Data regarding the quantitative expression of TCR Vβ subpopulations in children with autoimmune diseases provided interesting and sometimes conflicting results. The aim of the present study was to assess by comparative flow cytometric analysis the peripheral blood CD4+ TCR Vβ repertoire of children with an organ-specific autoimmune disorder, such as type 1 diabetes mellitus (T1DM), in comparison to children with a systemic autoimmune disease, such as Systemic Lupus Erythematosus (SLE) in comparison to healthy age-matched controls of the same ethnic origin. The CD4+ TCR Vβ repertoire was analysed by flow cytometry in three groups of participants: a) fifteen newly diagnosed children with T1DM (mean age: 9.2 ± 4.78 years old), b) nine newly diagnosed children with SLE, positive for ANA and anti-dsDNA, prior to treatment (mean age: 12.8 ±1.76 years old) and c) 31 healthy age-matched controls (mean age: 6.58 ± 3.65 years old), all of Hellenic origin.CD4 + TCR Vβ abnormalities (± 3SD of controls) were observed mainly in SLE patients. Statistical analysis revealed that the CD4 + Vβ4 chain was significantly increased in patients with T1DM (p < 0.001), whereas CD4 + Vβ16 one was significantly increased in SLE patients (p < 0.001) compared to controls.CD4 + Vβ4 and CD4 + Vβ16 chains could be possibly involved in the cascade of events precipitating the pathogenesis of T1DM and SLE in children, respectively. Autoimmunity involves complex pathophysiological processes; however, no comprehensive model of inflammatory induction has been unraveled as yet. Genetic and environmental immunologic factors, several hormones and stress have been implicated in the pathogenesis of autoimmune disorders -4. MoreoTCR Vβ repertoire is assessed with two methodologies, CDR3 spectratyping, which is a genetic assay that provides mainly qualitative information about TCR Vβ clonality ,9, and fThe aim of this study was to compare the peripheral blood CD4+ TCR Vβ repertoire in children with T1DM and SLE, in comparison to healthy age-matched controls of the same ethnic origin with the use of the flow cytometry assay.Complete blood count values, immunoglobulin levels and lymphocyte subpopulation counts were normal in all healthy children. Autoantibodies, either SLE-specific or diabetes speficic , were evaluated and they were negative in all controls tested. There was an unavoidable male preponderance in the tested children (boys: 80.6%), due to the higher frequency of anatomical abnormalities needing surgical reconstruction in boys.Mean values and standard deviations (SD) of each distinct Vβ family percentage on CD4+ lymphocytes of the 31 studied children are shown in Table The increased/decreased usage of a Vβ subfamily has arbitrarily been defined as mean value ± 2 or 3 standard deviations  ,11. Of tEnrolled infants and children were separated into three age groups, which were compared in order to find out possible differences in Vβ expression at different age groups. Table Assessment of the CD4+ TCR Vβ repertoire was performed in two ways: a) Vβ repertoire of each T1DM patient was compared with the Vβ mean values of the control group and b) statistical analysis was performed, in order to detect the presence of CD4 + Vβ lymphocyte clones in the group of T1DM patients with different quantitative expression in comparison to the control group.The increased/decreased usage of a Vβ subfamily was defined as control mean value ± 2 or 3 standard deviations ,11. FiveIn the SLE group, skewing in CD4 + Vβ subpopulations was detected in the majority of patients when compared to healthy individuals. In contrast to healthy children and T1DM patients, which presented with a similar CD4 + TCR Vβ repertoire pattern, 7 of the 9 SLE children tested displayed concurrently marked increased  and decreased usage  of several CD4 + Vβ subpopulations. This was noteworthy in one SLE patient who displayed increased usage of the CD4+ Vβ12 (9.44%), Vβ16 5.6%) and Vβ20 (9.17%) and a decreased one of Vβ2 (2.83%) and Vβ5.1 (0.12%) chains and in another one patient with high values of CD4+ Vβ3 (9%), Vβ12 (14.2%) and Vβ16 (7.3%) chains and very low values of CD4+ Vβ2 (0.2%), Vβ5.2 (0.2%), Vβ7.1 (0.1%) and Vβ22 (0.3%)  Increased expression of CD69 molecule in two SLE patients and in one T1DM patient, b) elevated percentage of the CD3+ TCRγδ lymphocyte population (20%) in two SLE patients, c) increased expression of the HLA-DR + cells in 2 of the nine SLE children tested and d) absolute number of B lymphocytes (CD19+) beyond normal values in one SLE and in one T1DM patient. No difference in the CD5 + CD19+ expression was noticed among the three groups and the values (%) of the CD5 + CD19+ populations ranged between 0.1 and 1.2% in all individuals tested.Flow cytometric analysis of the CD4 + TCR Vβ repertoire performed in three different groups of children of Greek origin, healthy, T1DM and SLE ones, provided results similar to previous published studies, but with some differences.As far as healthy children are concerned, the results of this study support the non-random usage of TCR Vβ subfamilies in Greek children, as observed in previous studies on normal adults and children ,16,17. TT1DM children had an almost same CD4 + Vβ repertoire pattern with healthy individuals, with the exception of the CD4 + Vβ4 chain which had increased expression in T1DM patients when compared to controls. Previous studies have shown similar results, that is either no differences between the two groups , or incrIn contrast to T1DM patients, in SLE patients skewing of the CD4 + Vβ repertoire is far more prominent. This finding is in accordance to previous studies published in adults ,15. CDR3Limitations of the present study are the limited number of the patients studied, but this is due to the rarity of lupus in children in the Greek population and to the fact that only patients upon the initial diagnosis were selected in order to avoid alterations in their immunological profile that medication could induce. Another parameter is the fact that evaluation of the Vβ repertoire in each patient is not influenced by the number of the patients investigated. Although genetic evaluation was not performed, all previous published studies in immune mediated diseases have showed that there are no discrepancies between the results from flow cytometric analysis and spectratyping, enhancing the reliability of flow cytometry. Finally, the CD8+ Vβ lymphocyte subpopulations were not analyzed, and therefore it is not known whether there are abnormalities in the repertoire of children with diabetes or lupus or not.As far as immunophenotyping analysis is concerned, no major differences among the groups were noticed. Regarding the role of the CD5 + CD19+ population in autoimmune process, its expression was quite similar in the three groups studied. This finding is in accordance with other studies in adult SLE patients, but not with studies in T1DM patients, in whom increased expression was observed ,21.This is the first comparative flow cytometric analysis between an organ-specific autoimmune disease, T1DM, and a systemic one, SLE, which underscores the importance of the TCR Vβ repertoire in patients with auto-immune diseases. This study was possible because of the availability of an easily applicable method at a clinical setting, which enabled TCR Vβ repertoire analysis prior to initiation of any treatment that could possibly be a confounding factor. Abnormalities of the CD4+ TCR Vβ repertoire quantitative expression were observed mainly in SLE children. Low usage of certain CD4 + TCR Vβ chains was also far more prominent in lupus patients. Only the CD4 + TCR Vβ4 and CD4 + TCR Vβ16 lymphocytes were significantly increased in T1DM and SLE children, respectively. However, the potential role of these lymphocyte clones in the pathogenesis of autoimmunity remains to be clarified.Samples were collected from a) Fifteen newly diagnosed children with T1DM  during their hospitalization upon diagnosis in the First Department of Pediatrics of the University of Athens in “Aghia Sophia” Children’s Hospital and b) Nine patients with SLE  upon diagnosis and prior to treatment initiation, all of Hellenic origin. Thirty one healthy Greek children  undergoing minor surgery at the First and Second Department of Surgery at “Aghia Sophia” Children’s Hospital were included as controls. Taking into account that several germ line polymorphisms of the TCR Vβ genes have been described in different ethnicities ,23, thisThe study was approved by the Ethical Committee of the “Aghia Sophia” Children’s Hospital and participants were included in the study only after informed consent was obtained from their parents or guardians.Blood (4 ml in EDTA tube and 3 ml serum) was taken from all participants for investigations, including full blood count, serum immunoglobulin levels assessment by nephelometry and screening for autoantibodies by indirect immune-fluorescence , as well as lymphocyte subpopulation immunophenotyping by flow cytometry.4Cl solution. At least 10.000 CD4+ lymphocytes were collected for analysis. CD4+ lymphocytes were gated using forward scatter, side scatter and FL4 fluorescence; whereas Vβ repertoire analysis used two additional fluorescence channels (FL1 and FL2).CD4 + TCR Vβ repertoire was analyzed using three-color flow cytometry with the IOTest Beta Mark TCR Repertoire Kit , which consists of fluorochrome conjugated monoclonal antibodies that identify 24 TCR Vβ subfamilies, covering about the 70% of the normal human CD4+ T cells. Since at the time of the study initiation, only three-color flow cytometry was in practice, and in order to assess the results with the same method, no changes in methodology were undertaken. For that reason only the CD4+ TCR Vβ repertoire and not the CD8+ one was assessed. Peripheral blood samples were collected in tubes containing anticoagulant (EDTA) and were stained within 2 hours. 100 μl of blood was incubated for 20 minutes, at room temperature with 20 μl of a mixture of three distinct anti-Vβ monoclonal antibodies and 10 μl of anti-CD4-PC5 monoclonal antibody (MoAb). Erythrocytes were lysed by NHData acquisition was performed initially on an EPICS XL (Beckman Coulter) flow cytometer and, later on, on an FC-500 (Beckman Coulter), instrument. Listmode analysis was performed using the CXP software.Lymphocyte subpopulations tested were: 1) CD3+ T cells, 2) helper T cells (CD3 + CD4+), 3) cytotoxic T cells (CD3 + CD8+), 4) natural killer cells (CD56+) and 5) B lymphocytes (CD19+). Moreover, the αβ and γδ expression on T cells, the expression of CD69+ and HLA-DR + on T cells as activation markers and the expression of CD5+ on CD19+ B cells were studied.11/12 years (n = 12), 5-911/12 years (n = 10) and ≥ 10 years (n = 9). The reason for this transformation was to reveal a possible difference in Vβ repertoire usage among specific age groups that roughly reflect the stages of child development and immune system maturation. Vβ repertoire expression variables are described as median and interquartile range IQR (75th-25th percentile) due to the small sample size and to their non-normal distribution. For the same reason, non parametric statistics were used; Mann–Whitney U statistic was used for the comparison of the aforementioned variables between two age subgroups and Kruskal-Wallis rank test when comparing the same variables among the three age subgroups of the study. All tests were two-sided at a significance level of p < 0.5. Data were analyzed using STATA™ .Statistical analysis among three age groups of the healthy individuals included in the study was performed in order to detect possible skewing among them. Age was transformed to categorical variables, thus constructing three age groups: 18 months-4th - 25th percentile) were used to describe the continuous variables. In addition, nonparametric statistical analysis (Mann–Whitney U) was used to test for any significant differences between the subgroups. Due to the multiple comparisons (n = 24), Bonferroni adjustment was performed in order to correct for inflation in type I error, setting the significance level from p < 0.05 to < 0.0021 (=0.05/24). Statistical analysis was also adjusted to a CV of 20%, which could characterize the results of Vβ chains with rare expression. The significant difference that was found between controls and T1DM patients in Vβ4 chain was also present when using Student’s t-test for the comparison. Although t-test is a parametric statistic and less powerful in our case due to the small number of patients, it allowed us to perform new hypothetical comparisons between the aforementioned groups in order to verify that the comparisons would remain significant even after increasing the mean value in the control group by 20% and decreasing the mean value in the T1DM group (for Vβ4 chain) by 20%.To assess the possible differences of CD4 + TCR Vβ repertoire expression, pediatric patients with newly diagnosed SLE and T1DM were compared with healthy age-matched controls. Due to the small subgroup sizes of SLE and T1DM , median and IQR  and has written the manuscript. MK has significantly contributed in the conception and design, the acquisition of data, the analysis and interpretation of data as well as in the drafting the article and in revising it critically for important intellectual content. She has also significantly contributed in the final approval of the version to be published. MT contributed in the analysis and interpretation of the data and revised the manuscript. CM has significantly contributed in the acquisition of data and the analysis and interpretation of data as well as in the drafting the article and revising it critically. More specifically due to his high qualifications in statistical analysis and interpretation he has contributed in the interpretation and presentation of the important findings of the study. EP has personally conducted the measurement of serum immunoglobulin levels and the assessment and screening for autoantibodies and she has contributed to the interpretation of data. GC revised the manuscript critically for important intellectual content. CKG contributed in the interpretation of the collected data and the preparation of the manuscript. Together with MK, she had the main contribution in the drafting of the article and in revising it critically for important intellectual content. She has also significantly contributed in the final approval of the version to be published. All authors read and approved the final manuscript.FT is the PhD candidate, who has conducted the study under the supervision of Dr. Maria Kanariou, the Director of the Department of Immunology & Histocompatibility, “Aghia Sophia” Children’s Hospital, Greece and the help of the members of the Department of Immunology & Histocompatibility. She holds master degree in “Molecular Medicine” (supervisor of the two-year training in basic research methodology is Professor Nikolaos Anagnou). She is Pediatrician and is currently Consultant at the 3rd Department of Pediatrics’ Clinics of “Attikon” University Hospital.MK is the Director of the Department of Immunology & Histocompatibility, Specific Center & Referral Center for Primary Immunodeficiencies - Paediatric Immunology, “Aghia Sophia” Children’s Hospital, Greece where all laboratory measurements have been conducted under her supervision.MT is a Medical Biopathologist with over a decade of experience in Flow Cytometry. She is currently responsible for the Flow Cytometry and Cell Culture Laboratories of the Department of Immunology & Histocompatibility, Specific Center & Referral Center for Primary Immunodeficiencies - Paediatric Immunology, “Aghia Sophia” Children’s Hospital.CM is GP, Consultant at Hospital of Kimi, Greece and holds a master degree in biostatistics and PhD diploma in epidemiology. He also has many publications in several studies in which he contributed as a biostatistician.EP is also a staff member of the Department of Immunology & Histocompatibility, “Aghia Sophia” Children’s Hospital, Greece.GC is the Medical Director of the First Department of Pediatrics and the Division of Endocrinology, Diabetes and Metabolism, of the National Kapodistrian University of Athens, “Aghia Sophia” Children’s Hospital, Greece.CKG is the supervisor of Mrs Tzifi’ PhD thesis and the scientific supervisor of the Diabetes Center, where the type 1 diabetes patients are followed. She is Associate Professor for Pediatric Endocrinology and Juvenile Diabetology in the First Department of Pediatrics and the Division of Endocrinology, Diabetes and Metabolism, of the Medical School of the National Kapodistrian University of Athens, “Aghia Sophia” Children’s Hospital, Greece.


Sir,British Journal of Cancer (α-trypsin inhibitor (IαI) and different members of the complement cascade. We focused our attention on the complement components and might have underestimated the potential importance of increased IαI levels.We appreciate the comments provided by αI proteins are a family of structurally related serine protease inhibitors with hyaluronan-binding capacities, assembled from a light chain and one of five homologous heavy chains (H1–H5). In correspondence, separation of IαI by two-dimensional gel electrophoresis results in different spots with a molecular weight of ∼80, 125 and 250 kDa  is influenced by the epirubicin/docetaxel therapy . The abundance of this spot increased in nearly all patients . Garantziotis referred in his letter to a recently found interaction of IαI with the complement system. In an experimental study, he could show that IαI attenuates in vitro complement activation and reduces in vivo complement-induced lung injury (αI (spot 405) with the total plasma C3 revealed that both parameters correlate negatively with each other . The total plasma C3 was measured by a routine nephelometric immunoassay that cannot distinguish between the different C3 isoforms. In our study, we also investigated the single isoforms of C3 by two-dimensional gel electrophoresis. They responded unequally to the therapy and some of them correlated with the tumour size at the end of treatment. However, none of these single isoforms showed a correlation with the IαI spots, and no 125 kDa IαI spot showed a correlation with the success of therapy.Irapy see . All theg injury . CompariαI and the complement system, several questions remain. Is the therapy-induced increase in plasma IαI the causative factor for C3 reduction? Is the decrease in C3 associated with reduced complement reactivity? How do the other IαI isoforms (80 and 250 kDa) react in response to therapy (they were not identified on our 2D gels)? Further studies are needed to find satisfactory answers.Although these findings corroborate the interaction of I


The commonality of many neurodegenerative disorders is the predictable temporal occurrence and progression of specific aggregated proteins in the brain. The hallmark proteopathy is Alzheimer’s disease in which aggregated amyloid-β peptide (Aβ) is deposited in the brain parenchyma as amyloid plaques. Multiple evidence suggests that β-amyloidosis is induced by aggregated Aβ which can spread within and among brain regions and act as corruptive templates (seeds) that induce a chain-reaction of Aβ misfolding and aggregation. The insight that the prion paradigm may also apply to cerebral β-amyloidosis and other proteopathies suggests new avenues in search of biomarkers and novel therapeutic strategies.


The π is missing from the 'Trp in V3 signature pattern 1 could form stabilizing cation- interaction with V2' paragraph of the Results and Discussion. The symbol should appear as follows in the first portion of the paragraph below:Trp in V3 signature pattern 1 could form stabilizing cation-π interaction with V2. We had marked earlier that position 317 (V3 position 22) is usually occupied by an aromatic residue, mostly F and in B’ W. Why should an aromatic amino acid interact with the positively charged region in V2 as suggested by DI ? This interaction could be explained by cation-π interactions between the cationic V2 region and the π-electron system of the aromatic F or W .


Substituted thiourea ligands are of interest because they possess various donor sites for metal ions and their application in separation of metal ions and as antimicrobial agents. The coordination of the sulfur donor atom led to interest in them as precursor for semiconductor nanoparticles. In this study, cadmium(II) complex of diethylthiourea was synthesized and characterized by elemental analysis, FTIR, and X-ray crystallography. Single crystal X-ray structure of the complex showed that the octahedral geometry around the Cd ion consists of two molecules of diethylthiourea acting as monodentate ligands and two chelating acetate ions. The thermal decomposition of the compound showed that it decomposed to give CdS. The compound was thermolysed in hexadecylamine (HDA) to prepare HDA-capped CdS nanoparticles. The absorption spectrum showed blue shifts in its absorption band edges which clearly indicated quantum confinement effect, and the emission spectrum showed characteristic band edge luminescence. The broad diffraction peaks of the XRD pattern showed the materials to be of the nanometric size. The data sets for the single crystal X-ray studies were collected with Mo Kα radiation (k = 0.71073) at 100(2) K on a Bruker SMART APEX ·H2O was dissolved in 10 mL of trinoctylphosphine and injected into 6 g of hot hexadecylamine (HDA) at 150°C. A subsequent decrease in temperature of 20–30°C was observed. The solution was heated to 150°C and stabilized at this temperature for 1 h. The solution was then allowed to cool to 70°C, an excess of methanol was added, and a flocculants precipitate was formed. The solid was separated by centrifugation and redispersed in toluene. The toluene was removed under vacuum to give HDA-capped CdS nanoparticles. About 0.5 g of  and O(4)–Cd(1)–O(3) [54.20(6)]. This also led to deviation of trans O(4)–Cd(1)–O(1), O(2)–Cd(1)–S(1) and O(3)–Cd(1)–S(2) bond angles significantly from 180° (ranges from 141.21(6)° to 151.48(4)°). The diethylthiourea ligands are cis to each other, and S–Cd(1)–S bond angle around the cadmium ion is 102.83(3)° which deviates considerably from 90°. This may be ascribed to the steric interaction between the substituents on the diethylthiourea. The C–S–Cd bond angles of 108.82(8)° and 111.59(8)° are slightly greater than the tetrahedral value.The molecular structure of the complex with atom numbering scheme is shown in The S–C–N and N–C–N bond angles ranges from 118.51(17)° to 121.62(17)° and are within the expected range of tetrahedral. The C–N bonds (1.327(3) Å to 1.458(3) Å) and C–S bonds 1.727(2) Å to 1.730(2) Å are intermediate between single and double bonds. This may be attributed to the delocalization of electron in the thioamide bonds , 21. The−1 experienced only very slight changes in the complex. The N–H band occurs at 3275 cm−1 with a shoulder at 3430 cm−1 in the spectrum of the complex. The bands overlap with the C–H band to form a generally broad band. This is due to the presence of H-bonds networks in the molecular structure of the complex as confirmed by the single crystal X-ray structure of the compound (ν(C=S) stretching vibrations occur at 747 cm−1, and the thioamide bond is observed as multiple band in the region 1554–1562 cm−1. This might be due to the overlap of the acetate ions with C–N bond stretching vibrations. The presence of multiple bands in the region 552–672 cm−1 can be attributed to the S–Cd–O interactions.The IR spectra of the ligand and complex were compared and assigned on careful comparison. The absorption band in the 3430–3200 cmcompound . The ν(C2(OOCCH3)2]·H2O, has been carried out to study the pyrolysis pattern in the temperature range 20–800°C. From the thermogram (TGA/DTG) in Thermogravimetric analysis of the compound, ·H2O. Recrystallization of the complex yielded well-defined crystals characterized by FTIR, elemental analysis, and single-crystal X-ray diffraction. Single crystal X-ray structure of the compound revealed that the coordination geometry around the Cd(II) is octahedron comprising of two diethylthiourea ligands and two acetate ions acting as bidentate chelating ligands. The single-source precursor route has been used for the preparation of CdS nanoparticles by thermolysis of the complex in hexadecylamine (HDA) to prepared HDA-capped nanoparticles. The absorption spectrum showed blue shifts in their absorption band edges which clearly indicated quantum confinement effect, and the emission spectrum showed characteristic band edge luminescence. The broad diffraction peaks of the XRD pattern showed the materials to be of the nanometric size with predominantly hexagonal phase. The TEM micrographs showed the CdS morphology to be almost spherical shape with particle sizes ranging between 5 and 19 nm.The reaction of cadmium acetate with diethylthiourea yielded the coordination complexes, [Cd(detu)


Pseudomonas aeruginosa is the major severe complication in cystic fibrosis (CF) patients, where P. aeruginosa persists and grows in biofilms in the endobronchial mucus under hypoxic conditions. Numerous polymorphonuclear leukocytes (PMNs) surround the biofilms and create local anoxia by consuming the majority of O2 for production of reactive oxygen species (ROS). We hypothesized that P. aeruginosa acquires energy for growth in anaerobic endobronchial mucus by denitrification, which can be demonstrated by production of nitrous oxide (N2O), an intermediate in the denitrification pathway. We measured N2O and O2 with electrochemical microsensors in 8 freshly expectorated sputum samples from 7 CF patients with chronic P. aeruginosa infection. The concentrations of NO3− and NO2− in sputum were estimated by the Griess reagent. We found a maximum median concentration of 41.8 µM N2O (range 1.4–157.9 µM N2O). The concentration of N2O in the sputum was higher below the oxygenated layers. In 4 samples the N2O concentration increased during the initial 6 h of measurements before decreasing for approximately 6 h. Concomitantly, the concentration of NO3− decreased in sputum during 24 hours of incubation. We demonstrate for the first time production of N2O in clinical material from infected human airways indicating pathogenic metabolism based on denitrification. Therefore, P. aeruginosa may acquire energy for growth by denitrification in anoxic endobronchial mucus in CF patients. Such ability for anaerobic growth may be a hitherto ignored key aspect of chronic P. aeruginosa infections that can inform new strategies for treatment and prevention.Chronic lung infection by  Pseudomonas aeruginosa is a Gram-negative, gamma proteobacterium, which dominates chronic lung infections in CF patients and is considered the most serious complication of CF P. aeruginosa lung infection in CF patients is characterized by presence of endobronchial biofilm aggregates surrounded by numerous polymorphonuclear leukocytes (PMNs) P. aeruginosa can withstand the bactericidal activity of the PMNs by forming biofilms of the protective mucoid phenotype 2) P. aeruginosa survives the attacking PMNs, but it is not known how P. aeruginosa acquires the energy required for the observed growth in endobronchial secretions 2 is absent. However, P. aeruginosa can grow anaerobically with alternative electron acceptors or by arginine fermentation P. aeruginosa can respire by denitrification in anoxic CF mucus utilizing nitrate (NO3−) and nitrite (NO2−), which are both present in sufficient amounts P. aeruginosa to utilize reduction of NOx for anaerobic respiration is well known 2O is a natural intermediate belonging to the gases defining denitrification 2 and N2O concentration gradients at high spatio-temporal resolution in freshly expectorated sputum from CF patients with chronic P. aeruginosa lung infection.Cystic fibrosis (CF) is an autosomal recessive disease. It is caused by mutations in the cystic fibrosis trans-membrane conductance regulator gene 3−) and nitrite (NO2−) turnover measurements in the sputum samples. These measurements provided important new insights to the micro-environmental conditions and chemical dynamics associated with persistent P. aeruginosa lung infections in CF patients and indicate that nitrogen compounds can play an important role in the interaction between pathogenic bacteria and an active immune response.Further evidence for denitrification was obtained from nitrate  from the sputum surface  (2O is related to P. aeruginosa we found significantly less N2O in three control sputum samples from 1 CF patient and from 2 primary ciliary dyskinesia (PCD) patients without detectable P. aeruginosa (p<0.030).Vertical profiles of O surface  suggesti, n = 8) . To veri2O profiles measured vertically through a sputum sample. The distribution of O2 is displayed at 0 hr. During the initial measuring period, N2O accumulated in the anoxic zone reaching a maximum concentration of 160 µM after 6.5 h incubation, which indicates ongoing production of N2O. Within the subsequent 4 hours the accumulated N2O decreased indicating consumption through reduction to N2.2O concentration dynamics over time in particular depths of a sputum sample showed an initial build-up of N2O in layers below 7 mm  for the reduction of nitric oxide (NO•) and N2O P. aeruginosanorCB) have been sequenced P. aeruginosa by consumption of NO• The depletion of O2O production in the sputum samples was followed by a period of net N2O consumption suggesting a depletion of NO• and a concomitant reduction of N2O to N2 by N2OR. The N2O consumption is in agreement with the demonstration of N2OR activity and the identification of the nos genes in P. aeruginosa2OR precursor in clinical isolates In our study, the initial phase of N2O production in sputum indicates ample presence of NO3− and NO2− that serve as electron acceptors for the denitrification pathway. We found high levels of NO3− and NO2− in the sputum, which are in agreement with previous findings 3− and NO2− in CF sputum originates from the rapid reaction between superoxide (O2-) and NO• 2-, while NO•, which is present in CF exhaled breath 3− and NO2− in sputum from CF patients Our demonstration of significant N2O production, we expected a consumption of the precursors NO3− and NO2−. Accordingly, NO3− was depleted in the sputum after incubation for 1 day, which likely is due to the membrane-bound nitrate reductase of P. aeruginosa3− consumption may also accompany assimilatory denitrification and ammonification resulting in the formation of ammonia (NH4+) 2O 4+ is also produced by several human cell types 2− was not changed during 1 day of incubation, but after 2 days of incubation the concentration of NO2− in the sputum was decreased significantly. This indicates that the production of NO• from NO2− is slower than the generation of NO2− resulting from reduction of NO3−. Indeed, during reduction of NO3− transient accumulation of NO2− is known from anaerobic cultures of P. aeruginosa growing by denitrification As a consequence of our demonstration of N2O production  As defined by the “Danish Act on Research Ethics Review of Health Research Projects” Section 2 the project does not constitute a health research project and was thus initiated without approval from The Committees on Health Research Ethics in the Capital Region of Denmark. Therefore, verbal informed consent was obtained using waiver of documentation of consent. The study was carried out on 21 anonymized samples of surplus expectorated sputum from 21 CF patients and 2 PCD patients . Chronic2-concentration profiles were recorded in the sputum with an amperometric O2 microsensor  mounted in a motorized PC-controlled profiling setup . Subsequently, vertical N2O concentration profiles were recorded at defined time intervals for up to 12 hours with an amperometric N2O microsensor 2O-25, Unisense A/S) mounted in the micromanipulator.Each of 8 different sputum samples (1–2 ml) was added to a glass vial (35×12 mm)  and allowed to settle for about 10 min. The glass vials were positioned in a heated metal rack, kept at 37°C. Vertical O2-microprofile and 5 seconds for the N2O-microprofile, before actual measurement and subsequent movement of the sensors to the next measuring depth. The interval between each cycle of profile measurements was 10 seconds.The microsensors (tip diameter 25 µm) were connected to a picoammeter  and positioned manually onto the upper surface of the sputum sample. Profile measurements were taken by movement of the sensor in vertical steps of 100 or 200 µm through the sputum sample. Positioning and data acquisition were controlled by dedicated software . The software was set to wait 3 seconds for the O2-microsensor was linearly calibrated by measuring the sensor signal in an alkaline sodium ascorbate solution (zero O2) and in air saturated free phosphate buffered saline (PBS) at experimental temperature and salinity. The O2 concentration in air saturated water was determined from the known temperature and salinity according to 2O -microsensor was linearly calibrated according to 2O free PBS at experimental temperature and salinity and in PBS with sequential addition of a known volume of N2O saturated PBS up to a final concentration of 100 µM N2O. The N2O concentration in saturated PBS was determined according to The O3− and NO2− in sputum was measured in 20 samples. From each sputum sample, 0.1 ml was aspired with a syringe and was immediately diluted 10x in PBS and stored at -20°C for later analysis. The remaining sample was incubated in a glass vial at 37°C for 24 h before dilution 10x in PBS and storage at −20°C. The NO3− and NO2− levels in the sputum were measured using the Griess colorimetric reaction  according to the manufacturer's recommendations. For this, sputum samples were transferred to a 96 well microtiter plate. NO2− concentration was estimated by addition of the Griess Reagent for 10 minutes, whereby NO2− was converted into a purple azo-compound, which was quantitated by the optical density at 540–550 nm measured with an ELISA plate reader . Total NO3− and NO2− levels were estimated by a two-step analysis process: The first step converted NO3− to NO2− utilizing NO3− reductase. After incubation for 2 hours, the next step involved the addition of the Griess Reagent, whereby NO2− was converted into a purple azo-compound. After incubation with Griess Reagent for 10 minutes, the optical density at 540–550 nm was measured with an ELISA plate reader . A NO3− standard curve was used for determination of total NO3− and NO2− concentration, while a NO2− standard curve was used for determination of NO2− alone. The concentration of NO3− was calculated as the difference between the NO3− concentration and the total NO3− and NO2− concentration.The concentration of NO2O fluxes in sputum samples were calculated from the measured N2O concentration gradient in the uppermost oxic sputum layer. It was assumed that no production or consumption of N2O occurred in the presence of O2. The flux was calculated using a modified version of Fick's 1st law of diffusion J is the flux of N2O (nmol N2O cm−2 min−1), D is the molecular diffusion coefficient of N2O in water at 37°C (2.76×10−5 cm2 s−1) 2O (µmol liter−1) at depth xn, where n = a, b or c denote 3 subsequent depths of measurement. The cumulated N2O emission was calculated by assuming linear changes between subsequent measurements. Net production and net consumption rates of N2O in particular sputum layers were calculated from the slopes of linear increase and decrease of N2O concentration at particular measuring depths in the sputum samples The local NStatistical significance was evaluated by Wilcoxon Signed Rank Test and by Students T-test. A p value <0.05 was considered statistically significant. The tests were performed with Prism 4.0c .


Moreover, monoubiquitination of PCNA does not affect TLS by pol λ while it appears to slightly stimulate TLS by pol η.DNA polymerase ε (pol ε) is believed to be the leading strand replicase in eukaryotes whereas pols λ and β are thought to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol ε is halted by an abasic site (apurinic/apyrimidinic (AP) site). We have previously reported that human pols λ, β and η can perform translesion synthesis (TLS) of an AP site in the presence of pol ε. In the case of pol λ and β, this TLS requires the presence of a gap downstream from the product synthetized by the ε replicase. However, since these studies were conducted exclusively with a linear DNA template, we decided to test whether the structure of the template could influence the capacity of the pols ε, λ, β and η to perform TLS of an AP site. Therefore, we have investigated the replication of damaged “minicircle” DNA templates. In addition, replication of circular DNA requires, beyond DNA pols, the processivity clamp PCNA, the clamp loader replication factor C (RFC), and the accessory proteins replication protein A (RPA). Finally we have compared the capacity of unmodified versus monoubiquitinated PCNA in sustaining TLS by pols λ and η on a circular template. Our results indicate that  At least three DNA polymerases (pols) are required for chromosomal DNA replication in eukaryotic cells: pol α, pol δ and pol ε. Pol α has an associated primase activity necessary for initiation of replication Abasic sites (AP sites) arise frequently by spontaneous hydrolysis of purines in DNA and are a common intermediate of numerous DNA repair systems. AP sites are among the most frequent endogenous DNA lesions in vitro by either Y or X family pols in vitro by pol α Translesion synthesis (TLS) of an AP site can be accomplished A widely accepted model of TLS is the polymerase-switching model, in which protein-protein interaction leads to a switch between the replicative pol arrested at a lesion and a pol capable of bypass. In eukaryotes, this switching appears to be mediated by a monoubiquitinated form of PCNA, although monoubiquitination appears to be important but not essential for TLS Conversely, another model, named gap-filling model, can be taken in consideration. This model could apply to TLS in DNA gaps resulting from re-priming events or processing of closely spaced lesions on opposite DNA strands Recently we have presented evidence that human pols λ, β and η can perform TLS of an AP site in the presence of pol ε, likely by extending the 3′OHs created at the lesion by the arrested pol ε However, since these studies were conducted exclusively with a linear DNA template, we decided to examine whether the structure of the template could influence the capacity of the pols ε, λ and β to perform TLS of an AP site. This was investigated by testing replication of damaged “minicircle” DNA templates. These circular templates-primers were engineered so that their replication by pol ε led to the formation of single–stranded gaps of different length downstream the AP site. Replication of circular DNA requires, beyond DNA pols, the processivity clamp PCNA, the clamp loader replication factor C (RFC), and the accessory proteins replication protein A (RPA). We have also compared the capacity of unmodified versus monoubiquitinated PCNA in sustaining TLS by pols λ, β and η.in vitro gap-directed TLS synthesis by pols λ and β in the presence of pol ε, RPA, RFC and PCNA is unaffected by the structure of the DNA template and that monoubiquitination of PCNA does not influence TLS by pol λ while it appears to stimulate TLS by pol η.Our results indicate that 2PO4 pH 8, 300 mM NaCl and protease inhibitor cocktail-Roche) and centrifuged at 20,000 rpm 1 hour in JA-20 rotor. The supernatant was mixed with Ni-DA beds in LEW buffer for 4 hours at 4°C and then poured into a column. Elution was performed with LEW plus 250 mM Imidazol. The 2 ml eluted fraction was diluted at 100 mM NaCl with buffer A  and charged on a mono Q column equilibrated with buffer A. Fractions of 0.4 ml were eluted with a gradient of 0.1 to 0.4 NaCl in buffer A and the protein peak fractions tested for the capacity to stimulate pol δ activity in presence of PCNA on a minicircular template (see Results and Discussion).Recombinant human pol λ, RPA and PCNA were expressed and purified as described 32P]-ATP according to the manufacturer's protocol. Each primer was mixed with the templates at a 1∶1 (M/M) ratio in the presence of 20 mM Tris HCl pH 8 and 50 mM KCl, heated at 90°C for 5 minutes and then slowly cooled. [γ-32P]-ATP was from Perkin Elmer, dNTPs were from Fermentas, and 20× Glycerol Tolerant Gel (GTG) buffer was from USB.The 100-mer oligonucleotide linear template, either undamaged or containing a synthetic AP site (tetrahydrofuran moiety) and the oligonucleotides used as scaffold for constructing circular DNA templates or as primers for extension assays, were from Eurogentec and were PAGE purified. For a detailed description of the method used to construct a mini-circular DNA template starting from a linear one see 2, 1 mM DTT, 200 μg/ml BSA, 2.5% Glycerol, 1.5 mM ATP and 50 μM each of dATP, dCTP, dGTP and dTTP. The incubation times and the amounts or of proteins used are indicated in the legends of the figures. The reactions were stopped by adding 7.5 μl of stop solution containing 0.1% xylene cyanol and 0.1% bromophenol blue in 90% formamide. Before loading onto the gel, samples were denatured by heating at 100°C for 3 min. The reaction products were resolved on denaturing polyacrylamide gel electrophoresis , run in GTG buffer , visualized and quantified using Molecular Dynamics PhosphorImager and ImageQuant software. The percentage of translesion synthesis (TLS) was calculated as the ratio of the intensity of bands present at the position opposite the lesion or beyond to the intensity of these bands plus the one present one nucleotide before the lesion.Reaction mixtures of 15 μl were incubated at 37°C and contained 0.15 pmoles of DNA templates, 50 mM Hepes KOH pH 7.5, 8 mM MgClE. coli replicase DNA polymerase III holoenzyme showed a limited capacity to replicate through an abasic site only when the lesion was present on a circular DNA substrate The structure of the DNA could affect the TLS activity of a DNA polymerase. Indeed, the We have recently shown that human pols λ, β and η can perform TLS of an AP site in the presence of pol ε; this TLS required, for pols λ and β, the presence of a DNA gap downstream from the product synthesized up to the lesion by pol ε Accessory replicative proteins such as the processivity clamp PCNA and the single-stranded DNA-binding protein RPA play a fundamental role in DNA replication, repair and recombination PCNA is a ring shaped homotrimeric protein that is loaded on the primer-template junction by a multiprotein clamp loader, replication factor C (RFC), which couples the hydrolysis of ATP with the opening and closing of the PCNA ring around the DNA In this study we have used RFC  and verified its functionality by monitoring its capacity to stimulate elongation by pol δ in the presence of PCNA on the undamaged minicircular template-primer A depicted in First, we checked the capacity of different concentrations of RFC to stimulate PCNA dependent pol δ synthesis. As it can be seen in We also performed a kinetic of stimulation and found that an increasing effect was observed up to 25 minutes of incubation , and thaTaken together, these data indicated that RFC substantially stimulated DNA synthesis by pol δ in the presence of PCNA, thus effectively acting as clamp loader under our experimental conditions.Next, we investigated whether the TLS catalysed at gaps in circular DNA by pols λ and β in presence of pol ε could be influenced by human PCNA, RPA and RFC. Previous data with linear DNA indicated that a 4 nucleotide gap is the optimal substrate to monitor TLS of the AP site by pol λ Monoubiquitination of PCNA at lys 164 (PCNA-Ub) appears to be necessary to make it capable of stimulating TLS by translesion pols in the cell in vitro TLS of an AP site by pol η remains controversial Interaction of the translesion pol η with monoubiquitinated PCNA has been suggested to mediate the polymerase switch at a lesion First we examined the replication of an AP site placed on a template bearing a gap of 33 nucleotides downstream the lesion. As expected, pol λ could not perform TLS on such template with either unmodified or PCNA-Ub (lanes 3 and 4 of Next the TLS capacity of the two enzymes at an AP site placed on a template bearing a gap of 4 nucleotides downstream the lesion was tested. Pol λ could perform TLS in the presence of pol ε and accessory proteins that resulted in filling of the gap with the same efficiency either with unmodified PCNA or PCNA-Ub (lanes 2 and 3 of Taken together these results suggest that, under our experimental conditions, pol η has the capacity to incorporate in front of an AP site in the presence of pol ε and accessory replicative proteins. However, this capacity, differently from the case of pol λ, is not gap directed and appears to be moderately enhanced by PCNA-Ub when a gap of 33 nucleotides is present.In conclusion, we show here that human pols λ and β can efficiently replicate, in the presence of pol ε, RPA and PCNA, an AP site placed in a gap situated downstream the lesion in a circular DNA template. These data are in agreement with those already reported for linear DNA templates containing an AP site Taken together, these results indicate that the known high affinity for gap binding of pols λ and β is the major if not unique biochemical determinant responsible for their gap-directed bypass capacity of lesions in the template strand.


A new attack against a novel certificateless aggregate signature scheme with constant pairing computations is presented. To enhance security, a new certificateless signature scheme is proposed first. Then a new certificateless aggregate signature scheme with constant pairing computations based on the new certificateless signature scheme is presented. Security analysis shows that the proposed certificateless aggregate signature scheme is provably secured in the random oracle. The AS scheme is very useful in real-world applications. For example, in the scenario of the secure Border Gateway Protocol (BGP)  ≥ (1−(1/qH1))qRPK, Pr[E2 | E1] ≥ ε, Pr[E3 | E1∧E2] ≥ (1/qH1), where qH1 and qRPK denote the numbers of H1 queries and RevealPartialKey queries separately. Then, the probability that 𝒞 solves the CDH problem isFrom the simulation we know that Pr≥(1 − (1/qH1))qRSK, Pr[E2 | E1] ≥ ε, Pr[E3 | E1∧E2] ≥ (1/qH1), where qH1 and qRSK denote the numbers of H1 queries and RevealSecertKey queries separately. Then, the probability that 𝒞 solves the CDH problem isFrom the simulation we know that Pr[𝒞 could solve the CDH problem with a nonnegligible probability since ε is nonnegligible. This contradicts the hardness of the CDH problem. Therefore, our CLS scheme is existentially unforgeable against a Type II adversary in random oracle model under the assumption that the CDH problem is hard.Then From the above two lemmas, we could get the following theorem.G1 is intractable.The CLS scheme is secure against adaptively chosen warrant attacks and chosen message and identity attacks in the random oracle model if the CDH problem in For the security of our CLAS scheme, we have the following theorem.G1 is intractable.The CLAS scheme is secure against adaptively chosen warrant attacks and chosen message and identity attacks in the random oracle model if the CDH problem in 𝒜 ∈ {𝒜1, 𝒜2} who could win G1 is intractable.Suppose there is an adversary TP: the time for executing a pairing operation;TS: the time for executing a scalar multiplications in G1.In this section, we compare our scheme with two latest CLAS schemes, that is, Zhang et al.'s scheme  and XionThe comparisons are listed in Recently, Xiong et al. proposed an efficient CLAS scheme. They claimed that both of their schemes are provably secure in the random oracle model. In this paper, we propose a new attack against their scheme. To overcome weakness, we also propose an improved CLAS scheme and show our scheme is provable in the random oracle model.


The stored Ca2+ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca2+, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca2+. We review here biochemical and biophysical evidence demonstrating that release of bound Ca2+ into the lumen of SR requires Ca2+/H+ exchange at the low affinity Ca2+ sites. Rise of lumenal Ca2+ above its dissociation constant from low affinity sites, or reduction of the H+ concentration by high pH, prevent Ca2+/H+ exchange. Under these conditions Ca2+ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca2+pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.The Ca In muscle cells, this transport activity plays an important role in lowering cytosolic Ca2+ as required for relaxation of contractile elements, and storing transported Ca2+ in the lumen of SR for subsequent release and contractile activation (Carafoli Sarcoplasmic reticulum (SR) membrane vesicles, originally referred to as “relaxing factor”, were first isolated from skeletal muscle by Ebashi and Lipmann , and HasCarafoli . GeneralCarafoli .2+ within the transmembrane region of the enzyme , leading to a new cycle.SERCA1 is a 996 amino acid membrane bound protein  steady state experiments in which oxalate is used for complexation of lumenal Ca2+ (Martonosi and Feretos b) pre-steady state experiments in which lumenal Ca2+ has yet to rise  while Ca2+ in the outer medium remains sufficiently high (μM) for ATPase activation  of active transport may be considered to be an intrinsic feature of the pump, if the ATPase reaction sequence allows an alternate pathway leading to hydrolytic cleavage of Pi without vectorial displacement of Ca2+  is likely to participate in Ca2+/H+ exchange  , vectorial translocation of bound Ca2+ in the direction of the SSM electrode is prevented, even though K+ is present in high concentration and may neutralize acidic residues at alkaline pH. This indicates a requirement for specific H+ binding at the Ca2+ sites, in order to obtain Ca2+ release.Further evidence for Ca+ exchange at the transport sites following phosphoenzyme formation occurs in Ca2+ ATPases, but does not occur in copper ATPases  preventing phosphoenzyme formation ). This indicates that if exchange is limited due to low H+ concentration, Ca2+ is less likely to dissociate from the phosphoenzyme. On the other hand, while Ca2+ translocation is reduced, steady state ATPase activity is increased as the pH is raised, and continues after maximal levels of Ca2+ uptake are reached. It is apparent that alkaline pH reduces Ca2+/H+ exchange and dissociation of bound Ca2+, whereby the phosphoenyme bypasses the Ca2+ release step and proceeds to hydrolytic cleavage of Pi. Therefore, reduction of the Ca2+/ATP transport ratio can be produced either by a high Ca2+ concentration or a low H+ concentration in the lumen of the vesicles.The importance of Ca2+/H+ exchange and a pathway for slippage of the Ca2+ pump, is given in Scheme A diagram of the sequential steps in the ATPase mechanism, derived from the original reaction diagram of de Meis and Vianna  and modi2+, yielding E1 · 2Ca2+. Utilization of ATP yields ADP · E1 ~ P · 2Ca2+, followed by release of ADP and utilization of the phosphorylation potential to change vectorial orientation and affinity of the Ca2+ sites. Bound Ca2+ is then released into the lumenal medium in exchange for H+. Hydrolytic cleavage of nH+ · E2-P and transition of nH+ · E2 to E1 finally yields closure of the lumenal gate and exposure of the Ca2+ sites to the cytosolic medium. Formation of E1 · 2Ca2+ then starts a new cycle.In the diagram on Scheme 2+ is higher than its dissociation constant, or lumenal H+ is too low to sustain exchange, Ca2+ release and formation of nH+ · E2 are prevented. Interference with completion of the ATPase cycle would then cause reversal to E1 ~ P · 2Ca2+ . On the other hand .Strong evidence for the role of protons and the nHand Fig. , further2+ binding, phosphoenzyme formation, occlusion and then dissociation of bound Ca2+, which are mechanically linked to specific bending and rotation patterns of each headpiece domain. These movements provide an explanation for the long range linkage of phosphorylation and Ca2+ binding domains, including the roles of critical amino acids in substrate binding, catalytic reactions, and Ca2+ transport. They also demonstrate that the states included in the reaction diagram are structurally distinct, and possess specific features that are functionally relevant. It is of interest that movement of M1-M2 causes displacement of membrane helix M4L from M5 and M6, thereby opening the lumenal gate and allowing outflow of Ca2+, following Ca2+/H+ exchange. The lumenal gate is then closed upon cleavage of phosphate and dissociation of H+, when reverse rotation of the A domain is accompanied by upward displacement of M4L and reduction of the space between the M4 and M6 helices.High resolution crystal structures of most states  comprising the ATPase reaction sequence have been obtained, and are described in detailed reviews (Toyoshima 2+ ATPase (SERCA1) of (at least human and rabbit) fast twitch skeletal muscle, and produces uncoupling of ATP utilization and Ca2+ transport, with a consequent thermogenic effect , a 31 amino acid polypeptide  of the Ca2+ sites on lumenal orientation would be significantly affected. The physiological interest of PLB and SLN is related to the reversibility of their effects upon phosphorylation catalyzed by signaling kinases (Koss and Kranias Both SLN and PLN reside within a groove surrounded by transmembrane helices M2, M4, M6 and M9, as shown by crystallographic studies (Toyoshima et al. 2+ pump uncoupling, an important question is whether uncoupling of SERCA1 interferes with reduction of cytosolic Ca2+ below the level allowing contractile relaxation of muscle fibers. The evidence presented above indicates that the Ca2+ pump is perfectly coupled when the SR lumen Ca2+in is low, even if cytosolic Ca2+out is relatively high. In the light of this information, we consider that relaxation of a muscle twitch occurs in less than 1 s and, within this time, lumenal Ca2+ does not reach a concentration higher than its dissociation from E2-P · 2Ca2+, as shown in experiments performed with rabbit native SR vesicles (Fig. 2+ is reduced to a level producing contractile relaxation, such a Ca2+ level will be also low with regard to SERCA activation, and the ATPase would then proceed at very low rates or remain inactive. Therefore during relaxation, the pump remains quiescent, mostly in the Mg2+ bound E1 state (Toyoshima et al. 2+ were to become higher than its dissociation constant from E2-P · 2Ca2+, and cytosolic Ca2+ were to remain above the level required for full ATPase activation. This may occur upon prolonged muscle activity, if cytosolic Ca2+ is maintained relatively high by multiple action potentials and Ca2+ flux through plasma membrane voltage sensitive channels, as expected in shivering thermogenesis. Alternatively, a rise of intracellular pH above 7.0, may affect intracellular Ca2+ signaling, as recently reported for G protein signaling (Isom et al. 2+ rise to high levels, while cytosolic Ca2+ remains sufficiently high. Most importantly, it was reported that in brown fat, in addition to uncoupling of the mitochondrial respiratory chain, uncoupled SERCA contributes to non shivering thermogenesis (de Meis et al. Considering the possibility of Ca


These results indicate the coexistence of Panx1 and P2X7 in, and Panx1‐dependent ATP release from, the human nasal mucosa, suggesting the possible participation of these molecules in the physiological functions of the upper airway.Pannexins are a family of transmembrane nonselective channel proteins that participate in the release of ATP into extracellular space. Previous studies have suggested that pannexin‐1 (Panx1) may constitute a local autocrine/paracrine system via transmitter ATP in association with the purinergic P2X7 receptor. In this study, we investigate the expressions of Panx1 and P2X7 in human nasal mucosa, together with hypotonic stress‐induced ATP release from this tissue. Twenty men and one woman ranging in age from 10 to 82 years with an average age of 44.2 ± 4.4 years participated in the study. Inferior turbinates were collected from patients with chronic hypertrophic rhinitis during endoscopic endonasal surgery. The expressions of Panx1 and P2X7 were examined by fluorescence immunohistochemistry and quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). We also examined hypotonic stress‐induced ATP release from the turbinate mucosa and the effects of channel blockers in an ex vivo experiment. Substantial expressions of both proteins were observed in human nasal mucosa. The immunoreactivity for Panx1 was stronger than that for P2X7. The presence of the transcripts of Panx1 and P2X7 was also shown by qRT‐PCR. Ten and 100  Pannexin‐1 (Panx1) participates in ATP release into the extracellular space. We demonstrated the existence of Panx1 and its ATP‐releasing effect in the human nasal mucosa. Panx1 may be a key molecular component in upper airway physiology. Pannexins are a family of transmembrane channel proteins in vertebrates homologous to innexins, which are invertebrate gap‐junction proteins  channel is the most thoroughly investigated member of the pannexin family and forms a large‐conductance (~500 pS) nonselective channel  is modulated by extracellular ATP through the activation of purinergic receptors on the epithelial cell surface , and air‐dried. The sections were hydrated in PBS with 0.3% Triton X‐100 (PBST) for 20 min, and treated with 1.5% normal donkey serum in PBST for 1 h. They were then incubated with rabbit anti‐human Panx1 polyclonal antibody (1:250)  at 4°C overnight. The fixed samples were transferred into 20% sucrose in 0.1 mol/L phosphate‐buffered saline of pH 7.4 (PBS), and incubated at 4°C for 2 nights with 3–4 changes of the solution. The samples were then embedded while frozen in the Tissue‐Tek O.C.T. Compound  and stored at −80°C before sectioning. Seven‐260/A280 ratio in the 1.9–2.1 range was considered acceptable). RNA concentration was determined from A260.For qRT‐PCR, the specimens were soaked in an RNA stabilization reagent  at 4°C overnight. Total RNA was extracted using an RNAqueous‐4PCR Kit  according to the manufacturer's instructions. The purity of the RNA was assessed by the ratio of light absorption at 260 to 280 nm  mRNA as a housekeeping gene, according to the manufacturer's specifications. The TaqMan Gene Expression Assays for Panx1 (assay identification number: Hs00209790_m1), P2X7 (assay identification number: Hs00175721_m1), and GAPDH (assay identification number: Hs02758991_g1) were purchased from Applied Biosystems. One‐hundred ng/μL of cDNA was mixed with TaqMan Fast Advanced Master Mix with AmpErase (uracil N‐glycosylase) and the primer/probe set of the TaqMan Gene Expression Assays. The mixture was then subjected to PCR amplification with real‐time detection. The thermal cycler conditions were as follows: holding at 50°C for 2 min followed by 95°C for 20 sec; this was followed by 40 cycles of two‐step PCR with 95°C for 1 sec and 60°C for 20 sec. Each sample was assayed in duplicate.Total RNA was reverse transcribed to cDNA with a High‐Capacity RNA‐to‐cDNA Kit , which uses random primers. The qRT‐PCR analysis was performed with an Applied Biosystems StepOnePlus real‐time PCR system using the TaqMan Fast Advanced Master Mix (Applied Biosystems) for T) was normalized by subtracting the CT for GAPDH of each sample from that for Panx1 and P2X7. From the obtained ∆CT, the ratio of Panx1 mRNA and P2X7 mRNA to GAPDH mRNA was calculated as follows:The measured threshold cycle (C−1 adjusted with mannitol) or hypotonic solution (100 mmol/L NaCl with 200 mOsmol L−1) in each well at room temperature for 10 min. One‐hundred microliter solution was collected by an ATP water‐testing device, AQUASNAP™ , and the ATP concentration was measured by a luciferin‐luciferase assay using luminometer SystemSURE Plus™ . To assess the contribution of the Panx1 channel to ATP release, the mucosal segment was also incubated in the presence of carbenoxolone , flufenamic acid , or gadolinium   , and washed with normal saline. The cutout mucosal segments were incubated in a 12‐well culture plate containing 4 mL isotonic solution . Substantial expressions of both proteins were identified, including in the epithelial layer. The immunoreactivity for Panx1 was stronger than that for P2X7.Panx1 mRNA/GAPDH mRNA and P2X7 mRNA/GAPDH mRNA ratios were 0.0165 ± 0.0011 and 0.0014 ± 0.0002, respectively , significantly higher than that of isotonic condition . In the presence of 10 and 100 μmol/L CBX, the ATP concentration significantly decreased to 108 ± 19  and 112 ± 19 fmol/L , respectively. In contrast, ATP concentration was not inhibited by FFA at a concentration of 300 μmol/L , which is considered sufficient to produce the maximum effect . These results suggest that ATP release from the human nasal mucosa under hypotonic condition is mediated, at least in part, by CBX‐sensitive and FFA‐insensitive channels – like the Panx1 channel – and is independent of stretch‐activated channels.The measurements of ATP release from nasal mucosa under various conditions are summarized in 3+ did not. These results suggest that the release of ATP from human nasal mucosa may in part be mediated by the Panx1 channel. Obviously, it would not have been ethical to obtain normal inferior turbinates for this study; we therefore collected the turbinates from patients with chronic hypertrophic rhinitis. Although hypertrophied turbinates histologically exhibit irreversible changes such as submucosal fibrosis, the epithelial layer of their lateral side generally shows almost normal histological findings  for their expertise.None declared.


Denitrification was the main driver of N2 production, with anammox only detected in two of the four ponds. No correlations were detected between the measured sediment variables  and denitrification or anammox. Furthermore, denitrification was not carbon limited as the addition of particulate organic matter  or methanol  did not stimulate production of N2. A simple mass balance model showed that only 2.5% of added fixed N was removed in the studied settlement ponds through the denitrification and anammox processes. It is recommended that settlement ponds be used in conjunction with additional technologies  to enhance N2 production and N removal from aquaculture wastewater.Settlement ponds are used to treat aquaculture discharge water by removing nutrients through physical (settling) and biological  processes. Nutrient removal through settling has been quantified, however, the occurrence of, and potential for microbial nitrogen (N) removal is largely unknown in these systems. Therefore, isotope tracer techniques were used to measure potential rates of denitrification and anaerobic ammonium oxidation (anammox) in the sediment of settlement ponds in tropical aquaculture systems. Dinitrogen gas (N The release of anthropogenic N to the coastal zone poses a threat to many shallow marine ecosystems 2). During denitrification, nitrate (NO3−) is reduced to nitrite (NO2−), nitric oxide (NO) and nitrous oxide (N2O), before eventually being converted to N2. Anammox also directly removes fixed N and couples NO2− reduction with ammonium (NH4+) oxidation to produce N2−2 d−1 and 61 mmol m−2 d−1 of N, respectively Denitrification and anammox are the major microbial processes removing fixed N from wastewater through the production of dinitrogen gas (N2 production in multiple zones of four settlement ponds on three farms (two prawn farms and one fish farm). We also investigated the relationship between the potential rates of N2 production with the geochemical characteristics of the ponds. Additionally, the effect of carbon on N2 production was tested since intensive aquaculture systems have N rich wastewaters where microbial N removal is typically limited by the supply of carbon as an electron donor The first step in optimizing the removal of fixed N through the denitrification and anammox pathways is to quantify their activity in settlement ponds and relate this to the environment of the ponds. Accordingly, the aim of this study was to determine if denitrification and anammox occur in sediments collected from tropical settlement ponds that are used to treat effluent from commercial production of prawns (shrimp) and fish. We used sediment slurry assays to investigate potential NPenaeus monodon) farms and one barramundi  farm. At Farm 1 sediment was collected from the two functional settlement ponds, this allowed comparison of N2 production over small spatial scales   . In all  and Z3)  section.2 production, sediments were collected at Z1, Z2 and Z3 in each of the four settlement ponds  (n = 3 per zone). The sediments were extruded, weighed and subsequently oven dried (60°C) and reweighed for porosity (ø) determination (n = 3). Dried sediment was then milled (Rocklabs Ring Mill) for total N determination . TOC was determined on a Shimadzu TOC-V Analyzer with a SSM-5000A Solid Sample Module. Solid phase S, P, Fe and Mn were also analyzed from milled sediment samples subjected to strong acid digestion. A THERMO Iris INTREPID II XSP ICP_AES was used to determine element content in triplicate sediment samples from each zone To investigate the spatial variation of sediment characteristics within and between settlement ponds, and their role in driving N2 zones) . Samplin2 (inclusive of both N2 and N2O) production through denitrification and anammox in March 2010 (Ponds B and C) or August 2010 (Ponds A and D). At the time of abiotic sample collection (see above), approximately 500 g of the most reactive sediments were collected from each zone in the four settlement ponds (n = 1 from each zone within each pond) (x is present or being reduced (denitrification) x penetrates but O2 does not, making conditions favorable for anammox 3− in the isotope assay. A known volume of sediment (3–6 g) was loaded into Exetainers  and ∼5 mL of degassed (flushed for 1 hr with ultra pure He), artificial seawater was added to form a slurry. Sediments were pre-incubated (overnight) under anoxic conditions to ensure all residual NO3, NO2− and O2 were consumed. Three different enrichment treatments  were added to the slurries. After the isotope amendment, the Exetainers were filled with the degassed seawater, capped without headspace and homogenized by inverting 2–3 times. Triplicate samples were sacrificed from each treatment at 0, 0.5, 17 h and 24 h by introducing 200 µL 50% w/v ZnCl2 through a rubber septum (n = 3). The 0 and 0.5 h time periods were chosen based on rapid turnover rates determined by Trimmer et al. 2 into or out of the Exetainers. A gas chromatograph (Thermo Trace Ultra GC) interfaced to an isotope ratio mass spectrometer  was used to determine 29N2 and 30N2 content of dissolved nitrogenous gas . Varying volumes (3–10 µL) of air were used as calibration standards.Slurry assays were conducted to test for the presence of Nch pond)  with a 32 production in the 24 h incubation trials (above) was calculated from the slope of the regression over the incubation period  based on Dalsgaard and Thamdrup 29N2 and 30N2 was non-linear and rates were calculated based on the first two production points. Therefore a subsequent slurry assay was run to investigate N2 production rates over short, regular time intervals (15 min) to gain a more accurate insight into potential process rates. Sediment for the additional assays was collected from settlement Pond D, Zones 1 (n = 1) and 3 (n = 1) in October 2010. These zones were chosen because the production of N2 was non-linear during the 24 h incubation assay (see The rate of Nssay see  section.2 production in some aquaculture systems 2 production in some systems previously in situ carbon source collected from Pond A. POM was collected by transporting settlement pond-influent water to the laboratory at the same time that sediments were collected. Suspended solids in influent water were concentrated by centrifugation (10 min at 3000 rpm). 400 µL aliquots of concentrated (∼100 mg L−1) POM were added to Exetainer vials prior to the addition of amendments. However, in the absence of a high total suspended solid load at Pond C, methanol (MeOH) was used as the carbon source as it stimulates denitrification but inhibits anammox in some circumstances The effect of an additional carbon source on the occurrence of denitrification and anammox was tested with a separate set of slurry assays because organic carbon limits N2 production rates calculated in the present study, and N inputs into the pond through the wastewater. Given the substantial contribution of N remineralized from sludge in shrimp grow-out ponds (often exceeding inputs of N originating from feeds imin). The following equation was used to calculate N removal and the parameters are further defined in A simplistic model was constructed to estimate the mean dry season N removal (NR) capacity (%) of the four settlement ponds. NR was estimated using the potential N2 = the mean total (inclusive of anammox) N2 production rate measured during the 24 h incubation ; t = 24 (h d−1); rA = atomic weight of N; Niww = mean rate of TN input (inclusive of particulates and dissolved) via the wastewater  monitoring data, quantified monthly by Farm 1; kg N d−1); Nimin = mean rate of N input via mineralisation (deduced from NH4+ and DON fluxes in Burford and Longmore −1).p-values from 9999 permutations based on Bray-Curtis distances. A 1-factor PERMANOVA was subsequently used to compare differences in N2 production rate data  between ponds with zones as replicates (n = 3). 9999 permutations were again used to calculate p-values based on Bray-Curtis distance. PRIMER version 6 and PERMANOVA+ version 1.0.4 were used to conduct both analyses.The sediment characteristics data was analysed as a 2-factor nested design, pond and zone(pond) using permutational multivariate analysis of variance (PERMANOVA) 2 production rate  and sediment characteristics was subsequently investigated using the BIOENV procedure in PRIMER. This procedure performs a rank correlation of the two similarity matrices (described above) and tests every combination of sediment characteristics to determine which set of variables best explains the observed N2 production rates 2 production rate data and sediment variable data was also used to conduct a hierarchical agglomerative cluster analysis which was superimposed on a multidimensional scaling (nMDS) plot. The nMDS plot provided a 2-D visualization of the relationship between sediment characteristics and N2 production rates.The relationship between N2 production rate in sediments was analyzed with paired t-Tests for each carbon source (POM and MeOH).The effect of carbon addition on potential NSurface water temperature (25.8°C±1.0) and pH (7.6±0.2) varied little similar across all ponds and zones. Surface water salinity in Pond C (Farm 2) was lower (17–18‰) than the other three ponds 31–35‰;  due to iPseudo F = 2.06, P = 0.028) and between zones within ponds . The variance in sediment characteristics at the finer scale (i.e. meters) between zones within ponds (52.4%) was greater than the variance between settlement ponds located kilometers apart (31.6%).Sediment at all zones was uniformly dark black with minor color variation shown in a narrow lighter band (∼3 mm oxic zone) at the surface of the sediment. The porosity ranged between 41–72%  and sedi2 production between ponds . The potential rate was highest in sediments collected from pond A, with denitrification the sole producer of N2  or the sediment variable matrix .Highly positive or negative loadings of the sediment characteristics appeared to have little influence on total Nfication  as thesefication , however15N labeling of nitrate only, the majority of 15N-NO3− converted to N2 was found in 30N2  or methanol  was added to the experimental sediment slurries  were within the range of those reported for a subtropical constructed wetland (1.1±0.2 to 13.1±2.6 nmol N cm−3 h−1) −3 h−1) Planctomycetes (anammox bacteria) are present in the ponds and that there is potential to stimulate N2 production rates and enhance N removal. To achieve this, an understanding of the mechanisms controlling N2 production is required. We therefore investigated the effect of carbon additions on N2 production rate and the relationship between the concentration of sediment elements and N2 production rates. However, there was no significant change in the rate of N2 production under carbon loading and there was no correlation between any of the measured sediment variables and N2 production rate via denitrification or anammox.Isotope tracer techniques confirmed the production of N4+, NO3− and NO2−. Similarly, methanol is a common additive to enhance denitrification for municipal wastewater treatment, increasing degradation of NO2− in activated sludge from 0.27 mg NO2− g−1 volatile suspended solids (VSS) h−1 to 1.20 mg NO2− g−1 VSS h−12 production was not enhanced through the addition of carbon, suggesting that there are additional controlling mechanisms driving N2 production. This concurs with the lack of significant correlation between measured sedimentary TOC and N2 production. The lack of stimulation of N2 production after the addition of carbon has also been demonstrated in the oxygen minimum zone of the Arabian Sea, where denitrification (and anammox) was only enhanced at one out of 11 depths 2− concentration, a factor which likely also plays a role in controlling denitrification in settlement pond systems but was not measured in the present study. NO3− concentration also regulates anammox activity in estuarine sediments 3−, NO2− and NH4+ with denitrification and anammox potentials to determine if these are driving process rates in settlement ponds.Denitrification is often limited by carbon in aquaculture ponds, as carnivorous marine species require high inputs of protein rich feeds. N removal can be enhanced through the addition of an exogenous carbon source, for example glucose and cassava meal x as a substrate 15NO3− only 7.9±2.7% was recovered as 15N2, so a large portion (i.e. ∼90%) of added 15NO3− could be rapidly consumed by competing pathways such as DNRA or assimilation. The prevalence of DNRA or assimilation over denitrification will determine the balance between N being removed from the system through gaseous N2 production, or conserved within the system It is also possible that the exogenous carbon source is instead stimulating nitrate ammonifiers (DNRA) and therefore competition for NO−2 d−1 and releasing free sulfides 2 production in the present study by reducing the amount of NO3− available to denitrifiers 3−2 production in tropical settlement ponds is largely unknown and further studies are needed to elucidate the potential of this factor on stifling N removal in settlement ponds.Another potential controlling factor may be the presence of free sulfides. Sulfur is cycled rapidly in tropical sediments 2 production pathway. In coastal, hyper-nutrified sediments, low N2 production through anammox has been attributed to the limitation of NO2−4+, total kilojoule nitrogen, TN, TP, salinity, redox state, and an inverse relationship with TOC 2 production is highly variable with values of 1–8% In our study denitrification was the dominant N2 production was exceptionally low. For example, during the 24 h incubation with sediment collected in pond B, N2 production was lower than in sediment collected from all other ponds, but anammox contributed 95% to N2 production. Low carbon oxidation rates and correspondingly low denitrification (and thus competition for substrate) have been proposed as the reason anammox contribution is high in environments where denitrification is low 2 production was carbon limited giving anammox the competitive advantage. In tropical estuary systems where high temperatures, low sediment organic content and low water column NO3− concentrations prevail, the order of NOx reduction pathways is proposed to be DNRA>denitrification>anammox Anammox was detected in sediment collected in ponds B (24 h incubation), C (carbon incubation) and D (1.5 h incubation), notably, where overall N15NH4+ was transformed through anoxic nitrification 15NO3− and the resulting 15N2 was produced as the result of denitrification.The apparent detection of anammox in the presence of MeOH in sediments collected from Pond C is unusual given that anammox is inhibited by MeOH 2 production has the potential to play a major role in removing N from aquaculture wastewater. However, we estimated that only 2.5% of total N added to the settlement pond via wastewater inputs and mineralization is removed through N2 production. It is likely that the noxious compounds of H2S and NH4+ are produced in settlement ponds when they are left unmanaged with no removal of settled particulate organic matter (sludge). These compounds have significant consequences for the inhibition of microbial processes that remove N from wastewater. In addition, H2S accumulation causes a shift in the species of gaseous N produced from N2 to N2O due to the inhibition of the last step of denitrification 2O is ∼300 times more potent than CO2 as a greenhouse gas whereas N2 is relatively inert 2S at which the last reductive step of denitrification is inhibited and relate this to the amount of sludge that has built up in the settlement pond. We recommend that sludge be extracted at this point to prevent H2S release and to prevent the recycling of soluble N through mineralization, DNRA or assimilation and subsequent senescence, as has been recommended for grow out ponds previously 2 production. If N2 production could be enhanced to the mean rate reported by Erler et al. −2 d−1, then 100% of total daily N inputs would be removed from settlement ponds every day. However, the estimates in the present study are based on a very simplistic understanding of the settlement pond functioning and the model requires better definition of the parameters. For example, accurate rates of NH4+ and DON production from the sediments are required to estimate N inputs accurately. Additionally, N2 production was measured in the dry season in the present study when rates are likely lower than in the wet season. Wet season precipitation lowers the salinity in the ponds to 5‰ in some cases, which favors higher denitrification, lower DNRA and lower NH4+ fluxes 3− concentrations from land run-off 2 production rates measured using intact core assays, instead of slurry assays, would also allow accurate predictions of N2 production rates. Slurry assays only generate potential rates of N2 production and we acknowledge that homogenizing sediments disrupts the sediment profile and can result in different nutrient availability than that which occurs in situMicrobial N