Petri Dishes Research Articles

Mating Assay: Plating Below a Cell Density Threshold is Required for Unbiased Estimation of Plasmid Conjugation Frequency of RP4 Transfer Between E. coli Strains

Mating assays are common laboratory experiments for measuring the conjugation frequency, i.e. efficiency at which a plasmid transfers from a population of donor cells to a population of recipient cells. Selective plating remains a widely used quantification method to enumerate transconjugants at the end of such assays. However, conjugation frequencies may be inaccurately estimated because plasmid transfer can occur on transconjugant-selective plates rather than only during the intended mating duration. We investigated the influence of cell density on this phenomenon. We conducted mating experiments with IncPα plasmid RP4 harbored in Escherichia coli at a fixed cell density and mating conditions, inoculated a serial dilution of the mating mixture on transconjugant-selective plates or in transconjugant-selective broth, and compared the results to a model of cell-to-cell distance distribution. Our findings suggest that irrespective of the mating mode (liquid vs solid), the enumeration of transconjugants becomes significantly biased if the plated cell density exceeds 28 Colony Forming Unit (CFU)/mm2 (or 1.68•105 CFU/standard 9 cm Petri dish). This threshold is determined with a 95% confidence interval of ± 4 CFU/mm2 (± 2.46•104 CFU/standard 9 cm Petri dish). Liquid mating assays were more sensitive to this bias because the conjugation frequency of RP4 is several orders of magnitude lower in suspension compared to surface mating. Therefore, if selective plating is used, we recommend to plate at this density threshold and that negative controls are performed where donors and recipients are briefly mixed before plating at the same dilutions as for the actual mating assay. As an alternative, a liquid enumeration method can be utilized to increase the signal-to-noise ratio and allow for more accurate enumeration of transconjugants.

Formation of linear arrays of holes in self-assembled collagen films

Collagen is one of the main constituents of mammalian extracellular matrix and is used extensively as a coating for tissue culture dishes and medical implants to promote cell growth and proliferation. By modulating the topography of the collagen coating at the nanometer to micrometer length scales, it is possible to achieve spatial control over cell growth and morphology. In this work, we are exploring the self-assembly of a thin collagen film on a glass substrate as a way to create new nanoscale surface features. By controlling the collagen concentration and adding an oscillatory flow, we are able to enrich the collagen film surface with a localized pattern of ripples oriented perpendicular to the flow direction. We propose that these ripples are the result of dewetting of the collagen film that leads to the formation of adjacent holes. We observe that individual holes form with an anisotropic rim due to the microstructure of the deposited collagen fibril network. This intrinsic anisotropy and the oscillatory flow yield new holes being formed in the film next to existing rims. As holes keep growing deeper, the rims extend along the flow direction, and the holes appear rectangular in shape, which gives the linear array of holes the apparent morphology of a ripple. Overall, we are able to create localized ripples at the surface of collagen films that would be difficult to produce via standard nanofabrication techniques.

Stress factors and cytotoxic and genotoxic action of ethanol in Saccharomyces cerevisiae

In industrial fermentation, Saccharomyces cerevisiae are exposed to different stress conditions. In this sense, the aim of this study was to evaluate the toxic action of ethanolic stress on Saccharomyces cerevisiae. Exploratory research was carried out on the stress factors that cause injuries in yeast. Fermentation tests were conducted with the Fleischmann® and Pedra-2 strains, cultivated in sugarcane juice at 22 ºBrix and pH 5.0, adding concentrations of 5, 10, and 15% of ethyl alcohol, and incubated at 30°C at 250 rpm for 10 hours. For the cytotoxic tests, 100 µl of samples were collected for evaluation of cell growth by spectrophotometric measurements at 570 nm, and 5 µl were dripped into Petri dishes containing 2% YPD solid medium and incubated at 30ºC for 72 hours for colony growth. For the genotoxicity test, the comet test was used with 0.5 µl of the samples added to slides previously prepared and subjected to electrophoretic running and subsequently stained in a 0.1% silver nitrate solution. 100 random nucleotides were evaluated, evaluating five DNA damage classes (0, 1, 2, 3, and 4) according to the intensity and pattern of genetic material entrainment. The results show that stress factors interfere with yeast performance. Fleischmann® showed sensitivity to ethanolic stress.

Co-culture of vascular endothelial cells enhances corticosterone production in steroid hormone-producing cells generated from adipose-derived mesenchymal stromal cells

Cell therapy for adrenocortical insufficiency can potentially provide steroid replacement in response to physiological stimuli. Previously, we reported that adipose tissue-derived stromal cells (ADSCs) are transformed into steroid-producing cells by overexpression of nuclear receptor subfamily 5 group A member 1 (NR5A1). The steroidogenic cells are characterized by the production of both adrenal and gonadal steroids. Cytotherapy for adrenocortical insufficiency requires cells with more adrenocortical characteristics. Considering the highly developed vascular network within the adrenal cortex, all adrenocortical cells are adjacent to and interact with vascular endothelial cells (VECs). In this study, NR5A1-induced steroidogenic cells derived from mouse ADSCs (NR5A1-ADSCs) were co-cultured with mouse VECs. Testosterone secretion in NR5A1-ADSCs was not altered; however, corticosterone secretion significantly increased while levels of steroidogenic enzymes significantly increased in the corticosterone synthesis pathway. Co-culture with lymphatic endothelial cells (LECs) or ADSCs, or transwell culture with NR5A1-ADSCs and VECs did not alter corticosterone production. VECs expressed higher levels of collagen and laminin than LECs. Culture in type-IV collagen and laminin-coated dishes increased corticosterone secretion in NR5A1-ADSCs. These results suggest that VECs may characterize ADSC-derived steroidogenic cells into a more corticosterone-producing phenotype, and VECs may be useful for generating adrenal steroidogenic cells from stem cells.

Double-Cannula Maneuver for the Double-Roll Configuration of Donor Descemet Membrane Outside Recipient Anterior Chamber During Descemet Membrane Endothelial Keratoplasty.

The purpose of this study was to present a new surgical technique to convert a single roll of Descemet membrane (DM) into a double roll using 2 cannulas in a balanced salt solution-filled Petri dish during DM endothelial keratoplasty. A single DM roll stained with trypan blue was placed in a balanced salt solution-filled Petri dish. Two cannulas (28G) were introduced from opposite ends of the single roll, inserted into the roll, and slowly spread apart to change the single roll into a double roll. The DM was aspirated into the modified Jones tube and loaded, maintaining a double-roll configuration with endothelium-down orientation in a bevel-up position. The modified Jones tube with the bevel down was inserted into the recipient anterior chamber through the main wound. The modified Jones tube was rotated to the bevel-up orientation. After checking the graft orientation, the DM was inserted into the recipient anterior chamber. The double-roll DM was easily unfolded by tapping the center of the cornea using a cannula. A 28G cannula was inserted under the DM, and the anterior chamber was filled with air. Three months after surgery, the patient's corrected visual acuity in the right eye was 6/7.5 and the endothelial cell count was 1095/mm2. The corneal thickness was 533 μm, and the cornea was clear. The double-cannula maneuver mechanically changes the single roll of the donor DM to a double roll outside the recipient anterior chamber, making DM unfolding easier and minimizing the risk of upside-down apposition of the donor DM.

Differentiation potential of SOX2-positive stem cells in the bovine pituitary gland

The pituitary gland is the master endocrine gland, harboring stem cells with various genetic characteristics; however, data from non-rodent and non-human sources are scarce. In this study, we isolated putative stem cells from the bovine pituitary gland and investigated their potential for differentiation into hormone-producing cells. Immunohistochemical analysis revealed that in calves and heifers, stem cell marker sex-determining region Y-box 2 (SOX2)-positive cells were widely present in the pituitary gland and partially co-localized with anterior pituitary hormones. Next, a single-cell suspension of primary anterior lobe cells from bovines aged 0 and 12 months was subjected to two-dimensional culture. Consequently, some cells proliferated in the culture dishes. The expression levels of Sox2 and several other stem cell markers were higher in these cells after culture. In addition, almost all proliferating cells were positive for SOX2, whereas all were negative for hormones. In three-dimensional cultures, SOX2-positive cells presented a spheroid-like morphology and differentiated into endocrine cells. These results provide evidence that SOX2-positive cells are pituitary stem cells with the potential to differentiate into hormone-producing cells, regardless of age. Our data lay a theoretical foundation for further studies on controlling fundamental processes, such as body growth, reproduction, and lactation.

(Invited) An in Vitro Model of Mechanical Strain Enhances Cellular Uptake of Single-Walled Carbon Nanotubes

Single-walled carbon nanotubes (SWCNTs) are ideal optical biosensors due to their intrinsic near-infrared (NIR) emission, which is highly sensitive, easily tunable, and indefinitely photostable. Due to such desirable properties coupled with appropriate functionalization strategies, SWCNTs have been used in numerous in vitro, live-cell, and in vivo applications. Depending on the type of functionalization as well as cell type, SWCNTs can be internalized by live cells via demonstrated energy-dependent endocytosis processes where they are retained within the endosomal pathway or are expelled by the cells. While this uptake and intracellular processing of SWCNTs has been recently examined, these studies are usually performed with standard cell culture techniques in petri dishes or 3D cultures. Unlike these cultures, cells in living organisms are continuously exposed to a wide variety of stimuli, e.g., periodic mechanical strain, during both normal and disease-related conditions. Thus, it is important to investigate how cells under physiological stretching compare to those grown in standard cultures. Herein, using a novel cell stretching platform to mimic physiological conditions, we investigate the uptake of SWCNTs into different human cancer cells.Our results indicate that cells exposed to uniform radial stretching display significantly higher rates of nanoparticle uptake, which can be correlated to their increased level of cellular activity. This approach recognizes the importance of studying cellular responses in environments that more closely resemble the complex conditions found in living organisms. It suggests a potential connection between mechanical stimuli and cellular processes related to SWCNT uptake. This finding has implications for understanding how nanotube uptake differs in different cellular stress environments and how cells respond to their dynamic and ever-changing microenvironment.Investigating how the mechanical strain influences the endocytosis processes and subsequent intracellular fate of SWCNTs can provide valuable insights into the interplay between nanomaterials and living cells under more realistic conditions. Further aiding us in the development of more accurate intracellular biosensors and effective drug delivery platforms using SWCNTs.

Self-regulating pen-needle-based micronozzle for printing array of nanoliter droplets under fluorinated liquid

High-throughput evaluation of materials and cells is crucial for scientific progress. Creating arrays of nanoliter (nL) droplets via contact printing offers an effective method for such evaluations. However, precise calibration between the printing tip and substrate is typically required, which can be time-consuming and affect printing quality. To address this issue, we developed a self-regulating microneedle based on a spring and pen needle and created an automated droplet printer using a Cartesian robot. The self-regulation mechanism eliminated the need for precise tip-substrate calibration. Nanoliter droplets were printed onto a Petri dish beneath a layer of perfluorocarbon (PFC) to maintain shape and prevent evaporation. Droplet volume (V) increased linearly with flow rate (Q) as V = 8.8 Q − 5.4 (nL, µL/min). Droplet volume decreased hyperbolically with robot speed (w) as V = 1613 w −1 + 14.3 (nL, mm/s), while the number of droplets produced per minute (N) increased linearly with speed as N = 2.0 w + 28.5. The system achieved a production rate of 42 droplets/min at 10 mm/s, scaling up to 180 droplets/min at 80 mm/s. This versatile platform enables nL-scale assays for various applications.

First Report of Phytophthora cinnamomi causing root rot of avocado trees in Greece.

In September 2023, thirty declining 30-year-old avocado (Persea americana) trees ('Hass' grafted on 'Zutano' seedlings) were detected in a 1.5-ha orchard in the island of Crete (Chania region). Crown symptoms encompassed wilting and leaf chlorosis, advancing to defoliation and extensive dieback. Tap and feeder roots decayed and brown discoloration of root tissues was evident on heavily infected trees. The disease was severe and widespread, resulting in a 5% mortality rate among 300 trees. The pathogen was isolated with a modified soil baiting technique (Ferguson and Jeffers, 1999). Surface disinfected avocado fruits were immersed in water containing soil samples. Following a period of 2 to 8 days, tissue fragments from the resulting necrotic lesions on the fruit surface were transferred on ΡΑRP medium and subsequently incubated at 20°C (Ferguson and Jeffers, 1999). Three isolates (AV2, AV12 and AV11a) were obtained by transferring single hyphal tips to new Petri dishes containing V8 juice agar. They were grown at 20˚C and used for identification after 10 days. Isolates formed coralloid colonies with abundant clustered spherical hyphal swellings and terminal or intercalary (ratio 1:5) thick-walled chlamydospores measuring 20 to 36 μm (avg 29±0.8 μm) with characteristic thick walls (avg 1.2±0.2 μm). Sporangia, produced in non-sterile soil-extract water, were ovoid to obpyriform, persistent, non-papillate, 32 to 81 μm (avg 56±4.8 μm) long and 20 to 42 μm (avg 31±3.2 μm) wide (n=100). Isolates were heterothallic as they did not produce oospores in single cultures. Based on the morphological traits the isolates were identified as Phytophthora cinnamomi (Erwin and Ribeiro 1996). The internal transcribe spacer region (ITS) including ITS1, 5.8S rDNA region, and ITS2 as well as the cytochrome c oxidase subunit I (coxI) gene of the three representative isolates wereamplified with ITS1/ITS4 and FM83/FM84 primers, respectively (White et al. 1990; Martin and Tooley 2003), and sequenced (GenBank acc. PP506613 to PP506615 and PQ063867 to PQ063869, respectively). BLAST search revealed almost 100% identity with the sequences of P. cinnamomi ex-isotype isolate (KC478663 and KU899315 respectively). Pathogenicity tests using isolate AV2 were conducted following the soil infestation method (Jung et al. 1996) using six-year-old avocado 'Zutano' seedlings. Six non-inoculated plants treated with vermiculite-multivitamin juice mixture were used as controls. Plants (1 m tall) were grown in pots under greenhouse conditions and watered regularly. Six weeks post inoculation, all inoculated trees showed chlorosis, wilting and root rot, while control plants remained symptomless. Symptoms were similar to those observed in the field and the pathogen was re-isolated and molecularly identified as previously described. This study presents the first documented occurrence of P. cinnamomi, widely regarded as the most destructive avocado pathogen globally, on avocado crops in Greece (Rodger et al. 2019). Additionally, this marks the first recorded presence of this pathogen on the island of Crete, regardless of the host species. The accurate identification of Phytophthora species associated with avocado root rot is essential for implementing an effective disease management strategy, particularly in the selection of appropriate disease-resistant rootstocks.

Bi-layered Adipose Mesenchymal Cell Sheets Improve Bladder Compliance in Spinal Cord-Injured Rats.

To improve bladder compliance in patients with low-compliance bladders, augmentation cystoplasty with the intestinal tract is performed. However, the use of the intestinal tract often leads to serious surgical complications. Tissue engineering technologies have the potential to improve bladder compliance without using the intestinal tract. In this study, we fabricated bi-layered adipose-derived mesenchymal cell (AMC) sheets and then determined whether the bi-layered AMC sheets could improve bladder compliance in rats with spinal cord injury (SCI). The abdominal adipose tissues of green fluorescence protein (GFP)-transfected Sprague-Dawley (SD) rats were harvested, and the attached and proliferating cells on type I collagen were used as AMCs. The AMCs were then cultured on temperature-responsive culture dishes. After reaching over-confluence, the AMCs that maintained cell-cell contacts were detached from the dishes and applied to a gelatin hydrogel sheet. Then, another detached AMC monolayer was accumulated on the AMC monolayer-applied gelatin. Prior to 4 weeks of transplantation, the levels of T8-9 in the spinal cords of recipient SD rats were partially transected. After producing the bi-layered AMC sheets and the rats with SCI, the detrusor muscles of the anterior bladder walls of the rats with SCI were incised, and the bi-layered AMC sheet was patch-transplanted onto the exposed bladder epithelium (n = 8). As a control, the sham operation was performed (n = 7). Four weeks after the transplantation, bladder capacity and bladder compliance in AMC sheet-transplanted SCI rats were significantly higher than those in sham-operated control SCI rats. The smooth muscle layers in AMC sheet-transplanted bladders were significantly larger than those in control bladders. In addition, the collagen fibers in the AMC sheet-transplanted bladders were significantly smaller than those in the control bladders. Some GFP-positive transplanted AMCs differentiated into smooth muscle actin- or desmin-positive cells. Furthermore, GFP-positive cells secreted transforming growth factor-β1 or vascular endothelial growth factor. Therefore, this study showed that bi-layered AMC sheets could improve bladder compliance and bladder tissues in SCI rats.

Effects of nutrition on recovery, mortality, and mobility of adult Tribolium castaneum after exposure to long-lasting insecticide-incorporated netting.

Long-lasting insecticide-incorporated netting (LLIN) has been used to deliver contact insecticides as an integrated pest management tool for stored product insect pests in food facilities. Although the presence of food is known to improve insect recovery after exposure, it is not clear whether food nutritional quality plays a role. Here, the red flour beetle Tribolium castaneum adults were exposed to two commercially available LLINs, Carifend (active ingredient α-cypermethrin) and D-Terrence (deltamethrin), then transferred to Petri dishes with foods with varying nutritional quality (e.g., 0-100% ratios of flour to non-nutritive cellulose). We investigated the effects of nutrition, LLIN type, and exposure time on post-exposure recovery, mortality, and mobility. After exposure for 2-168 h, the immediate mortality of T. castaneum adults ranged from 0.5% to 91.0% with Carifend and 0% to 75.3% with D-Terrence. Adult recovery and delayed mortality were significantly affected by nutritional quality, LLIN type, exposure time, and recovery time. For both LLINs, adult recovery increased over time, with a trend for higher recovery and lower mortality with increasing nutritional quality and decreasing exposure time. In addition, adult mobility decreased multiple-fold after Carifend or D-Terrence exposure for 30, 60 or 90 min compared to 10 min. This study shows nutrition significantly modulates the efficacy of LLIN against T. castaneum, and thus strengthens the rationale for implementing stringent sanitation protocols for food facility managers. © 2024 Society of Chemical Industry. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Patterned PVA Hydrogels with 3D Petri Dish® Micro-Molds of Varying Topography for Spheroid Formation of HeLa Cancer Cells: In Vitro Assessment

Cell spheroids are an important three-dimensional (3D) model for in vitro testing and are gaining interest for their use in clinical applications. More natural 3D cell culture environments that support cell–cell interactions have been created for cancer drug discovery and therapy applications, such as the scaffold-free 3D Petri Dish® technology. This technology uses reusable and autoclavable silicone micro-molds with different topographies, and it conventionally uses gelled agarose for hydrogel formation to preserve the topography of the selected micro-mold. The present study investigated the feasibility of using a patterned Poly(vinyl alcohol) hydrogel using the circular topography 12–81 (9 × 9 wells) micro-mold to form HeLa cancer cell spheroids and compare them with the formed spheroids using agarose hydrogels. PVA hydrogels showed a slightly softer, springier, and stickier texture than agarose hydrogels. After preparation, Fourier transform infrared (FTIR) spectra showed chemical interactions through hydrogen bonding in the PVA and agarose hydrogels. Both types of hydrogels favor the formation of large HeLa spheroids with an average diameter of around 700–800 µm after 72 h. However, the PVA spheroids are more compact than those from agarose, suggesting a potential influence of micro-mold surface chemistry on cell behavior and spheroid formation. This was additionally confirmed by evaluating the spheroid size, morphology, integrity, as well as E-cadherin and Ki67 expression. The results suggest that PVA promotes stronger cell-to-cell interactions in the spheroids. Even the integrity of PVA spheroids was maintained after exposure to the drug cisplatin. In conclusion, the patterned PVA hydrogels were successfully prepared using the 3D Petri Dish® micro-molds, and they could be used as suitable platforms for studying cell–cell interactions in cancer drug therapy.

Biophysical fluid dynamics in a Petri dish

The humble Petri dish is perhaps the simplest setting in which to examine the locomotion of swimming organisms, particularly those whose body size is tens of microns to millimeters. The fluid layer in such a container has a bottom no-slip surface and a stress-free upper boundary. It is of fundamental interest to understand the flow fields produced by the elementary and composite singularities of Stokes flow in this geometry. Building on the few particular cases that have previously been considered in the literature, we study here the image systems for the primary singularities of Stokes flow subject to such boundary conditions—the Stokeslet, rotlet, source, rotlet dipole, source dipole, and stresslet—paying particular attention to the far-field behavior. In several key situations, the depth-averaged fluid flow is accurately captured by the solution of an associated Brinkman equation whose screening length is proportional to the depth of the fluid layer. The case of hydrodynamic bound states formed by spinning microswimmers near a no-slip surface, discovered first using the alga , is reconsidered in the geometry of a Petri dish, where the power-law attractive interaction between microswimmers acquires unusual exponentially screened oscillations. Published by the American Physical Society 2024

Variation in Phenology and Morphological Traits of Seed-Propagated Laggera alata and Laggera crispata Forms

The phenological and morphological variation studies among forms of Laggera Sch. Bip. Ex. Benth. and Hook species are limited, despite the medicinal use of the genus. Therefore, this study aimed to document phenology and morphological variation in cultivated populations of Laggera alata and Laggera crispata forms from seedling to maturity. The forms were categorized as Laggera alata with a small capitulum (LA-SC), Laggera alata with a large capitulum (LA-BC), Laggera crispata from South Africa (LC-SA), and Laggera crispata from Zimbabwe (LC-ZIM). Seeds were germinated in Petri dishes, transplanted to plug trays, and later to field plots at 60 days. Phenological events were recorded when observed in at least one plant. Twelve qualitative and four morphometric traits were measured monthly on five plants per Laggera form. Analysis of variance and Tukey’s Honestly Significant Difference test (p < 0.05) were used for data analysis. Results indicated significant variation in phenology, qualitative traits, leaf traits, plant height, and stem diameter both within and between L. crispata and L. alata forms. Morphometric traits, such as leaf size and the number of leaves per plant, were identified as key descriptors for differentiating L. alata forms. These findings provide a foundation for the introduction of Laggera forms into farming systems for medicinal and commercial purposes.

Antibacterial Efficacy of Hiora: An Ayurvedic Mouthwash in Children.

Mouthwashes, as a form of antimicrobial delivery system, rank among the safest and most effective vehicles, particularly in the case of young children. This is attributed to their ability to distribute therapeutic components across all accessible oral surfaces, including interproximal areas. To evaluate the antibacterial efficacy of recently introduced Ayurvedic (Hiora) and triclosan-based mouthwashes among children. A total of 45 healthy children aged 10-15 years were randomly assigned to three groups: Herbal mouthwash (Hiora), triclosan-based mouthwash (Kidodent), and normal saline as the control group. Saliva samples were collected pre-rinse, 2 minutes, 30 minutes, and 60 minutes post-rinsing with the study mouthwashes. These samples were then inoculated onto Petri dishes containing blood agar culture media, followed by incubation under both aerobic and anaerobic conditions at 37°C for 48 hours. The resulting bacterial colonies of Streptococcus spp. and Lactobacillus spp. were counted (CFU/ml x 105). Statistical analysis, including ANOVA, Newman Keul's Post-hoc test, and a two-tailed 't' test, was conducted to determine the significance of the results. The Ayurvedic Hiora mouthwash demonstrated the most substantial reduction in salivary bacterial colony counts of Streptococcus spp. and Lactobacillus spp. with statistically significant results (p<0.01). The Ayurvedic Hiora mouthwash exhibited the highest antibacterial effectiveness, followed by the triclosan-based mouthwash in decreasing order, with saline showing the least efficacy.

Rapid counting of coliforms and Escherichia coli by deep learning‐based classifier

AbstractTo ensure that food has been handled hygienically, manufacturers routinely examine the numbers of indicator bacteria, such as coliforms and Escherichia coli. Using the deep‐learning algorithm YOLO, we developed a classifier that automatically counts the number of coliforms (red colonies) and E. coli (blue colonies) on a chromogenic agar plate. Using Citrobacter freundii IAM 12471T and E. coli NBRC 3301, we trained our YOLO‐based classifier with images of Petri dishes grown with each strain alone (10 images) and/or with a mixture of both strains (5 images). When the performance of the classifier was evaluated using 83 images, the accuracy rates for coliforms and E. coli reached 99.4% and 99.5%, respectively. We then investigated whether this classifier could detect other, non‐trained coliform species (22 species) and E. coli strains (13 strains). The accuracy rates for coliforms and E. coli were 98.7% (90 Petri dishes) and 94.1% (46 Petri dishes), respectively. Furthermore, we verified the practical feasibility of the developed classifier using 38 meats (chicken, pork, and beef). The accuracy rates for coliforms and E. coli in meat isolates were 98.8% (80 Petri dishes) and 93.8% (35 Petri dishes), respectively. The time required to count coliforms/E. coli on a single plate was ~70 ms. This novel method should enable users to rapidly quantify coliforms/E. coli without relying on a human inspector's color vision, leading to improved assurance of food safety.

Osteogenic Differential Expression of Rabbit Adipose-Derived Mesenchymal Stem Cells by PPARγ Transfection Coated with Nanoparticles

Adipose-derived stem cells (ADSCs) have advantages in utilization. Nanoparticles (NP) are degradable, nontoxic and have good biocompatibility, besides, they can encapsulate and transport water-soluble, fat-soluble and deoxyribonucleic acid. This study explored the influence of NP-coated PPARγ transfection on osteogenic differentiation of rabbit ADSCs. After preparing PPARγ transfection system coated with nanoparticles, Petri dishes containing the same amount of cells were divided at the beginning of cell culture into blank group (not transfected), control group and observation group (transfected with PPARγ coated NP). Alkaline phosphatase (ALP) staining and Western Blot were done to detect the contents of lipoprteinlipase (LPL), Runx2 and serum osteocalcin (BGP) protein. The levels of Runx2, BGP and PPARγ mRNA were measured by RT-PCR. After ADSCs cells were stained, a small number of cells adhered to each other, and the cells were mainly fibroblast-like and spindle-shaped, and proliferated in the form of colonies. The levels of Runx2 and BGP in the observation group were significantly lower than those in the control group (p &lt; 0.05) after the ADSCs cells were cultured for 7 and 14 days. The expression of LPL and PPARγ mRNA in the observation group was higher control group (p &lt; 0.05). Transfection of PPARγ coated with nanoparticles inhibited the osteogenic differentiation of rabbit ADSCs by inhibiting the expression of Runx2 and BGP and promoting the expression of LPL and PPARγ.

Modulation of dielectric and antibacterial properties of Zn0.5Mn0.5O nanoparticles by post growth annealing method

In this manuscript, we have investigated the dielectric and antibacterial potential of hydrothermally synthesized ZnMnO nanoparticles. The synthesized nanoparticles were annealed at various temperatures ranging from 450 to 650 °C with a step of 50 °C to modulate the structural, vibrational, dielectric, and antibacterial properties. XRD data confirmed the hexagonal structure of the synthesized samples and crystalline size was decreased to 4.8 nm at annealing temperature 600 °C. The lattice structure was further verified by Raman spectroscopy measurements, which strongly verified the XRD data due the presence of ZnMnO vibrational modes. The dielectric measurements revealed that the dielectric constant and los tangent were found to be increased with the increase annealing temperature and decreased with frequency, while a.c conductivity has an increasing trend with both parameters (temperature and frequency). The plot of real and complex parts of impedance against frequency demonstrated that both parameters decrease with the increased in frequency. But when we analyzed the behavior of the real part of impedance against the annealing temperature, a degradation in real part behavior is observed. The antibacterial activity of ZnMnO nanoparticles was determined by using the disc diffusion method against E. coli bacteria, which was grown on a Petri dish at room temperature for 24 h. This observation revealed that the samples annealed at 450 °C and 550 °C show remarkable antibacterial sensitivity as compared to other samples. It is concluded that crystalline size of 20 nm is found to be optimal value for good anti-baterial behavior.

Assessment of bisphenol accumulation from disposable devices used sequentially in IVF routine procedures

Research question: To investigate the release of bisphenols (BPs) from disposable devices (DDs) used in ART procedures and to determine whether they accumulate when several DDs are used sequentially under routine conditions.Design: A comprehensive assessment of 19 individual DDs (31 measurements) and 9 combinations of DDs replicating the main steps in an ART procedure was undertaken. The extraction of BPs followed routine use conditions (temperature and duration). The concentration of 10 BPs was determined using on-line solid phase extraction/liquid chromatography/mass spectrometry methodology.Results: Bisphenol S (BPS) was quantified consistently from 100 mm culture dishes (32 ± 20 pg) and from high security sperm straws (3 ± 1 pg). Also, BPS and Bisphenol A (BPA) were quantified consistently from spermicide-free condoms (95 ± 78 and 83 ± 49 pg, respectively). No other BPs were detected in individually tested DD.When DDs were used together, BPA and BPS were both detected consistently in the association of 13 DDs mimickin;g the sperm collection in condom and its preparation (46 ± 16 and 43 ± 32 pg, respectively). BPS was quantified consistently in the association of 14 DDs mimicking the sperm collection, its preparation and freezing (10 ± 4 pg), and in the association of 17 DDs mimicking the oocyte pick-up (37 ± 22 pg).Conclusions: BPA and BPS are released in small quantities from some DDs used in routine conditions during ART procedures, but do not clearly accumulate when these consumables are used together.

Matrix stiffness regulates the triad communication of adipocytes/macrophages/endothelial cells through CXCL13

Adipose tissue remodeling and plasticity are dynamically regulated by the coordinated functions of adipocytes, macrophages, and endothelial cells and extracellular matrix (ECM) that provides stiffness networks in adipose tissue component cells. Inflammation and fibrosis are crucial exogenous factors that dysregulate adipose tissue functions and drastically change the mechanical properties of the ECM. Therefore, communication among the ECM and adipose tissue component cells is necessary to understand the multifaceted functions of adipose tissues. To obtain in vivo stiffness, we utilized genipin as a crosslinker for collagen gels. Meanwhile, we isolated primary adipocytes, macrophages, and endothelial cells from C57BL/6J mice and incubated these cells in the differentiation media on temperature-responsive culture dishes. After the differentiation, these cell sheets were transferred onto genipin-crosslinked collagen gels with varying matrix stiffness. We found that inflammatory gene expressions were induced by hard matrix, whereas anti-inflammatory gene expressions were promoted by soft matrix in all three types of cells. Interestingly, the co-culture experiments of adipocytes, macrophages, and endothelial cells showed that the effects of soft or hard matrix stiffness stimulation on adipocytes were transmitted to the distant adipose tissue component cells, altering their gene expression profiles under normal matrix conditions. Finally, we identified that a hard matrix induces the secretion of CXCL13 from adipocytes, and CXCL13 is one of the important transmitters for stiffness communication with macrophages and endothelial cells. These findings provide insight into the mechano-transmission into distant cells and the application of stiffness control for chronic inflammation in adipose tissues with metabolic dysregulation.