Adrenergic stimulation is important for osteoclast differentiation and bone resorption. Previous research shows that this happens through β2-adrenergic receptor (AR), but there are conflicting evidence on presence and role of α2A-AR in bone. The aim of this study was to investigate the presence of α2A-AR and its involvement in neuro-endocrine signalling of bone remodelling in humans. Real-time polymerase chain reaction (PCR) and immunohistochemistry were used to investigate α2A-AR receptor presence and localization in bone cells. Functionality of rs553668 and rs1800544 single nucleotide polymorphism SNPs located in α2A-AR gene was analysed by qPCR expression on bone samples and luciferase reporter assay in human osteosarcoma HOS cells. Using real-time PCR, genetic association study between rs553668 A>G and rs1800544 C>G SNPs and major bone markers was performed on 661 Slovenian patients with osteoporosis. α2A-AR is expressed in osteoblasts and lining cells but not in osteocytes. SNP rs553668 has a significant influence on α2A-AR mRNA level in human bone samples through the stability of mRNA. α2A-AR gene locus associates with important bone remodelling markers . The results of this study are providing comprehensive new evidence that α2A-AR is involved in neuro-endocrine signalling of bone turnover and development of osteoporosis. As shown by our results the neurological signalling is mediated through osteoblasts and result in bone resorption. Genetic study showed association of SNPs in α2A-AR gene locus with bone remodelling markers, identifying the individuals with higher risk of development of osteoporosis. Each mechanism is contributing only little to the bone turnover ratio which is a reason why only little variability in BMD can be explained via a routine procedure at the University of Ljubljana’s Institute of Pathology. Immunohistochemistry was performed on the four samples that showed the highest (OP patient), an intermediate (OA patient) and the lowest (one autopsy participant and one OP patient) gene expression of α2A-AR, respectively. In addition, immunostaining was carried out on one cross-section of the femoral head from the autopsy participant as well. Paraffin sections were dewaxed, rehydrated and microwaved for 10 min. in 0.01 M sodium citrate buffer (pH 6) to release masked epitopes. The immunohistochemistry was hereinafter performed with a Mouse and Rabbit specific HRP/DAB detection IHC kit  in accordance with the manufacturer’s procedures. Tris buffered saline (TBS), with the addition of Triton X-100 , was used for washing purposes throughout the whole procedure. The primary rabbit polyclonal antibody to α2A-AR  was diluted 1:100 in TBS buffer and all control slides of each sample were treated with TBS only (negative controls). All slides were incubated overnight at 4°C in a humidified chamber. The specificity of the antibody used had been previously verified in our laboratory on HOS cell culture (data not shown). The tissue sections were counterstained with haematoxylin solution  and examined with an Olympus BX50 microscope . The intensity of staining in osteoblasts, lining cells and osteocytes was compared across all samples by two independent, blinded evaluators. Images were acquired using an Olympus XC50 camera and the CellSens Dimension program 1.6.0 (Olympus).Bone samples of intertrochanteric trabecular bone from 27 osteoporotic patients with femoral neck fracture (OP patients), as well as from 21 patients with osteoarthritis (OA) and 14 sex and age-matched autopsy participants (C) as controls including only those where the medical history did not include any disorders affecting bone were obtained (Table3) were collected during surgical procedures of femoral osteotomy from the trabecular bone at the metaphyseal cutting plane. A proportion of the bone samples was immediately frozen in liquid nitrogen and stored at −80°C until RNA and DNA extraction. The exclusion criteria for the enrolment of OP and OA patients, as applied through the questionnaire, laboratory results and interview, included the following: secondary OP or OA, liver and kidney diseases, endocrinological disorders and medical anamnesis on receiving medications with a known influence on bone metabolism. The study was approved by the Ethical Committee of Republic of Slovenia and all patients gave written informed consent.The expression of the α2A-AR gene was analysed by quantitative real-time polymerase chain reaction (qPCR) assay in bone samples of Slovenian patients undergoing either hemiarthroplasty or total hip arthroplasty as a consequence of low-energy femoral neck fracture (51 OP patients) or primary hip OA (75 OA patients). Bone tissue samples obtained from autopsy without any diagnosed systemic disease were used as controls (14 C subjects). Patients were included in the study in a consecutive manner over a period of 1.5 years as they were directed to arthroplasty at the Department of Traumatology in the General and Teaching Hospital Celje as a result of receiving a diagnosis of OP or OA. OP was diagnosed by radiologically confirmed low-energy femoral neck fracture and WHO criteria. The diagnosis of OA was established by clinical and radiographic criteria according to the Harris hip score RPLP0) using following primers: Forward 5′-TCTACAACCCTGAAGTGCTTGAT-3′ and Reverse 5′-CAATCTGCAGACAGACACTGG-3′. The reaction was performed under the following conditions: 95°C for 15 min., followed by 45 cycles of 95°C for 30 sec., 1 min. at 60°C; with the generation of the melting curve being performed at the end starting with 95°C for 5 sec. and following with increasing the temperature from 65 to 97°C with a heating rate of 2.5°C/sec. Cp values of each sample were normalized to the mean Cp value of the reference gene (α2A-AR/RPLP0 ratio = Cp(α2A-AR)/Cp (reference gene)). The geometric mean of relative gene expression in a group of individuals with the wt genotype of the rs553668 polymorphism of α2A-AR gene was set to 1.DNA and RNA were extracted using a TRI reagent (Sigma-Aldrich Chemie) in accordance with the manufacturer’s procedure. DNA and RNA concentration and purity were evaluated on a ND-1000 . With respect to RNA, the RNA integrity number (RIN) was evaluated using a Bioanalyzer 2100 . Where the RIN was very low, the extraction of RNA was repeated and confirmed by real-time analysis. For reverse transcription, the High Capacity RNA-to-cDNA Kit  was used. Complementary DNA (cDNA) from the total RNA extracted was synthesized according to our previously described procedure All experiments were performed on HOS cell lines (ATTC CRL-1543) grown on DMEM and supplemented with 10% FBS , 1% L-glutamate (Gibco) and 1% Antibiotic/Antimycotic (Gibco). Cell lines were passaged on a regular basis, as and when 80–90% confluence was achieved. No more than 20 passages were performed with a single cell line.luc gene and an α2A-AR promoter region upstream of the luc gene. All primers were designed based on the ENSEMBL genomic sequence of the α2A-AR gene (ENST00000280155). All PCRs were performed with a HiFi Polymerase Kit . First, an additional polyclonal site containing EcoR1 and two Ahd1 restriction sites followed by another EcoR1 site was inserted into the Xba1 site (GGAATTCGACCTTGAGTCGACAGATGGTCGAATTC) downstream of the luc gene to facilitate ADRA2A 3′ UTR cloning. Next, ATAAGATCTTGTTCGGAGATAGGAGAAGGC forward (containing a Bgl2 restriction site) and TTAAAGCTTGAACGATCAGCTCTCCAGGA (containing a Hind3 restriction site) reverse primers were used to amplify the 5′ promoter region of α2A-AR.Luciferase reporter assays were performed with pGl3-basic plasmids containing a 3′ UTR region of α2A-AR downstream of the EcoR1 and cloned downstream of the luc gene into the newly established EcoR1 restriction site. The final plasmid constructs were sequenced to verify the sequence identity and orientation upstream and downstream of the luc gene. GTCTTACCGGAAAACTCGAC forward and TGCATTCTAGTTGTGGTTTGTC reverse primers were used to produce forward- and reverse-sequenced clones downstream of the luc gene. RVprimer3 CTAGCAAAATAGGCTGTCCC and GLprimer2 CTTTATGTTTTTGGCGTCTTCCA primers were used to produce forward- and reverse-sequence clones upstream of the luc gene. Sequencing was performed with the DTCS quick sequencing kit  and reactions were separated on a GeXP Genomic Analyser (Beckman Coulter) in accordance with the manufacturer’s instructions.After obtaining the pGl3-basic with the α2A-AR promoter region, the 3′ UTR of α2A-AR from a heterozygous individual (polymorphism A/G rs553668) was amplified using the following primers: GTAGACTCACGCTGACTGCAG forward and GAAACTGTACAGTTTGGCAGGC reverse Functional assays were carried out by plating cells at a density of 35,000 cells/well in 24-well tissue culture plates. After 24 hrs, the cells were transfected in six replicates with a mixture of Fugene HD transfection reagents  at a ratio (ml reagent:ml DNA) of 3:2, DMEM and 475 ng DNA/well and an additional 25 ng/well of a pRL-TK control reporter vector. Cells were harvested 48 hrs post transfection, and the luciferase assay performed with a Dual-Luciferase Reporter Assay System . Luminescence was measured using a BIO-TEK Synergy HT multidetection microplate reader .We evaluated 661 Slovenian people who were referred to the outpatient departments of the University Medical Centre in Ljubljana, the General and Teaching Hospital in Celje or the University Medical Centre Maribor for BMD measurement. BMD measurements at the lumbar spine (L2-L4) BMD-ls, total hip BMD-hip and femoral neck BMD-fn were performed by dual-energy X-ray absorptiometry  in Ljubljana, Celje and Maribor. A cross-calibration study of the precision of measurements between the centres had previously been performed and a correction factor was not considered necessary. Each patient was examined clinically and routine biochemical tests were performed to exclude systemic and metabolic bone diseases other than primary OP. Subjects who had previously taken any drug known to influence bone metabolism were excluded from the study. Biochemical markers of bone turnover were measured in subgroups of subjects. Blood samples were collected between 8:00 a.m. and 10:00 a.m. after an overnight fast. The plasma and serum samples were analysed in a routine laboratory using standard procedures as outlined by the manufacturers. Osteocalcin (OC) in heparinized plasma was measured by means of a solid-phase, two-site chemiluminescent enzyme-labelled immunometric assay . Serum bone alkaline phosphatase (sBALP) was measured by radioimmunoassay . Serum C-terminal cross-linking telopeptides of type I collagen (CTX), osteoprotegerin and cathepsin K were measured by enzyme immunoassay . The study was approved by the Ethical Committee of the Republic of Slovenia and written informed consent was obtained from each patient participating in the study.DNA was isolated from peripheral blood leucocytes using a Flexigene kit (QIAgene) in accordance with the manufacturer’s protocol. Genotyping was performed with 5× HOT FIREPol® Probe qPCR Mix Plus  and 10 ng of genomic DNA and the TaqMan genotyping assays C__996424_20 for rs553668 and C___7611979_10 for rs1800544 (both Applied Biosystems) respectively, in line with the manufacturer’s protocol. Allelic discrimination was performed on LightCycler 480®  employing the following protocol: 1 cycle of 15 min. at 95°C, followed by 45 cycles at 95°C for 15 sec., 60°C for 60 sec. and 4 min. at 40°C. An end-point genotyping program was used to call genotypes automatically.D’ and r2, between both SNPs was calculated using EMLD software . The distribution of variables was assessed using the Shapiro–Wilk test and logarithmic transformation was used for variables that did not meet the normality assumption. The effects of genotypes, and alleles on BMD and biochemical markers of bone turnover were evaluated by means of the general linear model (GLM) adjusted for age, sex and body mass index (BMI) in all study subgroups. An LSD post hoc adjustment for multiple comparisons was used to find the differences within the genotype subgroups. The effects of the studied alleles on fracture risk were examined by logistic regression analysis in the group of all participants and, subsequently, in subgroups of elderly people, men and postmenopausal women. The model included the studied alleles , age (in years), BMI (in kg/m2) and femoral neck BMD (in g/cm2). The significance threshold was set at 0.05. SPSS Statistics version 21  was used for all statistical analyses. The power of our study was 61% to detect a 0.080 g/cm2 difference in BMD between the genotype and haplotype subgroups and 69.3–98.2% to discover a significant difference in the odds ratio of alleles between fracture patients and the no fracture controls Comparisons between genotype, and allele frequencies in subject subgroups were performed with a chi-squared test or Fisher’s exact probability test. The degree of linkage disequilibrium (LD), denoted as anova test once the normality of distribution had been confirmed.The association of α2A-AR genotypes with α2A-AR gene expression in the study group as a whole was evaluated using the TargetScan 6.2  To understand whether α2A-AR could be involved in neuro-endocrine signalling, its presence in human bones was investigated using immunohistochemistry. In addition, we has been suggested that since ARKO knockout mice show higher bone phenotype, we might see the difference in α2A-AR distribution in bones of different density. As a result of the qualitative and at best semi-quantitaive nature of immunohistochemistry, we used the method only to investigate α2A-AR localization within bone sample. Immunohistochemistry was performed on the four bone samples showing different α2A-AR expression according to real-time PCR see Table and C reP = 0.003). Statistical significant result was obtained also when no adjustment was made with slightly higher P-value of 0.006, showing that co-variables do not have much influence on α2A-AR gene expression. The highest expression was shown in AA genotype carriers, which was significantly different from the expression in GG genotype carriers; namely, by 10%. Heterozygote carriers showed the lowest expression  to draw complete conclusions. To clarify the effect of the A allele on gene expression, we continued with a Luciferase assay. Importantly no statistically significant difference in α2A-AR gene expression between male and female was observed. No statistically significant difference in α2A-AR gene expression was observed between disease groups although the OP group showed slightly but not statistically significant difference in comparison to OA or control group .The association between the gene expression of the α2A-AR and rs1800544 SNP was not significant in this study group (P < 0.012). This result supports the in vivo measurement of α2A-AR gene expression, where lower amount of mRNA was observed in individuals carrying G variant of rs553668 SNP.Luciferase reporter assay was performed to further support the evidence of rs553668 SNP functionality in osteoblasts. We constructed two novel plasmids containing 3′ UTR region of the α2A-AR cloned behind the luciferase reporter gene in pGl3-Basic to study the rs553668 SNP influence on mRNA stability. Each plasmid contained either G or A variant of rs553668 SNP. Transient transfections with two different plasmids were performed for an mRNA stability evaluation. Forty-eight hours post transfection, Renilla and firefly luciferase activities were measured and expressed as a ratio of firefly to Renilla activity. The relative ratio of luciferase activity was 1.000 and 0.896 in the A and G genotypes respectively Fig., with thA section of 3′ UTR was analysed using TargetScanHuman and miRANDA programs to evaluate if miRNA binding might play a role in α2A-AR mRNA stability. No binding site for miRNA was found in the vicinity of the polymorphic site. Next, the influence of rs553668 SNP on α2A-AR mRNA secondary structure formation was evaluated. Two distinct but equally stable ΔG = −2.4 kcal/mol) structures were noted  as a whole and in all subgroups. There were no significant differences in both SNPs genotypes between subgroups (P > 0.05). The genotype frequencies were similar to those in the HapMap for rs553668, while rs1800544 showed a higher frequency of the minor genotype GG (1.4–5.8% versus 0%) in our study groups compared to PubMed data for the European population . Both SNPs were in LD, with r2 = 0.56 and D’ = 0.986.The distributions of genotypes for both SNPs are shown in TableThe results of GLM analysis are presented in TableIn this study, the further evidence for involvement of α2A-AR signalling in neuro-endocrine regulation of bone remodelling is presented. This study is the first to provide evidence on the level of human bone tissue, using immunohistochemical, and functional characterization of genetic polymorphisms influence on the level of α2A-AR transcript. The research shows that not only β2-AR is involved in neurological signalization but also that α2A-AR will have to be considered in the future.First evidence of α2A-AR involvement in neuro-endocirne regulation of bone remodelling was obtained by identification of bone cells possessing α2A-AR. The results show that α2A-AR is present on osteoblasts and lining cells Fig. and C. TThe precise pathway of α2A-AR action is unknown. Our results suggest action through osteoblasts and/or lining cells because they carry the receptor. Several mechanisms of action might be envisaged. Signalling may either result in apoptosis and/or inhibition of osteoblast proliferation or in stimulation of osteoclast differentiation, tipping the bone remodelling balance in favour of bone resorption. Evidence from the study on ARKO mice shows latter might be more plausible, since decreased expression of RANKL was observed in mutant mice In conclusion, the study presents further evidence based on human samples that α2A-AR receptors are important in osteoblast neuro-endocrine signalling, bringing a new player repetition along with already known β2-AR. Although the precise pathway of events remains to be elucidated, it looks like the neurological signal of bone remodelling is mediated through osteoblasts and/or lining cells which seem to express most of the α2A-AR of bone remodelling cells. The study also shows that functional polymorphism in 3′ UTR of α2A-AR gene affects its mRNA stability and is associated with bone turnover markers.


Furthermore, elevated AR expression is positively correlated with α6β1 integrin levels and higher grades in patients. These findings can potentially serve as biomarker and therapeutic approach for controlling chondrosarcoma metastasis.Chondrosarcoma is a malignant tumor that produces cartilage matrix. The most lethal aspect is its metastatic property. We demonstrated that amphiregulin (AR) is significantly upregulated in highly aggressive cells. AR silencing markedly suppressed cell migration. Exogenous AR markedly increased cell migration by transactivation of α6β1 integrin expression. A neutralizing α6β1 integrin antibody can abolish AR-induced cell motility. Knockdown of AR inhibits metastasis of cells to the lung  Chondrosarcoma, the second most common type of bone cancer, is a heterogeneous group of malignancies that are characterized by the production of cartilage matrix. High-grade chondrosarcoma is more aggressive and is more likely to metastasize to other areas of the body. Although high-grade only occurs in approximately 5–10% of chondrosarcoma patients, it remains the major cause of death [Secreted proteins are responsible for the cross talking among cells, which may facilitate the progression of metastasis, particularly within the steps of migration and invasion . AmphirePrevious studies have demonstrated that EGF and AR are capable of promoting cell motility by increasing integrin expression –17. AR kAlthough the roles of AR have emerged as a pivotal factor in the regulation of cell motility across diverse cancer, the effect of AR on migration of chondrosarcoma cells still remains largely unknown. In the present study, we probed intracellular signal pathways involved in AR-induced integrin expression to regulate cell migration in human chondrosarcoma cells.To investigate whether AR is associated with migration activity in human chondrosarcoma cells, we first compared migration ability and the levels of AR secretion between JJ012 (S0) and JJ012 (S10). As shown in Fig. To examine the mechanism by which AR induces α6β1 expression, we directly measured the Ras activity and Raf-1 phosphorylation in response to AR. The results revealed that stimulation of cells to AR induced an increase in Ras activity and phosphorylation of Raf-1 in a time-dependent fashion Fig. . Pretreain vitro migration was measured by Transwell assay. The results revealed that AR elicited a significant rise in cell migration, which were drastically attenuated in the presence of AP-1 inhibitors , can be phosphorylated by MAPKs, leading to a significant increased in the activity of AP-1 –24. We tors Fig.  or knockors Fig. . Knockdoors Fig. , left. Sors Fig. . Moreoveors Fig. . We next126 Fig. . To furtin vivo, we monitored the metastatic potential of JJ012 (S10)-Luc cells stably expressing control shRNA or AR shRNA in mouse models of lung metastasis by using bioluminescence imaging. At day 0 post-injection, there were no differences between the control shRNA and AR shRNA groups, suggesting that mice were injected with the same number of cells. However, at day 60 after injection, tumor signals in the lung were significantly reduced when AR expression was knocked down , AR expression and integrin β1 , and integrin α6 and integrin β1   Fig. . Taken tAR is a member of the EGF family, and its increased expression has been reported in many cancers, including colorectal cancer , 7, breaGeneration of the soluble forms of AR is essentially mediated by the proteolytic activity of the transmembrane proteinase ADAM-17  also known as tumor-necrosis factor-alpha converting enzyme (TACE) . The ADAIn lung cancer, bone is a frequent target of metastasis. A previously study discovered that AR is able to activate expression of parathyroid hormone–related protein (PTHrP) that is a causative factor contributing to osteolytic metastases . With reDue the observation that AR is highly expressed in a variety of malignancies, insights into the mechanisms related to the anti-tumor activity of AR targeted therapy might help improve chondrosarcoma therapy. In chondrosarcoma, the novel therapeutic strategies targeting AR signaling by monoclonal antibodies, soluble receptor protein, multikinase inhibitors, and RNA interference could serve as therapeutic agents for clinical application in humans. More recently, a study further demonstrated that human epidermal growth factor receptor 2 (HER2) is the only marker suited for patient selection for the trastuzumab plus pertuzumab-based regimen in HER2-positive metastatic breast cancer . TherefoPrevious studies have indicated that integrins are implicated in cellular migrations in chondrosarcoma –46. Nota2.Human chondrosarcoma cell line (JJ012) was kindly provided by the laboratory of Dr Sean P Scully . The human chondrosarcoma cell line (SW1353) was obtained from the American Type Culture Collection. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/α-MEM supplemented with 10% fetal bovine serum and 100 units/ml penicillin/streptomycin at 37°C in a humidified chamber in 5% CO4 cells) suspended in 200 μl of serum-free medium were seeded onto the upper chamber and 300 μl of the same medium containing varying concentrations of AR  was placed in the lower chamber. After overnight incubation, cells were washed with PBS, fixed with 4% paraformaldehyde, and stained with 0.5% crystal violet. Cells remaining on the upper surface of filter membrane were completely removed by wiping with a cotton swab. Migrated cells were then counted from six random fields under light microscope.Cell migration was determined by using Transwell culture inserts  according to the manufacturer's instructions. In brief, cells . Original cells were designated as JJ012 (S0) .Conditioned medium was obtained from JJ012 cells grown in six-well plates. The concentrations of AR in medium were measured using Quantikine ELISA kits  according to the manufacturer's instructions.The pLKO.1-puro-based lentiviral vectors: TRCN0000117995 (shAR#1) and TRCN0000420315 (shAR#2), and pLKO.1-shscramble were obtained from National RNAi Core Facility at Academia Sinica, Taipei, Taiwan. Recombinant lentiviruses were packaged as per manufacturer's instruction. Cultured cells were incubated with lentiviral supernatants containing 8 μg/ml polybrene for 24 h, replaced fresh medium and incubated for another 48 h. For stable cell lines, cells were selected by puromycin (5 μg/ml) .To sequester downstream signaling, Cells were transfected with MEK1 dominant-negative mutant  or ERK2 dominant-negative mutant . To suppress gene expression, cells were transfected with ON-TARGET plus siRNAs targeting Ras, Raf-1, and c-Jun, and control from Dharmacon Research . Lipofectamine 2000  is used to introduce oligonucleotide according to the manufacturer's recommendations.Total RNA was extracted using TRIzol reagent  and reverse transcribed into cDNA using M-MLV reverse transcriptase, Oligo (dT), and dNTP Mix  as per manufacturer's instructions. Synthesized cDNA (1 μg) was used as template for quantitative PCR (qPCR) that was conducted using KAPA SYBR FAST qPCR Kits . Sequences for all target gene primers and probes were purchased commercially (Applied Biosystems). All primer used in qPCR were list in 6 cells collected and washed once with PBS, then fixed with 70% ethanol overnight. Fixed cells were washed twice by PBS and probed with mouse monoclonal antibody specific to α6β1 integrin . Cells were then washed again and incubated with fluorescein isothiocyanate isothiocyanate-conjugated goat anti-mouse secondary IgG  for 45 min and analyzed by flow cytometry using FACS Calibur and Cell-Quest software (BD Biosciences).Aliquots of 1 × 10Cells were treated with AR in a time-dependent fashion. Activation of Ras (Ras-GTP) was detected using the Ras-binding domain of Raf-1 to pull down active Ras  according to the manufacturer's recommendations. Following separation by SDS-PAGE, proteins were transferred to membranes which were probed with an anti-RAS antibody.Cells were lysed in RIPA buffer containing protease inhibitor cocktail. Protein concentration was determined by the Pierce BCA Protein Assay Kit . Equal amount of total protein was resolved by SDS-PAGE and transferred to PVDF membranes . Membranes were blocked with 5% bovine serum albumin and then probed with primary antibodies. After washes in tris-buffered saline with 0.05% Tween 20, membranes were subsequently incubated with appropriate peroxidase-coupled secondary antibodies. Membranes were then washed and bound antibodies were visualized using ECL reagents  and autoradiography.ChIP analysis was performed as described previously . BrieflyCells were co-transfected with luciferase reporter gene constructs and β-galactosidase using Lipofectamine 2000 (Invitrogen) as per manufacturer's instructions. At 24 h transfection, the cells were exposed to AR for 24 h or pre-treated with inhibitors for 30 min, followed by treatment with AR for 24 h. Luciferase activity was determined using the luciferase assay kit .6 cell per mouse) into the lateral tail vein of mice. Lung metastasis was monitored using an in vivo imaging system (Xenogen IVIS imaging system). At Day 60, mice were sacrificed by overdose with anesthetic. The lungs were then fixed in 10% formalin, embedded in paraffin and subsequently processed for IHC. All mice were handled in accordance with the Animal Care and Use Guidelines of the China Medical University  under a protocol approved by the Institutional Animal Care and Use Committee.Four-week-old male nude mice were used and randomly assigned to two groups (eight mice each group). Cancer cells (JJ012 (S10)/Luc or JJ012 (S10)/shAR-Luc) were resuspended in 100 μl of serum-free DMEM/α-MEM and subcutaneously injected . Fixed and paraffin-embedded tissues were deparaffinized with xylene, and rehydrated through a graded series of alcohols to water. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide Heat-induced antigen retrieval was carried out for all sections in 0.01 M sodium citrate buffer, pH 6 at 95°C for 20 min. Human AR, integrin α6, and integrin β1 antibodies were applied at a dilution of 1:200 and incubated at 4°C overnight. Bound antibodies were detected by NovoLink Polymer Detection System  and visualized with the diaminobenzidine reaction. The sections were counterstained with hematoxylin. IHC results were scored by taking into account the percentage of positive detection and intensity of the staining.t-test. Statistical comparisons of more than two groups were performed using one-way analysis of variance with Bonferroni's post hoc test. In all cases, P < 0.05 was considered significant.All data are presented as mean ± SEM. Statistical comparison of two groups was performed using the Student's


Augmented Reality is a new technological system that allows introducing virtual contents in the real world in order to run in the same representation and, in real time, enhancing the user's sensory perception of reality. From another point of view, Augmented Reality can be defined as a set of techniques and tools that add information to the physical reality. To date, Augmented Reality has been used in many fields, such as medicine, entertainment, maintenance, architecture, education, and cognitive and motor rehabilitation but very few studies and applications of AR exist in clinical psychology. In the treatment of psychological disorders, Augmented Reality has given preliminary evidence to be a useful tool due to its adaptability to the patient needs and therapeutic purposes and interactivity. Another relevant factor is the quality of the user's experience in the Augmented Reality system determined from emotional engagement and sense of presence. This experience could increase the AR ecological validity in the treatment of psychological disorders. This paper reviews the recent studies on the use of Augmented Reality in the evaluation and treatment of psychological disorders, focusing on current uses of this technology and on the specific features that delineate Augmented Reality a new technique useful for psychology. Augmented Reality (AR) is a new technological system that allows inserting virtual contents in the real world in order to run in the same representation and, in real time, enhancing the user's sensory perception of reality . Comparecombines real and virtual objects in a real environment,runs interactively and in real time,registers real and virtual objects with each other.More precisely, Azuma et al. , 5 definFurthermore, according to Milgram et al. , 8, AR pIn an AR system, users see an image made up of a real image and virtual elements that are superimposed over it. The addition of virtual elements may also inhibit the perception of real elements by overimposing the virtual elements on the real elements. Nevertheless, the most important aspect in AR is that the virtual elements provide the real world with remarkable and valuable information. The addiction of virtual elements may involve not only the view but also the hearing, smell, and touch .From the point of view of technology devices, AR can be defined as a set of techniques and tools that allow adding information to the physical reality. Various technologies are used in AR rendering including handheld devices, display system worn on one's user (nonhandheld devices), and projection displays.Modern handheld mobile computing like smartphones and tablets contain these elements, which include a camera and sensors such as accelerometer, Global Positioning System (GPS), and solid-state compass, making them a suitable AR platform. The smartphone or tablet take in real time the surrounding environment and the virtual elements are superimposed to the real world.An example of nonhandled device is the head mounted display (HMD), a display system worn on one user's head such as a helmet or glasses. HMD systems are characterized by sensors that receive input information about the environment by the user's head movements adding at a later stage various virtual contents.As regards the projection displays, the virtual elements are projected on the real objects in order to be augmented. The projection occurs with a single room-mounted projector without any display system worn on one user's head. The projector generates a virtual image on the room surface using an automated calibration procedure that takes into account the structure of the surface overlapping the virtual image.Furthermore, an AR platform requires a software application able to augment the real world by using one or more hardware devices. Marker-based and markerless systems are the two main software applications used in AR system. The AR marker-based systems are stylized pictures in black and white that are recognized by the computer webcam and which are superimposed in real time multimedia contents: video, audio, 3D objects, and so forth. Instead, in the markerless  AR systeTo date, AR has been used in medicine , entertaStarting from these premises, the aim of this paper is to review the recent studies on the use of AR in the evaluation and treatment of psychological disorders, focusing on current uses of AR in psychology and the various factors that make a new technique useful for the treatment of psychological disorders, expanding the possible fields of use of AR.We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines .*  OR assessment OR treatment). We excluded articles where the full text was not available or where the abstract lacked basic information for review. The first search was performed for publications in the English language, and then we decided to clean the results, considering only publications for the last ten years, from 2004 forward and eventually updated the search results through December 2014. Expert colleagues in the field were contacted for suggestion on further studies not considered in our search.A computer-based search in several databases was performed for relevant publications describing the use of AR in psychology. Databases used for the search were PsycINFO, PubMed/Medline, and Web of Science (Web of Knowledge). We searched using the string “Augmented Reality” AND  independently selected paper abstracts and titles and analyzed the full papers that met the inclusion criteria, resolving disagreements through consensus.The data extracted from each included study were sample type, study design, sample size, type of therapy or assessment, and selected findings.The flow chart of the systematic review is shown in Despite a large volume of studies on AR, little has been done specifically to psychological assessment or treatment. In the current review we present a broad range of experimental and clinical studies.In the area of clinical psychology, a few but remarkable studies have met the DSM-IV-TR criteria , showingIn the studies, the AR exposure therapy was based on Öst et al.'s “one-session treatment” guidelines , 42. IndThe first analysed study that used an AR system to assess and treat specific phobias was conducted by Juan et al. (2004) . A singlFear of Spiders Questionnaire (FSQ) [Botella et al. (2005)  assessedre (FSQ) , Spider re (FSQ) , and Subre (FSQ)  were usere (FSQ) .Juan et al. (2005) , for theIn Botella et al. (2010)  an AR syBretón-López et al. (2010)  evaluateIn 2011, Botella et al.  assessedIn 2011, Juan and Calatrava  comparedJuan and Joele (2011)  comparedWrzesien et al.  , 29 condThe first study  comparedIn the second study , five paIn a further study, Wrzesien et al. (2013)  experimeAll these studies represent new potentiality and possibility of assessment and treatment in the area of psychological disorders. However, they disclose some limitations. The majority of the disclosed studies , 31–35 iIn 2006, Juan et al.  advancedAnother study, conducted by Juan and Prez (2010) , compareIn the current scientific scenario, AR is a relevant issue and offers a viable alternative to traditional methods as the in vivo exposure therapy. This paper is the first systematic review that explores studies in the literature that use AR as a tool in the treatment of psychological disorders.To date, the analysis of the literature suggested that AR has been mainly used in the evaluation and treatment of specific phobias such as phobias for small animals (cockroaches and spiders) and acrophobia. Regarding the assessment and treatment of specific phobias, from the literature analysis, it has been observed that thirteen studies have used an AR system in order to reduce anxiety, fear, and avoidance behaviours. Specifically, eleven studies concerned the evaluation and treatment of cockroaches and spiders phobia, while two studies affected the acrophobia.As regards the cockroaches and spiders phobia, nine studies applied an AR-HMD system connected with a camera and a computer , 37–40. The last studies have tested two innovative technological AR systems , 33. In As mentioned above, the majority of the studies concerned the assessment and the treatment of small animals phobias: five studies have focused on patients with cockroaches' phobia , 39, 40 A remarkable feature refers to the number of subjects included in these studies and the psychological and experiential measures in the assessment of the AR exposure therapy. Three studies experimented with one case study , 35, 39,Furthermore, all these studies considered anxiety as psychological measure showing that AR elicits anxiety as soon as the stimulus appeared that decreased during the time of exposure , 37–39. Taking the two acrophobia studies , 43 intoRegarding the number of subjects, both of the studies included healthy volunteers and, specifically, in 2006, Juan et al.  assessedAmong these studies, just Juan and Prez (2010)  considerThe studies mentioned above suggest that AR offers many advantages such as the possibility to reproduce real objects in the environment , controlled situations, and ad hoc environments and objects in order to tailor the situations on the subject's needs and therapeutic purposes. However, the discussed studies are mostly preliminary researches that disclose some limitations due to the fact that AR exposure has only been recently tested for the evaluation and treatment of psychological disorders. The majority of the disclosed studies –35, 39 iInstead, the reaming studies focused on testing the AR system on healthy subjects in order to verify the efficacy, usability, and the quality of user's experience of the new platform using randomized controlled studies –38, 43.Apart from these studies, the literature is lacking studies of AR on the treatments of other several psychological disorders.Currently, other technological devices have proven effective in the evaluation and treatment of psychological disorders. In particular, VR has been demonstrated to be a very useful tool for the treatment of several psychological problems such as eating disorders and anxiety disorders –54. The In the last few years many studies have showed the efficacy of VR environments as therapeutic tool , 57–60. A feature that makes VR a useful tool for evaluation and treatment of psychological disorders is to elicit emotional responses commensurate with the real ones.The emotional engagement in virtual exposure depends on a number of factors such as the sense of presence. The sense of presence in virtual exposure is defined as the degree of “being there” in the virtual environment  and is mRecent studies have shown that virtual exposure is as effective as in vivo exposure –64 and, In the studies of Ferrer-García and Gutiérrez-Maldonado , 63 six Gorini et al.  started Finally, regarding the sense of presence, the results showed a significant degree of presence on the level of state anxiety in VR and real exposure conditions. As mentioned above, the VR gives the possibility of the subject to manipulate and interact with the environment and contents as in the real world.Similarly, AR could be considered a useful tool in the evaluation and treatment of psychological disorders providing a number of advantages as the VR. Indeed, the AR, as VR simulation, can be seen as an experiential process, and the experience is an essential component in dealing with critical situations. Furthermore, this experience could allow exploring environments and situations hard to reproduce in reality and, as the VR, the feeling of presence in the AR system could permit the user to assign the learned behaviours from the AR world to the real one.The AR simulation, therefore, could be considered an efficient method to act on real behaviours avoiding the risks and complications typical of real environments.The aim of this paper was to review the recent studies on the use of AR in the evaluation and treatment of psychological disorders, focusing on current uses of AR in psychology and the various factors that make a new technique useful for the treatment of psychological disorders, expanding the possible fields of use of AR.In general, the presented studies show that the AR seems to be a promising and useful tool for intervention in the treatment of specific phobias. Nevertheless, the small sample of subjects examined and the lack of control group and randomized controlled studies necessitate more randomized controlled experiments for exploring the AR efficacy in the clinical treatments. Despite these limitations, AR is proving to be a new technique useful to patients to experiment with technologically different and severe situations, as the exposure to fear or phobic stimuli, in a safe environment under the control of the therapist. Indeed, an AR system extends interactivity for assessing and supervising patient's reactions in real time and adaptability for creating controlled exposure settings based on the patient's needs or therapeutic purposes. Furthermore, it is to be noted that AR allows subjects/patients to manipulate and control the virtual elements, interacting with virtual objects placed in the real world in real time.As a consequence, the experience to amplify the physical world with virtual contents can improve the ecological validity of the “mixed reality”  on envirOverall, AR may represent a new challenge for the assessment and treatment of different kinds of psychological disorders, such as eating and anxiety disorders performing new studies based on systematic measures of psychological and neurophysiological effects.


Males develop kidney stones far more frequently than females with a ratio of 2–3:1, suggesting that androgen receptor (AR) signaling might play a key role in the development of nephrolithiasis. Using the cre-loxP system to selectively knock out AR in glyoxylate-induced calcium oxalate (CaOx) crystal mouse models, we found that the mice lacking hepatic AR had less oxalate biosynthesis, which might lead to lower CaOx crystal formation, and that the mice lacking kidney proximal or distal epithelial AR also had lower CaOx crystal formation. We found that AR could directly up-regulate hepatic glycolate oxidase and kidney epithelial NADPH oxidase subunit p22-PHOX at the transcriptional level. This up-regulation might then increase oxalate biosynthesis and oxidative stress that resulted in induction of kidney tubular injury. Targeting AR with the AR degradation enhancer ASC-J9 led to suppression of CaOx crystal formation via modulation of oxalate biosynthesis and oxidative stress in both in vitro and in vivo studies. Taken together, these results established the roles of AR in CaOx crystal formation. The incidence of kidney stones in adults has increased significantly over the last several decades. Up to 13% of men will have a kidney stone sometime in their lives 13. CalciuThe sex disparity of male to female patients with nephrolithiasis is up to 2–3:1. The mechanisms behind this greater proportion of male patients are not clear but may reasonably be expected to be due to differential concentrations of testosterone  or urinaEarly reports using the castrated rat model suggested that both testosterone and dihydrotestosterone might play important roles in the differential rates of CaOx crystal formation –10. Othe–Using cell-specific AR knockout (ARKO) mouse models  and the 12–2 environment at 37°C. When necessary, cells were treated with ASC-J9 at 5 μM for 48 or 72 hours or apocynin (Sigma-Aldrich) at 100 μM for indicated times. Dimethylsulfoxide or saline was used as a vehicle control.The human HepG2 and HEK-293 cell lines were purchased from American Type Culture Collection. The normal human kidney proximal epithelial cell lines, HKC5 and HKC8, were kindly provided by Dr. Syed Khundmiri of the University of Louisville . All of the cells were cultured in DMEM supplemented with 10% fetal bovine serum in a humidified 5% COTo generate AR-overexpressing or AR knocked-down stable clones of HepG2, HKC5, and HKC8 cells, HEK-293 cells were transfected with lentiviral vectors, pWPI-AR/pWPI-Vec or pLKO1-AR-si/pLKO1-sc, and with the pAX2 packaging plasmid and pMD2.G envelope plasmid at a 4:3:2 ratio using Lipofectamine 2000 (Invitrogen).flox/flox; C57/B6) mice with β-actin promoter-driven Cre , albumin promoter-driven Cre    or lackeoratory) , kidney oratory) , or cadhoratory)  mice, re2O (ddH2O) was injected ip every day for 7 days. All animals had free access to chow and water. The 24-hour urine samples was collected 1 day before killing, and mouse organs and serum were collected after killing.We established the CaOx crystal mouse model following the reported protocol . Glyoxyl2 (10 mM) and sodium oxalate (10 mM) solution with ddH2O  were mixed at room temperature. After 3 days of equilibration at 4°C, the crystals formed were washed with ddH2O and dried at 60°C, and a stock solution of COM (100 mg/mL) was obtained.CaCl2). After 24 hours of incubation, cells were fixed for imaging experiments, and media were collected for further experiments.The crystal imaging protocol was described by Khand et al  and LiesStudy protocols involving human materials were approved by the First Hospital of Xi'an Jiaotong University Ethics Committee. Seven young male patients with kidney stones and 10 young male healthy volunteers without a family history of urinary stone disease were included in the study. All of the patients underwent percutaneous nephrostolithotomy, and at the end of the procedure, ultrasound-guided puncture biopsy was done to acquire kidney tissues. The primary antibody rabbit anti-AR  was used for IHC staining. Orthotopic tumor tissues of LNCaP cells were used as positive controls; no primary antibody control was used as a negative control see . The GerTotal RNAs were isolated from cells or tissues using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Then 1 μg of total RNAs was subjected to reverse transcription using SuperScript III transcriptase (Invitrogen). qRT-PCR was conducted using a Bio-Rad CFX96 system with SYBR Green to determine the level of mRNA expression of a gene of interest. Expression levels were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA.Western blot assays were performed as reported previously . In brieN,N-dimethylacetamide) or ASC-J9 was injected ip  for 3 weeks. After 2 weeks of treatments, all mice were subjected to glyoxylate challenge ; ASC-J9 or vehicle was coinjected at this time. Seven days later, mice were killed, and kidneys were obtained for analyses.Eight-week-old B6 wild-type (WT) male mice were divided into 2 groups (6 mice/group) and either vehicle (2O2 (30%) and silver nitrate (5%) under a 60-W light at a 15-cm distance for 30 minutes. The H2O2 and silver nitrate mixture was replaced every 15 minutes. After the reaction, slides were washed with ddH2O and counterstained with Nuclear Fast Red staining solution (Sigma-Aldrich) for 5 minutes. To identify the positive Pizzolato-stained CaOx crystals in tissue sections (see Tissue sections (5 μm) were prepared from the paraffin block and stained with Pizzolato staining to detect CaOx crystals as follows. Tissue sections were dewaxed, dipped in water, and incubated with a mixture  of HWhen mice were killed, 0.6 to 0.8 mL of blood was collected from the inferior vena cava and immediately assayed for the serum testosterone level using the Coat-A-Count Total Testosterone radioimmunoassay    were measured with an H2O2 assay kit . The optical product was read at 595 nm.The HAfter cells were seeded in 96-well plates at the density of 104 cells/well and cultured for 24 hours, the confluent monolayers of cells were treated with COM crystals in serum-free DMEM for 24 hours. ASC-J9 (5 μM) or vehicle (dimethylsulfoxide) was also added as required by individual experiments. After centrifugation, the LDH amounts in supernatants were determined using a commercial kit . The optical products were read at 490 nm. Values were normalized to those for control group samples individually .2O2. The H2O2 generated was then allowed to react with 3-methyl-2-benzothiazolinone hydrazone and 3-(dimethylamino)benzoic acid in the presence of peroxidase to produce an indamine dye that can be detected on a spectrophotometer at 590 nm.Oxalate measurements in the HepG2 cell culture media (48 and 72 hours) and 24-hour mouse urine collections were determined using an Oxalate Kit (Trinity Biotech) according to the manufacturer's instructions. In brief, the oxalate was oxidized by oxalate oxidase to carbon dioxide and HA ChIP assay was performed as reported previously , 28. AR Human promoter regions of GO and NADPH oxidase subunit p22-PHOX were amplified and cloned into the pGL3 vectors, and the luciferase reporter assays were performed according to the methods used in a previous study .For the in vivo rescue experiment, apocynin (10 mg/kg/d) or saline was injected ip into 3 groups of mice every day for 7 days, followed by coinjection with glyoxylate (100 mg/kg/d) for 7 days: group 1 , saline control + glyoxylate; group 2 , saline control + glyoxylate; and group 3 , apocynin+ glyoxylate. After the treatment, the mice were killed, and kidneys were obtained.P value <.05 was considered statistically significant.Quantitative data are presented as means ± SE. Statistical significance among the control group and various treated groups was determined by ANOVA. A We performed clinical surveys by reviewing 167 patients with kidney stones seen in our hospital from 2011 to 2012 and found that 79% of patients with nephrolithiasis had oxalate stones, 11% had uric acid stones, and 10% had other stone subtypes A. FurtheP < .001, n = 25). Female patients with kidney stones also had higher testosterone concentrations than age-matched non–stone-forming patients .Using the ELISA for serum testosterone, we found that male patients with kidney stones had significantly higher concentrations than age-matched non–stone-forming males (P = .012 and P = .028).We next examined AR expression by IHC staining and qPCR assay and found strong AR-positive signals and relatively higher AR mRNA expression in male patients with kidney stones than in non–stone-forming patients .To test the hypothesis that the AR accounts, at least in part, for the sex difference in kidney stone formation we used the cre-loxP system to knock out the AR. Mice lacking systemic AR in the whole body (named TARKO) were generated from the mating of female floxAR mice with male ACTB-Cre mice . Taken together, these results support the human data demonstrating a positive role for AR signaling in promoting CaOx crystal formation.We assayed 24-hour urine oxalate excretion and found lower oxalate excretion in TARKO mouse urine than in the WT littermate control urine . As expected, there was also less urinary oxalate in these Alb-ARKO mice than in the WT littermate controls .Most oxalate is synthesized in the liver , 31. To P < .05 for TARKO/WT, P < .05 for Alb-ARKO/WT). Higher GO mRNA expression was then detected in human patients with kidney stones than in non–stone-forming male patients (P = .001).We then examined the expression of enzymes involved in the biosynthesis of oxalate in the liver and found that mRNA expression of GO , 34, butP < .05), and, consequently, higher oxalate secretion was detected in the culture media of these cells (P < .01 for 48 and 72 hours).The positive role of AR in promotion of GO expression was also shown in in vitro cell line studies. Human liver carcinoma HepG2 cells had higher GO mRNA expression when AR was incorporated into the cells (P = .03).The ChIP assay  demonstrTo investigate the role of AR in the mouse kidney, we developed 2 kinds of kidney-specific ARKO mice. AR was knocked out in either the proximal or distal/collecting tubular cells. Female floxAR mice were mated with male Kap-Cre mice  to generP = .037). Similarly, distal/collecting tubular cell–specific ARKO (CDH16-Cre) mice were developed using the CDH16-Cre mice .Kap-ARKO mice had less CaOx crystal formation in kidney tissues than the WT mice .Early reports suggested that the CaOx crystal formation in kidney is associated with OS-induced  renal tu2O2 detection, respectively, in their culture media (P = .03 for HKC5 and P = .007 for HKC8 cells). The consequences of the AR-altered H2O2-induced OS was also demonstrated by the decreased 8-OHdG level .We then confirmed this result in in vitro cell line studies. The addition of AR into the HKC5 cells or knockdown of AR in the HKC8 cells . These data explain why we observed significantly reduced CaOx crystal formation in the 2 types of kidney epithelial ARKO mice than in the WT mice in The increased OS might lead to renal cell injury. Therefore, we tested whether the LDH  level, wP = .008). Similar results were obtained in cell line studies. Adding AR into the HKC5 cells or knocking down AR in HKC8 cells led to altered expression of the p22-PHOX subunit at the mRNA level (P = .007 for HKC5 and P = .018 for HKC8 cells). We also detected p22-PHOX mRNA expression in human tissues and found higher p22-PHOX mRNA expression in patients with kidney stones than in non–stone-forming male patients (see P = .02).We also examined the expression of p22-PHOX, the subunit of NADPH oxidase , becauseents see ; P = .02P = .036).To further dissect the mechanism(s) by which the AR in kidney tubular cells modulates p22-PHOX subunit expression at the transcriptional level, we used the ChIP and luciferase assays. We found that AR could bind to the p22-PHOX subunit at the ARE (AGAACTnnnAGAGCC) site located from −1260 to −1246 bp in the 5′ promoter region of the p22-PHOX subunit gene C, and thP = .016), although the inhibitory level does not reach to the level shown in the TARKO mice (P = .007 compared with WT mice), suggesting that the inhibition of NADPH activity partially rescued the AR-promoted crystal formation in mouse kidney.Finally, to verify whether the AR-regulated p22-PHOX subunit expression influences CaOx crystal formation, we studied the effects of apocynin , an inhiTaken together, the results of All results from Figures 12O2 secretion into the culture media  or control vehicle for 2 weeks, followed by coinjection (ip) with the glyoxylate (100 mg/kg) for another week B-a. As sP = .003). The lower level of the excreted oxalate was also found in 24-hour urine collections (P = .041). Lower levels of urine H2O2 (P = .038) were also detected in the ASC-J9-pretreated mice than in the vehicle-injected control mice.We examined the CaOx crystal formation in mouse kidney using polarized microscopy after Pizzolato staining and found fewer CaOx crystals in the ASC-J9–pretreated mice than in the vehicle control mice (P = .027). The ASC-J9-treated mice also exhibited lower numbers of the 8-OHdG positively stained cells than the control mice (P = .002). Interestingly, the ASC-J9-treated mice also showed significantly lower GO mRNA expression in liver tissues (P = .007).We then examined the AR-modulated downstream genes in these mice and found less expression of the NADPH oxidase p22-PHOX in the ASC-J9-pretreated mice than in the vehicle control mice  have beOur findings here represent the first to identify the AR as a key player in promoting CaOx crystal formation via 2 distinct mechanisms. Theoretically, these findings provide a new potential target, the AR, that can be developed for new drug(s) to suppress this disease.ASC-J9 is a newly developed AR degradation enhancer that can selectively degrade AR in certain cell types. Mice that received 3 weeks of treatment with ASC-J9 showed no significant difference in appearance and had little change in serum testosterone concentrations , and sevIn conclusion, the current studies comprehensively revealed, for the first time, that enhanced AR signaling plays promoter roles in the early stages of CaOx crystal formation by increasing oxalate biosynthesis in hepatocytes and the OS–induced kidney tubular cell injury  and thatSupplementary data supplied by authors.Click here for additional data file.


PCGEM1 and PRNCR1 were implicated in progression of prostate cancer (PCa) as transcriptional co-regulators of the androgen receptor (AR). However, these findings were recently refuted asserting that neither gene physically binds to the AR. Despite evidence for differing AR transcriptional programs in vivo and in vitro, studies investigating AR-regulation of these genes hitherto have only been conducted in vitro. Here, we further examine the relevance of PCGEM1 and PRNCR1 in PCa, and their relationship with AR signaling, using patient-derived xenograft models.Long non-coding RNAs (lncRNAs) can orchestrate oncogenic or tumor-suppressive functions in cancer biology. Accordingly, PCGEM1 to be considerably expressed, while PRNCR1 showed scant basal expression. PCGEM1 was sharply down-regulated following castration and up-regulated upon AR activation in vivo. However, we found no parallel evidence following AR stimulation in vitro. A PCGEM1-associated gene expression signature (PES) was significantly repressed in response to androgen ablation therapy and in hormone-refractory versus hormone-naïve PCa patients. Furthermore, we found PCGEM1 was uniformly distributed in PCa cell nucleus and cytoplasm which remained unaltered upon AR transcriptional activation. PCGEM1 was up-regulated in primary PCa but not in metastasized PCa. Accordingly, the PES was significantly down-regulated in advanced and higher grade PCa patients from multiple independent studies.RNA sequencing of two distinct androgen-dependent models shows PCGEM1 as an in vivo androgen-regulated transcript with potential nuclear and/or cytoplasmic function(s). Importantly, the clinical expression profile of PCGEM1 implicates it in the early stages of PCa warranting further research in this direction.Our results demonstrate The online version of this article (doi:10.1186/s12943-015-0314-4) contains supplementary material, which is available to authorized users. PCAT1) [SChLAP1) [PCA3) [PCGEM1; aka PCAT9) [PRNCR1; aka PCAT8) [SChLAP1 has been extensively validated in the clinics as a biomarker of aggressive PCa [PCAT18  as a mediator of metastatic progression based on expression profiling of our patient-derived PCa xenograft models from the Living Tumor Laboratory (LTL) [PCGEM1, a highly prostate-specific transcript, was one of the first oncogenic lncRNAs to be described in PCa [PRNCR1 is not as well investigated, although its knockdown reportedly inhibits cell viability [In recent years, long non-coding RNAs (lncRNAs) have emerged as major contributors to cellular homeostasis as well as initiation and progression of numerous diseases , includiPCAT1) , second SChLAP1) , prostat) [PCA3) , prostata PCAT8) . Notablysive PCa  and PCA3sive PCa . Recentlry (LTL) . PCGEM1,d in PCa  and alsod in PCa . On the iability  – a claiiability .PCGEM1 as an AR regulated gene. Later on, PCGEM1 acting in complicity with PRNCR1 was shown to physically bind to the AR, thereby facilitating its ligand-independent transcriptional activity in castration resistant PCa (CRPC) [PCGEM1 nor PRNCR1 interacted with the AR to render androgen-independence, and that both genes had no prognostic relevance in PCa [PCGEM1 and PRNCR1 transcripts being AR regulated.AR is a ligand-responsive regulatory protein that mediates the effector functions of androgenic hormones in PCa. It is well established that sustained AR activity is indispensable for PCa cell survival and disease progression, even following androgen-deprivation therapy -20. Thisa (CRPC) . In conte in PCa . FurtherPCGEM1/PRNCR1 hitherto have been derived from in vitro experiments, using androgen-sensitive LNCaP cells [PCGEM1 and PRNCR1 are relevant in PCa and/or regulated by AR using our LTL patient-derived xenograft PCa models. The LTL has established a large panel of patient-derived PCa xenograft models that, unlike cell lines, retain key biological properties of the original malignancy, including histopathology, genomic profile, cellular heterogeneity, and invasive and metastatic ability [Notably, all published data on the relationship between AR and aP cells ,17. TheraP cells . In thisaP cells . This pu ability .PCGEM1 was considerably expressed , the expression of PRNCR1 was <8 FPKM in both models  for PCa xenotransplantation of the LTL-331 tumor line. Corresponding to the dosages, serum testosterone levels of 3.96 ± 0.679 ng/ml and 20.6 ± 14.1 ng/ml were achieved in the testosterone supplemented animals versus 1.95 ± 0.106 ng/ml in the intact hosts. Notably, the hosts supplemented with pure testosterone showed a dose-dependent up-regulation of PCGEM1, from 5-fold to greater than 22-fold, relative to the hosts without the augmented testosterone  of mice bearing the LTL-331 PCa xenograft. Tissue samples were collected just prior to castration and 3 weeks after castration, and were analyzed for gene expression. We observed a significant >500-fold decrease in in vitro studies investigating AR-regulation of PCGEM1 produced conflicting results. Srikantan et al. [PCGEM1 is AR-regulated, while Prensner et al. [PCGEM1 upon AR stimulation. The reason for this discrepancy could lie in the varied experimental conditions employed; in particular, the use of different agonists (viz. dihydrotestosterone (DHT) and synthetic R1881) for AR activation. Although the patterns of DHT-mediated and R1881-mediated gene expression changes are largely parallel, they do not completely overlap [PCGEM1, LNCaP and VCaP, were chosen for the in vitro studies . Similar observations were made in VCaP cells upon treatment with DHT  following stimulation of LNCaP cells with R1881 – a synthetic AR ligand  and MALAT1 (>80% for both) authenticate our findings. A similar sub-cellular distribution of PCGEM1 was observed in DHT treated and untreated LNCaP cells as well  (Additional file PCGEM1, the PES was also significantly up-regulated in PCa relative to other neoplasms and in PCa versus normal prostate tissue (Table PCGEM1 in early stages of PCa, PES was consistently repressed in metastatic and high Gleason PCa relative to primary and low Gleason neoplasms, respectively (Table Our results to this point indicate  samples . Here we to CRPC , our anaPCGEM1 is up-regulated in normal prostate epithelial cells of men with a family history of PCa [PCGEM1 gene are associated with a higher risk of PCa development in Chinese men [PCGEM1 is more important in the early stages, possibly initiation, of primary PCa. Interestingly, the PCGEM1 signature was also under-expressed in hormone-refractory PCa relative to hormone-naïve PCa (Table in vivo AR-regulation of PCGEM1.A review of literature also reveals that y of PCa  and certnese men . TogethePCGEM1. Simultaneously, it underscores the importance of using clinically relevant models that best represent the original malignancy. With mounting evidence for lncRNAs implicated in carcinogenesis and cancer progression, we regard lncRNAs as essential in decrypting cancer cell biology. Our study corroborates the irrelevance of PRNCR1 in PCa, and confirms PCGEM1 as an in vivo AR-regulated transcript as well as rationalizes its oncogenic involvement in early stages of the disease.Our work brings in light an even more pertinent concern with exploring AR transcriptional activity using cell line models. Recently, cell line models in accordance with their metastatic origin were demonstrated to exhibit the AR regulatory program that is active in metastatic advanced PCa as opposed to primary PCa . The AR


Prostate cancer growth is dependent upon the Androgen Receptor (AR) pathway, hence therapies for this disease often target this signalling axis. Such therapies are successful in the majority of patients but invariably fail after a median of 2 years and tumours progress to a castrate resistant stage (CRPC). Much evidence exists to suggest that the AR remains key to CRPC growth and hence remains a valid therapeutic target. Here we describe a novel method to inhibit AR activity, consisting of an interaction motif, that binds to the AR ligand-binding domain, fused to repression domains. These ‘engineered repressors’ are potent inhibitors of AR activity and prostate cancer cell growth and importantly inhibit the AR under circumstances in which conventional therapies would be predicted to fail, such as AR mutation and altered cofactor levels. The Androgen Receptor (AR) is a ligand-dependent transcription factor and like other members of the Nuclear Receptor family, has a modular structure consisting of the N-terminal domain, the central DNA-binding domain and the C-terminal ligand-binding domain . The recCoactivators are proteins that bind to and enhance the activity of transcription factors while not directly binding DNA themselves. Steroid receptor coactivators, such as the p160 family, often interact with the AF-2 surface of agonist-bound nuclear receptors, via an α-helical LxxLL motif , 8. HoweProstate cancer growth is almost always dependent upon the androgen-signalling axis and as a result, androgen deprivation therapy is a common treatment option for this disease. This involves one or both of chemical castration to ablate circulating androgens and antiandrogens – steroidal or non-steroidal ligands that bind to the AR LBD but do not promote subsequent target gene activation, either due to simple competition, by preventing receptor dimerization and/or nuclear translocation and DNA-binding, by promoting formation of a repressive complex at the androgen response element (ARE), or a mixture of mechanisms , 17. Altet al., for example, demonstrated that ectopic expression of a decoy androgen response element was successful at reducing AR activity [et al. performed a yeast 2-hybrid peptide screen against the full-length AR in the presence of the antiandrogen hydroxyflutamide [Due to the limited efficacy of current antiandrogens, novel methods to inhibit AR signalling are being devised that directly target different regions of the AR. Zhang activity . Other sactivity -22. Theslutamide . Fusion Here we describe the design and validation of AR engineered repressors that combine the desirable characteristics of coactivators and corepressors, in that they interact with the AR when it is in a holo conformation and block its activity. These consist of an interaction motif containing an FxxLF motif, fused to potent repression domains. Importantly, we demonstrate that these factors are successful in inhibiting the AR in circumstances thought to lead to castrate resistant prostate cancer.23FQNLF27 motif known to interact with the AR LBD (termed the interaction motif), to known repression domains from different proteins: MAD , KOX (aids 7-35 ) and PLZids 7-35 ). The re4 Figure . These r7-35-AR1-54 was fused to GFP and co-transfected into COS-1 cells with an AR expression vector. Confocal microscopy demonstrated that MAD7-35-AR1-54 is predominantly nuclear and appears to colocalise with the agonist bound AR  also pulled-down full-length AR , which should be specific for the AR. To investigate if the other closely related steroid receptors  were also inhibited by the engineered repressors, transcription assays were performed using these receptors. COS-1 cells were therefore transfected with plasmids encoding PLZFR Figure .7-35-AR1-54  and TIF2  were over-expressed in the majority of CRPCs [1-54) was unable to compete with this enhanced activity . To y Figure . By conty Figure .et al. found the T877A substitution in 25% of metastatic PCa samples analysed [1-54 for all ligand conditions.Mutations in the AR gene are rare in the early stages of prostate cancer, but their frequency increases significantly in advanced stages of the disease , 36. Foranalysed . Some mu1-54 had little effect upon proliferation . Exogenous expression of MAD7-35-AR1-54 and KOX1-75-AR1-54 significantly reduced colony formation, with no colonies emerging in any experiment using the latter construct  stage. Few therapeutic options exist for this stage of the disease, but much evidence exists to suggest that the AR continues to drive CRPC growth, and hence remains a valid therapeutic target .1-54), fused to known repression domains from a number of potent transcriptional repressors, namely KOX, MAD and PLZF [1-54 interaction motif generated potent repressors of AR activity. Mutation of the interaction motif (FQNLF to FQNAA) completely abolished repression, whereas mutation of the MAD7-35 repression domain significantly reduced, but did not abolish inhibitory potential, likely as a result of the intact interaction motif since this alone possesses some inhibitory activity  can successfully inhibit AR activity -23. We rand PLZF -26). Thectivity ( and data1-54) containing the 23FQNLF27 motif that mediates the N-/C-terminal interaction [1-452-AR1-54 repressor was found to be more specific for the AR but did also weakly inhibit the other steroid receptors. Previous studies using phenylalanine-rich motifs have also found weak cross-reactivity with these steroid receptors [Upon ligand binding, steroid receptors interact with coactivators, which enhance transcriptional activity . Despiteeraction . The reseceptors , hence rMultiple mechanisms have been described to explain therapy failure and CRPC, including alterations in AR cofactor levels/localisation. Transcriptional competition assays showed that, while the interaction motif alone was unable to compete with the effects of increased SRC1 expression, all of the engineered repressors were able to successfully inhibit AR activity under these conditions.Mutation of the AR is another mechanism believed to contribute to CRPC . IdentifWe have therefore demonstrated that fusion of repression domains with an AR-specific interaction motif generates potent inhibitors of the AR that are more potent than interaction motifs alone, and that these constructs are active in circumstances where conventional therapeutics would be predicted to fail. Growth assays demonstrated that these repressive effects upon the AR do translate to growth inhibition of prostate cancer cells. Therefore, the use of engineered repressors is a potential therapeutic approach to inhibit CRPC, and methods to refine and deliver such constructs, for example viral delivery or nanoparticles, merit further investigation.Mibolerone was purchased from Perkin Elmer . Oestradiol, Progesterone, Dexamethasone and Cyproterone Acetate were from Sigma Aldrich  and Bicalutamide and Hydroxyflutamide from Toronto Research Chemicals .XhoI and BamHI. Repression domains from KOX (a.a. 1-75), MAD (a.a. 7-35) and PLZF (a.a. 1-452) were subsequently PCR cloned upstream of AR1-54 using EcoRI and XhoI. To generate the pCDNA4.0  engineered repressor constructs, the repressors were PCR cloned into the KpnI and XbaI sites. Site directed mutagenesis  was performed on the pSG5-MAD7-35AR1-54 construct to generate constructs with mutation of the AR1-54 domain (FxxLF to FxxAA) or mutation of the repression domain (L12P and A16P). All constructs were confirmed by diagnostic digest and sequencing. The following plasmids have been previously described: pSG5-SRC1e [Amino acids 1-54 of the AR were PCR amplified and cloned into pSG5 using G5-SRC1e , pSVAR [G5-SRC1e , TAT-GREF a.a. 1-2 were su2. COS1 cells were grown in Dulbecco's modified Eagle's medium (DMEM) and LNCaP/LNCaP-C42 cells in Roswell Park Memorial Institute (RPMI) medium 1640 , both supplemented with 2mM L-Glutamine, 100units/ml penicillin, 100mg/ml streptomycin (PSG) and 10% foetal bovine serum. PC3-WTAR cells [COS1 and LNCaP cells were obtained from ATCC and grown at 37°C 5% COAR cells  were gro7-35AR1-54 using FuGENE 6  and incubated for 24hrs before treatment with ligand for 2hrs. Wells were washed 3x with PBS and incubated in 1% paraformaldehyde for 10 min, washed 3x in PBS and incubated for a further 10 min in 0.1% Triton X-100 in PBS. Wells were again washed 3x with PBS, incubated in blocking solution (5% BSA in PBS) for 30 min and a further 1 hr with an AR antibody  diluted 1:200 in blocking solution. Wells were washed 3x with PBS, re-blocked and incubated for 1hr with 594-Alexa Fluor conjugated secondary antibody . A final 3x PBS washes was performed before the coverslips were mounted onto glass slides . Images were obtained using a Zeiss Confocal Microscope, as previously described [COS1 cells were plated at 30% confluence on cover slips in 24 well plates. Cells were transfected with pSVAR and pGFP-MADescribed .7-35AR1-54 using FuGENE 6.0 . Cells were left for 24hrs, treated for 2hrs ± 10nM Mibolerone and lysed in IP buffer  containing freshly added protease inhibitors. Lysates were spun at 13,000 rpm , supernatants transferred to fresh tubes and protein concentration measured using the DC protein assay . Lysates were pre-cleared with 50 μl of sepharose beads , supernatant transferred to fresh tubes and incubated with anti-GFP antibody  for 1hr with rotation before addition of 50 μl of sepharose beads. After 1hr, beads were washed 3 times with IP buffer, laemmli loading buffer added to the beads and samples boiled before protein separation using immunoblotting, as previously described [COS1 cells were seeded at 70% confluence in 10cm plates in ‘stripped medium’ and transfected with plasmids encoding the AR and GFP-MAD1-54, pSG5-MAD7-35-AR1-54, KOX1-75-AR1-54, PLZF1-452-AR1-54, pSG5-MAD7-35-AR1-54(FQNAA)or pSG5-MAD7-35-AR1-54. PC3-WTAR cells were transfected with 50ng PDM-LACZ-β-GAL, 250ng TAT-GRE-E1B-LUC and 0-200ng of pSG5-AR1-54, pSG5-MAD7-35-AR1-54, KOX1-75-AR1-54, PLZF1-452-AR1-54 using FuGENE HD  following the manufacturer's instructions. Twenty-four hours after transfection, cells were treated with 10nM of ligand and cells incubated for a further 16 hrs. Luciferase and β-galactosidase expression was measured as previously described [COS1 cells were transfected using the calcium phosphate method as previously described . Cells wescribed .1-54, MAD7-35-AR1-54, KOX1-75-AR1-54 or PLZF1-452-AR1-54 using JetPrime , following the manufacturers instructions. 24hrs after transfection (set as day 0), GFP positive cells were visualised in 10 random fields of view from triplicate wells using an Axiovert Fluorescent Microscope  and the number of GFP positive cells assessed using Image J (NIH). Subsequent cell counting was performed on days 3 and 7.LNCaP cells were plated to 50% confluence in 6 well plates in ‘stripped media’ containing 10nM Mibolerone and co-transfected with pGFP-Empty and plasmids encoding ARet al. [2O and cells counted manually.Colony formation assays were based on Kawano et al. . Briefly


Jeotgalibacillus soli, a bacterium capable of degrading N-acyl homoserine lactone, was isolated from a soil sample in Portugal. J. soli constitutes the only Jeotgalibacillus species isolated from a non-marine source. Here, the draft genome, several interesting glycosyl hydrolases, and its putative N-acyl homoserine lactonases are presented. Jeotgalibacillus is under explored halophilic genus of family Planococcaceae. The cell wall peptidoglycan of members of this genus is of the A1α type, linked directly through l-Lys. The major quinones of Jeotgalibacillus spp. are MK-7 and MK-8 . Other species such as J. alimentarius, J. salarius, J. malaysiensis, and J. campisalis, for instance, are able to tolerate concentrations of 15 to 30% (wt/vol) ((wt/vol) .N50 of 525,494. The de novo assembly was performed using SPAdes  that act on α-1,4 and α-1,6 glycosidic bonds. In addition, our detection of the key glycogen-degrading enzyme oligo-1,4-1,6-α-glucosidase is consistent with the capacity of J. soli to grow on glycogen. Although β-glucosidase activity was not observed using a standard API 50 CHB/E test (bioMérieux), a gene encoding this enzyme was identified in the J. soli genome. In addition, the N-acyl homoserine lactone (AHL) degradation capability of J. soli was validated using an AHL inactivation assay performed with N-hexanoyl-l-homoserine lactone and N-(3-oxohexanoyl)-l-homoserine lactone, and several putative N-acyl homoserine lactonases (J. alimentarius, J. salarius, J. malaysiensis, and J. campisalis were found to be unable to degrade AHL.The G+C content of the ctonases  were ideJXRP00000000. The version described in this paper is the first version, JXRP01000000.This whole-genome shotgun project has been deposited in DDBJ/EMBL/GenBank under the accession no.


The satisfactory desorption capacity of 81.34 mg/g was obtained at a HNO3 concentration, desorption temperature, and desorption time of 0.2 mol/L, 60 ºC, and 30 min, respectively. The regeneration studies showed that the adsorption capacity of the LNC/MMT nanocomposite was consistent for five cycles without any appreciable loss in the batch process and confirmed that the LNC/MMT nanocomposite was reusable. The overall study revealed that the LNC/MMT nanocomposite functioned as an effective adsorbent in the detoxification of Ni(II)-contaminated wastewater.A new and inexpensive lignocellulose/montmorillonite (LNC/MMT) nanocomposite was prepared by a chemical intercalation of LNC into MMT and was subsequently investigated as an adsorbent in batch systems for the adsorption-desorption of Ni(II) ions in an aqueous solution. The optimum conditions for the Ni(II) ion adsorption capacity of the LNC/MMT nanocomposite were studied in detail by varying parameters such as the initial Ni(II) concentration, the solution pH value, the adsorption temperature and time. The results indicated that the maximum adsorption capacity of Ni(II) reached 94.86 mg/g at an initial Ni(II) concentration of 0.0032 mol/L, a solution pH of 6.8, an adsorption temperature of 70°C, and adsorption time of 40 min. The represented adsorption kinetics model exhibited good agreement between the experimental data and the pseudo-second-order kinetic model. The Langmuir isotherm equation best fit the experimental data. The structure of the LNC/MMT nanocomposite was characterized by X-ray diffraction (XRD) and transmission electron microscopy (TEM), whereas the adsorption mechanism was discussed in combination with the results obtained from scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), and Fourier-transform infrared spectroscopy analyses (FTIR). The desorption capacity of the LNC/MMT nanocomposite depended on parameters such as HNO Water contamination caused by heavy-metal ions generated from alloys, pigments, electroplating, mining, metallurgical activities, nuclear power plant operations, aerospace industries, electrical contacts, printing, and the manufacture of paper, rubber, plastics, and batteries , 2 is a Lignocellulose (LNC) is an ideal natural adsorbent for the removal of heavy-metal ions because of its specific structural characteristics . LNC, whThe objective of the present study was to evaluate the efficiency of lignocellulose/montmorillonite (LNC/MMT) nanocomposites in the removal of Ni(II) ions from model dilute aqueous solutions intended to mimic low-metal-concentration wastewaters. The effects of environmental parameters such as the initial Ni(II) concentration, pH level, adsorption temperature, and adsorption time on Ni(II) uptake were investigated, and the desorption of Ni(II) adsorbed onto the LNC/MMT nanocomposite was also examined. Different kinetic models were analyzed using pseudo-first-order and pseudo-second-order models. The Langmuir and Freundlich models are commonly used to describe adsorption equilibrium data and were applied to the experimental results. The behavior of the LNC/MMT nanocomposite was studied in continuous mode during adsorption-desorption recirculation experiments performed at the laboratory scale to investigate the feasibility of a cost-effective process for industrial applications. Moreover, the structure and presence of functional groups on the LNC/MMT nanocomposite, which may play an important role in the adsorption process, were confirmed by XRD, TEM, SEM-EDX, and FTIR to elucidate the interaction mechanism between Ni(II) ions and the LNC/MMT nanocomposite.−1) was purchased from Zhejiang Feng Hong Clay Chemical Co., China. MMT was washed using deionized water, dried overnight at 70°C, milled using a blender and finally sieved to 200 mesh particle size. Ni(NO3)2·6H2O was purchased from Shanghai Jinshan Chemical Co., China. All other chemicals and reagents used in the study were of analytical grade and used without further purification. All solutions were prepared using deionized water.LNC (SAM-100) was purchased from Beijing Huaduo Biotech, Ltd., China. MMT (CEC = 100 meq·(100 g)Reagents. A stock Ni(II) solution (1 mol/L) was prepared by dissolving a weighed quantity of analytical-grade Ni(NO3)2·6H2O in distilled deionized water and diluting to a concentration of 1 mol/L. In this study, the 1 mol/L Ni(II) stock solution was subsequently used to prepare solutions with initial Ni(II) concentrations of 0.0025 to 0.0036 mol/L, and the pH value of each Ni(II) solution was adjusted to the desired value using 0.1 mol/L HCl or NaOH solution. Fresh diluted solutions were used for each experiment.Adsorbents. The LNC/MMT nanocomposite was prepared as follows: a target amount of LNC was dissolved in NaOH solution [1:30 ratio of LNC weight (g) to NaOH volume (mL)] in batches and magnetically stirred, forming a uniform LNC suspension. A suspension of MMT (1.0 g suspended in 30 mL of distilled deionized water) was subsequently stirred for 0.5 h at 500 rpm, followed by addition to the LNC-NaOH suspension. The reaction mixture was heated to 60°C and stirred for 6 h, centrifuged using a centrifuge  and washed with HCl and NaOH solutions until the pH of the supernatant was 7.0. The pH of the solution was measured using a pH meter . Finally, the obtained material was dried under vacuum at 105°C in a vacuum oven  for 5 h until a constant weight was achieved. All samples were ground and sieved to a 200 mesh particle size and were subsequently stored in an airtight plastic container until used for the specific experiments.Characterization. X-ray diffraction (XRD) analysis of the powdered samples was performed using a PANanalytical X’pert PRO X-ray powder diffractometer equipped with a Cu anode operated at 40 kV and 30 mA; the samples were scanned at a rate of 3°/min from 4° to 18°. TEM image analyses of the samples were performed on a JEM-2010 electron microscope operated at 200 kV. The surface morphology of the samples was recorded by scanning electron microscopy . All of the samples were fixed onto aluminum stubs and coated with gold prior to SEM observation; the surface elemental analyses of Ni(II) on the LNC/MMT nanocomposite were conducted by energy-dispersive X-ray spectroscopy . The infrared spectra of the unloaded and Ni(II)-loaded LNC/MMT composites were obtained using Fourier-transform infrared spectrophotometry . The LNC/MMT nanocomposite was crushed, ground, and encapsulated in KBr pellets to prepare translucent sample disks; FTIR spectra were recorded in the range of 4000–500 cm-1 by averaging 20 scans at a maximum resolution of 4 cm-1.Adsorption experiments. LNC/MMT (0.1000 g) was accurately weighed and added to 50 mL of the Ni(II) solution. The suspension was stirred at a uniform speed of 120 rpm in a thermostatic shaker (SHA-C). Throughout the course of the experiments, the pH of each Ni(II) solution was adjusted to a constant value by periodic shaking after the addition of 0.1 mol/L HCl or NaOH solution. When adsorption equilibrium was reached, the mixtures were centrifuged at 6000 rpm for 5 min. Then, the Ni(II) concentration of the upper fluid was determined by the dimethylglyoxime method  are the rate constants of the pseudo-first-order and pseudo-second-order adsorption kinetic equations, respectively.The adsorption kinetic curve of the LNC/MMT nanocomposite was modeled by fitting the pseudo-first-order  and the R2) of the pseudo-second-order adsorption kinetic equation was higher than that of the pseudo-first-order kinetic equation. The adsorption amount obtained experimentally at equilibrium was similar to that determined from the pseudo-second-order adsorption kinetic equation , this diffraction peak correlates to a basal spacing of d = 1.52 nm. After the MMT was intercalated with LNC (2θ = 5.97°). Moreover, the intensity of the diffraction peak decreased and even disappeared which indicated the formation of a flocculated-intercalated nanostructure. Based on the XRD results, almost all of the LNC intercalated into the MMT interlayer, resulting in the destruction of the structure of MMT. XRD was an effective method for investigating the intercalation of LNC into MMT.with LNC , the typTEM analysis. SEM analysis. The SEM images of LNC/MMT before and after Ni(II) adsorption are shown in EDX analysis. The LNC/MMT was analyzed using EDX to explain the mechanisms of Ni(II) adsorption onto LNC/MMT. As shown in FTIR analysis. To determine the primary functional groups of the LNC/MMT nanocomposite involved in the adsorption of Ni(II) ions, FTIR spectra were recorded for the LNC/MMT nanocomposite after the adsorption and desorption of Ni(II) ions (−1 (−1 (−1 (−1 after the desorption of Ni(II) (−1 (−1 (−1 (−1 (−1 (II) ions . The resions  . The vib(II) -loaded LNC/MMT vs. desorption capacity are shown in 3 concentration. This observation is likely attributable to the increase in acid concentration leading to the accumulation of H+ in solution, thus increasing the concentration gradient of Ni(II) and H+ and resulting in an increased driving force for ion-exchange, favoring the desorption process. However, a higher concentration of HNO3 would dramatically increase the H+ concentration in solution and thus may enhance electrostatic repulsion among Ni(II) ions, inhibiting Ni(II) desorption [3 concentration of 0.2 mol/L.sorption . The maxEffect of temperature on desorption. The effects of different desorption temperatures on the Ni(II)-loaded LNC/MMT nanocomposite desorption capacity are shown in Effect of time on desorption. The effects of different desorption times on the Ni(II)-loaded LNC/MMT nanocomposite are shown in Recycling and reusability of LNC/MMT. The reuse of the LNC-MMT nanocomposite would decrease processing costs and may open the possibility of recovering the LNC/MMT nanocomposite after Ni(II) extraction from wastewater. Thus, the ability to recycle the LNC-MMT nanocomposite is a very important consideration with respect to its practical applications. The reusability of the LNC-MMT nanocomposite was investigated by six consecutive adsorption-desorption processes. The effects of the number of cycles on the Ni(II) ions adsorption/desorption capacity are shown in th process were 50.16 mg/g and 38.90 mg/g, respectively. The results of this study indicate that the LNC/MMT nanocomposite could be used as an adsorbent in practical applications for the removal of Ni(II). Therefore, the LNC-MMT nanocomposite has potential applications as a high-performance, inexpensive, and recyclable green adsorbent for wastewater treatment.R2 = 0.9980) and the Langmuir isothermal adsorption models (R2 = 0.9989), respectively, indicating that within the experimental range, the adsorption equilibrium was mainly dominated by monolayer chemical adsorption.A new LNC/MMT nanocomposite was prepared by chemical intercalation. The results of this study showed that the synthesized LNC/MMT nanocomposite could be used effectively for the adsorption of Ni(II) ions from aqueous solutions. The maximum Ni(II) ion adsorption capacity for the LNC/MMT nanocomposite reached 94.86 mg/g under an initial Ni(II) concentration, pH, adsorption temperature, and adsorption time of 0.0032 mol/L, 6.8, 70°C, and 40 min, respectively. The adsorption kinetics and isotherms were well fit to both the pseudo-second-order adsorption kinetic equation -loaded LNC/MMT nanocomposite were investigated using HNOThe adsorption-desorption experiments showed that the adsorption and desorption capacities of the LNC/MMT nanocomposite remained at a relatively high level after five adsorption-desorption cycles. Therefore, the LNC/MMT nanocomposite could be recycled if used to remove Ni(II) ions from Ni(II)-polluted wastewater.