The balance between bone forming cells (osteoblasts/osteocytes) and bone resorbing cells (osteoclasts) plays a crucial role in tissue homeostasis and bone repair. Several hormones, cytokines, and growth factors—in particular the members of the TGF-β superfamily such as the bone morphogenetic proteins—not only regulate the proliferation, differentiation, and functioning of these cells, but also coordinate the communication between them to ensure an appropriate response. Therefore, this review focuses on TGF-β superfamily and its influence on bone formation and repair, through the regulation of osteoclastogenesis, osteogenic differentiation of stem cells, and osteoblasts/osteoclasts balance. After introducing the main types of bone cells, their differentiation and cooperation during bone remodeling and fracture healing processes are discussed. Then, the TGF-β superfamily, its signaling via canonical and non-canonical pathways, as well as its regulation by Wnt/Notch or microRNAs are described and discussed. Its important role in bone homeostasis, repair, or disease is also highlighted. Finally, the clinical therapeutic uses of members of the TGF-β superfamily and their associated complications are debated. Bone tissue plays several essential physiological roles within the human body, in particular mechanical functions such as protection, rigid support, and an anchoring site for soft organs  and metabolic functions ,2. IndeeMany cytokines, growth factors, hormones (PTH), and vitamins are involved in the phases of bone repair and remodeling ,9,10,11.The TGF-β superfamily is an evolutionary conserved family of structurally related dimeric factors. They are secreted growth factors, which act as multifunctional regulatory peptides involved in a wide range of processes, including cell development, proliferation, and differentiation; wound healing; and carcinogenesis ,16. The In this review, the roles of bone-forming and bone-resorbing cells and their cooperation in healing and bone remodeling processes are presented. We will then introduce the role of the members of the TGF-β superfamily, their canonical/non-canonical signaling pathways and their respective regulations  and discuss the complexity of their mechanisms that influence homeostasis and bone disease. Finally, the controversial clinical uses of members of the TGF-β superfamily in orthopedic surgery is debated.−cells from the digested cortical bone fraction) were identified , is still under investigation (for review see .,81.80,81nd water . The relnd water ,84. Furteoclasts .2  BMP-2 or [125I] TGF-β1 [The canonical pathways activated by members of the TGF-β family can be inhibited by several mechanisms  203]. T. T203]. ] TGF-β1 . Interes] TGF-β1 . In the ] TGF-β1 .Another mechanism, preventing the signaling pathways of the TGF-β superfamily, is the use of antagonist proteins such as the Dan family (Gremlin), the Spemann organizer signal molecules  and follistatin ,159,203.Other regulatory mechanisms act directly in the cytoplasm. The deactivation of R-Smad can be obtained via their dephosphorylation by phosphatases, such as the protein phosphatase magnesium-dependent 1A (PPM1A). The canonical Smad pathway can also be blocked by intracellular molecules like Smad 6/7, also called I-Smad (Inhibitory Smad) . Unlike It was recently shown that microRNAs (miRNAs) can play a strong role in the regulation of the signal transduction induced by the members of the TGF-β superfamily. MicroRNAs, which possess 18–25 nucleotides, are small noncoding RNA molecules that can inhibit the translation of targeted mRNAs or induce their degradation (for review see ). Both mThe members of the TGF-β superfamily through binding to their preformed type I and type II receptors can first activate XIAP, then TAK1 and TAB1, which in turn initiates the p38, ERK, and JNK (c-Jun amino (N)-terminal kinases) MAPK cascades ,263,264.MAPK cascades can favor or prevent osteogenic differentiation. For example, MAPKs promote osteoprogenitor differentiation by upregulating the expression of Runx2 and Osterix ,267. MAPTo summarize, the description of the signal transduction induced by the members of the TGF-β superfamily can appear simple—hetero-oligomerization of limited number of Type I and Type II receptors leading to 2 canonical Smad pathways activation. However, it must be kept in mind that the ligand pro-domains, ligand heterodimerization, binding receptor affinities, structure of both ligand-receptor complexes, with or without third co-receptors, and R-Smad/Co-Smad complexes also have strong effects, which are still under investigation . FuWnt Signaling Pathwaysint-1 proto-oncogene in virus-induced C3H mouse mammary tumor model [wingless and its vertebrate homolog, integrated (int-1) [2+ pathways, which are considered to be non-canonical [The Wnt proteins were discovered in 1980s due to the work of Nusse and Varmus who identified the or model . Indeed, (int-1) . To dateanonical .The 3 major Wnt pathways act on cells through a family of seven-pass transmembrane Frizzled (Fz) receptors, which interact with Wnt, via their extracellular N-terminal cysteine-rich domain (CRD) (for review see  and 282282).Canonical Wnt pathwaysWhen the canonical Wnt pathway remains inactive, β-catenin forms a destruction complex with the adenomatous polyposis coli (APC), Axin, casein kinase I (CKI), and Glycogen synthase kinase-3 β (GSK3 β). Then, β-catenin is phosphorylated by these kinases (CKI then GSK3) leading to its ubiquitination by β-transducin repeat-containing protein (β-TrCP), an E3 ubiquitin ligase ,284. UpoWisp-1. The Wnt pathways can be antagonized by several proteins such as the Fz-related proteins (sFRPs), which are able to bind Wnt, preventing their recognition by the receptors. Other antagonists such as the members of the Dickkopf  interact with LRP5/6, inhibiting the canonical Wnt pathway [The canonical Wnt pathway is initiated by the binding of Wnt, such as Wnt1 and Wnt3A, to Fz receptor and a single-span transmembrane co-receptor called low-density-lipoprotein-related protein 5/6 (LRP5/6) . The co- pathway .The canonical Wnt pathway through the β-catenin is involved in the skeletal development, as well as the fracture healing process ,287. ForThe Wnt pathway activation states are, therefore, able to regulate the signaling of TGF-β superfamily members and vice-versa ,290,291.Some Wnt ligands can also promote a shift of the TGF-β signaling pathway from Smad2/3 towards Smad1/5/8. Using murine P2 chondrocytes, Van den Bosch et al. found that a Wnt3a (300 ng/mL) pretreatment is enough to decrease the amount of phosphorylated Smad2/3 induced by TGF-ß1 (5 ng/mL) for 30 min. In contrast, it increases the amount of phosphorylated Smad1/5/8, signaling involved in chondrocyte hypertrophy. Similar results were obtained with human G6 chondrocytes, and the effect on the shift in TGF-β-induced Smad phosphorylation is even stronger when Wnt3a is combined with WISP . The addSeveral studies showed that the osteoblastic differentiation of osteoprogenitor cells can also be enhanced by some BMP and Wnt combination ,295,296.Non-canonical Wnt signaling pathwaysXPAPC  via some transcription factors like ATF2 [The PCP pathway implies the binding of Wnt such as Wnt5A to Fz or ROR/PTK7 co-receptors, to activate JNK and members of the small Rho GTPase family like RhoA and Rac1 . The sigike ATF2 . It was ike ATF2 . Using oike ATF2 .2+ pathway involves Fz-mediated phospholipase C (PLC) activation via heterotrimeric G proteins. PLC in turn catalyzes the diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3) production [2+ release from intracellular endoplasmic reticulum to stimulate effectors such as calmodulin-dependent kinase II (CAMKII) and protein kinase C (PKC), which can activate, for example, the transcription factors NFκB and CREB (cyclic AMP response element-binding protein). Ca2+ and calcineurin can also activate the NFAT [The Wnt/Caoduction . IP3 indthe NFAT ,303.Notch Signaling PathwaysNotch are cell-surface receptors  that recognize Delta-like  and Jagged  single-pass transmembrane ligands on neighboring cells. The ligand-Notch receptor binding induces the intracellular cleavage of Notch by the TNFα-converting enzyme (TACE) or ADAM17 and γ-secretase complex. ADAM17 belongs to the ADAM  family of proteins, which are transmembrane metalloproteinases, possessing a catalytic extracellular domain, and are involved in ectodomain shedding of various cell surface proteins, including growth factors, cytokines, receptors, and adhesion molecules . The TβRThe intracellular cleavage of Notch induces the release of NICD (notch intracellular domain), which can then be translocated to the nucleus. Afterwards, NICD interact with a DNA-binding adaptor CBF1/RBPjk/Su(H)/Lag1, called CSL, to form a transcriptional activator complex . This coNotch receptors, as well as their ligands, can be expressed in bone-forming cells and bone-resorbing cells ,311,312.The role played by Notch in both osteoclastogenesis, as well as osteoblast differentiation, remains controversial due to discrepancy in the results obtained by several studies due to the experimental design, cell source, and operating conditions ,314,315.For example, Yamada et al. found that osteoclastogenesis, as shown by the TRAP positive cells, is decreased when precursors from the bone marrow, spleen, and peritoneal cavity are cultured on plates coated with human DLL1 for 6 days, with RANKL (25 ng/mL) and M-CSF (50 ng/mL). This inhibition depends on the tissue source of the osteoclast precursors varying from 23% to 100% for the bone marrow and the peritoneal cavity, respectively . In contBglap transcripts  in the presence of the Notch pathway inhibitors , as compared to BMP-9 alone [Several studies also highlighted the favoring role of the Notch pathway in osteoblast differentiation induced by BMPs ,317, whi-9 alone .The members of the TGF-β superfamily play a crucial role in the balance between bone formation and resorption. Indeed, the ability of the members of the TGF-β superfamily, especially BMPs such as BMP-2, BMP-4, BMP-6, BMP-7, and BMP-9, to induce the osteogenic differentiation of MSCs in vitro and bone formation in vivo is well documented ,153,154.Interestingly, several studies observed a higher osteogenic potential for BMP heterodimer compared to homodimer ,322,323.The member of the TGF-β family can act on osteoclast progenitor proliferation, osteoclastogenesis, bone resorption activity, as well as survival of mature osteoclasts through direct or indirect (via osteoblast/osteocytes secreted factors) mechanisms  59,171,,171,325.It was shown that BMP-9 (50 ng/mL) alone can increase the proliferation of mouse spleen macrophages after 3 days . HoweverFurthermore, several studies showed that some members of the TGF-β superfamily promote RANKL-induced osteoclast differentiation . For exaThe rhBMP-2/7 heterodimers (5–150 ng/mL) also enhance the RANKL-mediated osteoclastogenesis . The sam−4 or 2 × 10−4 ng/mL), while completely preventing it at high concentration (2 ng/mL) [Some members of the TGF-β superfamily can also inhibit osteoclastogenesis . Using m2 ng/mL) .Lee et al. also recently found that TGF-β1  dose-dependently decreases the number of TRAP-positive multinucleated cells incubated for 2 days with 20 ng/mL M-CSF and then 40 ng/mL RANKL for 6 days . HoweverThe members of the TGF-β superfamily were detected in all phases of the bone fracture healing, but their temporal expression patterns and amounts might vary in function of the animal model used ,335,336.TGFB2 and TGFB3 genes are only expressed after the fracture and in a smaller amount than TGF-β1 mRNA. Both TGF-β2 and TGF-β3 transcript levels remain high between days 1–7 and days 1–21, respectively. Thus, while TGF-β2 is mainly expressed during the fibrocartilaginous callus formation, TGF-β3 appears to act during both fibrocartilaginous and bony callus formation (bone repair phase). Indeed, the gene (COL2A1) encoding type II collagen specific to the cartilaginous matrix is highly expressed at day 7, while the expression of gene (COL1A1) encoding type I collagen specific to the osteoid matrix reaches a maximum, at days 14 and 21 [Cho et al. followed the mRNA levels of TGF-βs and BMPs in fractured mouse tibias for 4 weeks . They fo4 and 21 .It was also confirmed that human fracture hematoma contains a high concentration of TGF-β1 . InteresCho et al. also observed that among BMPs, BMP-2 is the earliest activated gene in fractured mouse tibias. The kinetic profiles of the BMP-2 transcripts reveal two peaks of similar intensity at day 1 (hematoma-inflammation formation phase) and day 21 (bone repair phase). The expression of genes encoding BMP-4, BMP-7, and BMP-8 reaches a peak between 14 and 21 days, corresponding to the bone repair phase . In contAge can affect the fracture healing and BMP gene expression profile obtained during the fracture healing process. Using a closed mid-diaphyseal fracture in 6 weeks and 1-year-old rats, Meyer et al. found that only younger rats present a fracture healing at 6 weeks . The fraAbnormal TGF-β signaling and polymorphisms in TGF-β1 are involved in widespread human disorders, such as fibrosis and cardiovascular diseases, as well as hereditary or sporadic cancers. Various heritable developmental disorders in humans are caused by mutations in the TGF-β system ,344. GivOsteoporosis is a systemic bone disorder characterized by low bone mass and microarchitectural deterioration, with consequent bone fragility and an increase risk of fractures. Bone loss occurs in postmenopausal women as a result of an increase in the rate of bone remodeling, and an imbalance between bone resorption, which is higher than bone formation . The larGenetic polymorphisms of members of the TGF-β family are associated with osteoporosis and low bone mass, such as polymorphisms in the genes encoding TGF-β, BMP2, and BMP4 .COL1A1, COL1A2). The altered quality of the bone matrix, composed mainly of type I collagen, could stimulate TGF-β signaling. In fact, the TGF-β produced by osteoblasts is secreted and included in the bone matrix in an essentially latent form. During osteoclastic bone resorption, this coupling factor is released and activated. However, the matrix environment of OI results in excessive TGF-β activation and signaling, which contributes to low bone mass [Osteogenesis imperfecta (OI) is an autosomal dominant form of osteoporosis most often caused by mutations in type I collagen genes  is a B cell malignancy characterized by the presence of an expanded monoclonal population of plasma cells secreting a monoclonal immunoglobulin in the bone marrow, and the development of an osteolytic bone disease .Many osteoclast activation factors were identified in myeloma bone disease , among tTargeting Activin A in MyelomaActivin A, produced after interaction of bone marrow cells with myeloma cells, stimulates osteoclastic resorption, and inhibits osteoblast formation. High levels of activin A, correlating with the extent of osteolysis and with poor survival, were reported in subjects with advanced MM . In a moACE-011 is a fusion protein composed of the extracellular domain of the human activin receptor type IIA linked to the Fc fragment of human IgG1, capable of binding activin. Administration of ACE-011 results in an increase in bone formation markers, and a decrease in bone resorption markers  . In a phTGF-β Family in Monogenic Developmental Bone DiseasesBMPR1B encoding the BMPR-IB receptor in acromesomelic chondrodysplasia [ACVR1 encoding ALK2 in progressive fibrodysplasia ossifying (FOP) [Mutations in genes of BMP receptors are implicated in human skeletal disorders, such as ysplasia  and ACVRng (FOP) .ACVR1 mutations do not affect the expression of ALK2 but result in an increase in ALK2 gain of function related to a R206H substitution in the intracellular GS-rich domain of the receptor linked to 95% of the patients [Acvr1 R206H/+ cells and might act synergistically with BMPs, to favor osteogenesis [FOP, a rare genetic disorder with an incidence of one in two million, is characterized by progressive ectopic bone formation in soft tissue (heterotopic ossification (HO)) such as skeletal muscle, tendon, ligament), either spontaneously or after trauma  ; and Mammalian cells: Chinese hamster ovary (CHO)) [The use of BMPs for therapeutic purposes necessarily involves large-scale production to meet market needs. The extraction and purification of small quantities of BMPs began from demineralized cadaveric bovine bone sources, a technique that required a very long production time and a contribution of several kilograms of bone at a very high cost  . Subsequy (CHO)) ,377,378.y (CHO)) . Indeed,y (CHO)) . Thus, gy (CHO)) .® and OP-1®) [® system  [®) (Stryker Biotech/Olympus) and with carboxymethylcellulose (OP-1 Putty®) (Stryker Biotech/Olympus).To date, the FDA approved the use of two types of rhBMPs (rhBMP-2 and rhBMP-7) associated with commercial delivery systems as an alternative to autologous bone graft in specific orthopedic applications (InFUSEd OP-1®) . RhBMP-2k, Inc.) ,384,385.k, Inc.) ,387. TheRecent studies demonstrated the benefits of using rhBMP-2 and rhBMP-7 for orthopedic treatments and surgeries, particularly in spinal fusion, lumbar fusion, and tibial fracture repair ,390,391 Furthermore, higher osteoinductive activity of BMP-6 and BMP-9, compared to BMP-2 or BMP-7, make them promising candidates for promoting bone repair or filling, as shown by several recent studies ,400,401.Therefore, the heterogeneity of the experimental approach  and the diversity of existing bone repair applications made the conclusions of most studies unclear . Future The members of the TGF-β superfamily are potent regulators of homeostasis and bone repair that act on cell proliferation, osteogenic differentiation, osteoclastogenesis, and osteoblast/osteoclast balance. A good understanding of their characteristics, biological functions, canonical, and non-canonical signaling pathways, as well as their respective regulation is essential for allowing their safe use in the restoration of injured or diseased skeletal tissues. Despite their incredible potential for improving bone repair, they are linked to various postoperative complications  pushing the FDA to limit their use to specific orthopedic application. The reduction of the applied BMP doses, as well as the development of delivery systems allowing a good control of their spatial and temporal release, seem to be the keys to their safe and effective clinical use. However, future well-designed studies are needed to get a better picture of the advantages and drawbacks associated with the use of the TGF-β superfamily members in clinical applications.


Circulating tumour cells (CTCs) from liquid biopsies are under current investigation in several cancers, including epithelial ovarian cancer (EOC) but face significant drawbacks in terms of non-standardised methodology, low viable cell numbers and accuracy of CTC identification. In this pilot study, we report that chemosensitivity assays using liquid biopsy-derived metastatic EOC CTCs, from 10 patients, nine with stage IIIC and one with stage IV disease, in progression after systemic chemotherapy, submitted for hypoxic isolated abdominal perfusion (HAP), are both feasible and useful in predicting response to therapy. Viable metastatic EOC CTCs , enriched by transient culture and identified by reverse transcription polymerase chain reaction (RT-PCR) and indirect immunofluorescence (IF), were subjected to flow cytometry-based Annexin V-PE assays for chemosensitivity to several chemotherapeutic agents and by RT-PCR for tumour gene expression profiling. Using a cut-off value of >80% cell death, CTC chemosensitivity tests were predictive of patient RECIST 1.1 responses to HAP therapy associated with 100% sensitivity, 50% specificity, 33% positive predictive, 100% negative predictive and 60% accuracy values. We propose that the methodology employed in this study is feasible and has the potential to predict response to therapy, setting the stage for a larger study. Epithelial ovarian cancer (EOC) is the 7th most common cancer and 8th leading cause of cancer mortality in women, with a cumulative 5-year survival rate below 45% , and in Treatment options for patients with platinum-resistant EOC include single agent chemotherapy with best supportive care, or a range of aggressive, multi-agent therapeutic regimens for asymptomatic patients comprised of taxanes, anthracyclines, gemcitabine, topotecan and/or trabectedin in various combinations and sequences, and non-platinum monotherapy combined with bevacizumab, followed by maintenance therapy, has also recently been approved, following a landmark trial . HoweverWithin this context, novel loco-regional chemotherapeutic procedures are under investigation to prolong survival and preserve quality of life (QoL). In stage III EOC, intravenous and intraperitoneal chemotherapeutic combinations prolong overall survival following primary cytoreductive surgery ,7,8. HowPressurised intraperitoneal aerosol chemotherapy (PIPAC) is another novel procedure for delivering pressurised aerosols of normothermic chemotherapeutic agents directly into the abdominal cavity. Cisplatin and doxorubicin PIPAC results in objective clinical responses from 62% to 88%, RECIST responses of approximately 50% and median overall survival from 1st PIPAC of approximately 12 months in patients with unresectable recurrent EOC in progression after >2 lines of systemic chemotherapy ,12.However, considering that drugs penetrate tumour tissues with HIPEC and PIPAC to depths of 1–3 mm  and EOC In addition, precision oncotherapy based upon tumour chemosensitivity assays has been under evaluation as an alternative therapeutic approach for the treatment of platinum-resistant EOC. Empiric therapies are chosen from the current literature based upon outcomes achieved for a particular tumour-type with single and combinations of chemotherapeutic agents, whereas drug-selection based upon chemosensitivity assays takes cues from the sensitivity of tumour tissues, tumour cell cultures ,25,26,27With respect to CTCs, flow cytometry studies have detected CTCs with a high degree of sensitivity and specificity in blood samples from melanoma, breast, prostate, pancreatic and colon carcinoma patients . As CTCsp = 0.010).Despite these drawbacks, molecular EOC CTC profiling has unveiled a myriad of biomarkers of potential diagnostic relevance . A studyConsidering that not all treatments are available in every institution and no single treatment strategy fits all, for reasons of lesion size and number, anatomical location, regional lymph node involvement, distant metastases, biomolecular aspects, concomitant disease and previous therapy, it is our considered opinion that treatment strategies for advanced EOC should be multidisciplinary and could benefit greatly from detailed biomolecular characterisation and chemosensitivity assessment of liquid biopsy derived CTCs from individual patients, already USA Food and Drug Administration (FDA)-approved for prognostics . FurtherRecently, we reported a method for liquid biopsy predictive oncotherapy based upon purified metastatic CTCs, transiently cultured in vitro, permitting both gene expression profile and chemosensitivity analysis ,39,40. HViable metastatic EOC CTCs  , were isCTCs were also assayed for epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), tumour protein p53 (p53), multidrug resistance (MDR1), thymidylate synthase (TYMS), dihydrofolate reductase (DHFR), serine hydroxy-methyltransferase 1 (SHMT1), DNA excision repair protein (ERCC1), and glutathione S-transferases (GST) mRNA expression  and, basRECIST 1.1 tumour responses to the drugs selected by CTC chemosensitivity assay in the 10 recurrent EOC patients submitted for HAP, are presented in Positive  and negative (stable disease or progression) RECIST 1.1 responses, following “two-drug” HAP, associated with CTC chemosensitivity of >80% or < 80% drug-induced cell death, are displayed in Additional multidisciplinary treatments were also based upon the results from CTC chemosensitivity assays, CTC gene expression profiles and BRCA mutational status. Based on gene expression assays, two patients received bevacizumab targeted-therapy (Patients 3 and 10). Patient 3 exhibited a CR of 48-month duration, subsequently received rucaparib for 36 months, in accordance with a mutated BRCA status. This patient is still alive and continues to exhibit a CR. In contrast, patient 10 exhibited a PR of 15-month duration and unfortunately died at 24 months. Three patients with locoregional and distant relapsed disease received systemic therapy, one patient received both locoregional and systemic therapy and four patients received best supportive care, only . The thrIn this study, we report that the methods employed for blood sampling, storage, transport and subsequent CTC purification and enrichment are feasible and reproducible, and that the subsequent CTC chemosensitivity and gene expression assays employed to select chemotherapeutic strategies are predictive of therapeutic response. Our results confirm that the numbers of EOC CTCs purified from liquid biopsies, expanded by temporary culture, are sufficient for flow cytometry-based Annexin V-PE chemosensitivity assays, which predict RECIST 1.1 responses to “two-drugs” locoregional HAP with 60% accuracy.The interval between blood sampling and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis did not exceed 80 h, previously reported to minimise alterations in gene and protein expression  and meta2, to densities sufficient for chemosensitivity assays. This raises the possibility for phenotypic alteration and enrichment of subpopulations less representative of the bulk tumour. Since, less-differentiated cancer stem cell-like subpopulations, present in the vast majority of CTC isolates [Considering the relatively low numbers of CTCs isolated from individual patients, isolates were expanded in appropriate media, at 37 °C and 5% COisolates , expand isolates , expandeFlow cytometry Annexin V-PE CTC chemosensitivity assays provided important information on several chemotherapeutic agents but in contrast to tissue-validated chemosensitivity assays, do not preserve cell-to-cell or cell-to-matrix interactions . For thiAccording to the confusion matrix displayed in With respect to tumour gene expression, CTCs from eight patients exhibited high-level p53 (>35%) expression, which is in line with reports that high P53 expression associates with minimal responses to platinum-compounds and better response to taxanes . MoreoveThe scope of this EOC small cohort pilot study was to provide a feasibility evaluation of our predictive oncotherapy CTC-based approach and was not designed to evaluate treatment safety, efficacy or effectiveness . This noThis project was performed in accordance with the Declaration of Helsinki and approved by the ethics committee of ASL n.1, Abruzzo, Italy . Ten patients were prospectively enrolled from 2011 to 2016 and written informed consent was obtained from each patient. Clinical characteristics of the 10 patients with advanced stage EOC are presented in g. CTCs, peripheral blood monocytes, lymphocytes, platelets and granulocytes were collected and washed with phosphate-buffered saline , incubated for 10 min in lysis buffer 154 mM NH4Cl , 10 mM KHCO3  and 0.1 mM EDTA in deionised water, to lyse erythrocytes, centrifuged, re-washed in PBS then sequentially incubated with magnetic beads conjugated with an antibody against leukocyte common antigen CD45 , followed by magnetic beads conjugated with a pan-cytokeratin antibody (pan-CK)  for 30 min at 4 °C. CD45 positive peripheral blood leukocytes were collected first, followed by cytokeratin positive/CD45-negative CTCs in a magnetic field, washed in PBS and cultured in 12-well plates  in RPMI-1640 plus 10% FBS for chemosensitivity, viability and qRT-PCR assays, and compared with CD45 positive peripheral blood leukocytes (PBMCs), purified from each patient, as non-cancer cell controls. CTCs were identified by qRT-PCR, using specific primers for CK19, and other cell types excluded by RT-PCR using primers for CD31 and N-cadherin. For chemosensitivity and gene expression assays, samples contained ≥5 viable CTCs/mL.Liquid biopsies consisting of 20 mL of blood, containing 7 mL of 0.02 M ethylenediaminetetraacetic acid (EDTA), were collected from each patient in sterile 50 mL Falcon tubes, placed in impact-resistant transportation containers at 2–8 °C, transported under refrigeration and subsequently analysed . CTCs weFor chemosensitivity assays, CTCs cultured in 12-well plates  were treated with either: 1 μM cisplatin , 10 μM 5-fluorouracil , 1.12 μM oxaliplatin , 1 μM carboplatin , 5 μM irinotecan , 10 nM paclitaxel , 10 nM docetaxel , 5 μΜ etoposide , 50 nM vinorelbine , 0.5 μM topotecan , 50 nM gemcitabine , 1 μM doxorubicin , 0.5–1 μΜ liposomal doxorubicin , 1 μΜ epirubicin  or 2 μM mitomycin C , and cell viability assessed by Annexin V-PE  flow cytometry  at 24-h intervals for 6 days and the percentage of living, dead and dying cells evaluated, using BD CellQuest Software (BD Instruments Inc) and corroborated by methyl-tetrazolium (MTT), crystal violet (CVE) and Sulfo-Rodhamine B (SRB) assays . The perFor tumour gene expression assays, RNAs were purified from EOC CTCs, using RNeasy Mini Kits, as directed . RNAs (1 µg) were reverse transcribed using a PrimeScript RT Reagent Kit, as directed  and subjected to KAPA SYBR Fast Master Mix (2×) Universal  real-time qPCR, in a final volume of 20 μL, using appropriate housekeeping genes and specific primers for epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), tumour protein p53 (p53), multidrug resistance gene-ABCB1 gene (MDR1), thymidylate synthase (TYMS), dihydrofolate reductase (DHFR), serine hydroxy-methyltransferase 1 (SHMT1), DNA excision repair protein (ERCC1) and glutathione S-transferases (GST), designed using Beacon Designer 8 . FollowiHAP procedures were performed under general anaesthesia, as previously described ,52,53. BTumour responses were assessed 30–45 days following HAP, using Response Evaluation Criteria in Solid Tumours version 1.1 , computeAccording to Connelly , a pilotWe conclude that the methodology for liquid biopsy, samples storage and transport, CTC purification and transient in vitro culture, and subsequent chemosensitivity and gene expression assays are both feasible and reproducible. However, issues that remain to be addressed, include: (i) the use of miniaturised single cell technology for low CTC numbers , (ii) te


Environmental stresses such as salinity, drought, heat, freezing, heavy metal and even pathogen infections seriously threaten the growth and yield of important cereal crops including wheat and barley. There is growing evidence indicating that plants employ sophisticated epigenetic mechanisms to fine-tune their responses to environmental stresses. Here, we provide an overview of recent developments in understanding the epigenetic processes and elements—such as DNA methylation, histone modification, chromatin remodeling, and non-coding RNAs—involved in plant responses to abiotic and biotic stresses in wheat and barley. Potentials of exploiting epigenetic variation for the improvement of wheat and barley are discussed. Triticum aestivum L. ssp. aestivum) and cultivated barley (Hordeum vulgare L. ssp. vulgare) are widely cultivated in the world and provide more than 20% of the caloric intake for one-half of the world’s population [+ can cause the clay to expand excessively when wet, severely restricting the movement of air and water, then resulting in poor drainage [−1) in soils could inhibit regular cell division, decrease photosynthesis and impair antioxidant enzyme activity [As two founding crops of the agricultural revolution that took place 10,000 years ago in the Fertile Crescent, bread wheat  and DRM2 [Arabidopsis and rice revealed that DNA methylation could take place in many chromatin regions, including intergenic transposable elements (TE), gene promoters and even gene-body [Arabidopsis [As a type of DNA chemical modification, DNA methylation regulates the chromatin structure, DNA stability, and even gene expression without changing the DNA sequence. Under the action of DNA methyltransferase, the cytosine C5 position is covalently bonded with a methyl group, which is one of the most common modifications of DNA in eukaryotic cells ,26,27. Iand DRM2 , while tand DRM2 , CHROMOMand DRM2 . High-reene-body ,33,34,35ene-body ,33,34,35bidopsis ,33,34,35Arabidopsis [Bgt (Blumeria graminis f. sp. tritici), the causal agent of wheat powdery mildew, in wheat diploid progenitor Aegilops tauschii [Arabidopsis, O’Malley et al. found that a regulatory relationship may exist between specific DNA methyltransferase and TF family [As an important epigenetic process, DNA methylation generally regulates plant responses to environmental stresses such as salinity and heavy metal stress in wheat and barley ,37. For bidopsis ,44. Eighbidopsis . Shafiq bidopsis ,46. Althbidopsis . It is ibidopsis . DNA dembidopsis . Meanwhibidopsis . Comparetauschii . Upon Bgtauschii . WGBS  in response to heat stress, and enriched at the promoters of CTL1, PGX3 and MYB54  under salt stress as well [HSFA3, UVH6, CTL1, PGX3, and MYB54 in Arabidopsis [HSFA3, UVH6, CTL1, PGX3, and MYB54 were constitutively increased in 35S:TaGCN5/gcn5 transgenic Arabidopsis, compared with wild-type and gcn5 plants, suggesting that GCN5-mediated histone acetylation responding to abiotic stress tolerance might be conserved in Arabidopsis and bread wheat , leaf rust-susceptible NIL and resistant NIL, were employed and the expression levels of six defense-related genes were analyzed as well [N-acetyltransferase is activated by enrichment of H3K4ac and H3K9ac at its promoter in leaf rust-susceptible NIL, and repressed by the histone deacetylation in leaf rust-resistant NIL [Peroxidase 12 in both NILs. The expression of other remaining four genes  was not correlated with histone acetylation [P. triticina infection, whose underlying mechanism remains further study. Recently, the wheat histone deacetylase TaHDA6 was found to interact with the wheat WD40-repeat protein TaHOS15 and was recruited to the promoters of defense-related genes, where TaHDA6 mediated histone deacetylation  and thereby bind to the promoter regions of wheat 3-KETOACYL-CoA SYNTHASE (TaKCS6), which encodes a key enzyme in the wheat cuticular wax biosynthesis  interacts with SWI3B physically, a core subunit of the putative SWI/SNF complex in Arabidopsis [Although the study of chromosome remodeling in response to stress in wheat and barley is very limited, the research in bidopsis . As a phbidopsis . Anotherbidopsis ,92. ThesArabidopsis, ARGONAUTE (AGO) family proteins such as AGO1 then bind the nascent mature miRNAs and guide the target-specific post-transcriptional gene silencing (PTGS) [Arabidopsis [As important epigenetic elements, non-coding RNAs (ncRNAs) widely regulate plant multiple processes, including growth, development and even responses to environmental stresses. Based on the size, ncRNA can be divided into short-chain non-coding RNAs and long-chain non-coding RNA ,94,95. Ig (PTGS) . Unlike g (PTGS) ,99. LncRg (PTGS) ,101. Lncg (PTGS) . Like mRg (PTGS) . More rebidopsis ,104.Arabidopsis studies revealed that ncRNAs such as siRNAs and lncRNAs regulate plant stress-responsive gene expression through multiple epigenetic mechanisms, including DNA methylation, histone modification and genome topology changes [Arabidopsis [Arabidopsis RNAPIV-generated siRNAs could load to Argonaute 4 (AGO4) and interact with lncRNAs generated by RNAPII to constitute a siRNA–AGO4–lncRNA silencing complex, which subsequently recruits the DMT domains rearranged methyltransferase 2 (DRM2) to mediate DNA de novo cytosine methylation [Arabidopsis mutant deficient in NRPD2, an essential subunit of RNAPIV were hypersensitive to heat stress, suggesting that RdDM pathway is essential to the regulation of plant stress responses [Arabidopsis [COOLAIR, a group of long antisense RNAs expressed from the FLOWERING LOCUS C (FLC) locus, promote the replacement of H3K36me3 with H3K27me3, as well as the H3K4me2 demethylation, at FLC locus during cold exposure [COLDAIR could interact with polycomb repressive complex 2 (PRC2) to facilitate H3K27me3 enrichment at FLC [Arabidopsis. For instance, Arabidopsis lncRNA APOLO promotes the chromatin loop formation at the PINOID (PID) locus, encoding a key regulator of polar auxin transport, which is further regulated by RdDM and ultimately determines the PID expression patterns [TalncRNA18, TalncRNA73, TalncRNA106, and TalncRNA108) exhibited differential expression upon infection of Puccinia striiformis f. sp. tritici (Pst), suggesting that these lncRNAs may get involved in regulation of wheat defense responses to Pst. However, detailed epigenetic mechanism in the regulation of wheat and barley stress responses remain to be explored in the future research.Increasing evidence from  changes . For insbidopsis . Arabidohylation . Arabidoesponses . Besidesbidopsis . For insexposure ,110. Simt at FLC ,112,113.patterns . With thpatterns ,117,118.In this review, we discuss the recent advance in the understanding of epigenetic regulation of plant responses to abiotic and biotic stresses in wheat and barley  populations were constructed and exhibited discernible phenotypic variation, including altered resistance against pathogen infection [Arabidopsis, Johnson et al. directed DNA methylation to target DNA sequences and caused expected phenotype changes through fusing ZFNs with the SRA domain-containing protein SUVH9—a protein integral to RNA-directed DNA methylation (RdDM) [With the advance of molecular technologies, our knowledge of the mechanisms of epigenetic responses to environmental stresses is rapidly growing, which could certainly lead to the more efficient improvement of cereal crops . In Arabnfection ,125,126.nfection ,125,126.nfection ,129,130.n (RdDM) . The dev


In humans, numerous cell stimulation pathways lead to the mobilization of sarco/endoplasmic reticulum (S/ER) stored Ca2+, resulting in the propagation of Ca2+ signals through the activation of processes, such as store-operated Ca2+ entry (SOCE). SOCE provides a sustained Ca2+ entry into the cytosol; moreover, the uptake of SOCE-mediated Ca2+ by mitochondria can shape cytosolic Ca2+ signals, function as a feedback signal for the SOCE molecular machinery, and drive numerous mitochondrial processes, including adenosine triphosphate (ATP) production and distinct cell death pathways. In recent years, tremendous progress has been made in identifying the proteins mediating these signaling pathways and elucidating molecular structures, invaluable for understanding the underlying mechanisms of function. Nevertheless, there remains a disconnect between using this accumulating protein structural knowledge and the design of new research tools and therapies. In this review, we provide an overview of the Ca2+ signaling pathways that are involved in mediating S/ER stored Ca2+ release, SOCE, and mitochondrial Ca2+ uptake, as well as pinpoint multiple levels of crosstalk between these pathways. Further, we highlight the significant protein structures elucidated in recent years controlling these Ca2+ signaling pathways. Finally, we describe a simple strategy that aimed at applying the protein structural data to initiating drug design.Calcium (Ca Under resting conditions, intracellular Ca2+ concentrations are maintained at ~100 nM by effectively segregating Ca2+ into the extracellular fluid and within intracellular stores, such as the mitochondria and sarco/endoplasmic reticulum (S/ER). Under basal conditions, Ca2+ levels within the cytosol and mitochondria are similar; conversely, in times of cellular stress or injury, mitochondria can uptake Ca2+ [2+ overload [2+ would lead to a cycle of higher respiratory chain activity and the generation of reactive oxygen species (ROS), resulting in oxidative stress, apoptosis, and/or necrosis [2+ is a cofactor that is required for mitochondrial dehydrogenase function, which are enzymes integral to the electron transport chain [2+ uptake is via the low affinity, high capacity mitochondrial Ca2+ uniporter (MCU) complex [2+/proton (H+) antiporter is involved in fine-tuning these levels [2+ uptake, dictating both mitochondrial and cellular function.Regulating cytosolic calcium ion Ca+ levels ake Ca2+ ,4 in ordoverload ,6,7. Pernecrosis ,9. Ca2+ rt chain . The pri complex ,13. Howee levels ,15. Thus2+ store, maintaining free Ca2+ in the ~400–700 µM range (depending on cell-type) [3Rs), respectively, serve as the predominant channels that release Ca2+ into the cytosol from the S/ER stores [2+ is critical for the activation of Ca2+-dependent signaling cascades that require more chronic elevations in cytosolic Ca2+ because S/ER Ca2+ stores are limited in capacity. Store-operated Ca2+ entry (SOCE) is the process whereby S/ER Ca2+ store depletion leads to the activation of plasma membrane (PM) Ca2+ channels for a sustained Ca2+ entry into the cytosol from the extracellular space [2+-sensing S/ER-membrane inserted stromal interaction molecules (STIMs) [2+ levels that are driven by SOCE not only signal downstream physiological and pathophysiological cellular responses, but also replenish the depleted intracellular Ca2+ stores.The S/ER is the largest intracellular Call-type) ,19,20,21ll-type) . In exciR stores ,24,25. Aar space ,27,28,29 (STIMs) ,31 and t (STIMs) ,34,35,363Rs and/or RyRs, leading to the depletion of the S/ER Ca2+ stores [2+ store release  [3 is a small diffusible second messenger that binds and activates IP3Rs, causing S/ER Ca2+ store release into the cytosol [3R-driven Ca2+ signaling mechanism is critical in platelets where thrombin binding to protease activated receptor (PAR)-1 or PAR4 mediates thrombosis [1 receptor (H1R) leads to bronchoconstriction [GPCRs constitute a major family of PM receptors that, when activated by the appropriate extracellular ligand, can serve to mediate the release of Caia IP3Rs ,42. Upon cytosol . This IPrombosis , in vascrombosis , and in triction , to name2+ release through IP3R activation. RTKs, such as insulin growth factor-1 receptor in skeletal myoblasts activate phospholipase C and IP3 production upon binding insulin-like growth factor-1, leading to ER Ca2+ release necessary for differentiation in myogenesis [3R activation, enabling humoral immunity signaling and apoptosis/elimination of self-reactive T-cells, respectively [3 generation and S/ER Ca2+ release [Nevertheless, it is not only GPCRs that can induce S/ER Caogenesis ,69. In sectively ,70. Likeectively ,17. Immu release ,17,55,563Rs and similarly function to release S/ER Ca2+ [2+ channels) open in response to membrane depolarization, resulting in the entry of Ca2+ into the cytosol from the extracellular space [2+ binds to S/ER RyRs, opening these channels, in contrast to IP3Rs, which require IP3. Remarkably, the RyR-mediated release of S/ER Ca2+ can propagate to adjacent channels and promote additional RyR activation in a process that is known as Ca2+-induced Ca2+ release [RyRs are much larger structural homologues of IP/ER Ca2+ . For exaar space ,61. This release ,61.2+ signaling pathway that has evolved to use S/ER Ca2+ depletion as an activation signal, facilitating further and more sustained extracellular Ca2+ influx in response to the release of intracellular S/ER Ca2+ [2+ sensor and SOCE activator [2+ changes  deactivates Orai1 channels to terminate SOCE [S-Glutathionylation, for example, is the covalent attachment of a glutathione (GSH) moiety to free cysteine (Cys) thiols. GSH modification of STIM1 at Cys56 has been shown to evoke constitutive Orai1 channel activation and Ca2+ entry independent of luminal Ca2+ concentrations [A process known as Caate SOCE ,86. Multate SOCE ,88,89,90ate SOCE ,102,103.trations . It is wSeveral heritable mutations in STIM1 and Orai1 cause disease. The loss of function mutations in STIM1 occur as frameshift i.e., E128RfsX9 ) and intRfsX9  anGain of function mutations occur in STIM1  potential or depletion of sodium (Na+) could inhibit SOCE [2+ uptake diminishers [2+ uptake near store-operated Ca2+ channels is believed to prevent Ca2+-dependent inactivation by limiting the formation of high Ca2+ level domains. Interestingly, one study showed that the distance between mitochondria and PM SOCE channels decreases upon SOCE activation, allowing for a more sustained Ca2+ entry due to reduced CDI [2+ uptake, but not mitochondrial translocation/motility, was required for STIM1 and Orai1-mediated SOCE [2+ levels [The first link between mitochondrial Cabit SOCE . Indeed,inishers . Mitochouced CDI . In contted SOCE . Further+ levels .2+ uptake following SOCE [+/Ca2+/lithium (Li+) exchanger (NCLX) on the IMM has been shown to be vital for clearance of mitochondrial Ca2+ . SOCE iand SOCE . The actits SOCE . It is bits SOCE .2+ uptake plays a central role in regulating this energy production and it is also instrumental in regulating apoptosis and shaping cytosolic Ca2+ transients [2+ can readily move through the voltage dependent anion channel (VDAC) of the outer mitochondrial membrane, Ca2+ uptake into the mitochondrial matrix is more precisely controlled [2+ uptake into the matrix, functioning as a multimeric protein complex that forms a Ca2+ selective pore through the IMM [2+ into the matrix, despite the highly negative IMM potential (~−180 mV); following a rise in cytosolic Ca2+ levels, the MCU channel open probability increases, allowing for Ca2+ to move into the matrix, being driven by the IMM potential [2+ uptake 1, 2, and 3  [Mitochondria are primarily recognized for their role in adenosine triphosphate (ATP) production, a process that requires an electrochemical gradient across the IMM . Mitochoansients . While Cntrolled . MCU pla the IMM ,13. Undeotential ,149,150.otential ,154,155,d MICU3) ,157; a sd MICU3) ; an MCU d MICU3) ; and a cd MICU3) .2+ selectivity and controls permeability through the channel due to binding of Ca2+ to the acidic residues [2+ uptake, which is likely due to the removal of these negative charges [2+-permeable pore [Caenorhabditis elegans MCU was suggested to exist as a pentamer, with the DIME motifs forming an unstructured loop at the opening of the channel [C. elegans NMR model have established metazoan MCU as a tetramer with the DIME motifs lining the pore as part of the helical transmembrane regions [The MCU pore forming subunit is an ~40 kDa protein  composed of two transmembrane domains spanning the IMM, with both the N- and carboxyl (C)-termini localized in the mitochondrial matrix ,161 FigA. An Aspresidues . Amino a charges . MCU subble pore . Initial channel . Several regions ,152,154 2+ uptake [Interestingly, the C-terminal domain of MCU alone can form a channel, because it contains the TM1 and TM2 helices that create the IMM channel pore ,162,163;+ uptake ,153,155 + uptake .2+-sensing properties to MCU channel activity. The presence of an Asp-rich C-terminus is thought to be important in MCU complex regulation [2+ uptake in mammals; subsequent overexpression of MCU is unable to recover the channel function [EMRE is a metazoan-specific ~11 kDa  single-pass membrane protein that spans the IMM . EMRE ingulation ,166; howgulation . The mosgulation   that shares ~50% sequence identity with MCU  Figure A. Much lble pore . Interesble pore . Mechanible pore . Once ineability ,180. Nev2+ uptake, reduces the IMM potential, and disrupts oxidative phosphorylation [2+ uptake, but also plays a key role in controlling cellular bioenergetics.MCUR1 has been identified as a positive regulator of MCU, functioning as a scaffold factor that binds to both MCU and EMRE ,181. MCUrylation . Collect2+ signaling has been implicated in a variety of pathophysiological disorders. Aberrant mitochondrial Ca2+ uptake not only affects processes reliant on maintaining tight cytosolic Ca2+ concentrations, but also impacts oxidative metabolism and can trigger cell death pathways [2+ overload and/or oxidative stress results in an opening of the mitochondrial permeability transition pore (mPTP) [2+ uptake has dire implications for cell survival and has been associated with various diseases, including cancer, neurodegenerative pathologies, diabetes, and learning and muscular disorders, among others [Mitochondrial Capathways ,147,183.e (mPTP) . High coe (mPTP) ,186. Ulte (mPTP) . Thus, dg others ,190,191.MICU1 mutations , which result in intronic insertions causing frameshifts, nonsense mediated mRNA decay, and loss of MICU1 protein [MICU1 exon 1 deletions  [MICU1 nonsense mutations  has been discovered, which eliminates full-length MICU2 protein [2+ uptake. Further, MICU2 protein abrogation also suppressed mitochondrial Ca2+ uptake rates [2+ levels due to either MICU1 or MICU2 protein downregulation [Patients have been identified with heritable  protein ,192,193.eotides)  and MICU:p.Q185* ) have be protein . All of  protein ,195,196. protein  rates ofgulation ,196, whi2+ uptake can suppress cytosolic Ca2+ signals in fibroblasts from MICU1-deficient patients [2+ signals [2+ uptake may be related to work showing deletion of MICU1 in mouse hepatocytes causes sensitization to Ca2+-overload-induced mPTP opening [Enhanced mitochondrial Capatients , which i signals ,199,200. opening . The ide2+ and K+ ion homeostasis, regulating key facets of mitochondrial physiology, such as osmotic balance and ATP production [LETM1 as one of ~2000 essential genes in human cell cultures [LETM1 is an essential IMM protein linked to mitochondrial ion transport, regulation of cell cycle, mitochondrial oxidative stress and bioenergetic function . Interesoduction ,207,208.oduction . Homozygoduction . Clinicaoduction ,210 , LETM1 has been shown to transport Ca2+ at ~nM cytosolic Ca2+ levels, suggesting that LETM1 provides cells with a high affinity/low capacity pathway necessary for precise mitochondrial Ca2+ homeostasis [2+/H+ exchange, independent of MCU [2+ uptake, decreases ATP production, and increases ROS production [In the context of mitochondrial Caeostasis ,214,215.t of MCU ,206,208.oduction ,216.2+-binding EF-hand motif [Human LETM1 is an ~83 kDa transmembrane protein  that localizes in the IMM ,207 FigA. LETM1 nd motif . This tond motif ,206,207.nd motif   [2+ ATPase (SERCA) pumps [2+ toolkit structural data, yet reinforces a major question at the nexus of the structural biology, cell biology, and clinical research fields. How can we use this wealth of protein structural information to design new diagnostic tools and drugs for research and treatment of disease?Over the past ~15 years, a number of protein structures involved in regulating cytosolic and stored Ca as SOCE ,31 and m+ uptake ,13, but tion NMR ,113, thetion NMR , and X-rtion NMR ,108  , S/ER CaA) pumps ,240, andA) pumps ,242. Thi2+ signaling proteins, there is a disconnect between applying this protein structural knowledge and rationally developing research tools or drugs. Perhaps this disconnect exists due to the relatively static insights provided by many of the resolved structures. Most  protein function involves dynamic changes in conformation and stability. Rationalizing effective drugs without a detailed understanding of the conformational ensembles present in the inactive, transition, and active states of proteins is practically challenging.Indeed, while obtaining structural information has been invaluable for informing the broad scientific community on the molecular and structural basis for the function of hundreds or thousands of Ca2+ uptake protein machinery are appealing drug targets for several reasons. First, mutations causing disease have been identified in the proteins mediating these processes (see above). Second, complexes of multiple different proteins regulate SOCE and mitochondrial Ca2+ uptake; hence, the modulation of function, in some cases, could be achieved by targeting native protein interaction partner components. Third, drugs targeting ion channels are demonstrated to be effective at treating a wide range of human diseases and acute conditions [2+ uptake molecular machinery provide a unique opportunity for the development of new diagnostics and/or therapeutics that can biasedly target different aspects of these pathways.The SOCE and mitochondrial Canditions ,244,245.nditions ,247). Finditions ,250,251.Saccharomyces cerevisiae as an MCU-null organism to screen for small molecule modulators of MCU function. Human MCU, human EMRE, and a mitochondrial-targeted Ca2+ indicator  were expressed in these yeasts to establish a reporter system for the screening of ~600 clinically approved drugs [2+.Small molecule screens targeting MCU function have already been performed. An initial study employed ed drugs . In a seed drugs . In the ed drugs . All of 2+ or NFAT translocation to the nucleus [Since the identification of STIM1 and Orai1, published drug screens targeting SOCE have largely involved the over-expression of STIM1 and Orai1 in mammalian cells or the use of cells with endogenous levels of STIM1 and Orai1, in combination with indicator dyes or proteins monitoring cytosolic Ca nucleus ,257,258. nucleus . In sili nucleus ,261.2+ uptake have been performed, these published approaches have not used the available protein structure information as initiation points to drug discovery.Thus, while drug screens targeting the protein machinery that mediates both store-operated and mitochondrial CaA starting point for drug development could be initiated by a simple high-throughput small molecule in silico screen of millions of compounds. This computational screen would use available protein structures and knowledge of the vital interfaces modulating activity. The structures of the small molecules/drugs would also need to be known. Such small molecule databases and computational docking programs exist and are freely available .2+ signaling, these types of FRET-based sensors have been engineered and used in screens for the identification of SERCA and RyR modulators. A green fluorescent protein (GFP) and red fluorescent protein (RFP) FRET pair was fused into the cytosolic headpiece of SERCA, which undergoes large scale structural changes during pump activity. This intramolecular FRET sensor was successfully used to identify the small molecule modulators of SERCA function [High resolution structural information can also be used to develop tools for high-throughput drug discovery. For example, fluorescence-resonance energy transfer (FRET)-based biosensors can be rationalized based on proximity information that is revealed by isolated and co-complexed structures. With respect to Cafunction ,266. Sinfunction ,268. In 2+ signaling axis, providing several possible initiating points to protein structure-based drug design. The elucidation of Ca2+ signaling protein structures is vital for understanding the fundamental mechanisms underlying cellular processes that are integral to life and death. The structural information can also provide an entry point for the development of tools and therapeutics targeting these pathways.Not discussed herein are the countless additional experimental , logistical, and administrative hurdles that are required to move any small molecule lead into clinical use. Nevertheless, we provide a simple strategy for applying high-resolution structural knowledge of proteins as a starting point to drug development. Excitingly, several key protein structures are available that inform on the mechanisms regulating the SOCE-mitochondrial Ca


Despite several RNA-Seq and microarray studies on differentially expressed genes (DEGs) between high- and low-abdominal fat deposition in different broiler lines, to our knowledge, gene coexpression analysis across multiple broiler lines has rarely been reported. Here, we constructed a consensus gene coexpression network focused on identifying consensus gene coexpression modules associated with abdominal fat deposition across multiple broiler lines using two public RNA-Seq datasets (GSE42980 and GSE49121).In the consensus gene coexpression network, we identified eight consensus modules significantly correlated with abdominal fat deposition across four broiler lines using the consensus module analysis function in the weighted gene coexpression network analysis (WGCNA) package. The eight consensus modules were moderately to strongly preserved in the abdominal fat RNA-Seq dataset of another broiler line (SRP058295). Furthermore, we identified 5462 DEGs between high- and low-abdominal fat lines (FL and LL) (GSE42980) and 6904 DEGs between high- and low-growth (HG and LG) (GSE49121), including 1828 overlapping DEGs with similar expression profiles in both datasets, which were clustered into eight consensus modules. Pyruvate metabolism, fatty acid metabolism, and steroid biosynthesis were significantly enriched in the green, yellow, and medium purple 3 consensus modules. The PPAR signaling pathway and adipocytokine signaling pathway were significantly enriched in the green and purple consensus modules. Autophagy, mitophagy, and lysosome were significantly enriched in the medium purple 3 and yellow consensus modules.Based on lipid metabolism pathways enriched in eight consensus modules and the overexpression of numerous lipogenic genes in both FL vs. LL and HG vs. LG, we hypothesize that more fatty acids, triacylglycerols (TAGs), and cholesterol might be synthesized in broilers with high abdominal fat than in broilers with low abdominal fat. According to autophagy, mitophagy, and lysosome enrichment in eight consensus modules, we inferred that autophagy might participate in broiler abdominal fat deposition. Altogether, these studies suggest eight consensus modules associated with abdominal fat deposition in broilers. Our study also provides an idea for investigating the molecular mechanism of abdominal fat deposition across multiple broiler lines.The online version contains supplementary material available at 10.1186/s12864-021-07423-6. Over the last few decades, broiler chickens have become a major animal protein source for the human diet , 2. ProdAt present, several studies have reported abdominal fat differentially expressed genes (DEGs) between high- and low-abdominal fat deposition in different broiler lines using RNA-Seq or microarray technology. There were 1687 DEGs identified between high- and low-abdominal fat line (FL and LL) broilers with a 2.8-fold divergence in abdominal fat percentage (AFP) at 7 weeks of age . In eachIn the current study, to explore the molecular mechanism of abdominal fat deposition across multiple broiler lines, we constructed a consensus gene coexpression network across GSE42980 (12 FL and 12 LL samples) and GSE49121 (8 HG and 8 LG samples) and detected consensus modules associated with abdominal fat deposition. Then, we tested the preservation of consensus modules in abdominal fat RNA-Seq data of another broiler line (SRP058295). The ClusterProfiler software package was used to predict Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) terms associated with the genes within consensus modules. Our study provides an idea for investigating abdominal fat deposition across multiple broiler lines.To reduce the influence of nongenetic factors, we collected public abdominal fat gene expression profile datasets of broilers under the same age, sex, and normal feed conditions. Broiler chickens were divergently selected over seven generations for either high (FL) or low (LL) abdominal fat at similar feed intake and body weight, and FL and LL broilers exhibited a 2.8-fold divergence in AFP at 7 weeks of age , 12. GSEThe average number of raw reads across the samples within GSE42980, GSE49121, and SRP058295 was 54.97 M , dihydrolipoamide S-acetyltransferase (DLAT), peroxisome proliferator-activated receptor gamma (PPARG), retinoid X receptor gamma (RXRG), and acyl-CoA oxidase 1 (ACOX1), all of which were significantly upregulated in the FL vs. LL comparison , acyl-CoA synthetase long chain family member 4 (ACSL4), succinate-CoA ligase ADP-forming beta subunit (SUCLA2), 7-dehydrocholesterol reductase (DHCR7), and acyl-CoA synthetase bubblegum family member 2 (ACSBG2), all of which were significantly upregulated in the HG vs. LG comparison , glycerol-3-phosphate acyltransferase 3 (GPAT3), glycerol-3-phosphate dehydrogenase 2 (GPD2), patatin like phospholipase domain containing 7 (PNPLA7), ethanolamine-phosphate phospho-lyase (ETNPPL), CD36 molecule (CD36), G protein subunit alpha i3 (GNAI3), fatty acid synthase (FASN), acyl-CoA dehydrogenase, C-2 to C-3 short chain (ACADS), palmitoyl-protein thioesterase 1 (PPT1), 3-hydroxyacyl-CoA dehydratase 1 (HACD1), fatty acid desaturase 2 (FADS2), fatty acid binding protein 3 (FABP3), stearoyl-CoA desaturase (SCD), 3-hydroxyacyl-CoA dehydratase 2 (HACD2), and malonyl-CoA-acyl carrier protein transacylase (MCAT) , autophagy related 3 (ATG3), autophagy related 12 (ATG12), autophagy related 9A (ATG9A), autophagy related 4B cysteine peptidase (ATG4B), microtubule associated protein 1 light chain 3 alpha (MAP1 LC3A), microtubule associated protein 1 light chain 3 beta (MAP1 LC3B), beclin 1 (BECN1), phosphate cytidylyltransferase 2, ethanolamine (PCYT2), and selenoprotein I (SELENOI)  within both pairwise comparisons and had the same expression trend across two pairwise comparisons, i.e., upregulated within both comparisons, and vice versa. A total of 2020 common DEGs were shared across the two pairwise comparisons, among which many lipid metabolism genes were significantly upregulated in the FL vs. LL and HG vs. LG, such as After genes with low expression were removed, a total of 13,626 genes were used to construct a consensus gene coexpression network across the two datasets (GSE42980 and GSE49121). A soft threshold (power) of 14 was selected to construct a scale-free network within the two datasets     were significantly correlated with abdominal fat deposition across the two datasets, and the correlation of the yellow consensus module was the highest  were detected in the eight consensus modules Fig. .Fig. 4VZsummary results showed that the turquoise, green, saddle brown, and yellow modules were strongly preserved (Zsummary > 10) and the dark green, purple, medium purple 3, and skyblue modules were moderately preserved (2 < Zsummary < 10) . The 8295. TheGPAT3, GPD2, PNPLA7, ETNPPL, and CD36, were identified. Steroid biosynthesis was significantly enriched in the medium purple 3 module, and relevant genes, DHCR7 and sterol-C5-desaturase (SC5D), were identified. Pyruvate metabolism, glyoxylate and dicarboxylate metabolism, and propanoate metabolism pathways were significantly enriched in the green module, and relevant genes of the three pathways, PDHA1, ACOX1, and SUCLA2, were identified. The PPAR signaling pathway was significantly enriched in the green consensus module, and relevant genes of this pathway, RXRG, ACOX1, and ACSL4, were identified. The fatty acid metabolism pathway was significantly enriched in the medium purple 3 and yellow consensus modules, and relevant genes of this pathway, ACSBG2, ACAT2, FASN, ACADS, PPT1, HACD1, FADS2, HACD2, and MCAT, were identified. Autophagy, lysosome, and mitophagy pathways were significantly enriched in the medium purple 3 and yellow consensus modules, and the relevant genes of the three pathways, ULK2, BECN1, MAP1 LC3A, MAP1 LC3B, ATG3, ATG4B, ATG12, and ATG9A, were identified in the consensus modules  and cholesterol esters . Based ochickens , 15. The pathway . In our GPAT3 is significantly upregulated during adipocyte differentiation in mice, and overexpression of GPAT3 results in increased TAG in mammalian cells [PNPLA7, GPAT3, DHCR7, DGAT2, and SC5D were identified in the three lipid metabolism pathways. PNPLA7, GPAT3, and DHCR7 were significantly upregulated in both the FL vs. LL and HG vs. LG, and DGAT2 and SC5D were significantly upregulated in the HG compared with the LG lines. Therefore, we inferred that the synthesis of fatty acids, TAG, and cholesterol in broilers with high abdominal fat deposition was greater than that in broilers with low abdominal fat deposition.The PNPLA protein family plays key roles in lipid droplet homeostasis, phospholipid metabolism, and triglyceride hydrolysis . PNPLA7 an cells . As the an cells . DHCR7 aan cells . In the PPARG is a well-known critical gene that is reported to positively regulate adipogenesis, lipid metabolism, adipocyte differentiation, and abdominal fat deposition [FABP3 and SCD [PPARG, RXRG, FABP3, SCD, and CD36, were identified and significantly upregulated in both the FL vs. LL and HG vs. LG.Abdominal fat deposition is a complex biological process that is controlled by a series of critical genes in signaling pathways. In chickens, position –24. RXRG and SCD . Active  and SCD . In the PCYT2) and ethanolamine phosphotransferase 1 (encoded by SELENOI) are the two critical enzymes for PE synthesis [SELENOI, PCYT2, ULK2, BECN1, MAP1 LC3A, MAP1 LC3B, ATG3, ATG4B, ATG12, and ATG9A, all of which were significantly upregulated in both the FL vs. LL and HG vs. LG. Therefore, we inferred that autophagy initiation might participate in broiler abdominal fat deposition.Autophagy is a process in which cytoplasmic macromolecules and organelles are degraded by lysosomes  and playynthesis , 34. In PDHA1, FASN, ACSBG2, FADS2, SCD, GPAT3, PNPLA7, DHCR7, PPARG, RXRG, FABP3, and CD36, all of which were identified in eight consensus modules and significantly upregulated in both the FL vs. LL and HG vs. LG. Based on lipid metabolism pathways enriched in eight consensus modules and overexpression of numerous lipogenic genes, we hypothesize that more fatty acids, TAGs, and cholesterol might be synthesized in broilers with high abdominal fat than in broilers with low abdominal fat. Autophagy, mitophagy, and lysosome were significantly enriched in the eight consensus modules, and the relevant genes of the three pathways, SELENOI, PCYT2, ULK2, BECN1, MAP1 LC3A, MAP1 LC3B, ATG3, ATG4B, ATG12, and ATG9A, were significantly upregulated in both FL vs. LL and HG vs. LG. Therefore, we inferred that autophagy initiation might participate in broiler abdominal fat deposition. These genes identified in eight consensus modules can provide targets for further research into abdominal fat deposition in multiple broiler lines. To the best of our knowledge, this is the first report on abdominal fat deposition across multiple different broiler lines via a consensus module analysis method.In our current study, we predicted eight consensus modules across four broiler lines , the preservations of which were verified in another broiler line dataset (SRP058295). Pyruvate metabolism, fatty acid metabolism, glycerolipid metabolism, steroid biosynthesis, the PPAR signaling pathway, and the adipocytokine signaling pathway were significantly enriched in the eight consensus modules. Relevant genes of these lipid metabolism pathways included To reduce the influence of nongenetic factors, such as analytical methods, versions of the genome and annotated files, experimental technologies (RNA-Seq or microarray), sex, diet condition, and weeks of age, we only collected abdominal fat RNA-Seq data of male broilers at 7 weeks of age with normal feed. In the current study, we downloaded GSE42980 , GSE4912GCF_000002315.6_GRCg6a) and chicken genome annotation files (GCF_000002315.6_GRCg6a_genomic.gtf.gz) were downloaded from the NCBI genome assembly website . Then, the read count of each gene was calculated using featureCounts (v2.0.1) with default parameters [To ensure high-quality reads for downstream analyses, quality control (QC) of the RNA-Seq reads was performed using fastp 0.19.7) with default parameters and workflow . After frameters . A summa9.7 with Low-expression genes within the GSE42980 and GSE49121 datasets were filtered using the filterByExpr function in the EdgeR package (3.24.3) , 39. A tp-value (FDR) ≤ 0.05. The significant DEGs in each pairwise comparison are listed in Additional file Within DESeq2, read counts from each library were normalized for differences in sequencing depth, used to estimate dispersions from the mean and to fit a linear model according to a negative binomial distribution for each pairwise comparison. Differentially expressed genes (DEGs) within the FL vs. LL (GSE42980) and HG vs. LG (GSE49121) were identified by testing the two factor variables of high- and low-abdominal fat deposition and batch using DEseq2 (1.22.2) with default parameters . The sigGene coexpression networks were constructed using the WGCNA (version 1.68) package in R . The genp-value ≤0.05).The module eigengene is defined as the first principal component of a gene module . To idenmedianRank was used to compare relative preservation among consensus modules, and a module with higher median rank exhibits weaker observed preservation statistics than a module with a lower median rank [Zsummary were set as follows: Zsummary > 10 means strong evidence of preservation; 2 < Zsummary < 10 means weak to moderate evidence of preservation; Zsummary < 2 means no evidence of preservation [To test the preservation of consensus modules significantly correlated with abdominal fat deposition in other broiler lines, abdominal fat RNA-Seq data of male broilers (SRP058295) were downloaded to construct a test network for preservation analysis. The preservation analysis of consensus modules was performed using the modulePreservation function in the WGCNA package with the parameters nPermutations = 200, randomSeed = 1, quickCor = 0, verbose = 3 . The median rank . The thrervation .p-value ≤0.05.Based on KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology) databases, the pathways and GO terms associated with the genes within significant consensus modules were predicted using the clusterprofiler software package (3.10.1) . The sigAdditional file 1: Table S1. Summary of RNA-Seq data, QC, and read mapping.Additional file 2: Figure S1. Hierarchical cluster of samples within GSE42980 and GSE49121.Additional file 3: Table S2. Summary of genes within consensus modules and DEGs within FL vs. LL and HG vs. LG.Additional file 4: Figure S2. Cluster dendrograms of consensus, GSE42980, and GSE49121 modules.Additional file 5: Figure S3. Heatmaps of consensus modules correlated with abdominal fat deposition within GSE42980 or GSE49121.Additional file 6: Table S3. Functional enrichment correlated with genes within eight consensus modules.


Surface phenotype and NanoString gene expression profiling indicated the closest steady-state counterpart of these cells to be monocyte-derived macrophages. In GVHD, however, there was upregulation of monocyte antigens SIRPα and S100A8/9 transcripts associated with leukocyte trafficking, pattern recognition, antigen presentation, and costimulation. Isolated GVHD macrophages stimulated greater proliferation and activation of allogeneic T cells and secreted higher levels of inflammatory cytokines than their steady-state counterparts. In HLA-matched mixed leukocyte reactions, we also observed differentiation of activated macrophages with a similar phenotype. These exhibited cytopathicity to a keratinocyte cell line and mediated pathological damage to skin explants independently of T cells. Together, these results define the origin, functional properties, and potential pathogenic roles of human GVHD macrophages.Myelopoiesis is invariably present and contributes to pathology in animal models of graft-versus-host disease (GVHD). In humans, a rich inflammatory infiltrate bearing macrophage markers has also been described in histological studies. In order to determine the origin, functional properties, and role in pathogenesis of these cells, we isolated single-cell suspensions from acute cutaneous GVHD and subjected them to genotype, transcriptome, and in vitro functional analysis. A donor-derived population of CD11c GVHD cohorts . Despite cohorts .Animal models previously demonstrated that immunocompetent donor myeloid cells enhance GVHD, without specifying a particular cell type , 11. MacIn humans, a number of reports highlight an increase in myeloid cells bearing macrophage markers, showing that the level of infiltration correlates with clinical severity and outcome , 20, 21.The role of (recipient) myeloid cells in responding to danger signals is integral to most models of GVHD, but it is not known whether human GVHD infiltrates bearing macrophage markers are recipient or donor in origin and immunogenic or antiinflammatory in activity.Although donor myelopoiesis usually dominates the peripheral blood compartment during GVHD, recipient dermal macrophages have very slow kinetics of turnover in humans  and pote+CD14+ myeloid cells with the genotype, phenotype, and transcriptional profiles of donor monocyte–derived macrophages. These cells have potent immunological functions that are likely to contribute to the pathogenesis of GVHD and may offer opportunities for therapeutic intervention.Here, we employ direct methods of isolation and testing to show that acute GVHD lesions in human skin are dominated by CD11chttps://doi.org/10.1172/JCI133909DS1). BMT control patients without GVHD were biopsied on median day 83 after transplantation . GVHD skin biopsies were taken at the onset of an acute onset erythematous rash, before initiation of therapy. Classical acute GVHD, immunosuppression withdrawal acute GVHD, and acute GVHD following donor lymphocyte infusion were all included. A pathological diagnosis of acute-type GVHD was confirmed by standard histological criteria in all cases, and patients with clinical or histological features of chronic GVHD were excluded. The median day of biopsy was day 53 . In situ analysis showed an increase in CD3+ T cells and CD11c+ myeloid cells in a perivascular and epidermal interface distribution in GVHD  low lymphocytes and HLA-DRescribed , 25. Surescribed . A relatthy skin . Myeloidthy skin –24, 26. +CD14+ myeloid cells were expanded more than 10-fold compared with healthy control skin or BMT skin without GVHD  and cDC1 , but showed upregulation of monocyte-associated antigens , CD11c+CD14+ GVHD cells were segregated with steady-state monocyte-macrophages and resident dermal macrophages, away from DC populations , recruitment (CCL24) and stimulation of lymphocytes (CD82), stimulation of proinflammatory cytokines (SPP1), and leukocyte extravasation (SELPLG)  . Differe(SELPLG) .+ cDC2, as described in GVHD skin , FCGR3A/B (Fc receptor/CD16), GNLY (granulysin), and IFN-response genes IFITM1 and GBP1  chemokine receptor-ligand pairs . The prohe blood .MRC1, CCR5, and PPBP, upregulated in GVHD macrophages in vivo, were also highly upregulated in MLR macrophages. MLR-activated macrophages also expressed cytotoxic molecules perforin, granzyme A, granulolysin, and TRAIL, similarly to GVHD macrophages . The cytokine milieu of an HLA-matched MLR was similar to that observed when GVHD skin was cultured , and a prophages . Many ofonocytes .The expression of cytotoxic molecules prompted us to test the possibility that MLR-activated macrophages might mediate cytotoxicity to epidermal cells. We observed that MLR-activated macrophages were directly cytotoxic to a keratinocyte cell line in vitro in a dose-dependent manner . In ordeIn this study, we have defined the role of myeloid cells in human cutaneous acute GVHD by characterizing mononuclear infiltrates from primary tissue, isolating the dominant myeloid cell, and defining its origin, transcriptional profile, and functional properties. The data indicate that human GVHD lesions are highly infiltrated with donor monocyte–derived macrophages with enhanced allostimulatory activity and the potential to mediate epidermal pathology.Myeloid cells found in GVHD have previously been characterized as macrophages based on histopathology describing a small number of surface antigens. These studies lack further details of the biological characteristics or potential pathogenic role of macrophages in GVHD , 20–22. Numerically, macrophages show the greatest fold increase in GVHD of any mononuclear cell and constitute the most consistent “cellular signature” of acute GVHD relative to those of recipients without GVHD or healthy control skin. The macrophage/DC ratio is sensitive and specific relative to BMT control skin without GVHD, increasing more than 100-fold in the presence of GVHD. Previous studies of human GVHD have placed emphasis upon the potential existence of a pathognomonic subset of T cells in GVHD, although none has been consistently identified –8. Unlik+CD14+ cells based on the most direct means of distinguishing the infiltrate from autofluorescent CD11c– resident macrophages by flow cytometry. Several lines of evidence point to a monocyte origin; most notably, they are donor derived and therefore unlikely to arise by proliferation of resident-recipient macrophages. Additional staining demonstrated high expression of monocyte antigens S100A8/A9 and SIRPA (CD172), consistent with recent emigration of monocytes from the blood . Biopsies were performed at the onset of patient rashes clinically compatible with GVHD. An independent clinical pathologist provided diagnosis and histological grading of GVHD. BMT recipient controls with no evidence of any rash were biopsied when seen for routine assessments at day 28 or day 100 after transplant. Biopsies were transported in serum-free medium  and analyzed within 24 hours. An independent clinical pathologist provided diagnosis and histological grading of GVHD in controls and GVHD biopsies. Healthy control skin was obtained from patients undergoing mammoplasty or abdominoplasty, as previously described , and 10% heat-inactivated FBS (Sera Lab). MLR macrophages were generated from coculture of PBMCs from HLA-matched BMT donor and recipient pairs. Recipient PBMCs were irradiated (20 Gy) and used as stimulators for donor PBMCs at a 1:1 ratio. Cultures containing 1 to 5 × 107 donor PBMCs were maintained in RPMI 1640 with 10% human AB serum (MilliporeSigma) for 7 days. HaCaT cells were obtained from Accegen.Unless stated otherwise, all cells were cultured in RPMI 1640 (Lonza) with 100 IU/mL penicillin, 10 μg/mL streptomycin, 2 mM Skin shave biopsies were used whole or split into dermis and epidermis by treatment with dispase 0.5–1.0 mg/mL in RPMI for 60 to 90 minutes at 37°C  before digestion in medium with 1.6 mg/mL type IV collagenase (Worthington) for 12 to 16 hours at 37°C in RPMI with 10% heat-inactivated FCS. Gentle dissociation and passage through a 100 μm filter generated single-cell suspensions.For some experiments, an enzyme-free preparation of leukocytes was required. Skin was cultured as above, but without collagenase. After 48 hours, migratory leukocytes were harvested from the supernatant. Supernatants were stored at –80°C and later used for cytokine analysis by Luminex assay (see below)Formalin-fixed and paraffin-embedded skin shave biopsies from GVHD diagnostic material were used. Sections of 4 μm were made. Antigen retrieval and staining were performed using the BenchMark autostainer (Ventana). CD3, CD163, CD11c, factor XIII, and Ki67 primary antibodies and the ultraView Detection Kit were used (Roche).Antibodies used for microscopy are listed in Antibodies used for flow cytometry are listed in Sorted cells were spun onto slides, fixed in methanol and acetic acid, and prepared with dual-labeled XY FISH probes on a ThermoBrite System (Abbott) in accordance with the manufacturer’s instructions.ASIP, C19orf59, CCL17, CD1C, CD207, CLEC10A, CLEC9A, CLNK, COBLL1, CXCL5, DBN1, F13A1, FGD6, FLT3, GCSAM, GGT5, MKI67, LPAR2, LYVE1, MAFF, MERTK, NDRG2, PACSIN1, PPM1N, PRAM1, S100A12, SIRPA, TMEM14A, UPK3A and ZBTB46). From 3000 to 10,000 sorted cells were pelleted and lysed in 5 μl RLT buffer (QIAGEN) plus 1% β-mercaptoethanol, yielding 50 to 150 ng total RNA for analysis. Hybridization was performed according to the manufacturer’s instructions using a NanoString Prep Station and Digital Analyzer. nSolver Analysis software,version 3.0, was used for background correction and normalization.Gene expression was quantified by NanoString, using the Human Immunology, version 2, panel with a custom 30-gene add-on targeting monocytes, macrophages, and DC genes  and labeled with 1 μM CSFE (Invitrogen). T cells were cocultured with sorted macrophages or DCs at a 25:1 ratio for 7 days, incubated with antibodies to CD3, CD4, CD8, HLA-DR, and CD69 , and anaE. coli (MilliporeSigma) and harvested at 10 hours. Supernatants were cryopreserved at –80°C and batch analyzed by Luminex assay  using QIAGEN LiquiChip 200 running Luminex 100 integrated system software, version 2.3. Procartaplex Analyst, version 1.0, was used to define standard curves.Quantification was performed on medium from isolated macrophage populations stimulated ex vivo, and medium was conditioned by explanted GVHD skin and by donor-recipient MLR. For macrophage stimulation, FACS-sorted skin macrophages were stimulated with 100 ng/mL LPS from 7 donor PBMCs were maintained in RPMI with 10% human AB serum (MilliporeSigma) for 7 days. Macrophages and T cells were sorted from MLR outputs as HLA-DR+CD14+CD11c+ cells and SSCloCD3+ cells, respectively. Shave biopsies of recipient skin were taken before BMT conditioning. Biopsies were divided and incubated with medium alone (negative control), 2 × 105 MLR lymphocytes, or 2 × 105 MLR macrophages for 3 days in RPMI 1640 with 20% heat-inactivated autologous serum. Explants were fixed in 10% buffered formalin, sectioned, and stained with H&E. Severity of histological damage was graded using the Lerner scale by an experienced assessor blinded to experimental conditions.Blood from BMT donor and recipient pairs was prospectively collected into EDTA before transplantation. Recipient PBMCs were irradiated (20 Gy) and used as stimulators for donor PBMCs at a 1:1 ratio. Cultures containing 1 to 5 × 10t tests were performed in MultiExperiment Viewer software, version 4.8 ; or Newcastle Biobank, Newcastle and North Tyneside Research Ethics Committee 1 (reference 17/NE/0361).LJ, MC, and AJS conceived the project. LJ, MC, VB, and SP devised methodology. LJ, UC, MG, KG, GR, SP, MP, CAL, AKL, and VB performed experiments. LJ and XNW performed formal analysis. MC and LJ wrote the original draft of the manuscript. MC and LJ revised and edited the manuscript. LJ, MC, and AJS acquired funding. MC, EH, AP, SN, GHJ, VB, GR, and MH provided resources. MC and AJS supervised the project.


Background: Zeta-Chain Associated Protein Kinase 70 kDa (ZAP-70) deficiency is a rare combined immunodeficiency (CID) caused by recessive homozygous/compound heterozygous loss-of-function mutations in the ZAP70 gene. Patients with ZAP-70 deficiency present with a variety of clinical manifestations, particularly recurrent respiratory infections and cutaneous involvements. Therefore, a systematic review of ZAP-70 deficiency is helpful to achieve a comprehensive view of this disease.Methods: We searched PubMed, Web of Science, and Scopus databases for all reported ZAP-70 deficient patients and screened against the described eligibility criteria. A total of 49 ZAP-70 deficient patients were identified from 33 articles. For all patients, demographic, clinical, immunologic, and molecular data were collected.Results: ZAP-70 deficient patients have been reported in the literature with a broad spectrum of clinical manifestations including recurrent respiratory infections (81.8%), cutaneous involvement (57.9%), lymphoproliferation (32.4%), autoimmunity (19.4%), enteropathy (18.4%), and increased risk of malignancies (8.1%). The predominant immunologic phenotype was low CD8+ T cell counts (97.9%). Immunologic profiling showed defective antibody production (57%) and decreased lymphocyte responses to mitogenic stimuli such as phytohemagglutinin (PHA) (95%). Mutations of the ZAP70 gene were located throughout the gene, and there was no mutational hotspot. However, most of the mutations were located in the kinase domain. Hematopoietic stem cell transplantation (HSCT) was applied as the major curative treatment in 25 (51%) of the patients, 18 patients survived transplantation, while two patients died and three required a second transplant in order to achieve full remission.Conclusion: Newborns with consanguineous parents, positive family history of CID, and low CD8+ T cell counts should be considered for ZAP-70 deficiency screening, since early diagnosis and treatment with HSCT can lead to a more favorable outcome. Based on the current evidence, there is no genotype-phenotype correlation in ZAP-70 deficient patients. ZAP70 gene consists of the kinase domain, Src homology 2 (SH2)-kinase linker, inter-SH2 linker, and two SH2 domains , presenting with recurrent infections, slightly milder than those with recessive forms of severe CID (SCID)  infection are common presenting illnesses. Occasionally, there are SCID patients who do not present with failure-to-thrive and thus, are not recognized to have immunodeficiency until late in the first year of life. SCID is fatal in the first 2 years of life unless the patient is treated with extremely restrictive isolation, hematopoietic stem cell transplant or therapy that replaces the abnormal gene or gene product.Protein-tyrosine kinases (PTKs) are known to have an integral role in T cell activation. Activation of T-cell antigen receptor (TCR) leads to tyrosine phosphorylation of a number of cellular proteins including Zeta(ζ)-Chain Associated Protein Kinase 70 kDa (ZAP-70), a member of the Syk family (non-receptor protein tyrosine kinase family), that co-precipitates with zeta upon TCR stimulation . The ZAP domains . ActivatZAP70 were identified initially in patients of Mennonite descent and subsequently in other ethnicities, including Hispanics, Japanese, Kurdish, Turkish, Portuguese, Caucasian, Mexican, Malagasy, and Iranian patients  pneumonitis are less common  G, while IgM and IgA levels are often normal ; the full-text version of all “unclear” articles was checked and subsequently classified in one of the two categories (include or exclude). All full-text manuscripts were assessed for eligibility criteria: written in English, conducted on human subjects, reporting at least one patient with ZAP-70 deficiency diagnosis, and detailed description of epidemiological, clinical, and immunological features associated with genetic mutations. Studies using animal models, reviews, congress abstracts, and articles in languages other than English were excluded. When necessary, the corresponding authors were contacted.In the first step, two researchers extracted the data from the studies included. The following data were collected from each article: publication year, the number of participants, and demographic, clinical, laboratory, and molecular data of the patients. The evaluation of immunologic data quality was based on the age-matched normal ranges and the normal ranges included in each article. Those patients appearing in more than one publication were identified and the duplicate data was removed. Two reviewers performed the selection process independently, while the third reviewer was consulted to resolve disagreements between two reviewers.Central and descriptive statistics were reported for quantitative data. For variables with abnormal distribution, median and interquartile ranges (IQR) were reported. Kaplan–Meier curve and log-rank test were utilized to compare different survival estimates. All statistical analyses were performed using the SPSS software .The literature search identified a total of 3,975 articles. Two thousand, five hundred forty-six articles were duplicated and 1,333 articles were excluded following title and abstract screening. Furthermore, eleven articles were excluded as they only reported general information regarding ZAP-70 deficient patients lacking specific details. As shown in In this systematic review, we included 49 ZAP-70 deficient patients  . Most ofZAP70 gene is located on chromosome position 2q11.2  “classical” group of 36 patients with amorphic mutations that abolish protein expression and/or function, 2) “leaky” group consisting of 5 patients with hypomorphic mutations that result in residual ZAP70 protein expression  reported, most patients had a SCID clinical diagnosis . n = 16, 48.5%) followed by dermatitis . Cutaneous manifestations were found to be the earliest and lymphoproliferative disorder the latest clinical presentation, with median (IQR) age of 3.5 (2.0–11.3) and 11 (5–16.0) months, respectively.Among patients with initial clinical diagnosis , diarrhea , and upper respiratory tract infections [sinusitis  and other ENT  infections ], including otitis media, bilateral otitis, and acute nasopharyngitis, were the most common infectious manifestations  , bacteria  (mainly BCG [18.4%]), fungal pathogens  [predominantly Candida albicans (28.9%)], and protozoa  . One patient developed lung tuberculosis at the age of 48 months  dermatitis  included types of silent brain infarct (2 patients), VZV encephalitis (1 patient), viral cerebellitis (1 patient), and facial paralysis (1 patient). Renal involvement (11.1%) in ZAP70 deficiency consisted of nephrotic syndrome (2 patients), one case of proteinuria with lack of progression to nephrotic syndrome, and IgA nephropathy (1 patient). Two patients (5.6%) were found to have cardiovascular problems, which manifested as persistent hypertension and atrioventricular block  patients. Hematologic disorders, consisting of non-immune hemolytic anemia, antibody to clotting factor VIII, hemophagocytic syndrome, immune thrombocytopenic purpura (ITP), autoimmune hemolytic anemia (AIHA), and bicytopenia, were reported in 6 (16.7%) patients. Neurologic involvements  was mentioned in different categories as autoimmune cytopenia (n = 2), autoimmune nephritis (n = 2), autoimmune enteropathy (n = 1), dermal involvements in the form of bullous pemphigoid (n = 1), and adrenal insufficiency (n = 1). Polyautoimmunity was present in 2 siblings, one with bullous pemphigoid, nephrotic syndrome, antibody to clotting factor VIII, and inflammatory bowel disease (P40) and a second patient with bullous pemphigoid and inflammatory bowel disease (P41). Both patients with polyautoimmunity had compound heterozygous mutations: R192W (LOF) and R360P (GOF), producing a weakly hyperactive ZAP70 protein (Autoimmunity ( protein .Three patients (8.1%) developed malignant diseases (Enteropathy (18.4%) and failure to thrive (43.6%) were other reported manifestations. Among patients with lymphoproliferative diseases, lymphadenopathy was the most frequent manifestation (32.4%), followed by hepatomegaly (19.4%), and splenomegaly (13.9%). Enlarged tonsils were also reported in 3 cases. diseases  includin+ cell counts were the most common profile (97.9%), followed by low numbers of NK cells (23.8%) and CD19+ B cells (11.1%). CD4+ T cell deficiency was reported in 12.2% of the ZAP-70 deficient patients. However, one patient was reported to have elevated CD8+ level, which deviates from the usual immunologic findings for ZAP-70 deficiency , IgA , and IgM . A decline in antibody response to polysaccharide and peptide vaccine was detected in 5 (55.6%) and 16 (80%) patients, respectively. Reduction in T-cell receptor excision circles (TREC) levels and phytohemagglutinin (PHA) response were observed in 5 (50%) and 38 (95%) of the cases with available data, respectively , peripheral blood , or cord blood  as their stem cell source. One of the patients received bone marrow for his first transplant then peripheral blood stem cells during his second transplant (Twenty-five patients (51%) underwent hematopoietic stem cell transplantation (HSCT), using bone marrow  and infections  were the most commonly reported complications after HSCT. One liver failure (n = 3) or died (n = 2). One patient went through an unsuccessful transplantation (P12) (p < 0.001).The median age of patients receiving a transplant was 10 6.7–17.2) months. Among the 21 cases with donor type mentioned, 13 (61.9%) had a matched sibling donor and 8 (38.1%) had a matched unrelated donor. Graft versus host disease (GVHD)   while an.7–17.2 mThe two deceased patients had received their HSCT at the age of 20 and 30 months old. Three patients at the age of 7, 9, and 18 months needed a second transplant to reach complete engraftment. One patient with an unsuccessful transplantation was 9 months at the age of HSCT. Eight out of twelve patients (66%) with the above mentioned complications post-HSCT were older than 6 months at the time of transplantation.Steroids and intravenous immunoglobulin (IVIG) have been tried as a prophylactic or to control adverse side effects of transplantation in 8 (32%) and 18 (36.7%) patients, respectively. Among the patients receiving steroids, five were clinically responsive .+ T cells, most of the patients were initially diagnosed as SCID . In fact, it was first described in 3 Mennonite patients  and sinc%. In fac as SCID , 17. Int as SCID .+ T cell (97.9%), normal CD4+ T cell, normal B cell, and normal NK cell counts. However, immunoglobulin levels varied individually with the majority being within the normal range in most cases. A notable decline in PHA response (95%), specific antibody production , and TRECs (50%) were detected in patients with ZAP-70 deficiency, which was in line with previous studies  presented with malignancy, cardiovascular and neurologic involvement in comparison to alive non-HSCT patients . There were no immunological differences between the above groups. Nonetheless, further experimental studies are recommended for the development of new therapeutic strategies such as gene therapy for patients with ZAP-70 deficiency.Allogenic HSCT seems to be the only curative therapy available for patients with ZAP-70 deficiency. Among the 25 patients who received HSCT, 22 patients (91.7%) ended up alive and well in a median follow up of 36 months; while, the survival rate for patients who did not undergo HSCT was 59.1% in median follow-up of 18 months. According to the Kaplan-Meier survival curve, HSCT significantly changes the patients' prognosis by decreasing the mortality rate. Unfortunately, it is not apparent whether the patients that had undergone HSCT were clinically less severe or the patients with severe disease had died before undergoing HSCT. Furthermore, some of the patients with “early deaths” may have been “transplant candidates” but judged to be “too sick” by their physician to undergo HSCT. We found that more patients who did not undergo HSCT and had died  with clinical complications post-HSCT were older than 6 months at the time of transplantation. It appears that younger patients undergoing HSCT, experience better outcomes and fewer complications; therefore, early screening and HSCT could lower the burden of the disease . Howeverhttps://www.ncbi.nlm.nih.gov/pubmed/.Publicly available datasets were analyzed in this study. This data can be found here: NS, MJ, MZ-D, MS, HM, FJ-N, SS, RY, HA, AA, and GA: substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data. BL, NS, MJ, MZ-D, MS, HM, FJ-N, SS, RY, HA, AA, and GA: drafted the article or reviewed it critically for important intellectual content and given final approval of the version.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.


Schizothorax griseus was sequenced. The mitogenome is 16,586 bp in length, composed of 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and two non-coding regions. The phylogenetic relationship constructed by Bayesian inference (BI) method showed that schizothorax is not a monophyly, species in the same river express a more close relationship. The results can contribute to explore the high level of diversification and the taxonomic conflict caused by morphologic characters of schizothorax, also, it will be an evidence for the biogeography and evolutionary mechanisms of Schizothoracinae fishes.In this study, the complete mitochondrial genome (mtDNA) of  Schizothorax griseus is distributed in upstreams of Wujiang river, Nanpanjiang river and Beipanjiang river , containing 13 protein-coding genes , two ribosomal RNA genes (12srRNA and 16srRNA), 22 transfer RNA (tRNA) genes, two non-coding regions: the control region (D-loop) and the origin of light-strand (rep-region: OL). Except ND6 and eight tRNA genes , all these genes were encoded on the H-strand. The overall nucleotide base composition base was: 25.4% T; 27.1% C; 17.8% G and 29.7% A, showing a weak anti-G bias which was nearly the same in 13 protein-coding genes (17.7% G). All protein-coding genes shared typical start codon ATG except for COI beginning with GTG, only six protein-coding genes terminated with the complete stop codon, the remaining had the incomplete stop codon. There were six regions of gene overlap totalling 21 bp (from 1 to 7 bp) and 10 intergenic spacer regions totalling 30bp (from 1 to 12 bp). The D-Loop, with 937 bp in length, was located between tRNAPho and tRNAPhe, and the base composition reflected a higher A + T-rich (66.9%) than the overall average mtDNA (55.1%).After assembling and alignment, the complete mitogenome of S. griseus and 25 complete mitogenome sequences downloaded from GenBank, Percocypris pingi, Ptychobarbus dipogon, and Aspiorhynchus laticeps were chosen as outgroup  has a more close relationship with species in genus Schizothorax and may contribute to origin of Schizothoracinae fishes for that we usually accepted Schizothoracinae was derived from Barbinae  method to construct phylogenetic tree between outgroup . The res