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Compared to other methods to monitor and detect cyanobacteria in phytoplankton populations, fluorometry gives rapid, robust and reproducible results and can be used in situ . Fluorometers capable of providing biomass estimates and physiological information are not commonly optimized to target cyanobacteria. This study provides a detailed overview of the fluorescence kinetics of algal and cyanobacterial cultures to determine optimal optical configurations to target fluorescence mechanisms that are either common to all phytoplankton or diagnostic to cyanobacteria. We confirm that fluorescence excitation channels targeting both phycocyanin and chlorophyll a associated to the Photosystem II are required to induce the fluorescence responses of cyanobacteria. In addition, emission channels centered at 660, 685 and 730 nm allow better differentiation of the fluorescence response between algal and cyanobacterial cultures. Blue-green actinic light does not yield a robust fluorescence response in the cyanobacterial cultures and broadband actinic light should be preferred to assess the relation between ambient light and photosynthesis. Significant variability was observed in the fluorescence response from cyanobacteria to the intensity and duration of actinic light exposure, which needs to be taken into consideration in field measurements., (© The Author(s) 2022. Published by Oxford University Press.)

Objectives: To study the population pharmacokinetics of micafungin in critically ill patients, evaluate and optimize dosage regimens., Methods: An HPLC-fluorescence bioassay for micafungin was developed, fully validated and applied to a pharmacokinetic study conducted in 14 ICU patients. Dense blood sampling was performed from days 1 to 7. A population pharmacokinetic model accounting for interindividual (IIV) and interoccasion variability (IOV) of the PK parameters was developed. Simulations were performed to estimate the probability of target attainment (PTA) for several dosing regimens., Key Findings: A two-compartment pharmacokinetic model best described the data, with population clearance CL = 1.31 L/h and central volume V1 = 14.2 L. The relatively high IOV observed (45% for CL, 27% for V1) sets limits for the dose individualization in this population. The low PTA on the first day of treatment suggests the need of a loading dose. PTA and CFR estimates show that the current micafungin dosage may be insufficient for the treatment of borderline susceptible Candida strains., Conclusions: A loading dose of up to 300 mg of micafungin is needed for the treatment of invasive candidiasis in ICU patients while a maintenance dose of up to 200 mg can be considered in empirical antifungal treatment., (© 2020 Royal Pharmaceutical Society.)

Background: The use of cell-free DNA (cfDNA) as a noninvasive biomarker to detect allograft damage is expanding rapidly. However, quantifying the low fraction of donor-derived cfDNA (ddcfDNA) is challenging and requires a highly sensitive technique. ddcfDNA detection through unique donor single nucleotide polymorphisms (SNPs) is a recent new approach, however there are limited data in pediatric solid organ transplant (SOT) recipients., Methods: We developed an assay using a combination of 61 SNPs to quantify the ddcfDNA accurately using a custom R script to model for both the patient and donor genotypes requiring only a single sample from the allograft recipient. Performance of the assay was validated using genomic DNA (gDNA), cfDNA and donor samples where available., Results: The R "genotype-free" method gave results comparable to when using the known donor genotype. applicable to both related and unrelated pairs and can reliably measure ddcfDNA (limit of blank, below 0.12%; limit of detection, above 0.25%; limit of quantification 0.5% resulting in 84% accuracy). 159 pediatric SOT recipients (kidney, heart, and lung) were tested without the need for donor genotyping. Serial sampling was obtained from 82 patients., Conclusion: We have developed and validated a new assay to measure the fraction of ddcfDNA in the plasma of pediatric SOT recipients. Our method can be applicable in any donor-recipient pair without the need for donor genotyping and can provide results in 48 h at a low cost. Additional prospective studies are required to demonstrate its clinical validity in a large cohort of pediatric SOT recipients., (© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The regulatory processes involved in eosinophil trafficking into tissues are poorly understood; therefore, it is crucial to elucidate these mechanisms to advance the quality of clinical care for patients with eosinophil-mediated diseases. The complex interactions between eosinophil integrin receptors and their corresponding ligands on the post-capillary venules of the bronchial endothelium result in distinct modifications to the cytoskeletal architecture that occur in coordinated, temporally regulated sequences. The current study utilizes real-time confocal microscopy and time-based immunofluorescence staining to further characterize the effects of physiologically relevant fluid shear stress on this novel phenomenon of perfusion-induced calcium response. We found that the mere perfusion of fluid over adhered human eosinophils induced a release of intracellular calcium observed in conjunction with changes in cell morphology (flattening onto the coverslip surface, an increase in surface area, and a loss of circularity), suggesting a previously unknown mechanosensing aspect of eosinophil migration out of the vasculature. Although changes in morphology and degree of calcium release remained consistent across varying perfusion rates, the latency of the response was highly dependent on the degree of shear stresses. Eosinophils were fixed post-perfusion at specific timepoints for immunofluorescence staining to track proteins of interest over time. The distribution of proteins was diffuse throughout the cell prior to perfusion; however, they quickly localized to the periphery of the cell within 5 min. The actin cytoskeleton became markedly built up at the cell edges rapidly after stimulation, forming punctate dots by 4 min, suggesting a pivotal role in directed cell motility., (©2020 Society for Leukocyte Biology.)

A single-chain variable fragment (scFv) antibody is a recombinant protein in which a peptide linker connects the variable regions of the heavy chain and light chain. Due to its smaller molecular size, an scFv can be expressed using Escherichia coli. The presence of two disulphide bonds in the molecule often prevents expression of correctly folded scFv in the E. coli cytoplasm, making a refolding process necessary to regenerate scFv activity. The refolding process is time-consuming and requires large amounts of expensive reagents, such as guanidine hydrochloride, l-arginine and glutathione. Here, to conveniently obtain scFv proteins, we devised a simple and systematic method to optimize the co-expression of chaperone proteins and to combine them with specially engineered E. coli strains that permit the formation of stable disulphide bonds within the cytoplasm. Several scFv proteins were successfully obtained in a soluble form from E. coli cytoplasm. Thermal denaturation experiments and/or surface plasmon resonance measurements revealed that the thus-obtained scFvs possessed a stable tertiary structure and antigen-binding activity. The combined use of engineered E. coli with the simplified and systematic chaperone optimization can be useful for the production of scFv proteins., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Ribonucleoprotein (RNP) complexes and RNA-processing enzymes are attractive targets for antibiotic development owing to their central roles in microbial physiology. For many of these complexes, comprehensive strategies to identify inhibitors are either lacking or suffer from substantial technical limitations. Here, we describe an activity-binding-structure platform for bacterial ribonuclease P (RNase P), an essential RNP ribozyme involved in 5' tRNA processing. A novel, real-time fluorescence-based assay was used to monitor RNase P activity and rapidly identify inhibitors using a mini-helix and a pre-tRNA-like bipartite substrate. Using the mini-helix substrate, we screened a library comprising 2560 compounds. Initial hits were then validated using pre-tRNA and the pre-tRNA-like substrate, which ultimately verified four compounds as inhibitors. Biolayer interferometry-based binding assays and molecular dynamics simulations were then used to characterize the interactions between each validated inhibitor and the P protein, P RNA and pre-tRNA. X-ray crystallographic studies subsequently elucidated the structure of the P protein bound to the most promising hit, purpurin, and revealed how this inhibitor adversely affects tRNA 5' leader binding. This integrated platform affords improved structure-function studies of RNA processing enzymes and facilitates the discovery of novel regulators or inhibitors., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Here, we describe the use of a fluorescence based lateral flow competition assay for the screening of four classes of drugs, viz, Δ9-tetrahydrocannabinol (THC), cocaine (through the detection of benzoylecgonine, BZE), opiates (through the detection of morphine, MOR) and amphetamine (AMP) present in the sweat of a fingerprint. The Drug Screening Cartridge was specifically developed for fingerprint sample collection and analysis. For this study, the cut-offs were set at: 190, 90, 68 and 80 pg/fingerprint for THC, BZE, MOR and AMP, respectively. Working with three UK coroners, the Drug Screening Cartridge, together with its fluorescence reader, was applied to the detection of drugs in the sweat of a fingerprint from deceased individuals. The study shows that there was sufficient sweat present on the fingertips to enable analysis and that the Drug Screening Cartridge could detect the presence, or absence, of each drug. The presence of the drugs was confirmed using LC-MS-MS analysis of a second fingerprint sample collected simultaneously. Excellent correlation was achieved between the results obtained from the Drug Screening Cartridge and the LC-MS-MS analysis of the fingerprint samples obtained from 75 individuals. The accuracy of the results was: 99% for THC; 95% for BZE; 96% for MOR and 93% for AMP. The results obtained using the Drug Screening Cartridge were also compared to toxicological analysis of blood and urine samples with good correlation. The accuracy of the results between the Drug Screening Cartridge and blood was: 96%, 92%, 88% and 97% for THC, BZE, MOR and AMP, respectively. The comparison with urine showed an accuracy ranging between 86% and 92%. This fingerprint sample method has a collection time of just 5 s and a total analysis time of <10 mins. These results show that the lateral flow Drug Screening Cartridge is an excellent screening test to provide information on drug use from the sweat in a single fingerprint sample., (© The Author(s) 2018. Published by Oxford University Press.)

The catalytic DNA circuits play a critical role in engineered biological systems and molecular information processing. Actually, some of the natural or synthetic DNA circuits were triggered by covalent modifications, where conformational changes were induced to facilitate complex DNA engineering functions and signal transmissions. However, most of the reported artificial catalytic DNA circuits were regulated by the toehold-mediated reaction. Therefore, it is significant to propose a strategy to regulate the catalytic DNA circuit not only by the toehold-mediated mechanism, but also by involving the conformational changes induced by the covalent modification. In this study, we developed the catalytic DNA logic circuits regulated by DNAzyme. Here, a regulation strategy based on the covalent modification was proposed to control the DNA circuit, combing two reaction mechanisms: DNAzyme digestion and entropy-driven strand displacement. The DNAzyme and DNA catalyst can participate into the reactions alternatively, thus realizing the cascading catalytic circuits. Using the DNAzyme regulation, a series of logic gates (YES, OR and AND) were constructed. In addition, a two-layer cascading circuit and a feedback self-catalysis circuit were also established. The proposed DNAzyme-regulated strategy shows great potentials as a reliable and feasible method for constructing more complex catalytic DNA circuits.

Objectives: Leishmaniasis, one of the most significant neglected diseases around the world, is caused by protozoan parasites of the Leishmania genus. Nowadays, the available aetiological treatments for leishmaniasis have variable effectiveness and several problems such as serious side effects, toxicity, high cost and an increasing number of resistance cases. Thus, there is an urgent need for safe, oral and cost-effective drugs for leishmaniases. Previously, our group has shown the effect of the ergosterol biosynthesis inhibitors on Leishmania amazonensis. Herein, we showed the effect of ravuconazole against L. amazonensis; ravuconazole is a second-generation triazole antifungal drug that has good bioavailability after oral administration and a long terminal half-life in humans, a broad activity spectrum, high effectiveness in treatment of mycosis and negligible side effects., Methods: Several methodologies were used: cell culture, fluorescence and electron microscopy, high-resolution capillary GC coupled with MS, fluorimetry and flow cytometry., Results: Our results showed that ravuconazole was able to inhibit the proliferation of L. amazonensis promastigotes and intracellular amastigotes in vitro, with single-digit to sub-micromolar IC50 values, causing several alterations in the morphology, ultrastructure, cell viability and physiology of the parasites. The mitochondrion was significantly affected by the treatment, resulting in a collapse of the mitochondrial transmembrane potential that consequently led to inhibition of ATP production, combined with an increase in reactive oxygen species and mitochondrial superoxide production; by transmission electron microscopy, the organelle displayed a completely altered ultrastructure. The treatment changed the lipid profile, showing a profound depletion of the 14-desmethyl endogenous sterol pool., Conclusions: These results suggest that ravuconazole could be an alternative option for the treatment of leishmaniasis.

Enteric viruses and bacteriophages are exposed to various inactivating factors outside their host, and among them chlorine and heat are the most commonly used sanitizer in water industry and treatment in the food industry, respectively. Using MS2 phages as models for enteric viruses, we investigated the impact of free chlorine and heat on their physicochemical properties. Free chlorine was first evaluated alone. No increase in either capsid permeability or hydrophobicity was observed. The negative surface charge slightly increased suggesting molecular changes in the capsid. However, a weakening of the capsid by chlorine was suggested by differential scanning fluorimetry. This phenomenon was confirmed when chlorination was followed by a heat treatment. Indeed, an increase in the inactivation of MS2 phages and the permeability of their capsids to RNases was observed. More interestingly, an increase in the expression of hydrophobic domains at the phage surface was observed, but only for phages remaining infectious. The chlorine-caused weakening of the capsid suggested that, for an optimal use, the oxidant should be followed by heat. The increased permeability to RNases and the expression of hydrophobic domains may contribute to the development or improvement of molecular methods specific for infectious enteric viruses.

Objectives: The ability of certain bacteria to form biofilms underlies their capacity to cause medical device-associated infections. Most bacteria need the metal iron for their proliferation but also to form biofilms. The aim of this in vitro study was to investigate whether iron restriction upon application of the iron chelator deferiprone (DFP) impacts on bacterial biofilm formation and whether such an intervention can exert synergistic effects towards the antibacterial activity of three antibiotic compounds against coagulase-negative staphylococci (CNS) residing on titanium plates., Methods: Bacteria were seeded on titanium discs and cultured to obtain biofilms. Biofilms were then exposed to DFP and/or antibiotic treatment with clindamycin, gentamycin or vancomycin. Fluorescence microscopy and scanning electron microscopy (SEM) were used for morphological analysis of the biofilms before and after treatment., Results: Whereas DFP alone had only a moderate inhibitory effect on biofilm growth, the combination of DFP with the respective antibiotics resulted in a significant decline of bacterial numbers by two to three logs as compared to the effect of antibiotics alone. Fluorescence staining and SEM demonstrated severe damage to even complete destruction of biofilms after combined treatment with DFP and antibiotics that was not the case upon sole treatment with antibiotics., Conclusion: Iron chelation is able to potentiate the antibacterial activity of conventional antibiotics by destroying bacterial biofilms that recommends this combination as a promising strategy for the treatment of chronic device infections with biofilm producing CNS.

Background: Deficiency of lysosomal acid lipase (LAL) causes Wolman disease and cholesterol ester storage disease. With the recent introduction of enzyme replacement therapy to manage LAL deficiency comes the need for a reliable assay of LAL enzymatic activity that can be applied to dried blood spots (DBS)., Methods: We prepared and tested a library of analogs of palmitoyl 4-methylumbelifferyl esters to find a highly active and specific substrate for LAL in DBS. The LAL assay was optimized leading to both LC-MS/MS and fluorometric assay of LAL. We tested the new assay on DBS from healthy and LAL-deficient patients., Results: The ester formed between palmitic acid and 4-propyl-8-methyl-7-hydroxycoumarin (P-PMHC) was found to be >98% selective for LAL in DBS based on the sensitivity of its activity to the LAL-specific inactivator Lalistat-2 and the fact that the activity was close to zero using DBS from patients previously shown to be LAL-deficient. Use of P-PMHC and heavy isotope-labeled internal standard with optimized assay conditions led to an approximately 2-fold increase in the specific activity of LAL compared with the previously reported LAL assay. Patients deficient in LAL were readily distinguished from normal persons with the new LAL assay using UPLC-MS/MS or fluorometric assay platforms., Conclusions: The new assay can measure LAL in DBS with a single measurement compared with the previous method involving 2 assays done in parallel., (© 2017 American Association for Clinical Chemistry.)

Response of rice (Oryza sativa) exposed to both biotic and abiotic stresses can be quantified by employing fast and accurate optical methods. In this study, the overall stress responses of (i) 12 near-isogenic lines (NILs) in the genetic background of the rice blast-susceptible cultivar Lijiangxintuanheigu (LTH) and (ii) four NILs in the genetic background of the bacterial blight-susceptible cultivar IR24, were inspected by means of Chl fluorescence (Chl-F) imaging. The distribution of the maximum and effective quantum yield of PSII (Fv/FM and QY) and steady-state Chl-F (Ft) were found to be effective in differentiating symptomatic leaf tissue for both rice blast and bacterial blight, which correlated well with 30 cycles of rice blast and six cycles of bacterial blight previously screened using classical (manual) approaches. Subsequently, identified Chl-F parameters allowing detection under ambient light (QY and Ft) were tested across both biotic and abiotic (drought) stress experiments, for rice cultivars contrasting for drought stress response (N22, IR64 and NSIC Rc 222). Their applicability has been proven for both rice blast and bacterial blight; however, QY failed to detect the effect of drought. In addition to Chl-F, the usefulness of 11 selected vegetation indices (Vis) was tested on these three cultivars exposed to particular stresses: (i) rice blast was detectable by Vis calculated from the visible spectrum; (ii) bacterial blight by near-infrared-related Vis; and (iii) drought by Vis calculated from the visible spectrum. The key Chl-F parameters and/or Vis have been summarized and discussed., (© The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

To establish optimal ultra-high-pressure (UHP)-assisted extraction conditions for procyanidins from lychee pericarp, a response surface analysis method with four factors and three levels was adopted. The optimum conditions were as follows: 295 MPa pressure, 13 min pressure holding time, 16.0 mL/g liquid-to-solid ratio, and 70% ethanol concentration. Compared with conventional ethanol extraction and ultrasonic-assisted extraction methods, the yields of the total procyanidins, flavonoids, and phenolics extracted using the UHP process were significantly increased; consequently, the oxygen radical absorbance capacity and cellular antioxidant activity of UHP-assisted lychee pericarp extracts were substantially enhanced. LC-MS/MS and high-performance liquid chromatography quantification results for individual phenolic compounds revealed that the yield of procyanidin compounds, including epicatechin, procyanidin A2, and procyanidin B2, from lychee pericarp could be significantly improved by the UHP-assisted extraction process. This UHP-assisted extraction process is thus a practical method for the extraction of procyanidins from lychee pericarp.

DNA and RNA aptamers have been extensively investigated as potential competitors for antibodies for a variety of applications including food safety testing. Ultrasensitive fluorescence detection of foodborne pathogenic bacteria as low as 1-10 cells/mL has been achieved using aptamers coupled to quantum dots in clear pristine buffers for environmental sample detection. Quantum dots offer other advantages, including single UV or blue light source multiplex (multicolored) detection. However, quantum dots can exhibit decreased fluorescence in some food matrixes and even completely fail to fluoresce in some fatty matrixes, as documented in this report. Given the need to detect substances in complex food matrixes (and from data reported elsewhere), aptamer-magnetic bead pull-down methods followed by enzymatic/fluorometric- or PCR-based detection methods may be more robust methods for testing in foods or enrichment cultures. Other lessons learned, including the initial choice of aptamer targets to enhance assay specificity, are also discussed.

Background: Deficiency of the lysosomal enzyme acid α-glucosidase (GAA) causes Pompe disease. Newborn screening for Pompe disease is ongoing, and improved methods for distinguishing affected patients from those with pseudodeficiency, especially in the Asian population, would substantially reduce the number of patient referrals for clinical follow-up., Methods: We measured the enzymatic activity of GAA in dried blood spots on newborn screening cards (DBS) using a tandem mass spectrometry (MS/MS) assay. The assay displayed a relatively large analytical range compared to the fluorimetric assay with 4-methylumbelliferyl-α-glucoside. DBS from newborns confirmed to have infantile-onset Pompe disease (IOPD, n = 11) or late-onset Pompe disease (LOPD) (n = 12) and those from patients bearing pseudodeficiency alleles with or without Pompe mutations, or Pompe disease carriers (n = 230) were studied., Results: With use of the MS/MS GAA assay in DBS, 96% of the pseudodeficiency newborns and all of the Pompe disease carriers were well separated from the IOPD and LOPD newborns. The fluorimetric assay separated <10% of the pseudodeficiencies from the IOPD/LOPD group., Conclusions: The relatively large analytical range MS/MS GAA assay but not the fluorimetric assay in DBS provides a robust approach to reduce the number of referrals and should dramatically facilitate newborn screening of Pompe disease., (© 2017 American Association for Clinical Chemistry.)

Organic, semiorganic, and conventional "Hayward" kiwifruits, treated with ethylene for 24 h and stored during 10 days, were assessed by UV spectrometry, fluorometry, and chemometrical analysis for changes in selected characteristics of quality (firmness, dry matter and soluble solid contents, pH, and acidity) and bioactivity (concentration of polyphenols via Folin-Ciocalteu and p-hydroxybenzoic acid assays). All of the monitored qualitative parameters and characteristics related to bioactivity were affected either by cultivation practices or by ethylene treatment and storage. Results obtained, supported by statistical evaluation (Friedman two-way ANOVA) and chemometric analysis, clearly proved that the most significant impact on the majority of the evaluated parameters of quality and bioactivity of "Hayward" kiwifruit had the ethylene treatment followed by the cultivation practices and the postharvest storage. Total concentration of polyphenols expressed via p-hydroxybenzoic acid assay exhibited the most significant sensitivity to all three evaluated parameters, reaching a 16.5% increase for fresh organic compared to a conventional control sample. As a result of postharvest storage coupled with ethylene treatment, the difference increased to 26.3%. Three-dimensional fluorescence showed differences in the position of the main peaks and their fluorescence intensity for conventional, semiorganic, and organic kiwifruits in comparison with ethylene nontreated samples.

Tolerance towards antibiotics of Pseudomonas aeruginosa biofilms is recognized as a major cause of therapeutic failure of chronic lung infection in cystic fibrosis (CF) patients. This lung infection is characterized by antibiotic-tolerant biofilms in mucus with zones of O2 depletion mainly due to polymorphonuclear leukocytic activity. In contrast to the main types of bactericidal antibiotics, it has not been possible to establish an association between the bactericidal effects of colistin and the production of detectable levels of OH ˙ on several strains of planktonic P. aeruginosa. Therefore, we propose that production of OH ˙ may not contribute significantly to the bactericidal activity of colistin on P. aeruginosa biofilm. Thus, we investigated the effect of colistin treatment on biofilm of wild-type PAO1, a catalase-deficient mutant (ΔkatA) and a colistin-resistant CF isolate cultured in microtiter plates in normoxic- or anoxic atmosphere with 1 mM nitrate. The killing of bacteria during colistin treatment was measured by CFU counts, and the OH⋅ formation was measured by 3(')-(p-hydroxylphenyl fluorescein) fluorescein (HPF) fluorescence. Validation of the assay was done by hydrogen peroxide treatment. OH⋅ formation was undetectable in aerobic PAO1 biofilms during 3 h of colistin treatment. Interestingly, we demonstrate increased susceptibility of P. aeruginosa biofilms towards colistin during anaerobic conditions. In fact, the maximum enhancement of killing by anaerobic conditions exceeded 2 logs using 4 mg L(-1) of colistin compared to killing at aerobic conditions., (© FEMS 2015.)

This study investigates the effect of sonic stimulation on Bacillus endospore germination. Germinating endospores in a microtiter plate were exposed to audible sound wave generated by an array of piezoelectric transducers. In situ germination kinetics was measured by terbium-dipicolinate fluorescence assay, optical density measurement and phase contrast microscopy. Fluorescence results revealed that sonic stimulation (5 kHz at 90 dB) promoted the germination speed by 43.7% ± 11.3% and final germination level by 61.7% ± 11.9% of Bacillus atrophaeus. This acoustic energy absorbed by endospores is postulated to change membrane permeability and increase enzyme activities; thereby, expediting the germination process. This also raises the likelihood of dormant endospores undergoing germination because of a rapid release of unidentified chemical mediators for quorum sensing. On the other hand, acoustic effect was not observed in B. subtilis endospores. This may be attributed to the different spore aspect ratio, 1.43 ± 0.05 for B. atrophaeus and 2.02 ± 0.08 for B. subtilis, which results in a difference in specific absorption rates towards audible sound waves. Our results demonstrate the modulation of endospore germination by an external field to shed light on germination mechanism and cell-wave interaction., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cytosine methylation is a widespread epigenetic regulation mechanism. In healthy mature cells, methylation occurs at CpG dinucleotides within promoters, where it primarily silences gene expression by modifying the binding affinity of transcription factors to the promoters. Conversely, a recent study showed that in stem cells and cancer cell precursors, methylation also occurs at non-CpG pairs and involves introns and even gene bodies. The epigenetic role of such methylations and the molecular mechanisms by which they induce gene regulation remain elusive. The topology of both physiological and aberrant non-CpG methylation patterns still has to be detailed and could be revealed by using the differential stability of the duplexes formed between site-specific oligonucleotide probes and the corresponding methylated regions of genomic DNA. Here, we present a systematic study of the thermal stability of a DNA oligonucleotide sequence as a function of the number and position of non-CpG methylation sites. The melting temperatures were determined by monitoring the fluorescence of donor-acceptor dual-labelled oligonucleotides at various temperatures. An empirical model that estimates the methylation-induced variations in the standard values of hybridization entropy and enthalpy was developed., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: There is interest in newborn screening and diagnosis of lysosomal storage diseases because of the development of treatment options that improve clinical outcome. Assays of lysosomal enzymes with high analytical range (ratio of assay response from the enzymatic reaction divided by the assay response due to nonenzymatic processes) are desirable because they are predicted to lead to a lower rate of false positives in population screening and to more accurate diagnoses., Methods: We designed new tandem mass spectrometry (MS/MS) assays that give the largest analytical ranges reported to date for the use of dried blood spots (DBS) for detection of mucopolysaccharidoses type II (MPS-II), MPS-IVA, and MPS-VI. For comparison, we carried out fluorometric assays of 6 lysosomal enzymes using 4-methylumbelliferyl (4MU)-substrate conjugates., Results: The MS/MS assays for MPS-II, -IVA, and -VI displayed analytical ranges that are 1-2 orders of magnitude higher than those for the corresponding fluorometric assays. The relatively small analytical ranges of the 4MU assays are due to the intrinsic fluorescence of the 4MU substrates, which cause high background in the assay response., Conclusions: These highly reproducible MS/MS assays for MPS-II, -IVA, and -VI can support multiplex newborn screening of these lysosomal storage diseases. MS/MS assays of lysosomal enzymes outperform 4MU fluorometric assays in terms of analytical range. Ongoing pilot studies will allow us to gauge the impact of the increased analytical range on newborn screening performance., (© 2015 American Association for Clinical Chemistry.)

Lot6p (EC 1.5.1.39; Ylr011wp) is the sole quinone oxidoreductase in the budding yeast, Saccharomyces cerevisiae. Using hexahistidine tagged, recombinant Lot6p, we determined the steady-state enzyme kinetic parameters with both NADH and NADPH as electron donors; no cooperativity was observed with these substrates. The NQO1 inhibitor curcumin, the NQO2 inhibitor resveratrol, the bacterial nitroreductase inhibitor nicotinamide and the phosphate mimic vanadate all stabilise the enzyme towards thermal denaturation as judged by differential scanning fluorimetry. All except vanadate have no observable effect on the chemical cross-linking of the two subunits of the Lot6p dimer. These compounds all inhibit Lot6p's oxidoreductase activity, and all except nicotinamide exhibit negative cooperativity. Molecular modelling suggests that curcumin, resveratrol and nicotinamide all bind over the isoalloxazine ring of the FMN cofactor in Lot6p. Resveratrol was predicted to contact an α-helix that links the two active sites. Mutation of Gly-142 (which forms part of this helix) to serine does not greatly affect the thermal stability of the enzyme. However, this variant shows less cooperativity towards resveratrol than the wild type. This suggests a plausible hypothesis for the transmission of information between the subunits and, thus, the molecular mechanism of negative cooperativity in Lot6p., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Herbivore injury has indirect effects on the growth and performance of host plants through photosynthetic suppression. It causes uncertain reduction in photosynthesis, which likely depends on the degree of infestation. Rapid light curves provide detailed information on the saturation characteristics of electron transport as well as the overall photosynthetic performance of a plant. We examined the effects of different intensities of infestation of the invasive mealybug, Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae), on the relative chlorophyll content and rapid light curves of tomato Solanum lycopersicum L. leaves using a chlorophyll meter and chlorophyll fluorescence measurement system, respectively, under greenhouse conditions. After 38 d of P. solenopsis feeding, relative chlorophyll content of tomato plants with initial high of P. solenopsis was reduced by 57.3%. Light utilization efficiency (α) for the initial high-density treatment was reduced by 42.4%. However, no significant difference between initial low-density treatment and uninfested control was found. The values of the maximum electron transport rate and minimum saturating irradiance for initial high-density treatment were reduced by 82.0 and 69.7%, respectively, whereas the corresponding values for low-density treatment were reduced by 55.9 and 58.1%, respectively. These data indicated that changes were induced by P. solenopsis feeding in the relative chlorophyll content and chlorophyll fluorescence of infested tomato plants. The results indicating that low initial infestation by P. solenopsis caused no change in relative leaf chlorophyll content or light utilization efficiency could have been because the plants rapidly adapted to P. solenopsis feeding or because of compensatory photosynthesis.

Some species of sacoglossan sea slugs can maintain functional chloroplasts from specific algal food sources in the cells of their digestive diverticula. These 'stolen' chloroplasts (kleptoplasts) can survive in the absence of the plant cell and continue to photosynthesize, in some cases for as long as one year. Within the Metazoa, this phenomenon (kleptoplasty) seems to have only evolved among sacoglossan sea slugs. Known for over a century, the mechanisms of interaction between the foreign organelle and its host animal cell are just now starting to be unravelled. In the study of sacoglossan sea slugs as photosynthetic systems, it is important to understand their relationship with light. This work reviews the state of knowledge on autotrophy as a nutritional source for sacoglossans and the strategies they have developed to avoid excessive light, with emphasis to the behavioural and physiological mechanisms suggested to be involved in the photoprotection of kleptoplasts. A special focus is given to the advantages and drawbacks of using pulse amplitude modulated fluorometry in photobiological studies addressing sacoglossan sea slugs. Finally, the classification of photosynthetic sacoglossan sea slugs according to their ability to retain functional kleptoplasts and the importance of laboratory culturing of these organisms are briefly discussed.

Objectives: Miltefosine, an alkylphosphocholine anti-cancer drug, exhibits direct activity against Leishmania donovani and also promotes anti-leishmanial host immunomodulatory functions. Nuclear factor-erythroid 2-related factor 2 (Nrf-2), a redox-sensitive transcription factor, regulates the host stress responses leading to effective microbial clearance by a positive effect on haem oxygenase-1 (HO-1) enzyme expression/activity. We aimed to investigate the role of miltefosine in regulating HO-1/Nrf-2-mediated oxidative responses in visceral leishmaniasis (VL) patients in vivo and in vitro., Methods: Splenic aspirate and bone marrow aspirate cells of VL patients (n = 23) were used in the study. RT-PCR of HO-1, Nrf-2 translocation analysis and HO-1 ELISA were used to investigate the HO-1/Nrf-2-mediated modulation of oxidative responses by miltefosine in vivo. Fluorometric measurement of reactive oxygen species (ROS) was performed, determination of glutathione peroxidase (GPx) activity was performed, and bilirubin and superoxide dismutase (SOD) levels were determined. The in vitro HO-1/Nrf-2-dependent anti-leishmanial effect of miltefosine was assessed by the use of specific inhibitors/inducers and subsequent microscopic measurement of parasite killing and Th1/Th2 cytokine regulation by ELISA., Results: Increased levels of transcript and serum HO-1, Nrf-2 nuclear translocation, serum bilirubin, GPx and SOD activity in untreated VL patients were reversed after miltefosine chemotherapy. The effectiveness of miltefosine for positive induction of ROS via NADPH correlated with a decrease in HO-1/ERK/Nrf-2-dependent parasite load. Furthermore, HO-1 blockade by miltefosine led to suppression of interleukin-10 and transforming growth factor-β, but enhanced interleukin-12 and tumour necrosis factor-α production, in VL patients., Conclusions: The antioxidant promoting property of L. donovani is crucial for protection against the mounting redox threat in the host. Therefore, these findings provide direct evidence for targeting HO-1/Nrf-2 as an anti-leishmanial approach for chemotherapy in human VL.

A renewed interest in non-coding RNA (ncRNA) has led to the discovery of novel RNA species and post-transcriptional ribonucleoside modifications, and an emerging appreciation for the role of ncRNA in RNA epigenetics. Although much can be learned by amplification-based analysis of ncRNA sequence and quantity, there is a significant need for direct analysis of RNA, which has led to numerous methods for purification of specific ncRNA molecules. However, no single method allows purification of the full range of cellular ncRNA species. To this end, we developed a multidimensional chromatographic platform to resolve, isolate and quantify all canonical ncRNAs in a single sample of cells or tissue, as well as novel ncRNA species. The applicability of the platform is demonstrated in analyses of ncRNA from bacteria, human cells and plasmodium-infected reticulocytes, as well as a viral RNA genome. Among the many potential applications of this platform are a system-level analysis of the dozens of modified ribonucleosides in ncRNA, characterization of novel long ncRNA species, enhanced detection of rare transcript variants and analysis of viral genomes.

Proton-driven Suc transporters allow phloem cells of higher plants to accumulate Suc to more than 1 M, which is up to ~1000-fold higher than in the surrounding extracellular space. The carrier protein can accomplish this task only because proton and Suc transport are tightly coupled. This study provides insights into this coupling by resolving the first step in the transport cycle of the Suc transporter SUT1 from maize (Zea mays). Voltage clamp fluorometry measurements combining electrophysiological techniques with fluorescence-based methods enable the visualization of conformational changes of SUT1 expressed in Xenopus laevis oocytes. Using the Suc derivate sucralose, binding of which hinders conformational changes of SUT1, the association of protons to the carrier could be dissected from transport-associated movements of the protein. These combined approaches enabled us to resolve the binding of protons to the carrier and its interrelationship with the alternating movement of the protein. The data indicate that the rate-limiting step of the reaction cycle is determined by the accessibility of the proton binding site. This, in turn, is determined by the conformational change of the SUT1 protein, alternately exposing the binding pockets to the inward and to the outward face of the membrane.

In mammalian brains, d-serine has been shown to be required for the regulation of glutamate neurotransmission as an endogenous co-agonist for the N-methyl-d-aspartate type glutamate receptor that is essential for the expression of higher-order brain functions. The exact control mechanisms for the extracellular d-serine dynamics, however, await further elucidation. To obtain an insight into this issue, we have characterized the effects of agents acting at the α-amino-3-hydroxy-5-methyl-4-isoxazolepropioinic acid (AMPA) type glutamate receptor on the extracellular d-serine contents in the medial prefrontal cortex of freely moving rats by an in vivo microdialysis technique in combination with high-performance liquid chromatography with fluorometric detection. In vivo experiments are needed in terms of a crucial role of d-serine in the neuron-glia communications despite the previous in vitro studies on AMPA receptor-d-serine interactions using the separated preparations of neurons or glial cells. Here, we show that the intra-cortical infusion of (S)-AMPA, an active enantiomer at the AMPA receptor, causes a significant and concentration-dependent reduction in the prefrontal extracellular contents of d-serine, which is reversed by an AMPA/kainate receptor antagonist, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt, and a calcium permeable AMPA receptor antagonist, 1-naphthyl acetyl spermine. The d-serine reducing effects of (S)-AMPA are augmented by co-infusion of cyclothiazide that prevents AMPA receptor desensitization. Our data support the view that a calcium permeable AMPA receptor subtype may exert a phasic inhibitory control on the extracellular d-serine release in the mammalian prefrontal cortex in vivo.

Objectives: To describe the plasma concentration-time profile of moxifloxacin after intravenous and enteral administration in intensive care unit (ICU) patients and to provide a pharmacodynamic (PD) evaluation with regard to pneumonia., Patients and Methods: Twenty-five adult patients from a cardiothoracic/mixed surgical ICU were enrolled. Moxifloxacin was given as a standard dose (400 mg once daily). Therapy was successfully switched to enteral administration on day 5 in 16 patients. A rich data sampling schedule was performed after intravenous (day 4) and enteral (day 8) administration. Moxifloxacin concentrations were analysed by HPLC. A population pharmacokinetic (PK) model was developed using NONMEM VII. Simulated concentration-time profiles were evaluated for their probability of attaining PK/PD target values relevant for community-acquired pneumonia (CAP) and hospital-acquired pneumonia (HAP)., Results: A linear-elimination two-compartment model described the data adequately. Parameter estimates (coefficient of variation of inter-individual variability) were: absorption rate constant, 1.09/h (135%); enteral bioavailability, 76% (20.0%); central volume of distribution, 55.6 L; peripheral volume of distribution, 59.6 L (15.3%); inter-compartmental clearance, 47.7 L/h; and clearance, 11.3 L/h (23.7%). Both intravenously and enterally administered standard-dose moxifloxacin reliably attained the PK/PD target values for pathogens with MICs ≤ 0.25 mg/L for CAP and ≤ 0.125 mg/L for HAP., Conclusions: Drug exposure to moxifloxacin in ICU patients was more variable than in healthy volunteers. The standard dosing provides sufficient drug exposure for treatment of CAP but for HAP it does so only when a highly susceptible pathogen is present. Intravenous/enteral sequential therapy may be considered for cautiously selected cases in ICU patients.

The Río Negro and Neuquén Valley is the most important apple and pear growing region in Argentina. Cydia pomonella L. (Lepidoptera: Tortricidae), the main fruit-tree pest is being controlled by azinphosmethyl (AzMe), acetamiprid (Acet), and thiacloprid (Thia) among other insecticides. The objective of this study was to evaluate the response of neonate larvae of codling moth to these three insecticides and on the role of cytochrome P450 monooxygenase in their toxicity. All field populations presented significantly lower mortality to a discriminating concentration (DC) of Acet and AzMe. In addition, 13 of the 14 populations showed significantly lower mortality to DC of Thia. Most of the field populations (71%) showed significantly higher 7-ethoxycoumarine O-deethylase activity compared with the laboratory-susceptible strain. While positive significant correlation (gamma = 0.59) was found between Thia and AzMe mortalities at the DC level, no significant correlations were detected between Acet and Thia (gamma = 0.35) or Acet and AzMe (gamma = 0.12). However, Acet and Thia mortalities were significantly correlated to the percentage of individuals exhibiting 7-ethoxy-coumarine O-deethylase activity activities higher than the mean upper 95% confidence limit of the susceptible strain (gamma = -0.52 and gamma = -0.63, respectively).

Objectives: There is a general lack of effective and non-toxic chemotherapeutic agents for leishmaniasis and there is as yet no study about the effect of HIV peptidase inhibitors (HIV PIs) on Leishmania/HIV-coinfected patients. In the present work, we performed a comparative analysis of the spectrum of action of HIV PIs on different Leishmania spp., including strains obtained from HIV-positive patients receiving or not receiving antiretroviral treatment., Methods: The effects of nelfinavir and saquinavir on Leishmania proliferation were assessed by means of a colorimetric assay (MTT). Subsequently, the effect of nelfinavir on aspartic peptidase activity from Leishmania spp. was assessed by following the degradation of the fluorogenic substrate MCA-G-K-P-I-L-F-F-R-L-K-DNP-Arg-NH(2)., Results: Nelfinavir was capable of significantly reducing the multiplication of many Leishmania reference strains and isolates obtained from HIV-positive patients receiving or not receiving antiretroviral treatment. Leishmania major growth was inhibited by ≈ 50%, while all other flagellates were strongly inhibited (at least 94%), except for a Leishmania chagasi strain obtained from an HIV-positive patient under treatment with highly active antiretroviral therapy (HAART). Culture of this isolate in the presence of nelfinavir induced a considerable reduction in the aspartic peptidase activity. In addition, nelfinavir was also capable of inhibiting the aspartic peptidase activity of all Leishmania strains tested., Conclusions: The present data contribute to the study of the effect of HIV PIs on Leishmania infection and add new insights into the possibility of exploiting aspartic peptidases as promising targets in order to generate novel medications to treat leishmaniasis.

Context: Trophoblast-derived human chorionic gonadotropin (hCG) promotes corpus luteum progesterone (P4) production, and wide ranges of serum P4 levels are noted in various pregnancy outcomes, despite similar hCG concentrations. There are five unique biologically active hCG variants in human pregnancy urine, and previous studies of P4 production in response to hCG have used only preparations containing all isoforms. Understanding exactly which hCG variant is primarily responsible for stimulating corpus luteum steroidogenesis may have great clinical and diagnostic implications, including in the setting of ectopic pregnancy., Objective: Our objective was to delineate the role of the standard and hyperglycosylated (H)-hCG isoforms in stimulating P4 production by luteinized granulosa cells., Design and Setting: Cell culture, ELISA, and fluorometric-based protein assays were done at Duke University Medical Center., Patients: Patients were anonymous oocyte donors., Intervention: Cultured luteinized granulosa cells were treated with 0.25, 0.5, and 1.0 ng/ml total hCG, which contains all isoforms, purified standard hCG (37.1 kDa), and purified H-hCG (42.8 kDa)., Main Outcome Measure: P4 produced per total cellular protein (nanograms per microgram) was measured via ELISA and fluorometric protein determination kits., Results: Both total hCG (P = 0.0003) and purified standard hCG (P < 0.0001) stimulated a dose-dependent increase in P4 production. Purified H-hCG did not change the P4 produced per total cellular protein response (P value not significant)., Conclusions: Standard hCG stimulated P4 production by cultured granulosa cells and likely supports corpus luteum function via interactions with the LH/hCG receptor. In contrast, H-hCG did not increase P4 production, which indicates a nonsteroidogenic role for this protein during early gestation.

Aims: To develop a simple, high-throughput and inexpensive procedure to detect and quantify aflatoxins into the culture media of growing mycelia., Methods and Results: Fungal conidia (Aspergillus flavus) were inoculated into the wells of a microplate containing 200 μl of different formulations of coconut-derived liquid medium. Time-dependent production of aflatoxins in the culture media was evaluated by a procedure relying on the UV-induced fluorescence emission by the toxin, using a microplate reader. These data were validated by comparison with the outputs of a conventional HPLC-based procedure. Determinations of aflatoxin concentration, according to the fluorimetric procedure, were performed either by withdrawing samples from the plates or by direct 'in situ' readings, the latter method reinforcing the high-throughput feature of the procedure. Fluorescence enhancers (cyclodextrins) did not ameliorate the sensitivity of the procedure to low concentrations of the toxin into the medium. The efficacy of the procedure was also validated by testing the effect on toxin yield of adding an antioxidant agent (α-lipoic acid) to the medium., Conclusions: We give evidence that our improved procedure is reliable and suitable to analyse aflatoxin accumulation time course in coconut-derived culture medium., Significance and Impact of the Study: This study shows that our procedure may profitably be used to give insights into the mechanisms of regulation of mycotoxin production and, consequently, to implement different strategies for the containment of aflatoxin contamination of food and feed commodities., (© 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.)

'Mexican' lime (Citrus aurantifolia Swingle) was transformed with constructs that contained chimeric promoter-gus gene fusions of phloem-specific rolC promoter of Agrobacterium rhizogenes, Arabidopsis thaliana sucrose-H(+) symporter (AtSUC2) gene promoter of Arabidopsis thaliana, rice tungro bacilliform virus (RTBV) promoter and sucrose synthase l (RSs1) gene promoter of Oryza sativa (rice). Histochemical β-glucuronidase (GUS) analysis revealed vascular-specific expression of the GUS protein in citrus. The RTBV promoter was the most efficient promoter in this study while the RSs1 promoter could drive low levels of gus gene expression in citrus. These results were further validated by reverse transcription real-time polymerase chain reaction and northern blotting. Southern blot analysis confirmed stable transgene integration, which ranged from a single insertion to four copies per genome. The use of phloem-specific promoters in citrus will allow targeted transgene expression of antibacterial constructs designed to battle huanglongbing disease (HLB or citrus greening disease), associated with a phloem-limited Gram-negative bacterium.

Background: Newborn screening for lysosomal storage diseases (LSDs) has been gaining considerable interest owing to the availability of enzyme replacement therapies. We present a digital microfluidic platform to perform rapid, multiplexed enzymatic analysis of acid α-glucosidase (GAA) and acid α-galactosidase to screen for Pompe and Fabry disorders. The results were compared with those obtained using standard fluorometric methods., Methods: We performed bench-based, fluorometric enzymatic analysis on 60 deidentified newborn dried blood spots (DBSs), plus 10 Pompe-affected and 11 Fabry-affected samples, at Duke Biochemical Genetics Laboratory using a 3-mm punch for each assay and an incubation time of 20 h. We used a digital microfluidic platform to automate fluorometric enzymatic assays at Advanced Liquid Logic Inc. using extract from a single punch for both assays, with an incubation time of 6 h. Assays were also performed with an incubation time of 1 h., Results: Assay results were generally comparable, although mean enzymatic activity for GAA using microfluidics was approximately 3 times higher than that obtained using bench-based methods, which could be attributed to higher substrate concentration. Clear separation was observed between the normal and affected samples at both 6- and 1-h incubation times using digital microfluidics., Conclusions: A digital microfluidic platform compared favorably with a clinical reference laboratory to perform enzymatic analysis in DBSs for Pompe and Fabry disorders. This platform presents a new technology for a newborn screening laboratory to screen LSDs by fully automating all the liquid-handling operations in an inexpensive system, providing rapid results.

Study Objectives: Sleep pattern and circadian rhythms are regulated via the retinohypothalamic tract in response to stimulation of a subset of retinal ganglion cells, predominantly by blue light (450-490 nm). With age, the transmission of blue light to the retina is reduced because of the aging process of the human lens, and this may impair the photoentrainment of circadian rhythm leading to sleep disorders. The aim of the study was to examine the association between lens aging and sleep disorders., Design: Cross-sectional population based study., Setting: The study was performed at the Research Center for Prevention and Health, Glostrup Hospital, Denmark and at the Department of Ophthalmology, Herlev Hospital, Denmark., Participants: An age- and sex-stratified sample of 970 persons aged 30 to 60 years of age drawn from a sample randomly selected from the background population., Interventions: Not applicable., Measurements and Results: Sleep disturbances were evaluated by a combination of questionnaire and the use of prescription sleeping medication. Lens aging (transmission and yellowing) was measured objectively by lens autofluorometry. The risk of sleep disturbances was significantly increased when the transmission of blue light to the retina was low, even after correction for the effect of age and other confounding factors such as smoking habits, diabetes mellitus, gender, and the risk of ischemic heart disease (P < 0.0001)., Conclusions: Filtration of blue light by the aging lens was significantly associated with an increased risk of sleep disturbances. We propose that this is a result of disturbance of photoentrainment of circadian rhythms.

Extent of resection is a major goal and prognostic factor in the treatment of gliomas. In this study we evaluate whether quantitative ex vivo tissue measurements of δ-aminolevulinic acid-induced protoporphyrin IX (PpIX) identify regions of increasing malignancy in low- and high-grade gliomas beyond the capabilities of current fluorescence imaging in patients undergoing fluorescence-guided resection (FGR). Surgical specimens were collected from 133 biopsies in 23 patients and processed for ex vivo neuropathological analysis: PpIX fluorimetry to measure PpIX concentrations (C(PpIX)) and Ki-67 immunohistochemistry to assess tissue proliferation. Samples displaying visible levels of fluorescence showed significantly higher levels of C(PpIX) and tissue proliferation. C(PpIX) was strongly correlated with histopathological score (nonparametric) and tissue proliferation (parametric), such that increasing levels of C(PpIX) were identified with regions of increasing malignancy. Furthermore, a large percentage of tumor-positive biopsy sites (∼40%) that were not visibly fluorescent under the operating microscope had levels of C(PpIX) greater than 0.1 µg/mL, which indicates that significant PpIX accumulation exists below the detection threshold of current fluorescence imaging. Although PpIX fluorescence is recognized as a visual biomarker for neurosurgical resection guidance, these data show that it is quantitatively related at the microscopic level to increasing malignancy in both low- and high-grade gliomas. This work suggests a need for improved PpIX fluorescence detection technologies to achieve better sensitivity and quantification of PpIX in tissue during surgery.

We studied the distribution of anoxygenic phototrophs in 23 steppe lakes in the Transbaikal region (Russia), in Uzbekistan (Central Asia) and in the Crimean peninsula (Ukraine). The lakes varied in their mineral content and composition (salinities from 0.2 to 300 g L(-1) ). The Transbaikal lakes were alkaline (pH>9), with high amounts of soda. The Uzbek and Crimean lakes were more pH neutral, frequently with high amounts of sulfates. The presence of anoxygenic phototrophs was registered by infrared epifluorescence microscopy, infrared fluorometry and pigment analyses. In mostly shallow, fully oxic lakes, the anoxygenic phototrophs represented 7-65% of the total prokaryotes, with the maxima observed in Transbaikal soda lakes Gorbunka (32%), Khilganta (65%), Zanday (58%) and Zun-Kholvo (46%). Some of the lakes contained over 1 μg bacteriochlorophyll L(-1) . In contrast, only small amounts of anoxygenic phototrophs were present in highly mineralized lakes (>100 g total salts L(-1) ); Borzinskoe, Tsagan-Nur (Transbaikal), Staroe (Crimea) and in the residual part of the south-west Aral Sea (Uzbekistan). The oxic environment and the specific diurnal changes of bacteriochlorophyll concentration observed suggest that the phototrophic community was mostly composed of aerobic anoxygenic phototrophs. The high abundances and bacteriochlorophyll concentrations point to an important role of aerobic anoxygenic phototrophs in the habitats studied., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Background: Tissue accumulation of advanced glycation end-products (AGE) is thought to be a contributing factor to the progression of cardiovascular disease (CVD). Skin autofluorescence, a non-invasive measure of AGE accumulation using autofluorescence of the skin under ultraviolet light, has shown associations with CVD in haemodialysis patients. The present study aimed to evaluate relationships of skin autofluorescence to renal function as well as CVD in pre-dialysis patients with chronic kidney disease (CKD)., Methods: Subjects in this cross-sectional analysis comprised 304 pre-dialysis CKD patients [median age, 62.0 years; median estimated glomerular filtration rate (eGFR), 54.3 mL/min/1.73 m(2); diabetes, n = 81 (26.6%)]. AGE accumulation in skin was assessed by skin autofluorescence using an autofluorescence reader. Relationships between skin autofluorescence, eGFR, CVD history and other parameters were evaluated., Results: Skin autofluorescence correlated negatively with eGFR (r = -0.42, P < 0.01) and increased as CKD stage advanced. Multiple regression analysis revealed significant correlations of skin autofluorescence with age, presence of diabetes, eGFR and CVD history in CKD patients (R(2) = 30%). Age, male gender, smoking history, skin autofluorescence and eGFR were significantly correlated with CVD history, and multiple logistic regression analysis identified age [odds ratio (OR), 1.09; 95% confidence interval (CI), 1.03-1.15; P < 0.01], history of smoking (OR, 6.50; 95%CI, 1.94-21.83; P < 0.01) and skin autofluorescence (OR, 3.74; 95%CI, 1.54-9.24; P < 0.01) as independent factors., Conclusions: Tissue AGE accumulation measured as skin autofluorescence increased as GFR decreased and was related to CVD history in CKD patients. Non-invasive autofluorescence readers may provide potential markers for clinical risk assessment in pre-dialysis CKD patients.