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Microorganisms Nov 2022induces heavy mortality in aquaculture. The detection of is often time consuming and uncertain, making it difficult to manage the disease. We validated a previously...
induces heavy mortality in aquaculture. The detection of is often time consuming and uncertain, making it difficult to manage the disease. We validated a previously published real-time quantitative PCR (qPCR) assay to confirm the presence of in fish and in water using environmental DNA (eDNA) quantification. Analytical sensitivity and specificity of the assay was assessed in silico, in vitro and the qPCR assay was compared with microbiological cultivation methods to detect and quantify in water samples from a controlled fish exposure experiment and from fish farms. Furthermore, we compared the use of an agar cultivation method and the qPCR assay to detect directly from mucus samples taken from the fish surface. The analytical sensitivity and specificity of the qPCR assay were high. The qPCR assay detected 100% of -positive water samples. In a field study, the qPCR assay and a microwell plate (MWP) enumeration method correlated significantly. Furthermore, the qPCR assay could be used to confirm the presence of in skin mucus. Thus, the qPCR assay could complement diagnostic methods in specifically detecting saprolegniosis in fish and used as a surveillance method for pathogen in aquaculture environments.
PubMed: 36363778
DOI: 10.3390/microorganisms10112186 -
RSC Advances Oct 2022Herein, we report a sensitive and selective enzyme-linked aptamer-based sandwich assay (ELASA) to detect lactate dehydrogenase (LDH), which is an attractive biomarker...
Herein, we report a sensitive and selective enzyme-linked aptamer-based sandwich assay (ELASA) to detect lactate dehydrogenase (LDH), which is an attractive biomarker for malaria diagnosis and antimalarial medication. We performed the sandwich assay with a single aptamer sequence, called 2008s, owing to the structural properties of the LDH tetramer instead of using a conventional sandwich assay with two different aptamers (or antibodies) for capturing and probing a target molecule. First, the biotinylated LDH aptamer was linked with immobilized streptavidin on a microwell plate for binding flexibility, and then LDH was bound to the aptamer. Next, a horseradish peroxidase-conjugated aptamer of the same sequence was used to analyze LDH quantitatively. Using this approach, the limit of detection (LOD) of LDH with the naked eye was 100 ng mL, and the LOD and limit of quantification from the absorbance measurements were 34.9 ng mL and 95.5 ng mL, respectively, based on LDH spiked blood samples. Our proposed method selectively binds LDH, not human lactate dehydrogenase. Therefore, this method may be a valuable tool for diagnosing, monitoring, and quarantining malaria cases easily and rapidly.
PubMed: 36320752
DOI: 10.1039/d2ra03796c -
International Journal of Molecular... Sep 2022Pancreatic lipase (PNLIP, EC 3.1.1.3) plays a pivotal role in the digestion of dietary lipids, a metabolic pathway directly related to obesity. One of the effective...
The In Vitro Inhibitory Effect of Selected Asteraceae Plants on Pancreatic Lipase Followed by Phenolic Content Identification through Liquid Chromatography High Resolution Mass Spectrometry (LC-HRMS).
Pancreatic lipase (PNLIP, EC 3.1.1.3) plays a pivotal role in the digestion of dietary lipids, a metabolic pathway directly related to obesity. One of the effective strategies in obesity treatment is the inhibition of PNLIP, which is possible to be achieved by specific phenolic compounds occurring in high abundance in some plants. In this study, a multidisciplinary approach is presented investigating the PNLIP inhibitory effect of 33 plants belonging in the Asteraceae botanical family. In the first stage of the study, a rapid and cost-efficient PNLIP assay in a 96-microwell plate format was developed and important parameters were optimized, e.g., the enzyme substrate. Upon PNLIP assay optimization, aqueous and dichloromethane Asteraceae plant extracts were tested and a cut-off inhibition level was set to further analyze only the samples with a significant inhibitory effect (inhibitory rate > 40%), using an ultra-high-performance liquid chromatography hybrid quadrupole time-of-flight mass spectrometry (UHPLC-q-TOF-MS) method. Specifically, a metabolomic suspect screening was performed and 69 phenolic compounds were tentatively identified, including phenolic acids, flavonoids, flavonoid-3-O-glycosides, and flavonoid-7-O-glycosides, amongst others. In the case of aqueous extracts, phytochemicals known for inducing PNLIP inhibitory effect, e.g., compounds containing galloyl molecules or caffeoylquinic acids, were monitored in Chrysanthemum morifolium, Grindella camporum and Hieracium pilosella extracts. All in all, the presented approach combines in vitro bioactivity measurements to high-end metabolomics to identify phenolic compounds with potential medicinal and/or dietary applications.
Topics: Asteraceae; Chromatography, High Pressure Liquid; Chromatography, Liquid; Flavonoids; Glycosides; Lipase; Lipids; Mass Spectrometry; Methylene Chloride; Obesity; Phenols; Phytochemicals; Plant Extracts
PubMed: 36232503
DOI: 10.3390/ijms231911204 -
Molecules (Basel, Switzerland) Jul 2022A sustainable downscaled procedure using smartphone-based colorimetric determination of manganese (Mn(II)) was developed. This novel Mn(II) determination procedure is...
Sustainable Downscaled Catalytic Colorimetric Determination of Manganese in Freshwater Using Smartphone-Based Monitoring Oxidation of 3,3',5,5'-Tetramethylbenzidine by Periodate.
A sustainable downscaled procedure using smartphone-based colorimetric determination of manganese (Mn(II)) was developed. This novel Mn(II) determination procedure is proposed using a simple, available microwell-plate platform and a smartphone as a detector. This approach is based on the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by periodate using Mn(II) as a catalyst. The catalytic kinetics of Mn(II) under different conditions was investigated to determine the optimum condition where the different catalytic activities of various concentrations of Mn(II) evince. Under the optimum condition, the bluish-green product of oxidized TMB, proportioned to the concentration of Mn(II), was monitored using a smartphone camera, and the color signals were processed using ImageJ Software. The developed procedure showed great selectivity and sensitivity as linearity ranged from 1.8 × 10 to 4.6 × 10 M (0.1 to 2.5 μg/mL). The limits of detection and quantitation were 3.6 × 10 and 1.1 × 10 M (0.2 and 0.6 μg/mL), respectively. The determination of Mn(II) in freshwater samples was demonstrated to assess environmental water quality as an initial model to more easily promote water management according to the United Nations Sustainable Development Goals (UN-SDGs). The intensity of the red could be successfully applied to evaluate Mn(II) in canals and river water with no significant differences compared with the reference method of Inductively Coupled Plasma Optical Emission Spectrometry at a confidence level of 95%.
Topics: Benzidines; Colorimetry; Fresh Water; Manganese; Periodic Acid; Smartphone
PubMed: 35956794
DOI: 10.3390/molecules27154841 -
Communications Biology Jul 2022Single cell RNA sequencing has the potential to elucidate transcriptional programs underlying key cellular phenotypes and behaviors. However, many cell phenotypes are...
Single cell RNA sequencing has the potential to elucidate transcriptional programs underlying key cellular phenotypes and behaviors. However, many cell phenotypes are incompatible with indiscriminate single cell sequencing because they are rare, transient, or can only be identified by imaging. Existing methods for isolating cells based on imaging for single cell sequencing are technically challenging, time-consuming, and prone to loss because of the need to physically transport single cells. Here, we developed See-N-Seq, a method to rapidly screen cells in microwell plates in order to isolate RNA from specific single cells without needing to physically extract each cell. Our approach involves encapsulating the cell sample in a micropatterned hydrogel with spatially varying porosity to selectively expose specific cells for targeted RNA extraction. Extracted RNA can then be captured, barcoded, reverse transcribed, amplified, and sequenced at high-depth. We used See-N-Seq to isolate and sequence RNA from cell-cell conjugates forming an immunological synapse between T-cells and antigen presenting cells. In the hours after synapsing, we found time-dependent bifurcation of single cell transcriptomic profiles towards Type 1 and Type 2 helper T-cells lineages. Our results demonstrate how See-N-Seq can be used to associate transcriptomic data with specific functions and behaviors in single cells.
Topics: High-Throughput Nucleotide Sequencing; Hydrogels; Microscopy; Porosity; RNA; Sequence Analysis, RNA
PubMed: 35908100
DOI: 10.1038/s42003-022-03703-3 -
F1000Research 2022Animal models have provided many insights into ocular development and disease, but they remain suboptimal for understanding human oculogenesis. Eye development requires...
Animal models have provided many insights into ocular development and disease, but they remain suboptimal for understanding human oculogenesis. Eye development requires spatiotemporal gene expression patterns and disease phenotypes can differ significantly between humans and animal models, with patient-associated mutations causing embryonic lethality reported in some animal models. The emergence of human induced pluripotent stem cell (hiPSC) technology has provided a new resource for dissecting the complex nature of early eye morphogenesis through the generation of three-dimensional (3D) cellular models. By using patient-specific hiPSCs to generate optic vesicle-like models, we can enhance the understanding of early developmental eye disorders and provide a pre-clinical platform for disease modelling and therapeutics testing. A major challenge of optic vesicle generation is the low efficiency of differentiation in 3D cultures. To address this, we adapted a previously published protocol of retinal organoid differentiation to improve embryoid body formation using a microwell plate. Established morphology, upregulated transcript levels of known early eye-field transcription factors and protein expression of standard retinal progenitor markers confirmed the optic vesicle/presumptive optic cup identity of models between day 20 and 50 of culture. This adapted protocol is relevant to researchers seeking a physiologically relevant model of early human ocular development and disease with a view to replacing animal models.
Topics: Animals; Cell Differentiation; Embryoid Bodies; Humans; Induced Pluripotent Stem Cells; Retina; Transcription Factors
PubMed: 35811797
DOI: 10.12688/f1000research.108829.1 -
Data in Brief Aug 2022SARS-CoV-2 pandemic opens up the curiosity of understanding the coronavirus. This demand for the development of the regent, which can be used for academic and...
SARS-CoV-2 pandemic opens up the curiosity of understanding the coronavirus. This demand for the development of the regent, which can be used for academic and therapeutic applications. The present data provide the biochemical characterization of synthetically developed monoclonal antibodies for the SARS-CoV-2 proteins. The antibodies from phage-displayed antibody libraries were selected with the SARS-CoV-2 proteins immobilized in microwell plates. The clones which bind to the antigen in Fab-phage ELISA were selected, and a two-point competitive phage ELISA was performed. Antibodies binding kinetic of IgGs for SARS-CoV2 proteins further carried with B.L.I. Systematic analysis of binding with different control proteins and purified SARS-CoV-2 ensured the robustness of the antibodies.
PubMed: 35789908
DOI: 10.1016/j.dib.2022.108415 -
Frontiers in Toxicology 2022The methods outlined here are part of a series of papers designed specifically for genotoxicity assessment of nanomaterials (NM). Common Considerations such as NM...
The methods outlined here are part of a series of papers designed specifically for genotoxicity assessment of nanomaterials (NM). Common Considerations such as NM characterization, sample preparation and dose selection, relevant to all genotoxicity assays, are found in an accompanying paper. The present paper describes methods for evaluation of mutagenicity in the mammalian (mouse) () gene occurring in L5178Y mouse lymphoma (ML) cells and in the designated gene in human lymphoblastoid TK6 cells. Mutations change the functional genotype from TK to TK, detectable as cells surviving on media selective for the lack of thymidine kinase (TK) function. Unlike cells with TK enzyme function, the TK cells are unable to integrate the toxic selection agent, allowing these cells to survive as rare mutant colonies. The ML assay has been shown to detect a broad spectrum of genetic damage, including both small scale (point) mutations and chromosomal alterations. This assay is a widely used mammalian cell gene mutation assay for regulatory purposes and is included in the core battery of genotoxicity tests for regulatory decision-making. The TK6 assay is an assay using a human cell line derived similarly via mutagenic manipulations and optimal selection. Details are provided on the materials required, cell culture methods, selection of test chemical concentrations, cytotoxicity, treatment time, mutation expression, cloning, and data calculation and interpretation. The methods describe the microwell plate version of the assays without metabolic activation.
PubMed: 35757197
DOI: 10.3389/ftox.2022.864753 -
European Journal of Cell Biology 2022Mesenchymal stem cells (MSCs) show a decline in pluripotency and differentiation with increased cell culture passages in 2D cultures. The 2D monolayer culture fails to...
Mesenchymal stem cells (MSCs) show a decline in pluripotency and differentiation with increased cell culture passages in 2D cultures. The 2D monolayer culture fails to correctly imitate the architecture and microenvironments of in-vivo cell models. Alternatively, 3D culture may improve the simulations of in-vivo cell microenvironments with wide applications in cell culture and drug discovery. In the present study, we compared various 3D culturing techniques such as 3D micro-well (3D-S), hanging drop (HD), and ultra-low attachment (ULA) plate-based spheroid culture to study their effect on morphology, viability, pluripotency, cell surface markers, immunomodulatory factors, and differentiation capabilities of Wharton's jelly-mesenchymal stem cells (WJ-MSCs). The cell morphology, viability, and senescence of 3D cultured WJ-MSCs were comparable to cells in 2D culture. The expression of pluripotency markers (OCT4, SOX2, and NANOG) was enhanced upto 2-8 fold in 3D cultured WJ-MSCs when compared to 2D culture. Moreover, the immunomodulatory factors (IDO, IL-10, LIF, ANG1, and VEGF) were significantly elevated in ULA based 3D cultured WJ-MSCs. Furthermore, significant enhancement in the differentiation potential of WJ-MSCs towards adipocyte (ADP and C/EBP-α), osteocyte (OPN and RUNX2), and definitive endodermal (SOX17, FOXA2, and CXCR4) lineages in 3D culture conditions were observed. Additionally, the osteogenic and adipogenic differentiation potential of WJ-MSCs over the time points 7 days, 14 days, and 28 days was also significantly increased in 3D culture groups. Our study demonstrates that stemness properties of WJ-MSCs were significantly enhanced in 3D cultures and ULA-based culture outperformed other methods with high pluripotency gene expression and enhanced differentiation potential. This study indicates the efficacy of 3D cultures to bridge the gap between 2D cell culture and animal models in regenerative medicine.
Topics: Animals; Cell Culture Techniques; Cell Differentiation; Cell Proliferation; Cells, Cultured; Mesenchymal Stem Cells; Osteogenesis; Wharton Jelly
PubMed: 35667339
DOI: 10.1016/j.ejcb.2022.151245 -
Stem Cell Research & Therapy May 2022Muscle denervation from trauma and motor neuron disease causes disabling morbidities. A limiting step in functional recovery is the regeneration of neuromuscular...
BACKGROUND
Muscle denervation from trauma and motor neuron disease causes disabling morbidities. A limiting step in functional recovery is the regeneration of neuromuscular junctions (NMJs) for reinnervation. Stem cells have the potential to promote these regenerative processes, but current approaches have limited success, and the optimal types of stem cells remain to be determined. Neural crest stem cells (NCSCs), as the developmental precursors of the peripheral nervous system, are uniquely advantageous, but the role of NCSCs in neuromuscular regeneration is not clear. Furthermore, a cell delivery approach that can maintain NCSC survival upon transplantation is critical.
METHODS
We established a streamlined protocol to derive, isolate, and characterize functional p75 NCSCs from human iPSCs without genome integration of reprogramming factors. To enhance survival rate upon delivery in vivo, NCSCs were centrifuged in microwell plates to form spheroids of desirable size by controlling suspension cell density. Human bone marrow mesenchymal stem cells (MSCs) were also studied for comparison. NCSC or MSC spheroids were injected into the gastrocnemius muscle with denervation injury, and the effects on NMJ formation and functional recovery were investigated. The spheroids were also co-cultured with engineered neuromuscular tissue to assess effects on NMJ formation in vitro.
RESULTS
NCSCs cultured in spheroids displayed enhanced secretion of soluble factors involved in neuromuscular regeneration. Intramuscular transplantation of spheroids enabled long-term survival and retention of NCSCs, in contrast to the transplantation of single-cell suspensions. Furthermore, NCSC spheroids significantly improved functional recovery after four weeks as shown by gait analysis, electrophysiology, and the rate of NMJ innervation. MSC spheroids, on the other hand, had insignificant effect. In vitro co-culture of NCSC or MSC spheroids with engineered myotubes and motor neurons further evidenced improved innervated NMJ formation with NCSC spheroids.
CONCLUSIONS
We demonstrate that stem cell type is critical for neuromuscular regeneration and that NCSCs have a distinct advantage and therapeutic potential to promote reinnervation following peripheral nerve injury. Biophysical effects of spheroidal culture, in particular, enable long-term NCSC survival following in vivo delivery. Furthermore, synthetic neuromuscular tissue, or "tissues-on-a-chip," may offer a platform to evaluate stem cells for neuromuscular regeneration.
Topics: Denervation; Humans; Induced Pluripotent Stem Cells; Neural Crest; Neural Stem Cells; Neurogenesis
PubMed: 35578348
DOI: 10.1186/s13287-022-02877-1