Arsenic, an environmental pollutant, decreases neuronal migration as well as cellular maturation and inhibits the proliferation of neural progenitor cells. Curcumin has been described as an antioxidant and neuroprotective agent with strong therapeutic potential in some neurological disorders. Human adipose-derived stem cells (hADSCs), a source of multipotent stem cells, can self-renew and differentiate into neural cells. The aim of the present study was to investigate the preventive effect of curcumin against arsenic toxic effects on the viability, telomerase activity, and apoptosis of neural stem/progenitor cells (NSPCs) derived from hADSCs.  The characteristics of human adipose tissue were identified by immunocytochemistry for surface markers namely, CD105, CD73, and CD90. Using neurosphere assay, hADSCs were differentiated into neuronal cells. To characterize neural cells, expression of nestin, SOX2, MAP2, and GFAP were assessed by immunocytochemistry. Cytotoxicity and viability of NSPCs were evaluated by MTT assay. Reactive oxygen species (ROS) generated by arsenic exposure, were measured and caspase 3/7 activity and caspase-3 processing as well as the telomerase activity were determined.The isolated hADSCs positively expressed CD105, CD73, and CD90. Nestin, Sox2, GFAP, and MAP2 were expressed in the neurospheres derived from hADSCs. Curcumin/arsenic co-treatment significantly increased telomerase activity of NSPCs compared to arsenic group. Furthermore, curcumin significantly reduced arsenic-induced apoptosis (via inactivation of caspases) as well as arsenic-associated ROS generation.Our findings revealed that curcumin has the potential to prevent harmful effects of arsenic on neurogenesis. Arsenic, an environmental pollutant, exists in soil and bedrock and may leach into ground- and surface-water , has anti-inflammatory, antioxidant, anti-proliferative, anti-carcinogenic, and anti-tumor properties in different types of cell lines and animals and is widely used for metal detoxification . It has been suggested that prevention of oxidative stress and induction of repair enzymes by curcumin, may be effective therapeutic strategies to reduce the adverse effects of arsenic  supplemented with fetal bovine serum 20 %  and seeded in T75 cm2 flasks. Adherent cells were observed after 24 hr.For isolation of mesenchymal stem cells, the obtained adipose tissue was washed by phosphate buffered saline (PBS). Adipose tissue was finely minced with the help of a scalpel, digested using collagenase 0.075 % (Sigma- Aldrich) and incubated at 37Immunophenotyping4 cells were seeded in a 12-well plate. After 24 hr, when cells were attached to the plate, one step of washing with PBS was performed and paraformaldehyde 4% (Sigma- Aldrich) was used for 15 min at room temperature to fix the cells. The fixed cells were exposed to blocking buffer that contained FBS, for 5 min at 4oC to increase the specificity of the primary antibody. Cells were then incubated overnight at 4oC with anti-mouse primary antibodies. Fluoresce in Isothiocyante was used as a secondary antibody (Millipore). For 2 h at 4oC, cells were observed by inverted fluorescence microscope . For detection of nuclei, propidium iodide 100 µg/ml (Sigma- Aldrich) was used.Immunocytochemistry was performed for surface markers CD105, CD73, CD90, CD56, and CD45 (Primary antibodies were purchased from Abcam) to identify the nature of the cells. For this purpose, 5×10Differentiation of hADSCs to neurosphereWhen cells reached 80-90% confluence, they were isolated through trypsinization and centrifugation. Cells were then cultured in T75 flasks using Dulbecco's modified essentialmedium with Ham's F-12  supplemented with basic fibroblast growth factor 20 ng/ml , B27 3% (Invitrogen), N2 1%, (Invitrogen) and epidermal growth factor 20 ng/ml . After 4-6 days neurospheres were formed  oxide, Sigma-Aldrich, Germany) and curcumin . According to the manufacturer instruction, curcumin was dissolved in DMSO to make a 15 mg/ml stock and stored at -20 °C. Then, an aliquot of the stock was further diluted in enriched DMEM to obtain the desired concentration. We first examined the cell viability at different concentrations of arsenic (III) oxide  after 4, 12, 18, 24, 36 and 48 h exposure to arsenic. The same procedure was performed for the optimal concentration of curcumin at 1, 2, 5, 10, 20, and 50 µM.ROS generation5 cells per ml was plated in 6-well plates and treated with curcumin 10 μM at the time of medium change. Then, Abcam's Cellular Reactive Oxygen Species Detection Assay Kit  was used according to manufacturer’s instruction to quantitatively assess ROS production in control, arsenic-treated, curcumin-treated and arsenic-curcumin co-treated cells after 4, 8, and 12 hr.A number of 3×10Caspase 3/7 assay2 , 10 mMKCl, 1 mM Na-EDTA, 68 mM sucrose, and 1 mM PMSF). Subsequently, the cells were located on ice for 15 min, and then, vortexed for 30–45 sec. The cell debris was pelleted by centrifugation at 4°C, 14,600 g for 30 sec. For luciferase assay, the supernatant of cell lysate was used for luciferase assay. The activity was immediately recorded upon addition of 20 ml of luciferase assay complex   and 2 mM ATP  to 20 ml of cell lysate using a luminometer ..To study the occurrence of apoptosis, caspase-Glo® 3/7 luminescent assay was used to measure caspase 3/7 activity. Cell lysate prepared by the cytosolic fractionation method with equal protein concentration was subjected to detect caspase-3/7 activity as described by manufacturer's instruction. Cells were harvested, washed with PBS, centrifuged and re-suspended in 400 ml of hypotonic buffer A , diluted at 1:1000. After incubation, the membrane was washed with PBS 0.05% tween solution three times and incubated for 1 hr at 1:1000 dilutions of horseradish peroxidase–conjugated goat anti-rabbit . Strips were washed three times with PBS 0.05% tween solution and the proteins of interest were detected using LI-COR imager.Immunoblotting was performed to further investigate the cleavage of caspase-3 in cytosol. Cells were lysed in Cell Culture Lysis Reagent buffer  containing 100 mM potassium phosphate (pH 7.8), 1 mM EDTA, 7 mM 2-mercaptoethanol, 1% (v/v) Triton X-100, and 10% (v/v) glycerol. Then, 100 μl of CCLR buffer was added to the collected cells and the mixture was stored at -80°C. To determine protein concentration in the cell lysate, standard Bradford method was used. After boiling for 5 min, 8% SDS polyacrylamide gels were prepared, and 50 µl samples were loaded into the wells. Gels were run for 90 min at 100 volt. Proteins were then transferred to a nitrocellulose membrane for 60 min by using a current of 250 mA. The membrane was blocked using blocking buffer for 1 hr at room temperature. Then, the membrane was incubated overnight at 4Detection of telomerase activity Telomerase activity was determined using the telomeric repeat amplification protocol (TRAP) with the TeloTAGGG PCR enzyme-linked immunosorbent assay ELISA kit  according to the manufacturer's instructions.Statistical analysispost-hoc test with ANOVA. Results were considered statistically significant if p<0.05. Where appropriate, the data were also analyzed by one-way ANOVA.Comparisons between groups were made using Tukey's HSD Isolation and characterization of hADSCsTo confirm the mesenchymal nature of the cells, cell surface markers namely, CD73 ،CD90, CD105, CD45, and CD56 were investigated. hADSCs showed high expression of CD105, CD7 and CD90 (>98%) and low expression of CD45 and CD56 (<1.5%). These findings demonstrated the mesenchymal nature of the cells .Generation and differentiation of neurospheres from hADSCs. Neurospheres were stained with GFAP, MAP2, SOX2, and nestin. The neurospheres were positive for MAP2 (mature neuron), GFAP (astrocyte), and both SOX2 and nestin  caspases was performed for caspase-3. Application of arsenic led to cleavage of caspase-3 in NSPCs as indicated by prominent detection of the p17 and p19 fragments. Although caspase-3 cleavage occurred in curcumin and arsenic/curcumin-treated NSPCs, a part of caspases was still in the form of procaspase-3. Procaspase-3 was dominated in arsenic/curcumin treated NSPCs and caspase cleavage fragment was at lower density compared to that of arsenic-treated tissue . Quantif; Lim et al., 2010Keeping in line with previous studies, our results indicated that neural stem/progenitor cells, astrocytes, and mature neurons could be obtained from the neurospheres derived from hADSCs (Kwon et al., 2011 ▶In line with our findings, it has been shown that curcumin alleviates arsenic-induced cell damage in skin stem cell (Poojan et al., 2015Arsenic induces ROS formation via activation of Nox2, a specific NADPH oxidase. This downregulates nitric oxidase synthase and leads to the accumulation of oxidants (Ellinsworth, 2015High levels of arsenic have been reported to be associated with aging process in which telomere length and telomerase activity decrease (Zhan et al., 2003Curcumin effect on increasing telomerase activity might be mediated via improving the stability of telomerase structure and in turn increasing the half-life of the enzyme (Xiao et al., 2014In conclusion, curcumin can be considered as a potential treatment to attenuate the harmful effect of ROS generation in arsenic-exposed neuronal stem cells. Furthermore, curcumin prevents the occurrence of apoptosis in neuronal stem cells via reduction of executioner caspase-3 activities and enhancement of telomerase activity . This su


Granular cell tumors (GCTs) are rare and benign tumors that can occur at any anatomical site. GCTs are thought to originate from nerve cells, particularly Schwann cells. Their name derives from the fact that an accumulation of cytoplasmic lysosomes imparts the tumor with a granular appearance. They are most commonly observed in the oral cavity, skin and subcutaneous tissue, breast, and respiratory tract. Granular cell tumors rarely affect the gastrointestinal tract. We report a 58-year-old female with a past medical history of hypertension, mitral valve prolapse, and depression who presented for surveillance colonoscopy. A single firm sessile polypoid lesion, with overlying pale tan color mucosa, measuring approximately 1 to 1.5 cm, was found in the ascending colon. Biopsy of the nodule followed by histopathology was positive for S100 and CD68, but negative for AE1/AE3, CD117, smooth muscle actin, and desmin, consistent with the diagnosis of GCT. We review the clinicopathologic features of GCTs. Granular cell tumors (GCTs) are rare and benign tumors that can occur along any anatomical site. GCTs are thought to originate from nerve cells, particularly Schwann cells . Their nA 58-year-old African-American female with a past medical history of hypertension, mitral valve prolapse, and depression presented for surveillance colonoscopy. Vital signs showed the heart rate at 72 bpm, temperature 98.3°F, respiratory rate 14 bpm, and blood pressure 134/73 mmHg. Her physical exam was unremarkable, and she denied alcohol consumption, smoking, or drug use. Laboratory findings showed hemoglobin 11.9 g/dL, hematocrit 38%, white blood count 4.6 x 103 K/µL, and platelet 136 x 103 K/µL. Diagnostic colonoscopy revealed a single firm sessile polypoid lesion with overlying pale tan color mucosa, measuring approximately 1 to 1.5 cm in the ascending colon , also known as granular cell myoblastoma or Abrikossof's tumor, is a benign proliferation of Schwan cells of soft tissues . It was Gastrointestinal GCTs are typically solitary and represent an incidental finding on screening colonoscopy. Additionally, these polyps are painless, nonulcerated, and firm. Although most case reports highlight that GCTs are benign neoplasms, some cases of malignancy have been reported in the literature, representing less than 2% of all GCTs -6. AdditThe diagnosis of GCT is confirmed by histopathology and includes (1) plump histiocyte-like, bland-looking neoplastic cells with abundant granular eosinophilic cytoplasms containing acidophilic, periodic acid Schiff (PAS) stain-positive, diastase-resistant granules, (2) small, uniform nuclei in which mitotic figures are absent, and (3) neural markers, including S-100 protein or neuron-specific enolase (NSE), expressed uniformly -9. In thMalignancy has been found to correlate with tumor size. Approximately more than 60% of metastatic GCTs were larger than 4 cm in diameter . HoweverWe report a case of GCT located in the ascending colon. The tumor was removed by endoscopic resection, and the patient was discharged without any complications. Clinicians should bear in mind the differential diagnosis of GCT when presented with submucosal colonic polyps. Additionally, endoscopists should be aware of the pathological characteristics associated with malignant GCTs in order to provide early diagnosis and treatment.


Interferon regulatory factors (IRFs) can play a critical role in the regulation of many facets of innate and adaptive immune responses through transcriptional activation of type I interferons, other proinflammatory cytokines, and chemokines. However, their roles in transplantation immunity still remain to be elucidated. To evaluate the time course of mRNA expression of all 9 members of IRFs family of transcription factors during liver allograft acute rejection. Blood samples of 19 patients with autoimmune hepatitis receiving liver transplants were collected on days 1, 3, 5, and 7 post-transplantation. The patients were followed for 6 months after transplantation and divided into two groups of acute rejection (AR) (n=4) and non-acute rejection (non-AR) (n=15). vs. 1.91±0.27, respectively); expression of other IRFs family members was not significantly different between the two groups on days 3, 5, and 7 post-transplantation.All of the studied transcription factors were down-regulated in AR-group on days 3, 5, and 7 post-transplantation compared to non-AR group. The mean±SEM IRF5 on day 7 post-transplantation was significantly (p=0.005) lower in AR-group than in non-AR group (0.7±0.21 IRF5 may have an important role during the acute rejection of liver transplants. Autoimmune hepatitis (AIH) is a chronic inflammatory disease with unknown etiology. It has a mean incidence of 1.9 cases per 100,000 people per year and a prevalence of 16.9 cases per 100,000 people . In spitWhereas T cell responses are both necessary and sufficient for acute allograft rejection, most of researchers have been focused on adaptive immune responses in transplantation immunology . During Involvement of the innate immunity in AR is arising from the important role of Toll-like receptors (TLRs) as the first responders to danger signals . In receSignaling through TLRs can be categorized into two pathways: the MyD88 (myeloid differentiation primary-response protein 88) dependent pathway and TRIF (TIR domain containing adaptor inducing IFN-β) dependent pathway . Both ofIRFs family of transcription factors consists of nine members in humans: IRF1, IRF2, IRF3, IRF4 , IRF5, IRF6, IRF7, IRF8 , and IRF9  . Each IRet al, demonstrated that single nucleotide polymorphism in promoter region of IRF5 encoding gene, is correlated with acute rejection of liver allograft [Several gene expression profiling studies have so far reported the role of IRFs  in acute allograft rejection -20. Alsollograft . In addillograft , 24-26. This prospective study tried to clarify the role of IRFs family of transcription factors, in acute rejection after liver transplantation in AIH patients. We evaluated the time course of mRNA expression levels of IRFs in peripheral blood mononuclear cells (PBMCs) of patients with AIH who had acute and non-acute rejection of liver transplants.From May 2012 to March 2014, 20 Iranian adult female patients, who satisfied the international criteria for AIH , and recThe study protocol was approved by the Ethics Committee of both Shiraz University and Shiraz University of Medical Sciences, based on the study protocol conformed to the ethical guidelines of the 1975 declaration of Helsinki. Immunosuppressant RegimenAll recipients received the routine immunosuppression regimen consisted of tacrolimus or cyclosporine (CsA) with mycophenolate mofetil and steroids. Briefly, the drug dosage was adjusted to maintain target therapeutic blood levels of 200 ng/mL for CsA (5 mg/kg/day), or 10 ng/mL for tacrolimus. The patients with AR episodes received high dose (500 mg) methylprednisolone for three consecutive days.RNA Extraction and cDNA Synthesisst strand cDNA Synthesis Kit , according to manufacturer’s protocol.Total RNA was isolated from 1.5 mL fresh whole blood of recipients, immediately after sample collection, using QIAamp RNA blood mini kit  according to the manufacturer’s instructions. The purity and integrity of RNA was evaluated through measuring the optical density 260/280 ratio by spectrophotometric analysis. Then 500 ng of RNA samples with ratio above the 1.8 were used for cDNA synthesis by PrimeScript 1Quantitative Real-time PCRver 7.8 . Genomic DNA amplification was not appeared in our qPCR reactions, containing not reverse transcribed RNA as template. Real-time PCR primer sequences and conditions are presented in The mRNA expression levels of IRFs were determined by StepOnePlus real-time PCR instrument , using SYBR Premix Ex Taq II kit . The gene-specific primer sets were designed to span introns or cross exon/exon junctions, using AlleleID software Statistical Analysisver 16 . Data are presented as mean±standard error of the mean (SEM). A p value <0.05 was considered statistically significant.The statistical significance of differences in the measured gene expression levels between AR and non-AR groups was evaluated by Mann-Whitney U test, using SPSS Among 19 patients enrolled in this study, four with a mean age of 35 (range: 24–51) years experienced one AR episode during the six months of OLT; 15 patients with a mean age of 34.8 (range: 14–46) years did not experience AR during the study period. All AR episodes occurred within the first month post-transplantation. There were no significant difference in age, BMI, MELD score, total cold ischemic time, warm ischemic time, donor age, and sex, between AR and non-AR groups . No signvs. Non-AR groupExpression of IRFs in AR vs. 1.91±0.27, respectively). Expression levels of other IRFs family members were not significantly different between the two groups on days 3, 5, and 7 post-transplantation  lower on day 7 post-transplantation in AR-group than in non-AR group , as well as other types of liver autoimmune diseases were excluded. Therefore, it might be assumed that the comparison of IRFs expression levels between AR and non-AR groups, with the same underlying disease, represented the functional impact of these transcription factors in AR after OLT.Our results demonstrated that the mRNA expression of all nine members of this family decreased in AR compared to non-AR group on days 3, 5, and 7 post-transplantation, but only the down-regulation of IRF5 on day 7 post-transplantation was significant. It is well established that danger signals, released from donor organ as a result of ischemia-reperfusion injury, tissue damages, and hepatic phase of the transplant procedure, can activate Toll-like receptors, especially TLR4 , 30-32. et al [et al, showed that activation of TLR4 invokes nuclear translocation of IRF5 [Testro, et al , reporteet al , 36. Sevet al . A previ of IRF5 . Unlike  of IRF5 . AlthougIn conclusion, despite of our small study sample size and limitation in pre-transplantation sample collection, this study, for the first time, tried to represent evidence for the role of IRF5 in liver transplant AR. Further studies are needed to elucidate the functional impact of this transcription factor on liver transplant AR.


Pheophytinase comprises a catalytic-triad of residues and shows limited but physiologically relevant substrate specificity towards magnesium-free chlorophyll derivatives due to its lid domain. in vivo; however, this was recently refuted. Instead, pheophytinase was discovered as a genuine in vivo phytol hydrolase. While chlorophyllase acts rather unspecifically towards different porphyrin substrates, pheophytinase was shown to specifically dephytylate pheophytin, namely Mg-free chlorophyll. The aim of this work was to elucidate in detail the biochemical and structural properties of pheophytinase. By testing different porphyrin substrates with recombinant pheophytinase from Arabidopsis thaliana we show that pheophytinase has high specificity for the acid moiety of the ester bond, namely the porphyrin ring, while the nature of the alcohol, namely the phytol chain in pheophytin, is irrelevant. In silico modelling of the 3-dimensional structure of pheophytinase and subsequent analysis of site-directed pheophytinase mutant forms allowed the identification of the serine, histidine, and aspartic acid residues that compose the catalytic triad, a classical feature of serine-type hydrolases to which both pheophytinase and chlorophyllase belong. Based on substantial structural differences in the models of Arabidopsis pheophytinase and chlorophyllase 1, we discuss potential differences in the catalytic properties of these two phytol hydrolases.During leaf senescence and fruit ripening, chlorophyll is degraded in a multistep pathway into linear tetrapyrroles called phyllobilins. A key feature of chlorophyll breakdown is the removal of the hydrophobic phytol chain that renders phyllobilins water soluble, an important prerequisite for their ultimate storage in the vacuole of senescent cells. Chlorophyllases had been considered for more than a century to catalyze dephytylation  Many attempts have been undertaken in the past to identify the underlying mechanism of Mg-dechelation  and it has been shown that absence of PPH in leaves results in a stay-green phenotype (a/b binding complexes in thylakoid membranes and loss of the phytol chain from pheophytin a is considered a prerequisite for the release of pheophorbide a from the membrane for further degradation by downstream enzymes (primary fluorescent chlorophyll catabolite (pFCC), the first colorless and non-photodynamic degradation product of chlorophyll. After subsequent site-specific hydroxylation by TIC55 , lacking the predicted transit peptide of 46 amino acids encoded by the first 138 bases (JXB online). The vector pMCSG29  was performed as published (-1 BSA (New England Biolabs) and 5 mM dithiothreitol. dTTP was added to a concentration of 1 mM to the PCR reaction and dATP at 1 mM to the vector reaction. Reactions were incubated for 90 min at 22°C and stopped by incubation for 20 min at 75°C. Subsequently, T4-treated vector and PCR product were combined and incubated for 40 min at room temperature. NEB 10-beta competent E. coli (High Efficiency) cells (New England Biolabs) were transformed with the construct, named ΔPPH-MBP. After verifying sequence accuracy by sequencing, ΔPPH-MBP was introduced into E. coli BL21 (DE3). Point mutations were introduced by PCR using primers as listed in Supplementary Table S1. All reactions were carried out using PCR Extender Polymerase (5Prime). The S221A mutant version of PPH was produced with Phusion polymerase (New England Biolabs) from a plasmid expressing MBP-ΔPPHS221A (E. coli DH5α. Point mutations were verified by sequencing and subsequently the constructs were transferred into E. coli BL21 (DE3) (Thermo Fisher Scientific) for protein expression.A truncated E. coli BL21 (DE3) harboring the different pMCSG29 constructs were grown at 37°C in Luria Broth (LB) medium supplemented with 2 g L-1 glucose. With an optical density at 600 nm of 0.5–0.6 protein expression was induced by adding 1 mM isopropyl β-D-1-thigalactopyranoside and cultures were grown overnight at 20°C. Cells were collected by centrifugation and resuspended in 20 mM Tris-HCl at pH 8 and 200 mM NaCl. Disruption of the bacterial cells was performed by using a French Press  at 150 MPa. Soluble protein fractions were separated by centrifugation, supplemented with 10% glycerin [v/v] and kept at -80°C until further use. As a control, cells containing the empty vector pMCSG29 were used. Presence of expressed proteins was analyzed by SDS-PAGE on 4–15% Mini-PROTEAN TGX gels (BioRad) using Coomassie blue staining or by immunoblot analysis. For the latter, membranes were hybridized with either anti-MBP antibodies (New England Biolabs) or custom-made anti-PPH antibodies, which were raised in rabbits against a keyhole limet hemocyanin-conjugated Arabidopsis PPH peptide, 127-VGSFHYEKQLTDLGRD-142 (AgriSera).For recombinant protein expression, a was produced from chlorophyll a (Livchem Logistics) as previously described  and bacteriochlorophyll a (Livchem Logistics), respectively, as previously described for pheophytin a (a methyl ester and Zn(II) pheophorbide a methyl ester were gifts from Bernhard Kräutler at the University Innsbruck, Austria; pyropheophorbide a methyl ester and protoporphyrin IX dimethyl ester were from Livchem Logistics. For calculating the KM values for pheophytin a, pheophorbide a methyl ester, bacteriopheophytin a, and pheophytin b different substrate concentrations, as shown in the Figures, were incubated with 150 µg ml-1 PPH for 30 min. The KM values were calculated with Prism (GraphPad).The activity of PPH towards different substrates was determined by incubating 150 µg ml-1 were incubated with 200 µM pheophytin a for 60 min.For testing the mutated PPH variants, expressed proteins at a concentration of 150 µg mlpph-1/clh1-1/clh2-1 triple mutant was produced by crossing a clh1-1/clh2-1 double mutant  .Modeling of PPH without the predicted N-terminal transit peptide (ΔPPH) and CLH1 was performed with the Protein Homology/analogY Recognition Engine V 2.0 . Accession numbers are as follows: Aquilegia coerulea: Acoe 1, 22028438; Acoe 2, 22028440; Acoe 3, 22028441; Arabidopsis lyrata: Alyr, 16059924; Arabidopsis thaliana: Atha (AT5G13800), 19671250; Amborella trichopoda: Atri, 31572443; Brachypodium distachon: Bdis 1, 32791837; Bdis 2, 32791838; Brassica rapa: Brap 1, 30607511; Brap 2, 30617286; Brachypodium stacei: Bsta, 32872976; Boechera stricta: Bstr, 30656110; Citrus clementina: Ccle, 20803863; Capsella grandiflora: Cgra, 28914601; Carica papaya: Cpap, 16412351; Capsella rubella: Crub, 20908496; Cucumis sativus: Csat 1, 16962141; Csat 2, 16962144; Citrus sinensis: Csin 1, 18102612; Csin 2, 18102614; Eucalyptus grandis: Egra 1, 32071411; Egra 2, 32071412; Eutrema salsugineum: Esal, 20205993; Fragaria vesca: Fves, 27256941; Glycine max: Gmax 1, 30485515; Gmax 2, 30548341; Gmax 3, 30535348; Gossypium raimondii: Grai 1, 26771977; Grai 2, 26816354; Kalanchoe marnieriana: Kmar 1, 32566857; Kmar 2, 32566858; Kmar 3, 32576249; Linum usitatissimum: Lusi 1, 23154596; Lusi 2, 23139227; Musa acuminata: Macu, 32295673; Malus domestica: Mdom 1, 22644010; Mdom 2, 22677109; Mdom 3, 22645276; Mimulus guttatus: Mgut 1, 28947539; Mgut 2, 28924057; Medicago truncatula: Mtru, 31060299; Oryza sativa: Osat 1, 33147660; Osat 2, 33147661; Osat 3, 33147662; Panicum hallii: Phal, 32519143; Physcomitrella patens: Ppat 1, 32980877; Ppat 2, 32980880; Ppat 3, 32980879; Prunus persica: Pper, 32074354; Populus trichocarpa: Ptri 1, 26988021; Ptri 2, 26988022; Panicum virgatum: Pvir 1, 30302578; Pvir 2, 30302579; Pvir 3, 30222197; Pvir 4, 30222200; Pvir 5, 30222198; Pvir 6, 30222199; Pvir 7, 30222201; Pvir 8, 30264911; Pvir 9, 30264913; Phaseolus vulgaris: Pvul 1, 27148885; Pvul 2, 27150655; Ricinus communis: Rcom, 16824129; Sorghum bicolor: Sbic, 32744955; Setaria italica: Sita, 32702516; Solanum lycopersicum: Slyc, 27302437; Spirodela polyrhiza: Spol, 31513730; Salix purpurea: Spur 1, 31398042; Spur 2, 31439261; Spur 3, 31439264; Solanum tuberosum: Stub 1, 24421437; Stub 2, 24421436; Setaria viridis: Svir, 32661223; Theobroma cacao: Tcac, 27432758; Vitis vinifera: Vvin, 17817618; Zea mays: Zmay 1, 30984753; Zmay 2, 30984754. Additional sequences for PPH-like proteins of Arabidopsis : At4g36530; At5g19850; At5g38520.Arabidopsis PPH orthologs were identified with BLASTP searching .Multiple sequence alignment of these proteins was generated and phylogenetic analysis  performed with the neighbor-joining method using MEGA7  and the optimal pH optimum to be 8.5 . As already published . This analysis confirmed that chlorophyll is indeed able to inhibit ∆PPH-MBP activity at low pheophytin a concentrations, indicating competition for binding by PPH; but at saturating substrate concentrations, this inhibition was out competed. Due to saturation of substrate and inhibitor solubility, the type of inhibition could not be determined.Arabidopsis PPH consists of 484 amino acids, including a predicted 46 amino acid long chloroplast transit peptide . The folished , which meant a KM value could not be determined. Thus, affinity of PPH for pheophytin b seems significantly lower than that for pheophytin a. Similar results were found for bacteriopheophytin a, which, in comparison to pheophytin a, has a reduced C7/C8 double bond and an acetyl group instead of a vinyl group at C3 of the pyrrole ring A. ∆PPH-MBP was able to form bacteriopheophorbide a  in its active site . Howevera and pheophorbide a formation was analyzed by HPLC  largely reduced activity. This indicates that this ‘southern’ region, which is rather close to the actual site of hydrolysis, is highly important for substrate recognition by the enzyme. Changes to the ‘northern’ part of the porphyrin ring, as in bacteriopheophytin a and pheophytin b, also reduced the activity of PPH, but to less extent, indicating that recognition of this part of the substrate is less specific.The lack of a crystal structure for PPH abolished the possibility to model the natural substrate of PPH, pheophytin finities  and 4. Fa hydrolysis activity of PPH , indicating competition for substrate binding, which can be overcome by increasing pheophytin a concentrations. Unfortunately, we were unable to determine the type of inhibition, but we propose that the two substrates compete for binding at the active site in a competitive manner. If this is true, we must assume that presence of Mg in chlorophyll a abolished the catalytic cycle. A reason for this could lie in the inability of the enzyme to properly close the lid domain, when chlorophyll a is bound, to fully engage the substrate in the active site as a prerequisite for catalytic activity , or a certain spatial orientation of the enzymatic machinery and respective substrates, conditions that are not met in the performed assays. It is interesting to mention in this respect that CCEs have been proposed to physically interact during catalysis and together with LHCII subunits to constitute a highly dynamic complex that allows metabolic channeling of chlorophyll degradation intermediates (pph mutants are not entirely blocked in chlorophyll breakdown and, accordingly, accumulate a certain amount of phyllobilins (cld1 mutants do not exhibit a stay-green phenotype (pph-1/clh1-1/clh2-1 triple mutant (An additional interesting aspect of PPH relates to the spatial organization of the first steps of chlorophyll olase (a . Surprise mutant . HoweverIn summary, our work determined important biochemical and structural properties of PPH, a key enzyme in chlorophyll breakdown during leaf senescence. Modeling of its 3-dimensional structure combined with site-directed mutagenesis allowed the identification of the most important amino acid residues that compose the catalytic triad of the enzyme. Comparison of PPH with CLH identified important structural differences between these two classes of hydrolases that might explain their distinct catalytic properties.JXB online.Supplementary data are available at Table S1. Primers used in this work.Fig. S1. Temperature and pH optima of ∆PPH-MBPa by chlorophyll a.Fig. S2. Partial inhibition of ∆PPH-MBP activity on pheophytin Fig. S3. Absorbance spectra of substrates and their hydrolyzed products.b.Fig. S4. Determination of the ∆PPH-MBP kinetics for pheophytin Fig. S5. Amino acid distribution and phylogenetic analysis of PPH proteins in plants.Fig. S6. The catalytic mechanism of PPH.E. coli extracts expressing recombinant PPH proteins as described in Fig. S7. Analysis of Fig. S8. Three-dimensional model of CLH1 and comparison of ΔPPH and CLH1 secondary structures.supplementary_Table_S1_Figures_S1_S8Click here for additional data file.


The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (Cas9) technology is revolutionizing genome editing approaches. Its high efficiency, specificity, versatility, flexibility, simplicity and low cost have made the CRISPR/Cas9 system preferable to other guided site-specific nuclease-based systems such as TALENs (Transcription Activator-like Effector Nucleases) and ZFNs (Zinc Finger Nucleases) in genome editing of viruses. CRISPR/Cas9 is presently being applied in constructing viral mutants, preventing virus infections, eradicating proviral DNA, and inhibiting viral replication in infected cells. The successful adaptation of CRISPR/Cas9 to editing the genome of Vaccinia virus paves the way for its application in editing other vaccine/vector-relevant orthopoxvirus (OPXV) strains. Thus, CRISPR/Cas9 can be used to resolve some of the major hindrances to the development of OPXV-based recombinant vaccines and vectors, including sub-optimal immunogenicity; transgene and genome instability; reversion of attenuation; potential of spread of transgenes to wildtype strains and close contacts, which are important biosafety and risk assessment considerations. In this article, we review the published literature on the application of CRISPR/Cas9 in virus genome editing and discuss the potentials of CRISPR/Cas9 in advancing OPXV-based recombinant vaccines and vectors. We also discuss the application of CRISPR/Cas9 in combating viruses of clinical relevance, the limitations of CRISPR/Cas9 and the current strategies to overcome them. Variola virus (VARV)—which caused smallpox, Monkey poxvirus (MPXV) and Cowpox virus (CPXV). Orthopoxviruses (OPXVs) are large (200 × 250 nm) brick shaped enveloped viral particles with a large genome (170–250 kb), which unlike most DNA viruses, replicate in the cytoplasm of the host cell. Whereas some OPXVs are host specific , others have broad host range and are also zoonotic .The genus Orthopoxvirus (OPXVs)  contains, among others, Vaccinia virus (VACV)—the type species of the genus, Orthopoxviruses (OPXVs) are suitable viral vectors because they have large transgene capacity (up to 25 kilobasepairs (kbp) ); broad The broad host range of OPXVs may represent a potential risk of spill-over to non-target species, which is not always beneficial, but also allows to reach multiple target species, such as during the deployment of Raboral V-RG (RVG), a VACV-based recombinant Rabies vaccine that targets multiple wildlife reservoir species for rabies control and eradication programs in Europe and North America ,10. A mathymidine kinase (TK) gene, haemagglutinin (HA) gene, intergenic region between the F12L and F13L genes as well as naturally occurring deletions sites in the OPXV genome  [Recombinant OPXVs can be constructed by homologous recombination between transfected heterologous DNA and replicating virus DNA , in vitr to MVA) . Clonal  to MVA) ,24, anti to MVA) ,26, tran to MVA) ,28, plaq to MVA) ,30 or co to MVA) ,31. SeveE. coli and (v) rescue of the genomic BAC clone with a helper fowlpox virus [en passant mutagenesis [Alternatively, in vitro ligation can be used to generate the transgene of interest with desired flanking regions by PCR, and this naked DNA sequence is transfected into cells infected with OPXV  or direcox virus ,34. For agenesis , Cre/Loxagenesis ,37.Generally, these systems require tedious laborious multi-steps with low efficiency among other limitations. Generating candidate recombinant vaccines by homologous recombination is limited by; (i) low recombination efficiency (<3%); (ii) time consuming processes such as generating the plasmid with the transgene and plaque purification of the recombinant virus; (iii) transgene instability upon virus expansion; (iv) requirement for 200–500 bp of flanking DNA sequence (which favour recombination into off-target sites); and (vi) lack of multiple editing of several genes in parallel ,39,40,41Streptococcus pyogenes Type II CRISPR/Cas system [cas gene targets and breaks DNA at specific sequences [cas gene and transcribing the CRISPR into, first a precursor CRISPR/RNA (crRNA), and then a mature crRNA; (3) interference—during which the target nucleic acid is recognized by its PAM (a conserved dinucleotide-containing Protospacer Adjacent Motif sequence upstream of the crRNA binding region) and nicked by the combined action of crRNA, a transactivating CRISPR RNA (tracrRNA) and Cas proteins [The CRISPR/Cas9 /associated protein 9) is one of the latest introductions to the tools of modern genome editing. Derived from the s system ,44 whereequences ,46, CRISequences ,44,47. Iproteins . In CRISproteins ,46, whicThe repair of the specific single double-stranded break made by CRISPR/Cas9 in a targeted region of the DNA by the preferred Non-Homologous End Joining (NHEJ) is error prone. Thus, during repair by this pathway, substitutions, insertions and deletions (indels) often occur leading to frameshift or premature stop codon that can inactivate the gene ,50. The TK gene—whose deletion mutants are restricted to replication in cancer cells, and the NIL and A46R genes that play important roles in VACV virulence and host immune response have been edited using CRISPR/Cas9 [N1L and A46R where the efficiency of specific mutations was ~10% [TK gene was targeted [N1L and A46R gene loci in the presence of a template for recombination  were achieved using a plasmid encoding Cas9 without nuclear localization signal [Viruses depend on host factors for their replication, thus, it was more challenging, compared to self-reproducing organisms, to adapt modern genome editing tools in editing virus genomes . The sucSPR/Cas9 , showingSPR/Cas9 . Howeverwas ~10% , unlike targeted . Both Adn signal . + T cells [Direct targeting and precise inactivation of proviral genomic regions of the human immunodeficiency virus (HIV-1), Epstein-Barr virus (EBV), JC polyomavirus (JCPyV), herpes simplex virus type 1 (HSV-1), hepatitis B virus (HBV), and human papilloma virus (HPV-16) in infected cells by CRISPR/Cas9 have been achieved with varying degree of success . For exa T cells , and fur T cells . Disrupt T cells ,75. Simi T cells ,88,89, r T cells , or redu T cells  have bee T cells  for a re T cells ,73,111,  T cells ,81, HBV  T cells , and EBV T cells ,68,81.Another strategy to interfere with viral infection is CRISP/Cas9-mediated targeting of host cell factors crucial for the viral life cycle. CRISPR/Cas9 mediated disruption of genes expressing receptors or co-receptors required for viral infection can protect host cells against infection. This has been demonstrated for HIV-1 co-receptors CCR5 ,141,142 CRISPR/Cas9 genomic editing can also be used to identify cellular proteins acting as anti-viral defense molecules. Disruption of the genes encoding IRF3, STAT1, IPS1 or STING showed that these proteins are important in anti-viral response to the alphaviruses Chikungunya virus, Venezuelan equine encephalitis virus, and Sindbis virus infection . STAT1 aAlternatively, CRISPR/Cas9 can be used to prevent that the virus evades inflammation and the immune response. This strategy has been successfully applied to enhance the immune response against respiratory syncytial virus (RSV). RSV can infect mesenchymal stem cells (MSCs), which are known to regulate immune response via immune regulatory factors, including cytokines, interferons, inducible nitric oxidase, and indoleamine-2,3-dioxygenase 1 (IDO-1). RSV infection of MSCs resulted in ~70-fold increase in IDO-1 protein levels . ConditiSAMHD1 gene, encoding the deoxynucleoside triphosphate triphosphohydrolase SAM domain- and HD domain containing protein 1 (a), increased HIV-1 infection [Genomic editing is an elegant technique to identify host cell proteins that are involved in viral replication. The CRISPR/Cas9 method allowed the identification of a luminal domain such as LAP1 (LULL1) as a crucial host protein in assembly and packaging of HSV-1 , while cnfection .Flaviviridae family. West Nile virus, Dengue virus, Zika virus, Yellow fever virus, Japanese encephalitis virus, and HCV replication all depended on genes whose products are associated with endoplasmic functions such as translocation, protein cleavage, and N-linked glycosylation , as well as in endocytosis , posttranslational modification , and in transmembrane processing and maturation  [COG1 and COG5 , and MGAT5  glycoprotein β-1,6-N-acetyl-glucosaminyltransferase) as essential host genes for enterovirus replication [Genomic editing has also identified several cellular proteins used by members of the nd SSR3) ,132,143.nd SSR3) . The calnd SSR3) . Genomiclication . The cellication . CRISPR/lication . +-dependent protein deacetylase SIRT1, a potential oncoprotein, suppressed proliferation and colony formation in soft agar of KSHV-transformed cells. These findings suggest that SIRT1 contributes to KSHV-induced tumorigenesis [EMC2, EMC3, SEL1L, DERL2, UBE2G2, UBE2J1, and SYVN1 whose products belonged to the endoplasmic reticulum-associated protein degradation pathway protected against West Nile virus- and Saint Louis encephalitis virus-induced cell death. However, knockout of these genes did not block viral replication [Finally, host cell genome editing unveiled cellular genes involved in the virulent properties of viruses. CRISPR/Cas9-mediated ablation of p53 and PTEN accelerated liver tumorigenesis in HBV transgenic mice , while Higenesis . The genlication .Several OPXV-based recombinant vaccines and vectors are currently at different stages of clinical trials. Many of the vaccines and vectors are based on VACV, MVA, NYVAC, Raccoon poxvirus, and modified Vaccinia Tian Tian (MVTT) OPXV strains, and they target malignancies  ,148,149)146R [153L [157L [159R [183R [184R [O19L [050L [TK gene to restrict virus replication to tumour cells is an additional strategy to ensure safety. A genome editing system such as CRISPR/Cas9:sgRNA that requires few virus multiplication cycles will reduce selection pressure on the vectored vaccine. For example, the TK gene was replaced (with the red fluorescent protein (RFP)) at a greater than 90% rate in VACV [Apart from arming the recombinants with the transgenes against the targeted disease, several strategies to increase the efficacy, e.g., immunogenicity, of OPXV-based recombinant vaccines and vectors include insertion of immunomodulatory and co-stimulatory genes, and gene deletion both to attenuate and increase immune induction. In MVA the immunomodulatory genes targeted for deletion include 146R , 153L [16R [153L , 157L [19R [183R , 184R [13R [184R , O19L [14R [O19L , and 0509L [050L . In many9L [050L ,155. For in VACV ; further in VACV ; but wit in VACV . Indeed, in VACV ,160,161;Apart from history of safe use, the good safety profile of MVA is predicated on the virus’s host range restriction—being unable to produce progeny viruses in human cells and most mammalian cell lines. However, the molecular basis for MVA’s host restriction has yet to be determined because the specific gene deletions and mutations that are responsible for the lack of full replication in most cells have not been identified , althougIn addition to lack of knowledge of the specific mutations that determine MVA host range restriction, knowledge gaps exist in the study of virus and host factors that influence rMVA vaccines/vectors. Research is currently underway to establish how transgene stability is affected by expression levels of transgene, timing of transgene expression, transgene insertion site, and sequence/structure of the transgene/flanking region ,165,166.Xanthomonas bacteria Type III secretion system [Streptococcus pyogenes Type II secretion system [Currently, CRISPR/Cas9 technology is being applied more extensively than TALENs (Transcription Activator-Like Effector Nucleases), ZFN (Zinc Finger Nucleases), ODM (Oligonucleotide Directed Mutagenesis), Cre/Lox and FLP-FRT recombination systems. TALENS and ZFN were previously considered the best programmable and precise techniques for genome editing . Like CRn system ; CRISPR/n system ,44). ZFNThe genomes of HIV-1, HPVs, HSV-2 and HBV have also been edited using TALENs and ZFN ,173,174,Streptococcus-derived CRISPR/Cas9 varies between 2% and 38% [N1L and A46R, the efficiencies were 60% and 94% respectively [gE gene locus was as high as 50%; and 47.5% in a recombinant adenovirus (Adv-EGFP) when enhanced green fluorescent protein (EGFP) was used as the target gene to be edited by Cas9 [Sub-optimal efficiency in eukaryotic cells and viruses: high efficiency (up to 100%) of CRISPR/Cas system has been obtained in bacteria in which the system is naturally expressed , but in  and 38% ,181, alt and 38% ,184,185. and 38% ,65. For  and 38% , althougectively ,65. Howe by Cas9 . The low by Cas9 , which a by Cas9 ,79. N1L and 85% efficiency of A46R) was more efficient than the simultaneous knock-in of both genes (60% efficiency) [Furthermore, sgRNA concentration and sensitivity ; number iciency) . Effortsiciency) . Also moiciency) .Streptococcus pyogenes Cas9 permits up to four nucleotide base mismatches between the sgRNA and complementary sequence of the target nucleic acid, thus resulting in non-specific binding which often leads to cleavage of non-target regions of the genome [9 in the human genome), fewer off-target effects can be expected. For example, when the sgRNA-175 sequence was aligned against the Adv and HSV-1 genomes, no significant homologous sequence was found [Target specificity and off-target effect: the sequence specificity of the widely used Cas9 from e genome ,187,188.e genome , concente genome . For exae genome . The offe genome . Howeveras found . Similaras found ,65. http://crispr.mit.edu) that predict potential off-target sites should be used to authenticate whether de novo mutations in genomes are caused by CRISPR/Cas9-mediated genome editing events. Moreover, the mutation frequency may also indicate whether the mutations are the result of CRISPR/Cas9 or occurred spontaneously because the estimated spontaneous mutation frequency of the human genome is around 1.5 × 10−9 [Several methods exist to detect off-target effects of the CRISPR/Cas9 technology (for a review see ). T7E1 c5 × 10−9 .Various strategies are being devised to increase specificity and minimize CRISPR/Cas9 off-target effects. One strategy is to generate Cas9 nickase mutants, which required two sgRNAs on opposite strands flanking the target site for its double strand break activity ,193,194.+ T cells expressing Cas9/sgRNA that targets the HIV-1 genome. The group showed viral escape from Cas9/sgRNA on the basis of Cas9-induced indels in the targeted viral sequence [Development of resistant escape virus variants: viruses have been reported to develop resistance to CRISPR/Cas9 or acquire revertant phenotypes over multiple infection cycles. Wang et al.  reportedsequence . The indsequence ,76,111. sequence .Similarly, in human cytomegalovirus (HCMV) and HSV-1 where essential genes were targeted for editing by CRISPR/Cas9, virus variants that harbour mutations but still express functional proteins were detected . This tyThe CRISPR/Cas9 system is revolutionizing genome editing approaches. Compared to other target-guided nuclease-based methods, the robustness, effectiveness, low cost and simplicity of CRISPR/Cas9 has made it easily adaptable to editing the genomes of almost any organism including viruses. The technique has been used to customize modifications in several viruses with the aim of generating recombinant mutants, inhibition of virus replication, excision of provirus genes or mutations in host cell receptors to prevent virus infection. The successful application of CRISPR/Cas9 to generate mutant VACV recombinants paves the way for its application in genome editing of other vaccine/vector-relevant OPXV strains. The technique can thus be applied in tackling some of the hindrances to approval of OPXV-based recombinant vaccines and vectors, which include sub-optimal immunogenicity, non-predictability of attenuation, reversion of attenuation, transgene/genome instability, potential of spread of transgenes to wildtype strains and/or close human and animal contacts of vaccinees or patients undergoing oncolytic therapies.However, the full potential of CRISPR/Cas9 will be realised when several of the limitations of the technique including off-target mutations, escape virus variants and sub-optimal efficiency are overcome. Several improved CRISPR/Cas9 systems are already being developed, e.g., the Cas9 nickase variants which improves specificity by requiring two sgRNAs on opposite strands flanking the target site; the Cas9 nickase-cytidine deaminase fusion, which is used to achieve site-specific single-base mutation without requiring double strand breaks; the Fok1-dead-Cas9 nuclease -a catalytically inactive Cas9 employed to minimize the endonuclease activity of Cas9; and inhibition of Cas9 by AcrIIA4 to reduce off-target activity of Cas9. CRISPR/Cas9 can also be coupled to synthetic biology techniques, thus, enabling genome manipulation to the extent which the classical molecular biology techniques cannot, thus opening new frontiers in vaccine and vector development. Further, the full potential of the CRISPR/Cas9 system will be harnessed for vaccine and vector development if, from the early stage, research in the area of safety, including biosafety related to the use of the technique is also taken into consideration. Presently, the focus of research on CRISPR/Cas9 is predominantly on improving specificity and efficiency, and limiting off- and on-target effects of the system. Research is also required on the biosafety implications of off- and on-target effects of the CRISPR/Cas9 mutations, in particular, characterizing the outcomes of such unintended effects by coupling them to phenotypic changes in the virus and host. The results of such research will contribute to advancing the use of the technique in advancing OPXV-based vaccine/vector development. It will also help in the on-going debate in the European Union and other regions of the world on how to regulate products of CRISPR/Cas9.


Connexins are integral membrane building blocks that form gap junctions, enabling direct cytoplasmic exchange of ions and low-molecular-mass metabolites between adjacent cells. In the heart, gap junctions mediate the propagation of cardiac action potentials and the maintenance of a regular beating rhythm. A number of connexin interacting proteins have been described and are known gap junction regulators either through direct effects  or the formation of larger multifunctional complexes . Most connexin partners can be categorized as either proteins promoting coupling by stimulating forward trafficking and channel opening or inhibiting coupling by inducing channel closure, internalization, and degradation. While some interactions have only been implied through co-localization using immunohistochemistry, others have been confirmed by biophysical methods that allow detection of a direct interaction. Our understanding of these interactions is, by far, most well developed for connexin 43 (Cx43) and the scope of this review is to summarize our current knowledge of their functional and regulatory roles. The significance of these interactions is further exemplified by demonstrating their importance at the intercalated disc, a major hub for Cx43 regulation and Cx43 mediated effects. Of note, these values are on the conservative side because proteins like ZO-1 and 14-3-3 have multiple modular domains and would have a larger diameter. For the Cx43CT, we combined the knowledge that the intrinsically disordered Cx43CT domain  domain plays a role in the trafficking, localization, and turnover of gap junction channels via numerous post-translational modifications and protein–protein interactions ,2,3,4,5.residue; ) can conresidue; . The ratIntercellular coupling is eventually determined by the number of open channels in gap junction plaques, which is governed by the synthesis, forward trafficking, and channel open probability. A number of protein partners affect these processes .Cx43 is translationally integrated into the endoplasmic reticulum (ER) and oligomerization occurs only after exit of the ER in the trans-Golgi network . One of Microtubular transport of connexons coincides with the recruitment of a number of protein interactors to the Cx43CT, a number of which have been implicated, however a direct interaction was not confirmed -binding protein Ezrin, binds the Cx43CT and enables PKA to phosphorylate Cx43CT serine residues. In particular, phosphorylation of S364 is a likely precursor to binding with the tight junction protein Zonula occludens 1 (ZO-1), another actin scaffolding protein . Functioapproach . Furtherapproach ,87. Workapproach ,87,88,89approach ,91. WhetCapture and incorporation of Cx43 containing vesicles at the plasma membrane (gap junction periphery) has been attributed to 14-3-3 ,93,94. LFully open channels require phosphorylation by casein kinase 1 (CK1) on residues S325, S328, S330 . InteresFinally, β-catenin is another protein identified to interact with Cx43. In response to Wnt signaling, β-catenin can interact with the Cx43 gene to increase transcription as well as modulate gap junction stability at the plaque ,111,112.Similarly, to facilitating coupling, down regulation of Cx43-mediated intercellular communication requires a number of direct protein interactions and phosphorylation events . Indeed,In the case of β-tubulin, at the gap junction plaque, this may be a mechanism in the disassembly process; at the trans-Golgi network, in cardiomyocytes this may re-route trafficking from the intercalated disc to lateral membranes; or inhibit trafficking to the plasma membrane altogether, leading to increased proteasomal and/or lysosomal degradation. For Drebrin, depletion in cells results in impaired cell–cell coupling, internalization of gap junctions, and targeting of Cx43 for degradation . While pIn addition to Src, another tyrosine kinase identified to directly interact with and phosphorylate the Cx43CT was the Janus kinase family member non-receptor tyrosine-protein kinase 2 . IntereDK pS279/282 585 µM vs non-pS279/282 1064 µM) of Nedd4 for Cx43 [One protein partner that undergoes recruitment following MAPK activation, is the E3 ubiquitin ligase Neural precursor cell expressed developmentally down-regulated 4 . Speciffor Cx43 . Our labfor Cx43 . Furtherfor Cx43 ). Importfor Cx43 ,134. In for Cx43 . A pointfor Cx43 .Phosphorylation of Cx43 by PKC occurs via indirect mechanisms following phosphorylation by Src . Cx43 re230VFF, S2-Y265AYF, and S3-Y286KLV; [AP2 is one protein member of a family of five adaptor protein complexes (AP1-5) that are involved in both clathrin and non-clathrin (AP4/5) mediated trafficking events (for review see ). AP2 asY286KLV; ,145). OnY286KLV; . S1 was Y286KLV; . AlthougY286KLV; , most liY286KLV; ).2+/CaM activates CaMKII leading to autophosphorylation and subsequent phosphorylation of target proteins, including Cx43 [2+ dependent manner and leads to gap junction channel closure, perhaps via occlusion of the pore  and CaM-dependent kinase 2 (CaMKII). Caing Cx43 ,148,149.ing Cx43 ). Whetheing Cx43 ). Howeveing Cx43 . This ocview see ). We recview see . It is tview see ). Duringview see . These cview see ,157,158.view see . Similarview see ,157.In addition to the phosphorylation-mediated changes in protein partner associations described above, new studies have begun to illustrate Cx43 as a potential target for proteolytic cleavage in various pathologies ,161,162.Cx43 is expressed in a large variety of cells , where iThe ubiquitin ligase Nedd4 interacts directly with Cx43  and bothV1.5 has recently been unraveled in some detail.In contrast to the binding of Nedd4 that primarily regulates the Cx43-dependent coupling, other binding partners may be important for the regulation of nearby partners. This has proven particularly crucial at the ID, as evidenced by the fact that mutations in a number of ID components lead to wide spread dysregulation of ID function . AlthougV1.5 [V1.5 expression were reduced in cardiomyocytes from D378stop mice [V1.5 expression at the membrane. The lack of NaV1.5 at the ID suggested that forward trafficking of NaV1.5 might be compromised. Using super resolution microscopy Agullo-Pascual et al. demonstrated that the plus end microtubule marker EB1 was partially dislocated from the ID in mice expressing Cx43-D378stop, which correlated with the presence of NaV1.5 clusters that came very close to the ID membrane without reaching it properly [Knock out of Cx43 in the heart leads to severe arrhythmias ,171, oriV1.5 ,172; andV1.5 , a resulV1.5 . IntriguV1.5 . As for top mice , showingproperly . This leproperly . Deletioproperly , demonstproperly . In contproperly , supportproperly , indicatThere is overwhelming evidence indicating that the functions of Cx43 extend beyond that of forming gap junction channels. Studies from various laboratories indicate that in fact, Cx43 is not only localized at the gap junction or in the perinexus , but alsIt has been over 30 years since the description by Beyer, Paul, and Goodenough of Cx43 as the major gap junction protein in the heart . Since t


Prostate cancer (PCa) is one of the most common cancers and among the leading causes of cancer deaths for men in industrialized countries. It has long been recognized that the prostate is an androgen-dependent organ and PCa is an androgen-dependent disease. Androgen action is mediated by the androgen receptor (AR). Androgen deprivation therapy (ADT) is the standard treatment for metastatic PCa. However, almost all advanced PCa cases progress to castration-resistant prostate cancer (CRPC) after a period of ADT. A variety of mechanisms of progression from androgen-dependent PCa to CRPC under ADT have been postulated, but it remains largely unclear as to when and how castration resistance arises within prostate tumors. In addition, AR signaling may be modulated by extracellular factors among which are the cysteine-rich glycoproteins WNTs. The WNTs are capable of signaling through several pathways, the best-characterized being the canonical WNT/β-catenin/TCF-mediated canonical pathway. Recent studies from sequencing PCa genomes revealed that CRPC cells frequently harbor mutations in major components of the WNT/β-catenin pathway. Moreover, the finding of an interaction between β-catenin and AR suggests a possible mechanism of cross talk between WNT and androgen/AR signaling pathways. In this review, we discuss the current knowledge of both AR and WNT pathways in prostate development and tumorigenesis, and their interaction during development of CRPC. We also review the possible therapeutic application of drugs that target both AR and WNT/β-catenin pathways. Finally, we extend our review of AR and WNT signaling to the mammary gland system and breast cancer. We highlight that the role of AR signaling and its interaction with WNT signaling in these two hormone-related cancer types are highly context-dependent. For the men in the United States, prostate cancer (PCa) is not only one of the most commonly diagnosed cancers, but also one of the most predominant causes of death from cancer ,2. The AWNT signaling is an evolutionary highly conserved signaling system throughout the eukaryotic kingdom. During embryonic and postnatal development, WNT signaling controls many cellular processes, including proliferation, survival and differentiation ,24,25,26Wingless (Wg) and integration 1 (Int1) are homologues in Drosophila and mouse, respectively [Wg was genetically characterized as a segment polarity gene in Drosophila in 1980 by Nüsslein-Volhard and Wieschaus [Int1 was first identified in 1982 by Nusse and Varmus as a preferential site for proviral integration of the mouse mammary tumor virus (MMTV) in a mouse mammary cancer model [Wnt1, genome sequencing has revealed the existence of 19 Wnt genes in mammals. All WNT proteins share common features that are essential for their function, including a signal peptide for secretion, many potential glycosylation sites, and WNT ligands interact with seven-pass transmembrane receptors of the Frizzled (FZD) family and/or single-pass transmembrane co-receptors, such as lipoprotein receptor-related protein 5/6 (LRP5/6), ROR2, and RYK [+  and WNT/JNK , WNT/Rho pathways, are β-catenin-independent and activate a variety of downstream intracellular signaling cascades [The term Wnt is an amalgam of Wg and Int , as the ectively ,46. Wg wieschaus . The proer model . Since t and RYK ,52,53,54 and RYK ,56,57. Rcascades ,58,59,60cascades ,24,25,26In this review, we will discuss the multifaceted manner with which both the canonical and non-canonical WNT pathways influence and modulate AR signaling in CRPC development. We will consider the possible therapeutic application of drugs that target both pathways. We will also discuss these under the context of recurrent mutations in both pathways identified from PCa genomes. Finally, we will extend our review of these two pathways to the mammary gland system and breast cancer.CTNNB1 gene [AR gene [http://www.stanford.edu/group/nusselab/cgi-bin/wnt/.The known molecular components and the cascade of the canonical WNT signaling pathway are summarized in NB1 gene , is a meNB1 gene . The N-tNB1 gene . β-CatenNB1 gene ,65,66,67NB1 gene ,70,71,72NB1 gene . CanonicNB1 gene ,75,76,77NB1 gene ,79. In cNB1 gene ,81. The NB1 gene ,65,66 anNB1 gene  and by rNB1 gene ,84, PygoNB1 gene ,86, CREBNB1 gene ,88 or HyNB1 gene , ensurinNB1 gene , Cyclin NB1 gene ,92, urokNB1 gene , CD44 [9NB1 gene , Cox-2 aNB1 gene , and the[AR gene ,97, as wLGR5 is a WNT target gene and although originally discovered as an intestinal stem cell marker [In addition to promoting the WNT activity, a series of biochemical experiments indicated that R-spondins (RSPOs) are able to synergize with the WNT pathway in the presence of canonical WNT ligands . Similarl marker , it has l marker ,100,101.l marker . Close rl marker ,102,103.The activation of canonical WNT signaling can also be blocked by extracellular proteins. These include the sFRP family  , WIF  and G protein, which activates phospholipase C (PLC), leading to production of 1,2-diacylglycerol (DAG); 1,2-DAG then activates protein kinase C (PKC), and inositol 1,4,5-triphosphate (IP3), thereby triggering intracellular calcium release from the endoplasmic reticulum [2+/calmodulin-dependent protein kinase II (CAMKII), which increase expression of nuclear factor of activated T cells (NFAT)-dependent genes and inhibit canonical WNT signaling through nemo-like kinase (NLK), respectively [In the WNT/Ca2eticulum ,113. Calectively ,115. Actectively ,117,118.ectively . In contectively ,60. In aectively ,121. Thiectively .In both human and rodents, the prostate gland surrounds the urethra at the base of the bladder and functions by contributing secretory proteins to the seminal fluid. In men, the prostate gland is a walnut-sized tissue with a zonal architecture, corresponding to central, periurethral transition, and peripheral zones, together with an anterior fibromuscular stroma . ImportaIn all species, formation of the prostate gland initiates during embryogenesis. During mid-gestation, the primitive urogenital sinus (UGS) is separated from the terminal region of the hindgut through division of the cloaca by the urorectal septum. The most rostral region  of the primitive UGS forms the urinary bladder, whereas the most caudal region  forms the penile urethra. The prostate gland originates from a sub-compartment of the lower urogenital tract (LUT), known as the definitive UGS ,128. TheSfrp2, Wnt4, Wnt5a, Wnt11, Fzd1, Fzd7, Fzd10, Lrp5, Axin1, Lef1, Nkd1, and RhoA [Sfrp1-overexpressing transgenic mice and Sfrp1-null mice confirmed that this WNT modulator stimulates prostate branching morphogenesis, epithelial cell proliferation and secretory gene expression [Wnt5a was found to be indispensable during the UGS development. High levels of Wnt5a expression has been observed at the distal tips and along the centro-distal periductal mesenchyme during the period of postnatal branching morphogenesis, with a rapid decline thereafter in the VP but not the DP and LP [Wnt5a impeded buds branching during morphogenesis [The use of transgenic mice combined with molecular analyses have demonstrated the importance of several developmental signaling pathways during prostate organogenesis, including bone morphogenetic protein (BMP), transforming growth factor beta (TGFβ), Notch, sonic hedgehog (SHH), and WNT pathways . Evidencand RhoA ,149. Accpression . Additiopression . This wapression . In addiP and LP . Anotherogenesis .Ctnnb1) in the mouse prostate during embryonic stages results in significantly decreased prostatic budding and abrogates prostatic development [R-spondin1-4 (Rspo1-4) in developing and neonatal mouse LUT. They found that Rspo3, together with Wnt4, Wnt10b, Wnt11 and Wnt16, appear to be more abundant in male versus female UGS and they stimulate prostatic development [β-Catenin has been identified in both epithelial and mesenchymal structures that undergo a budding program; its activation is necessary and/or sufficient for specification of hair follicle, mammary gland and tooth buds ,155,156.elopment . Furtherelopment . By in selopment .+ cells [+CD133+ basal cells also correspond to an enriched stem cell fraction in the human prostate epithelium [−Sca-1+CD133+CD44+CD117+ cells, which are predominantly basal in mice and are exclusively basal in humans [Nkx3-1 expressing cells (CARNs)) are bipotential and can self-renew in vivo [High cells, enriched in adult PSCs; (iii) Sca-1Low cells, that represent transit-amplifying cells; and (iv) Sca-1Negative cells representing terminally differentiated population with no regenerative potential [+ human cord blood-derived cells [−Sca−CD49fhigh mouse prostate stem and progenitor cells has been provided in the study by Lukacs et al. [Although development of the adult prostate is largely completed at puberty, it must possess a mechanism to assure the homeostasis of its epithelium. To achieve this, prostate, similar to other epithelial organs, sets aside a life-long reservoir of somatic stem cells that retain self-renewal. The regenerative capacity of prostate epithelial stem cells (PSCs) has been shown in the experiment with repeated rounds of androgen ablation and restoration; thus PSCs are androgen-sensitive but not dependent, are capable of self-regeneration, and give rise to transit-amplifying cells that differentiate into various specialized epithelial cells of the prostate . To date+ cells ,166,167.ithelium . Finallyn humans . In addi in vivo . Neverth in vivo ,171. To  in vivo . By usin in vivo . Of noteotential . Upregulotential . In anototential . Similarotential . Interesotential . This ised cells  and it sed cells . Furthers et al. . This grs et al. .The most critical molecular component of the androgen signaling pathway is the AR protein. Upon activation by androgens, AR mediates transcription of target genes that modulate growth and differentiation of prostate epithelial cells. AR plays a vital role in the development of male reproductive organs. Of note, its dysregulation contributes to the male pattern of baldness, development of prostatic hyperplasia, and later in life to PCa.AR gene is located on chromosome Xq11-12. It consists eight exons that encode an 110 kDa nuclear receptor that is a unique member of the nuclear steroid receptor gene family , Hsp40 (Ydj1), Hop (p60), Hsp90 and p23. The main role of this complex is to maintain AR in a conformation capable of ligand binding and to protect it from proteolysis ,187,188.Regulation of the AR activity occurs, in part, by posttranslational modifications, such as phosphorylation at several serine residues with or without a bound ligand . PreciseAR found in PCa, important recent studies have shown that AR can drive expression of oncogenes such as those encoding the ETS transcription factors  as a consequence of gene rearrangements [TMPRSS2-ERG gene fusion, resulting in expression of an N-terminally truncated ERG protein under the control of the androgen-responsive promoter of TMPRSS2  that miss the C-terminal LBD were detected in CRPCs [Prostate gland development and PCa are critically dependent on AR signaling. The ADT remains the most widely used treatment for patients with advanced PCa. In fact, androgen deprivation causes reduced AR expression, apoptosis and decreased tumor cell volume; however most PCas eventually develop the capacity for recurrent growth in the absence of testicular androgen  ,209,210.isoforms B. Anothe Xq11-12 ,220,221. tissues . This amy number . Therefoin CRPCs ,223,224.in CRPCs . Interesin CRPCs . Furtherin CRPCs . Of notein CRPCs .loss-of-function mutations in the tumor suppressor APC gene [APC or CTNNB1 (β-catenin) genes, which lead to constitutive activation of WNT signaling, similar to those found in colon cancer, have also been identified [The paradigm that PCa development and emergence of therapy resistance are a consequence of the restoration of embryonic developmental programs  has shed a new light on understanding the molecular mechanisms underlying epithelial invasion in prostate development and development of CRPC. While the (aberrant) AR signaling pathway is considered as the most critical player in CRPCs, as intracellular signaling pathways are often interconnected, other pathways, in particular, the WNT pathway, can also play key roles. As noted in the previous section, considerable evidence indicates that the WNT pathway plays a central role in the development of prostate tissues, by providing developmental growth inductive signals during embryonic/neonatal organogenesis. In PCa, studies by Schaeffer et al. have reported that androgen exposure regulates genes previously implicated in prostate carcinogenesis; these genes included those related to developmental pathways, such as WNT signaling, along with cellular programs regulating such “hallmarks” of cancer as angiogenesis, apoptosis, migration and proliferation . This obAPC gene ,231,232.entified ,235,236.+ PCa cell lines CWR22-Rv1 and LAPC-4 enhanced AR-mediated transcription of its target genes, in an androgen-dependent manner [AR gene expression via the TCF/LEF-1 binding sites in the AR promoter [Accumulating evidence has supported that the WNT/β-catenin pathway plays an important role in CRPC, by interacting with AR signaling ,238,239.t manner . Hence, t manner . Furtherpromoter . Thus, iIn the other hand, the effect of AR signaling on WNT/β-catenin signaling is more complicated. Early studies in gonadotropin-releasing hormone neuronal cells showed that in the presence of DHT, liganded AR repressed β-catenin/TCF-responsive reporter gene activity . In andrAR gene, as well as in genes encoding the main components of the WNT/β-catenin pathway, such as APC, β-catenin and R-spondins, leading to overactivation of WNT/β-catenin signaling [To add another layer of the complexity of interaction between AR and WNT/β-catenin signaling, it was shown that when PCa cells had been adapted to the low androgen environment , β-catenin could act as a co-activator of AR as well to enhance AR transcriptional activity in the presence of androstenedione, a weaker adrenal androgen remaining present in CRPC patients ,242,243.ignaling .WNT5A and WNT7B. Similarly, WNT5A or WNT7B mRNA was detected by RNA-ISH in a subset of CTCs from patients with CRPC [As described in the previous section, WNT ligands are highly conserved secreted molecules that play critical but pleiotropic roles in cell-cell signaling during embryogenesis. Interestingly, expression levels of several WNT ligands were found to be up- or down-regulated in advanced PCa. For instance, Chen et al. demonstrated that high levels of WNT1 and β-catenin expression were associated with advanced, metastatic, hormone-refractory prostate carcinoma, in which they could serve as markers for disease progression . In two ith CRPC . This deith CRPC . Interesith CRPC .WIF1 mRNA appears to be downregulated in a considerable percentage of PCa samples [Secreted WNT antagonists, including the sFRP family, DKK family, and Wnt inhibitory factor-1 (WIF1), are negative modulators of WNT signaling ,254,255. samples . Interes samples ,259. The samples ,255. Alt samples ,261. Alt+/CD133+ CSC-like subpopulation in these PCa cell lines. Previous studies have shown that PCa cells with these markers exhibited tumor-initiating and metastasis-initiation cell properties, although it was not absolutely clear whether the ALDH+/CD133+ subpopulation represented CSCs definitively [+/CD24− populations. Interestingly, they reported that it was the WNT/β-catenin signaling pathway that mediated upregulation of CD44 by DAB2IP knockdown. In this setting, CD44 not only served as a marker for CSCs, but also played a key role in facilitating the onset of prostate CSCs and increasing their chemoresistance [Cancer stem cells (CSCs) have been proposed to contribute to therapy resistance and cancer recurrence . In addinitively ,264,265.nitively , it was sistance . Importasistance . In anotsistance . By usinsistance . Similarsistance . Overallsistance .As to the non-canonical WNT pathway, the most recent clinical studies by Miyamoto et al. have shown that activation of this pathway in CTCs from patients with metastatic CRPC correlates with reduced effectiveness of antiandrogen treatment . In partTaken together, WNT signaling interacts with AR signaling using distinct mechanisms at different stages of PCa progression. In hormone-naïve PCa cells, WNT/β-catenin signaling promotes transcription of AR target genes, whereas AR signaling inhibits the transcription of WNT/β-catenin target genes A. HoweveWNT signaling plays key roles in both mammary gland development and breast cancer (BCa), largely through regulating mammary stem cell maintenance and basal mammary epithelial cell fate determination. An excellent review for this topic was published in this journal recently . As to tUnlike PCa, our understanding of AR signaling in BCa is still at its infancy. Some studies report that overexpression of AR is associated with better outcomes in BCa, while others illustrate a positive correlation of circulating androgens with high risk, recurrence and metastasis of BCa ,278,279.In breast tissues, androgen can be converted to DHT, which subsequently activates AR. The liganded AR direct or indirectly (possibly together with distinct co-regulators under different ERα settings) interacts with either ARE or ERE in its target genes . In the +/PR+/HER2−, ER+/PR+/HER2+, ER−/PR−/HER2+, and ER−/PR−/HER2− . Relating to the ER status, AR likely plays distinct roles in BCa in a subtype-specific manner.BCa is often classified clinically into four subtypes based on expression of ER, progesterone receptor (PR), and human epidermal growth factor receptor 2 : ER+, ER−/HER2+, and TNBC subtypes, respectively [Positive expression of AR was clinically defined as immunohistochemical (IHC) nuclear staining ≥1% or ≥10% according to various studies ,291,292.ectively ,297,298.n = 53) with testosterone therapy in patients with metastatic ER+ BCa [+ BCa, but this study is still at the beginning stage [Modulation of AR signaling, either inhibitory or stimulatory, exhibits somewhat contradictory observations in different subtypes of BCa, particularly when interacting with ER signaling ,299. Whe ER+ BCa . Androgeng stage ,305. Recng stage ,306,307.+ BCa with a frequency of ~70%–95% [+ BCa in a treatment-dependent manner: some studies indicated that higher AR expression is associated with better therapy outcomes, whereas others have reported that AR plays an oncogenic role in tamoxifen-resistant subjects [+ BCa initially, due to its ability to antagonize the growth-promoting role of ER signaling [+/AR+ BCas [AR is highly expressed in ER~70%–95% ,298,308.~70%–95% . AR and ~70%–95% ,310. AR subjects ,313,314.ignaling . AccordiAR+ BCas . But comAR+ BCas .− BCa and the functional crosstalk between AR and HER2 is critical for the tumor cell survival and expansion [AR transcription in a positive feedback loop [WNT7B, which activates β-catenin, resulting in HER3 transcriptional activation [AR is highly expressed in ERxpansion ,297,315.xpansion ,280. Mecack loop ,317. AR tivation , clinicativation .− breast tumors that were also AR+, was defined based on microarray expression profiling [+ TNBC was reported recently, in which a patient with this BCa subtype had progressive disease following six cycles of cytotoxic chemotherapy, but attained a 100% response to bicalutamide (an antiandrogen) [+ TNBC are being developed [The frequency of AR expression in TNBC is around 10% to 53% ,296,298.rofiling . Later orofiling . AR signrofiling ,321. Therofiling . An encondrogen) . With theveloped ,321.WNT1 is oncogenic for BCa [−/HER2+ BCa subtype. Using gene set enrichment analysis (GSEA), Ni et al. observed that androgen (DHT)-stimulated genes in ER−/HER2+ BCa cells were mainly those involved in WNT signaling [WNT7B transcription in a ligand-dependent manner. WNT7B is a canonical WNT ligand and may play roles in the normal mammary gland development during the stages of ductal formation and involution [WNT7B has been found in ~10% of BCa cases [−/HER2+ BCa cells by upregulating HER3, which encodes a key co-receptor of HER2 in HER2+ BCa [−/HER2+ breast tumor cells in vivo was inhibited [Overexpression of WNT induces aberrant activities of the WNT signaling pathway, which is a main driving force in BCa progression ,324. WNT for BCa . The intignaling . Furthervolution ,327. EleCa cases . In addiER2+ BCa . Importanhibited .+ BCa) and hormone-refractory cancers . Accordingly, both AR agonists and AR (and/or WNT) antagonists may be beneficial for BCa therapy, but in a BCa subtype and therapy stage-dependent manner. In particular, as both the AR and WNT signaling pathways drive progression and maintenance of AR+ TNBCs, inhibitors for these two pathways may prove to be useful for targeting this TNBC subtype. In addition, AR antagonists and anti-HER2 agents may also be used in combination to treat ER−/HER2+ BCa with AR expression, and inhibitors for WNT signaling may offer another therapeutic opportunity, particularly when ER−/HER2+ BCa cells develop resistance to the anti-HER2/AR agents.Thus, in both PCa and BCa, AR signaling appears to regulate distinct sets of target genes in hormone-dependent cancers  are refractory or unresponsive to hormone therapy, a better understanding of roles of AR and WNT pathways and their interactions in these hormone-refractory diseases should open a new avenue for improving their treatment and for combating the inevitable challenge of therapy resistance.As two key pathways regulating both normal development and tumorigenesis in hormone-responsive prostate and mammary glands, the context-dependent interplay of AR and WNT signaling pathways provides a unique opportunity to explore therapeutic options for treating prostate and breast cancers, particularly when under the setting of therapeutic resistance. As both CRPCs and ER


CYP2C19) on clopidogrel-mediated effects on platelet inhibition, inflammatory response and endothelial function, as well as risk of major adverse cardiovascular events (MACE), in coronary heart patients undergoing percutaneous coronary intervention (PCI) was investigated. To this end, we assessed the residual platelet aggregation rate (RPA), maximal aggregation rate (MAR) and plasma levels of sCD40L, sP-selectin, MMP-9, sVCAM-1 and sE-selectin after 24 h of PCI in 559 patients treated with clopidogrel and followed up for 1 year for evidence of MACE. CYP2C19*2 and *3 variants were identified using a clopidogrel-sensitive gene detection kit. Our results showed higher RPA and MAR as well as increased sE-selectin, sCD40L, sP-selectin, MMP-9, and sVCAM-1 levels in CYP2C19 intermediate metabolizer , poor metabolizer  and combined IM+PM groups, relative to those in extensive metabolizers . In total, 519 patients completed 1 year of follow-up, among which 69 (13.3%) experienced MACE. The risk of MACE in CYP2C19 IM+PM patients was 2.664 times higher than that in CYP2C19 EM patients (OR = 2.664 (1.397–5.193), P = 0.004). The data suggest that CYP2C19*2 and *3 variants modulate the drug efficacy of clopidogrel in coronary heart patients undergoing PCI and further enhance the risk of MACE. Accordingly, CYP2C19 pharmacogenetic profiling may be beneficial for coronary heart patients undergoing PCI to predict the efficacy of treatment with clopidogrel. We propose that IM and PM patients should benefit from treatment with higher clopidogrel doses to improve efficacy and reduce the incidence of MACE.The impact of pharmacogenetic variants of cytochrome P450 2C19 ( Owing to dietary and lifestyle changes, the number of coronary heart disease (CHD) patients worldwide has increased over the years. Percutaneous coronary intervention (PCI) is one of the most significant developments in the treatment of CHD. However, stent thrombosis (ST) is a major complication affecting the efficacy of PCI. Currently, anti-platelet drugs, such as clopidogrel and aspirin, are used to prevent the occurrence of ST after PCI  platelet receptor, P2Y12, is widely used to prevent thromboembolic events in patients  clopidogrel-mediated platelet effects, (2) clopidogrel-mediated inflammatory endothelial effects, and (3) risk of MACE.Considering this background, we aimed to investigate whether CYP2C19 variants on platelet reactivity, inflammatory and endothelial biomarkers and 1-year clinical outcomes in CHD patients undergoing PCI. Patients were eligible for study enrollment in the cases where (1) ages were higher than 18 and (2) they were diagnosed with CHD using coronary angiography and successfully treated with PCI. Exclusion criteria were as follows: (1) aspirin, clopidogrel and contrast agent allergy, (2) severe diseases, such as tumors and bleeding disorders, (3) hemodynamic instability (systolic blood pressure <90 mmHg and/or diastolic blood pressure <50 mmHg), severe ventricular dysfunction (LVEF <40%), (4) severe liver and kidney dysfunction, (5) blood disease or bleeding diathesis, platelet count <100 × 109/L and hematocrit <30%.Patients were enrolled from Fujian Provincial Hospital between March 2012 and March 2014. The ethics committee of the hospital approved the study and all patients provided written informed consent. Procedures were carried out in accordance with the approved guidelines. This prospective study was designed to determine the effects of After 24 h of PCI, 3 ml peripheral blood samples were collected and mixed with 3.8% trisodium citrate, and platelet aggregation was determined via the turbidimetric method in 2 h with a four-channel light transmission aggregometer . Platelet aggregation was stimulated with 5 μmol/L adenosinediphosphate (ADP). After agonist stimulation, the degree of light transmission was monitored. Results were recorded as light transmission at maximal aggregation. The platelet aggregation rate was recorded as a curve for 5 min and expressed as residual platelet aggregation rate (RPA).After 24 h of PCI, creatinine (Cr), lipids , platelet count (PLT), RPA, and fibrinogen (FIB) levels were tested in the hospital laboratory and plasma levels of sCD40L, sP-selectin, MMP-9, sVCAM-1, and sE-selectin were assessed via enzyme-linked immunosorbent assay (ELISA)  according to the manufacturer's instructions.PCI was performed in accordance with the standard of care. All patients were administered 300 mg clopidogrel and 40 mg atorvastatin prior to the operation. The operator decided on the choice of anticoagulant and the use of glycoprotein IIb/IIIa inhibitor (tirofiban), as well as the type of stent. After PCI, patients were treated with 100 mg/day aspirin indefinitely and 75 mg/day clopidogrel for 1 year. We defined the success of PCI as follows: (1) achievement of complete revascularization and residual stenosis of <20% in target vessels of patients, (2) level 3 TIMI blood flow, (3) partial or complete remission of symptoms of myocardial ischemia, and (4) no serious complications during hospitalization .CYP2C19 genotypes were identified using the clopidogrel-sensitive gene detection kit . DNA was extracted from whole peripheral blood, followed by stepwise evaluation involving single-base primer extension, PCR amplification chip spotting, mass spectrometry, DNA microarray and analysis of data with software to obtain CYP2C19 genotypes. All steps were performed in strict accordance with the BAIO genetic analyzer manual instructions. According to the CYP2C19*1, *2, *3 genotypes, patients carrying *1/*1 were grouped as extensive metabolizers (EM), *1/*2 or *1/*3 as intermediate metabolizers (IM), and *2/*2, *2/*3 or *3/*3 as poor metabolizers (PM).The clinical endpoints include the following MACE: cardiac death, myocardial infarction, and unstable angina. Cardiac death was defined as any death due to cardiovascular causes or not clearly attributable to non-cardiovascular causes. Myocardial infarction was defined as recent ischemic symptoms with ST-segment elevation or depression and abnormally elevated troponin. Unstable angina was defined as resting onset, duration greater than or equal to 20 min or more severe and longer duration of angina than before and higher frequency of onset. Diagnosis was performed during hospitalization according to serum creatine kinase levels, electrocardiogram findings and other indicators.t-test or univariate analysis of variance (ANOVA). Categorical variables were compared with the chi-square or Fisher exact test, as appropriate. Time-to-event outcomes were analyzed via Kaplan-Meier analysis and differences compared with the log-rank test. Logistic regression analysis was performed to assess independent predictors of MACE. P < 0.05 were considered statistically significant.Statistical analysis was carried out using SPSS software . Continuous data, presented as median and interquartile range, were compared using Kruskal-Wallis one-way analysis of variance or Mann-Whitney U test. Measurement data following normal distribution were expressed as means ± standard deviation (x ± s) and compared using P > 0.05). Levels of lipids, creatinine, platelet count and fibrinogen were comparable among the three groups (P > 0.05). Differences in usage of clinical medicines, including ACEI/ARB, β-blockers, CCB, statins, PPI, and tirofiban (P > 0.05) were not statistically significant. Moreover, left main coronary artery lesions, vascular lesions (≧3), frame lengths and bracket numbers were similar among the three groups (P > 0.05).A flow diagram of the study is shown in Figure P < 0.05).After 24 h of PCI, we measured levels of RPA, MAR, sE-selectin, sCD40L, sP-selectin, MMP-9, and sVCAM-1 in all patients. Significant differences were observed among the three groups using Kruskal-Wallis one-way analysis of variance (Table P = 0.009). The frequency of unstable angina differed among EM, IM and PM patients . We observed a strong trend toward higher frequency of unstable angina and MACE in IM patients, compared to PM patients. Comparison of the total frequencies of MACE and unstable angina in EM and IM+PM groups additionally revealed significant differences  . Notably, the risk of MACE in CYP2C19 IM+PM patients was 2.664 times higher than that in CYP2C19 EM patients (OR = 2.664 (1.397–5.193), P = 0.004) , vascular lesions ≥ 3, frame length, bracket number, levels of platelet activity indicators after PCI and CYP2C19 metabolizers (IM+PM and EM) as independent variables, we analyzed whether these relevant parameters influence the occurrence of MACE. Univariate logistic regression analysis showed significant association of CYP2C19 metabolizers with occurrence of MACE (OR = 2.318 1.251–4.297), .297, P =CYP2C19 genetic variants modulate clopidogrel-mediated antiplatelet effects and risk of MACE in patients undergoing PCI. Our main findings were as follows: (1) CYP2C19*2 and *3 variants influence RPA, MAR and levels of sE-selectin, sCD40L, sP-selectin, MMP-9, and sVCAM-1 in patients following PCI. The efficacy of clopidogrel is reduced in IM+PM, compared to EM patients. (2) CYP2C19 EM patients have lower risk of MACE, compared with IM, PM, and IM+PM groups. (3) CYP2C19*2 and *3 variants are independent risk factors of MACE in patients undergoing PCI.The current study focused on determining whether CYP2C19*2 polymorphism is associated with lower antiplatelet response and increases the rate of recurrent cardiovascular events in Caucasian, Korean, and Chinese patients , vascular lesions ≥3, frame length, bracket number and levels of platelet activity indicators , data from multivariate analysis showed that the risk of MACE in IM+PM patients is 2.664 times higher than that in EM patients. Our results are consistent with earlier studies showing that CYP2C19*2 and *3 variants influence the levels of RPA, MAR, sE-selectin, sCD40L, sP-selectin, MMP-9, and sVCAM-1 in coronary heart patients undergoing PCI and modulate the drug efficacy of clopidogrel, further increasing the risk of MACE. Based on these findings, we propose that CYP2C19 pharmacogenetic profiling may be beneficial for coronary heart patients undergoing PCI to predict whether treatment with clopidogrel is a feasible option. IM and PM patients may benefit from higher clopidigrel doses to improve efficacy and reduce the incidence of MACE.Taken together, our results suggest that HS, JQ, and HC conceived and designed the experiments. HS, ZC, and HZ performed the experiments. JQ, QQ, and HS analyzed the data. HC and JQ contributed reagents/materials/analysis tools. HS, JQ, ST, and HC wrote the paper. All authors reviewed the final version of the manuscript.This work was supported by the National Natural Science Foundation of China  and Health and Family Planning Commission of Fujian Province Medical Innovation project (No. 2014-CX-5); the National Scientific foundation of China  and the Youth Foundation of Xiangtan Hospital in Central South University (2014Q08).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.


The species-specific, as well as organ-specific expression of regulated necrosis (RN)-related molecules, is not known. We determined the expression levels of tumour necrosis factor receptor-1 (TNFR1), receptor activated protein kinase (RIPK)1, RIPK3, mixed lineage kinase domain-like (MLKL), CASP8, Fas-associated protein with death domain (FADD), cellular inhibitor of apoptosis protein (CIAP)1, CIAP2, glutathione peroxidase-4 (GPX4), cyclophilin D (CYPD), CASP1, NLRP3 and poly(ADP-ribose) polymerase-1 (PARP1) in human and mouse solid organs. We observed significant differences in expression of these molecules between human and mice. In addition, we characterized their expression profiles in acute as well as persistent tissue injury and chronic tissue remodelling using acute and chronic kidney injury models. We observed that the degree and pattern of induction of RN-related molecules were highly dependent on the trigger and disease pathogenesis. Furthermore, we studied their expression patterns in mice with lupus-like systemic autoimmunity, which revealed that the expression of MLKL, GPX4 and PARP1 significantly increased in the spleen along disease progression and CASP1, RIPK1, RIPK3 and CYPD were higher at the earlier stages but were significantly decreased in the later stages. In contrast, in the kidney, the expression of genes involved in pyroptosis, e.g. NLRP3 and CASP1 were significantly increased and TNFR1, RIPK1, RIPK3, CIAP1/2 and GPX4 were significantly decreased along the progression of lupus nephritis (LN). Thus, the organ- and species-specific expression of RN-related molecules should be considered during designing experiments, interpreting the results as well as extrapolating the conclusions from one species or organ to another species or organ respectively. For almost two decades apoptosis was considered to be the only programmed form of cell death and necrosis was felt to be an ‘accidental’ cell death passively induced by physiochemical insults . HoweverNecroptosis is triggered by death receptors and requires the receptor activated protein kinase (RIPK)3-dependent phosphorylation of mixed lineage kinase domain-like (MLKL) inducing plasma membrane pore formation ,5. Tumouc, using erastin, leading to depletion of intracellular generation of GSH, an antioxidant. Glutathione peroxidase-4 (GPX4) reduces hydrogen peroxide to water using GSH and therefore, suppression of GPX4 results in ferroptosis . The high confidence algorithm was used to reduce the risk of the false positive crossing point. All the samples that rise above the background fluorescence (crossing point Cp or quantification cycle Cq) between 5 and 40 cycles during the amplification reaction were considered as detectable. The melting curves were analysed for every sample to detect unspecific products and primer dimers. To visualize the similarity and differences in gene expression profiles among the samples, hierarchical cluster analysis was performed using algorithms incorporated in the open-source software MultiExperiment Viewer (MeV) version 4.9 [P-value) from the t test [noise-adjusted/standardized signal] [An equal amount of RNA (1 μg) was used to prepare cDNA . Complemsion 4.9 . DiffereThe harvested kidney tissues were fixed in 4% neutral-buffered formalin followed by dehydration in graded alcohols and embedded in paraffin. For periodic acid–Schiff (PAS) and haematoxylin and eosin (H&E) staining or immunostaining, 4 μm sections were deparaffinized, rehydrated, transferred into citrate buffer and either autoclaved or microwave treated for antigen retrieval and processed as described . Tubulart test. A P-value less than 0.05 indicated statistical significance.Data were expressed as mean ± S.E.M. Comparisons between groups were performed using unpaired Student's two-sided We quantified the mRNA expression levels of RN-related molecules involved in necroptosis, e.g. TNFR1, RIPK1, RIPK3, MLKL, CASP8, FADD and CIAP1 and CIAP2; ferroptosis, e.g. GPX4; MPT-RN, e.g. CYPD; pyroptosis, e.g. CASP1, NLRP3; parthanatos, e.g. PARP1 using qRT-PCR in healthy human organs. All of these molecules were constitutively expressed in the human spleen; however, the mRNA expression levels of MLKL, NLRP3 and FADD were generally low A. GPX4 aNext, we examined the same organs from healthy, 10–12 weeks old, C57BL/6 mice to quantify the mRNA expression levels of the RN-related molecules as listed earlier. All molecules were constitutively expressed in mouse spleen; however, the mRNA expression levels of RIPK3, MLKL, FADD, GPX4 and NLRP3 were low B. UnlikeWe further studied the changes in the expression of RN-related molecules in transient tissue injury. Since human and mice kidneys expressed relatively low mRNA levels of these molecules as in respective spleen, we selected three independent and well-characterized mouse models of acute kidney injury (AKI) viz. ischaemia-reperfusion injury (IRI), acute oxalate nephropathy and cisplatin nephropathy ,32,34,35Persistent and progressive tissue injury leads to the chronic tissue remodelling ,42. DuriFas-deficient MRL/lpr mice that displays spontaneous and similar to human systemic lupus erythematosus (SLE)-like systemic autoimmunity [Systemic autoimmunity is characterized by high levels of circulating autoantibodies, tissue inflammation and progressive tissue remodelling of multiple organ systems –48. To simmunity ,46,49. WThe most frequent complication of SLE is an immune complex glomerulonephritis called lupus nephritis (LN). The pathogenesis of progressive renal remodelling in LN involves a variety of extra- and intra-renal pathomechanisms . TherefoHistorically, necrosis was viewed as an uncontrolled form of cell death however, more recent studies showed that necrosis can be a highly regulated process ,3. BasedCypD-, Nlrp3- and Casp1-deficient mice show less ATN than wild-type mice [Necroinflammation is the central pathomechanism of ischaemic and toxic AKI ,34,35,52ype mice ,34. Furtype mice ,54. Therype mice ,19,35, hUnlike acute tissue injuries, the contribution of necroinflammation in the chronic tissue injury is not so clear. The presence of leucocytes rather represents tissue repair or fibrosis ,56. The Fas-deficient MRL/lpr mice as a model of SLE may explain the up-regulation of FADD mRNA in the spleen. We also observed an up-regulation in the mRNA expression of MLKL, GPX4 and PARP1 in the spleen. Interestingly, the peripheral blood mononuclear cells of patients with SLE as well as systemic sclerosis are known to express low levels and activity of PARP1 [SLE is characterized persistent exposure to autoantigens as a result of alterations in cell death pathways e.g. apoptosis, NETosis etc., and clearance deficiency of dead cells ,48. The of PARP1 –60. HoweThe present study did not focus on the gender-specific differences within the same species. However, several studies have addressed the possible role of gender as a biological variable–for example, lung and kidney injury, gut microbiota composition, haematopoiesis etc. –65. TherTogether, we have identified significant differences in the mRNA expression profiles of the RN-related molecules in humans and murine solid organs, as well as their regulation in murine autoimmunity, acute tissue injury and progressive tissue fibrosis. Therefore, it is important to take into account these species-specific differences to avoid misinterpretation and wrong conclusions. These findings warrant the need to validate the functional roles of these molecules, as identified in rodent studies, in human studies as well.