Six-well Plates Research Articles

Staphylococcus felis C4 exhibits invitro antimicrobial activity against methicillin-resistant Staphylococcus pseudintermedius in a novel canine skin explant model.

Canine superficial pyoderma is a common bacterial skin infection of dogs, generally caused by Staphylococcus pseudintermedius. The C4 strain of Staphylococcus felis was recently discovered to have strong antimicrobial activity against S. pseudintermedius in mice. We aimed to evaluate invitro if this antimicrobial activity was maintained using a novel canine skin explant model. Punch biopsies (8 mm) of skin from recently euthanised dogs were collected and placed into six-well plates on top of an agarose pedestal. Histological examination of the skin explants showed an intact dermal-epidermal organisation and a stratum corneum that was successfully colonised by S. pseudintermedius after topical application. The number of colony forming units of S. pseudintermedius showed a 2 log increase after 24 h colonisation, indicating that the explant supported bacterial growth. By contrast, co-treatment with S. felis C4 live bacteria and its sterile protein product significantly reduced the growth of a methicillin-susceptible (ST540, p = 0.0357) and a methicillin-resistant (MR) strain (ST71, p = 0.0143) of S. pseudintermedius. No detectable bacteria were recovered from or visualised on skin 24 h posttreatment with the S. felis C4 sterile protein product. Using a novel canine explant model, we demonstrate that the S. felis C4 strain inhibits the growth of S. pseudintermedius and that it is a promising candidate for a new probiotic therapy to treat cutaneous infections caused by S. pseudintermedius, including MR strains.

Silver nanoparticles exhibit in vitro anthelmintic and antimicrobial activities against Dactylogyrus minutus (Kulwieć, 1927), and Aeromonas hydrophila in Cyprinus carpio Koi

The study aimed to evaluate the in vitro anthelminthic and antimicrobial activity of silver nanoparticles (AgNPs) against Dactylogyrus minutus and Aeromonas hydrophila, pathogens of Cyprinus carpio Koi. Gill arches of the fish were removed and placed into six-well plates containing 10 mL of tank water with varying concentrations of AgNPs: 100, 400, 500, 600, and 800 mg/L, along with control groups using tank water and distilled water. Each group was tested in triplicate. Parasites were observed every 10 min for 300 min (5 h) using a stereomicroscope, and mortality rates were recorded. Anthelminthic efficacy was calculated at the end of the tests. For the in vitro antimicrobial test, the Minimum Inhibitory Concentration (MIC) of AgNPs was determined by adding 100 μL of Poor Broth (PB) culture medium to all 96 wells of a microplate. The first well was filled with 100 μL of AgNPs, followed by serial dilutions (1:2 ratio). Subsequently, 50 μL of A. hydrophila (1 × 107 CFU/mL) was added to all wells and incubated for 24 h at 28 °C. Results showed that 800 mg/L of AgNPs achieved 87% anthelminthic efficacy within 300 min, while 100 mg/L achieved 47% efficacy. The MIC showed bacterial growth inhibition at 125 mg/mL. Despite the 87% efficacy against parasites within 300 min, AgNPs did not reach 100% efficacy quickly, limiting their potential use in ornamental fish farming. Further studies are needed to assess the toxicity of AgNPs in fish.

Degradation of Microplastics by Zinc Oxide Nanoparticles Synthesized Using Piper longum Leaf Extract.

Background: The environmental hazards posed by microplastics have drawn considerable concern due to their buildup in ecosystems. Microplastics accumulate in human saliva, skin, and hair. Developing effective technology for managing and degrading microplastics remains a substantial challenge. In a concerted attempt to save the ecology, this study explores the photocatalytic breakdown of common microplastics like polystyrene (PS) microspheres and polyethylene (PE) using green-synthesized zinc oxide nanoparticles (ZnO NPs) under UV light exposure. Aim: To synthesize and characterize zinc oxide nanoparticles prepared using the extract of the leaves of Piper longumand qualitatively assess the photocatalytic degradation potential of the nanoparticles under light microscopy. Material and Methods:A fresh extract of P. longum leaves was used as a reducing agent to synthesize zinc oxide nanoparticles. Fourier transform infrared (FTIR) analysis, X-ray diffraction (XRD) analysis, UV-Vis spectra analysis, and scanning electron microscopy (SEM) analysis were performed to characterize the nanoparticles. Microplastics were isolated from the saliva of 50 healthy patients and were purified and filtered. In a six-well microtiter plate, 0.5 μg of varying concentrations of nanoparticles were added. After fixing with 15% formaldehyde, microplasticswere subjected to UV irradiation for 2 hours with different concentrations of ZnO nanoparticles (25, 50, 75,and 100 µg). Custom photoreactors activated the photocatalysts to degrade the microplastic pollutants. The six-well microtiter plate was viewed under 40x magnification in a light microscope to observe microplastic morphology after 24 hours of degradation. Results: The FTIR spectrum showed distinct peaks at 890.51 cm⁻¹, indicating the involvement of C-N in-plane vibrations of amino acids. XRD analysis revealed three distinct diffraction peaks at 31.68°, 34.39°, and 36.33°, corresponding to the hexagonal wurtzite structure of ZnO nanoparticles. The synthesized ZnO nanoparticles ranged from 50 to 90 nm in size, viewed at 100x magnification on SEM. The highest degradation of microplastics was observed ataZnO NPconcentration of 100 µL, with the ZnO NPs 50-90 nm in size. Conclusion: Zinc oxide nanoparticles synthesized using Piper longum leaf extract effectively degrade microplastics, with the highest degradation observed at a 100 µL concentration of ZnO nanoparticles and optimal degradation occurring at a concentration of 75 µL.

Study on the Effects of Low-Intensity Pulsed Ultrasound and Iron Ions for Proliferation and Differentiation of Osteoblasts

ObjectiveThis study involved the proliferation and differentiation of osteoblasts treated with low-intensity pulsed ultrasound (LIPUS) and iron (Fe3+) ions, respectively. The biological effects of LIPUS and Fe3+ ions on the proliferation and differentiation of osteoblasts were also evaluated. MethodsMC3T3-E1 cells were seeded in six-well plates with the medium, which contained different concentrations of Fe3+ (0, 100, 200, 300, 400, 500, 600 and 700 μg L-1, respectively). LIPUS treatment was directed at the bottom of the plate for 20 min at an intensity of 80 mW cm-2 every day. ResultsViability results showed that a dose of 400 μg L-1 Fe3+ ions had the best effect at promoting osteogenic proliferation in cell culture. The results of alkaline phosphatase staining and mineralization indicated that the differentiation of osteoblasts was promoted by LIPUS and Fe3+ ions. Fluorescence staining results showed that the number of cell nuclei in the LIPUS, Fe3+ and LIPUS-Fe groups increased by 37.20%, 55.81% and 89.76%, respectively. Migration data indicated that migration and proliferation rates were increased by LIPUS and Fe3+, and the results of protein expression indicated that LIPUS and Fe3+ may increase the expression of Wnt, β-catenin, and Runx2, hence promoting normal bone regeneration and development. ConclusionThe combination of LIPUS (1.5 MHz, 80 mW cm-2) and Fe3+ accelerates the proliferation and differentiation of osteoblasts significantly compared with single-factor treatment (stimulated by LIPUS and Fe3+ ions, respectively). This study could establish a foundation for LIPUS-responsive biomaterials in the repair and regeneration of bone tissues.

High-Throughput Bioprinting Method for Modeling Vascular Permeability in Standard Six-well Plates with Size and Pattern Flexibility.

Vascular permeability is a key factor in developing therapies for disorders associated with compromised endothelium, such as endothelial dysfunction in coronary arteries and impaired function of the blood-brain barrier. Existing fabrication techniques do not adequately replicate the geometrical variation in vascular networks in the human body, which substantially influences disease progression; moreover, these techniques often involve multi-step fabrication procedures that hinder the high-throughput production necessary for pharmacological testing. This paper presents a bioprinting protocol for creating multiple vascular tissues with desired patterns and sizes directly on standard six-well plates, overcoming existing resolution and productivity challenges in bioprinting technology. A simplified fabrication approach was established to construct six hollow, perfusable channels within a hydrogel, which were subsequently lined with human umbilical vein endothelial cells to form a functional and mature endothelium. The computer-controlled nature of 3D bioprinting ensures high reproducibility and requires fewer manual fabrication steps than traditional methods. This highlights VOP's potential as an efficient high-throughput platform for modeling vascular permeability and advancing drug discovery.

A Novel tetrahydrocannabinol (THC) analog, 3,10,10-Trimethyl-1,2,3,4,4a,6,7,8,10,10a-decahydro-9-oxa-5-phenanthrenone (TDP), mitigates the decrease in cell viability in oxidatively-stressed osteosarcoma bone-like cell cultures, UMR 106-01 BSP (UMR cells)

Cannabinoids have been extensively studied in the field of cancer research. Tetrahydrocannabinol (THC) has shown promising results in influencing cellular proliferation and viability when used in association with other cannabinoids. However, not many studies have been done to explore the effect of THC analogs on osteosarcoma cells when used in concert with oxidative stressors like camptothecin (CPT). In this study, it was hypothesized that TDP would mitigate the oxidative effect of CPT on UMR cells. Briefly, synthesis of the novel THC analog, TDP, was attempted via a Knoevenagel condensation and a Diels-Alder reaction, using 1,3-cyclohexanediol dissolved in methanol and citronellal with ethylenediamine diacetic acid (EDDA) at a temperature of 60°C. UMR cells were routinely passaged, counted, plated in six-well culture plates at 480,000 cells/mL, then treated with 10-fold dilutions of TDP. The plates were incubated for 72 hours in a humidified incubator at 37 degrees Celsius with 5% carbon dioxide infusion. At the end of the experiment, the cells were routinely washed with HANKS buffered saline solution (HBSS), then counted using the Luna II Automated Cell Counter. In another experiment, designated cells were treated with different concentrations of CPT alone, while others were co-treated with different concentrations of TDP+CPT, following the protocol above. F test ANOVA was used to compare variances and all values in the results were expressed as means ± SD. The results from the attempted cannabinoid analog synthesis yielded an active novel THC analog, TDP. Serial dilutions treatment of the UMR cells with TDP alone showed its ability to decrease cell viability in a concentration-dependent manner. The treatment of the UMR cells with different concentrations of CPT alone resulted in a decrease in cell viability in a concentration-dependent manner. Co-treatment of the UMR cells with different concentrations of CPT+TDP showed that TDP was able to mitigate the effect of the oxidative stressor (CPT) on the UMR cell cultures. Moreover, the F Test ANOVA showed robust statistical significance (p values <0.05) regarding TDP’s ability to decrease the oxidative effect of CPT on UMR cells. Overall, the outcomes of this study suggest that active forms of THC analogs can be synthesized and tested in concert with oxidative stressors like CPT. This study opens the door to explore the mechanisms by which TDP exerts its effect on oxidatively-stressed cells and how it can possibly be used to prevent or mitigate the effect of oxidative stress on a variety of cells in culture. Moreover, the hopes of suggesting therapeutic uses of THC analogs, upon future mechanistic experimentations, is a goal. La Sierra University Title V Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The toxicity assessment of phosmet on development, reproduction, and gene expression in Daphnia magna.

The use of pesticides to control pests, weeds, and diseases or to regulate plant growth is indispensable in agricultural production. However, the excessive use of these chemicals has led to significant concern about their potential negative impacts on health and the environment. Phosmet is one such pesticide that is commonly used on plants and animals against cold moth, aphids, mites, suckers, and fruit flies. Here, we investigated the effects of phosmet on a model organism, Daphnia magna using acute and chronic toxicity endpoints such as lethality, mobility, genotoxicity, reproduction, and gene expression. We performed survival experiments in six-well plates at seven different concentrations (0.01, 0.1, 1, 10, 25, 50, 100 μM) as well as the control in three replicates. We observed statistically significant mortality rates at 25 µM and above upon 24 h of exposure, and at 1 µM and above following 48 h of exposure. Genotoxicity analysis, reproduction assay and qPCR analysis were carried out at concentrations of 0.01 and 0.1 μM phosmet as these concentrations did not show any lethality. Comet assay showed that exposure to phosmet resulted in significant DNA damage in the cells. Interestingly, 0.1 μM phosmet produced more offspring per adult compared to the control group indicating a hormetic response. Gene expression profiles demonstrated several genes involved in different physiological pathways, including oxidative stress, detoxification, immune system, hypoxia and iron homeostasis. Taken together, our results indicate that phosmet has negative effects on Daphnia magna in a dose- and time-dependent manner and could also induce lethal and physiological toxicities to other aquatic organisms.

Platform development for high-throughput optimization of perfusion processes:Part I: Implementation of cell bleeds in microwell plates.

The promise of continuous processing to increase yields and improve product quality of biopharmaceuticals while decreasing the manufacturing footprint is transformative. Developingand optimizing perfusion operations requires screening various parameters, which is expensive and time-consuming when using benchtop bioreactors. Scale-down models (SDMs) are the most feasible option for high-throughput data generation and condition screening. However, new SDMs mimicking perfusion are required, enabling experiments to be run in parallel. In this study, a method using microwell plates (MWP) operating in semi-perfusion mode with an implemented cell bleed step is presented. A CHO cell line was cultivated in a 24-well MWP (Vw = 1.2 mL) and grown at four high cell density (HCD) setpoints. Quasi steady-state condition was obtained by manually performing cell bleeds followed by a total medium exchange after centrifugation. Further, two HCD setpoints were scaled up (VW = 30 mL), comparing a squared six-well deepwell plate (DWP) to shake flasks (SF). This evaluation showed comparable results between systems (DWP vs.SF) and scales (MWP vs.DWP + SF). The results show that the well-plate-based methods are suitable to perform HCD and quasi steady-state cultivations providing a robust solution to industrially relevant challenges such as cell clone and media selection.

Transcription Silencing and CpGs Hypermethylation as Therapeutic Gene Editing in Clinical Colorectal Adenocarcinoma Repression.

Colorectal cancer is the most common cancer in oncopathology, with an increasing incidence among the elderly during the last decade. Various genetic and environmental factors play important roles in the emergence of colorectal adenocarcinoma. Non-coding RNAs, approximately 20-22 nucleotides, are transcribed irregularly in many cancer cells and play a critical role in many metabolic pathways in clinical cancer cases. DNA methylation is a critical epigenetic alteration that controls gene expression. In the current study, transcriptional silencing and CpG hypermethylation were developed as a therapeutic gene editing strategy for the clinical repression of colorectal adenocarcinoma. A human colorectal adenocarcinoma cell line (Caco2) and a normal lung fibroblast cell line (Wi38) were utilized as the paradigms in this research to examine the effect of mir155 molecule transfection and CpGs-island (CGI) methylation. Cell counting was achieved using six-well and 24-well plates before transfection using a hemocytometer. The two cell lines were transfected with the mir155 agomir and antagomir molecules. The transfection efficiency, cell viability, cell IC50, and target gene expression were measured, and CGIs-methylation was achieved by bisulfate conversion. The outcomes revealed the downregulation of oncogenes (AKT1 and VCAM1 genes as cancer-associated genes) and the upregulation of tumor suppressor genes (TSGs, Tp53 and KEAP1). In addition, CpG-islands methylation showed significant blocking of the oncogene promoter regions, and the switch on of TSG promoter regions was continuous. miRNA-CGI-methylation led to the regression of Caco2 cell proliferation, suggesting the potential use of RNA silencing and DNA methylation in targeted gene therapy for colorectal cancer.

ERCC6L2 Disease and Shwachman-Diamond Syndrome Exhibit Alterations in Metabolomes in Patient Fibroblasts

Introduction Metabolomics is an emerging tool to study metabolic alterations in cancers. It has also the potential to identify clinically relevant biomarkers for diagnosis, risk appraisal, and targeted drug development. In hematology, metabolic reprogramming is essential for normal regulation of hematopoiesis. Association of discrete metabolic alterations with explicit somatic mutations have also been reported in hematological malignancies. We have previously observed glutaminase (GLS) overexpression and improved oxygen consumption rate with excess glutamine in low glucose conditions in patient-derived fibroblasts in ERCC6L2 disease (unpublished). This prompted us to study metabolomic alterations in ERCC6L2 disease and Shwachman-Diamond syndrome (SDS) induced strictly by the germline ERCC6L2 and SBDS mutations. Both inherited bone marrow failure (BMF) syndromes carry considerable tendency of somatic TP53 mutagenesis predisposing patients to a high risk myeloid malignancy. Our aim here was to identify potential drug targets by targeted metabolomics. Methods We examined patient-derived skin fibroblasts (ERCC6L2 disease, n=4; SDS, n=4) and healthy controls (n=4) and performed targeted metabolomics with U-13C-D-glucose and U-13C-L-glutamine labeling. Fibroblasts were seeded on six-well plates and on the following day exposed to 13C-glucose (25mM) or 13C-glutamine (2mM) label containing medium for 30 or 120 minutes. Next, metabolites were extracted and quantified with liquid chromatography mass spectrometer. The results were normalized to total metabolite intensity per sample. Results In 13C-glucose labeled samples, we detected statistically significantly lower intensity of pyruvate M+3 in both ERCC6L2 and SDS fibroblasts compared to control at 30 minutes, which persisted at 120 minutes in SDS cells. In the TCA cycle, malate M+3 labeling indicates pyruvate carboxylase (PC) activity and M+2 labeling pyruvate dehydrogenase activity (PDH) (Figure 1). We found a significantly higher portion of malate M+3 than M+2 in SDS cells at all time points, suggesting lower PDH and higher PC activity in SDS cells. Additionally, we detected higher levels of alanine M+3 in SDS samples. As pyruvate is a source for alanine synthesis, we suspect an alteration in pyruvate metabolism; potentially to overcome challenges in pyruvate oxidation due to either decrease in the respiratory chain function or PDH activity. In 13C-glutamine labeled samples, we detected statistically significantly higher intensity of glutamine M+5 and M+4 in ERCC6L2 samples at 30 minutes compared to control and SDS cells. Glutamine is first converted to glutamate by glutaminase (GLS), after which it enters the TCA cycle as ketoglutarate (KG) (Figure 1). In addition to glutamine, we detected an increase, although not statistically significant, in glutamate M+5 and M+4, as well as KG M+5, in ERCC6L2 cells at 30 minutes. A similar trend was observed in the subsequent TCA cycle intermediates succinic acid M+4, fumarate M+4, and malate M+4. At 120 minutes, there was no difference in metabolite intensities between the groups, suggesting a potentially faster glutamine uptake by ERCC6L2 cells. Conclusions Our analysis using non-hematopoietic tissue provides understanding of metabolic weak links not confounded by somatic events occurring early in the disease course in ERCC6L2 and SDS patients. Our findings imply increased glutamine utilization in ERCC6L2 cells and altered pyruvate metabolism in SDS cells. Therefore, these metabolic pathways are attractive drug targets (summarized in Figure 1). Further studies are needed to determine their potential in supporting the compromised hematopoiesis to reduce the stress for somatic mutagenesis.

Fabrication of high-crystallinity covalent organic framework and its nylon based membrane: Application in the enrichment and interception of dyes

Because of their high carcinogenicity and mutagenicity, dyes pose a great threat to the environment and humans, and the development of highly efficient dye removal methods is of great significance. Herein, a novel fluorine-rich COF (TPTF-COF) with high crystallinity was synthesized by connecting 1,3,5-tris(4-aminophenyl) benzene (TPB) and 1,3,5-tri(3-fluoro-4-formylphenyl) benzene (TF) at room temperature using acetonitrile as solvent. Owing to its large specific surface area (1084.61 m2·g−1), high crystallinity, high thermal stability, and rich functional group properties, TPTF-COF showed good adsorption capacities (20.63–386.30 mg·g−1) for six selected dyes. Based on TPTF-COF, a six-well plate assisted in-situ assembly method that can achieve the simultaneous synthesis of six sheets of TPTF-COF-based nylon membranes at a relatively low temperature (40 ℃) and within a short reaction time (48 h) was developed using aminodized nylon membrane as supports. The membrane with good uniformity showed high adsorption for the selected dyes, and thus showed good rejection, especially for Congo red (rejection >94%). Owing to the large gap between the aminodized nylon membranes, the TPTF-COF-based nylon membrane also exhibited superior water flux. The results of experimental and computational studies revealed that the pore size selectivity of TPTF-COF, π-π interaction, hydrogen bond and electrostatic interactions between TPTF-COF and dyes led to these selective adsorption effects. In this study, a new six-well plate assisted in-situ synthesis strategy for the preparation of imine-linked COF-based nylon membranes for dye removal, was developed. This approach may establish a viable route for the high-throughput and gentle fabrication of COF-based membranes for various applications.

The potential of suspended chitosan nanoparticles as a surgical irrigation fluid.

In this study, we sought to synthesize chitosan nanoparticles (CS-NPs) and characterize their morphology, efficacy in inhibiting bacterial attachment, and efficacy in eradicating bacteria established on implantable hardware. CS-NPs possess desirable properties, including antibacterial properties in biofilm-mediated infections. CS-NPs were produced using ionic gelation and characterized via scanning electron microscope imaging. Staphylococcus aureus was incubated with CS-NPs at various concentrations and compared to a 1% povidone-iodine with 1% H2 O2 control in 24-well plates. Stainless steel bone screws were placed in six-well plates and inoculated with S. aureus. After 24 h, the screws were transferred to one of three solutions (saline, 40 mg/mL CS-NP, or 1% povidone-iodine with 1% H2 O2 ). Four screws from each group were vortexed in saline and plated. The remaining screw from each group was prepped and imaged to map the location of persistent bacteria. Synthesized CS-NPs had a mean diameter of 0.39 ± 0.13 μm and circularity of 0.87 ± 0.05. The percent inhibition of bacterial attachment was 73% at 20 mg/mL, 73% at 30 mg/mL, 75% at 40 mg/mL, 79% at 50 mg/mL, and 78% at 60 mg/mL. When compared to saline, the 40 mg/mL CS-NP solution reduced bacteria on the screws by 76%. No bacteria were retrieved from the 1% povidone-iodine with 1% H2 O2 group. This study demonstrated that CS-NP solution effectively inhibited S. aureus bacterial attachment and was more effective than saline in eradicating bacteria from orthopedic hardware, suggesting that CS-NPs have the potential for prevention and treatment of musculoskeletal infections as a component of an intraoperative surgical irrigation solution.

Heterogenous morphogenesis of Caco-2 cells reveals that flow induces three-dimensional growth and maturation at high initial seeding cell densities.

Here, we introduce a customized hanging insert fitting a six-well plate to culture Caco-2 cells on hydrogel membranes under flow conditions. The cells are cultured in the apical channel-like chamber, which provides about 1.3 dyn/cm2 shear, while the basolateral chamber is mixed when the device is rocked. The device was tested by investigating the functional impact of the initial seeding density in combination with flow applied at confluency. The low seeding density cultures grew in two dimensional (2D) irrespective of the flow. Flow and higher seeding density resulted in a mixture of three dimensional(3D) structures and 2D layers. Static culture and high cell seeding density resulted in 2D layers. The flow increased the height and ZO-1 expression of cells in 2D layers, which correlated with an improved barrier function. Cultures with 3D structures had higher ZO-1 expression than 2D cultures, but this did not correlate with an increased barrier function. 2D monolayers in static and dynamic cultures had similar morphology and heterogeneity in the expression of Mucin-2 and Villin, while the 3D structures had generally higher expression of these markers. The result shows that the cell density and flow determine 3D growth and that the highest barrier function was obtained with low-density cultures and flow.

Analyses of Transcriptomics upon IL-1β-Stimulated Mouse Chondrocytes and the Protective Effect of Catalpol through the NOD2/NF-κB/MAPK Signaling Pathway

The overall objective of this study was to investigate the mechanism of inflammation on chondrocyte injury and the protective effect of catalpol on chondrocytes in an inflammatory environment. Chondrocytes were isolated and cultured from the knee joints of three-day-old newborn mice. Alcian Blue staining and the immunocytochemistry staining of type II collagen were used to identify the purity of chondrocytes. Primary chondrocytes were stimulated by IL-1β (10 ng/mL) and subjected to transcriptome analysis. Differentially expressed genes (DEGs) were further analyzed based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. In this experimental study, we performed the viability assay to determine the effects of different concentrations of catalpol on the cell viability of chondrocytes. Chondrocytes were seeded in six-well plates and exposed to 10 μM catalpol 2 h prior to treatment with IL-1β (10 ng/mL). Quantitative real-time (qPCR) and Western blotting were performed to evaluate the RNA and protein expression, respectively. Based on the results of transcriptomics analysis, we found the NOD2 signaling pathway, the NF-kappa B signaling pathway, and the MAPK signaling pathway showed significant changes in chondrocyte damage caused by inflammation. Catalpol (10 μM and 100 μM) could significantly reduce NO, IL-6, IL-1β, and TNF-α in supernatant of chondrocytes. Catalpol significantly inhibited the mRNA expression of IL-1, IL-6, and IL-12 in chondrocytes induced by IL-1β. Catalpol markedly inhibited MMP3, MMP13 mRNA, and protein levels. Catalpol could significantly reduce TNF-α mRNA levels in inflammatory chondrocytes. Inflammation causes significant increases in mRNA levels and protein levels of NOD2, mRNA levels, and protein levels were markedly suppressed by catalpol. In addition, catalpol could significantly increase IKBα protein levels and significantly lower intranuclear P65 levels. Catalpol significantly lowered the phosphorylation protein levels of ERK, p38, and JNK. Our transcriptomic analysis demonstrated that the activation of NOD2 and its downstream pathways, NF-κB and MAPK, is an important cause of the inflammatory injury to chondrocytes induced by IL-1β. Catalpol inhibited the activation of the NOD2 signaling pathway, which reduced the phosphorylation of ERK, p38, and JNK, inhibited the degradation of IκBα, inhibited p65 translocation into the nucleus, reduced the release of inflammatory cytokines, and attenuated the inflammatory damage to chondrocytes.

Cytotoxic and Anti-proliferative Effects of Moringa oleifera Lam. on HeLa Cells

Background:The aim of the study was to determine the mechanism of Moringa oleifera-induced apoptosis in HeLa cells. HeLa cells over-express cyclin E and cyclin B1, abrogate G0-G1 and G2-M cell cycle arrest, promoting tumorigenesis. Cyclin E, cyclin B1, E2F1 and telomerase expression, and caspase-3 and -7 activation were assessed after 24-treatment with M. oleifera leaf fractions.Material and methods:Apoptosis through caspase-3 and caspase-7 activation was determined quantitatively by the FAM FLICA™ Caspase-3/7 assay. Cyclin E, cyclin B1 and E2F1 were quantified by flow cytometry. Telomerase was evaluated by Telomeric repeat amplification protocol (TRAP reaction). The effects on colony formation were assessed by seeding treated cells in six-well plates for 7 days under culture conditions. The MTT assay was used to determine cell survival.Results:HeLa cells treated for 24 hours with M. oleifera leaf fractions showed dose-dependent cytotoxicity, activation of caspases-3 and -7, down regulation of cyclin E, cyclin B1, E2F1, and inhibition of telomerase expression. Cell cycle analysis of the dead cell population showed G2-M cellcycle arrest.Conclusion:M. oleifera leaf fractions triggered apoptosis through the mitochondrial pathway and cell cycle arrest at G2-M phase in HeLa cells after 24-hour treatment, through down regulation of cyclin E and cyclin B1 expression, and caspase-3 and -7 activation. In addition, M. oleifera leaf extract induces senescence in HeLa cells through the down-regulation of telomerase. Colony formation and cell proliferation were inhibited in a dose-dependent manner, corresponding with telomerase inhibition.

On-chip on-demand delivery of K+ for in vitro bioelectronics

Bioelectronic devices that interface electronics with biological systems can actuate and control biological processes. The potassium ion plays a vital role in cell membrane physiology, maintaining the cell membrane potential (Vmem) and generating action potentials. In this work, we present two bioelectronic ion pumps that use an electronic signal to modulate the potassium ion concentration in solution. The first ion pump is designed to integrate directly with six-well cell culture plates for optimal ease of integration with in vitro cell culture, and the second on-chip ion pump provides high spatial resolution. These pumps offer increased ease of integration with in vitro systems and demonstrate K+ concentration distribution with high spatial resolution. We systematically investigate the ion pump’s performance using electrical characterization and computational modeling, and we explore closed-loop control of K+ concentration using fluorescent dyes as indicators. As a proof-of-concept, we study the effects of modulating K+ concentration on Vmem of THP-1 macrophages.

Evaluation of the Anticoccidial Activity of Sheep Bile against Eimeria stiedae Oocysts and Sporozoites of Rabbits: An In Vitro Study.

Coccidiosis is one of the most common infectious diseases that causes digestive problems in rabbits, leading to global economic losses. This study was conducted to evaluate the effects of bile obtained from sheep gallbladder on the sporulation and morphology of Eimeria stiedae oocysts and sporozoites affecting rabbit liver cells and to determine the best concentration for sporulation inhibition. Sporulation inhibition per milliliter was measured in samples exposed to five concentrations of sheep bile (SB) in a 2.5% potassium dichromate solution: 12.5%, 25%, 50%, 75%, and 100% concentrations for oocysticidal activity and 125, 250, 500, 750, and 1000 μg/mL concentrations for antisporozoidal activity. A bioassay was performed to assess the in vitro anticoccidial activity of sheep bile against E. stiedae oocysts and sporozoite sporulation. In this assay, six-well plates with 5 mL of bile containing 1000 oocysts showed unsporulated oocysticidal activity after 48, 72, and 96 h and antisporozoidal activity after 12 and 24 h. A chemical assay was performed via infrared spectroscopy to investigate the presence of several anticipated active chemical compounds in sheep bile. Sheep bile was able to inhibit E. stiedae oocysts at 100% and 75% concentrations by about 91% and 81%, respectively. In addition, SB had the highest inhibition of E. stiedae sporozoite viability (92%) at a concentration of 1000 μg/mL and had the lowest inhibition of 8% at a concentration of 125 μg/mL. An increase in the incubation time and a higher dose generally increased the inhibition rate. The results showed that sheep gallbladder bile is effective due to its inhibitory potential and effect on the coccidian oocyst sporulation of E. stiedae. Further studies are needed to determine the precise active chemicals present in SB and their modes of action and application in vivo.

Epithelial Biological Response to Machined Titanium vs. PVD Zirconium-Coated Titanium: An In Vitro Study.

The aim of this study was to compare the epithelial biological response to machined titanium Ti-6Al-4V grade 5 and titanium Ti-6Al-4V grade 5 coated with zirconia (ZrN) by physical vapor deposition (PVD). Human keratinocytes were cultured in six-well plates. Machined titanium TiAl4V4 grade 5 (T1) and ZrN-coated titanium TiAl4V4 grade 5 (T2) discs were placed in two different wells. The remaining two wells served as control (C). Scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS) were performed to compare the T1 and T2 surfaces. Subsequent analyses were performed to explore the effect of T1 and T2 contact with human keratinocyte HUKE cell lines. Cell viability was evaluated using a trypan blue exclusion test and MTT assay. Cell lysates from C, T1, and T2 were Western blotted to evaluate E-cadherin and Integrin-α6β4 expression. SEM revealed that T2 was smoother and more homogeneous than T1. EDS showed homogeneous and uniform distribution of ZrN coating on T2. Cell viability analyses did not show significant differences between T1 and T2. Furthermore, E-cadherin and Integrin-α6β4 expressions of the epithelial cells cultured in T1 and T2 were similar. Therefore, titanium Ti-6Al-4V grade 5 surfaces coated with ZrN by PVD seem to be similar substrates to the uncoated surfaces for keratinocyte adhesion and proliferation.

TGF-β1 and -β3 for Mesenchymal Stem Cells Chondrogenic Differentiation on Poly (Vinyl Alcohol)-Chitosan-Poly (Ethylene Glycol) Scaffold.

Transforming growth factor-beta 1 (TGF-β1) has been reported to promote chondrogenic differentiation and proliferation in the multipotent stromal cell (MSCs), and the transforming growth factor-beta 3 (TGF-β3) tends to be exclusively in promoting cell differentiation alone. The objective of this study was to determine the effect of TGF-β1 and -β3 on the MSCs chondrogenic differentiation on the poly (vinyl alcohol)-chitosan-poly (ethylene glycol) (PVA-NOCC-PEG) scaffold, compared with that of monolayer and pellet cultures. In this study, P2 rabbit bone marrow-derived MSCs were seeded either on the untreated six-well plate (for monolayer culture) or onto the PVA-NOCC-PEG scaffold or cultured as a pellet culture. The cultures were maintained in a chemically defined serum-free medium supplemented with 10 ng/mL of either TGF-β1 or TGF-β3. Cell viability assay, biochemical assay, and real-time polymerase chain reaction were performed to determine the net effect of cell proliferation and chondrogenic differentiation of each of the growth factors. The results showed that the PVA-NOCC-PEG scaffold enhanced MSCs cell proliferation from day 12 to 30 (p < 0.05); however, no significant differences were observed in the cell proliferation between the cultures supplemented with or without TGF-β1 and TGF-β3 (p > 0.05). In terms of chondrogenic differentiation, the PVA-NOCC-PEG scaffold augmented the GAGs secretion in MSCs and the mRNA expression levels of Sox9, Col2a1, Acan, and Comp were elevated (p < 0.05). However, there was no significant difference between both the TGF-β1 and TGF-β3-treated groups (p > 0.05). In conclusion, TGF-β1 and TGF-β3 enhanced the chondrogenic differentiation of MSCs seeded on the PVA-NOCC-PEG scaffold; however, there was no significant difference between the effect of TGF-β1 and TGF-β3. Impact statement Transforming growth factor-beta (TGF-β) superfamily members is a key requirement for the in vitro chondrogenic differentiation of mesenchymal stem cells (MSCs). In this study, the effects of TGF-β1 and -β3 on MSC chondrogenic differentiation and proliferation on a novel three-dimensional scaffold, the poly(vinyl alcohol)-chitosan-poly(ethylene glycol) (PVA-NOCC-PEG) scaffold, was evaluated. In this study, the results showed both TGF-β1 and TGF-β3 can enhance the chondrogenic differentiation of MSCs seeded on the PVA-NOCC-PEG scaffold.

Effects of Electric-Toothbrush Vibrations on the Expression of Collagen and Non-Collagen Proteins through the Focal Adhesion Kinase Signaling Pathway in Gingival Fibroblasts

Electric-toothbrush vibrations, which remove plaque, are transmitted to the gingival connective tissue via epithelial cells. Physical energy affects cell function; however, the effects of electric-toothbrush vibrations on gingival extracellular matrix (ECM) protein expression remain unknown. We aimed to examine the effects of these vibrations on the expression of ECM proteins—type I collagen (col I), type III collagen (col III), elastin, and fibronectin (FN)—using human gingival fibroblasts (HGnFs). HGnFs were seeded for 5 days in a six-well plate with a hydrophilic surface, exposed to electric-toothbrush vibrations, and cultured for 7 days. Subsequently, the mRNA and protein levels of col I, col III, elastin, and FN were examined. To investigate the role of focal adhesion kinase (FAK) signaling on ECM protein expression in vibration-stimulated cells, the cells were treated with siRNA against protein tyrosine kinase (PTK). Electric-toothbrush vibrations increased col I, col III, elastin, and FN expression; promoted collagen and non-collagen protein production; and enhanced FAK phosphorylation in HGnFs. Moreover, PTK2 siRNA completely blocked the effects of these vibrations on the expression of col I, col III and elastin mRNA. The results suggest that electric-toothbrush vibrations increase collagen, elastin, and FN production through the FAK-signaling pathway in fibroblasts.