Antimicrobial resistance (AMR) is a major global public health concern and its surveillance is a fundamental tool for monitoring the development of AMR. In 1998, the Nepalese Ministry of Health (MOH) launched an Infectious Disease (ID) programme. The key components of the programme were to establish a surveillance programme for AMR and to develop awareness among physicians regarding AMR and rational drug usage in Nepal.Vibrio cholerae, Shigella spp., Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria gonorrhoeae.An AMR surveillance programme was established and implemented by the Nepalese MOH in partnership with the International Centre for Diarrhoeal Disease Research, Bangladesh  from 1998 to 2003. From 2004 to 2012, the programme was integrated and maintained as a core activity of the National Public Health Laboratory (NPHL) and resulted in an increased number of participating laboratories and pathogens brought under surveillance. The main strategies were to build national capacity on isolation, identification and AMR testing of bacterial pathogens, establish laboratory networking and an External Quality Assessment (EQA) programme, promote standardised recording and reporting of results, and to ensure timely analysis and dissemination of data for advocacy and national policy adaptations. The programme was initiated by nine participating laboratories performing AMR surveillance on Salmonella spp. was added to the surveillance programme in 2002 and extended spectrum β-lactamase producing Escherichia coli in 2011). From 1999 to 2012, data were available on 17,103 bacterial isolates. During the AMR programme, we observed changing trends in serovars/ species for Salmonella spp., Shigella spp. and V. cholerae and changing AMR trend for all organisms. Notably, N. gonorrhoeae isolates demonstrated increasing resistance to ciprofloxacin. Additionally, the performance of the participating laboratories improved as shown by annual EQA data evaluation.The number of participating laboratories was ultimately increased to 13 and the number of pathogens under surveillance was increased to seven (This Nepalese AMR programme continues and serves as a model for sustainable surveillance of AMR monitoring in resource limited settings. Antimicrobial resistance (AMR) in bacterial pathogens reduces the effectiveness of antimicrobial agents leading to increased morbidity, mortality and healthcare costs . While tNepal is a low-income country (as determined by the World bank) with a poorly organised healthcare delivery system, suffering from resource limitations. Political unrest and the Maoist insurgency from 2001 to 2008 affected many sectors of life including sustainable health care delivery. Furthermore, pharmaceutical manufacturing and importation is poorly regulated and drugs, including antimicrobials, are available without prescription, and antimicrobial consumption on sub-therapeutic dose, for sub-optimal duration is common. Additionally, pharmaceuticals are often stored in sub-standard conditions, compromising product quality. The public health care system in Nepal has a limited laboratory capacity for bacterial culture and AMR testing .Vibrio cholerae and Shigella spp.), respiratory pathogens (Streptococcus pneumoniae and Haemophilus influenzae) and sexually transmitted pathogen (Neisseria gonorrhoeae). The National Public Health Laboratory (NPHL) and the Epidemiology and Disease Control Division (EDCD) were identified as the national coordinating laboratory and the national focal point for the programme, respectively.In 1998, the Ministry of Health (MOH), Nepal and the United States Agency for International Development (USAID) launched an Infectious Disease (ID) control programme to strengthen national capacity for prevention and control of priority infectious diseases. The goals were to develop a sustainable national surveillance of AMR, develop awareness among physicians regarding AMR and rational drug use, to establish a microbiology quality assurance programme and to systematise record keeping system with data dissemination. The programme in Nepal was implemented by the International Centre for Diarrhoeal Disease Research, Bangladesh  Dhaka, Bangladesh in collaboration with Rational Pharmaceutical Management Project (RPM) Arlington, VA, USA. The programme was approved by ICDDR, B research review committee and ethical review committee. The programme started with a participatory planning workshop to build the programme team and to establish a forum for stakeholders ;ICDDR, B; and WHO/Nepal). The workshop focused on development of strategies and approaches of the programme. The programme was designed for selected enteric pathogens  holding annual refresher training workshops. Additionally, consensus workshops and annual meetings were organised during 1999 to 2004. Laboratory testing and procedures were standardised by developing and implementing Standard Operating Procedures (SOPs) for the isolation, identification and AMR testing of the selected pathogens, reporting results, data/record keeping systems, AMR reporting formats, documentation and communication protocols. Each participating laboratory was supported with bacterial culture media, antimicrobial susceptibility discs and antisera at the outset and was encouraged to incorporate the supplies for surveillance of AMR in their annual procurement list. NPHL was also supported with autoclaves, computers and a -86°C freezer. Each laboratory was also supplied with ATCC (American type culture collection) strains for internal quality control (QC).A baseline assessment of 13 laboratories  was conducted in November 1998 using a pre-defined  assessment tool and nine laboratories were selected to implement the programme. A national capacity building initiative through training and workshops were conducted by ICDDR, B and NPHL at NPHL, ICDDR, B and at the participating laboratories, through 1999–2012. The goal was to develop a pool of trainers to train technologists to compensate for transfer, replacement or separation and to facilitate expansion of trained staff. Training included i) the training of trainers, ii) bacterial isolation and Minimum Inhibitory Concentration (MIC) determination, iii) the isolation, identification and MIC determination of N. gonorrhoeae was established at specific STD clinics at Damak and Hetauda. To strengthen partnerships and networking among the laboratories, training visits between NPHL and participating laboratories were organised. Meetings were also organised among participating laboratories to discuss surveillance programme data, technical issues and sharing experiences and challenges in implementation of the programme. Additionally, ICDDR, B and NPHL team routinely visited the laboratories and provided technical assistance to improve the performance. A two-tier system of External Quality Assessment (EQA) programme and inter-laboratory comparison was implemented to ensure quality. The EQA programme included ICDDR, B (1999–2003)/NHPL (2003–2012) sending two isolates to each of the laboratories every three months, and the inter-laboratory comparison included retesting of 10% isolates at ICDDR, B/NPHL (1999–2003) and NHPL (2003–2012). The WHO scoring system was followed for the EQA and confidential evaluation reports were sent to the laboratories.Each participating laboratory isolated and identified selected pathogens  and performed AMR testing  and repSalmonella spp. was included in the surveillance in 2002, following an outbreak of enteric fever due to a multiple drug resistant (MDR) Salmonella Typhi [Escherichia coli was added to the programme in 2009. From 1999 through 2003, ICDDR, B procured and supplied antisera, bacterial culture media and antimicrobial susceptibility discs for the programme. After 2003, the procurement of supplies was integrated into the annual laboratory procurement programme of NPHL.The AMR surveillance programme was initiated by a network of nine laboratories in 1999. Lumbini Zonal Hospital (LZH), Butwal, Dhulikhel Hospital (DH), Kabhre, Kathmandu Model Hospital, Katmandu and Kathmandu Medical College and Hospital, Kathmandu, joined the programme in 2006, 2008, 2009 and 2011 respectively  incorporate local innovative ideas generated by local experts , and ii) encourage participating laboratory management in incorporating surveillance supplies in their respective laboratory procurement plan. Annual meetings were organised among key partners and stakeholders of the programme to i) communicate the progress and the key challenges ii) receive input from all key partners, iii) advocate for increasing the budget for the laboratories and iv) minimise the transfer of surveillance programme staff.During 1998 to 2003, the ICDDR, B provided technical support to NPHL to function as the national coordination laboratory, and from 2004, NPHL resumed both coordination and technical support responsibilities. Since 2008, NPHL integrated the surveillance of AMR as one of its core activities to ensure the sustainability of the programme.V. cholerae, 390 Shigella spp., 1,875 S. pneumoniae, 933 H. influenzae, 118 N. gonorrhoeae, 11,763 Salmonella spp., and 623 E. coli) were reported and had corresponding AMR data generated . The programme created a pool of trained technicians who can compensate for transfer and separation of staff and contribute to expansion of programme staff. Stakeholders’ meeting contributed in motivating the MOH, Nepal to increase the resources for laboratories, and incorporating the programme as a core activity of the NPHL and participating laboratories. The programme laid a solid platform for conducting laboratory- based surveillance in Nepal. Annual meetings and dissemination programme helped to establish a forum for discussion on surveillance of AMR in Nepal.S. pneumoniae and H. influenzae isolates were resistant to cotrimoxazole, the health authorities initiated discussion on suitable alternatives for cotrimoxazole for respiratory infections in Nepal. Ciprofloxacin is no longer considered as the drug of choice for treating salmonella infections, because of reduced susceptibility, even though it appears susceptible in vitro. In this example, laboratory technicians were guided to use nalidixic acid resistance as a proxy screening test to determine reduced fluoroquinolone susceptibility [The surveillance of AMR programme in Nepal contributed to some significant policy changes with regard to choice of antimicrobials and vaccination strategies. The national STD case management guidelines in Nepal recommended a change from ciprofloxacin to cefixime as the first-line therapy for the management of uncomplicated gonococcal infection. Also, as more than 20% of pneumococcal isolates were from children below the age of five years, this facilitated evidence-based decision by the MOH to formulate a strategy for pneumococcal vaccination for Nepalese children . Cotrimotibility  and physThis study illustrates the success of implementing a AMR surveillance programme in a resource poor developing country. To ensure the quality of the surveillance of AMR programme, regular feedback, refresher training, technical support, updating of SOPs, dissemination workshops and motivation of the laboratory staff are vital. Programme planning should pay attention to data collection, compilation and storage; an electronic data collection system and appropriate data backup system can reduce the workload of manual/semi electronic labour-intensive system. The surveillance of AMR has continued in Nepal with minimal external financial support; financial dependency on donors should be eliminated. The expansion of laboratory network, gradual incorporation of other pathogens as per local need and resistance testing for additional antimicrobials may be necessary. Analysis and timely feedback of surveillance of AMR data and findings can ensure the use of the data to guide local practices. Attention should be paid to human resources, training, standardisation, capacity building and ensuring the ownership of the programme (isolates and data) by the participating laboratories to ensure sustainability.AMR: Antimicrobial resistance; MOH: Ministry of Health; ICDDR B: International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka; NPHL: National Public Health Laboratory; EQA: External quality assurance; WHO: World Health Organisation; USAID: United States’ Agency for International Development; RPM: Rational Pharmaceutical Management Project; USCDC: United States’ Center for Disease Prevetion and Control; MDR: Multiple drug resistant; EDCD: Epidemiology and Disease Control Division; MIC: Minimum Inhibitory Concentration; ATCC: American type culture collection; QC: Quality control; STD: Sexually transmitted disease; LZH: Lumbini Zonal Hospital; DH: Dhulikhel Hospital.The authors declare that they have no competing interests.SM: AMR programme planning, implementation, data analysis, manuscript review. GS: AMR programme planning, implementation, data analysis, manuscript review. SPD: AMR programme, implementation, data analysis. PK: AMR programme implementation, data analysis. BR: AMR programme implementation, data analysis. AH: AMR programme planning, coordination, implementation, data analysis, manuscript review. GBN: AMR programme planning and coordination, data analysis, manuscript review. MJA: AMR programme planning and coordination, data analysis, manuscript review. DS: AMR programme planning, securing funding, donor coordination, implementation monitoring, data analysis, manuscript review. SB: Data analysis, manuscript review. MR: AMR programme planning, coordination and implementation, data analysis, manuscript drafting and manuscript review. All authors read and approved the final manuscript.Antimicrobial Susceptibility Surveillance Programme team, Nepal and participating laboratories. i) Dr. Sarala Malla, Dr. Geeta Shakya and Mr. Shyam P Dumre, NPHL, Kathmandu, ii) Dr. Chandrika Devi Shrestha and Ms. Jyotsna Shrestha, Bir Hospital Laboratory, Kathmandu, iii) Mr. Ram Babu Shrestha and Ms. Sabina Dangol, Patan Hospital Laboratory, Kathmandu, iv) Dr. T. P. Rajbhandari and Ms. Maiya Shrestha, Kanti Children’s Hospital Laboratory, Kathmandu, v) Mr. Nuchchhe Ranta Tuladhar and Mr. Nabaraj Banjade, Tribhuvan University Teaching Hospital Laboratory, Kathmandu, vi) Dr. Syamal Bhattacharya and Dr. Basudha Khanal, B. P. Koirala Institute of Health Sciences, Dahran, vii) Mr. Jagat Khadka and Mr. Boj Bdr Sunuwar, Western Regional Hospital Laboratory, Pokhara, viii) Dr. P.G. Shivananda and Dr. Joshy M Eason, Manipal Teaching Hospital Laboratory, Pokhara, ix) Mr. Hem Bdr Nepali, United Mission Hospital Laboratory, Palpa, x) Mr. Binod Kumar Gyawali, Lumbini Zonal Hospital, Butwal, xi) Mr. Ganesh Neupane, Dhulikhel Hospital, Kabhre and EDCD/Ministry of Health and Population.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/14/269/prepub


PROK1-V67I has been shown to play a role as a modifier gene in the PROK1-PROKR system of human early pregnancy. To explore the related modifier mechanism of PROK1-V67I, we carried out a comparison study at the gene expression level and the cell function alternation of V67I, and its wild-type (WT), in transiently-transfected cells. We, respectively, performed quantitative RT-PCR and ELISA assays to evaluate the protein and/or transcript level of V67I and WT in HTR-8/SV neo, JAR, Ishikawa, and HEK293 cells. Transiently V67I- or WT-transfected HTR-8/SV neo and HEK293 cells were used to investigate cell function alternations. The transcript and protein expressions were down-regulated in all cell lines, ranging from 20% to 70%, compared with WT. There were no significant differences in the ligand activities of V67I and WT with regard to cell proliferation, cell invasion, calcium influx, and tubal formation. Both PROK1 alleles promoted cell invasion and intracellular calcium mobilization, whereas they had no significant effects on cell proliferation and tubal formation. In conclusion, the biological effects of PROK1-V67I on cell functions are similar to those of WT, and the common variant of V67I may act as a modifier in the PROK1-PROKR system through down-regulation of PROK1 expression. This study may provide a general mechanism that the common variant of V67I, modifying the disease severity of PROK1-related pathophysiologies. Prokineticin 1 (PROK1), also known as endocrine gland-derived vascular endothelial growth factor (EG-VEGF), is a small, secreted peptide that belongs to the prokineticin family ,3,4, andIn recent years, PROK1 has been shown to play a role in female reproduction and human pregnancy. The temporal and spatial expression of PROK1, and its receptors in ovary and early gestational tissue, highlights their functions in follicular maturation, luteal angiogensis, embryo implantation, and uterine receptivity ,8. The dhttp://www.ncbi.nlm.nih.gov/pubmed/). The evolution of V67I is highly conserved, and the change of amino acid from V to I seems neutral. However, the impact of this genetic variant in the PROK1 system and its clinical relevance have not yet been explored.A common polymorphism of PROK1, V67I , located in the first nucleotide of exon 3, is a non-synonymous substitution resulting in amino acid change from valine (V) to isoleucine (I). The frequency of G to A transition varies among different ethnicities, and ranges from 43% to 64% in the general population  act as a genetic modifier in human early pregnancy . Previouhttp://www.ncbi.nlm.nih.gov/pubmed/). In general, the frequency of the G to A transition is around 50%, ranging from 39% to 64%, and varies among different populations . When comparing the protein levels in the cell lysate and supernatant of the culture medium, the protein expression of V67I was also down-regulated in the HEK293 (42.5%–47.5%), and HTR-8/SV neo (60%–71%) cells and showed similar results to those found for the transcript expression (p < 0.001). The basal protein concentration in non-transfected cells (Mock) were between 11 and 28 pg/mL in each cell line .In order to evaluate gene expressions of PROK1-V67I and -WT, we compared different cells transfected with V67I or WT plasmids in the transcript and protein levels using qRT-PCR and ELISA assays, respectively. The transcript expression of V67I compared with WT was 50.5% in HEK-293 and 64% in HTR-8/SV neo cells , but there was no difference in this enhancement between the PROK1 and V67I variant groups. We further evaluated the cell invasion ability in the prokineticin receptor 1 (PROKR1)- or prokineticin receptor 2 (PROKR2)-transfected cells, and both cell invasion abilities increased in a dose-dependent manner after treating with concentrated PROK1 and V67I  from conditioned medium  and their ligand activities. We evaluated PROK1-induced intracellular calcium mobilization by using a fluorescence-based assay in the prokineticin receptor 1 (PROKR1)- or prokineticin receptor 2 (PROKR2)-overexpressed cells. In this assay, HEK293 and HTR-8/SV neo cells were initially transfected with PKOKR1 or PROKR2 plasmid to enhance the amplitude of calcium signaling before investigation. The intracellular calcium influx was stimulated, but no significant differences were seen in the PROKR1- or PROKR2-transfected cells after treating with 5 nM PROK1 or V67I from conditioned medium (In a previous genetic association study of recurrent pregnancy loss (RPL), a PROK1 variant (V67I) was shown to be a genetic modifier gene in early human pregnancy . We evalThe biological interaction of PROK1 and PROKR1/2 is a ligand-receptor relationship. The impact of an amino acid change in PROK1 on the protein activity and the binding affinity between its receptors remain unclear, and no PROK1 variants have been reported before. We identified PROK1-V67I to be the only non-synonymous variant in the coding regions of PROK1 using Sanger sequence in 291 subjects  . The nonThe PROK1 level during pregnancy is dynamic, both in placental tissue and serum collected from pregnant women. Placental PROK1 expression gradually rises after embryo implantation and peaks at 8–11 weeks of gestation, subsequently decreasing until the end of the first trimester, and then this level is maintained throughout the remaining gestation ,7,23. PlDisease severity could be influenced by different genetic backgrounds, and genetic modifiers are known to alter the outcomes in various human diseases or animal models of disease, such as muscular dystrophy (MD), epidermolysis bullosa (EB), and some motor neuron diseases ,33,34,35V67I allele and genotype frequencies of our population, Han Chinese individuals in Taiwan, with those of other HapMap Populations  , and informed consents were obtained from all patients and controls. The clinical and molecular details of the RPL subjects examined in the current work were reported in a previous study ,18. In tulations .2 humidified incubator at 37 °C. After confluent growth, the attached cells were trypsinized and either cryopreserved or subcultured for further use.The human HTR-8/SVneo trophoblast cell line was a gift from Dr. Charles Graham . HTR-8/SVneo cells were grown in RPMI 1640 medium  supplemented with 10% fetal bovine serum (FBS) and 100 IU/mL penicillin-streptomycin. The human embryonic kidney cell line (HEK293) were grown in Dulbecco Modified Eagle’s Medium (DMEM) (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% penicillin-streptomycin solution. JAR cells were derived from human placental choriocarcinoma, which were purchased from Bioresource Collection and Research Center , and grown in RPMI 1640 medium with 10 mM HEPES, 1 mM sodium pyruvate, 10% FBS and 1% penicillin-streptomycin solution. Ishikawa cells were grown in Minimum Essential Media (MEM) (Invitrogen) supplemented with 1% non-essential amino acids (NEAA) + 5% FBS and 1% penicillin-streptomycin solution. These cells were cultured in a 5% CO5 cells) and HTR8/SVneo (3 × 105 cells) using 1 μg of pCMV-EGFP  expressing plasmid and 3 µL of TurboFect in six-well plates for 24 and 48 h. The transfection efficiency is 90% ± 10% in the two cell lines. The established constructs were transfected into HEK293 and HTR-8/SVneo cell lines by Turbofect for 24 h , and their expressions were confirmed by Western blotting. The PROKR1 and PROKR2 plasmids were constructed as reported in a previous study [The variant sequence of PROK1 (V67I) was introduced into the WT Myc-DDK-tagged cDNA in a pCMV6-Entry vector , which encodes the entire coding regions of human cDNA (GenBank NM_032414.2) of PROK1, using Stratagene’s Quik Change II Site-directed Mutagenesis Kit . A vector control, pCMV6 empty vector, was constructed from pCMV6-PROK1 plasmid using sgfI and MluI digestion and the T4 ligation method. All constructs were verified by nucleotide sequencing. pCMV6 and PROK1 constructs were propagated in JM109 Escherichia coli. Transfection efficiency was tested on HEK293  RNaseOut, dNTPs and random primers according to the manufacturer’s protocols. The qPCR reactions were carried out in an Applied Biosystems StepOne Plus system . The primers used were the PROK1 sense primer, 5’-CATGCTCCTCCTAGTAACTG-3’, the PROK1 antisense primer, 5’-TTTCCTGAAGAAGGGGAC-3’, and the internal reference, GAPDH sense primer, 5’-ACAGTTGCCATGTAGACC-3’, and the GAPDH antisense primer, 5’-TTTTTGGTTGAGCACAGG-3’, which amplifies fragments of 190 bp for PROK1 cDNA and 225 bp for GAPDH cDNA . A 10-μL reaction mixture containing cDNA, specific primers and Fast SYBR Green® Master Mix  was used in the PCR. Reactions were performed at 95 °C for 20 s, followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s. To calculate the relative expression for each gene, the 2Ct−ΔΔ method was used to relate the Ct values of PROK1 expression in each sample to the Ct values of GAPDH.We extracted total RNA from scraped cells using the Trizol reagents  according to the manufacturer’s instructions. The extracted RNA was spectrophotometrically quantified, and its quality was assessed by measuring the absorbance ratios at 260/280 and 260/230 nm using a GeneQuantTM Pro Spectrophotometer . Two micrograms of total RNA was reverse-transcribed using a SuperScript5) were transfected with pCMV vector, PROK1 wild type or V67I plasmid for 48 h. Cell culture supernatant and cell lysate (cells were lysed with RIPA buffer) were harvested for an assay using an ELISA kit to assess the PROK1 concentration following the manufacturer’s instructions . Briefly, supernatant or cell lysate were applied to the microplate coated with the capture antibody for 2 h at room temperature. Detection antibody conjugated to streptavidin-horseradish peroxidase was then applied, followed by the color development solution (tetramethylbezidine substrate) for 20 min. Color development was terminated by addition of sulfuric acid, and the optical density was determined at 450 nm by a microplate reader . A PROK1 standard  was used for control, and calculation of the results was performed using computer software capable of generating a four parameter logistic (4-PL) curve-fit.HEK293, HTR-8/SVneo, JAR, and Ishikawa cells (3 × 105) were transfected in six-well plates with PROK1-WT or PROK1-V67I plasmid in DMEM with 1% FBS for 48 h. We then collected the culture medium to concentrate secreted protein using a centrifugal filter device, Amicon® Ultra4-3000 MWCO , under 3500 rpm for 40 min. We calculated the concentrated protein level using the ELISA method, as described above. The concentrated protein medium (conditioned medium (CM)) was stored at −80 °C, and its protein activity (WT and V67I) was compared with that of a PROK1 standard  and tested for various cell functions, as described below.HEK293 cells . After adding 100 μL of cell proliferation reagent in each well for 6 h at 37 °C in a CO2 incubator, the absorbance at 570 nm , reflecting the number of viable cells, was measured with a microplate reader . All the treatments were carried out in quadruplicate, and each experiment was carried out at least three times.The effect of PROK1 variant (V67I) on cell proliferation was assessed when compared with that of PROK1 wild-type and pCMV6 control. Cell lines were grown at 2 × 104 cells/well) and HTR-8/SVneo (3.5 × 104 cells/well) cells were added and incubated at 37 °C in an atmosphere of humidified 95% air/5% CO2 for 4 and 6 h, respectively. Observations were made under an inverted photomicroscope to document the developmental stages. Tubal formation was assessed by measuring tubal length in four quadrants of high power fields in each well. Each assay was done in triplicate and each experiment was repeated at least three times. Quantification was measured with Image J software .The effects of PROK1 wild-type and variant (V67I) on tube organization were assessed by growing transfected cells (HEK293 and HTR-8/SVneo) on Matrigel. Approximately 80 μL ice-cold Matrigel  was layered into each well of a 96-well plate. The Matrigel was allowed to completely solidify at 37 °C for 1 h. The transfected HEK293 (5 × 105) or HTR-8/SVneo (1 × 104) cells were trypsinized and re-suspended in serum-free medium and placed in the upper chamber coated with Matrigel  in trans-well plates  with or without PROK1 or V67I conditioned medium . DMEM containing 10% FBS was placed in the lower chamber. The cells were incubated for 20 h in a humidified atmosphere with 95% air and 5% CO2 at 37 °C. Invaded cells on the bottom side of the membrane were fixed with 100% cold methanol and stained with Giesmsa’s azur eosin methylene blue solution  and counted from a minimum of four high-power fields per insert under light microscopy. The data were expressed as the averages of three independent experiments.To investigate the effects of PROK1 and its variant (V67I) on cell invasion, the transfected HEK293  supplemented with 0.1% bovine serum album. Cells were loaded with 4 μM calcium indicator Fluo-4 AM  for 1 h at 37 °C. After washing twice, the cells were re-suspended to a concentration of 1 × 106 cells/mL. The green fluorescence emission of Fluo-4 was analyzed using FACSCalibur flow cytometry (BD Immunocytometry System), as described previously [2+ baseline, the indicated level of concentrated PROK1, V67I or PROK1 standard   was added to the cell suspension to detect fluctuations in the green fluorescence.Twenty-four hours after transfection, cells were harvested from plates using EDTA-trypsin, and washed with HBSS-based buffer (20 mM HEPES, 1 mM MgSOeviously . Followit-test or one-way ANOVA, and a p value of less than 0.05 was considered statistically significant.All values of the experimental assays were expressed as means ± SEM. Differences between the groups were compared using the unpaired two-tailed We evaluated the function and possible regulatory mechanism of a common variant of PROK1 as a genetic modifier in early human pregnancy. Although PROK1-V67I has a similar functional effect to that of WT on cell behavior, gene expression at the transcript and protein levels is impaired. The results of this work provide an explanation for how a common, innocuous PROK1 variant could act epistatically with its receptor genes to interfere with the outcome of early human pregnancy. However, more research is needed to assess whether the clinical impact of the decreased protein production of PROK1-V67I is positive or negative. Considering the important role of PROK1 in pregnancy and the high prevalence of V67I in the general population, the findings of the present study may provide a common mechanism with regard to modulate the risk of various PROK1-related diseases.


P = 0.004, 0.007 and 0.039, respectively). The NTRG may thus be an independent prognostic factor for oncologic outcomes in rectal cancer patients after neoadjuvant therapy and radical surgery, but this conclusion must be validated in randomized trials.In this retrospective study, we defined a new tumor regression grade (NTRG), which we used to evaluate the prognosis of patients with locally advanced rectal cancer who received neoadjuvant therapy and then underwent radical surgery between June 2004 and October 2011. Calculated as the TRG plus a lymph node score, the NTRG was determined for 347 patients: NTRG 0, 46 patients (13.3%); NTRG 1, 63 (18.2%); NTRG 2, 183 (52.7%); NTRG 3, 30 (8.6%); NTRG 4, 25 (7.2%). Among this group, 45 (97.8%) NTRG 0, 56 (88.9%) NTRG 1, 148 (80.9%) NTRG 2, 24 (66.7%) NTRG 3, and 10 (40.0%) NTRG 4 patients experienced 5-year disease-free survival. We also found that NTRG is significantly associated with 5-year local recurrence, distant metastasis and disease-free survival ( Rectal cancer patients receiving neoadjuvant therapy (preoperative chemoradiotherapy or radiotherapy only) may experience late local recurrence and distant metastasis . Long-teP = 0.003)  after neoadjuvant therapy were identified in this retrospective study. With this group, each patient was assigned a new tumor regression grade (NTRG) as follows: NTRG 0, 46 patients 13.3%]; NTRG 1, 63 patients [18.2%]; NTRG 2, 183 patients [52.7%]; NTRG 3, 30 patients [8.6%]; NTRG 4, 25 patients [7.2%]). cT stage was significantly predictive of NTRG . No significant association was found between the NTRG and the degree of tumor differentiation after radical surgery. Given that pCR means there is no lymphatic invasion, venous invasion or tumor deposits, statistical analysis was performed with NTRG 1–4. And considering that NTRG 0 (pCR) corresponds to ypT0, ypT 4, ypN0 and ypN2, additional statistical analysis was restricted to NTRG 1–3 for those variables.The association of the NTRG with histopathologic factors is summarized in Table P = 0.035), but not 5-year local recurrence or DFS rates . By contrast, the NTRG was significantly associated with 5-year local recurrence, 5-year distant metastasis and 5-year DFS . Univariate analysis showed that the ypT and ypN stages, lymphatic invasion and venous invasion all correlated significantly with DFS   patients with NTRG 0, 56 (88.9%) with NTRG 1, 148 (80.9%) with NTRG 2, 24 (66.7%) with NTRG 3 and 10 (40.0%) with NTRG 4 experienced a 5-year DFS. TRG was significantly associated with 5-year distant metastasis (P = 0.146). We therefore combined the patients with NTRG 1 and 2 into a new group. The resultant modified NTRG system is shown in Table 2 = 45.965, P < 0.0001), NTRG 1 vs. NTRG 2  and NTRG 2 vs. NTRG 3 . Disease-free and overall survival curves for the modified NTRGs are shown in Figure When comparing the 5-year DFS rates among the five NTRG groups, we found that NTRG 1 and 2 gave similar results . Considering that overall radiation dose (50 Gy) and the interval between preoperative therapy and surgery (6–8 weeks) were similar for all patients in our study, this suggests differences in NTRG likely reflect the characteristics of the individual tumors.Tumor regression after neoadjuvant therapy for rectal cancer can vary considerably. Whereas some patients show a complete absence of tumor cells, others exhibit a mass of tumor cells with little or no regressive changes . Tumor rP < 0.001). Our findings, however, indicate that TRG may not be significantly associated with 5-year local recurrence or DFS. These differences may reflect an inadequate number of patients in the present study, the different regression scoring systems used and/or differences in the treatment regimens, including the radiation dose, medications used in chemotherapy and pathology practices, as well as differences in the duration between neoadjuvant therapy and surgery. In fact, Kalady et al. [We found that the TRG correlated with 5-year distant metastasis, but that it was unlikely that the TRG was a prognostic factor for 5-year local recurrence or DFS. Based on data from the CAO/ARO/AIO-94 trial, Rodel et al.  concludeThe widespread implementation of a number of grading systems has been hindered by a lack of standardization. In addition, interpretation of the TRG assigned by a pathologist may be challenging due to the subjective nature of the histological interpretation of the response, which could potentially lead to unreproducible findings. For example, one recent study reported poor agreement among experienced pathologists, irrespective of the TRG system used . What's However, reproducibility is a key issue with any medical test or procedure, and the utility of the NTRG remains unclear. The results from this study are constrained by all the inherent flaws of retrospective research, some of which could lead to bias. These include the limited number of patients enrolled and differences in the neoadjuvant therapy regimens and postoperative chemotherapy. The ideal trial design to assess the NTRG system would be a prospective and randomized clinical trial. Nevertheless, we believe our outcome data for the NTRG are encouraging, and our method may provide a new way to assess likely oncologic outcomes in rectal cancer patients after neoadjuvant therapy and radical surgery. We have therefore initiated a randomized clinical trial to carefully evaluate the validity of our findings. All of the trial participants have advanced and resectable rectal cancer, and we are using the same preoperative chemoradiotherapy and postoperative chemotherapy for all patients. But for those enrolled into Group A there is a 12-week interval between completion of the preoperative therapy and surgery, while the interval is 8 weeks for those in Group B. Our purpose is to assess the validity of our earlier conclusions, determine whether the NTRG is superior to the existing TRG systems, and assess and compare the accuracy of the NTRG as a predictor of rectal cancer patient outcome at two intervals between completion of neoadjuvant therapy and radical surgery.In sum, the TRG system is a prognostic factor for distant metastasis in rectal cancer after neoadjuvant therapy and radical resection, but it failed to predict the risk of local recurrence and distant metastasis in our study. Our results suggest the NTRG may be an independent risk factor predictive of oncological outcome in rectal cancer patients after neoadjuvant therapy and radical surgery, but the validity and reproducibility of this result must be tested in randomized trials.This study was not considered to constitute an additional risk for enrolled patients. Approval was obtained from the appropriate ethics committees at all the participating study centers before the study started.We examined the records of 347 patients with primary mid-rectal or distal rectal cancer who had received preoperative neoadjuvant therapy followed by radical surgery at four study sites between June 2004 and October 2011. The study inclusion/exclusion criteria were: (1) rectal adenocarcinoma confirmed by surgical resection with a total mesorectal excision; (2) locally advanced resectable disease  with an inferior tumor margin located no farther than 10 cm from the anal verge; (3) no evidence of distant metastasis; and (4) patients were administered neoadjuvant therapy.2/bid), and the rest received radiotherapy alone. All patients received the same capecitabine regimen  3 weeks after radical surgery, except 62 (17.9%) who rejected chemotherapy due to their older age, poor physical condition or side effects.There is currently no international consensus with regard to the indications for neoadjuvant chemoradiation therapy. Therefore, patients managed with preoperative radiochemotherapy or preoperative radiotherapy alone were identified in our retrospective study. All patients received preoperative radiotherapy (50 Gy/2 Gy/25 f). Among those, 213 (61.4%) patients were concurrently treated with chemotherapy .P value less than 0.05 was considered significant.Statistical analysis was performed using SPSS software (version 18). Local recurrence and distant metastasis were analyzed for all eligible patients who received R0 resection and who were without detectable distant metastasis at the time of surgery after neoadjuvant therapy. All time-to-event end points were measured from the date of radical surgery. DFS was calculated from the time of radical resection to the discovery of evidence of recurrence and/or distant metastasis. Differences were evaluated using the log-rank test. Local recurrence and distant metastasis were analyzed as cumulative incidences. Mutivariable analysis was performed using the Cox proportional hazards model. All significant variables in the univariable analysis were included in the multivariable Cox regression models in a forward-step procedure. The variables were entered into the regression models in order according to their clinical relevance with increasing complexity, and significance was assessed using analysis of variance. A two-sided


In this paper, we provide a state-of-the-art overview of the ethical challenges that arise in the context of antimicrobial resistance (AMR), which includes an introduction to the contributions to the symposium in this issue. We begin by discussing why AMR is a distinct ethical issue, and should not be viewed purely as a technical or medical problem. In the second section, we expand on some of these arguments and argue that AMR presents us with a broad range of ethical problems that must be addressed as part of a successful policy response to emerging drug resistance. In the third section, we discuss how some of these ethical challenges should be addressed, and we argue that this requires contributions from citizens, ethicists, policy makers, practitioners and industry. We conclude with an overview of steps that should be taken in moving forward and addressing the ethical problems of AMR. Numerous biological, behavioural, economic, environmental and social factors contribute to the production and propagation of antimicrobial resistance (AMR). After decades in which AMR was almost exclusively a topic for discussion among experts in the medical and microbiological profession, a recent push to broaden our approach and place it more prominently on the political agenda has resulted in wider public awareness of the threat it poses. As a part of this increase in political action and public awareness, the numerous ethical issues associated with AMR are also slowly gaining greater prominence.AMR has been described as one of the major threats to individual and population health in the 21st century, and national and international organizations have repeatedly underlined the urgent need for action . This yeGiven the enormous importance of antimicrobial drugs for the functioning and delivery of modern health care, the progressive exhaustion of effective antibiotics presents health care professionals and policy makers with a distributive dilemma that raises complex moral questions of justice, especially how to fairly allocate antimicrobial resources . On the While much has been written about causes and effects of AMR, the ethical issues of drug resistance have so far—with a few notable exceptions . These aPublic Health Ethics and believe that these contributions offer new and important insights that can help to inform and shape policy-making for AMR. These contributions will be discussed in the context of the wider ethical issues that AMR present.This paper seeks to provide a state-of-the-art overview of some of these current ethical challenges—focusing on three broad questions: (i) why AMR is an ethical issue, (ii) what specific moral questions it raises and (iii) how we should broadly approach the ethical issues raised by AMR. It also functions as an introduction for this journal symposium, which includes contributions exploring various philosophical, political and ethical issues raised by the problem of AMR. This symposium, which is the result of an international, multi-disciplinary workshop on the ethical implications of AMR hosted at the Brocher Foundation in Geneva on March 27–28 2014, seeks to address some of these questions in greater detail and provide a foundation for subsequent discussion. The event brought together experts from different academic disciplines, including medicine, public health, philosophy, economics and law, and provided a platform for discussion and the exchange of ideas across disciplinary boundaries. We are therefore happy to be able to present some of the proceedings of this symposium within This paper is divided into four sections. In the first section, we outline some of the reasons why AMR is a distinct ethical issue, and not merely a technical or medical problem. In the second section, we elucidate some of these more general themes and offer a more detailed account of a number of ethical challenges that AMR poses and why they need to be addressed as part of the response to AMR. Many of these issues are also discussed in greater detail in the articles contained in this symposium. Section three discusses how stakeholders should address some of these ethical aspects, and section four proposes steps that should be taken in moving forward.AMR is more than a problem that arises as a result of the complications of treating infectious diseases; it is a complex, multifaceted global challenge that affects the environment, human and animal health, agriculture and the economy. Given the multitude of persons, institutions and societies AMR impacts, it presents a distinct and significant ethical issue.AMR is putting current and future populations at substantial risk of injury, loss and death. It is going to require a redistribution of resources and a balancing of benefits and burdens, which in turn forces us to make a number of individual and collective sacrifices—often for people thousands of kilometres away and for future persons who have not come into existence yet. This will include questions about who is morally responsible for this predicament—and whether ascriptions of blame or sanctions should affect who should bare the costs of this problem. It is also going to require us to intentionally, and sometimes coercively, shape the institutional structures and individual behaviours of governments, corporations, scientists, clinicians and patients, which raises questions about important moral values such as solidarity, liberty, privacy, reciprocity, fairness and the common good.It is worth exploring some of the broad themes that make AMR a moral issue, and how these considerations contribute to why facing the problem of AMR requires recognition of its ethical implications and their need to be addressed as part of any successful policy response.The threat of progressing drug resistance puts us at a tangible risk of harm in our life time, and our failure to control and reduce AMR will impose risk on other people and on future generations . The scoIt is, however, not only the magnitude of the risk that current and future generations are faced with that creates ethical problems. Due to the speed and scale with which we must react in order to avert a post-antibiotic age, we are also faced with what constitutes a proportional response and, crucially, who bears responsibility to act. While AMR is a complex challenge with numerous causes, it is the broad use of antimicrobials in health care and agriculture that is the driving force behind the emergence of drug resistance . This meHowever, recognizing the large part that we play in the emergence of AMR also means that we must think seriously about who is accountable for the emergence of drug resistance. If the preservation of effective antimicrobials is in the interest of current and future generations, and indeed their lives depend on it, then we should also hold people blameworthy or sanctionable for the ignorant, unnecessary or wrongful use of antibiotics, or any other practice that is likely to hasten the emergence of AMR. This may mean that we will be morally justified in imposing greater burdens or costs for their contribution to the current state of AMR.At the same time, however, we must remember that there may be no truly sustainable way of using antibiotics in the long-run, as micro-organisms have shown to be almost infinitely adaptable since the first introduction of antibiotics. This means that our struggle to keep abreast of AMR will most likely be a continuous and vicious cycle of resistance and obsolescence . HoweverAMR not only uses more resources and increases the cost of health care delivery, by increasing the risk of complications or treatment failure and lengthening the recovery time, it also leads to productivity losses—which are commonly not fully taken into account and have led to an underestimation of the true cost of AMR in the past . MoreoveIn this symposium, Joanna Coast and Richard D. Smith discuss one of these problems with respect to the use of economic modelling for the prediction of costs associated with AMR. They show that the inclusion of economic models into AMR policy is becoming increasingly widespread, yet the findings of such models are not value-neutral. Depending on their ‘evaluative space’, that is, the parameters they seek to maximize, different models can produce very different recommendations with different distributive effects—a fact that should be noted by policy makers and lead us to discuss more openly which values we hold to be most important in this context .There are no current or future persons who will not be affected by AMR. The risks and costs associated with this problem reflect a shared vulnerability we all have. This fact highlights the need for solidarity between persons, institutions and nation states in responding to AMR. The importance of solidarity is gaining more recognition in the context of bioethics and public health ethics .Solidarity is important because it underlines the fact that we will only be able to effectively deal with AMR through collective and collaborative activities, but also because many of these population-level activities will often make it difficult to fulfil some individual-level preferences in order to achieve the public interest that arises out of mitigating AMR. This is not to understand AMR in simplistic terms as involving a conflict between liberty and the common good, or that population-level concerns should always win out over individual preferences. A focus on solidarity seeks to re-orient our ethical focus towards our common interests and vulnerabilities, individually and collectively, and how these considerations should make the distribution of health and risk a joint concern of all levels and sectors of our global society. The solidarity constitutive of a moral community of health justice will confer a mutual recognition, respect and reciprocal concern that requires us to, as Dawson and Jennings put the point, stand up for and stand besides those most affected by AMR .Following on from the previous point, any solution to the problem of AMR will require a fair balance of benefits and burdens among those affected by it. However, the global burden of infectious disease is distributed highly unevenly and low-income countries are disproportionately affected by AMR. This means that high-income countries will likely have to bear a much larger share of the response, e.g., through developing new drugs and technologies, enhancing surveillance and reporting systems and conducting research in areas that may not be aligned with their current national priorities. At the same time, a response to AMR will also require that we provide better access to high-quality drugs, diagnostic tools and expert care, since AMR is as much a problem of overuse as it is a problem of insufficient access in many low- and middle-income countries . AMR theHaving outlined the main reasons why AMR should also be understood as an ethical, and not merely as a technical or medical, problem we will briefly highlight some of the most significant ethical challenges that AMR and our response to it raise.The emergence of drug resistance is an inevitable by-product of widespread antibiotic use . Many caFrom an ethical perspective, the lack of new antibiotics poses a number of problems. Perhaps most pressing is the question of who bears responsibility for the development of new drugs and tests, and how these should be made available, once they enter the market. Since both the burden of infectious disease and the prevalence of AMR are disproportionately higher in low- and middle-income countries, there is an urgent need for the provision of low-cost, high-quality drugs and tests to be made widely available. Yet, these countries are also least likely to be able to finance the development of new diagnostic and therapeutic agents, or pay the high prices of novel, patent-protected drugs. There is thus an obvious conflict between medical needs and the current pharmaceutical business model, which seeks to recoup investments into upfront research and development costs through high price and high-volume sales throughout the period of patent protection  ethics, (ii) policy, law and regulation, (iii) public health practitioners and health care workers, and (iv) civil society and industry.Ethicists have so far largely ignored the issue of AMR. This is problematic for a number of reasons. First, in light of the projected consequences, AMR is one of the major threats to human health in the 21st century. Ethicists, especially those working on bioethics and public health ethics, should arguably be addressing the relevant issues that will affect millions or potentially billions of current and future people across the globe. A greater involvement of ethicists will also be necessary, because—as we have outlined in this paper—AMR creates numerous ethical challenges that are interesting in their own right. Some of them have been touched on in previous contexts. Here, ethicists can help with the transfer of knowledge and expertise.There are also ethical issues that arise in the context of AMR, however, which require new ideas and analysis. Much of the research that is needed in this area is complicated by the subject matter and the seemingly infinite contributing factors. AMR is thus not a problem that lends itself to the straightforward application of well-established basic principles of bioethics. As a result, further work will be needed from moral and political philosophers on translating agent-centred moral concepts to the population-level and elucidating the relational and socially embedded normative concepts that will underpin the AMR response, such as solidarity, reciprocity, stewardship, collective harm, trust, community, health justice and the common good. But in spite, perhaps even because, of this input from ethicists will be necessary to not only map out the challenges at hand, but also to suggest, how they can best be applied in practice. In addition to conceptual work that aims to clarify and analyse, the fruits of this moral and political thought must also be addressed in policy terms, as well as in public health and medical practice.Much of the response to AMR will be approached via the use of governance mechanisms within both public and private institutions, especially instruments such as policy, law and regulation . Going fThe progression of AMR in many countries suggests that current policy efforts are falling short of the goal to halt or even reverse the effects of AMR . The WHAFirst, as we discussed earlier, there is a clear need to acknowledge and address the fact that existing policy proposals imply a prioritisation of some interventions over others. How such a prioritisation is arrived at should be subject to open, transparent and accountable discussion. Secondly, policy and lawmakers have a responsibility to help ensure the reduction of antimicrobial misuse. These efforts will also need to be sensitive to the social, political and economic contexts in which AMR efforts are directed. The ethical analysis must be sensitive to, for instance, resource availability and drivers of inappropriate drug use in different regions of the world. The extent to which this is possible will be highly context-dependent; for instance, in countries where antibiotics are available without prescription more fundamental changes will be necessary. However, recent research suggests that lack of appropriate governance is one of the key driving factors in driving the emergence of AMR (As prescribers or dispensers, medical practitioners, health care workers and pharmacists play a crucial role not only in regulating access to antibiotics, but also in educating patients about AMR. This does not only pertain to a general understanding of drug resistance, but must also involve awareness of own prescribing or dispensing behaviour on the emergence of AMR. However, this requires that they themselves are sufficiently trained and aware of the challenge. Public health practitioners will also play an important role in this effort. Their focus on AMR at the population-level and ability to use the powers and resources of the State in these efforts will often involve navigating between societal and individual interests. If healthcare workers and public health practitioners are unaware or feel unable to address the ethical dilemmas that will arise in responding to AMR, they cannot be expected to respond appropriately. Ethics education, training and guidance need to be provided to these professionals to help them navigate the moral issues they will encounter.AMR strategies predominantly originate in governmental bodies and, as such, tend to focus on patients, professionals and public institutions. Nevertheless, we also cannot forget the role of civil society and industry—particularly in those areas that are not, at least currently, under some regulatory framework. We are going to require more public engagement and citizen action to ensure the success of our response. Not only can this involvement help in identifying the support and limits citizens will be prepared to accept in order to assist in AMR mitigation activities, but it also increases the likelihood of compliance with such activities when they are developed with input and buy-in from the wider community. Industry also needs to be more collaborative and willing to assist to a greater extent without waiting for government regulation to push them further. Current pricing strategies for antimicrobials, especially for drugs of last resort and new medicines that make them unaffordable for a majority of the world's population, would be a good place to start. Showing more concern and solidarity in these matters can only help industry’s public relations and stave off the feeling that increased regulation of industry should be undertaken to force them to do more.Successful responses to the problem of AMR will not only be a scientific or medical undertaking, it must also be an ethical undertaking. This means that an effective AMR mitigation strategy needs to be informed and guided by ethical analysis. As we have shown in this paper, every level of an AMR response strategy will inevitably involve making decisions with ethical implications. Reducing the use of antimicrobial drugs in humans and animals means instituting behaviour-changing interventions and restricting their choices, which are likely to limit preferences and potentially subjecting people to elevated risks of complications or infection and financial costs. Improving surveillance and reporting systems increases concerns about confidentiality and privacy. Preventing and controlling the spread of drug-resistant infections, especially if we have AMR-related epidemics/pandemics, can involve the increased use of restrictive measures, raising questions about constraints on liberty and human right derogations. Promoting research and innovation into different preventative, diagnostic and therapeutic interventions will require us to make funding and allocation decisions that prioritise AMR over other important projects and policies. What these examples entail for policy makers, practitioners and researchers alike is that ethical decisions in AMR policy cannot be avoided—and if such policies are to have any kind of normative legitimacy, they can no longer be ignored either.While the development of more extensive national and international responses to AMR is encouraging, it will also be hugely important to ensure that the underlying ethical issues will be addressed in the process. The fact that many countries will now begin to develop national response plans to AMR after the WHA, or make updates to existing ones, represents a great opportunity for the ethical significance of AMR to be included more widely into policy making, practice guidelines and training programmes. We believe that the articles in this symposium make a strong case for including ethics, which has not been traditionally associated with being central to infectious disease and global health policy, into the development of the policy responses to AMR that are so urgently needed.


CD99 is a transmembrane glycoprotein expressed in physiological conditions by cells of different tissues, including osteoblasts (OBs). High or low CD99 levels have been detected in various pathological conditions, and the supernatant of some carcinoma cell lines can modulate CD99 expression in OB-like cells. In the present work we demonstrate for the first time that two different human myeloma cell lines (H929 and U266) and, in a less degree, their conditioned media significantly downregulate CD99 expression in normal human OBs during the differentiation process. In the same experimental conditions the OBs display a less differentiated phenotype as demonstrated by the decreased expression of RUNX2 and Collagen I. On the contrary, when CD99 was activated by using a specific agonist antibody, the OBs become more active as demonstrated by the upregulation of Alkaline Phosphatase, Collagen I, RUNX2, and JUND expression. Furthermore, we demonstrate that the activation of CD99 is able to induce the phosphorylation of ERK 1/2 and AKT intracellular signal transduction molecules in the OBs. CD99 is a 32 kDa transmembrane glycoprotein, encoded by the MIC2 gene  which is+ cells of bone marrow, and all leukocyte lineages . Moreover, COLLI, RUNX2, members of AP1 complex , and mitogen-activated protein kinases (MAPKs) have been studied in hOBs after CD99 stimulation. COLLI, RUNX2, and JUND were also evaluated in hOBs treated for 30 minutes with 100 nM Wortmannin or for 60 minutes with 10 3VO4, 50 mM NaF, 100 mM phenylmethylsulfonyl fluoride, and a commercial protease inhibitor mixture.To detect the expression of caspases 3, 7, and 8 and Bid cleavage in hOBs after CD99 or hTRAIL stimulation, cells were lysed by incubation on ice for 30 min in lysis buffer containing 20 mM Tris-HCl (pH7.5), 1% Triton X-100, 150 mM NaCl, 10% glycerol, 1 mM Naμg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and subsequently transferred to nitrocellulose membranes . The blots were probed overnight at 4°C with the appropriate primary antibody.Cell proteins ; monoclonal anti-p-AKT, anti-p-JNK, and anti-caspase-8, polyclonal anti-total-AKT, anti-p-P38, anti-caspase-3, anti-caspase-7, and anti-Bid ; monoclonal anti-CD99 (12E7) (Santa Cruz Biotechnology); and polyclonal anti-RUNX2 .The following primary antibodies were used: monoclonal anti-COLLI, anti-p-ERK and anti-After incubation with the appropriate fluorescent-dye-conjugated secondary antibody , specific reactions were revealed with the LI-COR's Odyssey Infrared Imaging System .ALP was histochemically assessed in hOBs treated and nontreated for 6 days with anti-CD99 agonist antibody or with mouse IgG, using Leukocyte Alkaline Phosphatase Kit, a commercial kit based on naphthol AS-BI and fast red violet LB (Sigma).2, FRV-Alkaline Solution, Naphthol AS-BI Alkaline Solution) and finally washed with water.Cells were fixed with a citrate-acetone-formaldehyde fixative for 30′′ at room temperature. After being gently rinsed with distilled water, cells were incubated for 15′ in dark with alkaline-dye mixture . Three different experiments were performed for ALP evaluation.μg/mL of anti-CD99 agonist antibody for 24 and 48 hours. The cell viability experiments were performed in the presence of 10% FCS. MTT 0.5 mg/mL were added to the culture media followed by 4 hours incubation at 37°C in a humidified 5% CO2 atmosphere. The reaction was stopped by the addition of 150 μL of 0.04 N HCl in absolute isopropanol. The optical density was read at 570 nm using an automatic plate reader . The results were compared to cells incubated under control conditions.Mitochondrial dehydrogenases activity was determined by MTT assay. This assay is based on the ability of forming dye crystals to be developed only in living cells, providing an indication of the mitochondrial integrity and activity which, in turn, may be interpreted as a measure of cell viability. hOBs were cultured in 96-well tissue-culture plates as previously described. A part of the wells were used as control, while the others were treated with 2 Cell viability was evaluated in three independent experiments.t-test with the Statistical Package for the Social Sciences (spssx/pc) software . The results were considered statistically significant for P < 0.05.Statistical analyses were performed by Student's It was shown that some carcinoma cell line-conditioned media downregulate CD99 on human AHTO-7 OBs . Thus, aP < 0.013) inhibited hOB CD99 expression during the differentiation period reaching the maximum inhibition at the seventh day of culture (P < 0.013) inhibition of CD99 expression in the late phase of hOB differentiation (14 and 21 days) compared to what was observed in the presence of the cells (At this purpose we performed cocultures between two different HMCLs (H929 and U266) and undifferentiated normal hOBs cultured in the presence of osteogenic medium from 2 up to 21 days of culture. By western blot analysis we showed that in the coculture system both HMCLs significantly ( culture . Althoug culture . In parahe cells . These fP < 0.001)   higher ALP activity in hOBs treated for 6 days with 2 μg/mL of anti-CD99 agonist antibody compared to the control. In parallel, to exclude any nonspecific effect of the antibody, the cells were cultured for 6 days with mouse IgG and we did not find any difference  upregulated respect to untreated cells, and the treatment with the mouse IgG did not induce any effect  and no effect was exerted by mouse IgG . As shown in P < 0.001), RUNX2 (P ≤ 0.01), and JUND (P < 0.001) protein levels in hOBs.To identify the intracellular signal transduction molecules involved in CD99 signaling pathway(s) in our hOB culture system, the expression of several signaling mediators was examined after CD99 activation. In particular, we demonstrated that the anti-CD99 agonist antibody significantly induces the phosphorylation of signaling components such as AKT and ERK1/2 . In these experiments TRAIL stimulation, known to induce apoptosis in hOBs [Finally, due to the knowledge that the activation of CD99 causes T-lymphocyte and thymocyte apoptosis , 31, in  in hOBs , was useIn the present study we demonstrated that HMCLs or their conditioned media downregulate the expression of CD99 by hOBs during their differentiation process. Intriguingly, influenced by HMCLs, undifferentiated and differentiated hOBs, in addition to the reduced levels of CD99, display a less differentiated status. We further showed that CD99 is a critical molecule in the regulation of the physiological process of hOB differentiation and activity since the expression of ALP, COLLI, RUNX2 and JUND are upregulated by the activation of CD99 in hOBs. These findings suggested that the downregulated levels of CD99 could have a critical role in the well-known impairment of osteoblastogenesis and bone formation occurring in the osteolysis associated with MM.Although CD99 is largely expressed in normal tissues  and receIt is worth noting that the supernatants from different tumors associated with osteolytic lesions or osteosclerotic metastasis can vary CD99 expression in osteoblastic cells . HoweverIncreasing evidence demonstrate that MM cells can impair OB formation and activity through different cellular mechanisms including both secretion of soluble factors –29 and cIn this work we found that HMCLs, H929 and U266, which weakly express CD99, induce a significant inhibition of CD99 expression by normal hOBs during their differentiation process. We also demonstrated that this inhibition, although significant, is less evident in the presence of the conditioned medium of both HMCLs indicating that the cell contact and, in a less way, the possible release of soluble molecules in the media induce CD99 reduction in hOBs.On the basis of recent data demonstrating a high expression of CD99 in differentiated hOBs, we hypothesized that HMCLs through the reduction of CD99 could contribute to OB impairment. in vivo study with our in vitro data in which the downregulation of CD99 could be involved in the alteration of osteoblastic differentiation and activity taking part in the onset of osteolysis in MM.Indeed, these latter cells cocultured with the two HMCLs express less RUNX2 and COLLI amount in favor of the hypothesis that the modulation of CD99 by myeloma cells, in addition to other mechanism(s), could take part in the induction of a less differentiated OB phenotype. In addition, a further support for this hypothesis comes from a very recent paper showing that CD99 expression in extramedullary biopsies of MM patients correlates with longer overall survival suggesting CD99 a new marker for risk stratification of disease severity . These fTo prove that the reduction of CD99 we found could be directly responsible for the less differentiated status of OBs, it would be successful the use of a specific neutralizing antibody. However, a CD99 neutralizing antibody neither is at present available commercially nor is produced by some investigators. Thus, to overcome the difficulty of proving direct evidence that the CD99 reduction could take part in the impairment of osteoblastogenesis and bone formation occurring in the osteolytic process associated with MM, we investigated whether the activation of CD99 is able to modulate the activity of hOBs. At this purpose we performed different experiments on hOBs in the presence of a specific agonist antibody, widely used by other authors to achieve information on CD99 activities , 38, 39.We here demonstrated that in hOBs the activation of CD99 stimulates the activity of ALP, the most widely recognized biochemical marker for hOB, and doubled the levels of COLLI, the most abundant organic component of bone matrix –48. SuchOur results highlight an important role of CD99 in the differentiation and activity of hOBs in physiological and pathological conditions. HMCLs induce a reduction of CD99 expression in hOBs which display a less differentiated phenotype, suggesting a possible contribution of this molecule in the impairment of osteoblastogenesis occurring in MM bone disease. Indeed, by forcing the function of CD99 the hOBs result more active in the expression of their differentiation parameters.


Objective. Klinefelter syndrome (KS) has long-term consequences on bone health. However, studies regarding bone status and metabolism during childhood and adolescence are very rare. Patients. This cross-sectional study involved 40 (mean age: 13.7 ± 3.8 years) KS children and adolescents and 80 age-matched healthy subjects. For both patient and control groups, we evaluated serum levels of ionised and total calcium, phosphate, total testosterone, luteinising hormone, follicle stimulating hormone, parathyroid hormone (PTH), 25-hydroxyvitamin D (25(OH)D), 1,25-dihydroxyvitamin D, osteocalcin, bone alkaline phosphatase, and urinary deoxypyridinoline concentrations. We also calculated the z-scores of the phalangeal amplitude-dependent speed of sound (AD-SoS) and the bone transmission time (BTT). Results. KS children and adolescents showed significantly reduced AD-SoS (p < 0.005) and BTT (p < 0.0005) z-scores compared to the controls. However, KS patients presented significantly higher PTH (p < 0.0001) and significantly lower 25(OH)D (p < 0.0001), osteocalcin (p < 0.05), and bone alkaline phosphatase levels (p < 0.005). Interestingly, these metabolic bone disorders were already present in the prepubertal subjects. Conclusions. KS children and adolescents exhibited impaired bone mineral status and metabolism with higher PTH levels and a significant reduction of 25-OH-D and bone formation markers. Interestingly, this impairment was already evident in prepubertal KS patients. Follow-ups should be scheduled with KS patients to investigate and ameliorate bone mineral status and metabolism until the prepubertal ages.  Klinefelter syndrome (KS) is a common chromosomal disorder with a prevalence of 120–153 per 100,000 live-born male births , and theKS is an underdiagnosed condition; in fact, only 25% of patients are diagnosed, with only a minority of those diagnosed before puberty , 6, wherTestosterone (T) is an important hormone that plays a role especially during puberty to achieve bone maturation and adequate peak bone mass (PBM). In KS metabolic bone disorders have been reported , 8 and lIn fact, spine BMD in the lumbar region increased significantly after testosterone treatment in KS adults . On the Finally, a recent study showed that KS patients had lower total volumetric BMD, reduced trabecular density, and reduced cortical area and thickness at the tibia, compromising one strength at this site. These data suggest specific bone characteristics related to this genetic syndrome with picture similar to postmenopausal osteoporosis .Studies evaluating the bone status and metabolism of children and adolescents are very rare; however, the limited data available seem to suggest that this unfavourable metabolic bone profile may be present in childhood as well .Therefore, the purpose of our study was to evaluate bone mineral status and metabolism in a cohort of KS children and adolescents.Forty nonmosaic KS patients (mean age: 11.96 ± 3.97 years) were recruited consecutively from February 2013 to May 2015 at Anna Meyer Children's University Hospital in Florence, Italy. The cohort contained both children  and adolescents . None of the patients had ever received sex hormone therapy.The study was conducted according to the Declaration of Helsinki and the European Guidelines on Good Clinical Practice. Ethical approval was obtained from the Ethics Committee of our hospital. Written informed consent was obtained from parents and patients according to age and ability to consent.n = 15) and patients diagnosed during childhood and adolescence (n = 25). In all patients, chromosomal analysis was performed on peripheral blood lymphocytes, and karyotypes were established on 30 metaphases for each patient. In the presence of a prenatal diagnosis, the karyotype was confirmed postnatally. In some cases, array CGH (44 K array platform Agilent oligonucleotides with a resolution of approximately 100 kb) was also performed. As reported, patients with a mosaic karyotype were excluded.We examined both prenatally diagnosed patients with KS , waist circumference (WC), and pubertal staging.Participants or parents of both the KS patients and controls filled out a questionnaire reviewed by the medical staff during the baseline examination which was related to the pubertal history, the presence or absence of chronic health problems, current and past medications, vitamin D and/or calcium intake, familial and personal bone fracture history and osteoporosis, and physical activity. KS patients and controls showing already-known bone metabolic diseases, hyper/hypoparathyroidism, malabsorptive disorders, chronic renal insufficiency, cancer, and drug addiction  were excluded. None of the participants had a recent history of travelling to warmer, sunnier areas prior to and/or during the study.Physical activity was assessed with a modified activity score based on the sports/leisure activities . Outdoor exposure was quantified through questions regarding each subject's average number of daily outdoor hours and a prospective daily time-activity diary, as previously described .Calcium and vitamin D intake was assessed using the semiquantitative validated Food Frequency Questionnaire , 15. TheSerum 25(OH)D levels were stratified according to the brackets, ≤10, 11–20, 21–30, and >30 ng/mL, and were defined as severe deficiency, deficiency, insufficiency, and sufficiency, respectively, according to previously established guidelines for bone health  , 17.μs), which is the difference between the transmission time in the phalanx soft tissue and the bone and transmission time in the phalanx soft tissue. AD-SoS and BTT standard deviation scores (SDS) were automatically generated [Bone mineral status was evaluated with a DBM Sonic 1200 device . The device was equipped with two probes mounted on an electronic calliper. The emitter probe was positioned on the medial surface, and the receiver probe was positioned on the lateral side of the measured phalanx . The timenerated .The final result is the average AD-SoS and BTT over four fingers (digits II–V). All of the QUS measurements were carried out by the same operator on the patients' nondominant hands. The coefficients of precision, in vivo, were 0.8% and 1.5% for AD-SoS and BTT, respectively . z-scores [Low bone mineral status was defined in the presence of at least one densitometric parameter, such as AS-SoS or BTT ≤ −2.0z-scores .The data obtained were compared with an age-, sex-, and body-size-matched healthy subject control group . For every patient, we selected two control subjects that matched the following criteria: age ± 6 months, height ± 2.0 cm, weight ± 2.0 kg, and equivalent pubertal stage. Informed consent was obtained from all parents of the subjects. This healthy control group was randomly selected from a population survey of healthy Caucasian inhabitants in Tuscany with no endocrine or metabolic diseases; some of these subjects had been evaluated for noninflammatory musculoskeletal complaints.2). Waist circumference (WC) was assessed using inelastic anthropometric tape with a length of 2 m and an accuracy of 1 mm. Measurements were taken at the midpoint between the last rib and the iliac crest. However, height, BMI, and WC were normalised for chronological age by converting to SDS, as previously described [Height was measured using a wall-mounted stadiometer, and weight was measured to the nearest 0.1 kg. The measurements were performed by the same trained staff members. We calculated BMI as weight in kilograms divided by height in metres squared  < 10.4 nmol/L, and severe hypogonadism was defined as T < 8 nmol/L .All participants were examined in the morning after an overnight fast. Blood samples were collected, and serum and plasma were immediately separated and stored at −20°C in multiple vials for future analysis.Serum levels of total  = 2.10–2.50 mmol/L) and ionised calcium (NR = 1.10–1.32 mmol/L), phosphate (NR = 1.4–1.7 mmol/L for children aged 2–12 years and 1.1–1.4 mmol/L for youth aged 12–16 years), creatinine, and albumin were measured by a standard autoanalyser method routinely used for daily practice.μg/dL), intact PTH (NR = 12–72 pg/mL), and 25(OH)D (NR ≥ 30 ng/mL) were determined by chemiluminescent immunometric assays using commercial available kits for the IMMULITE 2000 Automated Analyser (Diagnostic Products Corporation (DPC), Medical System, Genova, Italy). The intra-assay and interassay coefficients of variation (CVs) in these hormone assays were all less than 8% in the full range.Serum levels of FSH , LH , E2 , total T , DHEAS  with a detection limit of 20 pg/mL. Intra-assay and interassay CVs were less than 13% and less than 19%, respectively.Serum 1,25(OH)2D was determined according to a competitive binding protein assay . The interassay CV was 8%. The normal range is stated as 19.9–67.0 pg/mL.A commercially available radioimmunoassay kit was used to measure serum osteocalcin levels . The sensitivity of the method was 0.50 ng/mL.Urinary deoxypyridinoline concentrations were measured using high-resolution chromatography in a fluid environment . We expressed deoxypyridinoline values in nM for mM of nocturnal 12 hr urinary creatinine. The intra-assay and interassay CVs were <9.8%.The serum level of bone-specific alkaline phosphatase (BSAP) was measured by immunoassay  with sensitivity of 0.7 U/L and a CV of 3.9–5.8%.t-test and Mann-Whitney U test depending on the distribution of the analysed variable. The chi-squared test and Fisher's exact test were used to examine associations between dichotomous variables. Spearman's (rank) correlation test was used to determine the correlation coefficients. We used multiple stepwise regression to determine the variables (age (years), serum PTH concentration, ionised and total calcium, phosphate, BSAP, 25(OH)D and 1,25(OH)2D, osteocalcin, LH, FSH, total testosterone levels, urinary deoxypyridinoline concentration, quantitative assessment of physical activity (hours per week), calcium intake (mg/day), and vitamin D intake (IU/day)) that might correlate independently with AD-SoS z-score values. p values < 0.05 were considered to be statistically significant.Statistical analyses were performed using SPSSX . Summaries of continuous variables are given as mean ± standard deviation . To compare differences, we used Student's Demographic, clinical, and laboratory characteristics of KS patients and healthy controls are summarised in p = NS)  . z-scores (−0.74 ± 0.91) compared to the controls  and adolescents   but not between children and controls (−0.28 ± 1.00 versus −0.02 ± 1.05) (Patients with KS showed significant differences regarding AD-SoS (−0.55 ± 1.13) and BTT < 0.05) . On the  ± 1.05) .z-score ≤ −2.0). This incidence was significantly higher than that observed in the control group . In particular, AD-SoS had a ≤−2.0 z-score in 4/40 patients  and BTT z-scores ≤ −2.0 in 3/40 patients , respectively. Of these, 6/7 KS patients showing AD-SoS and BTT z-score ≤ −2.0 were adolescents, while in KS children group the incidence was significantly lower .Overall, 7/40 KS patients (17.5%) had a low bone mineral status  and total calcium levels (2.38 ± 0.06 versus 2.35 ± 0.10 mmol/L) and phosphate levels (1.38 ± 0.15 mmol/L versus 1.34 ± 0.20 mmol/L) compared with controls. The ionised and total calcium and phosphate levels did not differ between KS children and adolescents.p < 0.005) and osteocalcin   (p < 0.0001) (p < 0.0001) and between KS population and control group  (p < 0.0001) were significantly higher in KS children and adolescents.The 25-OH-vitamin D levels were significantly lower  , whereas 0.0001) . Further 0.0001) . In addip < 0.0001; summer: 16.99 ± 4.88 versus 32.66 ± 8.71, p < 0.0001).Performing a seasonal analysis (winter = November to April), significantly lower 25(OH)D was found in both periods in KS patients compared to healthy subjects  2D in KS patients (59.35 ± 10.45 pg/mL) and controls  .β-Estradiol levels were significantly different in KS patients and controls (p < 0.05), and the ratio between total 17β-estradiol and total testosterone (E/T ratio) was significantly higher in KS patients than in healthy controls (data not shown).At the time of puberty, testosterone serum levels were normal in 10/21 (47.6%) KS adolescents and in the lower normal range in 11/21 (52.4%), whereas LH and FSH were in the normal range in 11/21 (52.4%) KS adolescents and elevated when compared to the normal reference ranges in 10/21 (47.6%) . No KS sA quantitative assessment of physical activity in patients with KS and controls did not reveal significant differences between the two groups: 0 hours per week , <2 hours per week , and >2 hours per week . z-scores correlated negatively with PTH  and with age  and positively with 25(OH)D levels . Both BSAP and osteocalcin levels also showed a significant correlation with total calcium values . PTH correlated significantly with ionised calcium , age , total calcium , phosphorus , magnesium , LH , FSH , total testosterone , vitamin D , 1,25(OH)2D , and osteocalcin .Correlation analysis showed that AD-SoS and BTT z-score, PTH levels (p < 0.001), and 25(OH)D levels (p < 0.005).The multiple regression analysis included age, sex, PTH, ionised and total calcium levels, phosphate levels, 25(OH)D and 1,25(OH)2D levels, serum osteocalcin levels, urinary deoxypyridinoline concentrations, quantitative assessment of physical activity, calcium intake, and BSAP levels and we demonstrated that they are all significant predictors of a lower AD-SoS z-scores when compared with healthy subjects. This aspect is particularly concerning since over 17% of KS children and adolescents had z-scores ≤ −2.0.Our results revealed that KS patients have an impaired bone mineral status that begins early in life. In fact, KS children and adolescents displayed significantly reduced AD-SoS and BTTKS children and adolescents also showed an impaired bone metabolism, with significantly reduced 25(OH)D levels and higher PTH levels compared with controls. Finally, KS children and adolescents seem to present reduced levels of bone formation markers, such as BPSA and osteocalcin levels.The aetiopathogenesis of this impaired bone mineral status in KS patients may be multifactorial, including the specific bone characteristics related to this genetic syndrome and environmental factors, that is, poor vitamin D levels, as reported by previous studies , 21. ParIn KS adults, testosterone deficiency is reported to be associated with a significant decrease in BMD; this deficiency is considered as the major risk factor for premature osteopenia and osteoporosis –25 and iIn KS, this relative androgen deficiency begins after pubertal onset, and since most KS patients show anticipated puberty , 29, it  receptor activator of NF-κB (Rank), tartrate-resistant acid phosphatase (Trap), matrix metalloproteinase-9 (Mmp-9), and Cathepsin K, in a dose-dependent manner, probably accelerating bone loss [It has also been reported that both serum levels of testosterone and LH show a significant association with osteoporosis or fractures –34. Inteone loss .Therefore, all these factors may have a role in reducing BMD and impairing the bone metabolism in KS patients. Nevertheless, these data do not explain our finding of an impaired bone metabolism also in children with KS, who usually do not have the typical pubertal problems related to their syndrome.Interestingly, some data showed a possible role of vitamin D pathway in KS. In adults, testosterone acts indirectly on the parathyroid hormone-vitamin D axis, because testosterone deficiency is related to a reduction in renal 1a-hydroxylase activity with a subsequent decrease in 1,25-hydroxyvitamin D concentration, the active form of vitamin D . MoreoveIt is well known that vitamin D is also an important factor in bone metabolism in prepubertal subjects and that vitamin D deficiency may play a role in worsening bone mass. Low vitamin D and high parathyroid hormone (PTH) levels are frequent findings in KS patients , 21, whiOur data interestingly seems to suggest that PTH levels were significantly higher in KS patients than in controls, even in absence of severe hypovitaminosis D, suggesting that KS patients tend to increase PTH levels more than the general population.In conclusion, in adults with KS, the hypogonadism, or the reduced testosterone levels, may represent one of the most important causes of reduced bone mineral status. However, possible new causes for this impairment in KS patients may be related to specific mechanisms of KS, such as an important hypovitaminosis D and higher PTH levels, aspects that are also present before the puberty onset. Additional studies are necessary to evaluate the bone metabolism in prepubertal KS patients. However, measures to prevent an impaired bone mineral status, such as ensuring adequate vitamin D intake, must be implemented early in life in KS patients.


Zea mays L.) towards adaptation to temperate climate has allowed the production of inbred lines withstanding cold springs with temperatures below 8 °C or even close to 0 °C, indicating that despite its tropical origins maize is not inherently cold-sensitive.Recent progress in selective breeding of maize  contains supplementary material, which is available to authorized users. Zea mays spp. indentata, Z. mays spp. indurata, Z. mays spp. saccharata and Z. mays spp. everta. For the breeding practice, however, more important are germplasm lineages represented by thousands of inbred lines used for production of hybrids in the public and private sectors. The strong physiological diversity of the inbreds reflects substantial differences at the molecular level. Two lines may differ in the expression pattern of hundreds of genes + to 1 mlwww.imagej.net). Corrected Total Fluorescence (CTF) was determined separately for vascular elements (VE), bundle sheath cells (BS), Kranz mesophyll cells (KMS), and for whole veins using the following equation:For microscopic observations, third-leaf fragments from plants grown as described above were fixed in 2 % (w/v) paraformaldehyde and 2 % (v/v) glutaraldehyde, dehydrated in a graded ethanol series and embedded in LR White. Thin sections (20 μm) were mounted on glass slides and stained with 1 mg/ml Calcofluor White (Sigma Aldrich) for 10 min. After rinsing, the sections were viewed in a Nikon A1R MP confocal microscope , and small and medium-size veins were observed. To allow comparison of results within a given maize line, scan conditions optimized for the sections from plants cold-treated for seven days, where the highest fluorescence was observed, were used for all samples. Fluorescence was analyzed using ImageJ software  and each inbred line CTF values were normalized against relevant mean CTF values from control plants. For each experimental variant, 7-15 veins from three different plants were analyzed.Arabidopsis were taken from the miRNA database at http://www.mirbase.org/. The best matches indicating likely true homology were obtained for seven miRNA species: miR162, 164, 167, 168, 169, 171, and 172.Nucleotide sequences of maize miRNAs orthologous to those known to require DCL1 for maturation in RNA was isolated from the third leaves of plants grown under control conditions or subjected to moderate chilling, as described above, for 1, 3, 5, and 7 days, and individual miRNAs were quantitated by RT-qPCR according to . Primers


The protein level of sEH in kidney was increased in UUO-treated mice group compared to nonobstructed group. Additionally, UUO-induced hydronephrosis, renal tubular injury, inflammation, and fibrosis were ameliorated in sEH−/− mice with the exception of glomerulosclerosis. Moreover, sEH−/− mice with UUO showed lower levels of inflammation-related and fibrosis-related protein such as monocyte chemoattractant protein-1, macrophage inflammatory protein-2, interleukin-1β (IL-1β), IL-6, inducible nitric oxide synthase, collagen 1A1, and α-actin. The levels of superoxide anion radical and hydrogen peroxide as well as NADPH oxidase activity were also decreased in UUO kidneys of sEH−/− mice compared to that observed in WT mice. Collectively, our findings suggest that sEH plays an important role in the pathogenesis of experimental obstructive nephropathy and may be a therapeutic target for the treatment of obstructive nephropathy-related diseases.Soluble epoxide hydrolase (sEH) is abundantly expressed in kidney and plays a potent role in regulating inflammatory response in inflammatory diseases. However, the role of sEH in progression of chronic kidney diseases such as obstructive nephropathy is still elusive. In current study, wild-type (WT) and Urinary obstruction, defined as functional or anatomic impedance to the flow of urine along the urinary tract leading to hydronephrosis and declining renal function, is a common cause of acute and chronic renal failure . It can  sEH both can increase the accumulation of EETs and other epoxy fatty acids which attenuate angiotensin II-induced hypertension and cardiac hypertrophy and fibrosis /37.8 × 1000).Briefly, deparaffinized sections were incubated with dye solution (0.1% Sirius Red and 0.1% Fast Green in periodic acid solution) (Polysciences) at room temperature for 30 min. The color was then eluted from the sections by incubation with 0.05 M NaOH in methanol and measured at OD 540 nm and OD 605 nm by spectrophotometer. The amount of collagen in tissue sections was calculated according to the manufacturer's instructions  and then viewed under a Motic TYPE 102M microscope. The scoring of microscopy assessments of tubular dilation, atrophy, leukocyte infiltration, and tubular interstitial volume and glomerulosclerosis was summarized in Tables μg/mL leupeptin, 10 μg/mL aprotinin, 1 mM PMSF, Tyr phosphatase cocktail I, and Ser/Thr phosphatase cocktail II on ice. After sonication, tissue extracts underwent centrifugation at 12000 ×g for 5 min at 4°C. The supernatants were collected as tissue lysates. All protein concentrations were examined by Bradford protein-binding assay. Aliquots (50 μg) of lysates were separated on 8% or 12% SDS-PAGE and then transblotted on an Immobilon-P membrane . After being blocked with 5% skim milk for 1 h, blots were incubated with primary antibodies and then with corresponding secondary antibodies. The protein bands were detected by use of an enhanced chemiluminescence kit  and quantified by ImageQuant 5.2 software .Tissues were lysed with phosphate buffered saline containing 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 1 2−) and hydrogen peroxide (H2O2) counts was performed as previously described with minor modification -cyclohexyloxy} benzoic acid or genetic deletion in mice prevents the renal inflammation and fibrosis [A role of the hydrolase activity of sEH in the metabolism of EETs, inflammation, and hypertension has been well documented. Pharmacological inhibition of hydrolase activity of sEH or genetic deletion oft models . For inst models , 11. Howfibrosis , 28. Col sEH activity stimulates the process of wound healing in experimental animals [ sEH promotes EMT in rat proximal tubular epithelial cells [ sEH decreased renal inflammation and led to attenuation of renal fibrosis. Taken together, these findings strongly suggest that sEH might play a key role in progression of tissue fibrosis. However, further investigation is still required to delineate how sEH regulates the cellular and molecular mechanisms underlying fibrosis.Leukocyte infiltration and cytokine production as indicators of inflammatory status are crucial in determining the stages of certain inflammatory diseases , 24. The animals , 31. Mor animals . On the al cells . In agreal cells . This no sEH−/− mice. These lines of evidence imply that sEH may have prooxidative and proinflammatory properties. Collectively, these observations strongly suggest the heavy involvement of sEH in the regulation of kidney physiologic function and the development of kidney diseases.Importantly, oxidative stress is known to be highly associated with the regulation of inflammation in a variety of inflammatory diseases , 18. Thi sEH gene ameliorates the renal injury by decreasing inflammation, renal tubular injury, and renal interstitial fibrosis. The mechanisms revealed in this study provide novel insights for delineating the role of sEH in inflammatory diseases. Our findings suggest that targeting sEH may have clinical implications and may be a valuable therapeutic strategy in treating obstructive nephropathy-related kidney diseases.In conclusion, we demonstrate the role of sEH in progression of obstructive nephropathy. Deletion of


Most ofThe signal of dopamine neurons in classical and instrumental conditioning has been explained in terms of the temporal-difference (TD) algorithm. However it is not clear whether and, if so, how reinforcement learning can account for the dopamine signals in complex decision-making tasks with noisy sensory information and temporal uncertainty of the relevant task events, as is the case in the detection task mentioned above . We haveThe dopamine phasic activity predicted by the model matches the experimental data and the prediction of the psychometric curve is consistent with the animal performance. Furthermore, the model provides an interpretation of the condition-dependent dopamine response to the go cue instruction in terms of reward prediction error. Using Bayesian inference the model constructs an internal belief about the presence of the somatosensory stimulus. This belief reflects the confidence about the sensory perception and thus the value assigned to this perceptual judgment. The large belief in stimulus-present decisions represents a high degree of confidence in the sensory perception and a great expectation for future rewards. On the contrary, stimulus-absent choices reflect a small belief and consequently a larger uncertainty about the decision and the future reward. This computational description of belief agrees with the previous interpretation of the data  and desc