined to be 1.98 h (=119 min), the Mut1 protein was more stable because its half-life was extended to be 2.84 h (=170 min) following CHX treatment .During the evolution by variation and selection, the diversity of living organisms increases their biological complexity of distinct species to survive in a changing environment . To meetd” and “a” positions of heptad repeats wheeled, respectively [a” position, they are conferred to drive heterodimerization of bZIPs in Arabidopsis thaliana, whereas the asparagine residue at the “a” position develops a tendency to form a homodimer [d” position preserved in the four subfamilies of CNC, Maf, ATF6, and OASIS, the histidine residues at this position are also conserved in another five subfamilies of Fos, Jun, ATF2, ATF3, and BATF, but the poor conservation occurs in other subfamilies. This implies the last “d” position is also responsible for distinct dimerization. Additional potential differences among distinct bZIP subfamilies are also postulated to determine their dimeric stability. This is evidenced by the finding that the asparagine residue at the third “a” position is highly conserved in most bZIP proteins, because it can also elicit a limitation of LZ dimerization [Within all distinct subfamilies of bZIP proteins, an essential conservative domain is composed of both BR and LZ regions . The dimectively ,44. Whenomodimer . Here, wrization ,47. g-proteobacteria [Arenosclera brasiliensis, which still retain Nach4, Nach5, and other 15 bZIP factors . The ba factors . Notably factors a. Cyprinid herpesvirus 1 and Gallid herpesvirus 2, respectively  to tesvirus, ,54, althCyprinid herpesvirus 1 and another homologous protein of BATF in Gallid herpesvirus 2, raises a few of interesting questions. It should be noted that viruses cannot be analyzed in the same manner as the cellular life, because they are polyphyletic . Since it is so, there are none of ancestral viral lineages; that is to say, not a single ancestral gene  has been identified so far for being shared by all viruses [Apart from the variant HBZ from the retrovirus HTLV-1, the identification of a homologous protein of Jun existing in  viruses . Rather, viruses . The par viruses ) from di viruses . This le viruses . superantigen gene  and South American herpesviruses infecting monkeys and rats) is horizontally captured by mammalian herpesviruses and integrated into their host genomes [sag twice from separate lineages that are distinctive from MMTV, but it was not reported whether the bZIP genes are horizontally transferred. Intriguingly, additional two bZIP homologous proteins of Skn-1, C/EBP, ATF2, Fos and Jun  of distinct life forms to be steadily transferred from the parents to off-springs . These pDrosophila melanogaster Cnc protein and Caenorhabditis elegans Skn-1, but none of their orthologues are identified in plants and fungi [CNC gene product from the Drosophila melanogaster [Interestingly, a subgroup of bZIP transcription factors with a unique conserved CNC domain  comprisend fungi . These Cnd fungi  or othernd fungi , in ordend fungi . Howevernogaster . Amphimedon queeslandica). This is because none of their orthologues have emerged in the unicellular protozoans or other prokaryotes beyond Endozoicomonas. Notably, only one or two Nach proteins are found in each of species such as ascidians, sea urchin, octopus, fly, and hydra. Overall, these findings indicate that the expansion and diversification of CNC-bZIP subfamily appear to have occurred only in the vertebrate. Along the early solitary evolutionary branch of the phylogenetic tree is budded by distinct species-specific Nach proteins from the marine bacteria to simple multicellular eukaryotes. This notion is in perfect agreement with an accepted fundamental concept in biology that the eukaryotes  have been evolved originally from the prokaryotes , and that their genomic DNA could be transferred among these species by a hierarchic evolutionary process [Nach could be transferred and then diversified hierarchically from marine bacteria to humans, but the details of possible vertical and/or horizontal gene transfer mechanisms remain obscure so far. Furthermore, it should be noted that, since a physical proximity is known as a prerequisite to promote possible horizontal gene acquisition, it is inferable that the potential transfer event could take place between the marine bacteria Endozoicomonas and its host animals standing aside from humans. This is due to the objective fact that not any one of the bona fide orthologuous proteins of the Nach/CNC-bZIP subfamily has emerged in the human-surrounding prokaryotes and viruses (including parasitic and infected ones), albeit these microbiomes could enable a potential physical proximity to marine bacteria and animals. Hence, such possible gene transfer mechanism is an interesting topic to warrant further studies.Fortunately, a novel subgroup of Nach1-8, with a high homology with all the known CNC-bZIP proteins, are herein identified to be present in the Echinodermata, Mollusca, Actiniaria, Placozoa, Porifera, and bacteria, respectively . This di process ,54,59. FThe evolution of early unicellular to multicellular organisms is also monitored by biosynthesis of membrane lipids, together with a proper assembly with the key membrane-embedded proteins ,64. NoneIn response to ER stress, both ATF6 and SREBP are allowed for a transport from the ER to the Golgi apparatus, in which ATF6 and SREBP-TM1 are enabled for successive proteolytic processing by SIP and S2P to yield their N-terminal releasable portions acting as activators e. As sucSince the complex relationship between an organismic genotype and phenotype is clearly mediated by many of certain interrelated biochemical networks , MetazoaSCF-dependent ubiquitin proteasome degradation [The present study has demonstrated that the Nach- and CNC-bZIP subfamilies with similar conserved, but slightly different, structural domains  and functional motifs  . Furtherradation . More exde facto present in the marine bacteria but not in the human genome, thereby in this human experimental setting, over-expression of Nach1/2 is artificial. This is suggestive of the main difference existing between Nach1/2 and the human Bach1/2, demonstrating that even if both have similar experimental effects as repressors, they rather play diverse roles in regulating distinct subsets of target genes in different species. According to the results . Such event enables their target genes to be rapidly recovered after their transcription is switched off.As such, the above suppression is also abolished by a deletion mutant of the canonical DSGxSL from Nach1, but not by another point-mutant DAGxAL . This obE-value = e−5). Meanwhile, the HMM search (http://hmmer.org/) was also employed to identify bZIP proteins with the default parameter . The resulting searched sequences were downloaded from the database of NCBI , of which the repeated and incomplete sequences were manually removed. In addition, some bZIPs that had been not identified in the past were herein denoted by a nomenclature rule “××_bZIP_TF× ” (The BLAST program was conducted to identify bZIP superfamily members with the parameter (ecies.)” . NotablyFor phylogenetic analysis of the BRLZ domains, they were extracted from all the selected bZIP proteins by using a local PERL SCRIPT program, and then aligned using DNAMAN 8.0 , T-Coffee Server (Comparative Bioinformatics Group Bioinformatics and Genomics Programme Center for Genomic Regulation (CRG), Barcelona, Spain) and ClustalX 2.0  with distinct default parameters. The multiple sequence alignments were manually refined and end-trimmed to eliminate the poor scored or divergent regions. Subsequently, the remaining unambiguously aligned sequences were subjected to construction of the neighbor-joining phylogenetic trees, by using the MEGA version 6.0  , which are displayed by the iTOL program . MoreoveThe above-selected bZIP proteins were used to predict TM-containing transcription factors by TMpred  and TMHMM Tools . The identified TM domains were subjected to the phylogenetic analysis, with multiple sequence alignment, and conservative analysis. In addition, six TM-folded α-helix properties of Nrf1, XBP1u, ATF6, OASIS, SREBP-TM1, and -TM2 were calculated with the HeliQuest tool .INteracting Genes and/or proteins, http://string-db.org/) [The interaction networks of between bZIP proteins in humans were also constructed with the STRING software  . Relativ+/+Nrf1), −/−Nrf1α , human embryonic kidney (HEK293) and HEK293CNrf1α (with stable tetracycline-inducible expression of Nrf1α established in human embryonic kidney) were cultured in a 37 °C incubator with 5% carbon dioxide, and allowed for growth in Dulbecco’s modified Eagle’s medium (DMEM) with 25 mmol/L high glucose, 10% (v/v) fetal bovine serum (FBS), 100 units/mL penicillin-streptomycin.Experimental cell lines, including human hepatocellular carcinoma HepG2 . The results were analyzed by the Bio-Rad CFX Manager 3.0 software (Bio-rad).Total RNAs was extracted from cell samples, by using an RNA extraction kit  and then subjected to reactions with reverse transcriptase  to synthesize the single-strand cDNAs. Subsequently, expression of the indicated bZIP genes at mRNA levels in different cell lines were measured by qRT-PCR with distinct pairs of primers as listed in After reaching 70% confluence of HepG2 cells that had been allowed for growth in 6-well plates for 24 h in DMEM containing 25 mmol/L glucose and 10% FBS, they were transfected with an expression construct for Nrf1, Nrf2, Nach2, Nach1, or its mutants  in a mixture of Lipofectamine 3000 . After transfection for 24 h, the cells were or were not treated with CHX (at 50 µg/mL) alone or plus MG132 (at 10 µmol/L) for additional 30 min to 4 h before being harvested in a lysis buffer . Total c5) of HepG2 cells were allowed for 24-h growth in each well of 12-well plates containing DMEM supplemented with 25 mmol/L glucose and 10% FBS. After reaching 70% confluence, the cells were transfected with expression constructs for Nrf1, Nrf2, Nach2, Nach1, or its mutants alone or in different combinations with one another, together with both GSTA2-6×ARE-Luc reporter and pRL-TK , in a mixture of Lipofectamine 3000 (Invitrogen Ltd.). Approximately 24 h after transfection, ARE-driven luciferase reporter activity was measured by Magellan7.1 SP1 systems and then calculated as fold changes (mean ± S.D), as described previously [Equal numbers . The data are shown as a fold change (mean ± S.D), each of which represents at least 3 independent experiments that were each performed in triplicates.Statistical significances of fold changes in the In an attempt to provide a better understanding of the origin of bZIP transcription factors with different subfamily evolutionary features , distinct sizes of four neighbor-joining phylogenetic trees have been established here, based on different conditional grounds. Among them, the first phylogenetic tree c has beebona fide function of Nach1/2 in the marine bacteria and its host animals.Overall, Nach1 to Nach8 are herein identified to comprise a novel subgroup of the CNC-bZIP subfamily, of which the marine bacterial Nach1 and Nach2 share higher homology with human activators of NF-E2 p45, Nrf1 and Nrf2, as well the repressor Bach1. As such, they indeed perform distinct transcriptional abilities to mediate differential expression of ARE-battery genes. Yet, it is required for further determination of a


Furthermore, we identified differentially co-expressed and co-regulated mRNA modules, which might be considered as potential systems biomarkers for NFPA prognosis and invasiveness. As a result, we have identified a novel 13-gene module, including CEACAM6, CYP4B1, EIF2S2, HID1, IFFO1, MYO18A, PDCD2, SGIP1, SWSAP1, and four unknown genes , which was able to categorize the patients into two groups as invasive and non-invasive NFPA with distinct prognosis. The prognostic core module genes were associated with progression and prognosis of brain and glandular based cancers as well. Furthermore, these module genes were also expressed in blood, salivary gland, and spinal cord tissues. These results may provide the evidence on featured gene module which might play a prominent role in NFPA prognosis and sub-typing as effective biomarkers and therapeutic targets in the future.Non-functioning pituitary adenomas (NFPAs) are tumors with clinically challenging features since they have insidious progression. A complex network of gene interactions is thought to have roles in tumor formation and progression. Therefore, revealing the genetic network behind NFPA tumorigenesis is not only essential to attain further knowledge of tumor biology, but also plays a fundamental role in the development of efficacious treatment strategies. Differential co-expression network analysis is an outstanding approach for elucidation of groups of genes which show distinct co-expression patterns among phenotypes. In this study, we carried out a differential co-expression network analysis of NFPA-associated transcriptome dataset ( Pituitary adenomas (PAs) are the second most frequently reported primary brain tumors that are associated with increased mortality and morbidity , 2. Non-In the classification of tumors of the endocrine system, World Health Organization (WHO) offers several markers associated with invasiveness such as the Ki-67 proliferative index, the number of mitotic figures, and the expression of p53  and H-cadherin (CDH13) were proposed as markers of invasiveness at mRNA level (MEG3) and Hox transcript antisense intergenic RNA (HOTAIR), were correlated with NFPA development and invasion  associated with the prognostic gene module were investigated, and expression levels of the module genes in various tissues were also screened.http://cancergenome.nih.gov/). Among three transcriptome datasets, GSE63357 (https://www.ebi.ac.uk/arrayexpress/). Gene expression profiles generated using Agilent whole human genome oligo microarrays were used as a training set to construct co-expression networks, identify hub genes, and differentially expressed genes (DEGs) in this study. Transcriptome data was composed of 40 samples classified as invasive (n = 22) or non-invasive (n = 18). NFPA samples were characterized in terms of clinical and pathological features including age, sex, hormonal secretion , immunohistochemical staining, tumor volume (varied between 0.5 and 77 cm3), tumor grade, follow-up time, and outcome . Immunostaining for FSH-β and LH-β and/or α-subunit was used for tumor characterization. According to WHO 2017 classification of tumors of the pituitary gland  downloaded from ArrayExpress database . The expP-values were obtained via Fisher's Exact Test. Benjamini-Hochberg's correction was used as the multiple testing correction techniques, and enrichment results with adjusted p < 0.01 were considered statistically significant. Comparison of the significant GO terms and KEGG pathways was performed to identify the different biological functions and processes between the invasive and non-invasive samples.The pathway and functional enrichment analyses were performed using ConsensusPathDB (Release 32)  to identp ≤ 0.05.Co-expression networks were constructed for invasive and non-invasive PA states as previously described . InitialTo identify differential co-expression profiles between two states, the following criterion was employed:NONINV and PCCINV are the PCC of a DEG pair in non-invasive and invasive states, respectively. Resultant gene pairs were selected to construct the co-expression networks, ICON (invasive co-expression network) and NICON (non-invasive co-expression network), representing invasive and non-invasive states of PA, respectively.where PCCTo elucidate highly connected network modules, co-expression networks were analyzed using MCODE  plugin oConsidering the expression profiles of module genes, Principal component analysis (PCA) was performed to cluster invasive and non-invasive samples. The principal components explaining at least 85% of the total variance was considered in the determination of clustering performance, i.e., the ability hereupon “sub-typing” of PA.p-value, the hazard ratio (HR), and its confidence intervals (CI).PCA was performed based on gene expression profiles of module genes, and samples were categorized into three clusters using k-means algorithm taking into consideration the first three principal components . Cox regression was performed via MedCalc Statistical Software  to elucidate survival-associated genes. Analysis results were visualized and compared through Kaplan-Meier plots using the follow-up times of invasive and non-invasive groups. The prognostic power of the module was determined using the log-rank test n = 77), breast invasive carcinoma , and ovarian serous cystadenocarcinoma; brain tumors including glioblastoma , meningioma , and medulloblastoma  (n = 497) and lung adenocarcinoma . For each dataset employed, samples were partitioned into low and high-risk groups according to their prognostic index, survival multivariate analyses, and risk assessments. The prognostic performance of modules in each dataset was determined using Kaplan-Meier plots, log-rank test p-values, hazard ratios (HR), and their confidence intervals (CI). Differences in gene expression levels between high and low-risk groups were presented by box-plots and statistical significance of the difference was calculated through t-test. Heatmaps were also created to clarify the correlation of the survival analysis with gene expression levels. Samples were sorted by their prognostic index and genes were clustered by using Euclidean distance.We used SurvExpress  web-base n = 76) , 30; andThe expression levels of modules genes in easily collectible tissues were obtained from The Genotype-Tissue Expression (GTEx) project , which p3), tumor grade (I-IV), follow-up time (3–10.5 years), and outcome . Tumor size and invasiveness were determined with regard to preoperative magnetic resonance imaging and perioperative findings, by employing the modified Hardy criteria (n = 22) and non-invasive (n = 18) groups.Transcriptome data (E-TABM-899) was composed of 40 NFPA samples, which were characterized in terms of clinical and pathological features including age, sex, hormonal secretion , immunohistochemical staining (FSH-β and LH-β and/or α-subunit), tumor volume  was observed between mean expression levels of invasive (88.49 ± 2.32) and non-invasive groups  as a criterion in the identification of DEGs and determination of their direction of regulation. This filtering resulted in 641 DEGs of which 145 were down-regulated and 496 were up-regulated.Following up a standard protocol including quantile normalization of raw data, statistical comparisons of expression levels through “limma” package, and multiple testing correction with Benjamini-Hochberg method, a total of 4,091 genes were identified as differentially expressed between two phenotypes, i.e., invasive and non-invasive pituitary adenoma, considering a statistical confidence level of p < 0.01) were axon guidance (17 genes), ABC family mediated transport (8 genes), SUMOylation of DNA damage response and repair (6 genes), estrogen receptor alpha signaling pathway (5 genes), imatinib pathway (3 genes), and warfarin pathway , disassembly of protein complexes and its regulation, endocytic recycling, and response to copper ion; whereas genes associated with responses to lipids, DNA damage stimulus, and cytokines, regulation of protein localization, proteasomal protein catabolism, endoplasmic-reticulum-associated protein degradation (ERAD) pathway, regulations of telomere maintenance and oxidoreductase activity, cell aging, and oxidative demethylation were up-regulated . The maj2 genes; .The possible co-expression patterns of 641 DEGs were analyzed through the employment of PCCs in invasive and non-invasive phenotypes separately to construct phenotype-specific co-expression networks. This resulted with a denser, centralized, and modular co-expression network in invasive phenotype (ICON) with 183 links among 162 genes  when comARL5, ASTN2, C11orf39, CTNNAL1, EIF2S2, NPTX2, RALGPS2, and SLC12A9) were enriched in cytolysis, Ras protein and Rho protein signal transduction and small GTPase signal transduction biological processes  were enriched in processes such as urogenital system development, epoxygenase P450 pathway, thyroid gland development, protein tyrosine kinase activity, drug metabolic processes, protein kinase B signaling, intracellular receptor signaling pathway , which were activated in non-invasive phenotype when compared to invasive. Differentially co-expressed gene modules were named as M1 (Module 1), M2, M3, M4, M5, M6, M7, M8, M9, and M10. Module sizes and their associations with biological processes and PA were tabulated . M1 was To determine the possible associations of the identified co-expression modules with clinicopathological features , we conducted the Principal Components Analyses (PCA) as well as Kaplan Meier survival analyses, and detected significant correlations with tumor grade and sub-type for several gene modules.CEACAM6, CYP4B1, EIF2S2, HID1, IFFO1, MYO18A, PDCD2, SGIP1, SWSAP1, A_24_P127621, A_24_P255786, A_24_P683553, A_24_P916979), which indicates higher discrimination accuracy, since clusters representing invasive and non-invasive states were clearly separable and distinguishable without any overlaps in PCA plots (p < 0.001). Notably, four genes  located in distinct chromosomes were not annotated with any molecular function yet , and the average survival probability of non-invasive and grade II tumors was significantly higher than that of invasive and grade IV tumors with a hazard ratio of 2.96 (p = 0.011).The genes in the core module indicated significantly different co-expression patterns between invasive and non-invasive phenotypes of PA . The cluGATA1, GATA2, ETS1, ESR1, and PRDM14 were top five regulators with highest degree values . Members of the core module were found in varied chromosomal locations. CEACAM6 and SWSAP1 were located on chromosome 19 whereas HID1 and MYO18A were on chromosome 17, CYP4B1, and SGIP1 were on chromosome 1, PDCD2 was on chromosome 6, A_24_P127621 was on chromosome 7, A_24_P683553 was on chromosome 9, A_24_P255786 was on chromosome 10, IFFO1 was on chromosome 12, EIF2S2 was on chromosome 20, and A_24_P916979 located on chromosome X.In order to elucidate the regulatory mechanism behind the co-expression pattern of module genes and to evaluate the condition-specific expression pattern alterations, we performed serial analyses to link the key regulators of transcriptional control, i.e., transcription factors (TFs) and microRNAs (miRNAs), to genes in the core module. A total of 10 TFs were found as regulators of the module genes and e values . The majt miRNAs . Among tp = 4.69 × 10−6), breast invasive carcinoma , and ovarian serous cystadenocarcinoma . Secondly, we tested the performance of core module in brain tumors (p < 10−20), meningioma , and medulloblastoma . Lastly, we tested the prognostic performance of the core module in two other unrelated tissue but highly prevalent cancers, namely lung, and prostate adenocarcinoma. This time survival analyses pointed out insignificant performance in both lung (p = 0.08) and prostate (p = 0.15) adenocarcinomas  in order to decipher whether these genes can be detectable at mRNA level. Core module genes have detectable expression values in varied tissue types like blood, kidney, lung, spleen, spinal cord, saliva, pancreas, and uterus. It was also difficult to obtain a cumulative behavior in expression patterns of the genes in the same tissue; however, we found that every core module genes have considerable expression values in three tissue types, which are blood, salivary gland, and spinal cord .There is a great need for interpretation of omics data to elucidate the multi-layered molecular mechanisms lay behind the invasion of PA. However, omics datasets associated with PA invasiveness is scarce in the publicly available databases. Even in TCGA, which is the most comprehensive and multi-dimensional database for cancer genomics with 33 cancer types, genomics and/or transcriptomics of PA were not considered. The scarcity of omics datasets indicates the urgent need for further efforts on mRNA profiling in NFPA.p53  separately. ICON hub genes were enriched mostly in signaling processes like Ras, Rho, and small-GTPase signaling. It was reported that Ras small GTPase had a key signal transduction role in activating MAPK cascade . Rho GTPCEACAM6, CYP4B1, EIF2S2, HID1, IFFO1, MYO18A, PDCD2, SGIP1, SWSAP1, and 4 unknown genes . The unknown genes, A_24_P127621, A_24_P683553, A_24_P255786, and A_24_P916979, have specific gene sequences and distinct chromosomal locations ; however, there was no known functional annotation of them in any of the annotation sources. Further in-depth experimental research studies might be performed to elucidate their function and association with PA tumorigenesis and/or invasiveness. The high prognostic power of the core module was not specific to NFPA but also observed in cancers with the same tissue origin such as glandular-origin cancers and brain cancers.The differential co-expression profiling followed by clustering and survival analyses resulted with a prognostic core module composed of 13 genes; namely, CEACAM6 encodes a protein that is a member of the carcinoembryonic antigen (CEA) family whose crew are cell surface glycoproteins  samples and cervical cancer vs. normal group . The loss of expression of HID1 was reported in breast, cervical, lung, thyroid, renal, and gastrointestinal cancer cell lines  gene encodes a nuclear protein expressed in a variety of tissues and its expression is controlled by transcriptional repressor BCL6. It plays an important role in cell death and/or in the regulation of cell proliferation. PCDC2 has been found as a tumor suppressor and involved in the pathogenesis of osteosarcoma. Aberrant expression of PDCD2 is associated with many tumors, such as leukemia and gastric cancer  functions as an endocytic protein that has effects on signaling in neuronal systems including energy homeostasis  gene is involved in homologous recombination repair by binding single-stranded DNA activity and it also has ATPase activity. Depletion of SWSAP1 contributes to defects in homologous recombination repair and Knockdown of SWSAP1 ends up with increased cell sensitivity to the DNA damaging  were unknown with their unknown functions. The present study may contribute to assigning function/ functions to these genes. Known module genes were briefly explained with their functions and roles in various types of cancers. But there was no research found that could report module members for PA progression and prognosis. Therefore, identified module genes and module itself are novel for PA prognosis and they might be considered as the indicator of the non-invasiveness.Remaining core module gene members  and glandular- originated tumors . Our findings are based on TCGA, ArrayExpress, and GEO data , so a critical extension of this work would be to learn whether the patterns can be recapitulated by clinical trials. All in all, future efforts should be carried out to incorporate this systems-level understanding of prognostic genes into the practice of construction of effective clinical prognostic vehicles.While our study provides some insight into the prognosis of PA in different states, more efforts should be performed to validate them clinically and extend our findings. We examined the prognosis of PA through core module genes in the context of gene co-expression networks. Results were CEACAM6, CYP4B1, EIF2S2, HID1, IFFO1,MYO18A, PDCD2, SGIP1, SWSAP1, and 4 unknown genes  were identified and in silico validated in association with the indicator of invasiveness and prognosis of PA, plus some related cancer types. The research not only increases the theoretical knowledge but also provides a novel prognostic module and therapeutic strategy for PA.Pituitary adenomas are the most common intracranial tumors in the central nervous system. They are accepted as benign in general but somehow tumors can exhibit gross invasion into surrounding tissues rarely. Invasion results in resistance to conventional treatment methods and leading to early and frequent recurrences. In order to shed light on the multi-layered molecular mechanism lay behind the invasion of PA, there is a great need for omics-level data and their integration into meta-analyses. Our study used gene co-expression analysis to construct a gene co-expression network, elucidate and validate network modules associated with the invasiveness and prognosis of PA. Eventually, a core module with 13 genes including https://www.ebi.ac.uk/arrayexpress/experiments/E-TABM-899/.Publicly available datasets were analyzed in this study. This data can be found here: BA and KYA designed the data analysis framework. BA performed the data analyses and evaluated the results. KYA conceived and directed the study. BA drafted the manuscript. KYA revised the manuscript. All authors read and approved the final 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.


SGLT-2 (sodium-glucose cotransporter-2) inhibitors are a novel class of oral hypoglycemic agents for the management of type 2 diabetes mellitus (T2DM). Herein, we aimed to assess the long-term effectiveness and safety of SGLT-2 inhibitors in a Southern Italy population of subjects affected by T2DM.  408 diabetic patients treated with one of the three SGLT-2 inhibitors currently available in Italy , either alone or in combination with other antidiabetic drugs, were retrospectively assessed at baseline, during, and after 18 months of continuous therapy. P < 0.001) in fasting plasma glucose. Variations in HbA1c occurred independently of the baseline clinical or biochemical characteristics. In addition, treatment with SGLT-2 inhibitors reduced body weight (P < 0.008) and decreased diastolic blood pressure (P = 0.004). With regard to safety outcomes, 66 patients out of 91 stopped SGLT-2 inhibitors during follow-up because of chronic or recurring genital infections, while the rest experienced other adverse events, such as urinary tract infections, polyuria, nausea, hypotension, dizziness, acute coronary event, worsening of glycemic control status, and rapid deterioration of renal function.  Treatment with SGLT-2 inhibitors resulted in a median decrease in HbA1c of 0.9%, with a percentage of decrement of 12 in relation to the baseline value, followed by a significant reduction ( In our patients' population, the glycometabolic effects of SGLT-2 inhibitors were durable and comparable to those observed in multicenter randomized controlled trials. This notwithstanding safety concerns must be raised regarding the frequent occurrence of genitourinary infections and the risk of a rapid decline of renal function in patients with evidence of volume depletion and/or receiving other medications which can adversely affect kidney function. Over the last four decades, the global prevalence of type 2 diabetes mellitus (T2DM) has quadrupled, in parallel to that of obesity , becauseβ-cell function [2), SGLT-2 inhibitors do not increase the urinary glucose excretion and, therefore, are not recommended. Beyond glycemic control, SGLT-2 inhibitors have also the potential of reducing weight, due to the calorie loss through glycosuria, and be beneficial for lowering blood pressure, due to their osmotic diuretic effect [SGLT-2 (sodium-glucose cotransporter-2) inhibitors are the last class of antidiabetic drugs approved by FDA and EMA regulatory agencies, which could be used in any stage of T2DM, irrespective of comedications. By lowering the renal threshold for glucose excretion, SGLT-2 inhibitors suppress renal glucose reabsorption with insulin-independent mechanisms and are therefore suitable for patients with long-standing diabetes and impairment of function . Howeverc effect . Furtherc effect –13 and Cc effect  have shoc effect , 16. Froc effect , with thc effect –22. Thisc effect , 24.The purpose of this study was to evaluate the long-term safety and efficacy of three SGLT-2 inhibitors  administered to Calabrian patients with T2DM, attending our endocrinology/diabetology outpatient clinics.Forxiga® and Xigduo®), empagliflozin 10 mg and 25 mg/day , and canagliflozin 100 mg and 300 mg/day (Invokana® and Vokanamet®), either alone or in combination with other antidiabetic drugs (including insulin), on the basis of the international clinical practice recommendations for the management of hyperglycemia in T2DM [Forxiga®) in Italy. Age, sex, body mass index (BMI), blood pressure (BP), lipid profile, fasting plasma glucose (FPG), HbA1c, aspartate aminotransferase/alanine aminotransferase (AST/ALT), serum creatinine, duration of diabetes, micro- and macrovascular complications of T2DM, and any concomitant pharmacological therapy were recorded at baseline for all patients.In this study, we retrospectively analyzed the safety and efficacy of SGLT-2 inhibitors in a Southern Italy population of subjects affected by T2DM. Data were collected from 408 diabetic patients who started treatment with one of the three SGLT-2 inhibitors currently available in Italy: dapagliflozin 10 mg/day . As the data were analyzed anonymously, there was no need for written informed consent. Study was performed in accordance with the Declaration of Helsinki.Data collection was approved by the ethics committee of All patients underwent periodical clinical and biochemical evaluation to monitor the safety and efficacy of SGLT-2 inhibitors therapy. The variables analyzed to assess efficacy included: HbA1c, FPG, body weight, BMI, systolic and diastolic BP, and total daily insulin dose (TDI). Safety variables included fasting lipid profile, AST and ALT liver enzymes, serum creatinine, estimated glomerular filtration rate (eGFR) using both the CKD-EPI creatinine equation and the MDRD study equation, genitourinary infections, hypoglycemic episodes, dehydration, and volume depletion symptoms. Any medical problems, including possible adverse events, were recorded on diary cards, and the entries were reviewed at each study visit.The primary outcome was to test the safety and tolerability of SGLT-2 inhibitors in our patients' population. The primary efficacy outcome measure was the change from baseline in HbA1c after 18 months of treatment. The secondary outcome measures included changes in body weight, BMI, BP, FPG, lipid profile, AST and ALT, TDI, and proportion of participants achieving HbA1c level < 7.0%. In addition, we searched for potential baseline predictors of a better response to therapy.P value of <0.05 (two-tailed) was considered statistically significant. Data were analyzed with SPSS 20.0 software .Initially, each quantitative trait was tested for normality of distribution, using the Shapiro-Wilk normality test. Continuous variables were expressed as median and interquartile range (IQR) and categorical variables as numbers and percentages. The nonparametric Wilcoxon signed-rank test was used for within-group quantitative differences, whereas the two-tailed Fisher's exact test was used for comparisons of proportions. Spearman's rank correlation analysis was used to explore the correlation between safety and efficacy of SGLT-2 inhibitors with clinical and biochemical parameters. A A total of 408 diabetic patients started the treatment with SGLT-2 inhibitors. Out of these patients, 246 (60.3%) were treated with empagliflozin, 107 (26.2%) with dapagliflozin, and 55 (13.5%) with canagliflozin. N = 66) discontinued treatment at 7.5 (3-12 IQR) months for chronic or recurring genital yeast infections, whereas 11 patients stopped therapy at 6 (3-12 IQR) months for persistent or recurrent urinary tract infections. 21 patients stopped using SGLT-2 medications for other adverse events, including polyuria (6 patients), nausea (1 patient), hypotension (1 patient), dizziness (1 patient), acute coronary event (1 patient), worsening of glycemic control status (3 patients), rapid deterioration of renal function as defined by a rising of serum creatinine levels (4 patients), and other side effects (4 patients). In 4 individuals, therapy was discontinued for lack of compliance. There were neither lower-extremity amputations nor episodes of stroke during the study period. In terms of percentage, women (31.2%) were more likely to discontinue SGLT-2 inhibitor therapy than men (18.7%) (P = 0.005), mainly because of chronic or recurring genital yeast infections (P = 0.020) . No other correlation was detected. As shown in 27 patients were lost to follow-up after a median duration of 3 months (0-9 IQR), while 98 (24%) individuals  stopped SGLT-2 inhibitors during follow-up because of adverse events. As reported in = 0.020) . On SpeaP < 0.001). In details, we observed a reduction of 0.9% (0.3-1.7), with a percentage of decrement of 12 (3.5-20.1) with respect to the baseline value , and this decrease paralleled the reduction in BMI: from 30.2 kg/m2 (27.8 to 33.0) to 29.4 kg/m2 (26.8 to 32.2) (P = 0.009). The decreasing trend in systolic BP, from 135 mmHg (120 to 150) to 130 mmHg (120 to 140), did not reach conventional levels of statistical significance (P = 0.111), whereas diastolic BP significantly decreased from 80 mmHg (70 to 80) to 70 mmHg (70 to 80) (P = 0.004). Instead, a significant increase emerged in HDL cholesterol after treatment: from 43 mg/dL (36 to 48) to 45 mg/dL (40 to 55) (P = 0.004). No significant differences were observed in other serum and urinary parameters, as well as in eGFR and TDI. When determinants of these differences were explored, only the use of sulfonylureas inversely correlated with body weight reduction . Finally, 25 further patients (26.3%) reached ADA target for glycemic control (HbA1c < 7%) . Among them, 11 patients (44%), who were on insulin therapy, held stable or lower daily insulin requirements following combination with SGLT-2 inhibitors: from 26 UI (17 to 32) to 20 UI (10 to 32).Considering the secondary outcomes, levels of FPG decreased from 182 mg/dL (160 to 208) to 144 mg/dL (121 to 168) (From the results of the present study, it can be concluded that treatment with SGLT-2 inhibitors can significantly lower FPG and HbA1c in T2DM patients over 18 months of follow-up in real-life clinical practice. As seen in our case, patients using SGLT-2 inhibitors showed a reduction in HbA1c of about 1% at the end of the study period, and over a quarter of participants achieved the ADA glycemic targets, thus closely resembling the efficacy results of randomized clinical trials with these agents , 27. ImpOnly the concomitant use of SGLT-2 inhibitors with sulfonylureas was inversely correlated with weight reduction, making this combination therapy less suitable for overweight and obese patients as it may encourage weight gain . Also, fConsistent with previous studies , our finCandida colonization of genital tissues [With regard to safety issues with SGLT-2 inhibitors, our data are in contrast to other postmarketing observational studies , 41. Chr tissues  and the  tissues .One strength of the present study is the large subset of adult patients with T2DM who received at least one dose of SGLT-2 inhibitors from a single tertiary care center, regardless of previous and/or concomitant antidiabetic agents, duration and severity of disease, comorbidities, and follow-up time. The study reflects real-life clinical practice, partially avoiding the selection bias that is common in randomized clinical trials. All patients were recruited in Calabria, Southern Italy, a region with limited genetic diversity , which lOur study indicates that the beneficial durable effects of SGLT-2 inhibitors in Calabrian patients with T2DM are comparable to those from multicenter randomized controlled trials. Safety concerns must be raised regarding the occurrence of genitourinary infections, which, although generally mild or moderate in intensity, tend to recur and eventually lead to treatment discontinuation. A warning should also be issued about the risk of rapid deterioration of renal function in patients prone to volume depletion and/or receiving medications, such as nonsteroidal anti-inflammatory drugs and renin-angiotensin-aldosterone system blockers. So, it is recommended that renal function should be monitored closely during SGLT-2 inhibitor therapy.


Transient Receptor Potential (TRP) channel consist in a superfamily of non-selective cation channels that are widely expressed within vascular endothelial cells. In addition, TRP channels are present in the two main endothelial progenitor cell (EPC) populations, i.e., myeloid angiogenic cells (MACs) and endothelial colony forming cells (ECFCs). TRP channels are polymodal channels that can assemble in homo- and heteromeric complexes and may be sensitive to both pro-angiogenic cues and subtle changes in local microenvironment. These features render TRP channels the most versatile Ca2+ entry pathway in vascular endothelial cells and in EPCs. Herein, we describe how endothelial TRP channels stimulate vascular remodeling by promoting angiogenesis, arteriogenesis and vasculogenesis through the integration of multiple environmental, e.g., extracellular growth factors and chemokines, and intracellular, e.g., reactive oxygen species, a decrease in Mg2+ levels, or hypercholesterolemia, stimuli. In addition, we illustrate how endothelial TRP channels induce neovascularization in response to synthetic agonists and small molecule drugs. We focus the attention on TRPC1, TRPC3, TRPC4, TRPC5, TRPC6, TRPV1, TRPV4, TRPM2, TRPM4, TRPM7, TRPA1, that were shown to be involved in angiogenesis, arteriogenesis and vasculogenesis. Finally, we discuss the role of endothelial TRP channels in aberrant tumor vascularization by focusing on TRPC1, TRPC3, TRPV2, TRPV4, TRPM8, and TRPA1. These observations suggest that endothelial TRP channels represent potential therapeutic targets in multiple disorders featured by abnormal vascularization, including cancer, ischemic disorders, retinal degeneration and neurodegeneration.Vasculogenesis, angiogenesis and arteriogenesis represent three crucial mechanisms involved in the formation and maintenance of the vascular network in embryonal and post-natal life. It has long been known that endothelial Ca Likewis (ECFCs) , which r (ECFCs) . Transie stimuli . Herein,The TRP channel superfamily of non-selective cation channels comprises 28 members that are subdivided in six subfamilies by sequence homology: TRP Canonical (TRPC1-7), TRP Vanilloid (TRPV1-6), TRP Melastatin (TRPM1-8), TRP Ankyrin 1 (TRPA1), TRP Mucolipin (TRPM1-3), and TRP Polycystin (TRPP) . The TRP2- and COOH-terminal tails. TRP channel subunits assemble into a tetrameric complex around a central cation conducting pathway lined by TM5, TM6 and their interconnecting pore loop  and STIM1 (2) , which fulfill numerous functions, including cytoskeleton interaction and channel opening by membrane deformation (“spring hypothesis”), channel gating upon covalent modifications induced by extracellular ligands, and channel tetramerization  α-helix segments, a reentrant pore loop between TM5 and TM6, and cytosolic NHore loop . This str in TM4 . The NH2nd STIM1 . For insnd STIM1 . The TRPnd STIM1 . TRP-boxTIM1 (2) , or is iTIM1 (2) . TRP-boxTIM1 (2) . In addirization . While Tnal tail . Coiled nd TRPPs . Moreover, STIM1 . Intrigur, STIM1 . TRPM6 ar, STIM1 . Moreover, STIM1 .All TRP subunits may preferentially aggregate into homomeric cation channels , althoug2O2)], intracellular ions , dietary agonists , synthetic ligands , gasotransmitters , proteins , mechanical perturbation , and temperature fluctuations  receptors (InsP3Rs) , arachidonic acid (AA), ADPr and hydrogen peroxide (Htuations . In addiInsP3Rs) .+ and K+) and divalent  cations, although their relative permeability to Ca2+ and Na+ (PCa/PNa) may widely change among the different subunits. For instance, TRPV5 and TRPV6 display the largest PCa/PNa (>100), while TRPM4 and TRPM5 are virtually impermeable to Ca2+ (PCa/PNa < 0.01), although they are gated by an increase in [Ca2+]i  up to 20 (TRPV1)  of vascular endothelial cells, which may scatter between −88 and −20 mV (+ and Ca2+ (ENa = ECa ≈ +60 mV), but more positive than the reversal potential for K+ (EK = −88 mV). Therefore, in response to extracellular stimulation, TRP channels conduct inward Na+ (and Ca2+ or Mg2+) currents, which exert a profound impact on intracellular Ca2+ dynamics. Accordingly, Ca2+ entry through Ca2+-permeable TRP channels may directly increase the [Ca2+]i, whereas Na+ influx induces membrane depolarization, thereby activating voltage-gated Ca2+ channels in excitable cells and modulating the driving-force for Ca2+ entry in non-excitable cells  microscopy   . Neverthd −20 mV , is morele cells . The funt of ∼5% . The capcroscopy . A recenductance , such asparklets . This fis (RyRs) .Endothelial cells line the inner lumen of blood vessels and are, therefore, exposed to a myriad of chemical  and physical  cues, which must be properly perceived for the maintenance of tissue homeostasis ,2019b. Tin vitro and may be further affected by serial passages of cultured cells , thereby causing a positive shift in VM that could be boosted by the Ca2+-dependent recruitment of TRPM4 (yet to be demonstrated) , TRP channels may result in endothelial-dependent hyperpolarization (EDH). EDH may in turn be electrotonically propagated to adjacent VSMCs, thereby promoting vasodilation and increasing local blood flow in mesenteric arteries and cerebral microcirculation  . Furtherstrated) . Notablychannels . Likewisal cells  as well al cells . Alternaculation .2O2-induced endothelial hyperpermeability in human pulmonary artery endothelial cells   , activat (NF-κB) , and nuc (NF-κB) . Endotheculation . Likewisculation , and by culation  and TRPVculation  in mouseculation . It is wculation . Finallyculation , whereasver 40°C . This sever 40°C . Besidesver 40°C .2+ entry in response to extracellular growth factors, such as VEGF and bFGF, that are liberated in peripheral circulation upon an ischemic insult or by growing tumors , and/or by detecting subtle changes in the composition of the local microenvironment, and/or in response to small molecule drugs , a minor phospholipid component of the plasma membrane, into the intracellular second messengers, InsP3 and DAG   that is detected by STIM1, a single-pass transmembrane protein that senses [Ca2+]ER  channels (2+ entry (SOCE). The pro-angiogenic role of SOCE has been mainly evaluated upon endothelial cell stimulation with VEGF, the master regulator of vascular remodeling  may fall below the resolving power of whole-cell patch clamp recording systems.An increase in endothelial ER . Once acchannels , therebymodeling . The phyn HUVECs  and HAECn HUVECs . Of note2+ current (known as ISOC) in HUVECs  approximately equal to 0 mV, and a single-channel conductance in the pS range . Notably, SOC was sensitive to 1 μM La3+, which selectively targets Orai1 rather than TRPC1  by binding to FGF receptor-1 (FGFR-1) and stimulating cytosolic phospholipase A2, phospholipase D (PLD) and PLC to release AA, which in turn recruits a heteromeric TRPC1/TRPC4 channel (2+ influx with carboxyamidotriazole (CAI) inhibit BAEC proliferation  were impaired by transfecting the cells with a specific siTRPC4  impaired spontaneous Ca26 cells . Consist26 cells . Oxygen 26 cells . Accordi26 cells . Earlierlls   and genetic (by using selective siRNAs) manipulation demonstrated that TRPC3-mediated Na+ entry could switch the Na+/Ca2+ exchanger (NCX) in the reverse (Ca2+ entry) mode  with diltiazem and amlodipine . PTEN was found to physically interact with TRPC6 through its tail domain (residues 394-403), thereby promoting the membrane translocation of TRPC6 and Ca2+ influx upon exposure to thrombin  . Subsequonstruct . Similarry) mode , 2017. Tn HUVECs , 2017. Tn HUVECs . Likewis in vivo . However to bFGF . Interessequence . Additiong TRPC3 . In addilodipine . It shoual cells  and thatal cells . TRPC6, al cells  and humaal cells . Interesthrombin . Conversthrombin . Therefo2+ entry downstream PLC activation, although the gating role of STIM proteins and ER Ca2+ store emptying remains to be fully elucidated  in an ERK- and NADPH oxidase-dependent manner, thereby inducing TRPC5 to translocate toward the plasma membrane and inhibit BAEC motility  and angiopoietin 1 expression  play clear roles in vascular function , while T and ∼15 . Converselective . TRPV1-4agonists . They aragonists . Herein,agonists . Furtheragonists .2S), and tarantula spider-derived vanillotoxins  TRPV1 phosphorylation, (2) physical interaction between eNOS and TRPV1; (3) eNOS phosphorylation and, ultimately, (4) NO release , was the first mammalian TRPV family member to be discovered and the most extensively studied . TRPV1 ilotoxins . Multipllotoxins , althouglotoxins . This st created at the femoral artery and vein . The fol2+ entry . These f2+ entry  and in t2+ entry .2+ than Na+ ions and mediate outward rectifier ionic currents  and is gated by the intracellular second messenger ADPr, which binds to its COOH-terminal NUDIX domain, H2O2 and compounds that generate reactive oxygen/nitrogen species , presents a significant fractional Ca2+ current, and is activated by dietary compounds, such as allicin and AITC, mechanical deformation, and thermal stimuli  in rodent MACs  expressed only TRPC1 and TRPC4, while umbilical cord blood-derived ECFCs were also endowed with TRPC3 . Geneticin ECFCs , which sse PAECs . SOCE ma in vivo  and by V in vivo , 2015b.  in vivo , whereasr, NF-κB . Similarent MACs . Furthertivation .2+ oscillations  pre-incubation with AA itself  and TRPV4 activation with AA or 4αPDD induced migration in B-TECs, but not control human cardiac microvascular endothelial cells  ConverseA itself . Further network . These din vitro by restoring their mechanosensitivity toward ECM stiffness through the blockade of basal Rho activity , which displayed enhanced motility due to their lower mechanosensitivity toward extracellular matrix (ECM) stiffness. This, in turn, resulted in excessive tumor growth and vascularization in a mouse model deficient of TRPV4 and subcutaneously injected with Lewis Lung Carcinoma (LLC) cells . Notablyactivity . Moreoveactivity . In addi in vivo . These fin vivo. Pharmacological (with selective agonists) and genetic (with specific siRNAs) manipulation further revealed that: (1) TRPA1 supported PCa-derived endothelial cell migration; (2) TRPC3 promoted PCa-derived endothelial cell chemoattraction toward tumor microenvironment; and (3) TRPV2 induced capillary-like formation in vitro and vascular development in a mouse model of postnatal retina in vivo .  in vivo . The proothelium . Therefon the ER . Genova 2+ oscillations in tumor-derived ECFCs -  and primed ECFCs , a featued ECFCs .2+ entry pathway in vascular endothelial cells, ECFCs and MACs. For instance, TRPC3 and TRPC6 mediate the pro-angiogenic response to VEGF, whereas TRPV4 senses the increase in laminar shear stress upon vascular occlusion, thereby promoting arteriogenesis. Nevertheless, endothelial TRP channels may also dampen angiogenesis, as reported for TRPM4 in HUVECs and TRPM7 in brain microvascular endothelial cells. Of course, a number of issues are yet to be clarified in order to fully appreciate and exploit the therapeutic potential of endothelial TRP channels. For instance, it is still unclear whether and how TRPC1 is recruited by VEGF in vascular endothelial cells and how it interacts with STIM1 and Orai1 to mediate VEGF-induced intracellular Ca2+ oscillations in ECFCs. Furthermore, while the role of endothelial TRP channels has been mainly investigated in vitro, the measurement of TRP channels-mediated endothelial Ca2+ signals in vivo remains a challenging issue. Recent investigations exploited a zebrafish model engineered to selectively express an endothelial genetic Ca2+ indicator (in vivo. The sensitivity of endothelial TRPV channels to a moderate to large increase in local temperature also represents a puzzling issue (Endothelial TRP channels serve a crucial role in vascular remodeling by regulating angiogenesis, arteriogenesis and vasculogenesis. The remarkable heterogeneity in their gating mechanisms, which can be further increased by the propensity of some isoforms to assemble into heteromeric complexes, render TRP channels the most versatile Candicator . Likely,ng issue . According issue  and hypeng issue , whereasng issue . The conng issue . An addiFM and SN drafted and finally edited the manuscript. PF, RB-R, and GG significantly contributed to revise the manuscript. All the authors read and approved the final version of 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.


To assess the association between plasma retinol-binding protein 4 (RBP4) levels both in the first trimester and second trimester and risk of gestational diabetes mellitus (GDM).Plasma RBP4 levels and insulin were measured among 135 GDM cases and 135 controls nested within the Peking University Birth Cohort in Tongzhou. Multivariable linear regression analysis was conducted to assess the influence of RBP4 levels on insulin resistance. Conditional logistic regression models were used to compute the odds ratio (OR) and 95% confidence interval (CI) between RBP4 levels and risk of GDM.P < 0.05). Plasma RBP4 concentrations in the first and second trimester were associated with fasting insulin, homeostasis model assessment for insulin resistance (HOMA-IR), and the quantitative insulin sensitivity check index (QUICKI) in the second trimester . With adjustment for diet, physical activity, and other risk factors for GDM, the risk of GDM increased with every 1-log μg/L increment of RBP4 levels, and the OR (95% CI) was 3.12 (1.08–9.04) for RBP4 in the first trimester and 3.38 (1.03–11.08) for RBP4 in the second trimester.The GDM cases had significantly higher levels of RBP4 in the first trimester than controls (medians: 18.0 μg/L vs 14.4 μg/L; Plasma RBP4 levels both in the first trimester and second trimester were dose-dependently associated with increased risk of GDM. RBP4 gene could enhance insulin sensitivity  [The primary outcomes of interest were insulin resistance and GDM. Homeostasis model assessment (HOMA) was used to estimate insulin resistance [HOMA-IR = insulin/(22.5elucose)] . The qualucose)] . GDM waslucose)] . Women w− 1) [Covariates were collected at the first prenatal visit, such as maternal characteristics regarding demographic information, pre-pregnancy weight, pregnancy history, smoking status, alcohol intake, family history of diabetes. Dietary intake was recorded for two non-consecutive days and average daily intake of calories was calculated. Physical activity was evaluated using the last 7-day, short form of the International Physical Activity Questionnaire and quantified using metabolic equivalents of task (MET-min week− 1) . Each su2 test. Multivariable linear regression analysis was conducted to estimate the associations of RBP4 levels in the first and second trimester with fasting insulin, HOMA-IR and QUICKI in the second trimester. Then, study subjects were divided into four groups  according to quartiles of RBP4 levels among control group, and the lowest quartile (Q1) was used as the reference group. Conditional logistic regression models were used to compute the odds ratio (OR) and 95% confidence interval (CI) between RBP4 levels and risk of GDM. Three models were established to assess the robustness of the results. In model 1, we adjusted for maternal age, education, occupation, gestational weeks of RBP4 measurements in the first trimester, pre-pregnancy BMI, systolic blood pressure (SBP), and diastolic blood pressure (DBP) at the first prenatal visit, and GWG before OGTT. We additionally adjusted for biochemical indicators involving total cholesterol, triglyceride, HDL, LDL, GFR, ALT, and AST in model 2. Further adjustment for daily intake of calories and weekly physical activity time was conducted in model 3. We also performed tests for linear trend by entering the median value of each RBP4 level as a continuous variable in the models. Restricted cubic spline regressions were used with three knots to examine possible nonlinear relationships between RBP4 levels and risk of GDM. In the sensitivity analysis, we converted all continuous covariates into categorical covariates  to test the robustness of our results. Moreover, we conducted a subgroup analysis to test if maternal age  could modify the association between RBP4 and GDM by adding an interaction term to the regression models. Unconditional logistic regression models were applied to compute the results. All analyses were performed with SAS 9.4 and two-sided P values < 0.05 was considered to be statistically significant.Continuous variables were presented as medians  and analyzed for between-group differences using Wilcoxon rank sum test. Categorical variables were analyzed for between-group differences using the χP > 0.05).The characteristics of the study subjects are presented in Table P < 0.05; Table P < 0.001), after adjustment for potential confounders, including biochemical indicators, daily intake of calories, and physical activity  and second trimester  after adjusting for maternal age, education, occupation, gestational weeks of RBP4 measurements in the first trimester, pre-pregnancy BMI, GWG before OGTT, SBP, and DBP. After additional adjustment for biochemical indicators, subjects in the highest quartile of RBP4 had 3.72-fold  and 3.25-fold  higher risk of developing GDM, respectively. In addition, further adjusting for daily intake of calories and physical activity didn’t attenuate this association, and the adjusted OR (95% CI) was 3.71 (1.27~10.84) for RBP4 in the first trimester and 3.30 (1.03~10.52) for RBP4 in the second trimester. We also estimated the risk of GDM associated with every 1-log μg/L increment in RBP4 levels, and the OR (95% CI) in model 3 was 3.12 (1.08~9.04) for RBP4 in the first trimester and 3.38 (1.03~11.08) for RBP4 in the second trimester. Findings of the sensitivity analysis showed that our results were robust after converting all continuous covariates into categorical covariates in the models . In the subgroup analysis, we found that maternal age did not modify the association between RBP4 levels and GDM . Another study also found RBP4 levels were significantly correlated with HOMA-IR in the first trimester in healthy pregnant women  [Mechanism studies found that RBP4 was involved in the pathogenesis of diabetes by inducing insulin resistance through several etiologic pathways. Increasing serum RBP4 could induce the expression of the phosphoenolpyruvate kinase from the liver and impair insulin signaling in muscle . Further< 0.001) . However< 0.001) . The con< 0.001) . FurtherThe relationship between RBP4 levels in early pregnancy and the risk of GDM have been assessed by limited studies and inconclusive results were yielded. A prospective study found that compared with the first quartile of RBP4 in the first trimester, adjusted ORs of GDM for the second, third and fourth quartile were 1.54, 3.05 and 6.36 respectively . AnotherSeveral limitations of the current study should be considered. First, although we adjusted for several potential confounders such as diet and physical activity, we cannot exclude the possibility of unmeasured confounders. Second, RBP4 is generally present in free and transthyretin-bound forms for the transport of retinol. Previous study reported that the RBP4:retinol ratio and the RBP4:transthyretin ratio might be more informative than RBP4 levels alone when assessing insulin resistance during pregnancy . We did In summary, we reported for the first time a dose-dependent association between RBP4 levels both in the first trimester and second trimester and GDM risk. In addition, plasma RBP4 in the first and second trimester is associated with insulin resistance in the second trimester. Further research is warranted to determine the underlying mechanism and examine if a reduction in RBP4 concentrations could lower the risk of GDM.Additional file 1: Figure S1. The dose-response analysis between GDM and RBP4 levels in the first trimester (A) and the second trimester (B) with restricted cubic spline. Conditional logistic regression models were adjusted for maternal age, education, occupation, gestational weeks of RBP4 measurements in the first trimester, pre-pregnancy BMI, GWG before OGTT, SBP, DBP, total cholesterol, triglyceride, HDL, LDL, GFR, ALT, AST, daily intake of calories, and weekly physical activity time. Bold lines are ORs and dashed lines are 95% CIs. The horizontal line is the reference line. Abbreviation: GDM, gestational diabetes mellitus; RBP4, retinol-binding protein 4.Additional file 2: Table S1. Comparison of the baseline characteristics between GDM cases included in the analysis and all GDM cases. Abbreviation: GDM, gestational diabetes mellitus; Gweek, gestational week; BMI, body mass index; GWG, gestational weight gain; OGTT, oral glucose tolerance test; PA, physical activity; SBP, systolic blood pressure; DBP, diastolic blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein; GFR, glomerular filtration rate; ALT, alanine transaminase; AST, aspartate aminotransferase.Additional file 3: Table S2. Comparison of the baseline characteristics between controls included in the analysis and all controls. Abbreviation: GDM, gestational diabetes mellitus; Gweek, gestational week; BMI, body mass index; GWG, gestational weight gain; OGTT, oral glucose tolerance test; PA, physical activity; SBP, systolic blood pressure; DBP, diastolic blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein; GFR, glomerular filtration rate; ALT, alanine transaminase; AST, aspartate aminotransferase.Additional file 4: Table S3. Odds ratio  of GDM associated with different levels of RBP4 in the first trimester and second trimester after converting all continuous covariates into categorical covariates in the models. Models were adjusted for maternal age , education , occupation , gestational weeks of RBP4 measurements in the first trimester , pre-pregnancy BMI , GWG before OGTT , SBP , DBP , total cholesterol , triglyceride , HDL , LDL , GFR , ALT , AST , daily intake of calories  and weekly physical activity time . Abbreviation: GDM, gestational diabetes mellitus; RBP4, retinol-binding protein 4.Additional file 5: Table S4. Odds ratio  of GDM associated with different levels of RBP4 in the first trimester and second trimester stratified by maternal age. Unconditional logistic regression models were adjusted for maternal age, education, occupation, gestational weeks of RBP4 measurements in the first trimester, pre-pregnancy BMI, GWG before OGTT, SBP, DBP, total cholesterol, triglyceride, HDL, LDL, GFR, ALT, AST, daily intake of calories, and weekly physical activity time. Abbreviation: GDM, gestational diabetes mellitus; RBP4, retinol-binding protein 4.