Because of their unique ability to modulate the immune system, mesenchymal stromal cells (MSCs) are widely studied to develop cell therapies for detrimental immune and inflammatory disorders. However, controlling the final cell phenotype and determining immunosuppressive function following cell amplification in vitro often requires prolonged cell culture assays, all of which contribute to major bottlenecks, limiting the clinical emergence of cell therapies. For instance, the multipotent Wharton’s Jelly mesenchymal stem/stromal cells (WJMSC), extracted from human umbilical cord, exhibit immunosuppressive traits under pro-inflammatory conditions, in the presence of interferon-γ (IFNγ), and tumor necrosis factor-α (TNFα). However, WJMSCs require co-culture bioassays with immune cells, which can take days, to confirm their immunomodulatory function. Therefore, the establishment of robust cell therapies would benefit from fast and reliable characterization assays. To this end, we have explored the metabolic behaviour of WJMSCs in in vitro culture, to identify biomarkers that are specific to the cell passage effect and the loss of their immunosuppressive phenotype. We clearly show distinct metabolic behaviours comparing WJMSCs at the fourth (P4) and the late ninth (P9) passages, although both P4 and P9 cells do not exhibit significant differences in their low immunosuppressive capacity. Metabolomics data were analysed using an in silico modelling platform specifically adapted to WJMSCs. Of interest, P4 cells exhibit a glycolytic metabolism compared to late passage (P9) cells, which show a phosphorylation oxidative metabolism, while P4 cells show a doubling time of 29 h representing almost half of that for P9 cells (46 h). We also clearly show that fourth passage WJMSCs still express known immunosuppressive biomarkers, although, this behaviour shows overlapping with a senescence phenotype. Immunosuppressive cells play crucial roles in regulating the human immune system, by secreting pro- and anti-inflammatory cytokines at ratio enabling desired specific immune response. A dysfunction of this equilibrium can lead to serious pathological disorders. For instance, the overexpression of pro-inflammatory cytokines has been identified as a main cause leading to Multiple Sclerosis  and grafl-arginine (L-ARG) and their production of nitric-oxide (NO), both phenomenon resulting in reduced T-cell proliferation and viability [Indeed, several cell types revealed a functional capacity to impact on this cytokine equilibrium, either by directly increasing or reducing their respective concentrations or by modulating the production or consumption rates of specific metabolites within the cell microenvironment, that indirectly affects cytokines secretion. For instance, macrophages exhibit specific behaviour depending on whether they are polarized towards the M1 or M2 phase . In the iability ,9. Interiability . For insiability . More reiability , have reiability ,14. Moreiability ,16,17. Tiability . Howeveriability ,19. Besiiability ,21, mostiability ,23.MSCs are a heterogeneous population and the International Society for Cell Therapy recognizes the need for standardized metrics to assess MSC immunoregulatory function to enable clinical development ,25. The Here, we present a study on the metabolic behaviour of Wharton’s Jelly mesenchymal stem cells (WJMSC), when comparing fourth (P4) to late ninth passage (P9) cells, wherein both cells presented slightly different immunosuppressive function under pro-inflammatory conditions. A dynamic metabolic flux analysis (dMFA) was performed to further characterize differences in the behaviour of both WJMSC cultures, using an in silico platform describing central carbon metabolism as well as pathways related to cell immunomodulatory mechanisms . A dynamt-test of 0.295 as compared to the reference value of 2.132 for the 95% confidence interval. However, although not statistically significant, one can observe a decreasing tendency of the cells immunomodulatory function between the 4th and the 9th passages  was performed, as proposed by Weiss et al. , who revpassages . This obTherefore, since both P4 and P9 cells do not significantly differ in their low immunosuppressive function under pro-inflammatory conditions , a dynamic metabolomic study was performed to specifically characterize and document biomarkers that are specific to the cumulative effect of successive passages.The metabolic model was first calibrated combining experimental data for P4 and P9 cells, as described in HO cells ,30,31. THO cells . InteresHO cells ,30,31.A first set of parameters were thus determined from minimizing simulation error, anchoring model structure and parameters on averaged WJMSCs behaviour combining P4 and P9 cell culture data sets. Then, a parameter sensitivity analysis to allow the specific identification of 31 highly sensitive parameters; a parameters subset enabling us to distinctively describe P4 and P9 cell behaviours in a second step, as shown in a previous work , keepingvmaxresp , which w−2 h−1  as compared to 1.36 × 10−2 h−1  for the late P9 passage cells , then rapidly reaches growth cessation at 85 h. However, the specific growth rate for P9 cells starts at 1.24 × 10−2 h−1, and then continuously decreases until 90 h (µ of 2.1 × 10−3 h−1), rapidly reaching growth cessation at 120 h. As indicated in Interestingly, model simulations, which cope with both P4 and P9 cell growth trends, allow for further analysing the effect of the cell passage number on WJMSC cells. Indeed, it was first intriguing to question the model for potential limiting nutrients that could have limited the culture post-confluency since cell cultures were both simultaneously stopped when P4 reached confluency at 72 h. Model simulations were thus prolonged from 72 h until simulating growth cessations in both cultures, under speculative prolonged cultures with no cell confluency phenomena . It was also possible to plot the cell specific growth rate with time B. Values−4 mmol·10−6 cells·h−1 was estimated for P4 cells as compared to 2.4 × 10−4 mmol·10−6 cells·h−1 for P9 cells, whereas the average lactate specific production rates were of 5.8 × 10−4 mmol·10−6 cells·h−1 for P4 cells and 5.6 × 10−4 mmol·10−6 cells·h−1, according to experimental data. Looking at intracellular metabolic intermediates of glycolysis, PYR concentration remained constant and at similar levels of 2 × 10−7 mmol·10−6 cells in P4 and P9 cells (−7 mmol·10−6 cells in P4 cells and 4 × 10−7 mmol·10−6 cells in P9 cells (Extracellular glucose (EGLC) uptake, extracellular lactate (ELAC) production, and intracellular pyruvate (PYR), and phosphoenolpyruvate (PEP) concentrations were measured with time whereas glucose-6-phosphate (G6P), fructose-6-phosphate (F6P) and glyceraldehyde-3-phosphate (GAP) concentrations were estimated from model simulations . Before P9 cells H, while P9 cells G.6 cells) glucose uptake rate than P9 cells after 54 h, but a higher specific lactate production rate during the whole culture. However, due to their higher specific cell growth rate, P4 cells culture lead to a higher final decrease of glucose concentration in the culture medium. Thus, P4 cells show a more active glycolytic metabolism during the fast growth period (0–54 h) with a similar specific glucose uptake rate, while a higher lactate specific production rate. P4 and P9 WJMSC cells present a glucose uptake and a lactate production rates before 54 h that are twice those reported for CHO cells. Indeed, our results agree with those of Moya et al. [−4 mmol·10−6 cells·h−1 and a lactate production rate of ~2 × 10−4 mmol·10−6 cells·h−1, according to our previous work [As mentioned above, a total of eight sensitive model parameters are part of glycolysis  and a sud by 50% , all of d by 50% , from whd by 50% . Globalla et al.  who obsea et al. . Higher a et al. . Furtherous work ,31 or toous work ,37,38.Model simulations suggest that most of the glycolytic flux feeds pyruvate synthesis. Indeed, the pentose phosphate pathway (PPP) accounts for less than 1% of the total glycolytic flux . From py−6 mmol·10−6 cells·h−1 as compared to a glycolytic global flux of ~10−4 mmol·10−6 cells·h−1 for both P4 and P9 cells  to values reported in literature for CHO cells [The pentose phosphate pathways (PPP) is consuming a minor part of the glycolytic flux, as mentioned above, with a glucose-6-phosphate dehydrogenase (G6PDH) flux of ~10P9 cells . Ribose-P9 cells . The traHO cells ,37.The analysis, by the use of model simulation, regarding the distribution of PPP fluxes to cell synthesis, nucleotides synthesis as well as returning to glycolysis, reveals that 55% of the incoming G6PDH flux returns to glycolysis in P4 cells, but this contribution reaches 65 to 75% in P9 cells, in their growth phase . However−7 mmol·10−6 cells·h−1, increasing briefly from 54 h in P4 cells. In both conditions, AKG increased from 8.6 × 10−8 to 1.3 × 10−7 mmol·10−6 cells·h−1 at 54 h decreasing slightly thereafter. FUM concentration showed a similar increasing behaviour in both P4 and P9 cells, although maintaining a higher concentration in P4 cells. In the case of MAL, its intracellular concentration stayed higher in P9 as compared to P4 cells. In the simplified metabolic network described in the model, FUM is synthesized by succinate dehydrogenase (SDH) and also by argininosuccinate lyase (ASL), a combination that may explain a higher FUM concentration level in P4 cells due to a higher activity of ASL in the Urea cycle in P4 when compared to P9 cells. The concentration of non-measured metabolites were estimated from model simulation, with higher concentrations for all simulated TCA intermediates in P9 cells except for OXA. Higher amounts of TCA intermediates, which suggests a more active TCA cycle in P9 cells, agrees with the above-mentioned observation of a respiratory metabolism in these late passage P9 cells as compared to P4 cells.The impact of successive cell passages on TCA cycle activity was also studied. The cell concentrations in major TCA intermediates, such as malate (MAL), succinate (SUC), alpha-ketoglutarate (AKG), and fumarate (FUM) were measured while model simulation enabled the determination of citrate (CIT), succinyl-CoA (SCOA), oxaloacetate (OXA) concentrations, as well as the TCA precursor acetyl-CoA (ACCOA) . SUC and−4 mmol·10−6 cells·h−1, whereas TCA metabolic intermediates show a concentration magnitude of ~10−7 mmol·10−6 cells  in P9 cells, results that are consistent with a respiratory metabolism, the expected low AMP-to-ATP ratio is observed (and simulated). Interestingly, both experimental ATP-to-ADP and AMP-to-ATP ratios tend to reach similar values at the end of the cultures, which coincided with P4 cell confluency and growth rate reduction (see above). Hardie et al. proposed a high AMP-to-ATP ratio as a marker of oxidative stress [In this work, energetic and redox nucleotides were quantified and simulated by the model, as concentrations , as wellll lines ,30,31, ae stress . Moreovee stress ,44. ThusThe urea cycle activity is known to be modulated with the immunosuppressive activity ,45. In tWhile ARGt activity in P9 cells remains higher and diverges from that in P4 cells, all other enzymes of urea cycle show a higher activity in the P4 cells when compared to P9 cells . Sensitiactivity . Thus, iactivity .Tryptophan metabolism is also well known as a key biomarker of an immunosuppressive phenotype ,47. IndeInterestingly, and as previously shown for other metabolic sub-pathways, model simulations allow for obtaining a clearer view on tryptophan metabolism. The model simulates a higher tryptophan specific consumption rate in P9 cells as compared to P4 cells , althoug−2 and cultivated in 75 cm2 T-flasks and 150 cm2 T-flasks  with 10 mL and 20 mL of α-MEM  supplemented with 20% of fetal bovine serum , 1% of Glutamax  and 1% of Antibiotic-Antimycotic , and cultured in a 5% CO2, 5% O2, and 37 °C humidified incubator. Cells were first amplified at the Ottawa Hospital Research Institute in order to furnish enough cells for further sets of experiments, leading to a 2nd passage (P2). A part of these P2 cells were transferred to Polytechnique on dry ice, whereas the other part was cultivated till 7 passage (P7) before sending. P2 (Second passage) and P7 (seventh passage) WJMSCs were thawed at Polytechnique and cultivated during 12h in the medium previously described to avoid thawing effects. After these 12 h, 15 ng mL−1 of TNFα, and 10 ng mL−1 of IFNγ were added to the culture medium and cells were cultivated 48 more hours in order to induce cells immunosuppressive properties, leading to P3 and P8 cells. After this 60 h-culture, the cells were passed in order to start the extraction experiment, leading to P4 and P9 cells. For early passage cells, the number of passage (4) was the smallest number possible in front of all the different constraints, whereas for late passage cells, a choice was made to use P9 cells. For each analysis, cells were detached using 50 μL cm−2 of TryplE , rinsed twice with PBS, centrifuged for 5 min at 300 g, and counted with a hematocytometer . Every 48 h, immunosuppressive properties of cells were maintained by adding 15 ng mL−1 of TNFα and 10 ng mL−1 of IFNγ in the culture medium. The protocol was the same for late and early cell cultures, however, the sample volume was adapted to the cultureF cell concentration. Because each sample has to contain at least 3 × 106 cells for adequate metabolite quantification, P4 passage cells were sampled at 0, 12, 24, 32, 40, 48, 56, 64, and 72 h, whereas P9 passage cells were sampled at 0, 18, 27, 36, 45, 54, 63, 72 h in order to obtain enough cells for analysis.WJMSCs were first isolated from human umbilical cords at the Ottawa General Hospital leading to a 1st passage (P1) and were transferred to the Ottawa Hospital Research Institute. During the entire process, the cells were inoculated at a cell density of 5000 cells cm+ T lymphocytes were isolated from umbilical cord blood samples  using a positive selection kit  and stained with 1 µM CFSE  for 5 min. Cells were washed twice with PBS , resuspended in OpTmizer media (Invitrogen), and 4 × 104 cells per well were seeded in triplicate in a 96 wells plate  and stimulated with CD3/28 Dynabeads (Invitrogen). MSCs were grown as described above in proinflammatory conditions induced with TNFα and IFNγ, 48 h prior to harvesting. MSCs were resuspended in OpTimizer media and 4 × 103 cells were added to the stimulated T cells. Co-cultures were placed at 37 °C in a humidified incubator, at 5% CO2 and 5% O2, for five days. To analyze T lymphocyte proliferation, cells were stained with anti-CD45 antibodies (clone 2D1) conjugated to AlexaFluor700  and 10 ng µL−1 propidium iodide (Sigma) and analyzed using a BDFortessa flow cytometer. Stimulated CFSE fluorescence of CD3+CD45+ cells, in the absence or presence of MSCs, were compared to determine T cell proliferation.CD3g and 4 °C to collect supernatants. The pellets were extracted a second and third time as described above with 200 μL of 50% cold methanol and 200 μL of ice-cold water, respectively. Supernatants were mixed and stored at −80 °C prior to analysis. Extracts were filtered through 0.2 μm PTFE filters  before analysis.The extraction protocol was adapted from Hammami et al.  based onN,N-dimethylhexylanine, Sigma) was used as an ion-pair reagent to improve the signal-to-noise ratio with positive ionization mode. The mobile phase consisted of Buffer A: 10 mM ammonium acetate, 15 mM DMHA at pH 7.0, and buffer B: 40/60% (v/v) acetonitrile. Mobile phase flow rate was set at 0.3 m/min with the following gradient: 0–10 min at 10% B, 10–20 min at linear gradient from 10 to 30% B, 20–21 min at linear gradient from 30 to 60% B, 21–26 min at 60% B, 26–27 min with a linear gradient from 60 to 10% B and 27–35 min at 10% B.Nucleotides in WJMSCs extracts were analysed following the method developed in Hammami et al. . A minimv/v) methanol in water. The flow rate was set at 0.3 mL/min, with the following gradient: 0–5 min at 10% A, 5–10 min at linear gradient from 10% to 20% A, 10–20 min at linear gradient from 20% to 100% A, 20–30 min at 100% A, 30–32 min at linear gradient from 100% to 10% A and 32–40 min at 10% A. Quantification of metabolites (nucleotides and organic acids) was performed by integrating peak areas and using calibration curves. Cell specific concentrations in metabolites were calculated by normalizing the quantity of metabolites in cell extracts to the number of extracted cells.As well as the previous described method, a minimum of 3 million cells were needed at each extraction in order for organic acids concentrations to be higher than the minimum threshold of this device. Organic acids concentrations were assessed using the above-mentioned UPLC-MS/MS system using a Hypercarb column  and a Hypercarb precolumn  . The mobile phase consisted of Buffer A, 20 mM ammonium acetate at pH 7.5, and Buffer B, 10%  before analysis.4  at pH 4. The mobile phase A contains 10% of the mobile phase buffer in water, and the mobile phase B contains 10% of the mobile phase buffer in acetonitrile (ACN) . The mobile phase B was linearly decreased from 90% to 35% in 19 min, and then was increased to 90% in one minute and held at 90% for 15 min at a flow rate of 0.1 mL min−1. The Agilent 6460 triple quadruple mass spectrometer (Agilent technologies), equipped with a Jet sheet stream electrospray ion source (Agilent technologies), was used for the analysis of amino acids in positive ion mode. The other parameters: gas temperature of 350 °C, gas flow rate of 9 L min−1, nebulizer pressure of 45 PSI, sheath gas temperature of 350 °C, sheath gas flow rate of 10 L min−1, and capillary votage of 3 kV. An internal standard solution that contains 2 µM homoarginine (Fisher cat.# AC169090010), 2 µM homophenylalanine (Sigma cat.# 294357), and 2 µM methionine-d3 (CDN isotope D1292) was used as internal standard for quantification. The MRM transition and retention time of each amino acid is listed in The analysis of amino acids concentration was performed on an Agilent 1290 UPLC system  coupled to an Agilent 6460 triple quadruple mass spectrometer , using previously described methods ,50. The Nitric oxide (NO) concentration in supernatant was assessed using a Nitrate/Nitrite Colorimetric Assay Kit . The absorbance was measured at 540 nm with a Victor 3V multilabel reader .ASTAV and AlaTAV), serine conversion to pyruvate (SDHHV), as well as amino acid conversion to alpha-ketoglutarate and succinate (respectively in AAtoSUCV and HISARGTAV). The urea cycle is considered, as well as the reaction involving indoleamine 2,3-dioxygenase (IDO) to account for well-known immunosuppressive-related metabolic pathways. In total, six metabolic reactions were considered to describe the urea cycle: the inducible nitric oxide synthetase (iNOS), aginase-1 (ARG1), aginase Tranferase (ARGt), argininosuccinate lyase (ASL), argininosuccinate synthase (ASS), and ornithine carbamoyltransferase (OCT). This set of reactions was taken from Morris et al. [−1 and the cell biomass a molar weight of 3.15 × 10−4 gDW·10−6 cells. Finally, enzymatic reactions were kinetically translated, as described in the The global modelling approach previously validated for Chinese Hamster Ovary (CHO) cell lines  has beens et al. . The TCAs et al. . Brieflys et al.  who deveIn addition to providing a wide new set of metabolomic data comparing the immunosuppressive phenotype of late passage WJMSC cells (P9) to earlier passage cells (P4), the present study has led to the development of a dynamic metabolic model that is specifically adapted to describe WJMSCs. The model metabolic network includes the central carbon metabolism, with the glycolysis, PPP, and TCA cycle, the energetic metabolism as well as pathways that are known to be differentially involved in immunosuppressive cells. Complementing the analysis of metabolomics data, model simulations enabled highlighting that P4 cells still exhibit an immunosuppressive-related metabolism. A series of coherent biomarkers were thus identified pointing out that immunosuppressive cells may favour a glycolytic metabolism, gradually switching to an oxidative phosphorylation metabolism while progressing with cell passages, from the forth (P4) to the ninth (P9) in the present study. The time-evolution of the AMP-to-ATP ratio also suggests that P9 cells have a lower tolerance to oxidative stress conditions. Moreover, even if P4 cells are not significantly immunosuppressive compared to P9 cells, we have demonstrated that P4 cells still express some biomarkers of an immunosuppressive phenotype that is normally seen at early passages WJMSCs (<P4). However, we have also clearly shown that the accumulation of cell passages leads to the overlap of immunosuppressive and senescence phenotypes, a result justifying the importance to better understand WJMSC metabolism specifically associated to the senescence phenotype. Indeed, more work is required with early passage cells, such as P2 and P3, to enable a clear characterization of WJMSC immunosuppressive metabolism. Finally, further work on the description of flux regulation ,54 is ex


GFP expression is controlled by PbrR in response to the presence of lead. The lead biosensor genetic element was cloned onto a low-copy number broad host range plasmid, which can stably exist in a range of laboratory and environmental isolates, including Pseudomonas, Shewanella, and Enterobacter. The biosensors constructed were found to be sensitive, rapid, and specific and could, as such, serve as monitoring tools for lead-contaminated water.The use of lead in manufacturing has decreased significantly over the last few decades. However, previous widespread use of lead-containing products and their incorrect disposal has resulted in environmental contamination. Accumulation of harmful quantities of lead pose a threat to all living organisms, through inhalation, ingestion, or direct contact, resulting in lead poisoning. This study utilized synthetic biology principles to develop plasmid-based whole-cell bacterial biosensors for detection of lead. The genetic element of the lead biosensor construct consists of  Lead po’s crust ,5. Damag’s crust . The Worng water . Detection of heavy metals in the environment requires use of analytical techniques, including atomic absorption spectrometry and inductively-coupled plasma-atomic emission spectrometry . These mpbrTRABCD) was identified on a mobile genetic element, the pMOL30 plasmid of Ralstonia metallidurans CH34  [pbrABCD operon is regulated by a transcriptional regulator, PbrR, belonging to the MerR family of metal ion-sensing regulatory proteins. Similar lead loci (pbrRABC) have also been identified on other plasmids, such as the pLVPK virulence plasmid from Klebsiella pneumoniae [Enterobacter cloacae [Bacteria respond rapidly to low concentrations of heavy metals in comparison to other members of soil biota; therefore, they are of increasing importance for use in toxicity tests of groundwater, soil, and sediments ,12. Leadns CH34) . The loceumoniae  and the  cloacae .pbrR and its native divergent promoter region, Ppbr, to allow expression of the luciferase operon (luxCDABE). However, in addition to lead, the biosensor was also responsive to mercury, cadmium, and zinc [Alcaligenes eutrophus CH34 [Escherichia coli and Pseudomonas fluorescens [Staphylococcus aureus and Bacillus subtilis [The understanding of resistance mechanisms has enabled synthetic biologists to re-design the system to enable expression of a detectable and measurable output signal, which is directly proportional to the concentration of a specific analyte . A bioseand zinc . A rangehus CH34 , Escheriorescens , and Grasubtilis ,20.pbrR and its divergent promoter region in lead biosensor constructs, and their potential for differential response in various Gram-negative bacteria. A plasmid-based, lead biosensor construct was developed and transferred into Pseudomonas aeruginosa PAO1, Shewanella oneidensis MR-1 and two wild-type Enterobacter sp. isolated from heavy metal contaminated soil. The latter two were selected because of their environmental relevance and bioremediation abilities [pbrR gene and its native divergent promoter region (Ppbr) from the pLVPK plasmid of K. pneumoniae CG43, in addition to a promoterless gfp gene. Quantitative analyses were utilized to determine the limit of detection, the response time and the specificity to lead nitrate (Pb(II)) of the five microbial biosensors. To determine the feasibility of the microbial biosensors for in situ studies, two spiked environmental samples, tap water and groundwater, were tested. Previously, lead biosensors have only been tested in response to lead in soil samples [The aim of this study was to develop lead microbial biosensors that could be used for ecotoxicological assessment of lead-polluted environmental samples. Furthermore, we aimed to increase knowledge of the use of bilities . The bio samples , therefoE. coli DH5α and P. aeruginosa PAO1 were grown at 37 °C, while S. oneidensis MR-1, Enterobacter sp. NCR3 and Enterobacter sp. LCR17 were grown at 28 °C. All strains were cultured on nutrient agar (NA) and in nutrient yeast broth (NYB) supplemented with antimicrobial agents, as necessary. NA contains 3.5% blood agar base (w/v) (1.5% (w/v) agar, 1% (w/v) ‘Lab-Lemco’ powder, 1% (w/v) peptone, 0.5% (w/v) NaCl) and 0.5% (w/v) yeast extract. NYB contains 2.5% (w/v) nutrient broth and 0.5% (w/v) yeast extract.The bacterial strains and plasmids used in this study are listed in 2O) at 104 µg·mL−1. Antimicrobial agents were freshly diluted in dH2O to appropriate concentrations as necessary. Selection for the transfer of pCR2.1®-TOPO® TA cloning vector  and the broad-host range plasmid, pBBR1MCS-5, was achieved on NA containing ampicillin (100 µg·mL−1) and gentamicin sulfate (10 µg·mL−1), respectively. A nalidixic acid resistant (7 µg·mL−1) laboratory strain E. coli DH5α was used for construction of the lead biosensor construct. Heavy metals used for testing of the biosensors included: sodium arsenite (As(III)), cadmium chloride (Cd(II)), copper chloride (Cu(II)), chromium oxide (Cr(VI)), lead nitrate (Pb(II)), mercury chloride (Hg(II)) and zinc chloride (Zn(II)). Antibiotics  were stored at 4 °C and heavy metal compounds were stored at room temperature. All antimicrobial agents were obtained from Sigma-Aldrich Pty Ltd. . Stocks of antibiotics and heavy metals were prepared with sterile distilled water (dHpbrR gene (435 bp) and the divergent promoter region  that it regulates from K. pneumoniae CG43 plasmid pLVPK  and a promoterless gfp gene , was synthesized by Integrated DNA Technologies. The synthesized fragment (1240 bp) was cloned into pCR® 2.1-TOPO® TA  (3931 bp) and subsequently subcloned into the EcoRI ) site of pBBR1MCS-5 (4768 bp). Blue-white screening was employed to detect the vector containing the recombinant DNA . Isolated plasmid DNA was subjected to EcoRI restriction digestion at 37 °C for 1 h and gel electrophoresis to visualise the release of the correct sized fragment. Both pTOPOpbrRgfp and pBBpbrRgfp were Sanger sequenced at the Australian Genome Research Facility to confirm the genetic element cloned.The genetic element containing the E. coli WM3064(pBBpbrRgfp)) and recipient  NYB-cultures (~109 cfu·mL−1) were mixed and filtered through a nitrocellulose membrane filter . The filter with the bacteria facing uppermost was then placed onto the surface of a pre-warmed NA plate. After incubation (2–3 h for P. aeruginosa PAO1 or 4–5 h for S. oneidensis MR-1 and Enterobacter spp.) the bacteria were resuspended in 2 mL NYB, serially diluted  and aliquots (100 µL) plated onto selective NA containing gentamicin sulfate at 10 µg·mL−1, allowed to dry and incubated under appropriate conditions. Aliquots (100 µL) of donor and recipient cultures were plated separately onto the same selective medium to serve as negative controls.Aliquots (1 mL) of exponential phase donor (600 nm = 0.6–0.8) of lead biosensors (E. coli DH5α(pBBpbrRgfp), P. aeruginosa PAO1(pBBpbrRgfp), S. oneidensis MR-1(pBBpbrRgfp), Enterobacter sp. NCR3(pBBpbrRgfp) and Enterobacter sp. LCR17(pBBpbrRgfp)) containing antibiotics were induced with a concentration of 100 µg·mL−1 Pb(II) for 24 h to allow expression of GFP. An identical NYB-culture was left uninduced and also incubated for 24 h to serve as a negative control. After 24 h, a drop of culture (10 µL) was placed onto a glass slide and covered with a cover slip. The cover slip was firmly pressed onto the slide and sealed using nail polish. Samples were viewed using a Nikon Eclipse Ti-E inverted microscope. Phase contrast was used to view the bacterial cells, and the GFP filter (excitation 485 nm and emission 535 nm) was used to visualise the fluorescent cells at ×1000 magnification with oil immersion. Exponential-phase NYB-cultures  spiked with either 0, 2, or 5 µg·mL−1 Pb(II). The groundwater and tap water used were collected from a lake system and the laboratory tap in Victoria, Australia. Following up to 4 h of incubation, the cells were pelleted and washed three times in dH2O. For time detection assays cells were pelleted and washed at consecutive time intervals after induction. After washing, cells were aliquoted (200 µL) into black 96-well plates with clear flat bottom which allowed successive measurement of the relative fluorescence units (RFU) and optical density (OD600 nm) of the bacterial cells. A Clariostar plate reader was used for measurements of RFU  and OD600 nm.Pb(II) at varying concentrations  was added to exponential phase NYB-cultures of lead biosensors and incubated for an appropriate period of time with shaking in the dark. For environmental assays, 900 µL of exponential phase NYB-cultures of lead biosensors were added to either dH600 nm of triplicate samples was calculated to obtain the mean RFU per OD600 nm of each biological replicate. Standard error was determined through the use of the students t-test (p < 0.05), whereby the mean RFU per OD600 nm of the three biological replicates of the uninduced sample was compared to the other samples being tested. The p value was calculated by comparing the variability between the mean RFU per OD600 nm of the lowest concentration used during the fluorescence assay ) or from the earliest time point during time assays (0 min) to that of other concentrations used or time points, respectively. In each fluorescence assay the mean RFU per OD600 nm of the three biological replicates of the uninduced sample was subtracted from that of the induced samples /OD600 nm(induced) − RFU(uninduced)/OD600 nm(uninduced)). For each assay, three independent biological replicates were performed in triplicate, unless otherwise stated. The mean RFU and ODpbrR, Ppbr and a promoterless gfp gene (1240 bp) was cloned into the pCR2.1®-TOPO® TA cloning vector   was transformed into E. coli WM3064 and transferred via conjugation to P. aeruginosa PAO, S. oneidensis MR-1 and Enterobacter sp. NCR3 and Enterobacter sp. LCR17. E. coli DH5α was used for cloning the genetic element and was, therefore, also included in the assessment. The synthesized biosensor genetic element containing CA, USA) a and sub4768 bp; b. The reE. coli DH5α(pBBpbrRgfp) was conducted using fluorescence microscopy to ensure functionality of the pBBpbrRgfp construct prior to its transfer into other Gram-negative bacteria. Pb(II) (100 µg·mL−1) was added to an exponential phase E. coli DH5α(pBBpbrRgfp) NYB-culture and incubated for 24 h. An identical exponential phase NYB-culture, lacking Pb(II), served as a negative control. No fluorescent cells were observed in the uninduced NYB-culture (−1 Pb(II)), as OD600 nm was comparable between the samples (~1.2 OD600 nm for both samples). It was also noted that the fluorescent cells in the induced NYB-culture were not fluorescing at the same intensity.Initial qualitative analysis of -culture a. In con-culture b. Differ−1) of Pb(II) for 4 h at their optimal growth temperatures. It was noted that there were several differences in sensitivity between the five biosensors (P. aeruginosa PAO1(pBBpbrRgfp) was found to respond to the lowest concentration of Pb(II) at 0.2 µg·mL−1 (Enterobacter spp. wild-type isolates detected 1 µg·mL−1 Pb(II) (S. oneidensis MR-1(pBBpbrRgfp) detected 0.5 µg·mL−1 Pb(II)  d,e while1 Pb(II) c.For the developed lead biosensors to complement current analytical techniques it is essential that they are able to rapidly detect Pb(II). As such the five lead microbial biosensors constructed were tested for their ability to detect varying concentrations of Pb(II) over different time intervals. The RFU were measured every 10–20 min intervals until a statistically significant increase from time point 0 was established. All five microbial biosensors were found to respond within 30–100 min after induction with Pb(II) .P. aeruginosa PAO1(pBBpbrRgfp) was also the most specific  was only able to detect Pb(II) at statistically significant levels following 4 h incubation. The other four lead biosensors detected Hg(II) at the same concentration as Pb(II). Enterobacter sp. NCR3(pBBpbrRgfp) was unexpectedly found to produce significantly higher expression levels in response to 1 µg·mL−1 of Hg(II), in comparison to Pb(II) (E. coli DH5α(pBBpbrRgfp) and Enterobacter sp. LCR17(pBBpbrRgfp) also detected Cr(VI) at 5 µg·mL−1.Previous studies have noted the lack of specificity of lead biosensors; therefore, the specificity of the five biosensors was investigated using a range of heavy metals. The most sensitive biosensor, specific b. At 0.2o Pb(II) d. Furthe−1 Pb(II). All five biosensors were able to detect 1 µg·mL−1 Pb(II), regardless of the water sample (S. oneidensis MR-1(pBBpbrRgfp) the biosensors were able to detect 5 µg·mL−1 Pb(II), however, it was noted that an increase in Pb(II) concentration did not correlate with an in increase in RFU. No significant difference (p > 0.05) was observed when comparing growth (OD600 nm) of the cells exposed to 0, 1, or 5 µg·mL−1 Pb(II) of a given water sample except for Enterobacter sp. LCR17(pBBpbrRgfp). The reason for the observed difference in cell density between the control and tested samples is unclear.To determine the effectiveness of the five lead biosensors to detect Pb(II) in environmental samples, tap water and groundwater, were spiked with 1 and 5 µg·mLr sample . With thpbrRgfp) was developed and introduced into several Gram-negative bacterial species, which were then exposed to a range of Pb(II) concentrations for various time periods. Like many other heavy metals, lead can also exist in a number of different forms, oxides and hydroxides, as well as oxyanion complexes that are released into the soil and groundwater. The most common form, Pb(II), was chosen for testing of the biosensors in addition to several other heavy metal ions to investigate the specificity of the developed biosensors.In this study, a lead biosensor construct (pBB−1 Pb(II) following 4 h incubation. P. aeruginosa PAO1(pBBpbrRgfp) was deemed the most sensitive. This may be due to a higher expression of GFP in Pseudomonas, as a consequence of reduced transcriptional repression by PbrR. Upon exposure to Pb(II) for 10 h, the sensitivity of the Pseudomonas lead biosensor increased from 0.2 to 0.05 µg·mL−1 (data not shown). It can, thus, be proposed that, upon further incubation, the Pseudomonas lead biosensor could detect below 0.01 µg·mL−1, which is the WHO guideline level for Pb(II) in drinking water [A. eutrophus luminescent lead biosensor detected ~331 µg·mL−1 Pb (compound not specified) [S. aureus and B. subtilis luminescent biosensors (pcadClucFF) detected 0.01 µg·mL−1 Pb(CH3COO)2 after ~2 h exposure [The five lead biosensors developed in this study were able to detect between 0.2 and 1 µg·mLng water . The limecified) . In a seexposure . Some ofexposure . Further−1) among the lead biosensors were further highlighted upon analysis of response times following exposure to Pb(II). Out of the five biosensors, E. coli DH5α(pBBpbrRgfp) and Enterobacter sp. LCR17(pBBpbrRgfp) responded quickest at 30 and 40 min, respectively. However, this may be attributed to differences in the growth rate of the microorganisms, as well as the different concentrations of Pb(II) used to test the response times. For example, E. coli DH5α(pBBpbrRgfp) was exposed to 5 µg·mL−1 whereas P. aeruginosa PAO1(pBBpbrRgfp) was exposed to 0.2 µg·mL−1. Response times are highly dependent on the concentration of the heavy metal used and the growth rate of the microorganism. Although a constant concentration could have been used, due to the variability of the lead biosensors’ response to Pb(II), the lowest detectable amount or above was chosen for testing the response times in this study. Previous studies have reported only induction time for lead biosensors tested [Slight variation of detection abilities  following exposure for 30–100 min. Additionally, the biosensors were able to detect at least one heavy metal ion other than Pb(II), in most instances Hg(II). E. coli DH5α(pBBpbrRgfp) and Enterobacter sp. LCR17(pBBpbrRgfp) were also responsive to Cr(VI). Interestingly, the response of lead biosensors to Cr(VI) has not previously been reported. Similarly, reported Cr(VI) microbial biosensors were not tested for response to Pb(II) [S. aureus and B. subtilis luminescent lead biosensors detected Cd(II), Hg(II), Mn(II), Sb(III), Sn(II), and Zn(II) [S. aureus and B. subtilis lead biosensors, The A. eutrophus luminescent lead biosensor only detected Pb (compound not specified) [−1) used for testing. Interestingly, Enterobacter sp. NCR3(pBBpbrRgfp) exhibited an elevated response to Hg(II) in comparison to the same concentration of Pb(II). As Hg(II) is generally more toxic than Pb(II), up-regulation of native Hg(II) resistance mechanisms may be responsible for increased uptake and binding of Hg(II) over Pb(II). In nature, the ability to sense a range of heavy metals in the surrounding environment may be hypothesized to provide microorganisms a competitive advantage, resulting in enhanced survival.All five lead biosensors developed could detect low levels of Pb(II) (0.2–1 µg·mLo Pb(II) . Nevertho Pb(II) . For exad Zn(II) ,20. The ecified) . This hi−1 Pb(II) in tap water and groundwater. Testing of biosensors in environmental samples which have not been treated via sterilization or filtering is useful to determine the effects of complex environmental samples with resident microbial community. Our findings indicate that short-term exposure of the biosensors to contaminated water samples yield a response to Pb(II). Organic compounds and resident microbial communities in the water samples did not affect the functionality of the lead biosensors. The difference in absolute RFU values between fluorescence assays of the same bacterial species may be explained by the natural variability of the cultures. Overall, the lead biosensors are sensitive, rapid, and generally specific for Pb(II). As such, they could serve as monitoring tools for in situ ecotoxicological assessment of lead-polluted environmental samples. The findings in this study have highlighted variations among biosensors utilizing the same genetic element and, as such, differences between bacterial species should be addressed in future microbial biosensor studies. Future biosensor research should focus on utilizing organisms involved in bioremediation processes, such as immobilization or phytoremediation, and incorporating the biosensor genetic element into their chromosomes. It would also be beneficial for future biosensor studies to develop a more high-throughput method for analysis of microbial biosensors and for detection of Pb(II) in other environmental samples, such as wet weather effluent. This may be performed in parallel to analytical methods for comparison of accuracy and feasibility of the lead biosensors to detect Pb(II) in various environmental samples. The potential of lead biosensors developed for in situ applications was assessed. All five lead biosensors detected 1 µg·mL


As low as micromolar range concentrations of puerarin significantly inhibited Kv7.1 and IKs. These inhibitory effects may due to the direct inhibition of puerarin upon channels not via the PKA-dependent pathway. These results provided direct preclinical evidence that puerarin prolonged APD via its inhibitory effect upon Kv7.1 and IKs, contributing to a better understanding the mechanism of puerarin cardioprotection in the treatment of cardiovascular diseases.Puerarin, a known isoflavone, is commonly found as a Chinese herb medicine. It is widely used in China to treat cardiac diseases such as angina, cardiac infarction and arrhythmia. However, its cardioprotective mechanism remains unclear. In this study, puerarin significantly prolonged ventricular action potential duration (APD) with a dosage dependent manner in the micromolar range on isolated rat ventricular myocytes. However, submicromolar puerarin had no effect on resting membrane potential (RMP), action potential amplitude (APA) and maximal velocity of depolarization (Vmax) of action potential. Only above the concentration of 10 mM, puerarin exhibited more aggressive effect on action potential, and shifted RMP to the positive direction. Millimolar concentrations of puerarin significantly inhibited inward rectified K Pueraria lobata) have been used as a Chinese herbal medicines to treat cardiovascular diseases for thousand years. Puerarin, a known isoflavone, was commonly found as an important component in the kudzu root. It is the 8-c-glucoside of daidzein245The kudzu root (2-kinase (JNK) signaling pathways to retard the progress of cardiac dependent NADPH oxidase activation and mediated downstream redox-sensiutive-AP-1 signaling pathways to achieve its anti-hypertrophic effect10+ channel by shifting the steady-state inactivation curve without alternation in the shape of the I-V curve of INaHowever, the mechanism of this anti-arrhythmia effect of puerarin remains unclear and less study is performed to illustrate the detailed mechanism of puerarin effect on cardiac electrical signal transmission. Puerarin was previously found to inhibit phosphatidylinositol 3-kinase (PI3 K)/protein kinase B (Akt) and c-jun-NHTherefore, in this study, we concentrate upon the effect of puerarin on cardiac action potential and related ion channels in ventricles.50 from (71.8 ± 11.8) mS to (100.5 ± 14.1) mS and APD90 from (164.6 ± 21.4) mS to (221.6 ± 25.7) mS (Puerarin is known as an isoflavone  that is 25.7) mS ; whereasThe results above indicated that puerarin apparently affected the repolarization phase of action potential, which are majorly contributed by IKs , IKr + blockage2+ blockage (data not shown). The ramp voltage was a classic protocol to clearly observe the inward rectification of K+ current below its reverse potential50 = 1.27 mM, Hill coefficient is 0.990 (n = 5 each point)  .Kir. Kir2.3 channels were expressed in HEK-293 cells to further clarify the effect of puerarin on Kir2. Puerarin at the concentrations of 10 mM, 50 mM, 100 mM, 200 mM significantly inhibited Kir2.3 currents illustrated by the ramp voltage protocol, respectively. This effect could also be reversed by washing-off compound  could augment Kv7.1 and IKs via a PKA pathwayIn the inside-out recording model, puerarin was applied directly to the intracellular side of ion channels and dissociated of puerarin in activities due to cellular barrier limitations. Single channel currents of Kv7.1 possessed the conductance of 4.4 pS elicited at the different voltages . The opeSingle channel currents of IKs possessed the conductance of 15.9 pS elicited at the different voltages . The ope50 = 8.02 μM, Hill coefficient is 0.983 (n = 5 each point)   . In addih point) , and thih point) .+ channels, and exhibited bigger effect upon Kir2.1 vs Kir 2.3. These results provided direct evidence and mechanism that puerarin prolonged APD majorly via its inhibitory effect upon Kv7.1 and IKs, supporting the widely clinical application of puerarin in treatment of cardiovascular diseases.In this study, puerarin significantly prolonged ventricular APD when the concentration as low as micromolar range and exhibited a dosage dependent inhibitory manner. However, submicromolar puerarin had no effect on resting RMP, APA and Vmax of action potential. Only above the concentration of 10 mM, Puerarin exhibited more aggressive effect on action potential, and shifted RMP to the positive direction. In addition, as low as micromolar range of concentrations, puerarin significantly inhibited Kv7.1 and IKs. These inhibitory effects were due to the directly inhibition of puerarin upon channels rather than the PKA pathway. In the millimolar range of concentrations, puerarin significantly inhibited inward rectified KWe also demonstrated that puerarin could reverse 10 nM ISO-mediated AP shortening on rat ventricular myocytes. Besides this cellular model of arrhythmia, several studies had announced puerarin could accelerate cardiac angiogenesis and improve cardiac function of myocardial infarction rats by upregulating VEGFA, Ang-1 and Ang-2+ channels50 and saturation. At a micromolar concentration, puerarin inhibited KvLQT1 and IKs and when application of milimolar puerarin, it affected Kir2.1, Kir2.3, KvLQT1 and IKs. Prolonged APD played a key role in slowing down heart rate and lengthening the refractory period of myocytes, these results might explain possible mechanism of action in the treatment of arrhythmia. Very recent comprehensive analysis of regional ion channel expression in normal and myopathic hearts documented that the ratio among Kir2.1, Kir2.2 and Kir2.3 was roughly around 13:7:8 in normal left ventricles and around 16:5:13 in myopathic left ventriclesV7.1/IKs in the procession of myopathic path might also occur, illustrating distinct pharmacology effects of puerarin in different ion channel expression profiles in this physiol-pathological process. In different patients, puerarin might exhibit different treatment efficiency due to the cardiac profiles with different ion channel composition.In addition to previous studies, which suggested that puerarin affected calcium-activated potassium channelsIn the heart, IKs had an important role in cardiac action potential repolarization39To date, nearly three hundred mutations in KCNQ1 and at least 20 mutations in KCNE1 have been identified, casing long QT syndrome (LQT), short QT syndrome (SQT), sinus bradycardia and atrial fibrillation (AF)2 to bind with Kir.Our results were consistent to a previous study in the conclusion that puerarin could inhibit inward rectified potassium channelsScutellaria baicalensis2+ -activated K+ channel (BK(Ca)) and inhibiting voltage-dependent Ca2+ channel (VDCC) in a concentration-dependent manner. The cGMP/PKG and cAMP/PKA pathways had been known in the regulation of these two channels. Therefore, further study was necessary to confirm the additional and disparate effect between puerarin and baicalin on cardiac arrhythmia.Interestingly, baicalin is a flavone, an analog of puerarin but extracted from another Chinese medicinal herb named In summary, the present study provides the novel inhibitory effect of puerarin on Kv7.1/IKs underlining the mechanism of extending APD and anti-arrhythmic activity. Puerarin may directly inhibit Kv7.1/IKs channel independent of PKA activation, and it certainly inhibits Kir2.1 and Kir2.3 channels in some extent, suggesting the rational and discreet application of puerarin in clinic treatment of cardiovascular diseases.2-ATP, HEPES, EGTA, baicalin, isoproterenol, forskolin, the cAMP analog 8-Bromoadenosine-3′, 5′-cyclic monophosphorothioate were purchased from Sigma-Aldrich . Collagenase (type II) and bovine serum albumin (BSA) were purchased from Invitrogen . All other reagents were of analytical grade. All drugs and reagents were dissolved in distilled water unless otherwise noted. Isoproterenol (1 mM) was dissolved in 0.1% ascorbic acid in distilled water. Baicalin (10 mM), forskolin (100 mM) was dissolved in DMSO. Final DMSO concentrations did not exceed 0.1%.Puerarin was obtained from Beijing Saisheng Pharmaceutial Company (98.4% purity), taurine, KFifty male Sprague-Dawley rats (10–12 weeks) were used for this experiment. Myocytes were prepared from ventricles by standard collagenase dissociation technique. All animals received humane care in compliance Guide for the Institutional Animal Care and Use Committee (IACUC) and was approved by the Ethics Review Board for Animal Studies of Institute of Molecular Medicine (IMM), Peking University). All methods were carried out in accordance with the approved guidelines.2, 1 MgCl2, 0.33 NaH2PO4, 10 HEPES and 10 glucose, pH 7.4 (adjusted with NaOH). The Ca2+-free Tyrode’s solution was prepared by removing CaCl2 from the Tyrode’s solution. The Kreb’s solution used for cell storage contained (mM): 70 glutamic acid, 30 KCl, 15 taurine, 0.5 MgCl2, 10 KH2PO4, 0.5 EGTA, 10 HEPES, 10 glucose, and 1% albumin, pH 7.4 (adjusted with KOH). The enzyme solution used for the rat cardiomyocyte isolation contained 0.08 g/L collagenase (type II) and 1.0 g/L BSA in Kreb’s solution. For action potential recording on cardiomyocyte, the pipette solution contained (mM): 100 potassium aspartate, 30 KCl, 1 MgCl2, 5 Mg-ATP, 5 EGTA, 5 HEPES, pH 7.3 (adjusted with KOH); the Tyrode’s solution was used as the standard bath solution. For whole-cell recording of IKs on myocytes, the pipette solution contained (mM): 125 potassium aspartate, 20 KCl, 1 MgCl2, 5 Mg-ATP, 10 EGTA, 5 HEPES, pH 7.3 (adjusted with KOH); the bath solution contained (mM): 132 NaCl, 4 KCl, 1.8 CaCl2, 1.2 MgCl2, 10 HEPES, 5 glucose, pH 7.4 (adjusted with NaOH). 3 μM nifedipine and 5 μM E-4031 were used to block the calcium current and IKr, respectively. For whole-cell recording on HEK-293 cells, the pipette solution contained (mM): 140 KCl, 2 MgCl2, 10 HEPES, 0.1 EGTA, 4 K2-ATP, pH 7.3 (adjusted with KOH); the bath solution contained (mM): 120 NaCl, 5 KCl, 1.5 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, pH 7.4 (adjusted with NaOH). For single-channel recording, the pipette solution (extracellular) contained (mM): 145 KCl, 10 HEPES, 10 glucose, pH 7.4 (adjusted with KOH); the bath solution (intracellular) contained (mM): 145 KCl, 1.2 MgCl2, 10 HEPES, 0.1 EGTA, pH 7.38 (adjusted with KOH).The Tyrode’s solution contained (mM): 126 NaCl, 5.4 KCl, 1.8 CaCl2+-free Tyrode’s solution for 5 min before the perfusate was switched to an enzymatic solution for 10–20 min. The perfusate was finally changed to Kreb’s solution for 5 min. The perfusates were bubbled with 95% O2 + 5% CO2 and maintained at 37 °C. The ventricles w ere cut into small chunks and gently agitated in Kreb’s solution. The cells were filtered through nylon mesh (pore size 200 μm) and stored in Kreb’s solution at 4 °C.Single ventricular myocytes were isolated from adult rat hearts via enzymatic dissociation as previously described50 and APD90), RMP, APA and Vmax were measured.Action potentials were elicited at 1 Hz by 2 ms current pulses applied using the patch pipette. The APD at 50% and 90% repolarization . The whole-cell recordings were performed using isolated single ventricular myocytes. Patch electrodes were pulled from a horizontal microelectrode puller  and fire polished to final tip resistance of 4.0–8.0 MΩ when filled with internal solutions. Signals were amplified with an Axopatch 200B amplifier  and filtered at 1 kHz. The pCLAMP 9.0 software was used to generate current and voltage clamp protocols, acquire data, and analyse the current traces. The whole-cell series resistance was compensated to more than 80%. All of the experiments were performed at room temperature (22 ± 1 °C). The whole-cell IK1 was elicited by a ramp voltage protocol from −140 to + 40 mV in 1 s, or at a 0 mV holding potential to test potentials from −140 to + 40 mV in 10 mV increments. The IK1 was calculated as the difference between the peak inward current and the holding current level. The whole-cell Kv7.1/IKs were elicited from a −30 mV holding potential to test potentials from −80 to + 40 mV in 10 mV increments.Single-channel activity was recorded at a series of holding levels ranging from 10 mV of 50 mV. The data were acquired at 20 KHz and lowpass filtered at 5 kHz. During post-analysis, data were further filtered at 300 Hz. Single-channel events were listed and analysed by pclampfit 9.0 . NPo, the product of the number of channels and the open probability, was used to measure the channel activity within a patch. In total, 50% threshold cross-method was used to determine valid channel openings. Initial (1–2 min) single-channel records were normally used as the control. The activity of Kv7.1/IKs during application of chemicals was normalized to activity during the control period to assess the effects of chemicals on Kv7.1/IKs activity.Human Kir2.1 cDNA, Kir2.3 cDNA, Kv7.1 cDNA and KCNE1 cDNA, was cloned in Human Embryonic Kidney 293 cells, respectively. In some cases, Kv7.1 cDNA and KCNE1 cDNA were simultaneously cloned in Human Embryonic Kidney 293 cells.All of the cDNAs above were confirmed with DNA sequencing .cAMP levels were measured in rat cardiomyocytes using a Mouse/Rat cAMP kit  according to the manufacturer’s instructions. The assay is based on the competitive binding technique. A streptavidin-coated plate is incubated with a biotinylated monoclonal antibody specific for cAMP. Following a wash to remove excess monoclonal antibody, cAMP present in a sample competes with a fixed amount of horseradish peroxidase (HRP)-labeled cAMP for sites on the monoclonal antibody. This is followed by another wash to remove excess conjugate and unbound sample. A substrate solution is added to the wells to determine the bound enzyme activity. The color development is stopped and the absorbance is read at 450 nm. The intensity of the color is inversely proportional to the concentration of cAMP in the sample. The level of cAMP in the sample was determined based on a standard curve and expressed as pmol/mg per each sample. Normalized cAMP was calculated by compared with the control group. In our study, we investigate the effect of puerarin on intracellular cAMP concentration and its effect upon the increase of cAMP induced by isoproterenol, forskolin and 8-Br-cAMP.The data were presented as the Mean ± SD. Dosage curves were fitted with pCLAMP 9.0 (Axon Instruments) and software Origin 7.0 . The statistical significance was determined using a t-test when comparing two groups and ANOVA when comparing multiple groups. A value of P < 0.05 was considered statistically significant.How to cite this article: Xu, H. et al. The Effects of Puerarin on Rat Ventricular Myocytes and the Potential Mechanism. Sci. Rep.6, 35475; doi: 10.1038/srep35475 (2016).


Penthorum chinense Pursh , a well-known Miao ethnomedicine, has been traditionally used to treat several liver-related diseases, such as jaundice and viral hepatitis. The aims of the present study were to evaluate the probable properties of the aqueous extract of PCP on carbon tetrachloride (CCl4)—induced acute liver injury in mice.4 exposure. Silymarin serves as a positive drug to validate the effectivenes of PCP.C57BL/6 mice were orally administered an aqueous extract of PCP (5.15 and 10.3 g/kg BW) or silymarin (100 mg/kg) once daily for 1 week prior to CCl4 exposure caused severe acute liver injury in mice, as evidenced by the elevated serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alanine phosphatase (ALP), and the increased TUNEL-positive cells in liver, which were remarkably ameliorated by the pretreatment of PCP. PCP was also found to decrease the levels of malondialdehyde (MDA), restore the glutathione (GSH) and enhance the activities of superoxide dismutase (SOD) and catalase (CAT) in the liver. In addition, the pretreatment of PCP inhibited the degradation of hepatic cytochrome P450 2E1 (CYP2E1), up-regulated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its target proteins in CCl4-treated mice.A single dose of CCl4-induced acute liver injury, which was comparable to efficacy of silymarin (100 mg/kg). This hepatoprotective effects might be attributed to amelioration of CCl4-induced oxidative stress via activating Nrf2 signaling pathway.Results indicated that the pretreatment of PCP (10.3 g/kg BW) effectively protected against CClThe online version of this article (10.1186/s13020-017-0153-x) contains supplementary material, which is available to authorized users. These free radicals may cause cellular DNA damage and the increased lipid peroxidation by reacting with cellular unsaturated lipids, leading to hepatocyte apoptosis and necrosis, which ultimately results in liver injury [Oxidative stress is well known to be involved in the pathogenesis of acute or chronic liver injury . The ove4) . As a chl models . CCl4 is4-induced liver injury.Antioxidant defense system, including non-enzymatic and enzymatic mechanisms, is principally responsible for protecting living organism from oxidative stress . Among tPenthorum chinense Pursh , has been traditionally used as the Miao ethnomedicine and folk remedy in the treatment of liver-related diseases, including jaundice and viral hepatitis for a long time [4-induced liver injury has not been adequately addressed. Therefore, in this study, we aimed to evaluate the probable protective properties of PCP against acute liver injury induced by CCl4, and further elucidate its underlying mechanisms with respect to Nrf2-mediated antioxidant response.ong time . PCP is ong time . Our preong time  and chroong time . Howeverd-glucoside, the chemical marker, was measured as 3.49 mg/g in raw PCP by HPLC–UV. The voucher specimen of PCP sample (No. GHX201401) was stored at Institute of Chinese Medical Sciences, University of Macau, Macau.Aerial part of PCP was provided by Sichuan New Lotus Traditional Chinese Herb Limited Company . Its botanical origin was identified by Dr. Chun-Feng Qiao from our university. The aqueous extract of PCP was prepared as previously described , 12. The4 group, silymarin-treated group , two PCP-treated groups (5.15 and 10.3 g/kg BW). The dose of PCP (10.3 g/kg) was calculated from the usage of Gan-Su-Ke-Li (WS3-B-2526-97), a drug approved by China Food and Drug Administration (CFDA), which was made from the aqueous extract of PCP for the management of viral hepatitis. Mice were gavaged silymarin or PCP once daily for 1 week prior to CCl4 challenge in treatment group. 24 h after last dosing, animals were intraperitoneally injected with 10% CCl4 diluted in olive oil  to induce acute liver injury [i.p.). After fasting for 12 h, all mice were anesthetized, and serum samples and the entire liver tissues were immediately collected. Animal protocol was conducted according to the animal procedure approved by Animal Ethics Committee, Institute of Chinese Medical Sciences, University of Macau (ICMS-AEC-2015-05). The Minimum Standards of Reporting Checklist  were housed in institutional individually ventilated cage (IVC) system. All animals were assigned randomly to five groups , i.e. control group, CClr injury , mice inSerum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alanine phosphatase (ALP) were examined through enzymatic colorimetric methods by their respective commercial assay kits  according to the manufacturer’s instructions. For the measurements of AST and ALT, serum was mixed well with corresponding matrix solution, and then reacted with 2,4-dinitro-phenylhydrazine for 20 min. NaOH solution (4 mol/L) was added to terminate the reaction. Results were measured by SpectraMax® M5 Multi-Mode Microplate Reader .v/v) phosphate-buffered formalin overnight, and embedded in paraffin. The cryostat sections were stained with hematoxylin and eosin according to a standard protocol [The right lobe of the liver tissue was fixed in 10%  on ice. The liver homogenates (10%) were centrifuged, and the final supernatants were subjected to measure the levels of malondiadehyde (MDA), reduced glutathione (GSH), oxidized glutathione (GSSG), the activities of superoxide dismutase (SOD) and catalase (CAT) by their respective assay kits (Jiancheng Bioengineering). Protein content in the homogenates was determined using a Pierce™ BCA Protein Assay Kit . The results were normalized per gram total protein.® Plus In Situ Apoptosis Fluorescein Detection Kit . Briefly, liver sections (4 μM) were fixed in 1% paraformaldehyde solution, then incubated in green fluorescein labelled dUTP solution at 37 °C for 1 h. After washing, the sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI) . The apoptotic cells were visualized on Zeiss Axio Imager A2 microscope .The apoptotic cells in liver cryostat section were evaluated by a commercial ApopTag® Green PCR Master Mix (Life Technologies). The mRNA expression was normalized to β-actin.The transcriptional expressions of CYP2E1, Keap1, HO-1 and GCLC in liver were determined by qPCR as previously described , 11. In The total protein was isolated from the left lobe of the liver tissues by the cold RIPA lysis buffer containing 1% phosphatase inhibitor cocktail (Beyotime Institute of Biotechnology). Approximately 60 μg of total proteins were loaded onto 10% SDS-PAGE, subsequently transblotted onto PVDF membranes . After blocking with 5% nonfat dry milk in TBST , the membranes were incubated with primary antibodies for 24 h at 4 °C, including CYP2E1 , Keap-1 , Nrf2 , HO-1 , GCLC  and GAPDH , then incubated with secondary antibodies conjugated to horseradish peroxidase (HRP) for 1 h. The proteins were visualized by Amersham ECL Select Western Blotting Detection Reagent .The value was expressed as mean ± SD. After verifying the distribution of the data by Kolmogorov–Smirnov test, the between-group comparison was conducted by ordinary one-way analysis of variance (ANOVA) using GraphPad 5.0 Software .4 exposure caused severe hepatotoxicity in mice , 42.2-fold (6.02 ± 3.61 vs. 260.3 ± 60.0 U/L) and 63.1% (101.2 ± 17.3 vs. 165.1 ± 23.8 U/L), respectively, compared with control group. However, these elevations were decreased significantly (p < 0.05) by pre-treatments of PCP at the doses of both 5.15 and 10.3 g/kg BW, and silymarin, a positive control, as well.The serum levels of AST, ALT and ALP, the commonly-used biomarkers of liver damage in the clinics , were meice Fig. , the ser4 challenge caused marked histopathological changes in the liver, characterized by apparent microvesicular and macrovesicular steatosis, massive inflammatory cells infiltration, and extensive hepatocyte necrosis. These histopathological changes were remarkably ameliorated by the pre-treatments of silymarin and PCP (10.3 g/kg BW), which were consistent with the results of serum parameters.Histological observations were performed to examine the pathological changes in liver. As shown in Fig. 4-induced liver injury [4 challenge, the number of TUNEL-positive cells in liver section was obviously increased over the control group, the apoptotic cells were greatly decreased in the pre-treatment of PCP (10.3 g/kg BW) or silymarin.As hepatocyte apoptosis also reflects the extent of CClr injury , a TUNEL4 exposure, the levels of MDA, GSH and GSSG, and the activities of SOD and CAT were examined in liver. As an end-product of lipid peroxidation (LPO), MDA was considered to be a useful marker of oxidative stress [4 exposure, which was decreased in both silymarin and PCP (10.3 g/kg BW)—treated groups. PCP (5.15 g/kg BW) showed a decreased concentration in hepatic MDA, but with no significant difference. CCl4 exposure also depleted endogenous antioxidants, as indicated by the level of GSH, and the activities of SOD and CAT in CCl4 group were significantly decreased to 50.5, 22.4 and 58.7%, respectively, compared to control group. These depletions were notably ameliorated by the pretreatment of silymarin and PCP (10.3 g/kg BW) , leading to the decrease in GSH/GSSG ratio (0.77 ± 0.23 vs. 0.25 ± 0.08)  or silymarin significantly reversed the expression of CYP2E1 in the protein level, but not the transcriptional level.As shown in Fig. 4-induced oxidative stress, the Nrf2 signaling pathway was measured by using immunoblot. Compared to control group, one single dose of CCl4 exposure decreased the protein expressions of Nrf2 in total, cytosol and nucleus, the pre-treatment of silymarin and PCP (10.3 g/kg BW) significantly normalized these reductions in Nrf2 expressions induced by CCl4 exposure  were comparable to silymarin (100 mg/kg BW), a positive control which is a herbal remedy for liver treatment with anti-inflammatory and antioxidant properties [4. This observation was also verified by histopathological examination and TUNEL assay as well.CClr injury . Serum lr injury . The eler injury . In our operties . These r4 is metabolized by CYP2E1 to produce the CCl4-derived free radical in the liver [4-induced oxidative stress also depletes the endogenous antioxidants, including the non-enzymatic group, such as GSH, and enzymatic antioxidants, such as SOD and CAT. It was documented that GSH is an important antioxidant in eliminating toxic free radicals and reactive toxic CCl4 metabolites [4 exposure caused severe oxidative stress in the liver where CCl4 is primarily metabolized, as evidenced by the increased hepatic levels of MDA and GSSG, the decreased GSH level, GSH/GSSG ratio and antioxidant enzyme activities of CAT and SOD. However, several compounds were identified from PCP, including flavonoids, flavonoid glycosides, polyphenols, steroids [4-induced oxidative stress by not only reducing hepatic MDA level but also enhancing endogenous non-enzymatic and enzymatic antioxidants.CClhe liver , 21. CClabolites , 23. Theabolites . In thissteroids . Among tsteroids , 27, whi4 to produce highly-reactive trichloromethyl free radicals [4-induced oxidative stress. CYP2E1 deficient mice are resistant to CCl4-induced hepatoxicity [4 exposure in rats [4-induced hepatotoxicity can be enhanced by the pretreatment of alcohol, a CYP2E1 inducer [4 challenge decreased the expression and activity of CYP2E1 [4 might labilize and inactivate CYP2E1, and enhance its degradation, which reveals ongoing oxidative damage [4 exposure, and the pretreatment of PCP at the doses of either 5.15 or 10.3 g/kg BW significantly elevated the CCl4-induced decrease of CYP2E1 expression in the protein level, but not in the transcriptional level, suggesting that PCP efficiently reduces the degradation of CYP2E1 induced by CCl4.Hepatic CYP2E1 is mainly responsible for the metabolism of CClradicals . Thus, Ctoxicity , CYP2E1  in rats . While,  inducer . Howeverf CYP2E1 –34. A mo4 exposure, the expressions of Nrf2 and its downstream genes in the liver were measured. Nrf2 plays a critical role in regulating antioxidant defense system in response to oxidative stress [4, ethanol, acetaminophen, pyrazole and arsenic [4 group, the hepatic Nrf2 expressions in total, cytosol and nucleus were significantly increased in PCP-treated group. As expected, the PCP-treated group showed higher expressions of Keap-1, HO-1 and GCLC in the liver.To understand how PCP reduces the oxidative stress induced by CCle stress . Nrf2-nu arsenic . In resp arsenic . The act arsenic . In the 4-induced acute liver injury, which was closely similar to efficacy of silymarin (100 mg/kg BW). These hepatoprotective effects might be associated with ameliorating CCl4-induced oxidative stress via activation of Nrf2 signaling pathway  can effectively protected against CClway Fig. .Fig. 8Sc


SLC26A2 gene cause a phenotype of multiple epiphyseal dysplasia-4 (MED-4). In the present study, we identified novel compound heterozygous mutations in the SLC26A2 gene in a Chinese family with two affected sibs with MED-4.Multiple epiphyseal dysplasia (MED) is a heterogeneous genetic condition characterized by variable phenotypes, such as short stature (mild to moderate), joint deformities, abnormal gait, scoliosis, and brachydactyly. Recessive mutations in the SLC26A2 c.824 T > C and SLC26A2 c.1198C > T were identified in two siblings in this family, which were inherited from both parents, one mutation from each.Radiographs revealed hip dysplasia, brachydactyly and scoliosis in patient 1. Radiological examinations in patient 2 also showed hip dysplasia recently. Both of them were diagnosed with MED-4. SLC26A2 mutations, and these results show that these novel compound heterozygous mutations in SLC26A2 contribute to MED-4.This is the first Chinese MED-4 family attributed to  Multiple epiphyseal dysplasia (MED) is a heterogeneous genetic condition characterized by variable phenotypes, such as short stature (mild to moderate), joint deformities, abnormal gait, and early-onset osteoarthritis . MED is COMP (MIM 600310) , C 120210) , COL9A2  120260) , COL9A3  MIM 12020 [MATN3  600972)  to the rclubfoot .SLC26A2. Further genetic studies and clinical evaluation of their parents revealed that these two mutations were from their father and mother, respectively. This study reported that compound heterozygous mutations in SLC26A2 contributed to MED-4.We present two siblings with MED-4 from an eastern Chinese family. Genetic analysis revealed compound heterozygotes for two novel heterozygous mutations in A 12-year-old girl was born normally to non-consanguineous, healthy eastern Chinese parents after a normal pregnancy. The body length at birth was 51 cm. She was referred to our hospital for diagnosis and treatment. There was no family history of endocrine diseases and musculoskeletal problems. Her parents noticed unequal leg lengths around the age of 6 years and radiography revealed coxa plana. Abnormal gait and limping were noticed at 9 years. The rotation function of the right leg was limited. For these reasons, she underwent right hip arthroplasty and resection of cartilago acetabularis in another hospital. Postoperative pathology revealed chronic synovitis. Half a year after the surgery, a lump with high skin temperature was noticed by her parents in the left femoribus internus. The flexion-extension function of the left leg was limited. Physical examination revealed the following: height 138 cm. Her intellectual development and hearing were normal. She had brachydactyly, bilateral skewfoot, and lumbosacral scoliosis. The movements of both hips were limited. She did not have a cleft palate, cephalofacial deformities, or respiratory insufficiency. Routine analysis for common skeletal dysplasia excluded any thyroid or growth hormone disorders and immunopathies. Upon analysis, bone metabolism appeared normal.Radiological documentation at the ages of 7 and 12 years revealed hip dysplasia with the following deformities: short femoral necks, flattened and irregular femoral heads, and early closure of epiphysis Fig. . Spinal A 6-year-old boy, the younger brother of patient 1, was also born to the same parents. The pregnancy was normal, and he was normal at birth without cleft palate or cephalofacial deformities. The body length at birth was 50 cm. An abnormal gait, waddling with short steps, was also noticed recently.Roentgenologic bone survey showed hip dysplasia with the following abnormalities: both femoral necks were short with flattened heads; acetabulum dysplasia, and secondary ossification center of femur dysplasia Fig. .Fig. 2HiCOL1A1, COL1A2, COL11A1, and other related genes, were analyzed by Targeted NGS in patient 1. The total size of the target regions of the capture array was 3.0 Mb. After filtering out common variants and neutral or benign mutations , the 1000 Genomes Pilot Project Data (http://www.1000genomes.org/) or BGI in-house database, which includes 1092 normal subjects.), two mutations, SLC26A2 c.824 T > C (NM_000112.3) and SLC26A2 c.1198C > T were identified  according to the manufacturer’s protocol. A total of 363 genes, including ied Fig. . Both vaied Fig. . Then saely Fig. .Fig. 3ThSLC26A2 mutation  in a 36-year-old man of tall-normal stature with MED-4 [COMP, COL9, and Matrilin-3.Multiple epiphyseal dysplasia is a heterogeneous group of skeletal dysplasias characterized by dysplastic epiphyses at multiple sites . Supertith MED-4 . Variablth MED-4 . A doublCOL1A1, COL1A2, COL11A1, SLC26A2, COMP, COL9A1, and other genes. Indeed, the younger brother harbored the same mutations in SLC26A2. We then suspected him to be at the early stage of MED-4, and we advised his parents to be cautious regarding the development of epiphyseal dysplasia in the future.In the present study, the presence of short stature, coxa plana, brachydactyly, abnormal gait, and scoliosis in Patient 1 led to the clinical diagnosis of MED-4. Very recently, the abnormal gait and coxa plana were noticed in the younger brother, Patient 2, at the same age as his elder sister did at 6 years. To verify the cause of the disease, we suggested their parents to authorize a genetic analysis using an available capture array, which covers 363 genes related to bone diseases, including SLC26A2 are related to a wide range of phenotypes, depending on the residual sulfate transporter activity. These phenotypes range in severity from the very severe achondrogenesis type IB, atelosteogenesis type II, and diastrophic dysplasia to the relatively mild recessive MED-4. The most common SLC26A2 mutation reported in several studies is homozygous c.835C > T (p.Arg279Trp) [Xenopus laevis oocyte expression system [SLC26A2 mutation, c.1957 T > A (p.Cys653Ser), in two unrelated patients with hip dysplasia, recurrent patella dislocation, and normal stature [SLC26A2 and a novel mutation, p.Ser522Phe, while her maternal grandfather was homozygous for the common mutation [SLC26A2 mutations were identified in Patient 1. These mutations, c.824 T > C (p.Leu275Pro) and c.1198C > T (p.Leu400Phe), are located in the extracellular loop (between amino acids 263 and 296) and the cytoplasmic loop (between amino acids 399 and 420), respectively. Both mutations were predicted to be functionally deleterious and missing in the above-mentioned databases. However, no functional studies have been undertaken for both mutations, so we do not know exactly if these are severe or mild mutations. Sanger sequencing confirmed that these mutations in Patients 1 and 2 came from their parents, one mutation from each. Clinical features and genetic analysis suggested that in this Eastern Chinese family, both patients were also compound heterozygotes for two novel SLC26A2 mutations.Mutations in g279Trp) . Karniskn system . Their r stature . Very remutation . Using aSLC26A2, c.824 T > C (p.Leu275Pro) and c.1198C > T (p.Leu400Phe).In conclusion, we present two patients of MED-4 with evolving clinical and radiological features. Skeletal surveys, joint complications, and genetic testing of their parents were found to be essential to understanding the mechanism. Both patients were compound heterozygotes for two unreported mutations in


All these interventions extend lifespan in multiple yeast backgrounds, revealing a poor correlation between rDNA silencing and longevity. In contrast, CR and deletion of the FOB1, HXK2, SCH9 and TOR1 genes, all significantly reduced rDNA recombination. This silencing-independent mechanism for suppressing rDNA recombination may therefore contribute to CR-mediated lifespan extension.Calorie restriction (CR) extends lifespan in yeast, worms, flies and mammals, suggesting that it acts via a conserved mechanism. In yeast, activation of the NAD-dependent histone deacetylase, Sir2, by CR is thought to increase silencing at the ribosomal DNA, thereby reducing the recombination-induced generation of extrachromosomal rDNA circles, hence increasing replicative lifespan. Although accumulation of extrachromosomal rDNA circles is specific to yeast aging, it is thought that Sirtuin activation represents a conserved longevity mechanism through which the beneficial effects of CR are mediated in various species. We show here that growing yeast on 0.05 or 0.5% glucose  does not increase silencing at either sub-telomeric or rDNA loci compared with standard (2% glucose) media. Furthermore, rDNA silencing was unaffected in the  In recent years, there has been an increasing recognition that similar fundamental cellular processes underlie aging in all eukaryotes . PerhapsSaccharomyces cerevisiae, have been at the forefront of recent efforts to understand the molecular mechanism of action of CR. Yeast lifespan can be measured in two ways. Replicative lifespan is defined as the number of buds produced by an individual yeast mother cell and is independent of calendar time  generation] downstream of Sir2  where the URA3 reporter gene is integrated in a nonsilenced genomic locus. Here, there was no difference in growth on selective media between the parent strain and its isogenic mutants: all grew similarly on −ura plates, none grew on FOA  and severe (0.05% glucose) CR produced a significant reduction in rDNA recombination of around 20%  Sirtuin-independent process. In both models, the longevity associated with reduced rDNA recombination is attributed to reduced ERC generation. While this remains a plausible mechanism, it is interesting to note that in fob1/sir2 double mutants, which have extremely low ERC levels, CR increases lifespan and reduces rDNA recombination  and Foremedium . 5-Fluoro-orotic acid (FOA) was obtained from Apollo Scientific . PCR primers were supplied by Sigma Genosys , genomic DNA isolation kits were from Invitrogen ; and PCR enzymes/reagents were from Promega . All other materials were obtained from Sigma-Aldrich.URA3 marker strain, AEY1017    (his3Δ200 leu2Δ1 ura3-167 ??::Ty1::mURA3)  . Silenci::mURA3) . As a co::mURA3) . Deletiostrategy . Success−1 and 1% DMSO final concentration. Single colonies were picked from a plate and grown overnight in 5 mL liquid YP media containing 2% glucose. The next morning, cultures were diluted to OD600 = 1 in sterile H2O in 1 mL. The adjusted cultures were serially diluted five times in sterile H2O at a ratio of 1:5 starting from OD600 = 1 in column 1 in a 96 well-plate to 100 μL final volume and plated with a replica plater onto SC, SC − ura and SC + FOA plates containing 0.05%, 0.5% and 2%, glucose and incubated at 30°C for 2 (0.5% and 2% glucose) to 4 days (0.05% glucose). Plates were imaged in a BioRad Universal Hood II Imager .For silencing assays, synthetic complete media (SC), uracil omission media (SC − ura) and FOA-supplemented media (SC + FOA) were prepared from 2× stock solutions and D-glucose added to 0.05%, 0.5% and 2% from a 50% stock solution. 5-Fluoro-orotic acid was prepared in DMSO and added to media to give a final concentration of 1 mg ml600 = 1 in 1 mL final volume. For silencing assays using the parent strains, cultures were then serially diluted in sterile H2O to 10−3, 5 × 10−3, 10−4, 5 × 10−4, and 100 μL of each dilution was plated on SC, SC − ura, or SC + FOA plates supplemented with the various glucose concentrations. The plates were incubated at 30°C for 2–4 days. For silencing assays using deletion strains, overnight liquid cultures were adjusted to OD600 = 1 in 1 mL, and serially diluted. On SC and SC − ura plates, 100 μL of a 10−4 dilution were plated, and on SC + FOA, 100 μL of a 10−3 dilution were plated. Colonies were counted either manually or with a BioRad Universal Hood II Imager and QuantityOne software (Biorad). Viability of the AEY1017 telomeric reporter strain on FOA medium is directly proportional to silencing activity (t-tests. Viability of the JS128 rDNA reporter strain on −ura medium is inversely proportional to silencing activity (t-tests.Cultures were grown overnight in 5 mL YPD, diluted with water to ODactivity . Viabiliactivity  and was URA3 gene in JS122 and JS128 strains was assayed using the method of URA3 marker loss by recombination was calculated by dividing the number of colonies that did not grow on −ura plates by the total number of colonies on replica SC or YPD plates. Marker loss per generation was calculated by dividing this value by the total number of mitotic divisions during the nine serial passages (t-tests.Mitotic stability of the


Streptococcus agalactiae, the Lancefield group B streptococcus (GBS) long recognized as a mammalian pathogen, is an emerging concern with regard to fish. We show that a GBS serotype Ia multilocus sequence type ST-7 isolate from a clinical case of human neonatal meningitis caused disease and death in Nile tilapia (Oreochromis niloticus). Streptococcus agalactiae, group B streptococcus (GBS), has a broad host range and is pathogenic to mammals, reptiles, amphibians, and fish (Tursiops truncatus)  appear to have a genetic relationship with human GBS. Human serotype Ia strains in the ST-23 lineage and human serotype III ST-17 strains are related to bovine ST-61 strains and frequently associated with neonatal infections (Oreochromis niloticus)  each were housed in 57-L aquariums at the Aquatic Animal Health Research Laboratory . All tanks were supplied with flow-through dechlorinated tap water and 2 submersible heaters and air stones to maintain desired water temperature and dissolved oxygen (DO) levels. Water quality  was measured daily by using a YSI 85 meter  and a Fresh Water Aquaculture Kit . Temperature was 32.1 ± 0.09°C, DO was 4.2 ± 0.14 mg/L, and ammonia concentration was 0.74 ± 0.08 mg/L. Fish were fed daily (4% of bodyweight) with Aquamax Grower 400 fish feed  and maintained and handled according to Institutional Animal Care and Use Committee–approved guidelines.7 CFU of GBS/fish. Specimens from a fish that died 3 days postchallenge were cultured on SBA, and GBS was recovered from nostrils, intestines, posterior kidney, and brain. One GBS colony isolated from brain was cultured on SBA, typed as GBS by Lancefield grouping (The serotype Ia ST-7 human GBS isolate (ID# 510012) was obtained from a patient in Japan who had neonatal meningitis. The isolate was cultured overnight on 5% sheep blood agar  at 32°C. Before the study, the isolate was passed through 5 Nile tilapia (weight 18.4 ± 0.48 g) 1 time each by intraperitoneal injection of 107, 106, 105, 104, 103, or 102 CFU/fish. Ten control fish were injected with 0.1 mL of TSB only. Fish were placed in separate 57-L aquariums at 32°C according to dose and monitored daily for signs of disease and death for 14 days postchallenge. Moribund fish were humanely euthanized by an overdose of tricaine methanesulfonate .Serial dilutions of the GBS isolate were prepared in tryptic soy broth , and 10 fish (weight 28.2 ± 0.51 g) were each injected intraperitoneally with 0.1 mL of inoculum at 10www.biolog.com/mID_product.html); a similarity index >0.50 and high probability (>90%) were considered a strong confirmation for GBS.Bacterial samples were obtained from nostrils and brains of all moribund or dead fish and cultured on SBA at 32°C for 24 h for GBS. Identification of GBS was performed by using methods of Evans et al.  (6 CFU. Sampled organs were negative for GBS and deaths were attributed to tank mate aggression to weakened fish. A linear dose response was not seen. Deaths occurred at low (102–103 CFU/fish) and high (107 CFU/fish) doses but not at median doses  . Overallan doses . DiseaseAll sampled organs from fish dying within 7 days of infection contained β-hemolytic, gram-positive, oxidase-negative, catalase-negative bacteria. BIOLOG analysis confirmed identification as GBS . None of the TBS-injected control fish showed signs of disease or died.2, 103, 106, and 107 CFU/fish, and GBS was reisolated from diseased fish. These experimental findings suggest GBS transmission between mammals and fish and that GBS-induced fish epidemics can originate from mammalian sources. Although we studied only 1 human isolate, other isolates or isolates repeatedly passed through fish may be more virulent. Susceptibility to GBS may also be enhanced by suboptimal environmental conditions, such as low DO, high ammonia levels, euthrophication, harmful algae, and changing or extreme water temperatures (Deaths among experimentally infected Nile tilapia indicate that an Ia ST-7 human GBS isolate can be pathogenic to fish. Such isolates have been associated with human (


To identify the underlying genetic defect in a north Indian family with seven members in three-generations affected with bilateral congenital cataract.Detailed family history and clinical data were recorded. Linkage analysis using fluorescently labeled microsatellite markers for the already known candidate gene loci was performed in combination with mutation screening by bidirectional sequencing.GJA8]), 2q33–36 , 3q21–22 , 12q12–14 (aquaporin 0 [AQP0]), 13q11–13 , 15q21–22, 16q22–23 , 17q11–12 , 17q24, 21q22.3 , and 22q11.2 . Crystallin, alpha B (CRYAB) at chromosome 11q23–24 was excluded by sequence analysis. However, sequencing the candidate gene, crystallin, gamma S (CRYGS), at chromosome 3q26.3-qter showed a heterozygous c.176G→A change that resulted in the replacement of a structurally highly conserved valine by methionine at codon 42 (p.V42M). This sequence change was not observed in unaffected family members or in the 100 ethnically matched controls.Affected individuals had bilateral congenital cataract. Cataract was of opalescent type with the central nuclear region denser than the periphery. Linkage was excluded for the known cataract candidate gene loci at 1p34–36, 1q21–25 (gap junction protein, alpha 8 [CRYGS associated with bilateral congenital cataract in a family of Indian origin. This is the third report of a mutation in this exceptional member of the β-/γ-crystallin superfamily and further substantiates the genetic and clinical heterogeneity of autosomal dominant cataract.We report a novel missense mutation, p.V42M, in  Congenital cataract is a clinically and genetically heterogeneous group of eye disorders that causes visual impairment and childhood blindness. It accounts for 10%–30% of blindness in children ,3.CRYAA; OMIM 123580], crystallin, alpha B , crystallin, beta A1 , crystallin, beta A4 , crystallin, beta B1 , crystallin, beta B2 , crystallin, beta B3 , crystallin, gamma C , crystallin, gamma D , crystallin, gamma S ), gap junctional molecules , lens major intrinsic proteins , beaded structural filament proteins , regulatory factors , glucosaminyl (N-acetyl) transferase 2 (GCNT2 [OMIM 600429]), chromatin modifying protein-4B (CHMP4B [OMIM 610897]), and transmembrane protein (TMEM114 [OMIM 611579]).At least 34 loci and mutations in 22 genes have been reported to be linked with different forms of congenital cataract. These genes encode for crystallins  was observed that resulted in the substitution of a highly conserved valine by methionine at codon 42 (p.V42M). This change co-segregated completely with the disease phenotype and was observed neither in unaffected family members nor in the 100 ethnically matched controls.We came across a three-generation north Indian family with individuals affected by bilateral congenital cataract at the Dr. Daljit Singh Eye Hospital . Using fluorescently labeled microsatellite markers, linkage was excluded to already known candidate gene regions at chromosome 1p, 1q, 2q, 3q21, 12q, 13q, 15q21, 16q, 17q, 17q24, 21q22.3, and 22q. The proband, a three-year-old child, was diagnosed with bilateral cataract. Family history revealed seven affected members in three generations . A detaiMarshfield linkage map. Seventy-five microsatellites from candidate gene regions at 1p34–36, 1q21–25 (GJA8), 2q33–36 , 3q21–22 (BFSP2), 12q12–14 (AQP0), 13q11–13 (GJA3), 15q21–22, 16q22–23 , 17q11–12 (CRYBA1), 17q24, 21q22.3 (CRYAA), and 22q11.2  were analyzed. Microsatellites were amplified by touchdown polymerase chain reaction (PCR) using fluorescently labeled primers as described elsewhere [LINKAGE program package [GENEHUNTER [Informed consent was obtained from all individuals studied. This study was approved by the Ethics Review Board of the Guru Nanak Dev University , consistent with the provisions of the Declaration of Helsinki. Blood was drawn and DNA was extracted by standard protocols. Linkage analysis based on semi-automated genotyping was performed with microsatellite markers on DNA samples of 14 clinically examined individuals (seven affected and seven unaffected including four spouses). Microsatellite markers and their distances were from the CRYAB (NM_001885) and CRYGS (NM_017541), which were localized at 11q23–24 and 3q26.3-qter, were analyzed by bidirectional sequence analysis. The primers were designed to amplify the coding regions and 30–50 bp flanking intronic regions using the PrimerSelect program of the Lasergene package . Amplification and sequencing were performed as described elsewhere [The candidate genes, S NM_01751, which The already known candidate gene regions for congenital cataract at 1p34–36, 1q21–25, 2q33–36, 3q21–22, 12q12–14, 13q11–13, 15q21–22, 16q22–23, 17q11–12, 17q24, 21q22.3, and 22q11.2 were tested using 75 fluorescently labeled microsatellite markers. Two-point LOD score values of less than −2.0 at θ=0.001 were obtained with all these markers, excluding these loci to be linked with cataract in this family.CRYAB (11q23–24) and CRYGS (3q26.3-qter), were analyzed by sequence analysis. Bidirectional sequence analysis of the coding regions of CRYAB in affected individuals  did not show any sequence alteration, excluding CRYAB to be associated with cataract in this family. However, bidirectional sequence analysis of CRYGS indicated a novel heterozygous c.176G→A change , beta (β-), and gamma (γ-) are the major structural proteins in the vertebrate eye lens, constituting approximately 90% of water soluble proteins. Their short range spatial order packing and stability are crucial to maintain lens transparency ,8. Cryststallins . γ-Cryststallins . They arstallins .CRYAB and CRYGS, at these respective loci, these genes were sequenced bidirectionally. No sequence alteration was found in the coding region of CRYAB in patients when compared to unaffected family members, thus excluding its association with ADCC in the present family. However, we observed a novel p.V42M substitution in the CRYGS polypeptide associated with bilateral congenital cataract. The p.V42M substitution is likely to cause cataract since it segregated with the phenotype and was not detected in either the unaffected family members or the 100 ethnically matched controls. The valine at codon 42 is highly conserved in different γ-crystallins  family with seven affected members in three generations, 12 known candidate gene loci harboring 17 candidate genes were excluded by linkage analysis using fluorescently labeled microsatellite markers. In addition, due to the unavailability of microsatellite markers with us at 11q23–24 and 3q26.3-qter and the availability of primers for sequence analysis of candidate genes, stallins .CRYGS linked with cataract (The present study is the third report of a mutation in cataract . Previoucataract  differs CRYGS have been reported to be associated with mice cataractogenesis as well. Sinha et al. [CRYGS lead to recessive nuclear cataract.Mutations in a et al.  have repa et al.  reportedMutations in the γ-crystallins tend to produce nuclear or zonular cataract consistent with their high level of expression in the lens nucleus, although the phenotype may vary significantly as documented in various reports. Similarly, mutations in BFSP2 are being linked with different phenotypes like juvenile-onset progressive cataract  and pulvMore than a dozen mutations in GJA8 have been linked with different cataract phenotypes in different families with diverse mutational mechanisms like a dominant negative effect for p.P88S and p.P88Q -24 or a The p.V42M substitution observed in the present cataract family represents a conservative amino acid change as both valine and methionine are uncharged non-polar amino acids. The other two missense mutations, p.G18V and p.S39C, also represent conservative amino acid changes. They are all located in exon 2, which constitutes domain 1 of CRYGS, and are obviously required for the maintenance of the tertiary structure of the protein and the morphology of the cortical fiber cells ,28. ThusIn summary, we describe a novel missense mutation, p.V42M, in CRYGS that causes congenital cataract in seven members in a three-generation north Indian family. The phenotype differs from that of two other mutations in the same domain. Therefore, these findings further substantiate the clinical and genetic heterogeneity of dominant congenital cataract.