Application of a novel fluorogenic polyurethane analogue probe in polyester‐degrading microorganisms screening by microfluidic droplet

Application of polyester‐degrading microorganisms or enzymes should be considered as an eco‐friendly alternative to chemical recycling due to the huge plastic waste disposal nowadays. However, current impranil DLN‐based screening of polyester‐degrading microorganisms is time‐consuming, labour‐intensive and unable to distinguish polyesterases from other protease‐ or amidase‐like enzymes. Herein, we present an approach that combined a novel synthetic fluorescent polyurethane analogue probe (FPAP), along with the droplet‐based microfluidics to screen polyurethane‐degrading microorganisms through fluorescence‐activated droplet sorting (FADS) pipeline. The fluorescent probe FPAP exhibited a fluorescence enhancement effect once hydrolysed by polyesterases, along with a strong specificity in discriminating polyesterases from other non‐active enzymes. Application of FPAP in a microfluidic droplet system demonstrated that this probe exhibited high sensitivity and efficiency in selecting positive droplets containing leaf‐branch compost cutinase (LCC) enzymes. This novel fluorogenic probe, FPAP, combined with the droplet microfluidic system has the potential to be used in the exploitation of novel PUR‐biocatalysts for biotechnological and environmental applications.

A complete FADS pipeline using FPAP as the fluorogenic probe for screening polyester‐degrading microorganisms was developed.     

Decomposition of polyurethane in a garbage landfill leakage water and by soil microorganisms

Summary The decomposition of polyurethane, measured gravimetrially or using infrared spectrophotometry, was found to be more complete in polyurethane based on polyester and only very small in polyurethane based on polyether. In the presence of clay minerals the decomposition was inhibited. If positive, the decomposition of polyurethane followed the sequence: remaining free isocyanatesurea and amide groupsurethane groupsisocyanuric acid rings.     

Growth kinetics of microalgae in microfluidic static droplet arrays

We investigated growth kinetics of microalgae, Chlorella vulgaris, in immobilized arrays of nanoliter‐scale microfluidic drops. These static drop arrays enabled simultaneous monitoring of growth of single as well as multiple cells encapsulated in individual droplets. To monitor the growth, individual drop volumes were kept nearly intact for more than a month by controlling the permeation of water in and out of the microfluidic device. The kinetic growth parameters were quantified by counting the increase in the number of cells in each drop over time. In addition to determining the kinetic parameters, the cell‐size distribution of the microalgae was correlated with different stages of the growth. The single‐cell growth kinetics of C. vulgaris showed significant heterogeneity. The specific growth rate ranged from 0.55 to 1.52 day^?1 for different single cells grown in the same microfluidic device. In comparison, the specific growth rate in bulk‐scale experiment was 1.12 day^?1. It was found that the average cell size changes significantly at different stages of the cell growth. The mean cell‐size increased from 5.99 ± 1.08 to 7.33 ± 1.3 µm from exponential to stationary growth phase. In particular, when multiple cells are grown in individual drops, we find that in the stationary growth phase, the cell size increases with the age of cell suggesting enhanced accumulation of fatty acids in older cells. Biotechnol. Bioeng. 2012; 109: 2987–2996. © 2012 Wiley Periodicals, Inc.     

Detection of l-alanylaminopeptidase activity in microorganisms using fluorogenic self-immolative enzyme substrates

A series of fluorogenic enzymatic substrates that incorporate a self-immolative spacer were synthesised for the purpose of identifying l-alanylaminopeptidase activity in microorganisms in agar media. These substrates resulted in the generation of fluorescent microorganism colonies with Gram-negative microorganisms.     

High-throughput screening of cell-free riboswitches by fluorescence-activated droplet sorting

Cell-free systems that display complex functions without using living cells are emerging as new platforms to test our understanding of biological systems as well as for practical applications such as biosensors and biomanufacturing. Those that use cell-free protein synthesis (CFPS) systems to enable genetically programmed protein synthesis have relied on genetic regulatory components found or engineered in living cells. However, biological constraints such as cell permeability, metabolic stability, and toxicity of signaling molecules prevent development of cell-free devices using living cells even if cell-free systems are not subject to such constraints. Efforts to engineer regulatory components directly in CFPS systems thus far have been based on low-throughput experimental approaches, limiting the availability of basic components to build cell-free systems with diverse functions. Here, we report a high-throughput screening method to engineer cell-free riboswitches that respond to small molecules. Droplet-sorting of riboswitch variants in a CFPS system rapidly identified cell-free riboswitches that respond to compounds that are not amenable to bacterial screening methods. Finally, we used a histamine riboswitch to demonstrate chemical communication between cell-sized droplets.     

Application of a microfluidic reactor for screening cancer prodrug activation using silica-immobilized nitrobenzene nitroreductase

The nitroreductase-catalyzed conversion of a strong electron-withdrawing nitro group to the corresponding electron-donating hydroxylamine is useful in a variety of biotechnological applications. Activation of prodrugs for cancer treatments or antibiotic therapy are the most common applications. Here, we show that a bacterial nitrobenzene nitroreductase (NbzA) from Pseudomonas pseudoalcaligenes JS45 activates the dinitrobenzamide cancer prodrug CB1954 and the proantibiotic nitrofurazone. NbzA was purified by affinity chromatography and screened for substrate specificity with respect to prodrug activation. To facilitate screening of alternate potential prodrugs, polyethyleneimine-mediated silica formation was used to immobilize NbzA with high immobilization yields and high loading capacities. Greater than 80% of the NbzA was immobilized, and enzyme activity was significantly more stable than NbzA in solution. The resulting silica-encapsulated NbzA was packed into a microfluidic microreactor that proved suitable for continuous operation using nitrobenzene, CB1954, and the proantibiotic nitrofurazone. The flow-through system provides a rapid and reproducible screening method for determining the NbzA-catalyzed activation of prodrugs and proantibiotics.     

Long-lasting smooth-muscle relaxation by a novel PACAP analogue in human bronchi

We compared the relaxant effect of original pituitary adenylate cyclase-activating peptide (PACAP)1-27 with that of a newly developed, synthetic PACAP1-27 analogue, [Arg15,20,21 Leu17]-PACAP-Gly-Lys-Arg-NH2, in human bronchi in vitro (n=4-5 in each group). Using precontraction by carbachol (0.1 microM), cumulative administration of PACAP1-27 and salbutamol caused concentration-dependent smooth muscle relaxation with similar potencies and maximum relaxant effects. Non-cumulative administration of the PACAP1-27 analogue and the original PACAP1-27 caused concentration-dependent relaxation with a similar maximum relaxant effect and potency as well. However, the onset and offset of action was markedly slower for the PACAP1-27 analogue than for the original PACAP1-27 (>90% versus <10% of peak relaxation remaining 5 h after administration). Peptidase inhibition by captopril (10 microM) and phosphoramidon (1 microM) significantly increased the maximum relaxant effect and duration of action of PACAP1-27 but not of the PACAP1-27 analogue, during the 3 h of observation in the human bronchi. We conclude that [Arg15,20,21 Leu17]-PACAP-Gly-Lys-Arg-NH2 produces significant concentration-dependent and sustained bronchial smooth muscle relaxation in vitro. The sustained relaxant effect is due, at least in part, to the synthetic PACAP1-27 analogue being less susceptible to cleavage by peptidases than the original peptide PACAP1-27.     

Novel type of general protein assay using a chromogenic and fluorogenic amine-reactive probe

We report on a new and simple one-reagent method for general protein assay. It makes use of one of two new reactive labeling reagents presented here (and referred to as pyrylium [Py] labels). These can be applied for both photometric and fluorometric protein assays at near neutral pHs at room temperature. The Py labels undergo a large spectral change on conjugation to the amino group of proteins and typically change their color from blue to red. Therefore, and unlike in other assays, there is no need to separate the unconjugated (blue) label from the red conjugate, which can be determined by direct photometry with a limit of detection of 1.2 microg/ml for human serum albumin. The assay can be extended to fluorometry because the fluorescence of the free Py label is weak (with a quantum yield of <1%) but increases strongly (to >40%) on conjugation. The strong fluorescence of the red conjugates can be determined directly and without interference by the blue (and weakly fluorescent) free label. The fluorometric assay resulted in a limit of detection of 60 ng/ml for bovine serum albumin (BSA). Validation of the fluorescence assay of blood plasma samples spiked with BSA gave recoveries in the range from 91 to 103%.     

A novel GFP-based approach for screening biocontrol microorganisms in vitro against Dothistroma septosporum

Dothistroma septosporum is the causal agent of Dothistroma needle blight of pine trees. A novel green fluorescent protein (GFP)-based screening method was developed to assess the potential of microorganisms for biocontrol of Dothistroma. The screen utilizes GFP expression as an indicator of metabolic activity in the pathogen and hygromycin resistance selection to determine if the interaction is fungistatic or fungicidal. Results suggested that six of eight Trichoderma isolates tested have the potential to control Dothistroma in vitro, via a fungicidal action. Because D. septosporum produces a broad-spectrum toxin, dothistromin, the inhibition of Trichoderma spp. by D. septosporum was determined by growth rate measurements compared to controls. Inhibition of the Trichoderma spp. ranged from no inhibition to 30% inhibition and was influenced by the assay medium used. The GFP screening method was also assessed to determine if it was suitable for screening bacteria as potential biocontrol candidates. Although a method involving indirect-contact had to be used, two of four Bacillus strains showed antagonistic activity against D. septosporum in vitro, via a fungistatic interaction. The four bacterial strains inhibited D. septosporum growth by 14.0 to 39.8%. This GFP-based method represents a novel approach to screening fungi and bacteria for antagonistic activity.     

Luteolytic potencin in a novel prostaglandin analogue

Estrus induction in cycling sows with exogenous prostaglandin is hindered by the extended refractory period and resistance of swine to the luteolytic action of PG's. The luteolytic potency of a novel PGF_2α analogue, Schering ZK 71677, was assessed at four different dosages in chronically cannulated cycling swine. Dosages totalling 1, 1.5, 3 and 10 mg of ZK 71677 were split into two injections given intramuscularly on day 13 of the estrous cycle. Onset of behavioural estrus and serum levels of progesterone and PGF metabolite were monitored. The three lowest doses did not advance estrus but did cause a transitory decline in serum progesterone concentration on day 13. The 10 mg level advanced estrus in three of four sows although the overall mean cycle length did not differ from pretreatment cycles (18.5 ± 1.3 vs 21.3 ± 0.6 days). Progesterone values droppd steadily in all four sows in the 10 mg treatment group. No level of ZK 71677 affected serum levels of PGF metabolite. The highest level tested of PGF_2α analogue ZK 71677 elicited an uninterrupted decline in serum progesterone concentrations but was inconsistent in its ability to promote early onset of behavioural estrus in swine.     

Use of a fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogenes.

A PCR-based assay for Listeria monocytogenes that uses the hydrolysis of an internal fluorogenic probe to monitor the amplification of the target has been formatted. The fluorogenic 5' nuclease PCR assay takes advantage of the endogenous 5' --> 3' nuclease activity of Taq DNA polymerase to digest a probe which is labelled with two fluorescent dyes and hybridizes to the amplicon during PCR. When the probe is intact, the two fluorophores interact such that the emission of the reporter dye is quenched. During amplification, the probe is hydrolyzed, relieving the quenching of the reporter and resulting in an increase in its fluorescence intensity. This change in reporter dye fluorescence is quantitative for the amount of PCR product and, under appropriate conditions, for the amount of template. We have applied the fluorogenic 5' nuclease PCR assay to detect L. monocytogenes, using an 858-bp amplicon of hemolysin (hlyA) as the target. Maximum sensitivity was achieved by evaluating various fluorogenic probes and then optimizing the assay components and cycling parameters. With crude cell lysates, the total assay could be completed in 3 h with a detection limit of approximately 50 CFU. Quantification was linear over a range of 5 x 10(1) to 5 x 10(5) CFU.     

Antinociceptive activity of a novel buprenorphine analogue

HS-599 is a didehydroderivative of buprenorphine that displays high affinity and good selectivity for mu-opioid receptors. We studied its antinociceptive properties after s.c. injection in mice with the tail-flick and hot-plate tests. In the tail-flick test HS-599 (AD50 = 0.2801 micromol/kg s.c.) behaved as a full agonist and was twice as potent as buprenorphine (AD50=0.4569 micromol/kg s.c.) and 50 times more potent than morphine (AD50 = 13.3012 micromol/kg s.c.). Whereas the mu-opioid receptor antagonists naloxone (1-10 mg/kg s.c.) and naltrexone (5-15 mg/kg s.c.) antagonized HS-599 induced analgesia, the delta-opioid receptor antagonist naltrindole (20 mg/kg s.c.) and the kappa-opioid receptor antagonist nor-binaltorphimine (20 mg/kg s.c.) did not. With the hot-plate test at 50 degrees C, HS-599 (AD50 = 0.0359 micromol/kg s.c.) was a full agonist about 130 times more potent than morphine (AD50 = 4.8553 micromol/kg s.c.). With a high intensity nociceptive stimulus (55 degrees C) HS-599 (AD50 = 1.0382 micromol/kg s.c.) remained 7 times more potent than morphine (AD50 = 7.0210 micromol/kg s.c.) but never exceeded the 55% of the maximum possible effect, behaving as a partial agonist able to antagonize morphine antinociception in a dose-dependent manner. HS-599 promises to be a potent and safe new analgesic, preferentially acting at spinal level.     

Ultrahigh-throughput screening of industrial enzyme-producing strains by droplet-based microfluidic system

Droplet-based microfluidics has emerged as a powerful tool for single-cell screening with ultrahigh throughput, but its widespread application remains limited by the accessibility of a droplet microfluidic high-throughput screening (HTS) platform, especially to common laboratories having no background in microfluidics. Here, we first developed a microfluidic HTS platform based on fluorescence-activated droplet sorting technology. This platform allowed (i) encapsulation of single cells in monodisperse water-in-oil droplets; (ii) cell growth and protein production in droplets; and (iii) sorting of droplets based on their fluorescence intensities. To validate the platform, a model selection experiment of a binary mixture of Bacillus strains was performed, and a 45.6-fold enrichment was achieved at a sorting rate of 300 droplets per second. Furthermore, we used the platform for the selection of higher α-amylase-producing Bacillus licheniformis strains from a mutant library generated by atmospheric and room temperature plasma mutagenesis, and clones displaying over 50% improvement in α-amylase productivity were isolated. This droplet screening system could be applied to the engineering of other industrially valuable strains.     

Selective and facile assay of human immunodeficiency virus protease activity by a novel fluorogenic reaction

A highly selective and facile assay of human immunodeficiency virus protease (HIV-PR) has been required for the screening of medicinal inhibitors and also for classifying the subtypes of HIV in the therapeutic treatment of acquired immune deficiency syndrome (AIDS). This article describes a novel assay method of HIV-PR based on the selective fluorogenic reaction of peptides. A peptide fragment generated from a substrate by the enzymatic digestion with HIV-PR could be selectively quantified by the spectrofluorometric detection after the fluorogenic reaction with catechol in the presence of sodium periodate and sodium borate (pH 7.0). This assay system uses an N-terminal acetyl peptide as the substrate and crude extracts from Escherichia coli expressing recombinant HIV-PR. The activity obtained by the proposed assay correlated with that obtained by a conventional HIV-PR assay based on fluorescence resonance energy transfer detection.     

Development of a sensitive long-wavelength fluorogenic probe for nitroreductase: a new fluorimetric indictor for analyte determination by dehydrogenase-coupled biosensors

Nitroreductase (NTR) is a flavin-containing enzyme that uses NADH as the electron source to reduce nitroaromatic compounds to the corresponding amines. Previous studies have shown that nitroreductase-targeted latent fluorophores exhibit low solubility in the aqueous media and fluoresce at lower wavelengths upon uncloaking, thus limiting their effective applications. Here, we have prepared a new switch-on long-wavelength latent fluorogenic substrate, NTRLF (4), for NTR. In the presence of NADH, NTR catalyzes the reduction of the nitroaromatic moiety in NTRLF (4), followed by the cascade reaction, 1,6-rearrangement-elimination reaction, cyclic urea formation, and concomitant ejects a long-wavelength fluorescence coumarin (8). However, this reaction was inhibited in the presence of nitroaromatic analogues. The fluorescence signal generated by the cascade reaction was specific and insensitive to various reductants. Accordingly, we propose that NTRLF and NTR in the presences of NADH constitute a useful switch-off high-throughput fluorescence sensor for screening nitroaromatic compounds. Furthermore, NTRLF in the NTR-coupled 3-hydroxybutyrate dehydrogenase and aldehyde dehydrogenase assay reactions was a sensitive fluorimetric indicator for the quantitatively measurement of 3-hydroxybutyrate and propionaldehyde, respectively within micromolar range. Our novel NTRLF and NTR-coupled dehydrogenase assay platform may thus be effectively applied for the quantitative estimation of a broad range of analytes.     

Application of MEN 203 as a polymorphic DNA probe in screening multiple endocrine neoplasia 2a.

Multiple endocrine neoplasia 2a (MEN 2a) is known to be genetically linked to a locus on chromosome 10. The application of polymorphic DNA probes for the region has made it possible to identify carriers of the disease susceptible gene. We performed DNA analysis for a newly found non-Caucasian MEN 2a family using MEN 203 as a probe. Data from DNA analysis of the family members were concordant with the results of conventional endocrinological tests. Furthermore, DNA analysis discriminated four individuals out of fifteen as non-carriers of the gene with a high degree of certainty. The results relieved these people from taking screening tests for years. DNA analysis employing suitable markers such as MEN 203 appears to be useful for a screening program of MEN 2a in Japanese as well as Caucasians.     

Application of a novel highly sensitive activity-based probe for detection of cathepsin G

Cathepsins are crucial in antigen processing in the major histocompatibility complex class II (MHC II) pathway. Within the proteolytic machinery, three classes of proteases (i.e., cysteine, aspartic, and serine proteases) are present in the endocytic compartments. The combined action of these proteases generates antigenic peptides from antigens, which are loaded to MHC II molecules for CD4+ T cell presentation. Detection of active serine proteases in primary human antigen-presenting cells (APCs) is restricted because of the small numbers of cells isolated from the peripheral blood. For this purpose, we developed a novel highly sensitive α-aminoalkylphosphonate diphenyl ester (DAP) activity-based probe to detect the serine protease cathepsin G (CatG) in primary APCs and after Epstein-Barr virus (EBV) exposure. Although CatG activity was not altered after short-term exposure of EBV in primary myeloid dendritic cells 1 (mDC1s), the aspartic protease cathepsin D (CatD) was reduced, suggesting that EBV is responsible for mitigating the presentation of a model antigen tetanus toxoid C-fragment (TTCF) by reduction of CatD. In addition, CatG activity was reduced to background levels in B cells during cell culture; however, these findings were independent of EBV transformation. In conclusion, our activity-based probe can be used for both Western blot and 96-well-based high-throughput CatG detection when cell numbers are limited.