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Prezygotic interspecific crossability barrier in the genus Cucumis is related to the ploidy level of the species (cucumber (C. sativus), x = 7; muskmelon (C. melo) and wild Cucumis species, x = 12). Polyploidization of maternal plants helps hybridization among other Cucumis species by overcoming prezygotic genetic barriers. The main objective of this paper is to compare the results of several methods supporting interspecific crosses in cucumber without and with polyploidization (comparison between diploid (2x) and mixoploid (2x/4x) cucumber maternal plants). Mixoploid plants were obtained after in vivo and in vitro polyploidization by colchicine and oryzalin. Ploidy level was estimated by flow cytometry. Embryo rescue, in vitro pollination, and isolation of mesophyll protoplast were tested and compared. Positive effect of polyploidization was observed during all experiments presented by higher regeneration capacity of cultivated mixoploid cucumber embryos, ovules, and protoplasts. Nevertheless, the hybrid character of putative hybrid accessions obtained after cross in vivo and in vitro pollination was not confirmed.
The microbial infection of the endodontic space occurs in a necrotic tooth as a result of dental caries, trauma, periodontal disease, or previous root canal therapy. The disruption of the biofilms and the reduction of the bacterial load inside root canals are crucial for the success of root canal therapy. The aim of this study was to compare, in vitro, the antibiofilm efficacy of a novel passive sonic irrigation (PSI) device with passive ultrasonic irrigation (PUI) and conventional needle irrigation (CNI). Forty-four single-rooted human teeth were inoculated with a culture of E. faecalis for 28 days. The specimens were randomly divided into three groups: PUI, CNI, and PSI (n = 12). The activation protocols were performed using both 17% EDTA and 5.25% NaOCl. Residual bacterial biofilm was taken by means of a canal brush and colony-forming unit (CFU) were counted. The data were analyzed using one-way ANOVA and Games-Howell's post hoc tests. A major reduction in CFU was observed in the PSI and PUI groups, in comparison with the CNI group. No difference was found (p > 0.05) in terms of CFU reduction between PSI and PUI. PSI could be as effective as PUI in the removal of bacterial biofilms from straight root canals.
Inhalation is the preferred route for the treatment of lung diseases. More recently, also formulations for systemic treatment have been developed. For efficient development of inhalation products it is necessary to identify a link between particle parameters and in vivo performance. Such a relation exists for oral drugs where dissolution and permeation across Caco-2 monolayers are correlated to in vivo absorption. By contrast, the only in vitro parameter with established link to absorption for inhalation is particle size. In vitro dissolution could be another important parameter because low solubility determines bioavailability of inhaled drugs. The review highlights the importance of dissolution for drug availability in general and lists important differences in dissolution testing of oral and inhaled formulations. Dissolution testing protocols are summarized with focus on the composition of the various fluids, which are used to mimic particle dissolution in the deep lung. Finally, a role of in silico modelling in identification of physiologically relevant dissolution fluids and in in vitro in vivo correlation is suggested.
Skin is easily accessible for transdermal drug delivery and also attractive for biomarker sampling. These applications are strongly influenced by hydration where elevated hydration generally leads to increased skin permeability. Thus, favorable transdermal delivery and extraction conditions can be easily obtained by exploiting elevated skin hydration. Here, we provide a detailed in vivo and in vitro investigation of the skin hydration dynamics using three techniques based on electrical impedance spectroscopy. Good correlation between in vivo and in vitro results is demonstrated, which implies that simple but realistic in vitro models can be used for further studies related to skin hydration (e.g., cosmetic testing). Importantly, the results show that hydration proceeds in two stages. Firstly, hydration between 5 and 10 min results in a drastic skin impedance change, which is interpreted as filling of superficial voids in skin with conducting electrolyte solution. Secondly, a subtle impedance change is observed over time, which is interpreted as leveling of the water gradient across skin leading to structural relaxation/changes of the macromolecular skin barrier components. With respect to transdermal drug delivery and extraction of biomarkers; 1 h of hydration is suggested to result in beneficial and stable conditions in terms of high skin permeability and extraction efficiency.
Cefiderocol is a siderophore cephalosporin active against MDR Gram-negatives including Stenotrophomonas maltophilia. Cefiderocol resistance remains uncommon and incompletely understood. We selected for cefiderocol-resistant S. maltophilia in vitro and characterized the genetic mechanisms and potential for cross-resistance to other antimicrobials.
Poor semen quality is increasingly the reason for human infertility. For couples who cannot be helped by conventional in vitro fertilization (IVF) treatment, micromanipulation methods and special sperm preparation techniques in in vitro fertilization hold considerable promise to enhance fertilization of the egg. "Partial zona dissection" (PZD n = 59) or "subzonal insemination" (SUZI n = 36) were performed in 88 IVF cycles obtaining a fertilization rate between 12.2% and 34.8% depending on the micromanipulation technique applied and a pregnancy rate of 19.2% per embryo transfer. So far ten ongoing pregnancies have occurred and five healthy children have been born. In certain cases of male factor infertility micromanipulation and special sperm preparation techniques therefore represent the only possibility of conceiving.
Multicellular tumor spheroids are powerful in vitro models to perform preclinical chemosensitivity assays. We compare different methodologies to generate tumor spheroids in terms of resultant spheroid morphology, cellular arrangement and chemosensitivity. We used two cancer cell lines (MCF7 and OVCAR8) to generate spheroids using i) hanging drop array plates; ii) liquid overlay on ultra-low attachment plates; iii) liquid overlay on ultra-low attachment plates with rotating mixing (nutator plates). Analysis of spheroid morphometry indicated that cellular compaction was increased in spheroids generated on nutator and hanging drop array plates. Collagen staining also indicated higher compaction and remodeling in tumor spheroids on nutator and hanging drop arrays compared to conventional liquid overlay. Consequently, spheroids generated on nutator or hanging drop plates had increased chemoresistance to cisplatin treatment (20-60% viability) compared to spheroids on ultra low attachment plates (10-20% viability). Lastly, we used a mathematical model to demonstrate minimal changes in oxygen and cisplatin diffusion within experimentally generated spheroids. Our results demonstrate that in vitro methods of tumor spheroid generation result in varied cellular arrangement and chemosensitivity.
Over the last decade, the African turquoise killifish, Nothobranchius furzeri, has emerged as an important model system for the study of vertebrate biology and ageing. Propagation of laboratory inbred strains of Nothobranchius furzeri, such as GRZ, however, can pose challenges due to the short window of fertility, the efforts and space requirements involved in continuous strain maintenance, and the risks of further inbreeding. The current method for long term strain preservation relies on arrest of embryos in diapause. To create an alternative for long term maintenance, we developed a robust protocol to cryopreserve and revive sperm for in vitro fertilization (IVF). We tested a variety of extender and activator buffers for sperm IVF, as well as cryoprotectants to achieve practical long-term storage and fertilization conditions tailored to this species. Our protocol enabled sperm to be preserved in a cryogenic condition for months and to be revived with an average of 40% viability upon thawing. Thawed sperm were able to fertilize nearly the same number of eggs as natural fertilization, with an average of ~ 25% and peaks of ~ 55% fertilization. This technical advance will greatly facilitate the use of N. furzeri as a model organism.
Additive Manufacturing is noted for ease of product customization and short production run cost-effectiveness. As our global population approaches 8 billion, additive manufacturing has a future in maintaining and improving average human life expectancy for the same reasons that it has advantaged general manufacturing. In recent years, additive manufacturing has been applied to tissue engineering, regenerative medicine, and drug delivery. Additive Manufacturing combined with tissue engineering and biocompatibility studies offers future opportunities for various complex cardiovascular implants and surgeries. This paper is a comprehensive overview of current technological advancements in additive manufacturing with potential for cardiovascular application. The current limitations and prospects of the technology for cardiovascular applications are explored and evaluated.
Herbal plants are traditionally utilized to treat various illnesses. They contain phytochemicals that can be extracted using conventional methods such as maceration, soxhlet, and boiling, as well as non-conventional methods including ultrasonic, microwave, and others. Carica papaya leaves have been used for the treatment of dengue, fungal, and bacterial infections as well as an ingredient in anti-aging products. Phytochemicals analysis detected the presence of kaempferol, myricetin, carpaine, pseudocarpaine, dehydrocarpaine I and II, ferulic acid, caffeic acid, chlorogenic acid, β-carotene, lycopene, and anthraquinones glycoside. Conventional preparation by boiling and simple maceration is practical, simple, and safe; however, only polar phytochemicals are extracted. The present study aims to investigate the effects of three different non-conventional extraction techniques (ultrasonic-assisted extraction, reflux, and agitation) on C. papaya phytochemical constituents, the antioxidant capacity, and wound-healing activities. Among the three techniques, the reflux technique produced the highest extraction yield (17.86%) with the presence of saponins, flavonoids, coumarins, alkaloids, and phenolic metabolites. The reflux technique also produced the highest 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging with an IC50 value of 0.236 mg/mL followed by ultrasonic-assisted extraction (UAE) (IC50: 0.377 mg/mL) and agitation (IC50: 0.404 mg/mL). At tested concentrations (3.125 µg/mL to 500 µg/mL), all extracts do not exhibit a cytotoxicity effect on the human skin fibroblast, HSF1184. Interestingly, reflux and UAE were active fibroblast proliferators that support 85% (12.5 µg/mL) and 41% (6.25 µg/mL) better cell growth, respectively. Additionally, during the early 24 h of the scratch assay, the migration rate at 12.5 µg/mL was faster for all extracts with 51.8% (reflux), 49.3% (agitation), and 42.5% (UAE) as compared to control (21.87%). At 48 h, proliferated cells covered 78.7% of the scratch area for reflux extract, 63.1% for UAE, 61% for agitation, and 42.6% for control. Additionally, the collagen synthesis was enhanced for 31.6% and 65% after 24 and 48 h of treatment for reflux. An HPLC-MS/MS-QTOF (quadruple time-of-flight) analysis of reflux identified nine phytochemicals, including carpaine, kaempferol 3-(2G-glucosylrutinoside), kaempferol 3-(2″-rhamnosylgalactoside), 7-rhamnoside, kaempferol 3-rhamnosyl-(1->2)-galactoside-7-rhamnoside, luteolin 7-galactosyl-(1->6)-galactoside, orientin 7-O-rhamnoside, 11-hydroperoxy-12,13-epoxy-9-octadecenoic acid, palmitic amide, and 2-hexaprenyl-6-methoxyphenol. The results suggested that reflux was the best technique as compared to ultrasonic and agitation.
Polymer-based eye drops are the most used drug delivery system to treat dry eye disease (DED). Therefore, the mucoadhesion between the polymer and the ocular mucin is crucial to ensure the efficacy of the treatment. In this context, the present study aimed to evaluate the potential use of in vitro methods to study the mucoadhesion of eye drop solutions and, specifically to evaluate the efficacy of two hyaluronic acid-based formulations (HA), HA 0.15% and 0.30% (w/v) to treat DED. Rheology methods and zeta potential determination were used to study the mucoadhesive properties of both eye drop solutions. All results indicated that interactions occurred between the mucin and the HA, being stronger with HA 0.30%, due to the physical entanglements and hydrogen bounding. In vitro tests on ARPE-19 cell line were performed using a 2D and a 3D dry eye model and the results have shown that pre-treated cells with HA showed a morphology more similar to the hydrated cells in both products, with a high survival rate. The in vitro techniques used in this study have been shown to be suitable to evaluate and predict mucoadhesive properties and the efficacy of the eye drops on relief or treatment of DED. The results obtained from these methods may help in inferring possible in vivo effects.
Vitamin B12 deficiency, which may result in anemia and nerve damage if left untreated, is currently treated by administration of cyanocobalamin via oral or intramuscular routes. However, these routes are associated with absorption and compliance issues which have prompted us to investigate skin as an alternative site of administration. Delivery through skin, however, is restricted to small and moderately lipophilic molecules due to the outermost barrier, the stratum corneum (SC). In this study, we have investigated the effect of different enhancement techniques, chemical enhancers (ethanol, oleic acid, propylene glycol), iontophoresis (anodal iontophoresis) and microneedles (soluble maltose microneedles), which may overcome this barrier and improve cyanocobalamin delivery. Studies with different chemical enhancer formulations indicated that ethanol and oleic acid decreased the lag time while propylene glycol based formulations increased the lag time. The formulation with ethanol (50%), oleic acid (10%) and propylene glycol (40%) showed the maximum improvement in delivery. Iontophoresis and microneedle treatments resulted in enhanced permeation levels compared to passive controls. These enhancement approaches can be explored further to develop alternative treatment regimens.
Hedera helix L. contains phytochemicals with good biological properties which are beneficial to human health and can be used to protect plants against different diseases. The aim of this research was to find the most suitable extraction method and the most favorable parameters for the extraction of different bioactive compounds from ivy leaves. Different extraction methods, namely microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), and conventional heating extraction (CHE), were used. The most suitable method for the extraction of saponins is MAE with an extraction efficiency of 58%, while for carbohydrates and polyphenols, the best results were achieved via UAE with an extraction efficiency of 61.7% and 63.5%, respectively. The antioxidant activity (AA) of the extracts was also determined. The highest AA was obtained via UAE (368.98 ± 9.01 µmol TR/gDM). Better results were achieved at 50 °C for 10 min of extraction, using 80% ethanol in water as solvent. In order to evaluate their in vitro cytotoxicity, the extracts richest in bioactive compounds were tested on NCTC fibroblasts. Their influence on the DNA content of RAW 264.7 murine macrophages was also tested. Until 200 µg/mL, the extracts obtained via UAE and MAE were cytocompatible with NCTC fibroblasts at 48 h of treatment. Summarizing the above, both MAE and UAE can be employed as green and efficient methods for producing extracts rich in bioactive compounds, exhibiting strong antioxidant properties and good noncytotoxic activity.
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