COL5A1 and COL5A2 genes encoding type V collagen (COLLV), which is a fibrillar COLL widely distributed in a variety of connective tissues. cEDS patients suffer from skin hyperextensibility, abnormal wound healing/atrophic scars, and joint hypermobility. Most of the causative variants result in a non-functional COL5A1 allele and COLLV haploinsufficiency, whilst COL5A2 mutations affect its structural integrity. To shed light into disease mechanisms involved in cEDS, we performed gene expression profiling in skin fibroblasts from four patients harboring haploinsufficient and structural mutations in both disease genes. Transcriptome profiling revealed significant changes in the expression levels of different extracellular matrix (ECM)-related genes, such as SPP1, POSTN, EDIL3, IGFBP2, and C3, which encode both matricellular and soluble proteins that are mainly involved in cell proliferation and migration, and cutaneous wound healing. These gene expression changes are consistent with our previous protein findings on in vitro fibroblasts from other cEDS patients, which exhibited reduced migration and poor wound repair owing to COLLV disorganization, altered deposition of fibronectin into ECM, and an abnormal integrin pattern. Microarray analysis also indicated the decreased expression of DNAJB7, VIPAS39, CCPG1, ATG10, SVIP, which encode molecular chaperones facilitating protein folding, enzymes regulating post-Golgi COLLs processing, and proteins acting as cargo receptors required for endoplasmic reticulum (ER) proteostasis and implicated in the autophagy process. Patients’ cells also showed altered mRNA levels of many cell cycle regulating genes including CCNE2, KIF4A, MKI67, DTL, and DDIAS. Protein studies showed that aberrant COLLV expression causes the disassembly of itself and many structural ECM constituents including COLLI, COLLIII, fibronectin, and fibrillins. Our findings provide the first molecular evidence of significant gene expression changes in cEDS skin fibroblasts highlighting that defective ECM remodeling, ER homeostasis and autophagy might play a role in the pathogenesis of this connective tissue disorder.Classical Ehlers-Danlos syndrome (cEDS) is a dominant inherited connective tissue disorder mainly caused by mutations in the  Classical Ehlers-Danlos syndrome (cEDS) MIM #130000) is an autosomal dominant connective tissue disorder with an estimated prevalence of 1:20,000 and is mainly characterized by abnormal skin texture and joint hypermobility (JHM). According to the 2017 EDS nosology [30000 is COL5A1 and COL5A2 genes encoding type V collagen (COLLV) [COL1A1 c.934C>T (p.Arg312Cys) variant is found [COL5A1 allele, whereas variants in COL5A2 affect its structural integrity and are generally associated with a more severe phenotype [COL5A1 are nonsense, frameshift or splice site mutations that generate a premature termination codon, with the consequent activation of the nonsense-mediated mRNA decay pathway, finally leading to COLLV haploinsufficiency [COL5A2 are missense or in-frame exon-skipping splice mutations, which result in the production of mutant α2(V) chains that exert a dominant negative effect on COLLV molecules [More than 90% of cEDS patients harbor a pathogenic variant in the  (COLLV) ,5. Besidis found . COLLV hhenotype ,8. The mi.e., “cauliflower”-shaped fibrils with an abnormal diameter distribution, and abnormal packing [COLLV, classified as a regulatory fibril-forming COLL, is a heterotrimer composed of two pro-α1(V) chains and a single pro-α2(V) chain . COLLV p packing .Col5a1- and Col5a2-deficient mice showed deficits in wound healing [COLLV has also been implicated in wound healing process given its upregulated expression during tissue healing ,13, alth healing ,18.To shed light on altered gene expression pattern and dysregulated biological processes involved in the molecular pathology of cEDS, we carried out transcriptome profiling on dermal fibroblasts from four cEDS patients with either haploinsufficient or structural mutations in both causative genes.S1 Table.Approval of the present research was obtained by the local Ethical Committee “Comitato Etico di Brescia, ASST degli Spedali Civili, Brescia, Italia”, registration number NP2658 and the study was conducted in accordance with the principles of the Declaration of Helsinki. cEDS patients  and healthy individuals  were evaluated in the Centre of Heritable Connective tissue disorders and Ehlers-Danlos syndromes of the Spedali Civili of Brescia. Mean age at examination was 40 years for the patients (range 34–46) and 42 years for the healthy subjects (range 32–49). Written informed consent to the study and for skin biopsy was obtained from all patients and healthy donors according to Italian bioethics laws. All of the patients fulfilled the cEDS criteria of the Villefranche nosology  and its COL5A1 pathogenic variants, respectively, and as AN_002534 carrying the c.2499+2T>C (p.Gly816_Pro833del) COL5A2 splice variant. Specifically, the c.2988del frameshift mutation and the c.1165-2A>G splice variant are null alleles leading to COLLV haploinsufficiency, whereas the c.4178G>A missense mutation and the c.2499+2T>C splice site variant are structural mutations that exert a dominant negative effect on the protein by interfering with the triple helix formation [S1 Table.The cEDS fibroblast strains here analyzed  were obtained from previously clinically characterized patients in Ritelli et al., [in vitro at 37° C in a 5% CO2 atmosphere in Earle’s Modified Eagle Medium (MEM) supplemented with 2 mM L-glutamine, 10% FBS, 100 μg/ml penicillin and streptomycin . Cells were expanded until full confluency and then harvested by 0.25% trypsin/0.02% EDTA treatment at the same passage number (from 2nd to 6th), as previously described [Skin fibroblast cultures from four cEDS patients and nine unrelated age-matched healthy donors were established in our lab from skin biopsies by standard protocols. Fibroblasts were grown escribed –16.Goat anti-COLLI, anti-COLLIII, and anti-COLLV polyclonal antibodies (Abs) were from Millipore-Chemicon Int. . Mouse anti-fibrillins (FBNs) (clone 11C1.3) monoclonal Ab (mAb) was from NeoMarkers . The rabbit Ab against human FN was from Sigma Chemicals . Rhodamine-conjugated anti-goat IgG Ab was from Calbiochem–Novabiochem INTL, and the Alexa Fluor 488 anti-rabbit and Alexa Fluor 594 anti-mouse secondary Abs were from Life Technologies.To analyze the COLLI-, COLLIII-, COLLV-, FN- and FBNs-ECM organization, cEDS fibroblasts were immunoreacted 48 h after seeding as described previously , 20–23. vs control cells, a one-way ANOVA analysis was applied as a criterion for the selection of DEGs. To assess significant differences in the gene expression profile in cEDS fibroblasts, we selected genes that had more than a 1.5-fold change and p-value <0.05. Multiple testing correction was applied to control the false-discovery rate (FDR) using the Benjamini–Hochberg (BH) procedure [Total RNA was purified from skin fibroblasts of four cEDS patients and nine healthy individuals using the Qiagen RNeasy kit, by standard protocol . Prior to microarray analysis, quality control of RNA was checked on the Agilent 2100 BioAnalyzer . Transcriptome analysis was carried out by using the Affymetrix GeneChip Human Gene 1.0 ST array as previously described in detail . The resrocedure . Genes wHPRT, GAPDH, ATP5B, and CYC1 reference genes were amplified for normalization of cDNA loading. Relative mRNA expression levels were normalized to the geometric mean of these reference genes and analyzed using the 2-(ΔΔCt) method. Results were expressed as the mean value of relative quantification ± SEM. Statistical significance between groups was determined using unpaired Student’s t-test with GraphPad Prism software.Relative expression levels of a series of selected DEGs identified by array analysis were confirmed by quantitative real-time PCR (qPCR) using different RNA extractions obtained from skin fibroblast cultures of all patients and 6 out of 9 healthy subjects. 3 μg of total RNA were reverse-transcribed with random primers by standard procedure. qPCR was performed with SYBR Green qPCR Master Mix (Life Technologies), 10 ng of cDNA, and with 10 μM of each primer set. qPCR was performed using the ABI PRISM 7500 Real-Time PCR System by standard thermal cycling conditions: initial denaturation for 30 s at 95° C, followed by 40 cycles of 95° C for 15 s and 60° C for 60 s. S2 Table). Fig 1A represents the volcano plot that illustrates the statistical significance (FDR) vs fold-change of DEGs, and Table 1 reports a selection of up- and down-regulated DEGs (FDR < 0.05 and fold change threshold ≥ 1.5). To group transcripts with similar expression profiles between patients and controls, hierarchical clustering of DEGs was conducted .To identify genes potentially involved in the molecular mechanisms associated with the pathogenesis of cEDS, transcriptome-wide expression analysis was carried out comparing gene expression pattern among patients’ and controls’ skin fibroblasts. Gene expression profiling identified 548 DEGs in cEDS patients’ cells when compared to controls: of these, 368 genes were up-regulated, and 180 were down-regulated . The most prominent group of up-regulated DEGs was implicated in DNA replication, cell cycle regulation, and DNA damage and repair responses, including different cell cycle protein-encoding genes. Specifically, different genes, i.e., CCNE2, CDC6, TYMS, CDC45, MCM8, POLE2, RRM2, MCM10, CDCA5, MCM4, MCM5, MCM6, are involved in the transition of G1/S phase of cell cycle. Many genes belong to the minichromosome maintenance complex , which is a key component of the pre-replication complex required for the initiation of eukaryotic DNA replication. We also found that GO categories related to “DNA synthesis involved in DNA repair” and “double-strand break repair via homologous recombination” were significantly enriched with different up-regulated genes . Another group of significantly up-regulated genes encode various histone proteins, i.e., HIST2H3A, HIST1H2AB, HIST1H2BB, HIST1H2BM, HIST1H2AE, which are essential for nucleosome core assembly.Functional analysis of the 368 up-regulated genes yielded 11 different GO clusters (Table 3 and S4 Table) were associated with genes related to the regulation of transcription . Many of these transcription-associated genes encode zinc finger domain-containing proteins that are involved in gene transcription and translation, mRNA trafficking, cytoskeleton organization, cell adhesion, protein folding, and chromatin remodeling. Different down-regulated genes, i.e., CDKN1A, MDM2, PTEN, GADD45A, GADD45B, are implicated in p53 and FOXO signaling pathways involved in the control of DNA replication, chromosome segregation and cell division.The most enriched GO clusters of the 180 down-regulated DEGs (S2 Table) including SPP1, POSTN, and EDIL3, which encode matricellular proteins primarily acting as mediators of cell–matrix interactions rather than as structural components. Specifically, osteopontin (SPP1) and the epidermal growth factor-like repeats discoidin I-like domain 3 protein (EDIL3), which contain an Arg-Gly-Asp (RGD) tripeptide integrin binding motif, and periostin (POSTN), containing an N-terminal EMI domain through which it binds to COLLI and FN, play an important role in cell proliferation and migration, cutaneous wound healing, cell cycle, and apoptosis. Likewise, IGFBP2 and C3 genes, showing up-regulated transcription in cEDS fibroblasts (S2 Table), encode insulin like growth factor binding protein 2 and complement C3, respectively, which are also involved in cell migration and wound repair process.Patients’ cells also showed decreased mRNA levels of several ECM-associated genes .Finally, in cEDS fibroblasts biological functions related to intracellular protein transport, endoplasmic reticulum (ER) proteostasis, and autophagy seem to be perturbed, as suggested by the reduced transcription of many related genes such as S5 Table, cell cycle was the most perturbed pathway in patients’ cells, as shown by a significant dysregulated expression of many cell cycle regulating genes. Some of these DEGs, i.e, CDKN1A, MCM4, MCM5, MCM6, MCM10, RRM2, CDC45, TYMS, POLE2, MCM8, CCNE2, and CDC6, are specifically involved in G1/S transition and G2/M checkpoint. DNA replication seems to be also induced, as indicated by the augmented transcription of different associated genes such as replication factor subunit 3 (RFC3), DNA polymerase epsilon 2 (POLE2), different members of the mini-chromosome maintenance complex , and the flap endonuclease 1 (FEN1), all playing a crucial role in DNA replication and damage repair response.To identify differentially expressed pathways in cEDS fibroblasts, an enrichment analysis was carried out on all DEGs by querying both ToppGene and DAVID databases and selecting a threshold of FDR-adjusted p-value <0.05. As shown in CCNE2), cyclin-dependent kinase inhibitor 1A (CDKN1A), ribonucleotide reductase M2 (RRM2), MDM2 proto-oncogene (MDM2), phosphatase and tensin homolog (PTEN), and growth arrest and DNA-damage-inducible alpha (GADD45A) and beta (GADD45B). Regarding the FOXO signaling pathway, apart from CDKN1A, MDM2, PTEN, and GADD45A, cEDS cells also showed a higher expression of other FOXO related genes including polo-like kinase 1 (PLK1) and 4 (PLK4), SMAD family member 3 (SMAD3), and homer scaffold protein 2 (HOMER2).Pathways analysis also indicated that p53 and FOXO signaling pathways seem to be perturbed in cEDS fibroblasts. These pathways are master regulators of cellular homeostasis in response to different stress signals including growth factor deprivation, metabolic stress, oxidative stress, and DNA mutations. Concerning the p53 signaling pathway, its perturbation is indicated by the dysregulated expression of several p53-regulated genes including cyclin E2 (GSTM3), monoamine oxidase B (MAOB), and different members of UDP glucuronosyltransferase family 2, such as member B17 (UGT2B17), member A2 (UGT2A2), and member B7 (UGT2B7).Drug metabolism-related biological process was also disturbed, as indicated by the altered expression of glutathione S-transferase mu 3 . qPCR confirmed the differential expression of genes related to ECM remodeling, wound healing, and connective tissues integrity, such as ITGA3, EDIL3, POSTN, SPP1, CCBE1, MMP11, PAPPA, ADAMTSL1, and IGFBP2 . We also verified the altered expression of many transcripts related to ER homeostasis and autophagy, i.e., ATG10, CCPG1, VIPAS39, SVIP, ALG13, and DNAJB7 . Significant changes of mRNA levels of several cell cycle regulating genes. i.e., KIF4A, CDKN1A, CCNE2, ASF1B, MKI67, CLSPN, DTL, and DDIAS, were also confirmed . qPCR also revealed altered transcriptional levels of different genes implicated in the cell adhesion and cytoskeleton organization, such as ENAH, ACTR10, MTSS1, SIPA1L3, and PODXL , and involved in inflammatory and immune response, i.e., ACKR3, IFNA16, IFNA6, CCL13, IL8, IL1RL1, COLEC12, VEGFD, GDF15, and C3 .We evaluated the differential expression of a selection of DEGs by qPCR. Genes were prioritized based on their expression pattern and GO functional classification that was significantly perturbed in cEDS fibroblasts. Specifically, we focused on genes related to ECM remodeling, ER protein folding homeostasis/autophagy, inflammatory and immune response, cell cycle regulation, cell adhesion/motility and actin cytoskeleton organization .In cEDS patients’ skin fibroblasts, we previously demonstrated abnormal synthesis and deposition of COLLV into the ECM together with the disarray of FN and the lack of COLLs- and FN-specific integrin receptors ,16,20. Mreatment . IF analFig 5).The present study reports the first molecular evidence of significant gene expression changes in dermal fibroblasts from cEDS patients. Although the reduction in the amount of COLLV is central to the pathogenesis of cEDS ,27, so fSPP1, POSTN, EDIL3, IGFBP2, PAPPA, CCBE1, ITGA3, MMP11, and ADAMTSL1, which are involved in ECM turnover, cell migration, and wound healing. After cutaneous injury, the wound healing occurs through several processes, including coagulation, granulation tissue formation, re-epithelialization, and ECM remodeling [SPP1 encodes osteopontin (OPN), a secreted phosphoprotein with an RGD motif through which it binds to cell surface receptors including αvβ3 integrin and CD44 [in vivo and in vitro studies indicated that OPN promotes cell proliferation, cell migration, and wound healing through inhibition of apoptosis [Opn-deficient mice show altered wound healing, with small COLL fibrils and disorganized ECM in the deep layers of the wound [in vivo wound studies that the knockdown of OPN improves the skin wound healing and reduces scarring in mice [Opn deficient mice. In addition, dermal fibroblasts isolated from Opn-/- mice show decreased migration compared to control cells, whereas treatment with OPN partially restores their migratory capability, suggesting an involvement of OPN in dermal fibroblasts migration [Transcriptome profiling revealed the differential expression of a subset of genes, such as modeling . ECM commodeling ,30. ECM modeling ,31. Amonand CD44 . OPN is and CD44 . Severalpoptosis –36. Intehe wound . Despite in mice . Wu and  in mice  also demigration .POSTN encodes the matricellular protein periostin that is a predominantly expressed in COLLs-rich tissues and contributes to proper COLLs assembly and homeostasis [eostasis ,40. It weostasis , whereaseostasis .in vivo wound repair capability that was improved by exogenous administration of periostin, indicating a role of this protein in the resolution of cutaneous wound repair. The authors also demonstrated that loss of periostin impairs proliferation and migration of healthy dermal fibroblasts that is rescued by exogenous supplementation of the protein, supporting its key role in the response to wounding. Moreover, loss of periostin affects COLLs stability and assembly in cultured human dermal fibroblasts, leading to higher amounts of COLLs fragments owing to proteases digestion [in vivo wound healing studies on a Col5a1+/- haploinsufficient murine model showing that these mutant mice heal significantly slower than the wild type [Col5a2 null alleles that show deficits in wound healing [Col5a1+/- mice exhibit reduced proliferation, decreased attachment of fibroblasts to wound matrix components, and impaired cell migration [Like other ECM proteins, periostin plays a fundamental role in tissue remodeling. Indeed, periostin interacts with αvβ3 and αvβ5 integrins on cell surfaces, influencing cell–matrix interactions, adhesion, proliferation, and differentiation . In addiigestion . Given tigestion  owing toigestion ,16,20,46ild type , as in m healing . In addiigration .EDIL3, which is the most down-regulated transcript in cEDS cells, encodes a matrix-associated matricellular protein with three epidermal growth factor (EGF)-like repeats, two discoidin-like domains, and an RGD motif in the second EGF-like repeat [anoikis through binding the RGD motif of the αvβ3 integrin [in vitro, EGF receptor-signaling mediated pathways, and induces cell cycle arrest at the G1 phase [Edil3-deficient mice develop more severe osteoarthritis compared to control mice that is associated with increased susceptibility of chondrocytes to apoptosis [e repeat . EDIL3 ce repeat . In endointegrin . In addiG1 phase . Moreovepoptosis .IGFBP2, which is most up-regulated transcript in cEDS fibroblasts, is a member of six similar proteins that bind insulin-like growth factors I and II (IGFs) modulating their activity. Other IGF-independent actions ascribed to IGFBP2 involve the C-terminal RGD motif, which mediates the interaction with the α5β1 integrin [The protein encoded by integrin . This inintegrin –56. Thesintegrin .i.e., CCL13, IL8, IL1RL1, COLEC12, GDF15, and C3. Of these, C3, showing an about 20-fold mRNA levels increase in patients’ fibroblasts, encodes a member of the complement system, which is composed of several plasma proteins interacting with numerous immune mediators involved in inflammatory response in several biological processes including wound healing [C3-/- mice C3 deficiency accelerates the initial rate of excisional wound healing, whereas wound healing is attenuated with sera from wild type animals or purified human C3 treatment. C3 deficiency also modulates the inflammatory infiltrate of healing by reducing the numbers of neutrophils and increasing the number of mast cells, thus accelerating the whole wound healing process [Furthermore, during tissue repair, an excessive inflammation delays healing and may lead to complications and chronic wounds . Complem healing . Complem healing . Specifi healing . Indeed, process . Additio process .Taken together, these gene expression changes suggest a potential involvement of different ECM-related proteins such as osteopontin, periostin, IGFBP2, and C3 in both the reduced migratory capability and wound repair of cEDS patients’ fibroblasts. More research is needed to establish the concrete role of these proteins in the process of cutaneous wound healing in cEDS cells.The ADAMTS  superfamily contains 19 secreted metalloproteinases involved in various biological processes including extracellular matrix (ECM) degradation, matrix assembly, angiogenesis, and cell migration.DNAJB7, CCPG1, ATG10, SVIP, and VIPAS39. Among them, the protein encoded by the DNAJB7 belongs to the DnaJ heat shock protein family, which are molecular co-chaperones acting as ER reductases that catalyze the removal of non-native disulfides and enhance protein folding [CCPG1) encodes an ER-resident transmembrane protein that acts as a non-canonical autophagy cargo receptor essential for ER-phagy and ER proteostasis by binding Atg8-family proteins, thus preventing the hyperaccumulation of insoluble protein within the ER lumen [ATG10) is an E2-like conjugating enzyme involved in two ubiquitin-like modifications essential for autophagosome formation, a double-membrane vesicle containing cellular debris that are degraded via lysosome-autophagy pathway [SVIP, encoding the small p97/VCP-interacting protein, acts as an inhibitor of the ER-associated degradation pathway (ERAD) by competing with the E3 ligase binding to p97/VCP to regulate its function [SVIP reduces the levels of the LC3 microtubule-associated protein, which is involved in the autophagic pathway [VIPAS39 encodes a core constituent of the multiprotein tethering complexes, namely HOPS and CORVET complexes, which are essential to coordinate endosome and lysosome fusion in eukaryotic cells [Vps33b and Vipas39 deficient mice [ER is a highly dynamic organelle in eukaryotic cells, which is deputed to lipid and protein biosynthesis, calcium storage, and detoxification of various exogenous and endogenous harmful compounds. Signaling sensors within the ER detect lumen perturbations and employ downstream cascades that engage effector mechanisms to restore homeostasis . The mos folding . Cell-cyER lumen . The pro pathway . SVIP, efunction . The p97function . The negfunction . On the  pathway . VIPAS39ic cells . Specifiic cells . Indeed,ic cells . Furtherent mice . Similarent mice .in vivo [anoikis [anoikis promotes cell survival, thus acting as a persistence strategy to mitigate the stresses of ECM detachment [anoikis cEDS fibroblasts by a cross-talk with EGFR [Both physiological and pathological changes in the composition of the ECM have been shown to alter autophagy activity. It is well known that many ECM components including COLLVI, decorin, and laminin α2 modulate autophagy both positively and negatively . For insin vivo ,77. Besi[anoikis . Reducedtachment . In lineith EGFR . These fith EGFR ,81. Addiith EGFR . Moreoveith EGFR . Conversith EGFR . Taken tCCNE2, KIF4A, DTL), S phase progression (MKI67), and S/G2 transition (CLSPN), as well as components of the DNA replication machinery , which ensure sequential progression through the cell cycle in an orderly fashion [CKDN1A), which encodes the p21 protein that is an inhibitor of cyclin-dependent kinase/cyclin complexes acting as a regulator of cell cycle progression at G1 [DDIAS), which has an anti-apoptotic function during DNA damage by regulating caspase-8 stability and promoting its proteasomal degradation [The prominent group of DEGs in cEDS cells was implicated in processes related to cell division, DNA replication, DNA repair, telomere organization, and associated with nucleosome and chromatin assembly. We also found significant expression changes of several transcripts encoding histone proteins that could reflect a perturbation of DNA synthesis, given that histones enter in nucleosome formation and proper DNA wrapping after the S phase of the cell cycle . Besides fashion . Converson at G1 . Overallon at G1 . In lineradation .In conclusion, our approach illustrates global mRNA profiling changes of several genes and related biological processes that could offer novel insights in the cEDS pathophysiology. Additional functional work is required to verify whether altered ER proteostasis and defective autophagy play a crucial role in the pathogenesis of this connective tissue disorder, thus offering future therapeutic options.S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


TB are due to the defects in bacterial recognition, phagocytic activity, and cellular activation which results in impaired production of chemokines and cytokines. The initiation of adaptive immunity is delayed by impaired antigen-presenting cell (APC) recruitment and function in hyperglycemic host, which results in reduced frequencies of Th1, Th2, and Th17 cells and its secretion of cytokines having a great role in activation of macrophage and inflammatory response of tuberculosis. In addition, impaired immune response and killing of intracellular bacteria potentially increase bacterial load, chronic inflammation, and central necrosis that facilitate bacterial dissemination and miliary tuberculosis. Understanding of the immunological and biochemical basis of TB susceptibility in diabetic patients will tell us the rational development of implementation and therapeutic strategies to alleviate the dual burden of the diseases. Therefore, the aim of this review was focused on the association between diabetes and tuberculosis, focusing on epidemiology, pathogenesis, and immune dysfunction in diabetes mellitus, and its association with susceptibility, severity, and treatment outcome failure to tuberculosis.The interaction between diabetes and major world infections like TB is a major public health concern because of rapidly rising levels of diabetes. The dual burden of tuberculosis (TB) and diabetes mellitus (DM) has become a major global public health problem. Diabetes mellitus is a major risk factor for the development of active and latent tuberculosis. Immune mechanisms contributing to the increased susceptibility of diabetic patients to  Globβ-cells , 6. It r2) cells , 8. Actita cells . The CD4ta cells , 11. T2Dta cells . Obesityta cells . In obesta cells , 14. CD8ta cells . Subsequing foot , 18 and ing foot , 19. Theing foot . In addiing foot , 18. Thuing foot .A person who has diabetes mellitus are approximately 3 times more likely to develop tuberculosis than nondiabetics . TubercuGlobally, there are an estimated 9.6 million new patients with active TB annually; from those, one million people have both TB and DM . DespiteMTB occurs by inhalation of bacilli that invade and replicate in alveolar macrophages and horizontally spread to macrophages, myeloid DCs, and neutrophils recruited from the periphery, which results in priming of adaptive immunity [M. tuberculosis by mononuclear phagocytes [High susceptibility to infections, including TB, is a major cause of morbidity and mortality in patients with diabetes, and the probable cause of increased prevalence and complication of the infections in DM patients is immune dysfunction , 41. Natimmunity , 43. In immunity , 44. Comagocytes , 46. Moragocytes .γ and cytotoxic T cell targeting of MTB-infected macrophages [MTB antigen in patients with DM.Despite this, antigen-specific T cells expand and travel to the lung where they promote an effective antimicrobial response as a result of activating macrophage through release of IFNrophages . But fewrophages , 43. Howrophages . The impMTB activates an innate response that recruits multiple myeloid cell types to the alveolar airspace [Mycobacterium tuberculosis infection and replication [MTB cell wall components that contribute reduced phagocytosis of MTB and increase tuberculosis susceptibility in diabetic hosts [Differences in innate immunity between diabetic and nondiabetic patients are more significant in the susceptibility and pathogenesis of infections including TB , 48. It airspace . Alveolalication . It has lication , 51. Alvic hosts , 52.MTB-infected AM is impaired in hyperglycemic mice resulting in a reduced expression of signals and chemokines that recruit macrophages, DCs, neutrophils, and innate lymphocytes to the airspace, and also, it produces a barrier to leukocyte transmigration into the airspace even if adequate recruiting signals are produced by infected AM [γ also determines the activation of macrophage, on which its production is mediated by the release of IL-1β, IL-12, and IL-18 from APCs upon stimulation with mycobacteria. Therefore, type 2 diabetes patients might be characterized by decreased secretion of IL-1β, IL-12, and IL-18 and respond with less IFNγ upon stimulation, leading to increased susceptibility to TB [An experimental study by aerosol challenging hyperglycemic mice and euglycemic control mice indicated that the function of ected AM , 50. In ty to TB . Dendritty to TB  are one ty to TB . The migty to TB , 55, 56.ty to TB , 47, 56.ty to TB , 48. Multy to TB . Moreovety to TB . The impty to TB   dependent killing activity of macrophages, while IL-2 is an essential cytokine for the development and proliferation of Th1 and CD8+ T cells, and Th17 cell secretes IL-17 and IL-23 that plays inflammatory response of TB [γ has been evaluated; however, results are contraindicated with studies showing either no difference [γ : IL-4, IFNγ : IL-5, and IFNγ : IL-10 and TNFβ : IL-4, TNFβ : IL-5, and TNFβ : IL-10) were lower in the diabetic TB patients than NDM-TB patients and healthy subjects. Consequently, lower Th1 : Th2 ratios may possibly contribute to susceptibility of MTB infection under diabetic conditions [β are regulatory cytokines with a broad spectrum of activity, predominantly anti-inflammatory cytokine which interferes mycobacterial antigen-specific Th1 and Th2 cytokine production [γ secretion, IL-10 can help mycobacteria to survive intracellularly and the elevated secretion of IL-10 may contribute to increased pathogenesis in diabetic TB patients [Adaptive immunity against esponses . T helpese of TB , 53, 59.se of TB . Severalfference  or highefference , 62, andfference , 59, 60.fference , 59. Irrfference . Since Tnditions . Anotheroduction . Even thpatients , 59, 61 γ (IFNγ) to enhance macrophage microbicidal activity, also facilitates intracellular bacterial persistence. Adaptive immunity is delayed by an impaired antigen-presenting cell (APC), and reduced frequency in diabetic hosts and dysregulation of the cytokine profile alter the activation and differentiation of T cell subsets. B cell activation and antibody production may also be impaired. The dysregulated inflammatory milieu affects granuloma formation, contributing to increased neutrophil recruitment and central necrosis that facilitates bacterial escape .2+ signaling and release of oxidants enhancing the intracellular killing, other mechanisms enhancing cell-mediated immunity, clearance of immunomodulatory mycobacterial antigens, direct antimycobacterial activity, and activation of complement [In humoral immune response, the role of antibody (Ab) either in pathogenesis or in protection against tuberculosis was controversial , 64. Mosmplement , 67, 68.mplement .Mycobacterium tuberculosis infection with diabetes is rapidly progressive with a shortened survival interval, more severe pulmonary and extrapulmonary pathologies, and a higher bacterial burden as compared to NDM controls [α and IL-1β and extrapulmonary bacterial burden than in NDM controls which lead to increasing the severity and disease progression of TB [MTB infection [MTB infection. Despite this, M. tuberculosis infection with a diabetic is failing to control bacterial growth due to increased anti-inflammatory cytokine levels and IL-4 which inhibits the expression of IFNγ, resulting in rapid disease progression before the onset of adaptive immunity to M. tuberculosis in diabetic patients [controls . Moreovecontrols , 70. Labcontrols . In chroon of TB . Therefonfection , 48, 71.nfection , 71. In patients , 71. Patpatients –74. A copatients , 76. Thepatients , 76–78.In conclusion, tuberculosis is one of the major causes of morbidity and mortality among infectious diseases worldwide, including Ethiopia. Diabetic patients induce an abnormal function in both innate and adaptive immune responses, which increased risk of the combined TB disease development, complication, outcomes of treatment failure, and death. The immunological mechanisms of diabetes to susceptibility of tuberculosis are complex and not fully elucidated. Research suggests that immune dysfunction in diabetes is more susceptible to tuberculosis. However, results from studies so far remain inconsistent. Therefore, further studies are required to fully understand the impact of DM patients on the immune response and increased susceptibility and treatment outcome of TB.


The objectives of this study were to 1) evaluate the influence of treatment with praziquantel on the inflammatory milieu in maternal, placental, and cord blood, 2) assess the extent to which proinflammatory signatures in placental and cord blood impacts birth outcomes, and 3) evaluate the impact of other helminths on the inflammatory micro environment.This was a secondary analysis of samples from 369 mother-infant pairs participating in a randomized controlled trial of praziquantel given at 12–16 weeks’ gestation. We performed regression analysis to address our study objectives. In maternal peripheral blood, the concentrations of CXCL8, and TNF receptor I and II decreased from 12 to 32 weeks’ gestation, while IL-13 increased. Praziquantel treatment did not significantly alter the trajectory of the concentration of any of the cytokines examined. Hookworm infection was associated with elevated placental IL-1, CXCL8 and IFN-γ. The risk of small-for-gestational age increased with elevated IL-6, IL-10, and CXCL8 in cord blood. The risk of prematurity was increased when cord blood sTNFRI and placental IL-5 were elevated.Our study suggests that fetal cytokines, which may be related to infectious disease exposures, contribute to poor intrauterine growth. Additionally, hookworm infection influences cytokine concentrations at the maternal-fetal interface.NCT00486863).ClinicalTrials.gov . Hookworm coinfection at 12 weeks’ gestation was, however, related to elevated levels of certain cytokines in the placenta . Adverse perinatal outcomes account for a substantial proportion of the global burden of disease  and lay Schistosoma japonicum endemic area, Kurtis and colleagues found increased concentrations of pro-inflammatory cytokines including interleukin-1 (IL-1β) and tumor necrosis factor (TNF) in placental and cord blood among women with S. japonicum infection [With respect to helminthiasis, less is known with regard to treatment strategies for pregnant women. In a non-interventional study conducted in a nfection . Furthernfection . This raHealthy pregnancies are characterized by a placental microenvironment that is biased toward a T-helper 2 (Th2) cytokine milieu , 13, andAs part of the aforementioned RCT conducted in Leyte, The Philippines, we investigated whether treatment for schistosomiasis at 12–16 weeks’ gestation and the presence of other helminth infections would influence the cytokine micro-environment. Specifically, the objectives of this study were to 1) examine the impact of treatment with praziquantel on the inflammatory milieu in maternal, placental, and cord blood, 2) assess the extent to which proinflammatory signatures in placental and cord blood impacts the risk for LBW, SGA, and prematurity, and 3) evaluate the impact of other helminths on the inflammatory micro environment.S. japonicum and soil transmitted helminths (STHs) eggs using the Kato-Katz method [S. japonicum, age 18 or older, otherwise healthy as determined by physician history, physical examination and laboratory studies, and pregnant at 12–16 weeks’ gestation with a live, singleton, intrauterine fetus. Women who met eligibility criteria (n = 370) were randomly assigned (1:1) to receive either over-encapsulated praziquantel (30 mg/kg × 2) or over-encapsulated placebo (dextrose), as a split dose over three hours in a double-blind fashion.This was a secondary analysis of data from a double blind placebo-controlled RCT examining the effects of praziquantel given at 12–16 weeks’ gestation for the treatment of schistosomiasis on pregnancy outcomes . The RCTz method , 24. TheAt 12–16 weeks’ gestation, a detailed demographic and medical history was collected and physical examination (including anthropometric measures) conducted. Weight, height and other anthropometric measures were made as described , 26. AntS. japonicum, low, moderate and heavy intensity infections were defined as 1–99, 100–399 and ≥400 eggs per gram (epg), respectively; Ascaris lumbricoides, low, moderate and heavy intensity infections were defined as 1–4,999, 5,000–49,999 and ≥50,000 epg, respectively; Trichuris trichuria, low, moderate and heavy intensity infections were defined as 1–999, 1,000–9,999 and ≥10,000 epg, respectively; hookworm, low, moderate, and heavy intensity were defined as 1–1,999, 2,000–3,999 and ≥4,000 epg, respectively [Stool samples were collected and intensity of helminth infection was determined as the mean of the three samples, and categorized using WHO criteria as follows: ectively , 24.th percentile for gestational age based on the INTERGROWTH standard [Following initial stabilization of the newborn and mother, placental samples (wedge biopsy and pooled blood) and cord blood were collected. Newborns were examined and weighed within 48 hours of delivery on a Tanita model BD 585 portable scale . LBW was defined as birthweight below 2500g, and SGA as birthweight below the 10standard . PretermMaternal 12-week, 32-week, placental, and cord blood serum samples were aliquoted and stored at -80°C prior to testing. All available samples at each timepoint were used for comprehensive biomarker testing–only 238 cord blood samples were available. Assessment of biomarkers in the blood samples was conducted at the Center for International Health Research Laboratory in Providence, RI, USA. Biomarkers measured include IL-1, 2, 4, 5, 6, 8, 10, 12 and 13, interferon gamma (IFN-γ), TNF, chemokine ligand-9 (CXCL9) and soluble TNF receptors I and II (sTNFRI and sTNFRII). Analytes were quantified using a multiplex bead-based platform  as described previously . The lowIn analyses examining the impact of praziquantel treatment and helminth infections on cytokine production in maternal, placental and cord blood, these biomarkers were outcome measures. These biomarkers were separately evaluated as predictors of adverse pregnancy outcomes. Cytokine production was considered as exposure or outcome in this analysis. Three different measures of cytokine production were also employed: (i) cytokine concentration in ng/L, (ii) the proportion of those with an 'elevated' cytokine concentration, and (iii) the proportion with cytokine present at a level above the assay detection limit. The means (±SE) of maternal cytokine concentrations at 12- and 32-weeks’ gestation were also estimated and the mean difference and 95% confidence interval (CI) estimated.P-values obtained from Fisher’s exact tests. Further, the means (±SE) of cytokine concentrations at 32-weeks’ gestation (with 95% CI) were estimated within treatment subgroups and compared using linear regression. The extent to which the ratios of placental blood cytokines to maternal 12-week cytokines, and placental blood cytokines to maternal 32-week cytokines differed by treatment was also evaluated using Wilcoxon rank-sum tests.To investigate the effect of praziquantel treatment on cytokine production, the proportions of participants with cytokine concentrations above detection limits in maternal 32 weeks’, placental, and cord blood samples were compared across treatment groups, and Generalized estimating equation regression models were used to assess the impact of each helminth infection at 12 weeks’ gestation on the proportion of participants with cytokines at a level above the assay detection limits in maternal 12- and 32-weeks’ gestation, placental and cord blood samples. Log-binomial models were used to evaluate the relationship between elevated maternal 32-week peripheral cytokines and placental and cord blood cytokines, and risk ratios (RRs) with 95% CI obtained. Log-binomial models were also used to examine the influence of elevated placental and cord blood cytokines on the risk of LBW, SGA, and prematurity. Log-binomial models provide RR estimates, which are intuitive and more appropriate for non-case control studies. The log-binomial model is however numerically unstable, and often fails to converge, and in those instances, log-Poisson models, which provide consistent but not fully efficient estimates of the RR and its CIs were employed .P-value <0.15, with no variables forced into the model. Regression models were adjusted for predictors as specified in the footnotes of the respective tables and figures. Variables included in the models were praziquantel treatment, maternal age , newborn sex , maternal height (cm), maternal weight at 12 weeks (kg), maternal underweight (body mass index <18.5kg/m2), parity (number), socioeconomic status (quartiles), reported smoking status , alcohol use , and detection of S. japonicum, A. lumbricoides, T. trichuria, and hookworm, at 12 and 32 weeks’ gestation .Potential confounders known to be related to cytokines and/or perinatal outcomes were considered for inclusion in multivariable models. In addition, potential confounders were identified through stepwise regression techniques, significant at P-values for effect modification were obtained by introducing an interaction term to the log-binomial regression model, in which praziquantel treatment status was multiplied by the biomarker category, and the model compared to the model without the interaction term using the likelihood ratio test. Possible effect modification by hookworm infection at 12 weeks’ gestation was also explored.P-values were 2-sided and statistical significance was defined as P-value <0.001, based on the Bonferroni correction for the familywise error rate , to account for multiple comparisons [α level. All data in our study were de-identified. Analyses were conducted using SAS 9.4 .parisons . CIs werThe study was approved by both the Rhode Island Hospital Institutional Review Board in Providence, RI, USA and the Ethics Review Board of the Research Institute of Tropical Medicine in Manila, The Philippines. This trial was registered with ClinicalTrials.gov, number NCT00486863.Participants included in this analysis were 369. Detailed information on the cohort’s participant characteristics have been previously presented . Most ofFig 1 details the selection of samples for cytokine quantification. Maternal cytokine concentrations significantly decreased from 12 to 32 weeks’ gestation (S2 Supporting Information) for sTNFRI . The concentration of sTNFRII , IL-6  and CXCL8  decreased while the concentration of IL-13  increased from 12 to 32 weeks’ gestation but the Bonferroni corrected P-values were not significant.Fig 2). Praziquantel treatment lowered the concentration of anti-inflammatory IL-10 by 32-weeks’ gestation ), though the difference was not significant after Bonferroni’s correction . Praziquantel treatment did not alter the concentration of other cytokines considerably. There was also no evidence that praziquantel significantly altered the likelihood of detecting cytokines in maternal serum at 12 and 32 weeks, or in placental or cord blood (Table 2).The proportion of participants with detectable cytokines varied widely from 1–100% but tended to be highest in cord blood. To examine the impact of praziquantel treatment on cytokine concentrations, the concentration of cytokines in maternal blood at 32 weeks’ gestation was compared by treatment group , most were of light intensity . Hookworm infection was associated with a 1.42 to 2.58-fold increased risk of elevated placental levels above detection limits for some cytokines : IL-1 , IL-5 , CXCL8  and IFN-γ  in multivariable models. Hookworm infection was not associated with an increased risk of detectable cytokines in maternal peripheral or cord blood . S7 Supporting Information reports the concentration of each cytokine at which the 90th percentile level was reached. Participants with elevated maternal 32-week IL-4 , IL-12 , and IFN-γ  were more likely to have elevated placental levels of the same cytokines. There were no significant associations in the level of maternal cytokines with the levels of the same cytokines in the cord blood.We investigated the extent to which cytokine levels in maternal peripheral blood was related to cytokine levels in placental and cord blood in multivariable log-binomial regression models (Tables  were associated with 2-fold increased risk of SGA: IL-10 (Th2)–RR = 1.80 , IL-6 –RR = 1.84 , and CXCL8 –RR = 1.84  . Elevated sTNFRI  and IL-5 in placenta  were associated with increased risk of prematurity . These associations were not significant following Bonferroni correction. There was no evidence that levels of other placental and cord blood cytokines were related to the occurrence of prematurity, LBW and SGA.The prevalence of LBW, prematurity and SGA were 14% (n = 52), 9% (n = 33) and 23% (n = 84), respectively. Elevated levels of certain cytokines in the cord blood Click here for additional data file.S2 Supporting Information(DOCX)Click here for additional data file.S3 Supporting Information(DOCX)Click here for additional data file.S4 Supporting Information(DOCX)Click here for additional data file.S5 Supporting Information(DOCX)Click here for additional data file.S6 Supporting Information(DOCX)Click here for additional data file.S7 Supporting Information(DOCX)Click here for additional data file.S8 Supporting Information(DOCX)Click here for additional data file.


Pancreatic cancer is often treatment-resistant, with the emerging standard of care, gemcitabine, affording only a few months of incrementally-deteriorating survival. Reflecting on the history of failed clinical trials, genetically engineered mouse models (GEMMs) in oncology research provides the inspiration to discover new treatments for pancreatic cancer that come from better knowledge of pathogenesis mechanisms, not only of the derangements in and consequently acquired capabilities of the cancer cells, but also in the aberrant microenvironment that becomes established to support, sustain, and enhance neoplastic progression. On the other hand, the existing mutational profile of pancreatic cancer guides our understanding of the disease, but leaves many important questions of pancreatic cancer biology unanswered. Over the past decade, a series of transgenic and gene knockout mouse modes have been produced that develop pancreatic cancers with features reflective of metastatic pancreatic ductal adenocarcinoma (PDAC) in humans. Animal models of PDAC are likely to be essential to understanding the genetics and biology of the disease and may provide the foundation for advances in early diagnosis and treatment. Pancreatic ductal adenocarcinoma (PDAC) is the most deadly of common human adult malignancies . The vasAmidst these dismal statistics, there are three areas of recent significant progress in understanding pancreatic carcinogenesis. The first is the observation that PDACs arise from the progression of non-invasive ductal epithelial neoplasms. Termed pancreatic intraepithelial neoplasms (PanIN), these lesions have progressively increasing architectural and cytological atypia, akin to intraepithelial neoplasms in other human tissues ,7. GradeKRAS is an oncogene that encodes a small GTPase transductor protein that, in its active, GTP-bound form, engages a broad series of kinase pathways relating to cell proliferation, survival, migration, metabolism, and many other biological processes. Numerous pathways, such as stimulated growth factor receptors, transduce their downstream effects through RAS guanine exchange factors (RAS-GEFs) that activate the RAS family proteins, KRAS, HRAS, and NRAS ,18. ConvIn addition to a role in tumor initiation, it appears that KRAS activation is required for maintenance of the tumorigenic growth of established PDAC, since disruption of KRAS activity—via RNA interference, antisense RNA, or expression of dominant-negative KRASN17—attenuates the tumorigenicity of PDAC cell lines . Hence, G12D mice from eight to 26 weeks of age, all showing pancreatic ductal lesions strongly reminiscent of human PanINs. Pdx1-Cre-mediated activation of the KrasG12D allele alone leads to PanIN formation, the constellation of mucinous transformation of the ductular epithelium with nuclear atypia, and papillary growth [G12D mice is relatively restricted to these abnormal ductal structures. Similar results were observed when crossing KRASG12D mice with animals harboring Cre recombinase under the control of pancreatic acinar cell-specific promoter, p48 (p48Cre), yields KRASG12D; p48Cre mice that develop pancreatic intraepithelial precursor lesions (PanINs) within four weeks of age [Activating K-Ras mutations are the first genetic changes detected in the progression series present in about 30% of lesions showing the earliest stages of histological disturbance . Increasy growth ,24. Agais of age ,24. Thuss of age . Thus, ts of age ,15,31.PI3K–AKT Pathway is an intracellular molecular pathway that plays a role in the regulation of cell proliferation, survival, and metabolism ,33. It cPDACs show elevated expression of EGF receptors and their ligands, consistent with the presence of autocrine EGFR signaling ,49. EGFRLox/Lox mice led to a centroacinar cell that possessed adult stem cell properties, the expansion of which produced metaplasias similar to the phenotypes of Pdx1-Cre, KrasG12D/+, PtenLox/+ mice. In addition to the intensive metaplastic phenotype, Pdx1-Cre Ptenlox/lox mice eventually develop invasive pancreatic adenocarcinomas at a low frequency [Lox/Lox, p53−/− compound mice tend to develop papillary ductal adenocarcinomas , sometimes mixed with small acinar carcinoma features [Aberrant activation of the PI3K–AKT pathway has been widely implicated in human cancers, including PDAC . The PTErequency . Meanwhifeatures .LKB1 (Liver kinase B1) is a tumor suppressor gene that activates the AMPKα signaling pathway and regulates cell polarity, survival, and metabolism ,65. InacG12D alleles in promoting tumors in several mouse models [12D, Pdx1-Cre, Ink4a/ArfLoxp/Loxp mice with documented efficient deletion of the Ink4a/Arf in the pancreas developed weight loss, ascites, jaundice, and a palpable abdominal mass between seven and 11 weeks of age [G12D mice with the Pdx1-Cre, LSL-KrasG12D, Ink4a/ArfLoxp/Loxp mice unequivocally proves a role of Ink4a/Arf in constraining the malignant progression of early-stage ductal neoplasms. Loss of INK4A function, brought about by mutation, deletion, or promoter hypermethylation, occurs in 85%–90% of sporadic PDACs . INK4A le models ,76. In tTGFβ is a potent inhibitor of epithelial cell growth and survival, although these effects are highly dependent on the cell context. In numerous epithelial cell lines and in epithelial tissue in vivo, TGFβ exerts a growth inhibitory program that involves modulation of cell cycle regulators, including the induction of p15INK4B and p21CIP1 expression, repression of c-Myc and ID family transcription factors, induction of apoptotic machinery, and repression of telomerase. The tumor suppressor role of TGFβ signaling is underscored by presence of inactivating TGFβ receptor mutations in a number of cancers . SignifiThe 4th most frequently mutated gene in PDAC is SMAD4 , encoding a transcriptional regulator that is a central component in the TGF-β superfamily signaling cascades ,84,85. TG12D expression (Ptf1a-cre KrasG12D Tgfbr2Lox/Lox mice) developed well-differentiated PDAC at 8–10 weeks of age with 100% penetrance. Heterozygous deletion of Tgfbr2 with KrasG12D expression also developed well differentiated PDAC with higher frequency of liver, duodenum, and lung metastases as compared to Ptf1a-cre; LSL-KrasG12D/+; Tgfbr2Lox/Lox mice [TGFβ is thought to promote PDAC desmoplasia , as well as contribute to the proliferation and invasion of the tumor cells in an autocrine manner; notably, the blockade of TGFβ signaling attenuates tumorigenicity of some xenografts ,96. In cLox/Lox, KrasG12D, Pdx1-Cre mice [TGFβ signal is an important biological tumor suppressor program that is based on the prevalent genomic deletion of the TGF-β superfamily gene in pancreatic cancer . RecentlCre mice .G12D and Pdx-1Cre, LSL-KrasG12D, p53Lox/Lox PDAC models, combined with KLF10 loss to dissect the molecular mechanism underlying KLF10 loss on pancreatic tumor development and progression [G12D leading to an invasive and widely metastatic phenotype of PDAC. Our studies further revealed that loss of KLF10 increased distant metastases and cancer stemness through activation of SDF-1/CXCR4 and AP-1 pathways in PDAC [KLF10, the zinc finger transcription factor, is a member of the Krüppel-like family of transcription factors. KLF10 can be induced by estrogen, TGF-βs, BMP, NGF, and EGF ,110,111.gression . In our  in PDAC . The TG-interacting factor 1 (TGIF1), a nuclear transcriptional corepressor of the TGFβ/Smad signaling, has been associated in the pathogenesis of numerous types of cancer . A recenL/+ mice. Apc modulates the canonical Wnt signaling pathway through mediating β−catenin degradation, thus inactivation of APC creates a permissive condition, whereby free unphosphorylated β-catenin is significantly more stable, and translocates into the nucleus to active Wnt signaling [G12D mutation and P53 loss in mice, leading to rapidly increased development of metastatic PDAC when compared with Pdx1-cre, KrasG12D, P53Lox/+ mice That evidence indicated a critical tumor suppressor role of APC in pancreatic cancer progression [Pdx1-Cre, KrasG12D/+, APCignaling ,127,128.ignaling ,130. Addignaling ,132. Furignaling ,134. Mutignaling ,136. Recignaling . Furtherignaling . These dgression .DNA damage sometimes results in multiple broken chromosomes, which can be caused by many factors, such as ionizing radiation (IR) and ultra violet light (UV), and by various metabolic compounds within cells (including reactive oxygen species (ROS) and nitric oxide (NO)) ,141. DNAG12D cooperate to promote PDAC progression and systemic metastasis in mice [G12D and a germline heterozygosity for Brca2 Tr/Δ11 background, and Pdx1-Cre, LSL-K-rasG12D mutant mice, and they found that the median survival for Pdx1-Cre, LSL-K-rasG12D, Brca2 Tr/Δ11 mice is significantly shorter by approximately 5 months. In contrast, in another study to demonstrate the function of Brca2 in the progression of pancreatic cancer, the mouse models that combined pancreas specific KrasG12D activation and TP53 deletion with BRCA2 inactivation were generated. Unexpectedly, their results indicated that the mutant model of TP53 deletion and Brca2 inactivation promoted pancreatic cancer development, but the mouse model of KrasG12D combined with BRCA2 inactivation caused chromosomal instability and cell apoptosis, and resulted in the inhibition of tumors growth [The BRCA1 and BRCA2, tumor suppressor genes, play a critical role in the repair of DNA double-strand breaks and homologous recombination (HR) pathway, and is involved in maintenance of structural and numeric chromosomal stability as well ,160. Not in mice . In thiss growth .G12D of pancreatic cancer induces more proliferative precursor lesions coupled with pancreatic fibrosis in the mice [ATM (ataxia telangiectasia-mutated) is the crucial DNA damage sensor of the response to DNA double strand breaks, which plays the tumor suppressor role in the DNA damage checkpoint pathway as p53 after DNA double-strand breaks ,169. Losthe mice . Furtherthe mice .The chromatin remodeler Switch/Sucrose nonfermentable (SWI/SNF)-complex functions as a tumor suppressor in many human malignant cancers ,173,174.The SWI/SNF complex functions as a crucial regulator of gene expression by recruiting to specific DNA regions and interacts with various transcription factors. The SWI/SNF complex was able to shift the position of histones of the chromatin in an ATP-dependent manner, and to give transcription factors access to bind the DNA, thereby regulating transcription ,180. It The P53 tumor suppressor gene is mutated in greater than 50% of PDAC samples . The p53PDAC is the deadliest of common human adult malignancies . The vas


Patients with idiopathic membranous nephropathy (IMN) can be categorized into phospholipase A2 receptor (PLA2R)-associated and non-PLA2R-associated cases, according to serum PLA2R antibody status. The present study aimed to determine whether clinical features differed between these.A total of 89 patients with IMN were retrospectively recruited for the present study. Serum PLA2R-Ab levels were determined by time-resolved fluoroimmunoassay. Furthermore, the relationship between serum PLA2R antibody levels and their responses to immunosuppressants among patients with a complete follow-up period, which was defined as at least 1 year, was analyzed.P = .04), proteinuria , and serum albumin , but had lower serum immunoglobulin G  and erythrocyte sedimentation rate , when compared to IMN patients without PLA2R. Furthermore, IMN patients without PLA2R exhibited a better response to immunosuppressants, when compared to patients with PLA2R-associated IMN , but the difference was not statistically significant.Among these enrollees, 71 (80.0%) patients were positive for serum PLA2R antibody. Furthermore, patients with PLA2R-associated IMN had significantly higher age (with vs without, 54.31 ± 14.03 vs 46.67 ± 13.30 years old; Patients with PLA2R-associated IMN had higher disease severity than IMN patients without PLA2R. Furthermore, PLA2R negative patients had a better response to immunosuppressive therapies than PLA2R-positive patients, but the difference was not statistically significant. Among all patients with MN, nearly 80% of these patients can be attributed to idiopathic MN (IMN), while the remaining patients are secondary MN (SMN)-related to diseases, including rheumatologic illnesses (such as systemic lupus erythematosus [SLE]), infections (such as hepatitis or syphilis), cancers, and medications. IMN can only be diagnosed after excluding other known etiologies based on clinical history, physical examinations, laboratory tests, and renal pathology.Membranous nephropathy (MN) is a common etiology of massive proteinuria. In China, the proportion of patients with MN has rapidly increased among patients with primary glomerulopathy, which ranged from 9.89% between 1979 and 2002 to 18.42% between 2003 and 2014. The most recent Kidney Disease Improving Global Outcomes (KDIGO) guideline recommends the use of the severity of proteinuria as a marker of treatment responsiveness. However, the amount of proteinuria is not a sensitive indicator of treatment responsiveness or outcome in patients with IMN. Therefore, biomarkers that can accurately estimate disease activity in addition to proteinuria are urgently needed to monitor responses to any treatment, and predict clinical outcomes in patients with IMN.However, the clinical features and prognosis of patients with IMN are highly variable. Furthermore, the spontaneous remission of nephrotic syndrome occurs in approximately 20% to 25% of patients with IMN, while approximately 40% of patients develop end-stage renal failure at 10 years after disease onset. The prevalence of serum PLA2R antibody (PLA2R-Ab) varies from 57% to 82% among patients with IMN from different geographic areas. The discovery of serum PLA2R-Ab substantially contributes to the advancement of our understanding of the pathogenesis of MN. Serum PLA2R-Ab levels have been reported to correlate with disease activity, treatment response and long-term outcomes.–11 However, the associations among serum PLA2R-Ab, clinical parameters, and remission rates in IMN patients remain to be investigated.In 2009, M-type phospholipase A2 receptor (PLA2R), a membrane glycoprotein localized to podocytes, was identified as a target antigen in patients with IMN. In the present study, IMN was classified as PLA2R-associated and non-PLA2R-associated IMN based on serum PLA2R-Ab positivity, and the clinical images and treatment responsiveness between these 2 groups of patients were compared.Different methods have been utilized to detect serum PLA2R-Ab, including enzyme-linked immunosorbent assay, western blot, and indirect immunofluorescence test. The investigators previously employed a highly sensitive time-resolved fluoroimmunoassay (TRFIA) to quantitatively detect serum PLA2R-Ab.22.1In the present study, a total of 89 patients with biopsy-proven IMN were identified between January 2014 and December 2016. The present study was conducted at the Affiliated Wuxi People's Hospital of Nanjing Medical University in China. Patients with secondary forms of MN, including autoimmune diseases (lupus nephritis and Sjogren syndrome), infection-related MN , and MN correlated to malignancies or exposure to toxic agents, were excluded. Sera and urine were collected from the study participants, and none of these patients received immunosuppressants before inclusion. The anti-hypertension and anti-proteinuric medications of these patients were continued.All study procedures were conducted according to the 2008 Declaration of Helsinki and good clinical practice guidelines. A written informed consent was obtained from each participant. The present study was approved by the Ethics Committee of the Affiliated Wuxi People's Hospital of Nanjing Medical University (no: Kyl2016001).2.2Serum creatinine (Scr), blood urea nitrogen, uric acid, albumin, total cholesterol, triglyceride, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, C3, complement 4 (C4), IgA, IgG, IgM, ESR, C-reactive protein, and hemoglobin were measured, and the estimated glomerular filtration rate was calculated using the Chronic Kidney Disease Epidemiology Collaboration equation. Urine was collected at the time of renal biopsy to assess the proteinuria amount using the average of 3 collections of 24-hour urinary protein and urine red blood cells. These patients were followed up for at least 1 year. The clinical data were recorded upon enrollment, and at 3, 6, and 12 months after immunosuppressant treatment. Patients were classified as having CR when they had proteinuria <0.3 g/d, and normal serum albumin and creatinine levels. Patients were classified as having PR when they had proteinuria <3.5 g/d and a 50% reduction in baseline levels, with improving serum albumin and creatinine, based on the KDIGO 2012 guidelines.2.3Light microscopy, immunofluorescence, and electron microscopy were harnessed to examine the renal biopsy specimens. Direct immunofluorescence for IgG, IgA, IgM, C3, C4, and C1q was performed on frozen tissue sections. These presented semi-quantitative results (0 to 4+). Furthermore, the dense deposits were also semi-quantitatively determined under electron microscopy (0 to 3+).2.4,13Recombinant PLA2R was coated onto 96-well plates for the purpose of capturing the antibody. A goat-anti-human IgG tracer with europium-chelate was prepared for subsequent use. After bound/free separation by washing, the fluorescence counts of the bound tracer were measured to quantify the serum anti-PLA2R-IgG levels. A purified anti-PLA2R-IgG calibrator was 1st prepared to ensure that consistent results could be obtained. The detection range of anti-PLA2R-IgG based on TRFIA was 0.02 to 340.00 mg/L. The intra- and inter-assay coefficients of variation were 3.2% and 5.6%, respectively. The cut-off point for normal anti-PLA2R was set at <0.91 mg/L, according to existing literature.2.5T test was used for the parametric analysis. Categorical variables were presented in frequencies or percentages, and the data were analyzed by Fisher exact test. The differences were considered statistically significant when the P-value was <.05.Statistical analysis was performed using the SPSS software version 18 . Data were presented as mean ± standard deviation. 33.1Eighty-nine patients with a pathologic diagnosis of IMN received tests for serum anti-PLA2R IgG. Among these patients, 71 (80.0%) patients had positive serum PLA2R-IgG (>0.91 mg/L).3.2P = .04), 24-hour proteinuria , and serum albumin . Furthermore, there were differences in serum IgG  and erythrocyte sedimentation rate  between these 2 groups of patients.The clinical features of the study participants at the time of renal biopsy are presented in Table 3.3A total of 53 patients were followed up for longer than 12 months after renal biopsy. Among these patients, 42 and 11 patients had positive and negative serum PLA2R-Ab, respectively. Within the 1st year of renal biopsy, immunosuppressants were prescribed to 32 (76.2%) PLA2R-associated IMN patients and 11 (54.5%) IMN patients without PLA2R. Furthermore, cyclophosphamide (CTX) plus glucocorticoids were given to 22 PLA2R-positive and 5 PLA2R-negative patients, while tacrolimus plus glucocorticoids were given to 10 PLA2R-positive and 1 PLA2R-negative patient. The findings are summarized in Table 3.4P = .340), and between patients who used CTX and tacrolimus .The complete remission (CR) and partial remission (PR) rates of proteinuria after 3, 6, and 12 months of immunosuppressive therapy were analyzed, and the results between IMN patients with and without PLA2R-Ab were compared. The immunosuppressive regimens comprised of 2 types: CTX and tacrolimus. The average proteinuric amount at the time of renal biopsy did not significantly differ between IMN patients . The findings are summarized in Table The outcome data at 3 months after starting the immunosuppressive therapies were available in 32 PLA2R-associated and 6 non-PLA2R-associated IMN patients. The remission rates were higher in patients with positive serum PLA2R-Ab , when compared to IMN patients with negative PLA2R , but the difference was not statistically significant  were higher in serum PLA2R-Ab-negative patients (66.7%) than in PLA2R-Ab-positive patients (62.5%), but the difference was not statistically significant Table .P = .435, Table Finally, the outcome data at 12 months after starting the immunosuppressive therapies in 32 PLA2R-associated and 6 non-PLA2R-associated IMN patients were analyzed. The remission rates (CR + PR) were higher in serum PLA2R-Ab negative patients (100.0%) than in serum PLA2R-Ab positive patients 87.5%), but the difference was not statistically significant (%, but th4By using TRFIA in the present retrospective, single-center cohort study, it was revealed that serum PLA2R-Ab was positive in 80.0% of patients with IMN. These present findings suggest that IMN patients with negative serum PLA2R had a significantly younger age, less proteinuria, and hypoalbuminemia, but had higher serum IgG, when compared to patients with positive serum PLA2R. In addition, IMN patients with negative PLA2R exhibited a better response to immunosuppressants after 6 and 12 months of immunosuppressive treatment. These observations improve our understanding of the clinical differences between IMN patients with and without PLA2R, and suggest that there may be potential differences in pathogenesis in these 2 groups of patients. the monitoring of treatment responses, prognosis stratification, and the design of personalized therapies. The prior study conducted by the investigators and others have shown that the TRFIA-based quantification of serum PLA2R-Ab exhibits high accuracy in the diagnosis and prognostication of patients with IMN. Researchers have even recommended the replacement of renal biopsy with serum anti-PLA2R tests in IMN patients with relative contraindications to renal biopsy due to the high sensitivity and specificity of the anti-PLA2R assay.The PLA2R has been recognized as an important antigen in patients with IMN, and contributes to a deeper understanding of its pathogenesis. The in situ formation of the immune complex, which comprises of PLA2R and its antibody, activates the complement and generates the membrane attack complex, leading to sublethal podocyte injury, the disruption of the glomerular filtration barrier, and proteinuria. The detection of serum PLA2R-Ab facilitates the diagnosis,,18,19 These findings suggest that serum PLA2R-Ab is closely associated with the severity of proteinuria. In the present cohort, it was similarly found that patients with PLA2R-associated IMN had significantly more proteinuria and more severe hypoalbuminemia, but had less serum IgG, when compared to IMN patients without PLA2R. Since MN has been recognized as an organ-limited autoimmune disease, non-PLA2R-associated IMN may exhibit a resemblance to immune system disorders. It is likely that the presentations of the enrolled MN patients in the present study occurred at the early phase of certain systemic immune disorders, such as SLE. Alternatively, the negative status of PLA2R-Ab can result from the absence of disease activity at the time of blood sampling. A report supported the present proposition by showing that PLA2R-Ab levels measured at IMN diagnosis are inversely associated with the probability of achieving spontaneous remission. In the present study, the low proteinuria and higher serum IgG levels found in patients with non-PLA2R-associated IMN might represent the low clinical activity among these patients. Furthermore, the remission of proteinuria occurred in all non-PLA2R-associated IMN patients after 6 and 12 months of immunosuppressant use. These data suggest that non-PLA2R-associated IMN may exhibit lower clinical activity, although these results require further confirmation with the underlying mechanisms investigated. Part of the participants had an elevated ESR, which could be due to their occult infection at the time of blood sampling. There was no significant correlation between the IMN and ESR reported from existing literature.The present data reveals that patients with PLA2R-associated IMN had significantly lower serum IgG, but had higher age, ESR and proteinuria, and more severe hypoalbuminemia. Previous studies have demonstrated that glomerular PLA2R is an important antigen involved in the pathogenesis of IMN, and that serum PLA2R-Ab can effectively target PLA2R. The optimal immunosuppressive regimen for treating IMN remains controversial. Given the potentially different pathogenesis of PLA2R-associated and non-PLA2R-associated IMN, the investigators planned to determine whether these 2 groups of IMN patients responded differently to immunosuppressants. It was found that there was no difference in the proportion of patients who responded to immunosuppressants during the 1st year after biopsy between patients with PLA2R-associated and non-PLA2R-associated IMN in the present cohort. The treatment decision was made by the physicians based on the individualized plan tailored to the clinical presentations of these patients. These present findings indicate that clinical remission can be achieved in non-PLA2R-associated IMN patients after 6 and 12 months of immunosuppressant use. Previous reports support these present findings by showing that PLA2R-Ab negative IMN patients appear to have a higher spontaneous remission rate, when compared to PLA2R-Ab positive IMN patients, and immunosuppressant use did not alter the chance to achieve remission in PLA2R-negative patients. More studies are needed to determine the necessity and long-term efficacy of immunosuppressants in treating PLA2R-negative IMN patients.Immunosuppressants, including corticosteroid alternating with alkylating agents and calcineurin inhibitors, are partially effective in reducing proteinuria in patients with MN. However, their use may be associated with the development of substantial adverse effects and relapse.The main limitation of the present retrospective study is the low sample size and relatively short follow-up duration. Hence, the interpretation of results correlated to long-term outcome, spontaneous remission and relapse rates can be difficult. Therefore, additional studies with a randomized prospective design and more enrolled patients should be conducted to validate the outcome associations found.In conclusion, PLA2R-associated IMN patients have lower serum IgG, but have higher age and ESR, more proteinuria, and more severe hypoalbuminemia, when compared to patients with negative PLA2R-Ab. Furthermore, patients with non-PLA2R-associated IMN responded better to immunosuppressants, when compared to patients with PLA2R-associated IMN, although the difference was not statistically significant. Further studies are needed to determine the long-term clinical outcome in non-PLA2R-associated IMN patients.Conceptualization: Qiuhua Zhang, Biao Huang, Yi Zhang, Zhen Qi.Data curation: Qiuhua Zhang, Xiaobin Liu, Weiwei Shan, Zhuxing Sun.Formal analysis: Qiuhua Zhang, Xiaobin Liu, Biao Huang, Yi Zhang, Bin Liu.Funding acquisition: Xiaobin Liu, Zhijian Zhang, Mian Wu, Bin Liu, Zhen Qi, Weiwei Shan, Zhigang Hu, Zhuxing Sun.Investigation: Xiaobin Liu, Zhijian Zhang, Mian Wu, Biao Huang, Yi Zhang, Bin Liu, Weiwei Shan, Zhigang Hu.Methodology: Zhijian Zhang, Mian Wu, Biao Huang, Bin Liu, Zhen Qi, Weiwei Shan, Liang Wang, Zhigang Hu.Project administration: Zhijian Zhang, Mian Wu, Yi Zhang, Zhen Qi, Liang Wang, Zhigang Hu.Resources: Liang Wang.Validation: Liang Wang.Writing – review & editing: Liang Wang.


It is well known that glutamate (Glu), a neurotransmitter in human body, is a protein amino acid. It plays a very important role in plant growth and development. Nowadays, Glu has been found to emerge as signaling role. Under normal conditions, Glu takes part in seed germination, root architecture, pollen germination, and pollen tube growth. Under stress conditions, Glu participates in wound response, pathogen resistance, response and adaptation to abiotic stress , and local stimulation (abiotic or biotic stress)-triggered long distance signaling transduction. In this review, in the light of the current opinion on Glu signaling in plants, the following knowledge was updated and discussed. 1) Glu metabolism; 2) signaling role of Glu in plant growth, development, and response and adaptation to environmental stress; as well as 3) the underlying research directions in the future. The purpose of this review was to look forward to inspiring the rapid development of Glu signaling research in plant biology, particularly in the field of stress biology of plants. The mGluRs can activate ion channels via coupling to G-protein signaling system by the following signaling pathways. 1) Ion channels can be directly activated by γ, β subunits of the G-protein; or 2) indirectly activated through triggering second messengers  , also known as α-aminoglutaric acid, is a key excitatory neurotransmitter. Glu mediates neural signaling transduction in the synapses, participates in cognition, learning, memory, and other physiological and pathological processes . Glu as ide: NO) . These iIt is well known that Glu is a protein amino acid, which is a precursor of the synthesis of proteins and polypeptides. Besides, Glu also is a common precursor of many organic compounds. These organic compounds include protein amino acids , non-protein amino acid , antioxidant tripeptide , heme, chlorophyll, and so forth . In addi2+, ROS, and electrical signaling /Glu synthase  cycle in the chloroplasts of photosynthetic tissue or non-photosynthetic tissue plastids and Glu dehydrogenase (GDH) in the mitochondria or cytoplasm. They regulate the homeostasis of Glu, Gln, 2-oxoglutarate (GO), and ammonia (NH3) in plant cells. In addition, plants also can produce Glu by Pro/pyrroline 5-carboxylate (P5C) cycle and transamination, which are alternative pathways (3) is toxic to plant cells mainly by interfering with energy metabolism (namely eliminating proton motive force by binding H+ to form ammonium) and/or disrupting pH balance  by binding with its putative receptors, which in turn regulating polyamine metabolism and plant growth (such as pollen tube growth and development) . Subsequently, the GABA is imported into mitochondria by GABA permease locating on the mitochondrial endomembrane, which in turn is converted into succinic semialdehyde (SSA) by GABA transaminase (GABA-T). Afterward, SSA dehydrogenase (SSADH) catalyzes the conversion of SSA to succinate , which in turn enters into tricarboxylic acid (TCA) cycle, further regulating redox balance and energy metabolism . GABA shlopment) .Arabidopsis, the Glu transporters include cationic amino acid transporters (CAT) family , γ-aminobutyric acid (GABA) permease-related (APC superfamily), and amino acid transporter family (ATF superfamily)  . Glu redrfamily) . TherefoArabidopsis thaliana leaves, the amount of Glu remains about 1 μmol g−1 fresh weight (FW), but in phloem cells, Glu content reaches 50 mM  and their interactions  and exogenous signals  . Becauseractions .2+ takes part in seed germination via across membrane into the cytosol, which is mediated by Ca2+ channels  was significantly up-regulated in germinating seeds, which in turn increased cytosolic Ca2+ concentration, eventually stimulating seed germination via counteracting the inhibiting effect of ABA . Also, Ca2+ could inhibit the gene expression of ABSCISIC ACID INSENSITIVE4 , which is involved in ABA response in seeds  Cachannels . In A. tt of ABA . Adversein seeds . These r2+, ROS, NO, H2S, Glu, and plant hormones , as well as their crosstalk  and 6,7-dinitroquinoxali-ne-2,3-dione acted (DNQX)] dramatically inhibited the growth of primary root and the density of lateral root by impairing root meristem size. On the contrary, the treatment with iGluR agonist Glu recovered the growth of Arabidopsis roots. In addition, a low amount of Ca2+ chelator  drastically inhibited the root elongation of Arabidopsis, while this inhibition was partially recovered by Glu , ROS, and auxin , the OsGLR3.1 knockout mutant plants also show a short root, and the further experiments indicate that OsGLR3.1 positively regulates cell proliferation and survival in the root apical meristem  plants, whereas the Glu-induced growth inhibition and curvature were restored in mutant MPK6 kinase (mpk6) seedlings, but aggravated in mutant mkp1 seedlings  play a very important role in the plant signaling transduction, which regulate most biological processes including root formation. In eedlings . These o2+, ROS, NO, H2S, and plant hormones, eventually forming a sophisticated signaling network controlling pollen germination and pollen tube growth  is a fundamental signaling transduction event  in pollen germination and pollen tube growth is not completely clear. Arabidopsis, GLRs can form Ca2+ channels and mediate Ca2+ influx across the plasma membrane, which in turn modulates apical [Ca2+]cyt gradient and consequently regulates pollen tube growth and morphogenesis. Adversely, iGluR antagonists CNQX, DNQX, and DL-2-amino-5-phosphonopentanoic acid (AP-5) show an inhibitory effect on Ca2+ signaling and pollen tube growth  done (AtCNIHs can enhance the activity of GLRs (mainly AtGLR3.3) by regulating their sorting, trafficking, location, and quantity without any ligands, which in turn contribute to cytosolic Ca2+ homeostasis, thus regulating pollen germination and pollen tube growth  in bidopsis , but alsHs) done . The spee growth .In addition to seed germination, root architecture, and pollen germination and tube growth mentioned above, Glu as a signaling molecule is also involved in the response and adaptation to salt, cold, heat, drought, pathogen, and wound stress  and late (ionic phase) stress phase. The former refers to the rapid production of osmotic stress within several minutes due to the quick decline in extracellular water potential, while the latter is ion and oxidative stress as well as nutrient imbalance as a result of accumulation of Naral days . Generaln plants .2+ and ABA regulate seed germination under NaCl stress. In A. thaliana, the results of AtGLR3.4-1 and AtGLR3.4-2 mutant plants were more sensitive to NaCl in both seed germination and post-germination seedling growth. In addition, in wild-type seedlings, NaCl triggered a striking increase in [Ca2+]cyt, but this increase was blocked by DNQX (GLR antagonist); whereas in both AtGLR3.4-1 and AtGLR3.4-2 mutants NaCl-triggered increase in [Ca2+]cyt was impaired (SOS3), SOS2, and SOS1, as well as more accumulation of Na+ than wild-type seeds  regulates seed germination under salt stress.As discussed above, seed germination is sensitive and fragile to environmental stress including NaCl stress, and Caimpaired . Also, upe seeds . In addil stress . Similarl stress . Recentl2+ channels and triggers Ca2+-centered signaling transduction pathways, eventually responding and adapting to cold stress  alleviated the sensitivity of AtGLR1.2 and AtGLR1.3 mutants to cold stress. In turn, the overexpression of AtGLR1.2 or AtGLR1.3 could improve the tolerance of mutants to cold stress by synthesizing endogenous JA (AtGLR1.2-OE [AtGLR1.2-GFP (green fluorescence protein)] and AtGLR1.3-OE [AtGLR1.2-GFP] was upregulated in transgenic plants (Solanum lycopersicum), GLR3.3 and GLR3.5, which in turn increased the resistance of tomato plants to a subsequent chilling stress at 4°C. On the contrary, the plants silenced GLR3.3 or/and GLR3.5 or supplemented DNQX all reversed the chilling tolerance induced by cold acclimation compared with the wild types. In wild types, Glu (as a GLR agonist) pretreatment increased the chilling tolerance, but GLR3.3- or GLR3.5-silenced or cosilenced plants failed (respiratory burst oxidase homolog1 (RBOH1) and its activity, further reduced the level of apoplastic H2O2 and the GSH/oxidized glutathione (GSSG) ratio, finally led to the decline in chilling tolerance (2O2 or GSH (RBOH1-silenced and GSH biosynthesis genes [γ-glutamylcysteine synthetase: GSH1 and GSH synthetase (GSH2)] also reduced the chilling tolerance, accompanying with reduced GSH/GSSG ratio. Pretreatment with DNQX had no significant effect on the GSH/GSSG ratio and the chilling tolerance in RBOH1-silenced plants and GSH1- and GSH2-cosilenced plants  seedlings treatment with Glu elevated the survival percentage of seedlings under heat stress. Also, heat tolerance induced by Glu was impaired by exogenous CaCl2 (may produce Ca2+ overloading), LaCl3 (plasma membrane Ca2+ channel blocker), EGTA (Ca2+ chelator), as well as trifluoperazine and chlorpromazine  (2)  . Similarsts) (2) . This woDrought stress caused by the decline in soil water potential interferes with many physiological processes at different levels, such as water uptake, transpiration, photosynthesis, seed germination, plant growth, and development . Under s2+, ROS, NO, H2S, and ABA (Arabidopsis and fava bean (Vicia faba L.), Glu treatment increased [Ca2+]cyt in guard cells, which in turn led to stomata closure. Glu-induced stomata closure was abolished by EGTA (extracellular Ca2+ chelator), BAPTA-AM (intracellular Ca2+ chelator), La3+ (plasma membrane Ca2+ channel blocker), and AP-5 (a specific antagonist of GLRs) (Arabidopsis ABA-deficient (aba2-1) and ABA-insensitive (abi1-1 and abi2-1) mutants, Glu treatment also induced stomata closure, further indicating this closure is ABA-independent manner (slac1) mutants, Glu treatment had no significant on stomata closure. Similar results could be observed in calcium-dependent protein kinase 6 (cdpk6) mutants, suggesting Glu induces stomata closure in a SLAC- and CDPK-dependent manner , which in turn increased the synthesis of salicylic acid. Then, which enhanced drought-induced proline accumulation, thus improving drought tolerance by regulating cellular redox potential  . In addit manner . Similart manner . For GLR pathway . In addiotential .Medicago truncatula seedlings, MtGLR genes , which were classified into four clades based on a phylogenetic analysis, and 17 of which showed specific domains consistent with iGluRs. Under drought stress, treatment with GLR competitive antagonists (AP-5 and DNQX) reduced the accumulation of NO induced by ABA, which was no significant effect on stomata closure  and their cross talks (Penicillium expansum in pear (Pyrus bretschneideri) fruit under normal or cold conditions , chitinase (CHI), phenylalanine ammonialyase (PAL), peroxidase, and polyphenol oxidase, as well as the gene expression of pathogenesis-related proteins1 (PR1), GLU, CHI3, CHI4, and PAL . In addition, in wild-type (Col-0) plants, pretreatment with DNQX was sensitive to B. cinerea and Hyaloperonospora arabidopsidis  and transmit this information to the whole plant body, thus triggering the defense responses, systemic acquired resistance (SAR), or systemic acquired acclimation (SAA) . Toyota Arabidopsis, wounding triggered and propagated the membrane depolarizations in a GLR-dependent manner, which resulted in the activation of defense genes. The GLR mutants attenuated leaf-to-leaf electrical signaling, but GLR3.3 mutants lost the defense against herbivores  propagation triggered by herbivores.Similarly, in rbivores . In addiibutions . The datArabidopsis wounding-triggered Ca2+ signaling promoted the biosynthesis of JA  by stimulating lipoxygenase and/or inhibiting phosphorylation of repressors JAV1 within the JAV1-JAZ-8-WRKY51 complex, finally activating plant defense response  and electrical signaling, which in turn takes shape a sophisticated signaling network, eventually regulating accurately plant growth, development, and defense response , especially H2S and methylglyoxal signaling, also needs to be further solved.Glu, however, as a novel signaling molecule in plants, the following open questions look forward to being uncovered in the future: 1) the detailed mechanisms of Glu-triggered abiotic and biotic tolerance, especially heat tolerance, will be illuminated at physiological, biochemical, molecular, and even omics levels. 2) Though the members and functions of GLRs have been expounded in many plant species, such as bidopsis , rice .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.


MMP-13 gene expression and COL2A1 gene expression. Results of these studies imply that microRNAs could become useful in the search for diagnostic biomarkers, as well as providing novel therapeutic targets for intervention in OA.An imbalance in gene expressional events skewing chondrocyte anabolic and catabolic pathways toward the latter causes an aberrant turnover and loss of extracellular matrix proteins in osteoarthritic (OA) articular cartilage. Thus, catabolism results in the elevated loss of extracellular matrix proteins. There is also evidence of an increase in the frequency of chondrocyte apoptosis that compromises the capacity of articular cartilage to undergo repair. Although much of the fundamental OA studies over the past 20 years identified and characterized many genes relevant to pro-inflammatory cytokines, apoptosis, and matrix metalloproteinases (MMPs)/a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS), more recent studies focused on epigenetic mechanisms and the associated role of microRNAs (miRs) in regulating gene expression in OA cartilage. Thus, several miRs were identified as regulators of chondrocyte signaling pathways, apoptosis, and proteinase gene expression. For example, the reduced expression of miR-146a was found to be coupled to reduced type II collagen (COL2) in OA cartilage, whereas MMP-13 levels were increased, suggesting an association between  The main focus of basic studies designed to unravel the pathology of osteoarthritis (OA) in humans recently employed non-surgical animal models with genetically modified mice as replicas of the human disease ,2. The rIn addition to the significance of molecular events that drive the progression of OA, there is now ample evidence that “chondroscenesence”, a term used to describe chondrocytes in the older individual, was linked to inflammation and was characterized by an abnormal interplay between autophagy and the inflammasome . Of noteRecent advances also increased our understanding of how the loss of articular cartilage extracellular matrix (ECM) proteins characteristic of OA synovial joints results from the action of pro-inflammatory cytokines , IL-6, IL-15, IL-17, IL-21) and several other interleukin family members ,9. In adBasic studies also implicated programmed cell death, also known as apoptosis, or in the chondrocyte context, “chondropotosis”, in the OA pathological process . The inc(TUNEL)/Hoechst 33258 staining revealed an elevated frequency of apoptosis in freshly isolated chondrocytes from OA cartilage , as wellGREM1, FRZB, DKK1, VEGF, EGF [GDF5 [In addition to those genes which effectively reduce chondrocyte viability, other specific genes and signaling pathways, include genes that encode bone morphogenetic proteins, the WNT/β-catenin proteins, leukemia inhibitory factor, hypoxia-inducible factor-1-α/2α (HIF-1α/2α) ,23, as wEGF, EGF ,25, GDF5GF [GDF5  genes, aGF [GDF5 . Each ofSignificant advances also occurred in defining the role that signal transduction pathways play in OA. For example, pro-inflammatory cytokine-mediated activation of mitogen-activated protein kinase (MAPK) and Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathways demonstrated how these signaling pathways control gene expression associated with pro-apoptosis protein synthesis and MMP synthesis, both of which were found to be dysregulated in OA articular chondrocytes ,31,32,33Epigenetics is a field of genetics wherein variations in cell phenotype are considered to result from external or environmental factors that affect how gene activity is regulated. This mechanism of gene regulation exists in contradistinction to genes regulated by changes in DNA sequence. Thus, in this way, epigenetic factors can regulate the dynamic potential of gene transcription.IL-1β, IL-6, and TNF-α, which were subject to epigenetic modifications during the OA process [GDF5, ASTN2, DOT1L genes), intrinsic factors , as well as extrinsic biomechanical factors , past joint injury) as particularly germane to the development of OA. Results of recent studies implicated epigenetics as an important contributor to the pathology and progression of cartilage ECM protein turnover and damage in OA. In that regard, genome-wide association studies (GWAS) identified several critical genes, including  process . In fact process . Of note process . In addi process . Just as process . This re process . Thus, GMicroribonucleic acids  are ~22-nucleotide non-coding RNAs which interact with cognate messenger RNAs (mRNAs) . Thus, iSeveral groups of investigators ,42,43,44Specifically, several miRs were identified as playing a role in OA pathology by virtue of their abnormal expression in OA. These included miR-9, miR-27, miR-34a, miR-140, miR-146a, miR-558, and miR-602 . In factKPNA3) gene expression, the latter identified as a mediator affecting the capacity of the NF-κB p65 subunit to translocate from the cytoplasm to the nucleus [One approach to recognizing the extent to which miRs may be involved in altering articular cartilage homeostasis was conducted by determining which miRs alter various aspects of chondrocyte signaling, apoptosis, and proteinase production . In that nucleus . Importa nucleus . In addi nucleus . Of note nucleus  also shohttps://www.ncbi.nlm.nih.gov/pubmed). This evidence demonstrated that many of the miRs  were implicated in targeting those genes that are involved in dysregulated signal transduction , ECM protein turnover , pro-inflammatory cytokines , methylation/demethylation , MMP and ADAMTS , and autophagy , many of which were shown to be critical in altering the chondrocyte phenotype characteristic of OA cartilage [Most of the targets shown in artilage ,57,58,59VEGF and decreased SMAD4 gene expression. Consequently, downregulation of miR-146a reduced human chondrocyte apoptosis, whereas upregulation of VEGF induced by miR-146a was shown to correlate with SMAD4 in chondrocytes stimulated by mechanical injury [Apoptosis is regulated by the balanced expression of pro-apoptotic and anti-apoptotic proteins, including the influential effects of a class of proteins termed cellular inhibitor of apoptosis proteins (c-IAPs) . Evidencl injury . More rel injury . Wu et al injury  showed tl injury , upregulCOL2A1 gene expression (type II collagen) were decreased in OA cartilage with low-grade OA, whereas MMP-13 expression was increased at a similar cartilage grade, suggesting that decreased miR-146a was associated with elevated MMP-13 levels. The results of this study [GAS5) in normal and OA regions of human cartilage removed at total knee replacement. In addition, overexpression of GAS5 increased the expression of MMPs, including MMP2, MMP3, MMP9, MMP13, and ADAMTS4, while also stimulating apoptosis and suppressing autophagy. Furthermore, miR-21 was also shown to regulate GAS5, as evidenced by its reduced level in OA and the capacity of miR-21 to significantly increase cartilage destruction in “OA mice”. Song et al. [histone deacetylase 4 (HDAC4). Thus, miR-222 was significantly reduced in OA chondrocytes, and overexpression of miR-222 suppressed apoptosis while also reducing HDAC4 and MMP13 gene expression. The biomarker of senescence, also known as p161INK4a, was shown to be a target for miR-24 [p161INK4a was over-expressed in chondrocytes, the levels of MMP1 and MMP13 increased. Thus, miR-24 was identified as a negative regulator of p161INK4a, as well as of cartilage development and OA. A few years ago, Meng et al. [MMP13, where NF-κB and MAPK activation was shown to downregulate miR-320 expression. This conclusion was later extended by Jin et al. [PBX3) gene, increased MMP-13, whereas PBX3 and the proteasome inhibitor, MG132, suppressed the effects of miR-320a on the expression of COL2A1, ACAN , sulfated glycosaminoglycans, and MMP-13. Moreover, miR-105 was also found to be important for the fibroblast growth factor-2 (FGF2)-induced gene expression of ADAMTS4, 5, 7, and 12 [Runx2 expression. Thus, the FGF2/p65/miR-105/ADAMTS axis appears to be important in OA pathogenesis. Mao et al. [ADAMTS4/ADAMTS-5 expression, which also involved NF-κB and MAPK activation. Additionally, miR-30a was also identified as a regulator of ADAMTS-5 [ADAMTS5 gene expression. A host of miRs were discovered to regulate the expression of several MMPs and ADAMTS which are relevant to the regulation of chondrocyte homeostasis, and to the pathogenesis and progression of OA. Thus, it was instructive in establishing a role for miRs in altering human OA cartilage ECM when Yamasaki et al.  showed tis study  also shois study  showed tg et al.  also shor miR-24 , and wheg et al.  also iden et al. , who sho, and 12 . These ao et al.  also repADAMTS-5 , wherebyMMP/ADAMTS gene expression [T. Thus, the results of these pre-clinical studies provide a compelling platform for considering the use of miRs in OA clinical safety trials going forward. Although persuasive evidence confirmed the critical role of pro-inflammatory cytokines as potent inducers of chronic inflammation, apoptosis, and proteinase gene expression in OA, the results of recent studies extended this analysis to demonstrate how various miRs regulate the effects of these cytokines, the balance between pro-apoptotic and anti-apoptotic proteins , and MMPpression , all of pression . Further