Microbial Reduction of Fumonisin B1 by the New Isolate Serratia marcescens 329-2

The mycotoxin fumonisin (FB) has become a major problem in maize products in southeastern Asia. Fumonisin can affect the health of humans and many animals. Fumonisin contamination can be reduced by detoxifying microbial enzyme. Screening of 95 potent natural sources resulted in 5.3% of samples yielding a total of five bacterial isolates that were a promising solution, reducing approximately 10.0–30.0% of fumonisin B1 (FB1). Serratia marcescens, one of the dominant degrading bacteria, was identified with Gram staining, 16S rRNA gene, and MALDI-TOF/TOF MS. Cell-free extract showed the highest fumonisin reduction rates, 30.3% in solution and 37.0% in maize. Crude proteins from bacterial cells were analyzed with a label-free quantification technique. The results showed that hydrolase enzymes and transferase enzymes that can cooperate in the fumonisin degradation process were highly expressed in comparison to their levels in a control. These studies have shown that S. marcescens 329-2 is a new potential bacterium for FB1 reduction, and the production of FB1-reducing enzymes should be further explored.

Simple and Fast DNA Based Sensor System for Screening of Small-Molecule Compounds Targeting Eukaryotic Topoisomerase 1

Background: Eukaryotic topoisomerase 1 is a potential target of anti-parasitic and anti-cancer drugs. Parasites require topoisomerase 1 activity for survival and, consequently, compounds that inhibit topoisomerase 1 activity may be of interest. All effective topoisomerase 1 drugs with anti-cancer activity act by inhibiting the ligation reaction of the enzyme. Screening for topoisomerase 1 targeting drugs, therefore, should involve the possibility of dissecting which step of topoisomerase 1 activity is affected. Methods: Here we present a novel DNA-based assay that allows for screening of the effect of small-molecule compounds targeting the binding/cleavage or the ligation steps of topoisomerase 1 catalysis. This novel assay is based on the detection of a rolling circle amplification product generated from a DNA circle resulting from topoisomerase 1 activity. Results: We show that the binding/cleavage and ligation reactions of topoisomerase 1 can be investigated separately in the presented assay termed REEAD (C|L) and demonstrate that the assay can be used to investigate, which of the individual steps of topoisomerase 1 catalysis are affected by small-molecule compounds. The assay is gel-free and the results can be detected by a simple colorimetric readout method using silver-on-gold precipitation rendering large equipment unnecessary. Conclusion: REEAD (C|L) allows for easy and quantitative investigations of topoisomerase 1 targeting compounds and can be performed in non-specialized laboratories.

Antagonistic Control of Genetic Circuit Performance for Rapid Analysis of Targeted Enzyme Activity in Living Cells

Genetic circuits have been developed for quantitative measurement of enzyme activity, metabolic engineering of strain development, and dynamic regulation of microbial cells. A genetic circuit consists of several bio-elements, including enzymes and regulatory cassettes, that can generate the desired output signal, which is then used as a precise criterion for enzyme screening and engineering. Antagonists and inhibitors are small molecules with inhibitory effects on regulators and enzymes, respectively. In this study, an antagonist and an inhibitor were applied to a genetic circuit for a dynamic detection range. We developed a genetic circuit relying on regulators and enzymes, allowing for straightforward control of its output signal without additional genetic modification. We used para-nitrophenol and alanine as an antagonist of DmpR and inhibitor of tyrosine phenol-lyase, respectively. We show that the antagonist resets the detection range of the genetic circuit similarly to a resistor in an electrical logic circuit. These biological resistors in genetic circuits can be used as a rapid and precise controller of variable outputs with minimal circuit configuration.

Isolation, Screening and Antimicrobial Activity of Aeromonas hydrophila from Spoiled Vegetables and Biochemical Analysis of Estuarine Microbial Sediments

Background: Aeromonas hydrophila is a heterotrophic, Gram-negative, rod-shaped, facultative anaerobic, non-spore forming bacteria that are autochthonous and widely dispersed in marine environments. The study aims at investigating the screening of Aeromonas hydrophila from spoiled vegetables and the sediment sample collected from three different estuaries located in the Bay of Bengal (Vedharanyam, Parangipettai and Pichavaram, Tamilnadu, India) for the presence of enzymes and antimicrobial activities. Objective: Isolation, enzyme screening, antimicrobial activity of Aeromonas hydrophila from spoiled vegetables and three different estuarine microbial sediment samples for the purpose of biochemical and enzymatic analysis. Methods: The bioactive compound produced by this strain was purified by using thin-layer chromatography. Results: The purified isolate of Aeromonas hydrophila strain produces good antimicrobial activity against Aspergillus niger, Candida albicans, Staphylococcus, Klebsiella and pseudomonas species. Conclusion: These isolates producing amylase, protease, lipase, and gelatinase enzymes, which are commercially very important and used in many industries and other biochemical sectors.

Toxic and Adverse Effects of Chemotherapy with 5-Fluoropyrimidine Drugs. Could Dihydropyrimidine Dehydrogenase Enzyme Screening Serve as a Prerequisite to Successful Chemotherapy?

Summary The article presents a detailed survey of recent publications in the literature concerning clinical expertise, existing guidelines, and differing opinions on Fluoropyrimidine chemotherapy-related toxicity and the implication of Dihydropyrimidine dehydrogenase (DPD) screening aiming to prevent severe 5-Fluorouracil-induced adverse drug reactions. The first section provides information on the mechanism of action, clinical application, pharmacokinetics and pharmacodynamics, and toxicity and adverse reactions of 5-Fluorouracil, Capecitabine, Floxuridine, and Flucytosine. The second section summarizes DPD phenol- and genotype data and provides reasons for determining a DPD life-threatening complete or partial enzyme deficiency. The pros and cons of the methodological approaches for DPD screening are analysed, and recommendations are made to introduce them into clinical practice. The third section includes a brief economic analysis of expenses for DPD screening of patients scheduled for 5-Fluorouracil chemotherapy. The costs are compared to those related to the treatment of patients suffering from 5-Fluorouracil-induced toxicity and unwanted adverse effects.

Genetically Encoded Biosensor-Based Screening for Directed Bacteriophage T4 Lysozyme Evolution

Lysozyme is widely used as a model protein in studies of structure–function relationships. Recently, lysozyme has gained attention for use in accelerating the degradation of secondary sludge, which mainly consists of bacteria. However, a high-throughput screening system for lysozyme engineering has not been reported. Here, we present a lysozyme screening system using a genetically encoded biosensor. We first cloned bacteriophage T4 lysozyme (T4L) into a plasmid under control of the araBAD promoter. The plasmid was expressed in Escherichia coli with no toxic effects on growth. Next, we observed that increased soluble T4L expression decreased the fluorescence produced by the genetic enzyme screening system. To investigate T4L evolution based on this finding, we generated a T4L random mutation library, which was screened using the genetic enzyme screening system. Finally, we identified two T4L variants showing 1.4-fold enhanced lytic activity compared to native T4L. To our knowledge, this is the first report describing the use of a genetically encoded biosensor to investigate bacteriophage T4L evolution. Our approach can be used to investigate the evolution of other lysozymes, which will expand the applications of lysozyme.

Microfluidic Pneumatic Printed Sandwiched Microdroplet Array for High-Throughput Enzymatic Reaction and Screening

High-throughput enzyme screening for desired functionality is highly demanded. This paper utilizes a newly developed microfluidic pneumatic printing platform for high-throughput enzyme screening applications. The novel printing platform can achieve distinct features including a disposable cartridge, which avoids crosstalk; a flexible cartridge design, allowing for integration of multiple channels; and fast printing speed with submicroliter spot size. Moreover, a polydimethylsiloxane (PDMS)-based sandwich structure has been proposed and used during the printing and imaging, which can lead to better results, including reduced evaporation as well as a uniform light path during imaging. Using this microfluidic pneumatic printed PDMS sandwiched microdroplet array platform, we have demonstrated the capability of high-throughput generation of a combinatorial droplet array with concentration and volume gradients. Furthermore, the potential for enzymatic study has been validated by quantified cellulose reaction implemented with the printing platform.

Investigation and comparison of G6PD enzyme screening test costs for all newborns with treatment costs of patients with favism admitted due to hemolysis

Objective. We compared the glucose-6-phosphate dehydrogenase (G6PD) deficiency screening costs in neonates and treatment costs for patients with favism who were referred to the government hospitals of Isfahan with acute hemolysis. Methods. In this retrospective study, 116 patients with favism were selected, and their clinical data and treatment costs were recorded in Iranian Rials (IRR) and United States Dollars (USD). The costs of neonatal G6PD screening tests were estimated based on quantitative and qualitative screening kits by referring to each hospital's laboratories. Approximately 84760 births occurred in Isfahan province from October 2014 to October 2015. Results. Based on our results, 73 patients (62.9%) were males, and 43 of them (37.1%) were females. The causes for hemolysis were the consumption of broad beans in 78.4%, infection in 19.8%, and drugs (erythromycin and sodium cephalothin) in 1.7% of patients. The majority of patients (46.6%) received a single blood transfusion, and 18.9% received two or more transfusions, while 34.5% had received no blood transfusions. The mean and standard deviation of patients' medical expenses were 7,472,005±7,329,847 IRR (276.74±271.47 USD). The total cost of treatment for patients was 869,162,324 IRR (32,191.19 USD). The screening cost for all newborns from October 2014 to October 2015 with qualitative kits was 754,364,000 IRR (27,939.40 USD) and with the quantitative kits was 960,330,800 IRR (35,567.80 USD). Conclusion. The costs of the qualitative screening test for G6PD deficiency are lower than the costs of treatments of favism patients in Isfahan.

Development of 96 Multiple Injection-GC-MS Technique and Its Application in Protein Engineering of Natural and Non-Natural Enzymatic Reactions

Directed evolution requires the screening of enzyme libraries in biological matrices. Available assays are mostly substrate or enzyme specific. Chromatographic techniques like LC and GC overcome this limitation, but require long analysis times. The herein developed multiple injections in a single experimental run (MISER) using GC coupled to MS allows the injection of samples every 33 s resulting in 96-well microtiter plate analysis within 50 min. This technique is implementable in any GC-MS system with autosampling. Since the GC-MS is far less prone to ion suppression than LCMS, no chromatographic separation is required. This allows the utilisation of an internal standards and the detection of main and side-product. To prove the feasibility of the system in enzyme screening, two libraries were assessed: i) YfeX library in an E. coli whole cell system for the carbene-transfer reaction on indole revealing the novel axial ligand tryptophan, ii) a library of 616 chimeras of fungal unspecific peroxygenase (UPO) in S. cerevisiae supernatant for hydroxylation of tetralin resulting in novel constructs. The data quality and representation are automatically assessed by a new R-script.