Revealing oxidative pentose metabolism in new Pseudomonas putida isolates.

The Pseudomonas putida group in the Gammaproteobacteria has been intensively studied for bioremediation and plant growth promotion. Members of this group have recently emerged as promising hosts to convert intermediates derived from plant biomass to biofuels and biochemicals. However, most strains of P. putida cannot metabolize pentose sugars derived from hemicellulose. Here, we describe three isolates that provide a broader view of the pentose sugar catabolism in the P. putida group. One of these isolates clusters with the well-characterized P. alloputida KT2440 (Strain BP6); the second isolate clustered with plant growth-promoting strain P. putida W619 (Strain M2), while the third isolate represents a new species in the group (Strain BP8). Each of these isolates possessed homologous genes for oxidative xylose catabolism (xylDXA) and a potential xylonate transporter. Strain M2 grew on arabinose and had genes for oxidative arabinose catabolism (araDXA). A CRISPR interference (CRISPRi) system was developed for strain M2 and identified conditionally essential genes for xylose growth. A glucose dehydrogenase was found to be responsible for initial oxidation of xylose and arabinose in strain M2. These isolates have illuminated inherent diversity in pentose catabolism in the P. putida group and may provide alternative hosts for biomass conversion.

Complete Genome Sequences of Five Isolated Pseudomonas Strains that Catabolize Pentose Sugars and Aromatic Compounds Obtained from Lignocellulosic Biomass.

We report on complete genome sequences of five Pseudomonas soil isolates that are capable of metabolizing pentose sugars and aromatic monomers. These complete genome sequence data provide insight into possible alternative hosts for the production of biofuels and bio-based chemicals from lignocellulosic feedstock.

Efficient Source Camera Identification with Diversity-Enhanced Patch Selection and Deep Residual Prediction.

Source camera identification has long been a hot topic in the field of image forensics. Besides conventional feature engineering algorithms developed based on studying the traces left upon shooting, several deep-learning-based methods have also emerged recently. However, identification performance is susceptible to image content and is far from satisfactory for small image patches in real demanding applications. In this paper, an efficient patch-level source camera identification method is proposed based on a convolutional neural network. First, in order to obtain improved robustness with reduced training cost, representative patches are selected according to multiple criteria for enhanced diversity in training data. Second, a fine-grained multiscale deep residual prediction module is proposed to reduce the impact of scene content. Finally, a modified VGG network is proposed for source camera identification at brand, model, and instance levels. A more critical patch-level evaluation protocol is also proposed for fair performance comparison. Abundant experimental results show that the proposed method achieves better results as compared with the state-of-the-art algorithms.

Metabolic Engineering of Cupriavidus necator H16 for Sustainable Biofuels from CO2.

Decelerating global warming is one of the predominant challenges of our time and will require conversion of CO2 to usable products and commodity chemicals. Of particular interest is the production of fuels, because the transportation sector is a major source of CO2 emissions. Here, we review recent technological advances in metabolic engineering of the hydrogen-oxidizing bacterium Cupriavidus necator H16, a chemolithotroph that naturally consumes CO2 to generate biomass. We discuss recent successes in biofuel production using this organism, and the implementation of electrolysis/artificial photosynthesis approaches that enable growth of C. necator using renewable electricity and CO2. Last, we discuss prospects of improving the nonoptimal growth of C. necator in ambient concentrations of CO2.

Response of Pseudomonas putida to Complex, Aromatic-Rich Fractions from Biomass.

There is strong interest in the valorization of lignin to produce valuable products; however, its structural complexity has been a conversion bottleneck. Chemical pretreatment liberates lignin-derived soluble fractions that may be upgraded by bioconversion. Cholinium ionic liquid pretreatment of sorghum produced soluble, aromatic-rich fractions that were converted by Pseudomonas putida (P. putida), a promising host for aromatic bioconversion. Growth studies and mutational analysis demonstrated that P. putida growth on these fractions was dependent on aromatic monomers but unknown factors also contributed. Proteomic and metabolomic analyses indicated that these unknown factors were amino acids and residual ionic liquid; the oligomeric aromatic fraction derived from lignin was not converted. A cholinium catabolic pathway was identified, and the deletion of the pathway stopped the ability of P. putida to grow on cholinium ionic liquid. This work demonstrates that aromatic-rich fractions obtained through pretreatment contain multiple substrates; conversion strategies should account for this complexity.

Engineering a nicotinamide mononucleotide redox cofactor system for biocatalysis.

Biological production of chemicals often requires the use of cellular cofactors, such as nicotinamide adenine dinucleotide phosphate (NADP+). These cofactors are expensive to use in vitro and difficult to control in vivo. We demonstrate the development of a noncanonical redox cofactor system based on nicotinamide mononucleotide (NMN+). The key enzyme in the system is a computationally designed glucose dehydrogenase with a 107-fold cofactor specificity switch toward NMN+ over NADP+ based on apparent enzymatic activity. We demonstrate that this system can be used to support diverse redox chemistries in vitro with high total turnover number (~39,000), to channel reducing power in Escherichia coli whole cells specifically from glucose to a pharmaceutical intermediate, levodione, and to sustain the high metabolic flux required for the central carbon metabolism to support growth. Overall, this work demonstrates efficient use of a noncanonical cofactor in biocatalysis and metabolic pathway design.

Glucuronide and Sulfate Conjugates of Bisphenol A: Chemical Synthesis and Correlation Between Their Urinary Levels and Plasma Bisphenol A Content in Voluntary Human Donors.

Bisphenol A (BPA) glucuronide and sulfate conjugates are major products of Phase II metabolism of BPA in humans. In the past, their determination in body fluids usually involves tedious enzymatic hydrolysis and multiresidual analysis. The recent availability of authentic standards of these conjugates enables our better understand of the human metabolism of BPA and the distribution of their metabolites in body fluids. In this work, we report the chemical synthesis and purification of BPA mono- and di-glucuronide and BPA mono- and di-sulfate. Their levels, as well as that of BPA, in 140 paired human plasma and urine samples collected randomly from voluntary donors in Hong Kong SAR, China, were determined by solid-phase extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS). BPA was found in more than 135 human plasma and urine samples. Its Phase II metabolites, ranging from N.D. to 36.7 µg g-1-creatinine, also were detected in 139 of the 140 urine samples. Good correlation (r = 0.911) between molar concentration of BPA in the plasma and that of "total urinary BPA" (i.e., ln [(BPA + ∑ BPA phase II conjugate)molar concentration]) was observed. Direct quantification of Phase II metabolites of BPA in human urine can be a useful assessment tool for population exposure to this potent endocrine disrupting chemical.

Glucuronide and sulfate conjugates of tetrabromobisphenol A (TBBPA): Chemical synthesis and correlation between their urinary levels and plasma TBBPA content in voluntary human donors.

3,3',5,5'-Tetrabromobisphenol-A (TBBPA) is an important brominated flame retardant in epoxy, vinyl esters and polycarbonate resins. Previous studies have already shown the occurrence of its Phase II metabolites, TBBPA-glucuronide and sulfate conjugates, in human urine, after oral administration of TBBPA. The main objective of this work is to examine correlations among level of TBBPA in human blood and those of its Phase II metabolites in human urine. Four water-soluble TBBPA conjugates were synthesized, purified and characterized. An analytical protocol using solid-phase extraction and liquid chromatography-electrospray tandem mass spectrometry (SPE-LC-MS/MS) quantification was developed for the simultaneous analysis of these glucuronide and sulfate conjugates in human urine samples. TBBPA and its Phase II metabolites in paired human plasma and urine samples collected randomly from 140 voluntary donors in Hong Kong SAR, China, were determined. One or more TBBPA conjugates were detected in all of the urine samples, with concentration ranging from 0.19 to 127.24µgg-1-creatinine. TBBPA was also quantified in >85% of the plasma and urine samples. Strong correlations were observed between TBBPA content in plasma and the total amount of TBBPA-related compounds in urine.

Measurement of serum aldosterone in picomolar level by LC-MS/MS using charge-tagged technique.

Aldosterone is a mineralocorticoid steroid hormone, the measurement of which in the clinical laboratory is principally performed for the investigation of primary hyperaldosteronism. Primary hyperaldosteronism is a specifically treatable and potentially curable form of hypertension, which typically presents as drug-resistant hypertension and, in up to 37% of cases, hypokalemia. Accurate measurement of aldosterone concentration is essential for correct diagnosis. The serum concentrations of aldosterone are in the picomolar range and therefore sensitive aldosterone assays are required. With the advancement in instrumentation of LC-MS/MS, the picomolar range of aldosterone can be easily measured by the newer models, but for those with a less sensitive instrument, special technique for sample preparation to enhance assay sensitivity is required. This work described the use of charge-tagging for the picomolar measurement of serum aldosterone in a less sensitive LC-MS/MS instrument. The assay was linear up to 3000 pmol/L with lower limit of quantitation at 80 pmol/L. The mean relative recovery was 96.5% with a range of 89.3-101.6% for aqueous calibrators and the mean relative recovery was 94.8% with a range of 87.5-101.4% for serum calibrators. Intra-assay CVs range from 8.2% to 11.3%, and inter-assay CVs ranged from 8.5% to 13.5% at concentration range from 229 to 1720 pmol/L. The LC-MS/MS method compared well (y = 1.04x + 8.97) with the in-use radioimmunoassay method. There was no significant difference found (p = 0.7135) between results determined by LC-MS/MS and radioimmunoassay method.

Azacytidine sensitizes acute myeloid leukemia cells to arsenic trioxide by up-regulating the arsenic transporter aquaglyceroporin 9.

BACKGROUND: The therapeutic efficacy of arsenic trioxide (As2O3) in acute myeloid leukemia (AML) is modest, which is partly related to its limited intracellular uptake into the leukemic cells. As2O3 enters cells via the transmembrane protein aquaglyceroporin 9 (AQP9). Azacytidine, a demethylating agent that is approved for the treatment of AML, has been shown to have synergistic effect with As2O3. We tested the hypothesis that azacytidine might up-regulate AQP9 and enhances As2O3-mediated cytotoxicity in AML. METHODS: Arsenic-induced cytotoxicity, the expression of AQP9, and the intracellular uptake of As2O3 were determined in AML cell lines and primary AML cells with or without azacytidine pre-treatment. The mechanism of AQP9 up-regulation was then investigated by examining the expression of transcription factors for AQP9 gene and the methylation status of their gene promoters. RESULTS: As2O3-induced cytotoxicity in AML cell lines was significantly enhanced after azacytidine pre-treatment as a result of AQP9 up-regulation, leading to increased arsenic uptake and hence intracellular concentration. Blocking AQP9-mediated As2O3 uptake with mercury chloride abrogated the sensitization effect of azacytidine. AQP9 promoter does not contain CpG islands. Instead, azacytidine pre-treatment led to increased expression of HNF1A, a transcription activator of AQP9, through demethylation of HNF1A promoter. HNF1 knockdown abrogated azacytidine-induced AQP9 up-regulation and almost completely blocked intracellular As2O3 entry, confirming that azacytidine enhanced As2O3-mediated cell death via up-regulation of HNF1A and hence increased AQP9 and As2O3 intracellular concentration. Azacytidine sensitization to As2O3 treatment was re-capitulated also in primary AML samples. Finally, azacytidine did not enhance arsenic toxicity in a liver cell line, where HNF1A was largely unmethylated. CONCLUSIONS: Azacytidine sensitizes AML cells to As2O3 treatment, and our results provide proof-of-principle evidence that pharmacological up-regulation of AQP9 potentially expands the therapeutic spectrum of As2O3. Further clinical trial should evaluate the efficacy of azacytidine in combination with As2O3 in the treatment of AML.

Urinary bromophenol glucuronide and sulfate conjugates: Potential human exposure molecular markers for polybrominated diphenyl ethers.

One possible source of urinary bromophenol (BP) glucuronide and sulfate conjugates in mammalian animal models and humans is polybromodiphenyl ethers (PBDEs), a group of additive flame-retardants found ubiquitously in the environment. In order to study the correlation between levels of PBDEs in human blood plasma and those of the corresponding BP-conjugates in human urine, concentrations of 17 BDE congeners, 22 OH-BDE and 13 MeO-BDE metabolites, and 3 BPs in plasma collected from 100 voluntary donors in Hong Kong were measured by gas chromatograph tandem mass spectrometry (GC-MS). Geometric mean concentration of ΣPBDEs, ΣOH-BDEs, ΣMeO-BDEs and ΣBPs in human plasma were 4.45 ng g(-1) lw, 1.88 ng g(-1) lw, 0.42 ng g(-1) lw and 1.59 ng g(-1) lw respectively. Concentrations of glucuronide and sulfate conjugates of 2,4-dibromophenol (2,4-DBP) and 2,4,6-tribromophenol (2,4,6-TBP) in paired samples of urine were determined by liquid chromatography tandem triple quadrupole mass spectrometry (LC-MS/MS). BP-conjugates were found in all of the parallel urine samples, in the range of 0.08-106.49 µg g(-1)-creatinine. Correlations among plasma concentrations of ΣPBDEs/ΣOH-BDEs/ΣMeO-BDEs/ΣBPs and BP-conjugates in urine were evaluated by multivariate regression and Pearson product correlation analyses. These urinary BP-conjugates were positively correlated with ΣPBDEs in blood plasma, but were either not or negatively correlated with other organobromine compounds in blood plasma. Stronger correlations (Pearson's r as great as 0.881) were observed between concentrations of BDE congeners having the same number and pattern of bromine substitution on their phenyl rings in blood plasma and their corresponding BP-conjugates in urine.

LC-MS/MS in the routine clinical laboratory: has its time come?

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been increasingly used in routine clinical laboratories during the last two decades. The high specificity, sensitivity, and multi-analyte potential make it an ideal alternative to immunoassays or conventional high-performance liquid chromatography (HPLC). It also provides higher throughput than gas chromatography-mass spectrometry (GC-MS). LC-MS/MS also offers higher flexibility than immunoassays because LC-MS/MS assays are typically developed in-house. In addition, abundant information can be obtained from a single LC-MS/MS run which can produce a large amount of quantitative or qualitative data. In this review, typical LC-MS/MS clinical applications are presented, personal experiences are shared, and strengths and weakness are discussed. It is foreseeable that LC-MS/MS will become a key instrument in routine clinical laboratories.

Determination of plasma cholesterol sulfate by LC-APCI-MS/MS in the context of pediatric autism.

Cholesterol sulfate (CS) has various biological functions. Previously, plasma CS was measured primarily as a means to diagnose X-linked ichthyosis; however, a recent hypothesis suggests that CS deficiency might be related to autism. As such, an assay capable of measuring both very high (in the case of X-linked ichthyosis) and very low (in the case of autism) plasma CS levels is required. Here we describe a novel LC-APCI-MS/MS method for the determination of CS in human plasma, and we propose normal CS ranges for children, based on studies of a local population of normal Chinese children between the ages of 2 and 10. In addition, we have used this method to measure plasma CS in autistic children. CS was isolated by solid-phase extraction, and quantified by isotope-dilution LC-APCI-MS/MS in negative ion mode monitoring 465.3>97.1 m/z (CS) and 472.3>97.1 m/z (CS-d7). Mean recovery of the assay ranged from 88.1 to 112.7%; within- and between-run imprecisions have CVs less than 7.2 and 8.1%, respectively. The assay was linear up to at least 100 µmol L(-1). The reference interval of plasma CS in males (range: 1.16-4.23 µmol L(-1)) was found to be higher than in females (range: 0.86-3.20 µmol L(-1)). Comparison of normal and autistic children showed no statistically significant difference in the plasma CS level. In conclusion, a robust LC-APCI-MS/MS method for plasma CS was developed, and a pediatric reference interval was derived from applying the method to normal and autistic children.

Combination of arsenic trioxide and chemotherapy in small cell lung cancer.

INTRODUCTION: Small cell lung cancer (SCLC) carries high mortality despite standard chemotherapy. Arsenic trioxide (ATO) has demonstrated clinical efficacy in leukemia and in vitro activity in various solid tumors. This study was conducted to determine the in vitro and in vivo combination effects of ATO and chemotherapy in SCLC. MATERIALS AND METHODS: The in vitro model consisted of 5 SCLC cell lines (H187, H526, H69, H841 and DMS79) and the anti-proliferative effects of ATO, cisplatin, etoposide or combinations thereof were measured. Synergism was determined by calculation of the combination index (CI) according to Chou and Talalay. Assays for apoptosis, intracellular glutathione (GSH) content, and mitochondrial membrane depolarization (MMD) were performed. Arsenic content was measured by inductively coupled plasma-mass spectrometry. Expression level of MRP1, MRP2 and pH2AX was detected by Western blot while cellular pH2AX level was monitored by immunofluorescent staining. An in vivo xenograft model in nude mice was established with a H841 cell line to test the effects of drug combinations. RESULTS: All 5 SCLC cell lines were sensitive to ATO, with IC(50) values (48 h) 1.6-8 µM. Synergistic or additive effects were obtained by combining cisplatin with ATO in all 5 cell lines. Combination of etoposide with ATO resulted in antagonistic or barely additive effects. Apoptotic assays and pH2AX immunofluorescent staining corroborated the synergistic combination of ATO and cisplatin. In addition, the ATO/cisplatin combination enhanced MMD, depleted GSH, downregulated MRP2 and elevated intracellular ATO content compared with either ATO or cisplatin alone. In vivo combination of ATO and cisplatin also demonstrated synergism in the H841 xenograft model. CONCLUSIONS: There was clinically relevant in vitro activity of ATO in a panel of 5 SCLC cell lines. Significant synergism was demonstrated with the ATO/cisplatin combination, while antagonism was noted with the ATO/etoposide combination in both in vitro and in vivo models.

Synthesis and characterization of bromophenol glucuronide and sulfate conjugates for their direct LC-MS/MS quantification in human urine as potential exposure markers for polybrominated diphenyl ethers.

Bromophenol glucuronide and sulfate conjugates have been reported to be products of mammalian metabolism of polybrominated diphenyl ethers (PBDEs), a group of additive flame-retardants found ubiquitously in the environment. In order to explore their occurrence in human urine, four water-soluble bromophenol conjugates, namely, 2,4-dibromophenyl glucuronide, 2,4,6-tribromophenyl glucuronide, 2,4-dibromophenyl sulfate, and 2,4,6-tribromophenyl sulfate, were synthesized, purified, and characterized. An analytical protocol using solid-phase extraction and ion-paired liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) quantification has been developed for the direct and simultaneous determination of these glucuronide and sulfate conjugates in human urine samples. The limit of detections for all analytes were below 13 pg mL(-1), with 73-101% analyte recovery and 7.2-8.6% repeatability. The method was applied to analyze 20 human urine samples collected randomly from voluntary donors in Hong Kong SAR, China. All the samples were found to contain one or more of the bromophenol conjugates, with concentration ranging from 0.13-2.45 µg g(-1) creatinine. To the best of our knowledge, this is the first analytical protocol for the direct and simultaneous monitoring of these potential phase II metabolites of PBDEs in human urine. Our results have also suggested the potential of these bromophenol conjugates in human urine to be convenient molecular markers for the quantification of population exposure to PBDEs.

Association of lower habitual physical activity level with mitochondrial and endothelial dysfunction in patients with stable coronary artery disease.

BACKGROUND: Exercise training improves endothelial function in patients with coronary artery disease (CAD) through unclear mechanisms. We hypothesized that mitochondrial dysfunction related to a lower habitual physical activity level (PAL) is associated with endothelial dysfunction. METHODS AND RESULTS: We assessed habitual PAL by a validated International Physical Activity Questionnaire, brachial flow-mediated dilation (FMD) and serum lactate, pyruvate, fasting glucose and lipid profiles in 105 CAD patients (age 68±10; 87% men). As defined by the lactate/pyruvate ratio (LP ratio) ≥75(th) percentile of the age-and sex-matched controls (ie, ≥18), mitochondrial dysfunction was observed in 33/105 (31%) patients. With decreasing PAL tertiles, there were significant linear trends of lower FMD (P=0.004) and higher LP ratio (P=0.009). Multivariate logistic regression found that the lowest compared with the highest PAL tertile (adjusted odds ratio=3.78, P=0.02) had more patients with high LP ratio. After adjustment for cardiovascular risk factors and medications, the lowest compared to the highest PAL tertile had significantly lower FMD (absolute decrease 1.25%, P=0.03); and high LP ratio was associated with impaired FMD (absolute reduction 1.09%, P=0.03). CONCLUSIONS: In CAD patients, a lower level of habitual PAL is associated with impaired FMD and increased prevalence of mitochondrial dysfunction as defined by high LP ratio. Moreover, high LP ratio predicts a lower FMD, suggesting that the occurrence of mitochondrial dysfunction with lower habitual PAL is associated with endothelial dysfunction in CAD patients.

Quantification of acylglycines in human urine by HPLC electrospray ionization-tandem mass spectrometry and the establishment of pediatric reference interval in local Chinese.

Urinary organic acids, plasma amino acids and acylcarnitine profile analyses are the main tools used to diagnose inborn errors of metabolisms (IEMs). However, without metabolic decompensation, these parameters are often not helpful. On the other hand, in cases of IEM, acylglycines are consistently raised even when patients appear to be in remission. This study aims to set-up a simple liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the determination of urine acylglycines, complementary to organic acid and acylcarnitine profiles, for the diagnosis of IEM. In addition, local reference intervals for various acylglycines are established by using this method. Acylglycines were isolated by solid-phase extraction, derivatized with n-butanol, separated by HPLC, and detected by ESI-MS/MS. Acylglycines were quantified with deuterated internal standards. Mean recoveries of acylglycines ranged from 90.2 to 109.3%. Within- and between-run imprecisions for all acylglycines have CVs less than 10%. Linear regression coefficients were greater than 0.99. Reference intervals were established according to CLSI guidelines by analyzing 204 samples from apparently healthy individuals less than 18 years of age. The distributions of AG in the "normal" urine were skewed towards the right. After log transformation, all the results were normally distributed. Partitioning into age group reference intervals was not indicated, according to the Harris and Boyd approach. In this context, a single reference interval for each acylglycine could be used. This method of urine acylglycines analysis is a powerful diagnostic tool, complementary to urine organic acids and plasma acylcarnitine profiling, for detecting certain inborn errors of metabolism.

Effect of exercise training on vascular endothelial function in patients with stable coronary artery disease: a randomized controlled trial.

BACKGROUND: We aim to investigate the effect of exercise training on endothelial function and exercise capacity in patients with coronary artery disease. METHODS AND RESULTS: A randomized, controlled trial was conducted to determine the effects of an 8-week exercise training programme (n = 32) vs. controls (n = 32) on brachial flow-mediated dilation (FMD) in patients with stable CAD. After 8 weeks, patients received exercise training had significant improvements in FMD (1.84%, p = 0.002) and exercise capacity (2.04 metabolic equivalents, p < 0.001) compared with controls. The change in FMD correlated inversely with baseline FMD (r = -0.41, p = 0.001) and positively with the increase in exercise capacity (r = 0.35, p = 0.005). After adjusting for confounders, every 1 metabolic equivalent increase in exercise capacity was associated with 0.55% increase in FMD. Furthermore, patients received exercise training had significantly increased high-density lipoprotein cholesterol and decreased diastolic blood pressure and resting heart rate compared with controls. However, exercise training did not alter high-sensitivity C-reactive protein, oxidative stress measured as superoxide dismutase and 8-isoprostane, and CD34/KDR + endothelial progenitor cell count. Subgroup analysis showed that FMD was significantly improved only in CAD patients with baseline low exercise capacity (