Heat shock enhances the expression of cytotoxic granule proteins and augments the activities of tumor-associated antigen-specific cytotoxic T lymphocytes

Focal inflammation causes systemic fever. Cancer hyperthermia therapy results in shrinkage of tumors by various mechanisms, including induction of adaptive immune response. However, the physiological meaning of systemic fever and mechanisms of tumor shrinkage by hyperthermia have not been completely understood. In this study, we investigated how heat shock influences the adaptive immune system. We established a cytotoxic T lymphocyte (CTL) clone (#IM29) specific for survivin, one of the tumor-associated antigens (TAAs), from survivin peptide-immunized cancer patients’ peripheral blood, and the CTL activities were investigated in several temperature conditions (37–41 °C). Cytotoxicity and IFN-γ secretion of CTL were greatest under 39 °C condition, whereas they were minimum under 41 °C. To address the molecular mechanisms of this phenomenon, we investigated the apoptosis status of CTLs, expression of CD3, CD8, and TCRαβ by flow cytometry, and expression of perforin, granzyme B, and Fas ligand by western blot analysis. The expression of perforin and granzyme B were upregulated under temperature conditions of 39 and 41 °C. On the other hand, CTL cell death was induced under 41 °C condition with highest Caspase-3 activity. Therefore, the greatest cytotoxicity activity at 39 °C might depend on upregulation of cytotoxic granule proteins including perforin and granzyme B. These results suggest that heat shock enhances effector phase of the adaptive immune system and promotes eradication of microbe and tumor cells.

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Efficient Production of Lactic Acid from Sweet Sorghum Juice by a Newly Isolated Lactobacillus salivarius CGMCC 7.75

Sweet sorghum juice was a cheap and renewable resource, and also a potential carbon source for the fermentation production of lactic acid (LA) by a lactic acid bacterium. One newly isolated strain Lactobacillus salivarius CGMCC 7.75 showed the ability to produce the highest yield and optical purity of LA from sweet sorghum juice. Studies of feeding different concentrations of sweet sorghum juice and nitrogen source suggested the optimal concentrations of fermentation were 325 ml l⁻¹ and 20 g l⁻¹, respectively. This combination produced 142.49 g l⁻¹ LA with a productivity level of 0.90 g of LA per gram of sugars consumed. The results indicated the high LA concentration achieved using L. salivarius CGMCC 7.75 not only gives cheap industrial product, but also broaden the application of sweet sorghum.

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Epidemiological profiles between equol producers and nonproducers: a genomewide association study of the equol-producing phenotype

Equol is a daidzein (a phytoestrogen isoflavone) metabolite of gut bacteria, and the ability to produce equol varies between individuals and reduces the risks of several diseases. We tested the effects of equol production on health in Koreans and identified the genetic factors that determine the equol-producing phenotype. In 1391 subjects, the equol-producing phenotype was determined, based on measurements of serum equol concentrations. The anthropometric and blood biochemical measurements between equol producers and nonproducers were analyzed by LC-MS/MS. Genetic factors were identified in a genomewide association study (GWAS), and the interaction between genetic factors and the equol-producing phenotype was examined. We observed that 70.1 % of the study population produced equol. Blood pressure was significantly lower in equol producers (beta ± SE = −1.35 ± 0.67, p = 0.045). In our genomewide association study, we identified 5 single-nucleotide polymorphisms (p < 1 × 10⁻⁵) in HACE1. The most significant SNP was rs6927608, and individuals with a minor allele of rs6927608 did not produce equol (odds ratio = 0.57 (95 % CI 0.45–0.72), p value = 2.5 × 10⁻⁶). Notably, the interaction between equol production and the rs6927608 HACE1 SNP was significantly associated with systolic blood pressure (p value = 1.3 × 10⁴). Equol production is linked to blood pressure, and HACE1, identified in our (GWAS), might be a determinant of the equol-producing phenotype.

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Genetic predisposition to type 2 diabetes is associated with impaired insulin secretion but does not modify insulin resistance or secretion in response to an intervention to lower dietary saturated fat

Genome-wide association studies have identified SNPs reproducibly associated with type 2 diabetes (T2D). We examined the effect of genetic predisposition to T2D on insulin sensitivity and secretion using detailed phenotyping in overweight individuals with no diagnosis of T2D. Furthermore, we investigated whether this genetic predisposition modifies the responses in beta-cell function and insulin sensitivity to a 24-week dietary intervention. We genotyped 25 T2D-associated SNPs in 377 white participants from the RISCK study. Participants underwent an IVGTT prior to and following a dietary intervention that aimed to lower saturated fat intake by replacement with monounsaturated fat or carbohydrate. We composed a genetic predisposition score (T2D-GPS) by summing the T2D risk-increasing alleles of the 25 SNPs and tested for association with insulin secretion and sensitivity at baseline, and with the change in response to the dietary intervention. At baseline, a higher T2D-GPS was associated with lower acute insulin secretion (AIRg 4% lower/risk allele, P = 0.006) and lower insulin secretion for a given level of insulin sensitivity, assessed by the disposition index (DI 5% lower/risk allele, P = 0.002), but not with insulin sensitivity (Si). T2D-GPS did not modify changes in insulin secretion, insulin sensitivity or the disposition index in response to the dietary interventions to lower saturated fat. Participants genetically predisposed to T2D have an impaired ability to compensate for peripheral insulin resistance with insulin secretion at baseline, but this does not modify the response to a reduction in dietary saturated fat through iso-energetic replacement with carbohydrate or monounsaturated fat.

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Astemizole Synergizes Calcitriol Antiproliferative Activity by Inhibiting CYP24A1 and Upregulating VDR: A Novel Approach for Breast Cancer Therapy

Background

Calcitriol antiproliferative effects include inhibition of the oncogenic ether-à-go-go-1 potassium channel (Eag1) expression, which is necessary for cell cycle progression and tumorigenesis. Astemizole, a new promising antineoplastic drug, targets Eag1 by blocking ion currents. Herein, we characterized the interaction between calcitriol and astemizole as well as their conjoint antiproliferative action in SUM-229PE, T-47D and primary tumor-derived breast cancer cells.

Methodology/Principal Findings

Molecular markers were studied by immunocytochemistry, Western blot and real time PCR. Inhibitory concentrations were determined by dose-response curves and metabolic activity assays. At clinically achievable drug concentrations, synergistic antiproliferative interaction was observed between calcitriol and astemizole, as calculated by combination index analysis (CI <1). Astemizole significantly enhanced calcitriol’s growth-inhibitory effects (3–11 folds, P<0.01). Mean IC₂₀ values were 1.82±2.41 nM and 1.62±0.75 µM; for calcitriol (in estrogen receptor negative cells) and astemizole, respectively. Real time PCR showed that both drugs alone downregulated, while simultaneous treatment further reduced Ki-67 and Eag1 gene expression (P<0.05). Astemizole inhibited basal and calcitriol-induced CYP24A1 and CYP3A4 mRNA expression (cytochromes involved in calcitriol and astemizole degradation) in breast and hepatoma cancer cells, respectively, while upregulated vitamin D receptor (VDR) expression.

Conclusions/Significance

Astemizole synergized calcitriol antiproliferative effects by downregulating CYP24A1, upregulating VDR and targeting Eag1. This study provides insight into the molecular mechanisms involved in astemizole-calcitriol combined antineoplastic effect, offering scientific support to test both compounds in combination in further preclinical and clinical studies of neoplasms expressing VDR and Eag1. VDR-negative tumors might also be sensitized to calcitriol antineoplastic effects by the use of astemizole. Herein we suggest a novel combined adjuvant therapy for the management of VDR/Eag1-expressing breast cancer tumors. Since astemizole improves calcitriol bioavailability and activity, decreased calcitriol dosing is advised for conjoint administration.     

Levels and trends in cardiovascular risk factors and drug treatment in 4837 elderly Dutch myocardial infarction patients between 2002 and 2006

Background

It is important to gain insight into opportunities for secondary prevention of cardiovascular disease. Our aim was to investigate levels and trends in cardiovascular risk factors and drug treatment in Dutch post-myocardial infarction (MI) patients between 2002 and 2006 and to make comparisons with the EUROASPIRE surveys (1999–2007).

Methods

We analysed data from 4837 post-MI patients (aged 69 years, 78% men) from 32 Dutch hospitals, using baseline cross-sectional data from the Alpha Omega Trial.

Results

Between 2002 and 2006, significant declines were found in the prevalence of smoking (23% to 16%, p < 0.001), hypercholesterolaemia (≥5 mmol/l; 54% to 27%, p < 0.0001) and hypertension (≥140/90 mmHg; 58% to 48%, p < 0.001). The prevalence of antithrombotic drugs was high (97%). The prevalence of lipid-modifying drugs and antihypertensives was high, and increased (74% to 90%, p < 0.0001 and 82% to 93%, p < 0.001, respectively). The prevalence of obesity (27%) was high in 2002 and decreased to 24% in 2006, albeit not significantly. Diabetes prevalence was high and increased between 2002 and 2006 (18% to 22%, p = 0.02). In comparison with EUROASPIRE patients, who were on average 8–10 years younger, our study in 2006 included patients with lower levels of obesity, hypertension, hypercholesterolaemia, diabetes and lower use of antiplatelets and β-blockers, but similar levels of lipid-modifying drugs.

Conclusions

This study showed that older Dutch post-MI patients were adequately treated with drugs, and that risk factors reached lower levels than in the younger EUROASPIRE patients. However, there is room for improvement in diet and lifestyle, given the high prevalence of smoking, obesity, and diabetes.

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Real-life use of vitamin D3-fortified bread and milk during a winter season: the effects of CYP2R1 and GC genes on 25-hydroxyvitamin D concentrations in Danish families,the VitmaD study

Common genetic variants rs10741657 and rs10766197 in CYP2R1 and rs4588 and rs842999 in GC and a combined genetic risk score (GRS) of these four variants influence late summer 25-hydroxyvitamin D (25(OH)D) concentrations. The objectives were to identify those who are most at risk of developing low vitamin D status during winter and to assess whether vitamin D₃-fortified bread and milk will increase 25(OH)D concentrations in those with genetically determined low 25(OH)D concentrations at late summer. We used data from the VitmaD study. Participants were allocated to either vitamin D₃-fortified bread and milk or non-fortified bread and milk during winter. In the fortification group, CYP2R1 (rs10741657) and GC (rs4588 and rs842999) were statistically significantly associated with winter 25(OH)D concentrations and CYP2R1 (rs10766197) was borderline significant. There was a negative linear trend between 25(OH)D concentrations and carriage of 0–8 risk alleles (p < 0.0001). No association was found for the control group (p = 0.1428). There was a significant positive linear relationship between different quintiles of total vitamin D intake and the increase in 25(OH)D concentrations among carriers of 0–2 (p = 0.0012), 3 (p = 0.0001), 4 (p = 0.0118) or 5 (p = 0.0029) risk alleles, but not among carriers of 6–8 risk alleles (p = 0.1051). Carriers of a high GRS were more prone to be vitamin D deficient compared to carriers of a low GRS. Furthermore, rs4588-AA carriers have a low but very stable 25(OH)D concentration, and interestingly, also low PTH level.

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Long-term,large scale cryopreservation of insect cells at −80 °C

Standard tissue culture methods advise freezing cells in small aliquots (≤1 × 10⁷ cells in 1 mL), and storing in liquid nitrogen. This is inconvenient for laboratories culturing large quantities of insect cells for recombinant baculovirus expression, owing to the length of time taken to produce large scale cultures from small aliquots of cells. Liquid nitrogen storage requires use of specialized cryovials, personal protective equipment and oxygen monitoring systems. This paper describes the long-term, large scale cryopreservation of 8 × 10⁸ insect cells at −80 °C, using standard 50 mL conical tubes to contain a 40 mL cell suspension. Sf9, Sf21 and High 5 cells were recovered with a viability > 90 % after storage for one year under these conditions, which compared favorably with the viability of cells stored in liquid nitrogen for the same length of time. Addition of green fluorescent protein encoding baculovirus demonstrated that cells were “expression ready” immediately post thaw. Our method enables large scale cultures to be recovered rapidly from stocks cryopreserved at −80 °C, thus avoiding the inconvenience, hazards and expense associated with liquid nitrogen.

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Biomarkers in outpatient heart failure management; Are they correlated to and do they influence clinical judgment?

Aims

Heart failure (HF) management is complicated by difficulties in clinical assessment. Biomarkers may help guide HF management, but the correspondence between clinical evaluation and biomarker serum levels has hardly been studied. We investigated the correlation between biomarkers and clinical signs and symptoms, the influence of patient characteristics and comorbidities on New York Heart Association (NYHA) classification and the effect of using biomarkers on clinical evaluation.

Methods and results

This post-hoc analysis comprised 622 patients (77 ± 8 years, 76 % NYHA class ≥3, 80 % LVEF ≤45 %) participating in TIME-CHF, randomising patients to either NT-proBNP-guided or symptom-guided therapy. Biomarker measurements and clinical evaluation were performed at baseline and after 1, 3, 6, 12 and 18 months. NT-proBNP, GDF-15, hs-TnT and to a lesser extent hs-CRP and cystatin-C were weakly correlated to NYHA, oedema, jugular vein distension and orthopnoea (ρ-range: 0.12–0.33; p < 0.01). NT-proBNP correlated more strongly to NYHA class in the NT-proBNP-guided group compared with the symptom-guided group. NYHA class was significantly influenced by age, body mass index, anaemia, and the presence of two or more comorbidities.

Conclusion

In HF, biomarkers correlate only weakly with clinical signs and symptoms. NYHA classification is influenced by several comorbidities and patient characteristics. Clinical judgement seems to be influenced by a clinician’s awareness of NT-proBNP concentrations.

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The brown seaweed Sargassum hemiphyllum exhibits α-amylase and α-glucosidase inhibitory activity and enhances insulin release in vitro

Diabetes is one of the most prevalent chronic diseases globally. In this study, major polyphenols (17.35 ± 0.93–36.66 ± 2.01 mg/g) and minor fucoxanthin (non detected 15.12 ± 0.09 mg/g) were isolated from water, ethanol, and acetone extracts (WES, EES, and AES, respectively) of Sargassum hemiphyllum. Inhibition of α-amylase, α-glucosidase, sucrose, and maltase activities and stimulation of insulin secretion was greater with AES than with WES or EES and correlated with polyphenol and fucoxanthin concentrations in extracts. Moreover, 250 μg/ml EES and AES significantly increased insulin secretion in the presence of 25 mg/ml glibenclamide to higher levels than those obtained with 50 mg/ml glibenclamide. None of the extracts exhibited cytotoxicity, exacerbated the side effects of glibenclamide, or inhibited glibenclamide-induced insulin secretion. These results suggested that the S. hemiphyllum extracts WES, EES, and AES could be used as pharmaceuticals and functional foods to reduce dosages of synthetic diabetes drugs.     

H2O2 pretreated rice seedlings specifically reduces arsenate not arsenite: difference in nutrient uptake and antioxidant defense response in a contrasting pair of rice cultivars

The study investigated the reduction in metalloid uptake at equimolar concentrations (~53.3 μM) of As(III) and As(V) in contrasting pair of rice seedlings by pretreating with H₂O₂ (1.0 μM) and SA (1.0 mM). Results obtained from the contrasting pair (arsenic tolerant vs. sensitive) of rice seedlings (cv. Pant Dhan 11 and MTU 7029, respectively) shows that pretreatment of H₂O₂ and H₂O₂ + SA reduces As(V) uptake significantly in both the cultivars, while no reduction in the As(III) uptake. The higher growth inhibition, higher H₂O₂ and TBARS content in sensitive cultivar against As(III) and As(V) treatments along with higher As accumulation (~1.2 mg g⁻¹ dw) than in cv. P11, unravels the fundamental difference in the response between the sensitive and tolerant cultivar. In the H₂O₂ pretreated plants, the translocation of As increased in tolerant cultivar against AsIII, whereas, it decreased in sensitive cultivar both against AsIII and AsV. In both the cultivars translocation of Mn increased in the H₂O₂ pretreated plants against As(III), whereas, the translocation of Cu increased against As(V). In tolerant cultivar the translocation of Fe increased against As(V) with H₂O₂ pretreatment whereas, it decreased in the sensitive cultivar. In both the cultivars, Zn translocation increased against As(III) and decreased against As(V). The higher level of H₂O₂ and SOD (EC 1.15.1.1) activity in sensitive cultivar whereas, higher, APX (EC 1.11.1.11), GR (EC 1.6.4.2) and GST (EC 1.6.4.2) activity in tolerant cultivar, also demonstrated the differential anti-oxidative defence responses between the contrasting rice cultivars.

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Hydrolyzing Proficiency of Keratinases in Feather Degradation

The keratinase degrade highly rigid, cross linked structural polypeptides with different efficiency depending on the type of source. Two newly isolated strains of Bacillus subtilis (RSE163 and RSE165; NCBI Accession no {"type":"entrez-nucleotide","attrs":{"text":"JQ887983","term_id":"453198585","term_text":"JQ887983"}}JQ887983 and {"type":"entrez-nucleotide","attrs":{"text":"JQ887982","term_id":"453198574","term_text":"JQ887982"}}JQ887982) were found to be efficient keratinase producers with unusual catalytic activity result in different morphological changes in degradation pattern of feather, confirmed by their scanned electron micrographs. Maximum keratinolytic activity of both the strains B. subtilis RSE163 and RSE165 were found to be 366 ± 15.79 and 194 ± 7.26 U after 72 h of incubation. While the disulphide reductase activity of RSE163 and RSE165 estimated 0.24 ± 0.05 and 0.15 ± 0.03 U/ml of enzyme after 24 h of incubation. A total of 16 free amino acids of variable concentration were also analyzed in the cell free supernatant of hydrolyzed feather from two strains. Present study demonstrates the action of two different keratinases in feather degradation.

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Comparative study of genotoxicity and antimutagenicity of methanolic extracts from Teucrium chamaedrys and Teucrium montanum in human lymphocytes using micronucleus assay

Since Teucrium chamaedrys and Teucrium montanum are the most popular plants used in the treatment of many diseases, we evaluated genotoxic potential of their methanolic extracts on cultured human peripheral blood lymphocytes (PBLs) using cytokinesis-block micronucleus (MN) assay. Cultures were treated with four concentrations of both plants (125, 250, 500 and 1,000 μg/ml), both separately and in combination with mitomycin C (MMC). The results revealed that extract of T. chamaedrys administered at the tested concentrations did not significantly affect the mean MN frequency in comparison to untreated cells. Methanolic extract of T. montanum increased the mean MN frequency in PBL at the tested concentrations, but significantly only at the concentration of 1,000 μg/ml. In all tested concentrations, the extract of T. chamaedrys significantly reduced the MMC-induced MN frequency, in a dose dependent manner (r = − 0.687, p < 0.01). The extract of T. montanum decreased the MMC-induced MN frequency at the tested concentrations, but statistically only at 125 μg/ml. Both extracts administered alone did not significantly affect the nuclear division index (NDI) at the tested concentrations. In the combined treatments with MMC, the extract obtained from T. chamaedrys in the concentrations of 500 and 1,000 μg/ml significantly decreased NDI values in comparison to MMC-treated cells alone, while the extract of T. montanum significantly decreased NDI at all tested concentrations. Both extracts nonsignificantly decreased NDI at all tested concentrations in comparison to untreated cells. Our results suggest the important function of T. chamaedrys extract in cancer therapy, this methanolic extract may prevent genotoxic effects of chemotherapy in PBLs.     

Antithrombotic therapy in patients undergoing TAVI: an overview of Dutch hospitals

Purpose

To assess current antithrombotic treatment strategies in the Netherlands in patients undergoing transcatheter aortic valve implantation (TAVI).

Methods

For every Dutch hospital performing TAVI (n = 14) an interventional cardiologist experienced in performing TAVI was interviewed concerning heparin, aspirin, thienopyridine and oral anticoagulation treatment in patients undergoing TAVI.

Results

The response rate was 100 %. In every centre, a protocol for antithrombotic treatment after TAVI was available. Aspirin was prescribed in all centres, concomitant clopidogrel was prescribed 13 of the 14 centres. Duration of concomitant clopidogrel was 3 months in over two-thirds of cases. In 2 centres, duration of concomitant clopidogrel was based upon type of prosthesis: 6 months versus 3 months for supra-annular and intra-annular prostheses, respectively.

Conclusions

Leaning on a small basis of evidence and recommendations, the antithrombotic policy for patients undergoing TAVI is highly variable in the Netherlands. As a standardised regimen might further reduce haemorrhagic complications, large randomised clinical trials may help to establish the most appropriate approach.

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Concentration-dependent dual effects of hydrogen peroxide on insulin signal transduction in H4IIEC hepatocytes

Background

Oxidative stress induced by the accumulation of reactive oxygen species (ROS) has a causal role in the development of insulin resistance, whereas ROS themselves function as intracellular second messengers that promote insulin signal transduction. ROS can act both positively and negatively on insulin signaling, but the molecular mechanisms controlling these dual actions of ROS are not fully understood.

Methodology/Principal Findings

Here, we directly treated H4IIEC hepatocytes with hydrogen peroxide (H2O2), a representative membrane-permeable oxidant and the most abundant ROS in cells, to identify the key factors determining whether ROS impair or enhance intracellular insulin signaling. Treatment with high concentrations of H2O2 (25–50 µM) for 3 h reduced insulin-stimulated Akt phosphorylation, and increased the phosphorylation of both JNK and its substrate c-Jun. In contrast, lower concentrations of H2O2 (5–10 µM) enhanced insulin-stimulated phosphorylation of Akt. Moreover, lower concentrations suppressed PTP1B activity, suggesting that JNK and phosphatases such as PTP1B may play roles in determining the thresholds for the diametrical effects of H2O2 on cellular insulin signaling. Pretreatment with antioxidant N-acetyl-L-cysteine (10 mM) canceled the signal-promoting action of low H2O2 (5 µM), and it canceled out further impairment of insulin of insulin signaling induced by high H₂O₂ (25 µM).

Conclusions/Significance

Our results demonstrate that depending on its concentration, H2O2 can have the positive or negative effect on insulin signal transduction in H4IIEC hepatocytes, suggesting that the concentration of intracellular ROS may be a major factor in determining whether ROS impair or enhance insulin signaling.     

Freeze-Dried Targeted Mannosylated Selenium-Loaded Nanoliposomes: Development and Evaluation

The aim of this investigation was to develop and evaluate freeze-dried mannosylated liposomes for the targeted delivery of selenium. Dipalmitoylphosphatidylcholine, distearoylphosphatidylglycerol, and cholesterol were dissolved in a chloroform and methanol mixture and allowed to form a thin film within a rotatory evaporator. This thin film was hydrated with a sodium selenite (5.8 μM) solution to form multilamellar vesicles and homogenized under high pressure to yield unilamellar nanoliposomes. Se-loaded nanoliposomes were mannosylated by 0.1% w/v mannosamine (Man-Lip-Se) prior to being lyophilized. Mannosamine concentration was optimized with cellular uptake studies in M receptor expressing cells. Non-lyophilized and lyophilized Man-Lip-Se were characterized for size, zeta potential, and entrapment efficiency. The influence of liposomal composition on the characteristics of Man-Lip-Se were evaluated using acidic and basic medium for 24 h. Thermal analysis and powder X-ray diffraction were used to determine the interaction of components within the Man-Lip-Se. The size, zeta potential and entrapment efficiency of the optimum Man-Lip-Se were observed to be 158 ± 28.9 nm, 33.21 ± 0.89 mV, and 77.27 ± 2.34%, respectively. An in vitro Se release of 70–75% up to 24 h in PBS pH 6.8 and <8% Se release in acidic media (0.1 N HCl) in 1 h was observed. The Man-Lip-Se were found to withstand gastric-like environments and showed sustained release. Stable freeze-dried Man-Lip-Se were successfully formulated with a size of <200 nm, ∼75% entrapment, and achieved controlled release of Se with stability under acidic media, which may be of importance in the targeted delivery of Se to the immune system.

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The online version of this article (doi:10.1208/s12249-013-9988-3) contains supplementary material, which is available to authorized users.Key words: mannosylation, nanoliposome, selenium, thermal properties     

Detection of Ammonia-Oxidizing Archaea in Fish Processing Effluent Treatment Plants

Ammonia oxidation is the rate limiting step in nitrification and thus have an important role in removal of ammonia in natural and engineered systems with participation of both ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). However, their relative distribution and activity in fish processing effluent treatment plants (FPETPs) though significant, is hitherto unreported. Presence of AOA in sludge samples obtained from FPETPs was studied by amplification and sequencing of thaumarchaeal ammonia monooxygenase subunit A (AOA-amoA) gene. Different primer sets targeting 16S rRNA and AOA-amoA gene were used for the detection of AOA in FPETPs. Phylogenetic analysis of the gene revealed that the AOA was affiliated with thaumarchaeal group 1.1a lineage (marine cluster). Quantitative real time PCR of amoA gene was used to study the copy number of AOA and AOB in FPETPs. The AOA-amoA and AOB-amoA gene copy numbers of sludge samples ranged from 2.2 × 10⁶ to 4.2 × 10⁸ and 1.1 × 10⁷ to 8.5 × 10⁸ mg⁻¹ sludge respectively. Primer sets Arch-amoAF/Arch-amoAR and 340F/1000R were found to be useful for the sensitive detection of AOA-amoA and Archaeal 16S rRNA genes respectively in FPETPs. Their presence suggests the widespread occurrence and possible usefulness in removing ammonia from FPETPs which is in line with reports from other waste water treatment plants.

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Simultaneous quantification of 12 different nucleotides and nucleosides released from renal epithelium and in human urine samples using ion-pair reversed-phase HPLC

Nucleotides and nucleosides are not only involved in cellular metabolism but also act extracellularly via P1 and P2 receptors, to elicit a wide variety of physiological and pathophysiological responses through paracrine and autocrine signalling pathways. For the first time, we have used an ion-pair reversed-phase high-performance liquid chromatography ultraviolet (UV)-coupled method to rapidly and simultaneously quantify 12 different nucleotides and nucleosides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, uridine triphosphate, uridine diphosphate, uridine monophosphate, uridine, guanosine triphosphate, guanosine diphosphate, guanosine monophosphate, guanosine): (1) released from a mouse renal cell line (M1 cortical collecting duct) and (2) in human biological samples (i.e., urine). To facilitate analysis of urine samples, a solid-phase extraction step was incorporated (overall recovery rate ≥ 98 %). All samples were analyzed following injection (100 μl) into a Synergi Polar-RP 80 Å (250 × 4.6 mm) reversed-phase column with a particle size of 10 μm, protected with a guard column. A gradient elution profile was run with a mobile phase (phosphate buffer plus ion-pairing agent tetrabutylammonium hydrogen sulfate; pH 6) in 2–30 % acetonitrile (v/v) for 35 min (including equilibration time) at 1 ml min⁻¹ flow rate. Eluted compounds were detected by UV absorbance at 254 nm and quantified using standard curves for nucleotide and nucleoside mixtures of known concentration. Following validation (specificity, linearity, limits of detection and quantitation, system precision, accuracy, and intermediate precision parameters), this protocol was successfully and reproducibly used to quantify picomolar to nanomolar concentrations of nucleosides and nucleotides in isotonic and hypotonic cell buffers that transiently bathed M1 cells, and urine samples from normal subjects and overactive bladder patients.

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