Top Agar Research Articles

Gene function characterization in Aspergillus niger using a dual resistance marker transformation system mediated by Agrobacterium tumefaciens

Aspergillus niger is a well-known workhorse for the industrial production of enzymes and organic acids. This fungus can also cause postharvest diseases in fruits. Although Agrobacterium tumefaciens-mediated transformation (ATMT) based on antibiotic resistance markers has been effectively exploited for inspecting functions of target genes in wild-type fungi, it still needs to be further improved in A. niger. In the present study, we re-examined the ATMT in the wild-type A. niger strains using the hygromycin resistance marker and introduced the nourseothricin resistance gene as a new selection marker for this fungus. Unexpectedly, our results revealed that the ATMT method using the resistance markers in A. niger led to numerous small colonies as false-positive transformants on transformation plates. Using the top agar overlay technique to restrict false positive colonies, a transformation efficiency of 87 ± 18 true transformants could be achieved for 106 conidia. With two different selection markers, we could perform both the deletion and complementation of a target gene in a single wild-type A. niger strain. Our results also indicated that two key regulatory genes (laeA and veA) of the velvet complex are required for A. niger to infect apple fruits. Notably, we demonstrated for the first time that a laeA homologous gene from the citrus postharvest pathogen Penicillium digitatum was able to restore the acidification ability and pathogenicity of the A. niger ΔlaeA mutant. The dual resistance marker ATMT system from our work represents an improved genetic tool for gene function characterization in A. niger.

Ketoconazole-loading strategy to improve antifungal activity and overcome cytotoxicity on human renal proximal tubular epithelial cells

Ketoconazole (KTZ), an antifungal agent used to treat localized or systemic fungal infections by inhibiting ergosterol synthesis, exhibits restricted efficacy within eukaryotic cells owing to its elevated toxicity and limited solubility in water. This study aims to improve the biological activity and overcome cytotoxic effects in the renal system of the hydrophobic KTZ by incorporating it into poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) utilizing biomaterial nano-engineering techniques. KTZ-loaded PLGA NPs (KTZ-NPs) were prepared by single emulsion solvent evaporation method and characterized by using dynamic light scattering (DLS), electrophoretic light scattering (ELS), Fourier transform-infrared (FT-IR) spectroscopy and scanning light microscopy (SEM). Particle size and zeta potential of KTZ-NPs were determined as 182.0 ± 3.27 nm and −27.4 ± 0.56 mV, respectively. Antifungal activity was analyzed with the time-kill and top agar dilution methods on Candida albicans (C. albicans) and Aspergillus flavus (A. flavus). Both KTZ and KTZ-NPs caused a significant decrease in A. flavus cell growth; however, the same effect was only observed in time-killing analysis on C. albicans, indicating a methodological difference in the antifungal analysis. According to the top agar method, the MIC value of KTZ-NPs against A. flavus was 9.1 μg ml−1, while the minimum inhibition concentration (MIC) value of KTZ was 18.2 μg ml−1. The twofold increased antifungal activity indicates that nanoparticular drug delivery systems enhance the water solubility of hydrophobic drugs. In addition, KTZ-NPs were not cytotoxic on human renal proximal tubular epithelial cells (HRPTEpCs) at fungistatic concentration, thus reducing fungal colonization without cytotoxic on renal excretion system cells.

Agar plate-based activity assay for easy and fast screening of recombinant Pichia pastoris expressing unspecific peroxygenases.

Unspecific peroxygenases (UPOs) are promising biocatalysts that catalyze oxyfunctionalization reactions without the need for costly cofactors. Pichia pastoris (reclassified as Komagataella phaffii) is considered an attractive host for heterologous expression of UPOs. However, integration of UPO-expression cassettes into the genome via a single cross-over yields recombinant Pichia transformants with different UPO gene copy numbers resulting in different expression levels. Selection of the most productive Pichia transformants by a commonly used screening in liquid medium in 96-well plates is laborious and lasts up to 5 days. In this work, we developed a simple two-step agar plate-based assay to screen P. pastoris transformants for UPO activity with less effort, within shorter time, and without automated screening devices. After cell growth and protein expression on agar plates supplemented with methanol and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), an additional top agar layer supplemented with ABTS and peroxide is added. UPO activity is visualized within 15min by formation of green zones around UPO-secreting P. pastoris transformants. The assay was validated with two UPOs, AbrUPO from Aspergillus brasiliensis and evolved PaDa-I from Agrocybe aegerita. The assay results were confirmed in a quantitative 96-deep well plate screening in liquid medium.

Enumeration of Bacteriophages by Plaque Assay.

Enumeration of bacteriophages by plaque assay requires the mixing of host-specific bacteria with a lytic bacteriophage of interest in a soft agar overlay (top agar) to prevent the spread of viral infection in the medium; the mixture is then spread on a solid bottom agar. An infection of a single lytic phage particle with a bacterium results in the lysis of the host bacterium and the release of new phage progeny. The new phage progeny released from each bacterium will infect/lyse neighboring bacteria to form a "plaque," which is a clear visible area (that can be counted) with the naked eye. If phages are not present in the mixture, the host bacterium will form a "lawn" in the soft agar overlay and grow to a stationary phase.

Tracing ΦX174 bacteriophage spreading during aerosol-generating procedures in a dental clinic

ObjectiveThe aim of this study was to test the plausibility of using the ΦX174 bacteriophage as a tracer of viral aerosols spreading in a dental aerosol-generating procedure (AGP) model.MethodsΦX174 bacteriophage (~ 108 plaque-forming units (PFU)/mL) was added into instrument irrigation reservoirs and aerosolized during class-IV cavity preparations followed by composite fillings on natural upper-anterior teeth (n = 3) in a phantom head. Droplets/aerosols were sampled through a passive approach that consisted of Escherichia coli strain C600 cultures immersed in a LB top agar layer in Petri dishes (PDs) in a double-layer technique. In addition, an active approach consisted of E coli C600 on PDs sets mounted in a six-stage cascade Andersen impactor (AI) (simulating human inhalation). The AI was located at 30 cm from the mannequin during AGP and afterwards at 1.5 m. After collection PDs were incubated overnight (18 h at 37 °C) and bacterial lysis was quantified.ResultsThe passive approach disclosed PFUs mainly concentrated over the dental practitioner, on the mannequin’s chest and shoulder and up to 90 cm apart, facing the opposite side of the AGP’s source (around the spittoon). The maximum aerosol spreading distance was 1.5 m in front of the mannequin’s mouth. The active approach disclosed collection of PFUs corresponding to stages (and aerodynamic diameters) 5 (1.1–2.1 µm) and 6 (0.65–1.1 µm), mimicking access to the lower respiratory airways.ConclusionThe ΦX174 bacteriophage can be used as a traceable viral surrogate in simulated studies contributing to understand dental bioaerosol’s behavior, its spreading, and its potential threat for upper and lower respiratory tract.Clinical relevanceThe probability to find infectious virus during AGPs is high. This suggests the need to continue characterizing the spreading viral agents in different clinical settings through combination of passive and active approaches. In addition, subsequent identification and implementation of virus-related mitigation strategies is relevant to avoid occupational virus infections.

New possibilities of the Ames test for evaluation of mutagenicity of technical products of active ingredients of pesticides

Introduction. The Ames test is the one of the most popular methods for mutagenicity evaluation of environmental factors. In some cases, this method is suggested to be the only and sufficient assay for the first stage of the equivalence assessment of pesticide technical grade active ingredients (TGAI) to the original products. A limitation of the Ames test is related to the impossibility of an objective equivalence assessment of some cytotoxic TGAIs, in particular, sulfonylureas, and triazolpyrimidines. Based on the mode of action of the pesticides belongs to these chemical classes, we suggested a modification of the plate incorporation method protocol of the Ames test to the increase of maximal non-cytotoxic concentration up to the 5 mg/plate recommended by regulatory documents. Materials and methods. The five strains of Salmonella typhimurium TA98, TA100, TA1535, TA97, TA102 were used. The modification of the protocol included a supplementation of the top agar with isoleucine (1-5 mM). Results. The maximum non-cytotoxic concentrations of thifensulfuron-methyl and florasulam using the standard top agar did not exceed 0.05-0.125 mg/plate. The enrichment of the top agar with isoleucine allowed evaluating the mutagenicity of the substances up to the maximal recommended concentration of 5.0 mg/plate. The number of spontaneous revertants was within the historical limits of the laboratory control obtained under standard conditions. Positive controls showed pronounced mutagenic effects in case of all strains with and without metabolic activation (p≤0.05). Limitations. Mutagenicity was evaluated only for TGAIs, which are acetohydroxyacid synthase inhibitors. Conclusion. The application of the modified Ames test protocol for mutagenicity assessment of TGAIs from the classes of sulfonylureas and triazolpyrimidines under supplementation of the top agar with isoleucine is a more objective way to evaluate their mutagenicity. The proposed protocol expands the possibilities of revealing dangerous mutagenic impurities that may occur in TGAIs in the small quantities, and after entering the environment can cause the gain in the mutation level in living organisms.

Morphological and Genetic Characterization of Eggerthella lenta Bacteriophage PMBT5.

Eggerthella lenta is a common member of the human gut microbiome. We here describe the isolation and characterization of a putative virulent bacteriophage having E. lenta as host. The double-layer agar method for isolating phages was adapted to anaerobic conditions for isolating bacteriophage PMBT5 from sewage on a strictly anaerobic E. lenta strain of intestinal origin. For this, anaerobically grown E. lenta cells were concentrated by centrifugation and used for a 24 h phage enrichment step. Subsequently, this suspension was added to anaerobically prepared top (soft) agar in Hungate tubes and further used in the double-layer agar method. Based on morphological characteristics observed by transmission electron microscopy, phage PMBT5 could be assigned to the Siphoviridae phage family. It showed an isometric head with a flexible, noncontractile tail and a distinct single 45 nm tail fiber under the baseplate. Genome sequencing and assembly resulted in one contig of 30,930 bp and a mol% GC content of 51.3, consisting of 44 predicted protein-encoding genes. Phage-related proteins could be largely identified based on their amino acid sequence, and a comparison with metagenomes in the human virome database showed that the phage genome exhibits similarity to two distantly related phages.

Effect of Antibiotics and Glycerol on Improving Bacteriophage Detection and Enumeration

Antibiotic-resistant bacteria are a growing issue in aquaculture, and phage therapy offers an alternative approach to the use of antibiotics. Phages for phage therapy can be isolated, identified and enumerated using a Double-Layer Agar (DLA) technique. While some phages form large and well-defined plaques that can be enumerated when plated with the DLA technique, some produce small and turbid plaques that are difficult to detect and count. A phage, Aeromonas phages-T65, was isolated from Aeromonas hydrophila, which formed small and turbid plaques making it hard to count. The use of antibiotics and chemicals have been suggested as a way to increase plaque size for easy detection and enumeration of phage plaques. To increase the plaque size of Aeromonas phages-T65, antibiotics and glycerol were added to the culture media, and tested at different concentrations. Application of penicillin-streptomycin and/or glycerol showed an increase in plaque size and the biggest improvement in plaque size were observed using either 40 U/μl penicillin-streptomycin or a 5 % glycerol in the top agar layer of the DLA technique. These findings suggest that such modifications could be implemented into the standard DLA overlay method to investigate new Aeromonas phages for the development of phage therapy against Aeromonas spp. in aquaculture.

Double-Layer Agar (DLA) Modifications for the First Step of the Phage-Antibiotic Synergy (PAS) Identification

The research carried out so far for phage-antibiotic synergy (PAS) differs as regards the technique of modifying the double-layer agar (DLA) method to show the PAS effect on Petri plates, which may contribute to non-uniform research results. Therefore, there is a need to unify the method to effectively detect the PAS effect, at its most basic in vitro test. In this study, bacteriophage T45 and 43 antibiotics belonging to different antibiotic classes were used. Seven different DLA method modifications were tested, in terms of antibiotic addition placement and presence or absence of the base agar. The overall number of phage plaques per plate mainly depended on the antibiotic used. Differences in plaque quantity depended on the type of the DLA method modification. The largest total number of plaques was obtained by the addition of an antibiotic to a bottom agar with the presence of a top agar. This indicates that even though an antibiotic could manifest the PAS effect by a standard disk method, it would be worth examining if the effect is equally satisfactory when applying antibiotics directly into the agar, with regards to using the same bacteriophage and bacterial host.

Screening Bacterial Colonies Using X-Gal and IPTG: α-Complementation.

Many plasmid vectors (e.g., the pUC series, Bluescript, pGem, and their derivatives) carry a short segment of Escherichia coli DNA containing the regulatory sequences and the coding information for the first 146 amino acids of β-galactosidase. Vectors of this type are used in host cells that express the carboxy-terminal portion of β-galactosidase. Although neither the host-encoded fragments nor the plasmid-encoded fragments of β-galactosidase are themselves active, they can associate to form an enzymatically active protein. This type of complementation, in which deletion mutants of the operator-proximal segment of the lacZ gene are complemented by β-galactosidase-negative mutants that have the operator-proximal region intact, is called α-complementation. The lac+ bacteria that result from α-complementation are easily recognized because they form blue colonies in the presence of the chromogenic substrate X-Gal. However, insertion of a fragment of foreign DNA into the polycloning site of the plasmid almost invariably results in production of an amino-terminal fragment that is no longer capable of α-complementation. Bacteria carrying recombinant plasmids therefore form white colonies. To screen bacterial colonies, the chromogenic substrate X-Gal and the gratuitous inducer IPTG are mixed with suitable dilution of a culture, combined with molten top agar, and then spread on agar plates containing the appropriate antibiotic. The efficiency of transformation is slightly higher when the bacteria are plated in top agar rather than on the surface of agar plates. Perhaps the transformed bacteria prefer the slightly anaerobic state within the soft agar or the isosmolarity provided by the agar medium.

Выбор оптимальных параметров получения сбивных изделий без яичного белка

Development of new types of functional whipped confectionery of souffle type in which animal proteins are replaced with vegetable ones is promising and of high priority. The author proposed to introduce top grade wheat flour into the recipe of the whipped product and lemon zest as an enriching additive. Experimental studies were carried out under laboratory conditions of the department of technology of bakery, confectionery, macaroni and grain processing industries of Voronezh State University of Engineering Technologies. The whipped product was obtained using an experimental whipping device developed by the staff of the department. The author justified the choice of the components in the recipe. The optimum modes of obtaining the whipped product which includes top grade wheat flour, water, citric acid, agar, white sugar, molasses, and lemon zest were selected using the experimental-statistical approach. The main cooking parameters are the duration of whipping (seconds); rotation frequency of the whipping parts (minutes-1), an output parameter (the volume weight of the final product, g/cm3). The results are the following: duration of the mass whipping x1 = 379 s-1, rotation speed of the whipping parts x2 = 651 min-1. The author determined quality indicators of the final whipped product. It has a good form-retaining ability, uniform and fine-porous structure, slightly creamy color, and slight taste of lemon zest. Mass fraction of dry matter is 76%, density – 0.43 g/cm3. The obtained product has original organoleptic parameters and higher nutritional value. The author did not include any food color additives and flavoring agents. For that reason the product can be recommended not only to people suffering from egg white intolerance, but also to all categories of consumers.

Top Starch Plating Method for the Efficient Cultivation of Unicellular Red Alga Cyanidioschyzon merolae.

The unicellular red alga Cyanidioschyzon merolae has been used as a model photosynthetic eukaryote for various basic and applied studies, and several of these molecular genetics techniques have been reported. However, there are still improvements to be made concerning the plating method. The conventional plating method often generates diffuse colonies and single colonies cannot be easily isolated. To overcome these problems, we established a novel plating method for C. merolae, making use of melted cornstarch as the use of top agar plating in bacterial genetics. This method improved the formation of defined colonies in at least 4-fold higher efficiency than the conventional method, and made the handling procedure much easier than the previous method.

Recipes and Tools for Culture of Escherichia coli.

In this article, we provide information about culture media, including minimal liquid media, rich liquid media, solid media, top agar, and stab agar. We also provide descriptions and useful information about tools used with growth media such as inoculating loops, sterile toothpicks, and spreaders. © 2018 by John Wiley & Sons, Inc.

Plating Bacteriophage M13.

A plaque of bacteriophage M13 derives from infection of a single bacterium by a single virus particle. The progeny particles infect neighboring bacteria, which, in turn, release another generation of daughter virus particles. If the bacteria are growing in semisolid medium (e.g., containing agar or agarose), then the diffusion of the progeny particles is limited. Cells infected with bacteriophage M13 are not killed, but have a longer generation time than uninfected Escherichia coli In consequence, plaques appear as areas of slower-growing cells on a faster-growing lawn of bacterial cells. This protocol describes plating of bacteriophage M13 stocks. Plaques are readily detectable on top agar after 4-8 h of incubation at 37°C.

High throughput selection of antibiotic-resistant transgenic Arabidopsis plants

Kanamycin resistance is the most frequently used antibiotic-resistance marker for Arabidopsis transformations, however, this method frequently causes escape of untransformed plants, particularly at the high seedling density during the selection. Here we developed a robust high-density selection method using top agar for Arabidopsis thaliana. Top agar effectively suppressed growth of untransformed wild-type plants on selection media at high density. Survival of the transformed plants during the selection were confirmed by production of green true leaves and expression of a firefly luciferase reporter gene. Top agar method allowed selection using a large amount of seeds in Arabidopsis transformation.

Enhancement of Antifungal Activity of Juglone (5-Hydroxy-1,4-naphthoquinone) Using a Poly(d,l-lactic-co-glycolic acid) (PLGA) Nanoparticle System

This study aimed to synthesize and characterize juglone-entrapped poly(d,l-lactic-co-glycolic acid) (PLGA) nanoparticles and compare the antifungal properties of free juglone with its PLGA nanoparticle formulation for the first time. The juglone-loaded nanoparticles prepared using the oil-in-water (o/w) single-emulsion solvent evaporation method were characterized by the reaction yield (RY), encapsulation efficiency (EE), polydispersity index (PDI), particle size, zeta potential (ZP), FT-IR, and in vitro release properties and evaluated for their morphological features using SEM. The nanoparticle formulation had size, RY, ZP, EE, and PDI values of 212 nm, 66.91 ± 2.4%, -16.3 ± 0.7 mV, 70.66 ± 3.1%, and 0.083 ± 0.024, respectively. In vitro release showed a triphasic pattern with initial burst followed by sustained release and dormant phase over the study period, releasing about 72.8% in total after 42 days. The antifungal studies against Aspergillus flavus, Candida albicans, and Fusarium spp. using agar dilution and top agar dilution methods indicated that the juglone-encapsulated nanoparticle was more effective than free juglone. This study showed that the top agar method, which was applied for the first time on antifungal activity, is more suitable for the nanoparticular system based on sustained release. Therefore, PLGA nanoparticle formulations may be an important tool for application in many areas for the effective and beneficial use of hydrophobic compounds such as juglone.

Sprouty2 regulates proliferation and survival of multiple myeloma byinhibiting activation of the ERK1/2 pathway invitro and invivo.

Multiple myeloma (MM) is an incurable disease, and its pathogenesis remains unclear. MicroRNA (miR)-21 was detected at a high level in MM and plays a key role in the pathogenesis of MM. However, Sprouty2 (spry2), a downstream target of miR-21, has low expression, and its mechanism in MM is unknown. We investigated whether spry2 could exert an antimyeloma effect and further studied the potential pathogenesis and progression of MM. To address the functional consequences of spry2, we assessed the expression levels of spry2 in several myeloma cell lines and detected low expression levels in MMcells. Overexpression of spry2 suppressed growth and colony formation ability and decreased the phosphorylation of extracellular signal-regulated kinases 1 and 2. Spry2 also decreased secretion of vascular endothelial growth factor and partially enhanced the sensitivity of MMcells to an inhibitor of mitogen-activated protein kinases 1 and 2. Additionally, spry2 inhibited the tumorigenesis and angiogenesis of MMcells invivo. In summary, we report for the first time that spry2 can inhibit MMcell growth and survival with a concomitant reduction in phosphorylation of extracellular signal-regulated kinases 1 and 2 invitro and invivo.

Antibacterial activity of bacteriophages isolated from poultry against Shiga-toxic strains of Esherichia coli isolated from calves

The aim of the study was to evaluate the antibacterial activity of selected bacteriophages obtained from poultry against selected Shiga-positive Escherichia coli strains isolated from calves under in vitro conditions. Eight bacteriophages of the families Myoviridae and Siphoviridae, isolated from different poultry housing sys-tems in south-eastern Poland, were used in the experiment. Bacteriophage morphology was examined with a transmission electron microscope on negative-stained slides with 2% silicotungstate. The range of lytic activity against Shiga-toxin-producing strains of E. coli was determined by a plaque assay on double-layer top agar plates. Twelve Shiga-toxin-producing E. coli strains isolated from calves were obtained from the National Veterinary Research Institute in Puławy (Department of Hygiene of Food of Animal Origin), and nine of our own strains were collected from calves with clinical signs of diarrhoea in the Lublin region. The results obtained showed that the phages specific to E. coli isolated from poultry express strong lytic anti-bacterial properties against Shiga-positive E. coli isolated from calves. These phages exhibited complete lysis of E. coli strains on double-layer agar plates. This result confirms the lack of a species barrier in the antibacte-rial activity of bacteriophages, which suggests their usefulness in eliminating E. coli infections in cattle.

A double layer plaque assay using spread plate technique for enumeration of bacteriophage MS2

Bacteriophage MS2 is used widely as a model organism to estimate pathogenic virus survival in various environments, and is usually quantified by plaque assay. Although current plaque assays work well in enumeration of MS2 in environmental samples, quantification of MS2 calls for better visibility and higher consistency. In an attempt to improve the visibility and consistency of the current plaque assay, spread plate technique was introduced, instead of the pour plate technique used commonly in existing methods. Other parameters that influence the outcome of the plaque assay were also compared. Using spread plate technique resulted in an increase of plaque size by approximately 50% and contributed to a better visibility. Addition of supplements (glucose, CaCl2 and thiamine); reduction of agar thickness and hardness, also contributed to enhanced plaque visibility and increased plaque count. Among all the conditions tested, a supplemented thin bottom agar (10ml 1% agar) and a supplemented thin top agar (10ml 0.45% agar) with spread plate technique gave the maximum countable plaques with a minimum standard deviation. When compared to other methods, it produced significantly higher plaque count and lower variation. The optimized plaque assay significantly improved visibility and consistency of the existing plaque assay methods and could be used in quantification of MS2.

Activation of the Leukotriene B4 Receptor 2-Reactive Oxygen Species (BLT2-ROS) Cascade following Detachment Confers Anoikis Resistance in Prostate Cancer Cells

The majority of prostate cancer-related deaths are associated with advanced and metastatic malignancies. Although anoikis resistance has been recognized as one of the hallmarks of metastatic prostate malignancies, the molecular events that cause anoikis resistance are poorly understood. In this study, we found that the detachment of PC-3 prostate cancer cells caused a time-dependent increase in the expression level of the leukotriene B4 receptor-2 (BLT2) and that BLT2 played a critical role in establishing anoikis resistance in these cells. Blocking BLT2 with the pharmacological inhibitor LY255283 or with RNAi knockdown clearly abolished anoikis resistance and resulted in severe apoptotic death. Additionally, we demonstrated that the activation of NADPH oxidase (NOX) and subsequent generation of reactive oxygen species (ROS) were downstream of BLT2 signaling and led to the activation of NF-κB, thus establishing anoikis resistance during cell detachment. Furthermore, we observed that the ectopic expression of BLT2 in normal prostate PWR-1E cells rendered the cells resistant to anoikis and apparently diminished apoptotic cell death following detachment. Taken together, our results suggest that BLT2-NOX-ROS-NF-κB cascade induction during detachment confers a novel mechanism of anoikis resistance in prostate cancer cells and potentially contributes to prostate cancer progression.