Petri Dishes Research Articles

Allelopathic potential of invasive alien plant species, Xanthium strumarium L., water and methanol extracts on germination and seedling growth of Guizotia abyssinica (L.f.) Cass., and Linum usitatissimum L.

The objective of this study was to investigate the allelopathic impact of Xanthium strumarium on the seedling growth and germination of Guizotia abyssinica and Linum usitatissimum. Plant specimens and samples of Xanthium strumarium were collected from the Minjar-Shenkora district in Ethiopia. In the laboratory, a series of dilutions of extracts from the leaves and roots of Xanthium strumarium were prepared using distilled water and methanol (99.85%) as solvents. Twenty-five grams of root and leaf samples were soaked separately in 250 mL of the specified solvent to create the stock solution. The experiment was carried out using Petri dishes in a completely random manner, with three replications repeated twice. Various concentrations of the extracts (25, 50, 75, and 100%) were applied as treatments, while distilled water served as the control. Each petri dish contained 25 seeds of each test crop and 5 mL of the corresponding extract. The number of Linum usitatissimum and Guizotia abyssinica seeds germinating was recorded every 24 hours until 12 and 14 days, respectively. Both descriptive and inferential analyses were conducted. When Guizotia abyssinica and Linum usitatissimum seeds were treated with 100% concentrations of water extract from leaf samples, germination percentages were inhibited by 72 and 77.4%, respectively. In methanol extracts, the entire inhibition of germination was observed in both test plant species at ≥ 50% and ≥ 75% concentrations of leaf and root samples, respectively. Therefore, the inhibitory effect of water and methanol extracts showed a concentration-dependent pattern, with increasing concentrations resulting in greater inhibition. The inhibitory effect of leaf extracts was more pronounced compared to root extracts, suggesting the presence of herbicidal activities. Further phytochemical and molecular studies are recommended to identify the active compounds in Xanthium strumarium. Additionally, utilizing isolated allelopathic substances as a means of controlling invasive alien species is suggested.

The study on 4D culture system of squamous cell carcinoma of tongue

Traditional cell culture methods often fail to accurately replicate the intricate microenvironments crucial for studying specific cell growth patterns. In our study, we developed a 4D cell culture model-a precision instrument comprising an electromagnet, a force transducer, and a cantilever bracket. The experimental setup involves placing a Petri dish above the electromagnet, where gel beads encapsulating magnetic nanoparticles and tongue cancer cells are positioned. In this model, a magnetic force is generated on the magnetic nanoparticles in the culture medium to drive the gel to move and deform when the magnet is energized, thereby exerting an external force on the cells. This setup can mimic the microenvironment of tongue squamous cell carcinoma CAL-27 cells under mechanical stress induced by tongue movements. Electron microscopy and rheological analysis were performed on the hydrogels to confirm the porosity of alginate and its favorable viscoelastic properties. Additionally, Calcein-AM/PI staining was conducted to verify the biosafety of the hydrogel culture system. It mimics the microenvironment where tongue squamous cell carcinoma CAL-27 cells are stimulated by mechanical stress during tongue movement. Electron microscopy and rheological analysis experiments were conducted on hydrogels to assess the porosity of alginate and its viscoelastic properties. Calcein-AM/PI staining was performed to evaluate the biosafety of the hydrogel culture system. We confirmed that the proliferation of CAL-27 tongue squamous cells significantly increased with increased matrix stiffness after 5 d as assessed by MTT. After 15 d of incubation, the tumor spheroid diameter of the 1%-4D group was larger than that of the hydrogel-only culture. The Transwell assay demonstrated that mechanical stress stimulation and increased matrix stiffness could enhance cell aggressiveness. Flow cytometry experiments revealed a decrease in the number of cells in the resting or growth phase (G0/G1 phase), coupled with an increase in the proportion of cells in the preparation-for-division phase (G2/M phase). RT-PCR confirmed decreased expression levels of P53 and integrinβ3 RNA in the 1%-4D group after 21 d of 4D culture, alongside significant increases in the expression levels of Kindlin-2 and integrinαv. Immunofluorescence assays confirmed that 4D culture enhances tissue oxygenation and diminishes nuclear aggregation of HIF-1α. This device mimics the microenvironment of tongue cancer cells under mechanical force and increased matrix hardness during tongue movement, faithfully reproducing cell growthin vivo, and offering a solid foundation for further research on the pathogenic matrix of tongue cancer and drug treatments.

Pain research in a petri dish? Advantages and limitations of neuro-glial primary cell cultures from structures of the nociceptive system

How can we learn more about pain without causing pain in humans or animals? This short review focuses on neuro-glial primary cell cultures as models to study neuro-immune interactions in the context of pain and discusses their advantages and limitations.The field of basic pain research places scientists in an ethical dilemma. We aim to understand underlying mechanisms of pain for an improved pain therapy for humans and animals. At the same time, this regularly includes the induction of pain in model animals. Within the field of psychoneuroimmunology, the examination of the complexity of neuro-immune interactions in health and disease as well as the bi-directional communication between the brain and the periphery make animal experiments an inevitable part of pain research. To address ethical and legal considerations as well as the growing societal awareness for animal welfare, scientists push for the identification and characterization of complementary methods to implement the 3R principle of Russel and Burch. As such, methods to replace animal studies, reduce the number of animals used, and refine experiments are tested. Neuro-glial primary cell cultures of structures of the nociceptive system, such as dorsal root ganglia (DRG) or the spinal dorsal horn (SDH) represent useful in vitro tools, when research comes to a cellular and molecular level. They allow for studying mechanisms of neuronal sensitization, glial cell activation, or the role of specific inflammatory mediators and intracellular signaling cascades involved in the development of inflammatory and neuropathic pain. Moreover, DRG/SDH-cultures provide the opportunity to test novel strategies for interventions, such as pharmaceuticals or cell-based therapies targeting neuroinflammatory processes. Thereby, in vitro models contribute to a better understanding of neuron-glia-immune communication in the context of pain and in the advancement of pain therapies. However, this can only be one piece in a large puzzle. Our knowledge about the complexity of pain will depend on studies in humans and animals applied in vitro and in vivo and will benefit from clear and open-minded interdisciplinary communication and transparency in public outreach.

Characterization of Freeze-dried Pumpkin Powders: Powder Quality and Nutrient Stability

Being a good source of nutrients, viz., ascorbic acid, β-carotene, phenols, and antioxidants, pumpkin pulp (Cucurbita moschata) can be utilised as a functional ingredient in many food products by converting them into a more stable product like pumpkin powder. Hence the present study is focused on the production of freeze-dried pumpkin powder by two different methods (drying in petri plates with 5mm sample thickness and in 250 ml flask). The moisture content and water activity were reduced by 92.50% and 74.73%, respectively, compared to the fresh pumpkin pulp, indicating safe storage limits. Freeze-dried pumpkin powders exhibited a decrease of 25% ascorbic acid, 21.58% β-carotene, 39.39% reduction in total polyphenols and 72.98% in antioxidant content. The powder properties of the developed freeze-dried powders exhibited water activity of 0.4aw to 0.35aw bulk density of 0.15 to 0.12 g cm-3, and flow ability of 18.39° to 22.74°.

Pseudochrobactrum asaccharolyticum mitigates arsenic induced oxidative stress of maize plant by enhancing water status and antioxidant defense system

BackgroundOxidative stress mediated by reactive oxygen species (ROS) is a common denominator in arsenic toxicity. Arsenic stress in soil affects the water absorption, decrease stomatal conductance, reduction in osmotic, and leaf water potential, which restrict water uptake and osmotic stress in plants. Arsenic-induced osmotic stress triggers the overproduction of ROS, which causes a number of germination, physiological, biochemical, and antioxidant alterations. Antioxidants with potential to reduce ROS levels ameliorate the arsenic-induced lesions. Plant growth promoting rhizobacteria (PGPR) increase the total soluble sugars and proline, which scavenging OH radicals thereby prevent the oxidative damages cause by ROS. The main objective of this study was to evaluate the potential role of Arsenic resistant PGPR in growth of maize by mitigating arsenic stress.MethodologyArsenic tolerant PGPR strain MD3 (Pseudochrobactrum asaccharolyticum) was used to dismiss the ‘As’ induced oxidative stress in maize grown at concentrations of 50 and 100 mg/kg. Previously isolated arsenic tolerant bacterial strain MD3 “Pseudochrobactrum asaccharolyticum was used for this experiment. Further, growth promoting potential of MD3 was done by germination and physio-biochemical analysis of maize seeds. Experimental units were arranged in Completely Randomized Design (CRD). A total of 6 sets of treatments viz., control, arsenic treated (50 & 100 mg/kg), bacterial inoculated (MD3), and arsenic stress plus bacterial inoculated with three replicates were used for Petri plates and pot experiments. After treating with this MD3 strain, seeds of corn were grown in pots filled with or without 50 mg/kg and 100 mg/kg sodium arsenate.ResultsThe plants under arsenic stress (100 mg/kg) decreased the osmotic potential (0.8 MPa) as compared to control indicated the osmotic stress, which caused the reduction in growth, physiological parameters, proline accumulation, alteration in antioxidant enzymes (Superoxide dismutase-SOD, catalase-CAT, peroxidase-POD), increased MDA content, and H2O2 in maize plants. As-tolerant Pseudochrobactrum asaccharolyticum improved the plant growth by reducing the oxidation stress and antioxidant enzymes by proline accumulation. PCA analysis revealed that all six treatments scattered differently across the PC1 and PC2, having 85.51% and 9.72% data variance, respectively. This indicating the efficiency of As-tolerant strains. The heatmap supported the As-tolerant strains were positively correlated with growth parameters and physiological activities of the maize plants.ConclusionThis study concluded that Pseudochrobactrum asaccharolyticum reduced the ‘As’ toxicity in maize plant through the augmentation of the antioxidant defense system. Thus, MD3 (Pseudochrobactrum asaccharolyticum) strain can be considered as bio-fertilizer.

Self‐cleaning films of biopolymer polyhydroxybutyrate with different semiconductors incorporation: Development, characterization, and photocatalytic activity evaluation

AbstractSelf‐cleaning hydrophobic films were developed using polyhydroxybutyrate (PHB), enhanced with titanium dioxide (TiO2), bismuth oxyiodide (BiOI), and a BiOI/α‐Fe2O3 heterojunction. This is the first study to analyze the photoactivity of BiOI and BiOI/α‐Fe2O3 immobilized into PHB. Alternative solvents for dissolving PHB were investigated. Additionally, formation methods such as casting in Petri dishes, as well as controlled thickness casting through non‐solvent‐induced phase separation (NIPS), and evaporation‐induced phase separation (EIPS), were employed and compared. Despite differing morphologies, both NIPS and EIPS films exhibited similar photocatalytic activity, suggesting that only the catalyst on the external surface of the film is active. The highest pseudo first‐order apparent kinetic constant (0.1468 ± 0.0015 min−1 g−1) was found in films with the lowest polymer and catalyst concentrations analyzed (5% and 3% ). A double‐layer film preparation method was proposed to enhance photocatalytic activity, resulting in a 70% increase in the kinetic constant (0.2506 ± 0.0019 min−1 g−1). Double‐layer composite films with BiOI and BiOI/α‐Fe2O3 showed photocatalytic activity comparable to TiO2 under UV–visible light. Furthermore, their photocatalytic activity under visible light was confirmed, unlike TiO2, which exhibits a negligible kinetic constant in this wavelength range.

Assessing the effects of allelopathic properties of organic mulches on liverwort control in container-grown ornamentals

Liverwort (Marchantia polymorpha) is a problematic weed in ornamental crop production. The major limitation of liverwort control is lack of herbicide options inside greenhouses and their potential to cause injury. The current research was undertaken to study the effectiveness of allelopathic properties of six different organic mulches including rice hull (RH), cocoa hull (CH), pine bark (PB), maple leaf (ML), shredded cypress (SC), and red hardwood (HW) for liverwort control. Mulch extracts were prepared and used to impregnate agar media at an increasing dose at 1X (2 mL mulch extract), 2X (4 mL), 3X (6 mL), and 4X (8 mL) rates, in a randomized complete block design. Ten liverwort gemmae were transferred to each petri dish and they were maintained inside a growth chamber. The number of gemmae germinating in each petri dish after 1 week and gemmae surviving at the end of 2 weeks was recorded. In greenhouse study, mulch extracts were applied to containers filled with substrate for assessing liverwort control. The mulch extracts or no extract (control) were applied to the containers uniformly at 1X (15 mL), 2X (30 mL), 3X (45 mL) or 4X (60 mL) rates, in a randomized complete block design. The percentage of substrate surface covered by liverwort thalli was visually estimated bi-weekly for 10 weeks. Fresh biomass of the thalli and gemmae cup counts in each pot were also recorded. After 1 week in the growth chamber, ML followed by SC, PB and RH extracts showed maximum suppression of liverwort gemmae germination. At 2 weeks, ML applied at either of the rates provided complete inhibition of liverwort growth. In greenhouse, all the mulch extracts were able to provide complete liverwort control for the first two weeks. All the mulches and rates of applications were significantly different from the control after 6, 8, and 10 weeks. PB and HW mulches showed excellent liverwort control and minimum fresh biomass of liverwort after 10 weeks as compared to other mulches. The allelopathic potential of the organic mulches can be a promising option for biopesticidal control of liverwort and a component of integrated liverwort management.

Effects of Ozone Treatments on in vitro Seed Germination of Ruscus aculeatus, Ruscus hypoglossum and Danae racemosa

Ruscus aculeatus, R. hypoglossum and Danae racemosa are cut foliage and valuable for ornamental plants, they are also very valuable in terms of their medicinal aromatic properties. Propagating these plants using their seeds are very challenging due to deep dormancy and required pre-treatments before sowing. Ozone is a colorless gas with a pungent odor, made up of three oxygen atoms and it offers eco-friendly solution to break dormancy in seed. In this study, stored seeds for four years were treated to ozone gas for 0, 15 and 30 minutes using a ozone generator has the capacity to produce 6 grams of ozone per hour. Then the seed cultured in Petri dishes containing Murashige and Skoog medium without plant growth regulators. In vitro seed germination rates were recorded at 30, 40, 50 and 60 days after culture initiation. According to statistical analysis effects of species, duration of ozone treatments and interaction of species and duration of ozone treatments on in vitro germination rates of seeds were statistically significant. The highest in vitro germination rates with 42%, 28% and 24% were recorded at 30 min ozone treatment in R. aculeatus, R. hypoglossum and D. racemosa respectively. These results indicate that ozone application positively affects the germination of plant seeds.

Discovering Novel Bioherbicides: The Impact of Hemp-derived Phytocannabinoid Applications on Zea mays L. and Relevant Weeds

AbstractIn addition to competition, phytotoxic plant metabolites contribute to the weed-suppressing properties of cover crops, which could be the basis for the development of novel bioherbicides. We investigated the impact of five Cannabis sativa L. -derived neutral phytocannabinoids and an aqueous C. sativa tissue extract (HE) at six concentrations on the germination rate (GR) and seedling root length (RL) of Zea mays L., two monocotyledonous and two dicotyledonous weed species in laboratory Petri dish bioassays. Additionally, the effect of pre-emergence applications of HE, cannabidiol (CBD), and cannabidivarin (CBDV) formulations on GR and shoot dry matter (SDM) were examined in greenhouse pot studies. The effects of phytocannabinoids and HE were analyzed in dose-response curves. For the highest rates, the effects on GR, RL and SDM were calculated by ANOVA and HSD test (p < 0.05). HE exhibited the greatest suppression on GR and RL for all plant species in the Petri dish bioassay, with RGR, RL exceeding −90%. Phytocannabinoids reduced mainly RL of all plants and decreased the GR of most weed species. Effects varied among plants and phytocannabinoids, with CBDV and CBD showing similar high inhibitory effects on RL as HE in the Petri dish bioassay. All pre-emergence applications resulted in a positive RGR across all studied plants and in a positive RSDM in Z. mays and Echinochloa crus-galli (L.) P. Beauv, whereas in the other weed species the RSDM was negative. In conclusion, phytocannabinoids play a major role in weed suppression of HEs. CBDV and CBD are the most promising candidates for bioherbicide development especially against annual dicotyledonous weed species.

Microbiological evaluation of ultraviolet C light-emitting diodes for disinfection of medical instruments

BackgroundDespite the many guidelines for reprocessing of medical instruments, challenges persist such as microbial resistance to biocides, corrosive effects on materials, and time-consuming reprocessing procedures. Ultraviolet (UV) C light-emitting diode (LED) chambers might provide a solution but the integration in healthcare is still in its infancy. Here, we evaluated the efficacy of a novel ZAPARAY™ UVC LED chamber as a time and energy-efficient alternative for reprocessing of medical instruments for which current disinfection protocols exhibit limitations. MethodsWe verified the disinfection efficacy of the UVC LED chamber on a Petri dish and contaminated several medical devices with Staphylococcus aureus ATCC 25923. The bacterial reduction was assessed after five minutes of UVC LED exposure. Additionally, we investigated the impact of rinsing before UVC exposure. ResultsWe demonstrated a bacterial reduction of 9 log10 on a Petri dish. Non-rinsed dental tools exhibited varied reduction levels ranging from a 3.23 log10 to a 6.25 log10 reduction. Rinsing alone yielded an average reduction of 2.7 log10 and additional UVC exposure further reduced the bacterial load by an average of 3.65 log10. We showed an average 4.90 log10 reduction on thermistors, 2 log10 or less on orthodontic pliers, and no reduction on handpieces. ConclusionsThis study demonstrates that UVC LED chambers may be used as a standardized substitute for specific (manual) disinfection procedures of certain medical devices, offering a time-efficient and more sustainable alternative. However, its use should be preceded by efficacy testing for each specific type of instrument.

Proliferation of SH-SY5Y neuroblastoma cells on confined spaces

Background:Microfluidics offers precise drug delivery and continuous monitoring of cell functions, which is crucial for studying the effects of toxins and drugs. Ensuring proper cell growth in these space-constrained systems is essential for obtaining consistent results comparable to standard Petri dishes. New method:We investigated the proliferation of SH-SY5Y cells on circular polycarbonate chambers with varying surface areas. SH-SY5Y cells were chosen for their relevance in neurodegenerative disease research. Results:Our study demonstrates a correlation between the chamber surface area and SH-SY5Y cell growth rates. Cells cultured in chambers larger than 10 mm in diameter exhibited growth comparable to standard 60-mm dishes. In contrast, smaller chambers significantly impeded growth, even at identical seeding densities. Similar patterns were observed for HeLaGFP cells, while 16HBE14σ cells proliferated efficiently regardless of chamber size. Additionally, SH-SY5Y cells were studied in a 12-mm diameter sealed chamber to assess growth under restricted gas exchange conditions. Comparison with existing methods:Our findings underscore the limitations of small chamber sizes in microfluidic systems for SH-SY5Y cells, an issue not typically addressed by conventional methods. Conclusions:SH-SY5Y cell growth is highly sensitive to spatial constraints, with markedly reduced proliferation in chambers smaller than 10 mm. This highlights the need to carefully consider chamber size in microfluidic experiments to achieve cell growth rates comparable to standard culture dishes. The study also shows that while SH-SY5Y and HeLaGFP cells are affected by chamber size, 16HBE14σ cells are not. These insights are vital for designing effective microfluidic systems for bioengineering research.

Toxicity and Synergistic Effect of Native Plants Extracts against the Cowpea Weevil (Callosobruchus maculatus Fab.) (Coleoptera: Bruchidae)

Callosobruchus maculatus causes loss of cowpea seeds and its weight after six months of unprotected storage. This study aimed to evaluate the toxicity and synergistic effects of plant extracts on the biological parameters of C. maculatus from Bobo Dioulasso in order to reduce its damage on cowpea stocks in Bobo Dioulasso city. Seven treatments including an untreated control and doses of three powdery extracts of Cassia nigricans, Zanthoxylum zanthoxyloides and Hyptis suaveolens, and their combinations were compared in a Fischer block with four completely randomized repetitions. One hundred healthy cowpea seeds were mixed with doses of each extract in Petri dishes then submitted as food substrates to ten pairs of C. maculatus for 15 days. The synergistic effect was evaluated following the formula of Chou and Talalay. Extracts of C. nigricans, Z. zanthoxyloides and H. suaveolens was toxic against the weevil. The extract of Z. zanthoxyloides was more toxic to adults (21.16 mg/g) followed by H. suaveolens (51.84 mg/g) and C. nigricans (52.09 mg/g). The infestation rates of batches treated with C. nigricans and Z. zanthoxyloides extracts did not exceed 1.00% compared to 2.00% for H. suaveolens at 20 mg/g. All combinations were synergistic and resulted in 100% mortality and less than 14.98 eggs/female in H. suaveolens coupled with C. nigricans. The application of Z. zanthoxyloides extract alone or the combination of H. suaveolens and C. nigricans extracts would be an alternative for the sustainable management of C. maculatus in cowpea stocks.

Preparation of Collagen-Coated Compartmented Culture Dishes for Neuronal Cell Culture

Preparation of Collagen-Coated Compartmented Culture Dishes for Neuronal Cell Culture

Preparation of Collagen-Coated Compartmented Culture Dishes for Neuronal Cell Culture

Preparation of Collagen-Coated Compartmented Culture Dishes for Neuronal Cell Culture

Infiltrating monocytes drive cardiac dysfunction in a cardiomyocyte-restricted mouse model of SARS-CoV-2 infection.

Cardiovascular manifestations of coronavirus disease 2019 (COVID-19) include myocardial injury, heart failure, and myocarditis and are associated with long-term disability and mortality. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and antigens are found in the myocardium of COVID-19 patients, and human cardiomyocytes are susceptible to infection in cell or organoid cultures. While these observations raise the possibility that cardiomyocyte infection may contribute to the cardiac sequelae of COVID-19, a causal relationship between cardiomyocyte infection and myocardial dysfunction and pathology has not been established. Here, we generated a mouse model of cardiomyocyte-restricted infection by selectively expressing human angiotensin-converting enzyme 2 (hACE2), the SARS-CoV-2 receptor, in cardiomyocytes. Inoculation of Myh6-Cre Rosa26loxP-STOP-loxP-hACE2 mice with an ancestral, non-mouse-adapted strain of SARS-CoV-2 resulted in viral replication within the heart, accumulation of macrophages, and moderate left ventricular (LV) systolic dysfunction. Cardiac pathology in this model was transient and resolved with viral clearance. Blockade of monocyte trafficking reduced macrophage accumulation, suppressed the development of LV systolic dysfunction, and promoted viral clearance in the heart. These findings establish a mouse model of SARS-CoV-2 cardiomyocyte infection that recapitulates features of cardiac dysfunctions of COVID-19 and suggests that both viral replication and resultant innate immune responses contribute to cardiac pathology.IMPORTANCEHeart involvement after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection occurs in multiple ways and is associated with worse outcomes in coronavirus disease 2019 (COVID-19) patients. It remains unclear if cardiac disease is driven by primary infection of the heart or immune response to the virus. SARS-CoV-2 is capable of entering contractile cells of the heart in a culture dish. However, it remains unclear how such infection affects the function of the heart in the body. Here, we designed a mouse in which only heart muscle cells can be infected with a SARS-CoV-2 strain to study cardiac infection in isolation from other organ systems. In our model, infected mice show viral infection, worse function, and accumulation of immune cells in the heart. A subset of immune cells facilitates such worsening heart function. As this model shows features similar to those observed in patients, it may be useful for understanding the heart disease that occurs as a part of COVID-19.

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.

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.