The distal convoluted tubule (DCT) is an important nephron site for parathyroid hormone (PTH) and calcitonin regulation of urinary divalent cation excretion. These hormones exert their effects on the DCT in substantial part through activation of adenylyl cyclase (AC); however, it is unknown which AC isoforms are involved.To examine this, two mouse DCT cell lines were studied: 209 and D1 cells. AC isoform mRNA expression was analyzed by real-time PCR. Cyclic AMP was measured using enzyme immunoassay.Calcitonin, but not PTH, stimulated cAMP accumulation in 209 cells, while PTH, but not calcitonin, increased cAMP content in D1 cells. Both cell types expressed AC3, AC4, AC6, AC7, and AC9 mRNA; in both cell types, AC6 mRNA was most abundant, followed by AC9, then AC3 and AC7, with relatively very small amounts of AC4 mRNA. Microdissected mouse DCT had a similar pattern of AC isoform mRNA expression although AC5 mRNA was detected. Individual siRNA knockdown of AC6 and AC9 reduced calcitonin-stimulated cAMP accumulation in 209 cells and PTH-induced cAMP levels in D1 cells. Knockdown of AC3 had no effect on hormonal augmentation of cAMP in either cell line. Surprisingly, knockdown of AC7 increased calcitonin-induced cAMP accumulation in 209 cells as well as PTH-stimulated cAMP content in D1 cells.Taken together, these findings indicate that AC6 and AC9 mediate calcitonin- and PTH-stimulated cAMP accumulation in DCT cells, while activation of AC7 may paradoxically reduce the stimulatory effects of PTH and calcitonin on cultured DCT cAMP levels. Althougderstood . DCT traderstood . While tsorption , 2. Somesorption  and ostesorption , while Psorption . ConsequAll experiments were carried out in accordance with and after approval by the University of Utah Health Sciences Center Institutional Animal Care and Use Committee. All mice were fed standard chow and water ad lib. No experimental procedures were performed on live animals. At the time of sacrifice for tissue harvest, mice were euthanized with enflurane and when breathing was stopped for 1 min, kidneys were harvested.+ and Cl− uptake, thiazide-stimulated Ca2+ uptake, and absence of an effect of bumetanide (inhibits Na+/K+/2Cl− transporter) [2 incubator at 37 °C.Two mouse DCT cell lines, 209 and D1, were provided by Dr. Peter Friedman at the University of Pittsburgh. Both cell lines were initially derived from primary cultures of DCT cells that were simian virus transformed and cloned by limiting dilution, termed 209 cells . The DCTsporter) . The D1 sporter) . Both ceBoth cell lines were grown to 50% confluence. Cells were treated for 24 h with 100 μl Opti-MEM Reduced Serum Medium  containing 1.5 μl Lipofectamine® RNAiMAX Transfection Reagent (Life Technologies) and 10 pmoles scrambled or AC isoform siRNA. Media was then removed and cells incubated with DMEM:F12 containing 1% FBS for 24 h  or for 48 h . The siRNA  was: AC3 - SR422209A, AC6 - SR422280A-C, AC7 - SR422059A-C, AC9 - SR422722A-C, and scrambled controls.Cells used for dose-response studies or in siRNA studies were treated with Hanks Balanced Salt Solution + HEPES containing calcitonin or PTH  for 15 min at 37 °C. Cells were then incubated with 70% ethanol at -20 °C for 3 h, the cells scraped, the mixture dried, the pellet re-suspended in water, and total cell cAMP determined by enzyme immunoassay . Total cell protein was determined by the Bradford assay .RNA was measured from both cell lines at baseline and after the relevant siRNA treatment. In addition, RNA was measured from DCT that were microdissected from C57BL6 mice, aged 2–3 months. The RNA was isolated using the RNeasy Mini Kit and reverse transcribed with Omniscript RT Kit . In addition, AC isoform and GAPDH mRNA levels were determined by real-time PCR  using the Taqman Gene Expression Assay for AC1 , AC2 (Mm00467874_m1), AC3 (Mm00460371_m1), AC4 (Mm01323891_m1), AC5 (Mm00674122_m1), AC6 (Mm00475773_g1), AC7 (Mm00545780_m1), AC8 (Mm00507722_m1), AC9 (Mm00507743_m1) and GAPDH (Mm99999915_g1). The same amount of cDNA is used for each amplification and GAPDH values from each run are all within 0.5 cycles of one another.The presence of AC isoform mRNA was determined in 209 cells, D1 cells, and mouse brain (as a positive control). For mouse brain, frozen mouse tissue stored in the laboratory from past experiments was used; no mice were sacrificed for the purposes of these studies. Specific primers utilized and expected product sizes are shown in Table P < 0.05 was taken as significant.Data are presented as mean ± standard error. Changes in cAMP responses after siRNA treatment were analyzed by the unpaired Student’s t-test. +/Cl− cotransporter expression and regulation, plasma membrane Ca2+ ATPase and Na+/Ca2+ exchanger-1 expression, as well as lack of thick ascending limb transporter activity [The 209 and D1 cell lines are well characterized cell lines with respect to their retaining the in vivo DCT cell characteristics of Naactivity , 8. Howeactivity . ConsequThe presence of AC isoform mRNA was initially assessed using non-quantitative PCR Fig. . 209 celTo compare the DCT cell lines with intact DCT AC mRNA expression, DCT were microdissected from mice and AC mRNA abundance determined Table . SimilarN = 4 each data point. Treatment with siRNA only reduced the targeted siRNA species in 209 cells, i.e., AC3, AC6, AC7 and AC9 mRNA levels were not reduced by siRNA that did not specifically target them (data not shown). In the absence of calcitonin, 209 cells treated with vehicle, scrambled siRNA, or AC isoform-specific siRNA had no detectable cAMP. We next examined the effects of siRNA on calcitonin-stimulated cAMP. 10 nM calcitonin was used for these studies since this concentration of calcitonin elicited ~50–60% of maximal cAMP accumulation in the dose-response studies. Treatment with AC6 and AC9 siRNA reduced calcitonin-stimulated cAMP accumulation by roughly comparable degrees (45–60%) . In the absence of PTH, D1 cells treated with vehicle, scrambled siRNA, or AC isoform-specific siRNA had barely detectable cAMP levels . AC6 and AC9 siRNA reduced PTH-stimulated cAMP accumulation; AC6 siRNA reduced cAMP levels by 60%, while AC9 siRNA elicited a 30% decrease , indicating that even incomplete targeting of the AC isoforms achieves a clear biologic effect. These observations are important because they provide insights into signaling systems that modulate the effects of calcitonin and PTH on DCT cells - each AC isoform is uniquely regulated by G proteins, divalent cations, small molecules, post translational modification, and subcellular localization . Adenyly and Gαi , 20. By  and Gαq . Of note and Gαq ; the sub and Gαq . In contd Gαq [i , 23. ThuWe did not observe an effect of siRNA knockdown of AC3 on PTH- or calcitonin-stimulated cAMP. This could be due to incomplete targeting of AC3 in the DCT cells. However, a recent study by Blanchard et al.  reportedThe present study did not specifically explore the intracellular signaling pathways that are involved in PTH and/or calcitonin regulation of DCT AC isoform activity; such studies are important but were beyond the scope of these initial observations. Another key finding that needs further clarification is how AC7 knockdown increases hormonal-stimulated cAMP levels in DCT cells. We could not find examples in the literature of an interplay between AC isoforms wherein activation of one isoform reduced the increase in cAMP content catalyzed by other isoforms. As noted above, AC-stimulated cAMP accumulation occurs in discrete cellular microdomains, so it is conceivable that activation of a given AC isoform could modulate the activity of another through locally induced signaling molecules. While the nature of such pathways is entirely speculative, one possibility is that AC7 might activate a cAMP-dependent phosphodiesterase that reduces AC6 and/or AC9 induced cAMP accumulation. Clearly, this represents an important area in need of further study. Finally, these studies were conducted in DCT cell lines; conclusions about the in vivo role of DCT cell AC isoforms cannot be directly drawn. Unfortunately, there is no way to specifically target the entire DCT in vivo, hence such studies will await future technological developments.This study identifies the specific AC isoforms mediating the cAMP-stimulating effects of calcitonin and PTH in cultured DCT cells. We report that AC6 and AC9 mediate the stimulatory effects of calcitonin and PTH on cAMP accumulation, while AC7 paradoxically inhibits hormone-stimulated cAMP content accumulation. These studies are important for at least 3 reasons: 1) since many of the cell signaling pathways have been described by which specific AC isoforms are regulated, the current studies, by identifying which of these AC isoforms mediate hormonal action in at least cultured DCT, will facilitate future studies looking at how these hormones exert their biological effects; 2) these studies have uncovered an unusual inhibitory effect of AC7 on hormone-stimulated cAMP accumulation; and 3) these studies will potentially identify a possible role for specific AC isoform agonists and/or antagonists in modulating electrolyte handling by the distal tubule; such agents are in active development.


Escherichia coli. Here, we present a complete and vetted genetic analysis of functional amyloid fiber biogenesis. The Keio collection of single gene deletions was screened on Congo red indicator plates to identify E. coli mutants that had defective amyloid production. We discovered that more than three hundred gene products modulated curli production. These genes were involved in fundamental cellular processes such as regulation, environmental sensing, respiration, metabolism, cell envelope biogenesis, transport, and protein turnover. The alternative sigma factors, σS and σE, had opposing roles in curli production. Mutations that induced the σE or Cpx stress response systems had reduced curli production, while mutant strains with increased σS levels had increased curli production. Mutations in metabolic pathways, including gluconeogenesis and the biosynthesis of lipopolysaccharide (LPS), produced less curli. Regulation of the master biofilm regulator, CsgD, was diverse, and the screen revealed several proteins and small RNAs (sRNA) that regulate csgD messenger RNA (mRNA) levels. Using previously published studies, we found minimal overlap between the genes affecting curli biogenesis and genes known to impact swimming or swarming motility, underlying the distinction between motile and sessile lifestyles. Collectively, the diversity and number of elements required suggest curli production is part of a highly regulated and complex developmental pathway in E. coli.Curli amyloid fibers are the major protein component of the extracellular matrix produced by Enterobacteriaceae during biofilm formation. Curli are required for proper biofilm development and environmental persistence by  Escherichia coli and Salmonella typhimurium  and andrffA,nduction . These rWT cells . Mutantsoduction . The oppoduction . ECA mutre curli . SimilarE levels  and wereor of σE ,76,77, poduction . The σE t levels ,79. E induction . Gl. GlsucA,E. coli (UPEC) [d-glucose which is converted from glucose-6P through the action of Pgm and GalU  . Like urand GalU  and addeent ions B. Indeed mutants .E. coli to persist as a pathogen [oprF mutants in Pseudomonas aeruginosa have impared anaerobic respiration and as a result, have significantly reduced anaerobic biofilm production [sdhA–D) produced altered CR binding phenotypes  [. Similar to an rpoS mutant, an nlpD mutant completely lacks curli, probably due to loss of the major rpoS promoter within nlpD [S [relA spoT double mutant, which is defective in ppGpp synthesis [rpoS, we found that deletion of the sigma factor genes rpoN and rpoZ decreased curli production  ,114,115.hin nlpD . Since D nlpD [S , we testynthesis , was ligoduction , underscqseB, qseC, aaeR, lsrF, ygiU (mqsR), sdiA, and flgM. The qseC strain produced more curli than WT; however, qseB had mostly light pink colonies with a few dark red suppressor colonies    and CsgDRT-qPCR) . Twenty- from WT . There w protein . MutantsD levels . Of 19 mt levels  and all PCR data . There wnagA and dksA, had significantly increased csgD transcript levels  [fabH or overexpression leads to decreased fatty acid chain length and abundance [The CR screen also revealed possible post-translational control of CsgD . For exatability . ConsistA (mRNA) . The Fabbundance ,139,140.ddpD, glvG, and php, which had altered CR binding phenotypes, yet normal csgD transcript and CsgD protein levels  with 100 μg/mL CR or 50 μg/mL CR and 10 μg/mL CBB were sometimes used. For the experiments looking at the effect of divalent cations, salts were added to CR indicator media prior to autoclaving. To remove residual salts in these experiments, strains were grown to saturation in LB overnight, washed and diluted five-fold in YESCA, and spotted onto the appropriate plates. LPS mutants were always surrounded by other strains due to differences in CR binding near the colony edge. Antibiotics were added when appropriate in the following final concentrations: 25 µg/mL kanamycin; 25 µg/mL chloramphenicol; or 100 µg/mL ampicillin.Typically, bacteria were grown for 48 h at 26 °C on YESCA plates . Congo red indicator plates are YESCA media amended with 50 μg/mL CR and 10 μg/mL Coomassie Brilliant Blue (CBB). CFA agar . To solubilize CsgA, samples were briefly treated with formic acid as described [N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) and 10% Methanol. Western blots were developed using the chemiluminiscent Pierce super signal detection system . LPS was extracted as described [600 of 10 cells grown on YESCA plates for 48 h at 26 °C. Samples were normalized to 10 ng of keto-deoxy-d-manno-8-octanoic acid (KDO) per lane using the Thiobarbituric Acid Assay and were separated and silver stained in a 14% Tricine SDS-PAGE gel [Bacteria were scraped off YESCA plates and resuspended in phosphate buffered saline (PBS) (pH 7.4) before normalization of optical density at 600 nm :YESCA. Cells were pelleted via centrifugation: 7000× g for 5 min at room temperature. Cell pellets were immediately frozen and stored at −80 °C. Pellets were thawed at room temperature and resuspended in RNA extraction solution (18 mM ethylenediaminetetraacetic acid (EDTA), 0.025% SDS, 1% β-mercaptoethanol, 95% formamide). Cells were lysed by incubation at 100 °C for 7 min. After centrifugation: 16,000× g for 5 min at room temperature, the supernatant was transferred to a new microcentrifuge tube. The solution was diluted with 4 volumes RNase-free H2O, and then 0.1 volume 3 M sodium acetate pH 5.2 and 2 volumes ethanol. The solution was placed at −80 °C for at least 1 h or overnight. Precipitated nucleic acids were pelleted via centrifugation: 16,000× g for 20 min at 4 °C and washed with ice-cold 75% ethanol. The pellets were resuspended in 1× DNase I buffer  and incubated at 55 °C for 10 min. The solutions were clarified via centrifugation: 16,000× g for 5 min at room temperature and the supernatant was transferred to a new microcentrifuge tube and digested with DNase I (NEB) for 45 min at 37 °C. RNA was precipitated by adding 0.1 volume 8 M LiCl and 3 volumes ethanol and placing at −80 °C overnight. RNA was pelleted via centrifugation: 16,000× g for 15 min at 4 °C and washed with ice-cold 75% ethanol. The RNA pellet was dried and resuspended in RNase free H2O.RNA extraction was performed as described  with modcsgD were normalized to 16s levels.Reverse transcriptase complementary DNA (cDNA) synthesis was performed using Promega Go-Script (A5003)  according to the manufacturer’s instructions with random primers . Real-time quantitative PCR was performed using Invitrogen Power Sybr green master mix  according to the manufacturer’s instructions. Briefly, reverse transcriptase reactions were diluted 1000-fold for reactions using csgD primers and 10,000-fold for reactions using 16s primers. The data were analyzed using the Pfaffl method . The amp600 of 1 and 30 uL were spread onto YESCA plates and allowed to grow at 26 °C. After 24 h, bacteria were resuspended in 1.5 mL of RNAprotect (Qiagen) vortexed and incubated for 5 min. Cells were pelleted at 5000× g for 10 min and treated with lysozyme  (200 μL of freshly prepared 1mg/mL stock in 30 mM Tris-HCl 10 mM EDTA pH 8.0) for 5 min at room temperature. RNA was purified using the RNeasy Mini kit  and included an in column treatment with RNase free DNase . The RNA yield and purity were quantified in an Infinite 200 Pro NanoQuant Tecan reader  and only samples with a 260/280 nm ratio equals or over 2 were further used. Aliquots of 2 ug/mL of mRNA were used to synthesize the cDNA using the Promega ImProm II reverse transcription kit and adding betaine 5 mM solution to the pre-annealing step with the random primers. Three serial dilutions  by duplicate of each sample were analyzed using the ABsolute Blue QPCR SYBR Green Low ROX supermix from Thermo Scientific  (3:5), the absolute quantification plate type and a standard 7500 run mode in an Applied Biosystems 7500 Fast Real-Time PCR System . The primers were designed using Primer 3 software, sequences are indicated in the rpoA gene expression were measured, both worked similarly but 16s was more consistent and abundant, so 16s was used in the experiments described in this work. After confirming that the 16s primers and the target gene primers had similar efficiencies using the cycle threshold values generated from the ABI 7500 Fast System software analysis , all the calculations for the fold gene expression data were done applying the ∆∆Ct standard calculation.The mRNA extraction was based on protocols for the RNeasy Mini Kit (Qiagen) with minor changes. Briefly, bacteria were normalized to an OD600 of 0.3–0.6). Strains were normalized to 0.2 OD600 in YESCA and 2 uL were inoculated into 0.2% Agar YESCA motility plates. Plates were grown for 8 h at 26 °C. The strains tested for motility were also tested for growth rates in YESCA at 26 °C using a Klett meter . No appreciable growth rate differences were measured.Cells were grown overnight in YESCA with appropriate antibiotics. Saturated cultures were diluted 1/100 in fresh YESCA and grown to mid log phase (ODBacteria were grown on YESCA plates for 48 h at 26 °C. Samples were resuspended in PBS and stained with 2% uranyl acetate as previously described . Grids wcsg transcription. General themes arose for two major stress response systems, σS and Cpx. Mutations that that result in elevated σS levels produced more curli, while, those that induced σE or the Cpx system, produced less curli. An intact cell envelope was required to support curli biogenesis, as mutations in LPS and OPG resulted in less extracellular amyloid. Curli amyloid production was tied to key metabolic pathways such as the TCA cycle, the nucleotide synthesis pathways, and the catabolite utilization pathways. Collectively, the number and diversity of mutations that result in altered CR binding demonstrate that amyloid biogenesis is a complex and highly regulated developmental pathway in E. coli.More than 300 gene deletions that altered curli amyloid levels were identified in our CR screen. Several of the mutants focused on in this study are part of regulatory cascades, including stress response systems, which affect


In the present study, by using Florite® PS-10 (119.1 mg) and Vivapur® 105 (105.6 mg) as solid carriers, VST-loaded solidified SuSMEDDS (S-SuSMEDDS) granules were successfully developed, which possessed good flow properties and rapid drug dissolution. By introducing croscarmellose sodium (31 mg) as a superdisintegrant, S-SuSMEDDS tablets were also successfully formulated, which showed fast disintegration and high dissolution efficiency. Preparation of granules and tablets was successfully optimized using D-optimal mixture design and 3-level factorial design, respectively, resulting in percentage prediction errors of <10%. In pharmacokinetic studies in rats, the relative bioavailability of the optimized granules was 107% and 222% of values obtained for SuSMEDDS and Diovan® powder, respectively. Therefore, we conclude that novel S-SuSMEDDS formulations offer great potential for developing solid dosage forms of a liquefied formulation such as SuSMEDDS, while improving oral absorption of drugs with poor water solubility.In order to improve the dissolution and oral bioavailability of valsartan (VST), and reduce the required volume for treatment, we previously formulated a supersaturable self-microemulsifying drug delivery system (SuSMEDDS) composed of VST (80 mg), Capmul Use of a self-microemulsifying drug delivery system (SMEDDS) is widely known as one of the most effective approaches to overcome problems associated with low solubility and poor oral absorption of water-insoluble drugs. Previously, we have demonstrated that a supersaturable SMEDDS (SuSMEDDS) greatly contributed to enhanced dissolution and oral absorption of valsartan (VST), a drug with poor solubility in water . HoweverVarious types of solid carriers have been extensively investigated to solidify SMEDDS, including silica-based water-insoluble adsorbents , cellulose-based hydrophilic diluents , and saccharide-based water-soluble diluents  –11. Alth® CL-SF as a disintegrant showed maximum drug release of over 90% within 30 min, whereas tablets without disintegrant resulted in poor drug release of less than 5% after 2 h ·100/ρ(tapped), where ρ(bulk) and ρ(tapped) are the bulk and tapped densities, respectively . Appareng for 10 min to remove the water-insoluble solids. The size of dispersed droplets in the supernatant was determined using a photon correlation spectrometer .Solidified mass  was dispersed in 250 mL of distilled water (DW). The mixture was gently vortexed  and centrifuged  at 16,000 ®), and the filtrate was appropriately diluted with methanol for HPLC analysis. VST concentration was computed from the validated calibration curve of the drug in methanol , and drug contents were expressed as percentages of the theoretical quantity.Solidified mass (100 mg) was dispersed in methanol (10 mL), mixed thoroughly, and sonicated for 30 min with a bath type sonicator  to extract VST. Samples were filtered through a membrane filter , ultraviolet detector , data station , and chromatographic C18 column , maintaining a flow rate of 1.0 mL per minute at 25 °C. The isocratic mobile phase was composed of acetonitrile and distilled water (60:40 [v/v]). The pH was adjusted to 3.0 using 10% phosphoric acid. Finally, 20 μL of each sample was injected into the column, and the absorbance was measured with ultraviolet detection at 247 nm.In vitro dissolution tests were performed using the USP apparatus II (paddle) method with a Vision® Classic 6™ Dissolution Tester and Vision® heater . A pH 1.2 medium was prepared by dissolving 2 g of sodium chloride in 7 mL of hydrochloric acid and diluting with DW to 1000 mL. Each formulation, containing 80 mg of VST, was introduced into the pH 1.2 dissolution medium (500 mL) at 37 ± 0.5°C, and stirred at 100 rpm. Samples (5 mL) were taken at predetermined time points  and filtered through a 0.45-μm polyvinylidene difluoride membrane. After appropriate dilution of the filtrate with methanol, the VST concentration in each sample was assayed using HPLC as described above. For comparison of dissolution profiles, DE was calculated using the trapezoidal rule as follows:where y is the percentage of dissolved product .® PS-10 and Vivapur® 105) were selected and used in the solidification process at different ratios by blending with SuSMEDDS as described above. The final mixture, a free-flowing powder with a non-greasy appearance, was passed through a 500-μm sieve, and the resultant S-SuSMEDDS granules were stored at ambient temperature in an airtight container.Based on the screening results, two solid carriers  was used for developing and evaluating experimental design. The experiment was designed using the two components as independent variables: Florite® PS-10 (X1) and Vivapur® 105 (X2) were set within ranges of 0–100%. CSR (Y1) and D15 (Y2) were evaluated as response variables to determine the optimal S-SuSMEDDS granule formulation, for maximal drug release with minimal quantity.D-optimal mixture design was used to optimize the ratio of Florite® PS-10, Vivapur® 105, and VST-loaded S-SuSMEDDS granules were investigated using a scanning electron microscope : samples were fixed on a metal plate, sputtered for 90 s with platinum , and photographed at an excitation voltage of 5 kV. DSC measurements were performed using a DSC-Q20 : samples (2–5 mg) were placed in an aluminum pan, and measurements were taken over a temperature range of 30–300 °C, at a heat rate of 5 °C/min under nitrogen flow (20 mL/min).Solid-state properties of the drug powder, solid carriers, and optimized S-SuSMEDDS granules were investigated using SEM and DSC. Morphological features of VST powder, Florite® GmbH, Heusenstamm, Germany) for 5 min. The blended mixture was directly compressed into tablets on a single-punch tablet machine  at a compression force of 500–2000 kgf using 12 mm standard circular concave punches, and the resultant S-SuSMEDDS tablets were stored at ambient temperature in an airtight container.S-SuSMEDDS granules were blended with selected superdisintegrants  and lubricant (magnesium stearate) using a cube mixer  and concentration of superdisintegrant  were used as numerical factors, and were set within ranges of 500–2000 kgf and 2–8%, respectively; the type of superdisintegrant (X3) was used as a categorical factor, and included CS, KC, and SSG. Tablet hardness (Y1), Disint% (Y2), DE (Y3), and the total mass of S-SuSMEDDS tablet (Y4) were evaluated as the response variables, to determine the optimal formulation for desirable physiochemical characteristics.3-LFD was used to optimize the conditions for S-SuSMEDDS tablet preparation. The experiment was designed using three components as independent variables: the compression force . The thickness and diameter of ten tablets were measured individually using Vernier calipers . Tablet friability was calculated as the percentage of weight loss  using a friability tester . A hardness tester  was used to determine tablet hardness. Ten tablets from each formulation were tested.*(Wi-Wf)/Wi, where Wi and Wf are the masses (mg) of samples before and after the disintegration test, respectively.Disintegration tests were performed in 900 mL distilled water at 37°C for 30 min using a disintegration tester . After the disintegration test, the remaining sample was taken and dried in an oven at 40°C for 24 h to determine Disint%, calculated by 100® Kit. Test subjects were randomly divided into three groups (n = 5–7): Group 1 received Diovan® , Group 2 received SuSMEDDS, Group 3 received optimized S-SuSMEDDS granules. In all treatments, a dose equivalent to 10 mg/kg VST was accurately weighed and dispensed into hard gelatin capsules (Torpac capsule size 9)  by means of a stand, funnel, and tamper . All capsules were administered directly into the stomach by using a dosing syringe plunger . Blood samples (approximately 0.3 mL) were collected from the retro-orbital plexus into heparinized tubes at predetermined time points  and were centrifuged at 16,000 g for 15 min. Plasma samples were stored at -80°C until analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS).After rats had undergone overnight fasting (12–18 h), VST treatments were administered using a Torpacg for 5 min, 20 μL of the supernatant was carefully transferred to a test tube and was evaporated using nitrogen. The dry residue was reconstituted in 480 μL of DW, and the mixture was vortexed and centrifuged at 16,000 g for 5 min. Finally, 100 μL of the supernatant was transferred to autosampling vials for introduction into the LC-MS/MS system.Whole plasma samples (50 μL) were mixed with 700 μL of methanol and 20 μL of internal standard (IS) solution  and were vortexed for 3 min. After centrifugation at 16,000 Liquid chromatographic separation was performed using an Agilent 1260 autosampler . The temperature of the autosampler was maintained at 7°C, and 5 μL of each reconstituted sample was separated into components using a Waters Atlantis dC18 column  at 35°C. An isocratic mobile phase was used, containing 10 mM ammonium formate (pH 2.7) and methanol , at a flow rate of 0.3 mL per min.The components eluted from the column were delivered into an API 4500 triple quadrupole mass spectrometer  with electrospray ionization in positive ion mode for ion production. The ion spray voltage was set at 5.5 kV, and the source temperature was set at 550°C. Multiple reaction monitoring was performed using nitrogen as the collision gas. Analytes were detected by monitoring the transitions 436.2 (Q1) → 291.0 (Q3) and 439.2 (Q1) → 294.0 (Q3) m/z, with a declustering potential of 28 V and collision energies of 23 V, for VST and IS, respectively. Nebulizer gas (Gas 1) at 40°C and heater gas (Gas 2) temperatures were both set at 70°C. For quantifying VST in the plasma samples, each peak area of VST was divided by that of the internal standard, and the ratio was compared with a calibration curve obtained using VST standard solution in the same manner.max and Tmax were determined directly from the concentration-time data. RBA was calculated by dividing the AUCs of the test samples with those of the VST suspension.Data analysis was performed using the BA Calc 2007 pharmacokinetic analysis program . The AUC from 0 to 24 h was calculated using the linear trapezoidal rule. The Ct-test, with p < 0.05 considered statistically significant. Design-Expert software was used to determine the simultaneously assigned statistical values of all responses.All data are expressed as mean ± standard deviation (SD). Statistical significance was determined using Student’s ® MCM, Tween® 80, Transcutol® P, and Poloxamer 407 was efficiently solidified with Florite® PS-10 and Vivapur® 105, and the resultant granules showed good flow properties and rapid drug dissolution. By introducing CS as a superdisintegrant, S-SuSMEDDS tablets were successfully formulated, resulting in fast disintegration and high dissolution efficiency. In PK studies in rats, the RBA of the optimized granules was 107% and 222% of the values obtained for SuSMEDDS and Diovan® powder, respectively. Therefore, we suggest that the optimized S-SuSMEDDS formulations offer great potential for developing solid dosage forms, with improved oral absorption of poorly water-soluble drugs such as VST.Novel formulations of VST-loaded S-SuSMEDDS granules and tablets were successfully developed using D-optimal mixture design and 3-LFD, respectively, resulting in percentage prediction errors of <10%. SuSMEDDS composed of VST, Capmul


Nitrososphaera, Nitrosotalea, and Nitrosoarchaeum; AOB genes related to Nitrosomonas and Nitrosospira; and NOB genes related to Nitrospira. The overall abundance of AOA, AOB and NOB was examined using quantitative PCR (qPCR) amplification of the amoA and nxrB functional genes and 16S rRNA genes. Gene copy numbers ranged from 3.2 × 104 – 4.9 × 107 archaeal amoA copies ∗ μg DNA-1, 1.5 × 103 – 5.3 × 105 AOB 16S rRNA copies ∗ μg DNA-1, and 1.3 × 106 – 7.7 × 107Nitrospira nxrB copies ∗ μg DNA-1. Overall, Nitrospira nxrB genes were found to be more abundant than AOB 16S rRNA and archaeal amoA genes in most of the sample sites across 2013 and 2014. AOB 16S rRNA and Nitrospira nxrB genes were quantified in sediments with pH as low as 3.2, and AOA amoA genes were quantified in sediments as low as 3.5. Though pH varied across all sites (pH 3.2–8.3), pH was not strongly correlated to the overall community structure or relative abundance of individual OTUs for any gene (based on CCA and Spearman correlations). pH was positivity correlated to the total abundance (qPCR) of AOB 16S rRNA genes, but not for any other genes. Metals were not correlated to the overall nitrifier community composition or abundance, but were correlated to the relative abundances of several individual OTUs. These findings extend our understanding of the distribution of nitrifying microbes in AMD-impacted systems and provide a platform for further research.Extremely acidic and metal-rich acid mine drainage (AMD) waters can have severe toxicological effects on aquatic ecosystems. AMD has been shown to completely halt nitrification, which plays an important role in transferring nitrogen to higher organisms and in mitigating nitrogen pollution. We evaluated the gene abundance and diversity of nitrifying microbes in AMD-impacted sediments: ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and nitrite-oxidizing bacteria (NOB). Samples were collected from the Iron Springs Mining District  during early and late summer in 2013 and 2014. Many of the sites were characterized by low pH (<5) and high metal concentrations. Sequence analyses revealed AOA genes related to  Nitrobacter and Nitrospira lineages. Recent discoveries documented the complete oxidation of ammonia to nitrate (comammox) by Nitrospira  (nxrB gene (encoding the beta subunit of the nitrite oxidoreductase enzyme) , while nitrite oxidation is mediated by nitrite-oxidizing bacteria (NOB) commonly from the trospira . The mictrospira  or funct enzyme)  and nxrB enzyme) . Many ni enzyme) .Nitrification has been documented in virtually every environment across the earth  . NonetheNitrobacter and Nitrospira cultures significantly decreased below pH 6.5  to examine the abundance and diversity of AOA, AOB, and NOB in AMD-impacted sediments in the Iron Springs Mining District near Ophir, CO, United States.Figure 1 and Supplementary Figure The Iron Springs Mining District located in Ophir, CO, United States consists of several abandoned mines. From 1877 to 1960, the Iron Springs Mining District was predominantly mined for metals such as silver, gold and lead, and also to some extent for iron (pigment) and tungsten . AMD froIn Situ Multiparameter meter. Metal analyses (TRW and DM) of the Iron Springs water samples were done at the EPA Region 8 lab  using Inductively-Coupled Plasma Mass Spectrometry (ICP-MS) following EPA method 200.8, and Inductively-Coupled Plasma Optical Emission Spectrometry (ICP-OES) following EPA method 200.7. The analytes measured for TRW and DM included aluminum (Al), antimony (Sb), arsenic (As), barium (Ba), cadmium (Cd), calcium (Ca), copper (Cu), iron (Fe), lead (Pb), magnesium (Mg), manganese (Mn), nickel (Ni), selenium (Se), silver (Ag), sodium (Na), strontium (Sr), thallium (Tl), vanadium (V) and zinc (Zn).Temperature, pH, conductivity, and dissolved oxygen were measured at the sediment surface using a Thermo Scientific Orion 5-Star Multiparameter Meter Kit  and an n = 38) based on gel electrophoresis, DNA quantification, and PCR amplification were selected for further analyses .The PowerMax Soil DNA Isolation Kit  was used to isolate total DNA from subsamples of mechanically homogenized saturated composite sediment (∼10 g) from each sample site. DNA extracts were quantified using the Qubit dsDNA HS Assay Kit with the Qubit 2.0 Fluorometer . High quality samples . AOA ch-amoAR . AOB werch-amoAR . AOB amo/amoA–2R  or amoA–/amoA–2R . NitrospnxrB638R . Three p/nxrB-1R , Nitrobaro-1423r , and NitGPS1269r .amoA  for archaeal amoA; 10 μl of Failsafe Green Premix F , 0.3 μM of each primer, 19.8 ng ∗ μl-1 of BSA, and 1 Unit of AmpliTaq DNA polymerase  for AOB 16S rRNA; and 10 μl of Failsafe Green Premix F , 1 μM of each primer, and 0.5 Units of AmpliTaq DNA polymerase  for Nitrospira nxrB. The amount of DNA used in each reaction ranged from 0.32 to 53.3 ng of DNA for all samples .qPCR conditions and reactions (with modifications pertaining to the current study) were carried out as previously described for archaeal amoA , and foramoA  with theira nxrB  were appamoA and Nitrospira nxrB assays were generated by gene synthesis (Life Technologies Corporation) from Nitrosoarchaeum limnia SFB1 amoA and Nitrospira defluvii nxrB sequences. The standard gene copies for the assays ranged from 13 – 1.2 × 107 for archaeal amoA, 16 – 3.3 × 107 for AOB 16S rRNA, and 15 – 1.5 × 107 for Nitrospira nxrB. Duplicate or triplicate reactions were carried out for all the samples and standards, and average values were calculated. Melt curves were generated after each SYBR assay to check the specificity of amplification. For the standard curves, PCR efficiencies averaged 79–91% and the correlation coefficients (R2) were >0.99. Samples that amplified but fell below the lowest standard  were identified as “below detection limit.” “Non-specific amplification” was identified in the melt curve analysis.Standard curves for the 16S rRNA assay were generated using linearized plasmid DNA from a cloned Iron Springs AOB 16S rRNA gene sequence. Standards for the archaeal Nitrobacter 16S rRNA  to amplify the products from eight different PCR primer sets: Arch349F/Arch806R for archaeal 16S rRNA , V4_515F16S rRNA , nxrB169ira nxrB , and ArcrB  High Fidelity Fast Start Kit and 20x Access Array loading reagent according to Fluidigm protocols. Into each well of a PCR plate, 1 μL of each sample was mixed with 1 μL of Fluidigm Illumina linkers and unique barcodes mix, 0.5 μL of 10X FastStart Reaction Buffer without MgCl2, 0.9 μL of 25 mM MgCl2, 0.25 μL of DMSO, 0.1 μL of 10 mM PCR grade nucleotide mix, 0.05 μL of 5 U ∗ μL-1 FastStart High Fidelity enzyme blend, 0.25 μL of 20X Access Array Loading Reagent, and 0.95 μL of water. In a separate plate, 20X primer solutions were prepared by adding 2 μL of each forward and reverse primer , 5 μL of 20X Access Array Loading Reagent, and 91 μL of water.Samples were diluted to a final concentration of 2 ng ∗ μLOnce the sample mixture was complete, 4 μL was loaded into the sample inlets and 4 μL of the primer solution was loaded into the primer inlets of a primed Fluidigm 48.48 Access Array IFC. The IFC was then placed in a Fluidigm AX controller for microfluidic mixing of each primer and sample combination before being loaded into the Fluidigm Biomark HD PCR machine. Amplicons were generated using the following Access Array cycling program without imaging: 50°C for 2 min, 70°C for 20 min, 95°C for 10 min; 10 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 1 min; 2 cycles of 95°C for 15 s, 80°C for 30 s, 60°C for 30 s, and 72°C for 1 min; 8 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 1 min; 2 cycles of 95°C for 15 s, 80°C for 30 s, 60°C for 30 s, and 72°C for 1 min; 8 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 1 min; and 5 cycles of 95°C for 15 s, 80°C for 30 s, 60°C for 30 s, and 72°C for 1 min.After amplification, 2 μL of Fluidigm Harvest Buffer was added to each sample inlet, and the IFC loaded onto the AX controller to harvest all PCR products from each sample . The PCR products were quantified using Qubit and stored at -20°C. The samples were run on a Fragment Analyzer  to confirm the expected sizes of amplicons. All of the 48 samples  were then pooled together in equal DNA concentrations into one tube. The pooled product was size selected on a 2% E-gel (Life Technologies Corporation), then recovered based on expected fragment size with a Qiagen  gel extraction kit. Cleaned, size-selected products were run on an Agilent Bioanalyzer to confirm the expected profile and determine the average product size.The size-selected pool was qPCR quantitated and loaded onto one MiSeq flowcell using a MiSeq 600-cycle sequencing kit, version 3 for 300 bp paired-end sequencing using a MiSeq FGx system in RUO mode. After sequencing, read data was translated into FASTQ files using the Illumina bcl2fastq 1.8.4 software with an ASCII offset of 33. PhiX DNA reads (used as a spike-in control) were removed by alignment to the PhiX genome. The Roy J. Carver Biotechnology Center used in-house scripts for sorting the reads  and demultiplexing .Pseudomonas. One of these OTUs had 7,000 sequence reads , but all other OTUs had low read counts (2–186 reads per OTU). No Archaeal 16S rRNA, AOA amoA, or Nitrospira nxrB OTUs were present in the blank. One sample (Carib03Sept14) was sequenced twice to evaluate reproducibility. Bacterial 16S rRNA gene community membership and relative abundance were highly correlated (2R > 0.99) between the replicate samples.Sequence reads were evaluated for each gene set in a no-template control sample (nuclease free water). A total of 24 Bacterial 16S rRNA OTUs were found in the blank, all of which were related to amoA were done based on sequencing read one due to the lack of overlap between read one and read two (gene amplicon length: 635 bp). Sequencing reads were clustered into operational taxonomical units (OTUs) at 97% sequence identity. For 16S rRNA genes, the final OTUs were checked for chimeras using the DECIPHER web tool with the short sequences option , and Bacteria were filtered from the total 16S rRNA reads.Relative sequence abundance and diversity analyses were conducted using QIIME   and UPARs option  and putas option  for 16S Nitrosomonadales taxa-specific AOB 16S rRNA, 60 sequences for archaeal 16S rRNA, 60 sequences for Thaumarchaeota taxa-specific archaeal 16S rRNA, 70 sequences for archaeal amoA, and 500 sequences for Nitrospira nxrB (low sequence thresholds were set in order to maximize OTU recovery). Rarefaction was performed at multiple sequence depths between one and the rarified depth (set to the median number of sequences for each gene). Chao1 richness estimates were determined at rarified depth for each gene.The number of observed OTUs for each gene was determined using QIIME. The sequence threshold cutoffs were set to 15,680 sequences for the combined V4-V5 region of bacterial 16S rRNA, 650 sequences for Nitrospira nxrB OTUs were aligned to the curated alignment created by amoA, 480 bp for AOB 16S rRNA, and 445 bp for Nitrospira nxrB. Maximum Likelihood trees were constructed for representative sequences of observed OTUs using FastTree v2.1.5 package  corrected using Bonferroni correction . Spearman correlations between environmental parameters and the relative abundance of individual OTUs were carried out using QIIME (observation_metadata_correlation.py) with p-values corrected using the Bonferroni correction. The environmental parameters from surface sediments included in the statistical analyses were: pH, temperature (°C), conductivity (μS ∗ cm-1) and dissolved oxygen (mg ∗ L-1). Environmental parameters from surface water included in statistical analyses were: TRW (in μg ∗ L-1) including Al, As, Ba, Cd, Ca, Cu, Fe, Pb, Mg, Mn, Ni, Na, Sr, Tl, and Zn; and DM (in μg ∗ L-1) including Al, Ba, Cd, Ca, Cu, Fe, Pb, Mg, Mn, Ni, Na, Sr and Zn.Spearman correlation between environmental parameters and total gene abundances (based on qPCR) was performed using the IBM SPSS Statistics software package version 23  with critical amoA, AOB 16S rRNA, and Nitrospira nxrB genes) and environmental parameters were analyzed using canonical correspondence analysis (CCA) with the Canoco 5 v. 5.04 .Correlations between nitrifier community composition . The measured environmental parameters varied by sampling date and sampling location . The Caribbeau Mine sites had circumneutral pH at all time points (pH 7.3 on average).Sample sites covered four different mines/regions within the greater Iron Springs Mining District: Caribbeau Mine , Iron Bog/Fen , New Dominion Mine , and Howard Fork River (HF04)  were measured for all samples sites and time points . Archaeal amoA genes were most abundant in the Iron Bog/Fen sites (HFIBE and FenIB01) during both 2013 and 2014, ranging from 5.4 × 105 – 4.9 × 107 copies ∗ μg DNA-1. Archaeal amoA gene copy numbers were lowest at the New Dominion Mine FenND03 site in 2013 with 4.8 × 104 copies ∗ μg DNA-1, with a ∼1,000-fold difference when compared to the most abundant sites.Archaeal amoA genes did not amplify in our samples . The ovamoA genes were more abundant than AOB 16S rRNA genes at most sites and time points, which may suggest that AOA are more abundant than AOB since both genes are thought to exist in single copies within each genome .The qPCR results indicated that archaeal h genome . ArchaeaNitrospira nxrB gene copies ranged from 1.3 × 106 – 7.7 × 107 copies ∗ μg DNA-1 across 2013 and 2014 . During 2013, Nitrospira nxrB genes were most abundant at the Howard Fork River sites ranging from 1.6 × 107 – 2.9 × 107 copies ∗ μg DNA-1 and Caribbeau Mine sites ranging from 5.3 × 106 – 2.8 × 107 copies ∗ μg DNA-1. In 2014, Nitrospira nxrB genes were most abundant at the Iron Bog/Fen sites (FenIB01 and HFIBE) ranging from 3.8 × 107 – 7.7 × 107 copies ∗ μg DNA-1 followed by Caribbeau Mine sites (Carib02 and Carib03) ranging from 8.8 × 106 – 6.5 × 107 copies ∗ μg DNA-1. Nitrospira nxrB gene copy numbers were the lowest at New Dominion Mine sites (1.3 × 106 – 3.8 × 107 copies ∗ μg DNA-1) when compared across all sample regions during 2013 and 2014.Nitrobacter genes did not amplify in any of the samples using nxrB-1F/nxrB-1R primers , Bacteroidetes (10%), Chloroflexi (9%), Acidobacteria (8%), and Actinobacteria (7%). Nitrospira accounted for 0.4% and Nitrosomonadaceae accounted for <0.1% of the total bacterial 16S rRNA community. No Nitrobacter sequences were found in the bacterial 16S rRNA gene dataset. A total of 2286 unique bacterial 16S rRNA gene OTUs were identified (at 97% sequence identity), with 181-1291 OTUs observed within each sample  including the Thaumarchaeota (42%), Euryarchaeota (16%), and Parvarchaeota (26%). A total of 66 unique archaeal 16S rRNA gene OTUs were identified, with 1–22 OTUs observed within each sample , Nitrosotenuis (1 OTU), Nitrosotalea (6 OTUs), and soil clones that had 82.7% sequence identity to Nitrososphaeara (6 OTUs) , 8 OTUs were recovered across all samples, with 1–5 OTUs observed within each sample , and ranged from 80 to 94% nucleotide identity to Nitrosopumilus maritimus—the first cultured AOA.For archaeal Nitrosomonadales, with 1–11 OTUs observed within each sample .A total of 20 unique AOB 16S rRNA gene OTUs were recovered belonging to the order Nitrospira nxrB sequences had the highest number of observed OTUs (compared to AOA and AOB), with a total of 93 unique OTUs and 4-43 OTUs found at each individual site  and ‘Candidatus Nitrospira nitrosa’ (91.6% pairwise sequence identity), organisms recently implicated in the complete nitrification of ammonia to nitrite (comammox) (nxrB database sequences.omammox) . HoweverNitrobacter 16S rRNA gene sequence analyses revealed that one OTU was closely related (99% sequence identity) to sequences from a municipal wastewater Nitrobacter clone. One Nitrobacter OTU was also present in another Iron Springs sample used as technical sequencing replicate, as well as other OTUs with low read counts (2–69 per OTU). Nitrobacter sequences were not further analyzed due to the low number of sequences and samples with hits.amoA, AOB 16S rRNA, Nitrospira nxrB), we evaluated the relationship between environmental parameters and the total gene abundances (qPCR), relative gene abundance for individual OTUs (based on gene sequence counts), and overall community composition (CCA of relative gene abundance). All correlation analyses used corrected p-values to account for the large number of comparisons.For each nitrification gene dataset , pH was not strongly correlated to the overall community structure or relative abundance of individual OTUs for any gene (based on CCA and Spearman correlation). pH was positivity correlated to the total abundance (qPCR) of AOB 16S rRNA genes, but not for any other genes . Nonetheless, the relative abundances of several individual Nitrospira nxrB OTUs (OTU 28)  in several ways. CCA showed that the overall composition of the AOB 16S rRNA and Nitrospira nxrB communities was strongly correlated with conductivity  was positively correlated with Ca  and nitrite (NO2-) concentrations are very low due to the chemical equilibrium in solution . At low pH, the number of protons in solution increases and the equilibrium is shifted away from the energetic substrate (ammonia and nitrite). Here, AOA amoA and Nitrospira nxrB genes were present at most sites and time points regardless of pH. Archaeal amoA and Nitrospira nxrB gene abundances were high (103 – 107 copies per μg DNA) at sites with pH ≤ 4.5, and genes were detected at sites with pH as low as 3.5. Because pH reduces the bioavailability of ammonia, acidic habitats may favor AOA dominance over AOB due to their high affinity for ammonia  at all of the sites exceeded the allowable concentrations as determined by the Colorado Department of Public Health and Environment (CDPHE) Regulation 31. Sites with pH <4 had the highest concentrations of metals, perhaps because low pH can increase metal dissolution. Heavy metals are known to inhibit nitrification in some systems ; yet, we for AOA . Specifi for AOA . For ins for AOA . Though  for AOA , it is cNitrospira nitrite oxidizers were also abundant across all sample regions. When ammonia oxidizers and nitrite oxidizers were compared together, we observed that the Nitrospira nxrB genes were more abundant than archaeal amoA and AOB 16S rRNA genes at many sites . However, gene copy numbers vary for each organism: AOA have one amoA copy per cell, AOB have one 16S rRNA copy per cell, and Nitrospira have 2-6 nxrB copies per cell , possibly suggesting that AOA, AOB and NOB were absent in those selected sites, present in low numbers, or did not properly amplify with the PCR primers. Further analyses would be necessary to confirm low nitrifier abundance and its ecological implications .Nitrifier gene abundance showed interesting patterns in the Iron Springs mining region. Gene abundance showed a clear seasonal pattern: AOA, AOB, and NOB genes were more abundant in late summer than in early summer (∼5 times more abundant in late summer for ∼80% of samples where abundance was measured in both time points for a given year). Overall, AOA were more abundant than AOB across most sample regions irrespective of varying environmental conditions, which may be due to differences in AOA and AOB physiology. per cell . At a nuNitrosotalea associated with acidic soil environments   , which migure 4) .Nitrosomonadaceae, which are generally well distributed in both terrestrial and aquatic environments . The majority of OTUs dominating the Caribbeau Mine region clustered with Nitrosospira found in soil environments . Although Nitrobacter are typically observed along with Nitrospira in aquatic systems, Nitrospira are often numerically dominant in sediments , that the available primer sets do not amplify the particular Nitrobacter found at these sites, or that biases in DNA extraction or PCR amplification prevented recovery of Nitrobacter sequences.NOB in these AMD-impacted sites were related to phylogenetically diverse ediments  and wateediments . These famoA or Nitrospira nxrB genes, or to the overall community structure or relative abundance of individual OTUs for any gene. Heavy metals, which had high concentrations in the Iron Springs sediments, were correlated with the relative abundances of several individual OTUs, but not the overall nitrifier community composition or abundance. It is important to note that many other factors not measured here could impact nitrification, including other chemical variables or organismal interactions. In addition, the combined effect of multiple, simultaneous stresses  on gene abundance and community composition is difficult to tease apart. This study provides a foundation for future work to determine rates of nitrification and the role of comammox bacteria in this AMD-impacted system, and to cultivate acidophilic and metal-tolerant AOA and NOB.In summary, we found interesting patterns of gene abundance and diversity of AOA, AOB, and NOB in AMD-impacted sediments with low pH and high metal concentrations. Sediment pH was correlated with the total abundance of AOB 16S rRNA genes, but not the total abundance of archaeal Conceived and designed the experiments: AM and TR. Sampling, site information, and chemistry: TR. Performed the experiments: BR. Analyzed the data: BR, AB, TR, and AM. Contributed to the manuscript: BR, AB, TR, and AM. Funded the research: AM and TR.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sclerocarya birrea (A.Rich.) Hochst (Anacardiaceae) is a savannah tree that has long been used in sub-Saharan Africa as a medicinal remedy for numerous ailments. The purpose of this study was to increase the scientific knowledge about this plant by evaluating the total content of polyphenols, flavonoids, and tannins in the methanol extracts of the leaves and bark , as well as the in vitro antioxidant activity and biological activities of these extracts. Reported results show that MLE is rich in flavonoids , whereas MBE has the highest content of tannins . The antioxidant activity was measured using four different in vitro tests: β-carotene bleaching (BCB), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), O2−•, and nitric oxide (NO•) assays. In all cases, MBE was the most active compared to MLE and the standards used (Trolox and ascorbic acid). Furthermore, MBE and MLE were tested to evaluate their activity in HepG2 and fibroblast cell lines. A higher cytotoxic activity of MBE was evidenced and confirmed by more pronounced alterations in cell morphology. MBE induced cell death, triggering the intrinsic apoptotic pathway by reactive oxygen species (ROS) generation, which led to a loss of mitochondrial membrane potential with subsequent cytochrome c release from the mitochondria into the cytosol. Moreover, MBE showed lower cytotoxicity in normal human dermal fibroblasts, suggesting its potential as a selective anticancer agent. Plants are considered to be producers of natural compounds that have been used by humans in medicines to treat diseases since ancient times. Nowadays, a large number of drugs used against various diseases have been isolated or derived from natural sources, often starting from their use in traditional medicine .The biological activities from natural sources are due to the variety of chemicals that are widely distributed in nature ,3,4,5. ISclerocarya birrea (A.Rich.) Hochst (Anacardiaceae), commonly known as marula, has long been used in sub-Saharan Africa as a medicinal remedy for numerous ailments: indeed, the stem bark, roots, and leaves of S. birrea are used to treat hypertension, diabetes, dysentery, and inflammation [ammation .S. birrea roots inhibit the growth of Candida spp. and Cryptococcus neoformans [S. birrea and it was found that polyphenols, including phenolic acid, flavonoids and their glycosides and procyanidins, were the main compounds. Considering the antioxidant and proapoptotic activity of the methanol extract of S. birrea root [S. birrea bark and leaf extracts on the human hepatocarcinoma cell line (HepG2) vs. fibroblasts.It has been reported that extracts from oformans ; the roooformans ,12. Moreoformans . Previouoformans ,15,16 inS. birrea leaf extracts ranged from 30.2 ± 1.3 mg GAE/g  to 62.6 ± 0.8 mg GAE/g , whereas the TPC values of the bark extracts ranged from 31.3 ± 0.2 mg GAE/g  to 241.3 ± 8.9 mg GAE/g . The tannin content of the leaf and bark extracts are expressed as milligram (mg) of tannic acid equivalents (TAE) per gram (g) of dried extract and ranged from 74.2 ± 5.5 mg TAE/g  to 175.8 ± 5.5 mg TAE/g (CLE) in S. birrea leaves and from 158.7 ± 1.6 mg TAE/g (CMBE) to 949.5 ± 29.7 mg TAE/g (MBE) in bark extracts. The highest TFC  per g) was observed in MLE (132.7 ± 10.4 mg QE/g of dried extract) and MBE (57.7 ± 3.5 mg QE/g of dried extract), whereas the lowest values were observed in n-hexane extracts (8.3 ± 1.2 mg TAE/g in HLE and 4.7 ± 0.5 mg TAE/g in n-hexane bark extract (HBE)) (t (HBE)) .50 = 11.3 ± 0.2 µg/mL). The inhibition was dose dependent  (ABTS) radical) and physiological radicals . Methanol extracts showed a high dose-dependent ABTS scavenging activity . Antioxependent A,B and mactivity A,B. At t50 values of 19.9 ± 2.9 and 15.6 ± 1.0 µg/mL for nitric oxide and superoxide anion, respectively. MLE showed an IC50 value of 31.1 ± 3.3 µg/mL for superoxide anion, but a low inhibition of nitric oxide reaching 39.9 ± 0.5% inhibition at 200 µg/mL. In both assays, ascorbic acid was used as the standard showing IC50 values of 35.6 ± 2.4 and 199.3 ± 20.1 µg/mL for nitric oxide and superoxide anion, respectively.Both vegetal parts showed antiradical activity against the investigated physiological radicals in a dose-dependent manner A,B, but S. birrea were tested on HepG2 and normal human dermal fibroblast cells treated with different concentrations of both extracts for 24 h.Using the Calcein AM viability assay, the cytotoxic effects of MBE and MLE from 50 = 180 and 270 µg/mL, respectively), although its activity was lower than that of the methanol extract from roots (MRE) that was previously demonstrated [As indicated in nstrated . 50 > 400 µg/mL), suggesting their use as a potential anticancer agent  of each extract for 24 h; cells were analyzed by flow cytometry after staining with Annexin V/7-AAD . UntreatTo investigate the role of ROS in extract-mediated apoptosis, we determined ROS production in HepG2 cells after 3 h of treatment with MLE or MBE by measuring the oxidation of a non-fluorescent probe ) to its reduced fluorescent form ).m) and mitochondria-mediated apoptosis, we evaluated mitochondrial membrane polarization in HepG2 cells treated for 3 h with different concentrations of MLE and MBE; the cationic fluorescent probe tetramethylrhodamine methyl ester (TMRM), which is easily incorporated into mitochondria of viable cells, was used. As shown in m), leading to a decrease in TMRM fluorescence of about 50% at a concentration of 200 µg/mL, while MLE did not alter ΔΨm significantly.Since excessive oxidative stress could lead to mitochondrial damage, causing a loss of mitochondrial membrane potential  for 3, 6, and 24 h. Immunoblot analysis highlights a progressive decrease in the mitochondria-enriched heavy membrane (HM) fractions and a consequent increase in the cytosolic fractions of cytochrome c in HepG2 treated cells vs. untreated control cells  and it is well known, in fact, that an increased consumption of antioxidant-rich foods and food supplements are obtained often from natural sources. Antioxidants are molecules having several chemical structures that can act as inhibitors or quenchers of free radical reactions, delaying or inhibiting cellular damage. Plant species can be considered as a rich source of antioxidant molecules. S. birrea extracts [Phenolic compounds are the major constituents of extracts ,15,16 anextracts ,3; howevextracts ,19,20,21•+, O2−•, and NO• assays. Our results have shown that both of the investigated extracts (MLE and MBE) have a dose-dependent activity, similar to the reference standards . The lipid peroxidation of both extracts by the β-carotene bleaching assay (BCB) led to a lower value than the standard (BHT). Phenolic compounds including tannins and flavonoids are known as hydrophilic antioxidants and in aqueous systems they have the highest activity. However, BCB instead provides information about the level of lipophilic compounds [In the present study, the radical scavenging activity was performed by in vitro BCB, ABTStandards . The comompounds .S. birrea leaves and bark were tested by the Calcein AM assay. HepG2 and normal fibroblast cells were treated with different concentrations of MLE and MBE for 24 h and the cells’ viability decreased in a dose-dependent manner. MBE had the highest cytotoxicity in HepG2 cells, whereas both extracts showed an IC50 value higher than 400 µg/mL in fibroblasts, demonstrating their potential anticancer effect. The higher cytotoxic activity of MBE was confirmed through more pronounced alterations in cellular morphology  and galloylated glycosides of quercetin and kaempferol were identified mainly in leaf extracts, whereas procyanidins (condensed tannins), including epicatechin 3-O-gallate, epigallocatechin3-O-gallate, and galloylepicatechin-epigallocatechin-3-O-gallate, were found in bark extracts of S. birrea. These results agree with spectrophotometric data reported in this work, where the flavonoid content (TFC) was dominant in leaves, whereas tannins (TTC) were abundant in bark.Overall, considering the obtained results, the effects of cellular exposure to MBE clearly demonstrates both a higher radical scavenging activity and a greater cytotoxicity of this extract compared to MLE. This difference could be explained by the different phytochemical contents. Indeed, previous studies have reported the chemical composition of bark and leaf extracts using HPLC-MS analysis ,16. PhenS. birrea root extract in which the content of procyanidins was very high [The classes of flavonoids and procyanidins include a wide range of molecules with several chemical structures that affect their biological activity. As previously reported , the degery high . Our finFurther studies will be directed to identifying the compounds responsible of the biological activities.2CO3), aluminum chloride (AlCl3), sodium nitrate (NaNO3), sodium hydroxide (NaOH), bovine serum albumin (BSA), sodium dodecyl sulfate (SDS), triethanolamine, iron(III) chloride (FeCl3), ABTS ), potassium persulfate, β-carotene, linoleic acid, Tween 20, ascorbic acid, sodium nitroprusside (SNP), sulfanilamide, naphthylethylenediamine, nicotinamide adenine dinucleotide (NADH), phenazinemethosulfate (PMS), nitroblue tetrazolium (NBT), potassium phosphate monobasic (KH2PO4), gallic acid, quercetin, tannic acid, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), butylated hydroxytoluene (BHT), Dulbecco’s Modified Eagle Medium (DMEM), dimethyl sulfoxide (DMSO), Calcein AM was purchased from Sigma Aldrich . All solvents were purchased from Carlo Erba Reagents . Trypsin-EDTA solution, FBS, glutamine, penicillin-streptomycin, and PBS were purchased from Euroclone . Tetramethylrhodamine methyl ester  was a kind gift from Massimo Lasorsa .Folin–Ciocalteu reagent, sodium carbonate  [n-hexane (HE), chloroform (CE), chloroform-methanol 9:1 (CME), and methanol (ME).Dried leaves and bark of . (2013)  obtaininThe total content of polyphenols (TPC), flavonoids (TFC), and tannins (TTC) were evaluated using three different spectrophotometric assays, as reported by Armentano, et al. (2015) . TPC was•+). For the analysis of the extracts, the ABTS radical solution was diluted with distilled water to an absorbance of 1.00 at 734 nm. Extracts were tested at different concentrations (0–200 µg/mL) and the results were expressed as the percentage of radical inhibition and then converted to IC50, which is the concentration of sample required to inhibit 50% of the ABTS radicals. Trolox was used as the reference standard.The free radical scavenging capacity of each plant extract was studied using the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical assay. Experiments were performed according to the previous report  with sli•) that is generated by the oxidation of linoleic acid; the addition of an effective antioxidant reduces the discoloration, which can be quantified spectrophotometrically at 470 nm. BHT was used as a positive control and the results were expressed as the percentage of antioxidant activity (% AA) [The inhibition of lipid peroxidation was monitored by the β-carotene/linoleic acid system. The assay is based on the discoloration of β-carotene, which is caused by an attack of the lipid peroxide radicals (LOOy (% AA) .Nitric oxide scavenging activity can be estimated by using the Griess reaction. The compound sodium nitroprusside is known to decompose in aqueous solution at physiological pH 7.2, producing NO, which is able to react (under aerobic conditions) with oxygen to produce stable products (nitrate and nitrite) that can be determined using the Griess reagent. Scavengers of NO compete with oxygen and leads to reduced production of nitrite ions as reported by a previous study . ResultsS. birrea extracts was determined as previously reported [l-Ascorbic acid was used as a control. The decreased absorbance of the reaction mixture indicates increased superoxide anion scavenging activity. Results were expressed as the percentage inhibition of superoxide anion generation and it was calculated using the following formula:0 is the absorbance of the control and A1 is the absorbance of the extract and standards.Measurement of superoxide anion scavenging activity of reported . SuperoxHuman hepatoma (HepG2) and normal human dermal fibroblast  cell lines were cultured and maintained under the same conditions as previously reported . Bark anS. birrea were tested against HepG2 and normal human dermal fibroblast cell lines using the Calcein AM viability assay. Cells were seeded in 96-well black-walled plates (1 × 104 cells/ well) and after 24 h were incubated with various concentrations of MBE or MLE  at 37 °C for 24 h. Human fibroblasts were incubated with up to 400 µg/mL of MBE/MLE. Thereafter, the media were discarded from the wells and the cells were incubated with 100 µL of 1 µM Calcein AM in PBS for 30 min at 37 °C. Cellular Calcein fluorescence was detected using a plate reader  at excitation and emission wavelengths of 490 and 510–570 nm, respectively.Leaf and bark methanol extracts of 5 cells/well and treated with increasing concentrations  of MBE and MLE for 24 h. The untreated cells served as a control. Changes in cell morphology were imaged using inverted phase contrast microscopy at 40× magnification .HepG2 cells were cultured in standard conditions in 24-well tissue culture plates at a seeding density of 2 × 105 cells/well and treated with MBE and MLE  for 24 h. At the end of the treatment, cells were harvested and suspended in 500 µL of binding buffer containing 5 μL Annexin V and 5 μL 7-AAD and then incubated in the dark for 15 min. Finally, at least 1 × 104 stained cells per sample were analyzed in a BD FACSCanto II  flow cytometry analyzer.The percentage of apoptotic cells was determined using the Annexin V/7-AAD double staining assay, according to the manufacturer’s protocol . In brief, HepG2 cells were plated in a 12-well culture plate at a density of 2 × 104 cells/well and incubated for 24 h at 37 °C. Following treatment with MLE or MBE  for 3 h, cells were stained with 10 µM DCFH-DA for 30 min at 37 °C in the dark. The fluorescence was measured by the GLOMAX Multidetection System  using a blue filter .The intracellular ROS level was measured using a fluorescent probe, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), as previously reported . Briefly5 cells were cultured in 12-well plates and treated with MLE and MBE at different concentrations  for 3 h. The cells were then detached, washed in ice-cold PBS, and incubated for 20 min at 37 °C in darkness with 150 nM TMRM in PBS. After dilution with PBS, cells were analyzed by FACS with an excitation wavelength of 488 nm and an emission wavelength of 575 nm.Changes in mitochondrial membrane potential (MMP) were monitored by flow cytometry (FACSCanto II) with the mitochondrial tracking fluorescent dye TMRM. In brief, 2 × 1050 concentration of MBE (180 µg/mL) for 3, 6, and 24 h and then harvested and washed twice with ice-cold PBS. Mitochondria enrichment was obtained as previously described [HepG2 cells were exposed to an ICescribed . The memp < 0.05).All the results are presented as mean ± SD of three independent experiments performed in triplicate. In the viability assays, the percentage survival values were normalized by an arcsine square root transformation and then compared using analysis of variance (ANOVA) and Tukey’s HSD test. In the measurement of reactive oxygen species and mitochondrial membrane potential, statistical significances were analyzed by one-way analysis of variance (ANOVA) and Tukey’s HSD test. Both analyses were performed using software R version 2.8.1 . Different letters denote significant differences (


Escherichia coli ABWA45, a 16S rRNA methyltransferase-producing wastewater isolate. Assembly and annotation resulted in a 5,094,639-bp circular chromosome and four closed plasmids of 145,220 bp, 113,793 bp, 57,232 bp, and 47,900 bp in size. Furthermore, a small open plasmid  was assembled.We present the complete genome sequence of  The World Health Organization (WHO) defines antimicrobial resistance as one of the major threats to the health and welfare of both humans and animals . AminoglEscherichia coli isolate collected from wastewater near Basel, Switzerland, in January 2016  that included the MLST-1.8 server  and belongs to the IncF incompatibility group. Interestingly, this plasmid revealed no good hits to any other plasmid compared to the NCBI database . pABWA45_2 is a 113,793-bp nontypeable plasmid  and resembles a Klebsiella oxytoca plasmid, pKPC_UV02 , but is missing the KPC-encoding Tn4401. pABWA45_4  could not be assigned to any incompatibility group by PlasmidFinder but shows substantial homologies to the backbone structure of pBK31567 (accession number JX193302), which belongs to the IncX5 group , CP022155 (pABWA45_1), CP022156 (pABWA45_2), CP022157 (pABWA45_3), CP022158 (pABWA45_4), and CP022159 (pABWA45_5). This is the first version of this genome.Sequence and annotation data of the genome have been deposited in GenBank under accession numbers


Syntrophaceae sublineages were previously reported to be responsible for syntrophic alkane degradation. However, limited information is currently available on their physiological capabilities in nature because it is very challenging to cultivate these as-yet uncultured microbes. We herein performed metagenomic sequencing of the methanogenic hexadecane-degrading culture M82 and recovered a nearly complete genome  belonging to Syntrophaceae sublineage II. The assembly genome was tentatively named “Candidatus Smithella cisternae strain M82_1”. Genes encoding alkylsuccinate synthase for alkane activation were identified, suggesting that this organism is capable of oxidizing alkanes through fumarate addition. This capability was further supported by the detection of methyl pentadecyl succinic acid and methyl tetradecyl succinic acid in cultures amended with hexadecane and pentadecane, respectively. Genes encoding enzymes for the β-oxidation of long-chain fatty acids and butyrate were also identified. The electron transfer flavoprotein/DUF224 complex is presumed to link electron flow from acyl-CoA dehydrogenase to a membrane hydrogenase or formate dehydrogenase. Although no indications of Rnf complexes were detected, genes encoding electron-confurcating hydrogenase and formate dehydrogenase were proposed to couple the thermodynamically favorable oxidation of ferredoxin to generate H2 and formate from NADH. Strain M82_1 synthesized ATP from acetyl-CoA by substrate-level phosphorylation or F1F0-ATP synthases. These results provide an insight into the potential metabolic traits and ecophysiological roles of the syntrophic alkane degrader Syntrophaceae.The methanogenic degradation of hydrocarbons plays an important role in hydrocarbon-contaminated environments in the absence of an external electron acceptor. Members of  Alkanes consist of hydrogen and carbon atoms only, all their bonds are single bonds, and these molecules are major constituents of natural gas and petroleum . AlkanesSyntrophus spp. were detected as the dominant bacterial phylotypes , the genus Smithella was proposed with Smithella propionica as a cultured representative  analogues were detected in the metagenome, suggesting that hydrocarbon degradation may be initiated through the addition of fumarate. Embree et al.  for 30 min. Control cultures without hexadecane and pentadecane addition were also collected at the same time. The cultures were then preserved at pH<2 by the addition of 6 M HCl prior to extraction for putative metabolites. Samples were extracted three times with 70 mL of ethyl acetate. The extracts were dried over anhydrous Na2SO4 and concentrated to a volume of 30 to 50 μL by rotary evaporation under a flow of N2. The organic extracts were then allowed to react with 400 μL of N,O-bis(trimethylsilyl)trifluoroacetamide  and were incubated at 65°C for 45 min. Trimethylsilyl (TMS) derivatives were analyzed using GC-MS  with a DB-5MS capillary column (30 m×0.25 mm×0.25 μm). Oven temperature remained at 40°C for 2 min, was increased to 270°C at a rate of 4°C per min, and was then held at 270°C for 10 min. The mass transfer line temperature was 280°C. Mass spectral data were generated using a mass spectrometer  at an electron energy of 70 eV in the SCAN/SIM mode. These data were initially used to search the NIST11 mass spectral library in order to obtain a reference. Mass spectral metabolites were also referenced to previously reported fragments, which represented either fumarate addition  amended with hexadecane or pentadecane were transferred to clean glass vials under the protection of N0–100 mL addition , 17, 39 addition  of alkan−1. gDNA was purified through agarose gel electrophoresis, and DNA concentrations were measured using a NanoVue spectrophotometer . Purified gDNA was stored at –80°C until used.gDNA was extracted from 8- to 12-mL cultures using a modified beating method  without Taq I and Msp I were applied for archaeal and bacterial DNA digestion, respectively , 2 μL of 10×buffer, 1.6 μL of dNTPs, 0.4 μL of each primer, 1 μL of bovine serum albumin (BSA), 1 μL of template DNA, and 13.1 μL of water. The PCR conditions used for bacterial amplification were as follows: 5 min at 95°C; 26 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and primer extension at 72°C for 2 min and a final extension at 72°C for 10 min. PCR conditions for archaea were as follows: 94°C for 4 min, 25 cycles of 94°C for 1 min, 52°C for 1 min, and 72°C for 2 min, and a final extension at 72°C for 7 min. Purified PCR products were quantified by fluorimetry with a Qubit® dsDNA HS Assay kit  and were then pooled and sequenced using a GS FLX Titanium LV emPCR Kit on a GS FLX+ Instrument  according to the manufacturer’s instructions.Fragments of bacterial and archaeal 16S rRNA genes were amplified using the primers B27f/B533r  and A344f/A915r , respectively, with Roche 454 sequencing adapters  (44). ThRaw pyrosequencing sequences were analyzed using Mothur v.1.31.2, mainly according to the standard operating procedure . Brieflyca. 23 kb, 8–10 μg) and was sequenced using the Illumina HiSeq 2000 system at BGI-Shenzhen, generating ca. 41 Gb of raw data. Sequence quality trimming was performed by Trimmomatic with a minimum quality score of 20 and minimum sequence length of 36 bp (De novo assembly was conducted using IDBA-UD (v. 1.1.1) with kmers of 20 (min) and 90 (max) and a min-contig size of 200 bp , Methanosaeta , and Thermofilum (2%) , unclassified Parcubacteria bacteria (19%), Kosmotoga (8%), unclassified Marinimicrobia bacteria (4%), Treponema (2%), and Desulfovibrio (1%)  (Smithella sp.) is affiliated with Syntrophaceae sublineage II, which has been proposed as a candidate species for syntrophic alkane degradation bacteria  . The bacrio (1%) . The micrio (1%) . The typF126282) , 30. Thibacteria . Howeverneage II  has not 50 size=4,619 bp, Proteobacteria  with clone HB1_11, which represents a syntrophic hexadecane degrader through the DNA stable isotope probing technique  of the TNF of the post-binning sequences showed that Smithella scaffolds were tightly clustered , Smithella sp. ME-1 (287), Smithella sp. SCADC (204), and Smithella sp. SC_K08D17 (165). A total of 1,937 ORFs (69%) were categorized into 21 functional COGs representing energy production and conversion , cell wall/membrane/envelope biogenesis , and replication recombination and repair  database using the best BLAST hit with a score of E<e–5, we found that the closet associations were with syntrophic microorganisms , such asThe draft genome of strain M82_1 has an incomplete Embden–Meyerhof–Parnas pathway (EMP pathway) because no gene encoding pyruvate kinase (phosphoenolpyruvate to pyruvate) was identified . The penSmithella spp. are known to degrade alkanes in cooperation with methanogens under strictly anoxic conditions  and carboxyl transferase 65_11), respectively. This degradation mechanism may be similar to that by which , respectns AK-01 .+ and (M-15)+ ions of silylated methyl pentadecyl succinic acid (MPA) metabolites occurred at m/z 471 and m/z 355, respectively. Other major ions were observed at m/z 262, 217, 172, 147, and 73  and MTA  were not detected in the hexadecane or pentadecane culture, respectively (data not shown). Metabolites that had molecular ions at m/z 262, 457, and 471 were not detected in the control culture with similar retention times to those amended with hexadecane and pentadecane, respectively  in the methylmalonyl-CoA pathway in the binning genome indicated that M82_1 is incapable of propionate oxidation  clustered in the scaffold of 227 (227_17:23), which encode the structural subunits of molybdenum-dependent nitrogenase . This result indicates that some sulfatereducing bacteria, rather than the syntrophic alkane degrader M82_1, possess the capacity to perform anaerobic respiration using sulfate as a terminal electron acceptor.Genes responsible for reducing inorganic sulfur species, such as sulfate, elemental sulfur, thiosulfate, and sulfite, were not detected. Only genes  for the conversion of sulfide to cysteine were detected in the M82_1 genome, suggesting the fixation of inorganic sulfide into a carbon skeleton . HoweverS. aciditrophicus, which has been proposed to synthesize ATP from acetyl-CoA , electron transport phosphorylation (ETP), and flavin-based electron bifurcation (FBEB) . Acetyl-etyl-CoA  In addit was 1:3 . The oxaon pump” . The deton pump” . In addi2 and formate are electron sinks in syntrophic bacteria  revealed a high degree of homology at the amino acid level (S. fumaroxidans likely confurcate electrons from NADH and ferredoxin (Fd) to protons and carbon dioxide to produce H2 and formate, respectively  may be generated by heterodisulfide reductase/methyl-viologen-reducing hydrogenase (HdrABC/MvhD)-like compounds, encoded by a gene cluster (437_38:51), which was named Hdr/Flox proteins in syntrophic bacteria  and S. wolfei (Swol_0697:6). This set of ETFs was adjacent to a gene (108_1) predicted to encode the transmembrane FeS-binding oxidoreductase “DUF224” with unknown function, 73% sequence identity to SYN_02638, and 45% homology to Swol_0698. In S. wolfei, the Bcd/EtfAB/DUF224 complex is presumed to link electron flow from butyryl-CoA dehydrogenase to the membrane. The co-expression of the gene cluster (Swol_0696:8) confirmed that electrons derived from butyryl-CoA are transferred through a membrane-bound EtfAB:quinone oxidoreductase (DUF224) to a menaquinone cycle and further via a b-type cytochrome to an externally oriented formate dehydrogenase (The M82_1 genome has two gene clusters (108_2:3 and 155_20:21) that encode ETFs . One setrogenase , 75.Chromobacterium violaceum ATCC 12472 (CRISPR id: NC_005085_1). The 23-spacer CRISPR repeat sequence in scaffold 536 showed 95% sequence similarity with the hyperthermophilic archaeum Thermoproteus tenax Kra 1 (CRISPR id: NC_016070_6). The 25-spacer exhibited 86% homology with the piezophile Photobacterium profundum SS9 (CRISPR id: NC_005871_1). The M82_1 genome also harbored two gene clusters encoding cas proteins: one (536_53:54) is related to a type V CRISPR-associated endonuclease, and the other (519_19:21) encodes proteins of the Repeat-Associated Mysterious Proteins (RAMP) superfamily.The M82_1 genome has five CRISPR regions distributed in scaffolds 437, 536, and 592 . The thrATP-binding cassette (ABC) transporters are an important class of transport proteins that use the energy derived from the hydrolysis of ATP to ADP, are widely distributed in all domains of life, and are involved in a large variety of processes . Genes eSyntrophaceae phylotype represent some of the most abundant syntrophic alkane-degrading bacteria and appear to play an important role in the carbon cycle in hydrocarbon-contaminated environments  to acetate were revealed. ATP was synthesized by substrate-level phosphorylation from acetyl-CoA or F1F0-ATP synthases. Moreover, strain M82_1 may employ electron-confurcating FDH and HYD or ETF and transmembrane FeS-binding oxidoreductase to overcome the energy barrier during the production of H2/formate using electrons derived from NADH or β-oxidation. In addition, an Hdr/Mvh-like complex, instead of an Rnf complex, was proposed to produce Fdred. The Candidatus Smithella cisternae strain M82_1 genome provides another genetic reference to deepen our understanding of the diverse ecological functions of this important genus and sheds light on its applications in alkane degradation in hydrocarbon-contaminated environments.Members of the ronments . In the Syntrophaceae (Candidatus Smithella cisternae” because of the absence of a pure culture. Smithella cisternae refers to the isolation of the organism from an oil field (cisternae) .We propose the provisional taxonomic assignment of sublineage II of the Family ophaceae  as “Cand


Lactobacillus plantarum strain A6, a strong acid producer, was isolated from a Vietnamese fermented sausage (nem chua). Here, we report the genome sequence of this strain . L. plantarum in food fermentation has received recent attention, and attempts to systematically define genetic loci associated with wider probiotic properties have been made (L. plantarum WCFS1)  are microorganisms widely used in food fermentation as starter cultures because of their beneficial influence on food preservation and the organoleptic characteristics that they create in fermented foods , 2. Foodeen made . Genome een made  will assm WCFS1) . L. planentation . Here, wde novo assembly was performed on the A5-miseq pipeline, an open source for genome assembly from Illumina data  with 2 × 300-bp paired-end reads. The genomic library preparation was performed using the Nextera XT DNA library preparation kit. The ina data , and conina data , 13.L. plantarum A6 is 3,368,579 bp in length, with a GC content of 44.2%. PGAP identified 3,218 genes, including 3,028 coding sequences, 92 RNAs, and 98 pseudogenes. There are 22 rRNAs, 66 tRNAs, and 4 noncoding RNAs (ncRNAs). RAST identified 3,261 coding sequences and 91 RNAs, including 68 tRNAs and 23 rRNAs. Several native plasmids are likely to be present in the strain; however, further investigation is required to define these precisely.PGAP revealed that the genome size of L. plantarum strain B21 (GenBank accession number NZ_CP010528) (L. plantarum WCFS1 (GenBank accession number AL935263), a reference strain isolated from human saliva (A comparison of the A6 genome with that of the previously published The significant number of transpositions relative to its nearest neighbors, B21 and WCFS1, confirm the well-known plasticity of the LAB genome and this Vietnamese isolate.L. plantarum strain A6 has been deposited in NCBI GenBank under the accession number LRUO00000000.The complete genome sequence of


Pteris vittata L. growing in a lead-zinc mining area with different As contamination levels.Alleviating arsenic (As) contamination is a high-priority environmental issue. Hyperaccumulator plants may harbor endophytic bacteria able to detoxify As. Therefore, we investigated the distribution, diversity, As (III) resistance levels, and resistance-related functional genes of arsenite-resistant bacterial endophytes in P. vittata with different As concentrations. Based on the 16S rRNA gene sequence analysis of representative isolates, the isolates belonged to Proteobacteria, Actinobacteria, and Firmicutes. Major genera found were Agrobacterium, Stenotrophomonas, Pseudomonas, Rhodococcus, and Bacillus. The most highly arsenite-resistant bacteria (minimum inhibitory concentration > 45 mM) were isolated from P. vittata with high As concentrations and belonged to the genera Agrobacterium and Bacillus. The strains with high As tolerance also showed high levels of indole-3-acetic acid (IAA) production and carried arsB/ACR3(2) genes. The arsB and ACR3(2) were most likely horizontally transferred among the strains.A total of 116 arsenite-resistant bacteria were isolated from roots of P. vittata plants with high As concentrations may select diverse arsenite-resistant bacteria; this diversity might, at least partly, be a result of horizontal gene transfer. These diverse endophytic bacteria are potential candidates to enhance phytoremediation techniques.The results of this study suggest that  Chronic weight) . HoweverP. vittata. L (Chinese brake fern) has potential to be used in the phytoremediation of As-contaminated soils, since it is able to accumulate more than 1000 mg kg− 1 of As in its fronds [P. vittata and their roles in As tolerance and transformation is severely limited.Phytoremediation employs plants to accumulate, transfer, stabilize, and remove trace elements from polluted soils and water systems. It has received considerable attention due to its economic and environmental benefits . Howevers fronds . Plant-as fronds , 8. Theys fronds . HoweverarsB and Acr3p have been identified in various soil bacterial species [P. vittata is crucial to improve phytoremediation strategies for As-contaminated sites.Endophytic bacteria, which have great potential in enhancing phytoremediation processes , 10, can species , knowledP. vittata from a Pb-Zn mine soil for analysis of the 16S rRNA gene and arsenite transporter gene (e.g. arsB and Acr3p) diversity. The objectives of this study were to (1) evaluate the distribution and diversity of arsenite-resistant endophytic bacteria in P. vittata with different arsenic contamination levels; and (2) investigate the arsenite transporter genes and correlate their presence to the arsenic resistance level of the bacteria. We hypothesized that the distribution and diversity of endophytic bacteria and their arsenite transporter genes are related to the As concentration in P. vittata roots.In this context, we isolated endophytic bacteria from No specific permits were required for the described studies. No specific permissions were required for these locations/activities because sample collection did not involve endangered or protected species or privately owned location.P. vittata is the dominant species in this area [Sampling was performed in the Tangjia Pb-Zn mine in Hanyuan, Sichuan , 890 m above sea level. The area has a mean annual temperature of 18.5 °C, with a mean annual precipitation of 486 mm and an average annual evaporation of 1553 mm. Based on the findings of a previous field survey, P. vittata plants were sampled from each subplot. The samples from the subplots were pooled and homogenized to form a composite plot-level sample.With regard to the distribution of the slag heaps, four different soils within the mining area, with different arsenic contamination levels, were selected: an ore outlet (site S2) and a tailing dam with smelting wastes and wastewater (site S3) with high As-contamination levels, and an ore charge heap (site S4) and a slag heap (site S5) with intermediate levels. A field used to cultivate summer rice and winter wheat, approximately 10 km from the mine area, was chosen as a non-contaminated control site (site S1). Soil and ferns samples (reproductive stage) were collected in August 2016 with the permission of the land owner. For this, three plots of 16 m × 10 m were randomly established in each site; each plot was divided into four 8 m × 5 m sampling subplots. Fresh roots and the corresponding rhizosphere soils from three individual 3/HClO4 (87/13 v/v). Soil samples were air-dried at ambient room temperature (25 °C) and sieved through a 6-mm sieve. Then metals in the soil samples were extracted with aqua regia. Total concentrations of As, Pb, Zn, Cu, and Cd in root extractant and aqua regia were determined using an inductively coupled-plasma optical emission spectrometer  [Root and soil samples were placed in polyethylene bags at 4 °C and transported to the laboratory for analyses. For metal analysis, roots were washed with distilled water and dried to constant weight at 55 °C. Dried roots were ground into a fine powder and digested with HNOon, USA) . Soil pHon, USA) . Soil waon, USA) . Soil toon, USA) .P. vittata root samples were washed with tap water, followed by three rinses with deionized water and sterilization by sequential immersion in 70% (v/v) ethanol for 2 min and 1% mercuric chloride for 1 min; subsequently, samples were rinsed three times with sterile water. Water from the last rinse was plated on Luria–Bertani’s (LB) agar to test whether the root surface was successfully disinfected. Surface-sterilized roots (approximately 0.5 g) were ground by a mortar and pestle in 5 ml of ddH2O. Sterile quartz sand was added to the mortar to improve cell wall disruption. Serial dilutions were plated on LB medium containing 800 μM NaAsO2 and incubated at 28 °C for 7 days. Single colonies were picked and restreaked several times to obtain pure isolates. Isolates were stored on LB medium at 4 °C.The 2, 2.5 U Taq DNA polymerase , 1× PCR buffer, 100 pmol of the primers BSF 8/20: 5′-AGAGTTTGATCCTGGCTCAG-3′; BSR1541/20: 5′-AAGGAGGTGATCCAGCCGCA-3′ [w/v) agarose gel with 0.5 μg mL− 1 ethidium bromide; subsequently, obtained PCR products (1.5 kb) were purified with a Gel Extraction Kit .Genomic DNA of the isolates was extracted from isolates grown in 5 mL of LB liquid medium at 28 °C with 150 rpm for 18 h, using the standard phenol-chloroform method described by Chang et al. . The 16SCCGCA-3′ , and 200HhaI, HaeIII, MspI, and TaqI. Based on colony morphology and the 16S rRNA gene ARDRA pattern, representative isolates were selected for 16S rRNA gene sequencing in an ABI3730XL automatic sequencer at Sangon Biotech .In the amplified rDNA restriction fragment analysis (ARDRA), purified PCR products (about 5 μl) were digested at 37 °C for 4 h, using the restriction enzymes arsB genes were amplified with the degenerate primers darsB1F (5’-GGTGTGGAACATC- GTCTGGAAYGCNAC-3′) and darsB1R (5’-CAGGCCGTACACCACCAGRTACATNCC-3′) [ACR3 (2) genes were amplified with the primers dacr5F (5’-TGATCTGGGTCATGATCTTCCC- VATGMTGVT-3′) and dacr4R (5’-CGGCCACGGCCAGYTCRAARAARTT-3′) [The TNCC-3′) . The ACRARTT-3′) . The PCRarsB/Acr3p. Sequences from the isolates and reference sequences from GenBank were checked manually and edited to the same lengths, using the software package ClustalX 2.0 [All amplified products were sequenced using an ABI3730XL automatic sequencer at Sangon Biotech . Similar sequences were searched using BlastN for 16S rRNA gene and BlastX for talX 2.0 . PhylogetalX 2.0 . The rel2 that inhibited growth in chemically defined medium (CDM) broth, were tested in triplicate. Stock solutions of NaAsO2 were prepared in ddH2O and sterilized. Isolates were inoculated in 3 mL of CDM broth supplemented with 6, 8, 10, 15, 20, 25, 30, 36, 42, and 48 mM NaAsO2 and incubated at 28 °C for 4 days. Low salt phosphate (LSP) agar plates without NaAsO2 were used as controls. The IAA production was measured as described by Sheng et al. [The minimum inhibitory concentrations (MICs), defined as the lowest concentrations of arsenic as NaAsOg et al. , 27.arsB, and MF185799–MF185803 for ACR3(2).The sequences obtained in this study were deposited in the NCBI GenBank database under accession numbers MF185755–MF185785 for 16S rRNA genes, MF185786–MF185798 for P < 0.05) were used to compare treatment means. All analyses were performed using SPSS 13.0 for Windows .Analysis of variance and the Student–Newman-Keuls (SNK) test , Gammaproteobacteria , Actinobacteria , and Firmicutes  , while the Rhodococcus sp. isolates showed low MICs (≥ 15 mM). Among the Pseudomonas isolates, the isolates containing arsB showed higher MICs than those without arsB. The average MIC of the 10 Acr3(2)-carrying isolates (40.9 ± 4.5 mM) was higher than that of the five arsB-carrying isolates (25.8 ± 2.3 mM). Five of the 19 highly arsenite-resistant isolates (MIC > 42 mM) were obtained from P. vittata roots with high arsenic levels  test for arsenite, all strains grew in 15 mM, 36 in 36 mM, 19 in 42 mM, and 5 in 48 mM NaAsO− 1 IAA. Isolates L8 (Rhizobium sp.), L11 (Rhodococcus equi), L31 (Agrobacterium tumefaciens), and X10 (Bacillus megaterium) produced over 90 μg ml− 1 of IAA. Two-thirds of the sequenced endophytes produced siderophores. Isolates L8 (Rhizobium sp.), K5 (Bacillus subtilis), and X4 (Bacillus sp.) produced more siderophores than the other isolates.In total, 28 out of the 46 isolates produced both IAA and siderophores Table . The majarsB genes and 11 ACR3(2) genes, were successfully amplified  formed separate clusters  sequences of the isolates were similar to those from Gammaproteobacteria and Alphaproteobacteria, respectively. The Acr3(2) sequences were divided into separate subgroups, similar to either Acr3(2) from Agrobacterium sp. or from Ensifer sp. Agrobacterium sp. L5, L8, L9, and L19 carried Acr3(2) similar to those of Ensifer sp.   and arsB genes, were obtained from isolates from the highly As-contaminated mining area.A total of 16 arsenite transporter genes, including five d 11 ACR3 genes, wd 11 ACR3 genes, wP. vittata, an arsenic hyperaccumulator growing in the Pb-Zn mine tailings in Southwest China [Plants that accumulate As can provide a specific environment for bacterial endophytes to adapt to high As concentrations . In thisst China .Pseudomonas, Stenotrophomonas, Rhodococcus, Agrobacterium, and Bacillus. Of these, Agrobacterium, Bacillus, and Pseudomonas species have previously been reported as metal-resistant endophytes in other As-contaminated sites [Stenotrophomonas strains have been isolated from As-contaminated soil and water [Stenotrophomonas and Rhodococcus have not been isolated earlier. Most of the representative isolates belonged to Agrobacterium (phylum Proteobacteria) and Bacillus (phylum Firmicutes). Similarly, Ni-resistant bacterial endophytes from Alyssum bertolonii belonged mainly to Firmicutes and Actinobacteria [Sedum alfredii belonged mainly to Proteobacteria (57%) and Firmicutes (43%) [Firmicutes, Proteobacteria, and Actinobacteria [In our study, we isolated 116 As-resistant bacterial endophytes. Despite the small number of isolates, the As-resistant bacterial endophyte community was diverse. The representative isolates were affiliated with ed sites –31. Arsend water , but, tobacteria . Moreovees (43%) . Taken tbacteria . These hbacteria .P. vittata have been reported to produce siderophores [Endophytic bacteria can increase the growth and development of plants growing in contaminated sites and their metal resistance by producing the phytohormone IAA and siderophores –39. Siderophores .P. vittata roots from sites S2 and S3 (with high As-concentrations) hosted a higher diversity of arsenite-resistant bacteria. In addition, the resistance levels of the isolates were also higher than those among isolates from sites with intermediate and low As concentrations. The 19 strains with arsenite MICs over 40 mM were all isolated from P. vittata growing in the Pb-Zn mining area. Previous studies have proposed that high As-contamination levels are likely to exert a strong selective pressure, thereby decreasing microbial diversity [P. vittata was widely distributed in the Pb-Zn mine area [P. vittata. Moreover, Huang et al. [P. vittata had had sufficient time to adapt to metal and/or metalloid stress. Zhu et al. [P. vittata after long-term exposure to high As-levels [In our study, iversity , 42. Howine area , which mg et al.  reportedu et al.  found ths-levels .ACR3(2) genotypes were previously reported to be predominant over arsB in bacterial strains isolated from As contaminated soil [Brevibacterium linens AE038–8 contained three ars operons  and two copies of the arsO gene [Thiomonas sp. possessed two operons (aio and ars system) [Rhodopseudomonas palustris CGA009 carried three sets of arsenic resistance determinants  on the chromosome [ACR3 may have a higher affinity to arsenite and a higher rate of arsenite transport than arsB, which makes it more effective [arsB gene-containing strains, strains having ACR3(2) mostly showed higher arsenite resistance in our study.Exploring the relations among the degree of arsenite resistance, the distribution and diversity of the arsenite transporter gene families of the bacterial endophytes is a key goal of microbial ecology. The ted soil , which wrsO gene , Thiomon system) , while Rromosome . Previouromosome . Surprisffective . AccordiACR3(2) appeared to be more easily transferred than arsB, and the transfer would be possibly stimulated by the exponential growth of environmental pollution as proposed in a previous study [Aeromonas, Stenotrophomonas, and Comamonas in highly As-contaminated soils. Similarly, the phylogenetic discrepancies between 16S rRNA genes and ACR3(2)/arsB indicate that ACR3(2) might have been horizontally transferred, especially in the isolates from P. vittata roots containing high As-levels. The HGT process may have occurred under the high arsenic pressure and resulted in increased functional and species diversity [Horizontal gene transfer (HGT) plays an important role in allowing a microbial community to rapidly adapt to a new environmental stress like heavy metal contamination, and thus could play an important role in the adaptation of the endogenous endophytic community . In our us study . Cai et us study  have repiversity , 50, whiP. vittata roots, collected from five soils with different levels of As-contamination, and studied the arsenite resistance and arsenic transport genes of the isolates. Proteobacteria, Actinobacteria, and Firmicutes were the predominant taxa. The distribution and diversity of cultivable endophytes were affected by the arsenic concentration in P. vittata. A high number of the isolates were resistant to high concentrations of As and multiple heavy metals and showed plant growth-promoting characteristics. Horizontal gene transfer of ACR3(2) and arsB was detected in some of the isolates from P. vittata roots with high As concentrations. Overall, this study provides valuable information about endophytic bacterial species in relation with As transport and As-resistance genes. Our results contribute to the knowledge on the diversity and distribution of As-resistant endophytic bacteria that may be applied in the phytoremediation of As-contaminated sites.We investigated the distribution and diversity of arsenite-resistant endophytic bacteria in