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Cold Spring Harbor Protocols Apr 2022is a powerful model system for cell and developmental biology in part because frogs produce thousands of eggs and embryos year-round. In vitro fertilization (IVF) is...
is a powerful model system for cell and developmental biology in part because frogs produce thousands of eggs and embryos year-round. In vitro fertilization (IVF) is ideal for obtaining developmentally synchronized embryos for microinjection or when natural mating has failed to produce a fertilization. In IVF, females are induced to ovulate, and then eggs are collected by manual expression. After testes are collected from a euthanized male frog, the eggs are fertilized in vitro. The embryos are then treated with cysteine to remove the sticky protective jelly coat. Dejellied embryos are much easier to manipulate during microinjection or when sorting in a Petri dish. The jelly coat is also very difficult to penetrate with an injection needle. After microinjection, embryos are maintained in Petri dishes until desired stages are reached. Although in vitro fertilization in and is similar, critical differences in solutions, handling of testis, response of fertilized eggs directly after introduction of sperm, and developmental timing are required for successful fertilization in .
Topics: Animals; Female; Fertilization; Fertilization in Vitro; Male; Spermatozoa; Xenopus; Xenopus laevis
PubMed: 34031212
DOI: 10.1101/pdb.prot106351 -
PHAGE (New Rochelle, N.Y.) Dec 2021Bacteriophage plaque enumeration is a critical step in a wide array of protocols. The current gold standard for plaque enumeration on Petri dishes is through manual...
Bacteriophage plaque enumeration is a critical step in a wide array of protocols. The current gold standard for plaque enumeration on Petri dishes is through manual counting. However, this approach is not only time-consuming and prone to human error but also limited to Petri dishes with countable number of plaques resulting in low throughput. We present OnePetri, a collection of trained machine learning models and open-source mobile application for the rapid enumeration of bacteriophage plaques on circular Petri dishes. When compared against the current gold standard of manual counting, OnePetri was ∼30 × faster. Compared against other similar tools, OnePetri had lower relative error (∼13%) than Plaque Size Tool (PST) (∼86%) and CFU.AI (∼19%), while also having significantly reduced detection times over PST (1.7 × faster). The OnePetri application is a user-friendly platform that can rapidly enumerate phage plaques on circular Petri dishes with high precision and recall.
PubMed: 36159886
DOI: 10.1089/phage.2021.0012 -
Journal of Fungi (Basel, Switzerland) Nov 2018has several advantages as an experimental host for the study of infectious diseases. Worms are easily maintained and propagated on bacterial lawns. The worms can be... (Review)
Review
has several advantages as an experimental host for the study of infectious diseases. Worms are easily maintained and propagated on bacterial lawns. The worms can be frozen for long term storage and still maintain viability years later. Their short generation time and large brood size of thousands of worms grown on a single petri dish, makes it relatively easy to maintain at a low cost. The typical wild type adult worm grows to approximately 1.5 mm in length and are transparent, allowing for the identification of several internal organs using an affordable dissecting microscope. A large collection of loss of function mutant strains are readily available from the genetic stock center, making targeted genetic studies in the nematode possible. Here we describe ways in which this facile model host has been used to study , an opportunistic fungal pathogen that poses a serious public health threat.
PubMed: 30405043
DOI: 10.3390/jof4040123 -
European Journal of Pharmaceutics and... Apr 2018The features rendering orodispersible films (ODFs) patient-centric formulations are widely discussed in the scientific literature. However there is a lack of research...
The features rendering orodispersible films (ODFs) patient-centric formulations are widely discussed in the scientific literature. However there is a lack of research studies exploring ODF characteristics with a potential impact on end-user acceptability. The aim of this study was to identify the key ODF characteristics affecting end-user acceptability by developing in vitro test methods for the prediction of ODFs acceptability and correlate these formulation characteristics with the data obtained from a human panel study. Four drug-free single-polymer films were prepared by solvent casting. Solutions of poly(vinyl) alcohol (PVOH) 39 KDa (P1), PVOH 197 KDa (P2), carboxymethylcellulose (CMC) 395 KDa (C1), and CMC 725 KDa (C2) were prepared. Texture analysis and Dynamic Mechanical Analysis (DMA) were used to assess film tack. Petri dish and drop methods were used to assess disintegration time. A human panel of 24 healthy young adults was employed to identify end-user acceptability criteria of the four study film samples. Texture analysis data of ODF tack were not found to be in agreement with the in vivo perceived stickiness in the mouth. However, measurement of the area under the adhesive force curve obtained by DMA correlated with in vivo perceived stickiness data for all samples. The disintegration times obtained by drop method were more comparable to human panel data than the petri dish method. Hence DMA and drop methods proved to be promising methodologies for the prediction of the end-user acceptability. The type and molecular weight of the film-forming polymer had a strong influence on stickiness perception, whereas only polymeric molecular weight influenced perceived disintegration time. The human panel study showed that Participant Reported Outcomes (PROs) for the perceived stickiness in the mouth and disintegration time of test films received significantly different scores between samples, and thus were identified as the key attributes with the potential to affect the end-user acceptability. ODF stickiness and disintegration time should therefore be evaluated at an early stage of the drug product design.
Topics: Administration, Oral; Adolescent; Adult; Carboxymethylcellulose Sodium; Cross-Over Studies; Drug Compounding; Drug Delivery Systems; Humans; Patient Preference; Pilot Projects; Polyvinyl Alcohol; Single-Blind Method; Solubility; Solvents; Young Adult
PubMed: 29355687
DOI: 10.1016/j.ejpb.2018.01.003 -
Lab on a Chip Feb 2021The selection of high quality sperm is critical for intracytoplasmic sperm injection (ICSI), a prevalent assisted reproduction technology. However, standard selection...
The selection of high quality sperm is critical for intracytoplasmic sperm injection (ICSI), a prevalent assisted reproduction technology. However, standard selection methods are time-consuming and fail to recover the most viable sperm, thereby limiting the ICSI success rate. Microfluidics enables rapid selection of viable sperm in a manner representing in vivo processes, however, existing platforms lack clinical applicability. Here, we present FertDish, which integrates the clinically established ICSI Petri dish with a film featuring an array of sperm-selecting microchannels for selection of sperm directly from semen. The FertDish format mimics the clinician-familiar ICSI dish setup, and provides rapid (<10 min) single stage sperm preparation that circumvents standard labour-intensive multi-stage sperm processing steps. Tests with human donor and patient semen samples show that FertDish enables the selection of a high quality sperm sub-population, featuring improvements in DNA fragmentation index of more than 91% (donor) and 74% (patient) versus raw semen and 50% (donor) and 63% (patient) versus standard methods, and a distribution of more than 97% sperm with viable and high level DNA. The FertDish enables a high sperm recovery rate (>3.3 × 105 sperm per mL), and is readily adaptable to the clinical workflow with potential to improve ICSI outcomes.
Topics: Humans; Male; Microfluidics; Sperm Injections, Intracytoplasmic; Spermatozoa
PubMed: 33507191
DOI: 10.1039/d0lc00874e -
Integrative Biology : Quantitative... Jul 2018Improving fluorescent proteins through the use of directed evolution requires robust techniques for screening large libraries of genetic variants. Here we describe an...
Improving fluorescent proteins through the use of directed evolution requires robust techniques for screening large libraries of genetic variants. Here we describe an effective and relatively low-cost system for screening libraries of fluorescent protein variants for improved photostability in the context of colonies on a Petri dish. Application of this system to the yellow fluorescent protein mCitrine, led to the development of Citrine2 with improved photostability and similar high fluorescent brightness. The photobleaching robot was constructed using a Lego Mindstorms Ev3 set and a xenon arc lamp, which together create even and high irradiance over an entire Petri dish through patterned illumination.
Topics: Bacterial Proteins; Crystallography, X-Ray; Escherichia coli; HeLa Cells; Humans; Light; Luminescent Proteins; Models, Biological; Mutation; Photobleaching; Photochemistry; Plasmids; Robotics
PubMed: 29897363
DOI: 10.1039/c8ib00063h -
HardwareX Apr 2022Multispectral imaging is at the forefront of contactless surface analysis. Standard multispectral imaging systems use sophisticated software, cameras and light filtering...
Multispectral imaging is at the forefront of contactless surface analysis. Standard multispectral imaging systems use sophisticated software, cameras and light filtering optics. This paper discloses the building of a customizable and cost-effective multispectral imaging and analysis system. It integrates a web camera, light emitting diodes (LEDs) lighting, a semisphere for even lightening, an open-source Arduino™ development board and a free Python application to automatically obtain and visually analyze multispectral images. The device is hereafter called MEDUSA and its optical performance was tested for repeated Imaging consistency, visible and near infrared band sensitivity and lighting evenness. Four proof of concept tests were run in order to understand the advantageous use of this system, as compared to a simple visual score of diverse samples. Each of three qualitative tests used sets of 12 LED band spectral images to analyze ink changes in a counterfeit bill, surface bruises on Hass avocado fruits and transient changes in petri dish grown bacterial colonies. A fourth test used single band imaging in a set of standard laboratory analyzed plant samples, to quantitatively relate a red band light reflectance to its nitrogen content. These tests indicate that MEDUSA made images may yield qualitative and quantitative spectral information unseen to the naked eye, suggesting potential use in currency counterfeit tests, food quality analyses, microbial phenotyping and agricultural plant chemistry. MEDUSA can be freely reproduced and customized from this research, making it a powerful and affordable analytical tool to analyze a wide range of subtle chemical properties in samples at industrial and science fields.
PubMed: 35509904
DOI: 10.1016/j.ohx.2022.e00282 -
Annals of Translational Medicine Sep 2017
PubMed: 28936460
DOI: 10.21037/atm.2017.07.07 -
Frontiers in Plant Science 2022Coumestrol (CMS) derivatives are unique compounds, which function as phytoalexins; they are derived from soybean roots, following abiotic and biotic stresses. As a...
Coumestrol (CMS) derivatives are unique compounds, which function as phytoalexins; they are derived from soybean roots, following abiotic and biotic stresses. As a phytoalexin, CMS forms a defense system that enables plants to maintain their viability. However, it is still challenging to achieve the mass production of phytoalexins, which exhibit pharmacological values, plant breeding. Here, the synthesis of CMS derivatives from the seedling, plant, and adventitious root (AR) of were investigated under artificial light, as well as a chemical elicitor treatment. In the presence of constant light, as well as under treatment with methyl jasmonate, the CMS monoglucoside (coumestrin; CMSN) and malonyl CMSN (M-CMSN) contents of the AR culture (4 weeks) increased drastically. The two CMS derivatives, CMSN and M-CMSN, were obtained as a mixture of isomers, which were identified nuclear magnetic resonance analysis. These derivatives were also observed in a soybean plant that was grown on artificial soil (AS; 5 weeks) and a Petri dish (9 days) although in considerably lesser quantities than those observed in the AR culture. Compared with the two other media (AS and the Petri dish), the AR culture achieved the superior synthesis of CMSN and M-CMSN within a relatively short cultivation period (<1 month) in laboratory-scale (3 L) and pilot-scale (1,000 L) bioreactors. The isoflavone content of AR under the constant light conditions was three-fold that under dark conditions. Significant quantities of malonyl daidzin and malonyl genistin were produced in the root of AS and the seedling of Petri dish, respectively. Flavonol glycosides were not produced in the AR culture under the dark and light conditions, as well as in AS under the dark condition. However, significant contents of kaempferol glycosides were produced in the leaves of AS and seedling of Petri dish, following the light treatment. Thus, we proposed that the established soybean AR-cultivation approach represented a better method for biosynthesizing phytoalexins, such as the CMS derivatives, as plant-derived functional materials.
PubMed: 35800610
DOI: 10.3389/fpls.2022.923163 -
Nature Reviews. Molecular Cell Biology Jan 2017Human pluripotent stem cells (hPSCs) provide an unparalleled opportunity to establish in vitro differentiation models that will transform our approach to the study of... (Review)
Review
Human pluripotent stem cells (hPSCs) provide an unparalleled opportunity to establish in vitro differentiation models that will transform our approach to the study of human development. In the case of the blood system, these models will enable investigation of the earliest stages of human embryonic haematopoiesis that was previously not possible. In addition, they will provide platforms for studying the origins of human blood cell diseases and for generating de novo haematopoietic stem and progenitor cell populations for cell-based regenerative therapies.
Topics: Animals; Cell Culture Techniques; Cell Differentiation; Hematopoiesis; Humans; Induced Pluripotent Stem Cells; Mice; Pluripotent Stem Cells
PubMed: 27876786
DOI: 10.1038/nrm.2016.127