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Methods in Molecular Biology (Clifton,... 2012Gel electrophoresis is an important methodology employed for protein analysis. It is often necessary to elute and recover proteins separated by sodium dodecyl sulfate... (Review)
Review
Gel electrophoresis is an important methodology employed for protein analysis. It is often necessary to elute and recover proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The procedure involves localizing the protein of interest on the gel following SDS-PAGE, eluting the protein from the gel, removing SDS from the eluted sample, and finally renaturing the protein (enzymes, for example) for subsequent analysis. Proteins are extracted from gels by several methods. These include dissolution of the gel matrix, passive diffusion, and electrophoretic elution. Proteins eluted from gels have been used successfully in a variety of downstream applications, including protein chemistry, proteolytic cleavage, determination of amino acid composition, polypeptide identification by trypsin digestion and matrix-assisted laser desorption ionization-time of flight mass spectroscopy, as antigens for antibody production, identifying a polypeptide corresponding to an enzyme activity and other purposes. Protein yields ranging from nanogram levels to 100 μg have been obtained.
Topics: Acetone; Chemical Precipitation; Diffusion; Electrophoresis, Polyacrylamide Gel; Proteins
PubMed: 22585504
DOI: 10.1007/978-1-61779-821-4_33 -
Toxics Apr 2019Phthalates (diesters of phthalic acid) are widely used as plasticizers and additives in many consumer products. Laboratory animal studies have reported the... (Review)
Review
Phthalates (diesters of phthalic acid) are widely used as plasticizers and additives in many consumer products. Laboratory animal studies have reported the endocrine-disrupting and reproductive effects of phthalates, and human exposure to this class of chemicals is a concern. Several phthalates have been recognized as substances of high concern. Human exposure to phthalates occurs mainly via dietary sources, dermal absorption, and air inhalation. Phthalates are excreted as conjugated monoesters in urine, and some phthalates, such as di-2-ethylhexyl phthalate (DEHP), undergo secondary metabolism, including oxidative transformation, prior to urinary excretion. The occurrence of phthalates and their metabolites in urine, serum, breast milk, and semen has been widely reported. Urine has been the preferred matrix in human biomonitoring studies, and concentrations on the order of several tens to hundreds of nanograms per milliliter have been reported for several phthalate metabolites. Metabolites of diethyl phthalate (DEP), dibutyl- (DBP) and diisobutyl- (DiBP) phthalates, and DEHP were the most abundant compounds measured in urine. Temporal trends in phthalate exposures varied among countries. In the United States (US), DEHP exposure has declined since 2005, whereas DiNP exposure has increased. In China, DEHP exposure has increased since 2000. For many phthalates, exposures in children are higher than those in adults. Human epidemiological studies have shown a significant association between phthalate exposures and adverse reproductive outcomes in women and men, type II diabetes and insulin resistance, overweight/obesity, allergy, and asthma. This review compiles biomonitoring studies of phthalates and exposure doses to assess health risks from phthalate exposures in populations across the globe.
PubMed: 30959800
DOI: 10.3390/toxics7020021 -
BioTechniques May 2011We have developed a quick and low-cost genomic DNA extraction protocol from yeast cells for PCR-based applications. This method does not require any enzymes, hazardous...
We have developed a quick and low-cost genomic DNA extraction protocol from yeast cells for PCR-based applications. This method does not require any enzymes, hazardous chemicals, or extreme temperatures, and is especially powerful for simultaneous analysis of a large number of samples. DNA can be efficiently extracted from different yeast species (Kluyveromyces lactis, Hansenula polymorpha, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris, and Saccharomyces cerevisiae). The protocol involves lysis of yeast colonies or cells from liquid culture in a lithium acetate (LiOAc)-SDS solution and subsequent precipitation of DNA with ethanol. Approximately 100 nanograms of total genomic DNA can be extracted from 1 × 10(7) cells. DNA extracted by this method is suitable for a variety of PCR-based applications (including colony PCR, real-time qPCR, and DNA sequencing) for amplification of DNA fragments of ≤ 3500 bp.
Topics: Acetates; Candida albicans; DNA, Fungal; Kluyveromyces; Pichia; Polymerase Chain Reaction; Saccharomyces cerevisiae; Schizosaccharomyces; Sodium Dodecyl Sulfate; Yeasts
PubMed: 21548894
DOI: 10.2144/000113672 -
Molecular Cancer Therapeutics Apr 2023Monomethyl auristatin E (MMAE) is a potent tubulin inhibitor that is used as the payload for four FDA-approved antibody-drug conjugates (ADC). Deconjugated MMAE readily...
Monomethyl auristatin E (MMAE) is a potent tubulin inhibitor that is used as the payload for four FDA-approved antibody-drug conjugates (ADC). Deconjugated MMAE readily diffuses into untargeted cells, resulting in off-target toxicity. Here, we report the development and evaluation of a humanized Fab fragment (ABC3315) that enhances the therapeutic selectivity of MMAE ADCs. ABC3315 increased the IC50 of MMAE against human cancer cell lines by > 500-fold with no impact on the cytotoxicity of MMAE ADCs, including polatuzumab vedotin (PV) and trastuzumab-vc-MMAE (TvcMMAE). Coadministration of ABC3315 did not reduce the efficacy of PV or TvcMMAE in xenograft tumor models. Coadministration of ABC3315 with 80 mg/kg TvcMMAE significantly (P < 0.0001) increased the cumulative amount of MMAE that was excreted in urine 0 to 4 days after administration from 789.4±19.0 nanograms (TvcMMAE alone) to 2625±206.8 nanograms (for mice receiving TvcMMAE with coadministration of ABC3315). Mice receiving 80 mg/kg TvcMMAE and PBS exhibited a significant drop in white blood cell counts (P = 0.025) and red blood cell counts (P = 0.0083) in comparison with control mice. No significant differences, relative to control mice, were found for white blood cell counts (P = 0.15) or for red blood cell counts (P = 0.23) for mice treated with 80 mg/kg TvcMMAE and ABC3315. Coadministration of ABC3315 with 120 mg/kg PV significantly (P = 0.045) decreased the percentage body weight loss at nadir for treated mice from 11.9%±7.0% to 4.1%±2.1%. Our results demonstrate that ABC3315, an anti-MMAE Fab fragment, decreases off-target toxicity while not decreasing antitumor efficacy, increasing the therapeutic window of MMAE ADCs.
Topics: Humans; Animals; Mice; Immunoconjugates; Oligopeptides; Trastuzumab; Disease Models, Animal; Therapeutic Index; Cell Line, Tumor; Xenograft Model Antitumor Assays
PubMed: 36723609
DOI: 10.1158/1535-7163.MCT-22-0440 -
Astrobiology Dec 2022For exploring nearby stars, let us consider the challenges of a picogram- to nanogram-scale probe to land, replicate, and produce a communications module based on...
For exploring nearby stars, let us consider the challenges of a picogram- to nanogram-scale probe to land, replicate, and produce a communications module based on biominerals at the destination. A billion such probes could be launched for similar cost as a single gram-scale probe. One design is a highly reflective light sail, traveling a long straight line toward the gravitational well of a destination star, and then photo-deflected to the closest nonluminous mass-ideally a planet or moon with exposed liquid water.
PubMed: 36475966
DOI: 10.1089/ast.2022.0008 -
Environmental Chemistry Letters 2016Many analytical techniques have been used to monitor environmental pollutants. But most techniques are not capable to detect pollutants at nanogram levels. Hence, under... (Review)
Review
Many analytical techniques have been used to monitor environmental pollutants. But most techniques are not capable to detect pollutants at nanogram levels. Hence, under such conditions, absence of pollutants is often assumed, whereas pollutants are in fact present at low but undetectable concentrations. Detection at low levels may be done by nano-capillary electrophoresis, also named microchip electrophoresis. Here, we review the analysis of pollutants by nano-capillary electrophoresis. We present instrumentations, applications, optimizations and separation mechanisms. We discuss the analysis of metal ions, pesticides, polycyclic aromatic hydrocarbons, explosives, viruses, bacteria and other contaminants. Detectors include ultraviolet-visible, fluorescent, conductivity, atomic absorption spectroscopy, refractive index, atomic fluorescence spectrometry, atomic emission spectroscopy, inductively coupled plasma, inductively coupled plasma-mass spectrometry, mass spectrometry, time-of-flight mass spectrometry and nuclear magnetic resonance. Detection limits ranged from nanogram to picogram levels.
PubMed: 32214934
DOI: 10.1007/s10311-015-0547-x -
Journal of Pain Research 2020Ceruletide (CRL) is a decapeptide, originating from the skin of a tropical frog, and is many times more potent that cholecystokinin (CCK) in a number of assays. The... (Review)
Review
Ceruletide (CRL) is a decapeptide, originating from the skin of a tropical frog, and is many times more potent that cholecystokinin (CCK) in a number of assays. The compound was first isolated and characterized around 50 years ago, and its analgesic properties were subsequently identified. Since the 1980s it has been available in the clinic as a parenteral solution and is used as a diagnostic tool to characterize pancreas and gall bladder malfunctions. Its analgesic properties were evaluated in a number of indications: cancer pain, burns, colic pains and migraine. Preclinically, CRL reduces pain in low microgram dose range and promotes clear and long-lasting analgesic activity in nanograms when applied centrally. CCK is amongst the most widely expressed neuropeptides in the brain. CCK-induced analgesic effects in response to persistent and inflammatory pain have recently been associated with CCK2 receptor signaling. CRL, a potent CCK agonist, might be worthwhile to rediscover as a putative analgesic drug and could represent a potential analgesic intrathecal strategy to patients with cancer-related pain.
PubMed: 32021401
DOI: 10.2147/JPR.S232714 -
Pharmaceutics Aug 2019Bone morphogenetic protein-2 (BMP-2) is a potent growth factor affecting bone formation. While recombinant human BMP-2 (rhBMP-2) has been commercially available in cases... (Review)
Review
Bone morphogenetic protein-2 (BMP-2) is a potent growth factor affecting bone formation. While recombinant human BMP-2 (rhBMP-2) has been commercially available in cases of non-union fracture and spinal fusion in orthopaedics, it has also been applied to improve bone regeneration in challenging cases requiring dental implant treatment. However, complications related to an initially high dosage for maintaining an effective physiological concentration at the defect site have been reported, although an effective and safe rhBMP-2 dosage for bone regeneration has not yet been determined. In contrast to protein delivery, BMP-2 gene transfer into the defect site induces BMP-2 synthesis in vivo and leads to secretion for weeks to months, depending on the vector, at a concentration of nanograms per milliliter. BMP-2 gene delivery is advantageous for bone wound healing process in terms of dosage and duration. However, safety concerns related to viral vectors are one of the hurdles that need to be overcome for gene delivery to be used in clinical practice. Recently, commercially available gene therapy has been introduced in orthopedics, and clinical trials in dentistry have been ongoing. This review examines the application of BMP-2 gene therapy for bone regeneration in the oral and maxillofacial regions and discusses future perspectives of BMP-2 gene therapy in dentistry.
PubMed: 31387267
DOI: 10.3390/pharmaceutics11080393 -
BMC Biology Oct 2022Shotgun metagenomic sequencing has greatly expanded the understanding of microbial communities in various biological niches. However, it is still challenging to...
BACKGROUND
Shotgun metagenomic sequencing has greatly expanded the understanding of microbial communities in various biological niches. However, it is still challenging to efficiently convert sub-nanogram DNA to high-quality metagenomic libraries and obtain high-fidelity data, hindering the exploration of niches with low microbial biomass.
RESULTS
To cope with this challenge comprehensively, we evaluated the performance of various library preparation methods on 0.5 pg-5 ng synthetic microbial community DNA, characterized contaminants, and further applied different in silico decontamination methods. First, we discovered that whole genome amplification prior to library construction led to worse outcomes than preparing libraries directly. Among different non-WGA-based library preparation methods, we found the endonuclease-based method being generally good for different amounts of template and the tagmentation-based method showing specific advantages with 0.5 pg template, based on evaluation metrics including fidelity, proportion of designated reads, and reproducibility. The load of contaminating DNA introduced by library preparation varied from 0.01 to 15.59 pg for different kits and accounted for 0.05 to 45.97% of total reads. A considerable fraction of the contaminating reads were mapped to human commensal and pathogenic microbes, thus potentially leading to erroneous conclusions in human microbiome studies. Furthermore, the best performing in silico decontamination method in our evaluation, Decontam-either, was capable of recovering the real microbial community from libraries where contaminants accounted for less than 10% of total reads, but not from libraries with heavy and highly varied contaminants.
CONCLUSIONS
This study demonstrates that high-quality metagenomic data can be obtained from samples with sub-nanogram microbial DNA by combining appropriate library preparation and in silico decontamination methods and provides a general reference for method selection for samples with varying microbial biomass.
Topics: DNA; Decontamination; Endonucleases; Gene Library; High-Throughput Nucleotide Sequencing; Humans; Metagenomics; Reproducibility of Results; Sequence Analysis, DNA
PubMed: 36209213
DOI: 10.1186/s12915-022-01418-9 -
Nanomaterials (Basel, Switzerland) Sep 2023Micro- and nanoplastics are emerging pollutants with a concerning persistence in the environment. Research into their environmental impact requires addressing challenges...
Micro- and nanoplastics are emerging pollutants with a concerning persistence in the environment. Research into their environmental impact requires addressing challenges related to sensitively and selectively detecting them in complex ecological media. One solution with great potential for alleviating these issues is using radiolabeling strategies. Here, we report the successful introduction of a Cu radiotracer into common microplastics, namely polyethylene, polyethylene terephthalate, polystyrene, polyamide, and polyvinylidene dichloride, which allows the sensitive detection of mere nanograms of substance. Utilizing a Hansen Solubility Parameter screening, we developed a swelling and in-diffusion process for tetraphenylporphyrin-complexed Cu, which permits one-pot labeling of polymer particles.
PubMed: 37836327
DOI: 10.3390/nano13192687