Detection of superoxide radical in all biological systems by Thin Layer Chromatography.

The study presents a new method that detects O2•-, via quantification of 2-hydroxyethidium (2-ΟΗ-Ε+) as low as ∼30 fmoles by High-Performance Thin Layer Chromatography (HPTLC). The method isolates 2-ΟΗ-Ε+ after its extraction by the anionic detergent SDS (at 18-fold higher than its CMC) together with certain organic/inorganic reagents, and its HPTLC-separation from di-ethidium (di-Ε+) and ethidium (Ε+). Quantification of 2-OH-E+ is based on its ex/em maxima at 290/540 nm, and of di-E+ and E+ at 295/545 nm. The major innovations of the present method are the development of protocols for (i) efficient extraction (by SDS) and (ii) sensitive quantification (by HPTLC) for 2-OH-E+ (as well as di-E+ and E+) from most biological systems (animals, plants, cells, subcellular compartments, fluids). The method extracts 2-ΟΗ-Ε+ (by neutralizing the strong binding between its quaternary N+ and negatively charged sites on phospholipids, DNA etc) together with free HE, while protects both from biological oxidases, and also extracts/quantifies total proteins (hydrophilic and hydrophobic) for expressing O2•- levels per protein quantity. The method also uses SDS (at 80-fold lower than its CMC) to extract/remove/wash 2-ΟΗ-Ε+ from cell/organelle exterior membrane sites, for more accurate internal content quantification. The new method is applied on indicative biological systems: (1) artificially stressed (mouse organs and liver mitochondria and nuclei, ±exposed to paraquat, a known O2•- generator), and (2) physiologically stressed (cauliflower plant, exposed to light/dark).

Isolation of autophagic fractions from mouse liver for biochemical analyses.

Isolation of autophagosomes, autolysosomes, and lysosomes allows mechanistic studies into the pathophysiology of autophagy-a lysosomal quality control pathway. Here, we outline a Nycodenz density gradient ultracentrifugation approach for high-yield isolation of autophagic fractions from mouse liver. These fractions can be used for immunoblotting, transmission electron microscopy, and proteomic and lipidomic analyses. For complete details on the use and execution of this protocol, please refer to Toledo et al. (2018).

Fluorescent Peptide Biosensors for Probing CDK Kinase Activity in Cell Extracts.

Fluorescent biosensors can report on the relative abundance, activity, or conformation of biomolecules and analytes through changes in fluorescence emission. A wide variety of genetically-encoded and synthetic biosensors have been developed to monitor protein kinase activity. We have focused on the design, engineering and characterization of fluorescent peptide biosensors of cyclin-dependent kinases (CDKs) that constitute attractive cancer biomarkers and pharmacological targets. In this chapter, we describe the CDKACT fluorescent peptide biosensor technology and its application to assess the relative kinase activity of CDKs in vitro, either using recombinant proteins or cell extracts as a more complex source of kinase. This technology offers a straightforward means of comparing CDK activity in different cell lines and evaluating the specific impact of treatments intended to target kinase activity in a physiologically relevant environment.

Cervidins A-D: Novel Glycine Conjugated Fatty Acids from the Tarsal Gland of Male Whitetail Deer, Odocoileus virginianus.

Sexually mature male deer are known to rub-urinate, a process where urine is deposited on the tarsal gland. The resulting mixture of compounds from urine and secretions from the tarsal gland are used to signal sex, age, maturation status, and other information at close distance. We examined the difference in metabolites of tarsal gland extracts from male and female whitetail deer, Odocoileus virginianus, harvested during the mating season. Using NMR spectroscopy and high-pressure liquid chromatography linked to high resolution mass spectrometry (HPLC/HR-MS) we identified a homologous series of four male-specific compounds. The compounds are novel glycine conjugates of 10-hydroxy-6,9-oxido fatty acids, which we term cervidins A-D. Cervidins were deemed to possess the absolute configuration 6S,9R,10R through comparison of their spectroscopic data with those of known compounds. In addition, cholesterol 3-sulfate and 3-(3-hydroxyphenyl)-propanoic acid were found to be present in the extracts. Our results clearly demonstrate the diversity of potential semiochemicals contained in the mammalian integument.

Assessment of variation of inline cartridge extraction mass spectra.

Recently, mass-spectrometry methods show its utility in tumor boundary location. The effect of differences between research and clinical protocols such as low- and high-resolution measurements and sample storage have to be understood and taken into account to transfer methods from bench to bedside. In this study, we demonstrate a simple way to compare mass spectra obtained by different experimental protocols, assess its quality, and check for the presence of outliers and batch effect in the dataset. We compare the mass spectra of both fresh and frozen-thawed astrocytic brain tumor samples obtained with the inline cartridge extraction prior to electrospray ionization. Our results reveal the importance of both positive and negative ion mode mass spectrometry for getting reliable information about sample diversity. We show that positive mode highlights the difference between protocols of mass spectra measurement, such as fresh and frozen-thawed samples, whereas negative mode better characterizes the histological difference between samples. We also show how the use of similarity spectrum matrix helps to identify the proper choice of the measurement parameters, so data collection would be kept reliable, and analysis would be correct and meaningful.

Bioactive potential of endophytic fungus Chaetomium globosum and GC-MS analysis of its responsible components.

The recent exploration of various medicinal plants for bioactive potential has led to the growing interest to explore their endophytes for such bioactive potential which may turn out to be better option than the plants. In the present study, Chaetomium globosum, an endophytic fungus isolated from Moringa oleifera Lam has been explored for its various biological activities. The chloroformic extract of C. globosum showed good antimutagenicity against the reactive carcinogenic mutagen, 2-aminofluorene (2-AF) in Ames test. The antiproliferative activity against various cell lines such as HCT-15, HeLa and U87-MG was found to be dose dependent and the viability reduced to 9.26%, 15.7% and 16.3%, respectively. Further, the chloroformic fungal extract was investigated for free radical scavenging activity using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethyl-benzthiazolin-6-sulfonic acid) assay which showed the IC50 value of 45.16 µg/ml and 50.55 µg/ml, respectively. The fungal extract also showed good ferric reducing power. Total phenolic and flavonoid content was found to be in linear relationship with the antioxidant potential of the fungal extract. High performance liquid chromatography showed the presence of phenolics which may help to combat the free radicals. The presence of various bioactive compounds was analysed by GC-MS which endorsed Chaetomium globosum to be a promising candidate for drug development.

Development of ion pairing LC-MS/MS method for itaconate and cis-aconitate in cell extract and cell media.

Accumulation of Immune Responsive Gene 1(IRG1) in macrophage induced by lipopolysaccharide (LPS) and interferon gamma (IFN-γ) leads to production of itaconate by decarboxylation of cis-aconitate. The biology associated with IRG1 and itaconate is not fully understood. A rapid and sensitive method for measurement of itaconate will benefit the study of IRG1 biology. Multiple HPLC and derivatization methods were tested. An ion pairing LC-MS/MS method using tributylamine/formic acid as ion pairing agents and a HypercarbTM guard column we proposed demonstrated better peak shape and better sensitivity for itaconate. The current protocol allows baseline separation of itaconate, citraconate, and cis-aconitate without derivatization and direct analysis of analytes in 80% methanol/water solution to avoid the dry-down step. It provides the limit of quantitation (LOQ) of 30 pg itaconate on column with a 4.5-minute run time. This method is validated for measurement of itaconate and cis-aconitate in RAW264.7 cell extract and cell media in a 96-well plate format. We applied this method to successfully measure the increase of itaconate and the decrease of cis-aconitate in RAW cell extract and cell media after LPS/IFN-γ treatment.

A comparative study of extraction techniques for maximum recovery of ß-galactosidase from the yogurt bacterium Lactobacillus delbrueckii ssp. bulgaricus.

The study reported in this research communication evaluates the chemical (solvents) and mechanical (sonication, bead-beater) extraction methods to determine the maximum recovery of ß-galactosidase from L. bulgaricus spp. Among all extraction techniques, sonication-assisted extraction yielded the highest amounts of enzyme activity (between 1892-2156 Miller Units) in cell-free extract (supernatant). Interestingly, solvent extracted enzyme activities were found to be very low (between 83-153 Miller Units) in supernatant. SDS-polyacrylamide gel electrophoresis and the total protein determination showed that mechanical methods can completely lyse the cells. Our results thus demonstrated that the mechanical extraction method of sonication is the best one for recovering the maximum amount of lactase from L. bulgaricus strains.

The origin identification method for crude drugs derived from arthropods and annelids using molecular biological techniques.

We evaluated whether the origins of crude drugs derived from arthropods and annelids could be identified using molecular biological techniques. DNA was extracted from 20 crude drugs prepared from different animals using a commercial kit with added phenol treatment. The target regions used to identify origin were the mitochondrial 16S ribosomal RNA (rRNA), 12S rRNA, and cytochrome oxidase subunit I (COI) gene regions. Extracted DNA was amplified by polymerase chain reaction, and then sequenced by the Sanger method. The aligned sequences were compared with all available sequences using BLAST to estimate the origins of the crude drugs. The origin of crude drugs used in this study could be estimated using this method. The COI region was the best for identifying origin among three regions examined, based on the success rate of PCR amplification and analysis. Moreover, the 12S rRNA region was also useful for origin identification, with the exception of the earthworm. However, the origin of some crude drugs could not be strictly identified due to matches to various species in all three regions. One likely cause was that the species of origin of a crude drug has not been registered in DNA databases. We found that even the same crude drug from the same pharmaceutical company had different origins by production lot or import source country. Therefore, this method is useful not only for DNA-based origin identification but also quality control of production lots.

Site- and Structure-Specific Quantitative N-Glycoproteomics Using RPLC-pentaHILIC Separation and the Intact N-Glycopeptide Search Engine GPSeeker.

Site- and structure-specific quantitative N-glycoproteomics characterization of differentially expressed N-glycosylation at the intact N-glycopeptide level with distinct chromatographic separation and structure-specific fragment ions has become possible with the recent development of RPLC-pentaHILIC 2DLC separation and use of the intact N-glycopeptide search engine GPSeeker. Here we provide a detailed protocol for this GPSeeker-centered structure-specific isotopic-labeling quantitative N-glycoproteomics pipeline. The protocols include sample preparation of a 1:1 mixture of light (-CH3 )2 and heavy (-13 CD2 H)2 dimethylated intact N-glycopeptides from LO2 and HepG2 cells, RPLC-pentaHILIC 2DLC separation of the mixture, intact N-glycopeptide database search and identification using GPSeeker, and quantitation of differentially expressed intact N-glycopeptides using the quantitation module GPSeekerQuan. © 2019 by John Wiley & Sons, Inc.

A simple mix-and-read bacteria detection system based on a DNAzyme and a molecular beacon.

Here, we describe a simple mix-and-read method for the detection of specific bacterial strains that uses a DNAzyme and a molecular beacon to generate a signal. We have greatly improved upon the previously described DNAzyme-based bacteria detection method by eliminating a tedious preparation step while maintaining detection sensitivity.

Peroxisome protein import recapitulated in Xenopus egg extracts.

Peroxisomes import their luminal proteins from the cytosol. Most substrates contain a C-terminal Ser-Lys-Leu (SKL) sequence that is recognized by the receptor Pex5. Pex5 binds to peroxisomes via a docking complex containing Pex14, and recycles back into the cytosol following its mono-ubiquitination at a conserved Cys residue. The mechanism of peroxisome protein import remains incompletely understood. Here, we developed an in vitro import system based on Xenopus egg extracts. Import is dependent on the SKL motif in the substrate and on the presence of Pex5 and Pex14, and is sustained by ATP hydrolysis. A protein lacking an SKL sequence can be coimported, providing strong evidence for import of a folded protein. The conserved cysteine in Pex5 is not essential for import or to clear import sites for subsequent rounds of translocation. This new in vitro assay will be useful for further dissecting the mechanism of peroxisome protein import.

Response of Paracoccidioides lutzii to the antifungal camphene thiosemicarbazide determined by proteomic analysis.

AIM: To perform the proteomic profile of Paracoccidioides lutzii after treatment with the compound camphene thiosemicarbazide (TSC-C) in order to study its mode of action. METHODS: Proteomic analysis was carried out after cells were incubated with TSC-C in a subinhibitory concentration. Validation of the proteomic results comprised the azocasein assay, western blot and determination of the susceptibility of a mutant to the compound. RESULTS: Proteins related to metabolism, energy and protein fate were regulated after treatment. In addition, TSC-C reduces the proteolytic activity of the protein extract similarly to different types of protease inhibitors. CONCLUSION: TSC-C showed encouraging antifungal activity, working as a protease inhibitor and downregulating important pathways impairing the ability of the fungi cells to produce important precursors.

A gel-free proteomic analysis of Taenia solium and Taenia crassiceps cysticerci vesicular extracts.

The taeniasis/cysticercosis complex is a zoonosis caused by the presence of the parasite Taenia solium in humans. It is considered a neglected disease that causes serious public health and economic problems in developing countries. In humans, the most common locations for the larval form are the skeletal muscles, ocular system, and the central nervous system, which is the most clinically important. Several glycoproteins of T. solium and Taenia crassiceps cysticerci have been characterized and studied for their use in the immunodiagnosis of neurocysticercosis and/or the development of synthetic or recombinant vaccines against cysticercosis. The aim of this study was to perform a gel-free shotgun proteomic analysis to identify saline vesicular extract (SVE) proteins of T. solium and T. crassiceps cysticerci. After solubilization of the SVE with and without surfactant reagent and in-solution digestion, the proteins were analyzed by LC-MS/MS. Use of a surfactant resulted in a significantly higher number of proteins that were able to be identified by LC-MS/MS. Novel proteins were identified in T. solium and T. crassiceps SVE. The qualitative analysis revealed a total of 79 proteins in the Taenia species: 29 in T. solium alone, 11 in T. crassiceps alone, and 39 in both. These results are an important contribution to support future investigations and for establishing a Taenia proteomic profile to study candidate biomarkers involved in the diagnosis or pathogenesis of neurocysticercosis.

Screening of intact yeasts and cell extracts to reduce Scrapie prions during biotransformation of food waste.

Yeasts can be used to convert organic food wastes to protein-rich animal feed in order to recapture nutrients. However, the reuse of animal-derived waste poses a risk for the transmission of infectious prions that can cause neurodegeneration and fatality in humans and animals. The aim of this study was to investigate the ability of yeasts to reduce prion activity during the biotransformation of waste substrates-thereby becoming a biosafety hurdle in such a circular food system. During pre-screening, 30 yeast isolates were spiked with Classical Scrapie prions and incubated for 72 h in casein substrate, as a waste substitute. Based on reduced Scrapie seeding activity, waste biotransformation and protease activities, intact cells and cell extracts of 10 yeasts were further tested. Prion analysis showed that five yeast species reduced Scrapie seeding activity by approximately 1 log10 or 90%. Cryptococcus laurentii showed the most potential to reduce prion activity since both intact and extracted cells reduced Scrapie by 1 log10 and achieved the highest protease activity. These results show that select forms of yeast can act as a prion hurdle during the biotransformation of waste. However, the limited ability of yeasts to reduce prion activity warrants caution as a sole barrier to transmission as higher log reductions are needed before using waste-cultured yeast in circular food systems.

METLIN: A Technology Platform for Identifying Knowns and Unknowns.

METLIN originated as a database to characterize known metabolites and has since expanded into a technology platform for the identification of known and unknown metabolites and other chemical entities. Through this effort it has become a comprehensive resource containing over 1 million molecules including lipids, amino acids, carbohydrates, toxins, small peptides, and natural products, among other classes. METLIN's high-resolution tandem mass spectrometry (MS/MS) database, which plays a key role in the identification process, has data generated from both reference standards and their labeled stable isotope analogues, facilitated by METLIN-guided analysis of isotope-labeled microorganisms. The MS/MS data, coupled with the fragment similarity search function, expand the tool's capabilities into the identification of unknowns. Fragment similarity search is performed independent of the precursor mass, relying solely on the fragment ions to identify similar structures within the database. Stable isotope data also facilitate characterization by coupling the similarity search output with the isotopic m/ z shifts. Examples of both are demonstrated here with the characterization of four previously unknown metabolites. METLIN also now features in silico MS/MS data, which has been made possible through the creation of algorithms trained on METLIN's MS/MS data from both standards and their isotope analogues. With these informatic and experimental data features, METLIN is being designed to address the characterization of known and unknown molecules.

Autocatalytic microtubule nucleation determines the size and mass of Xenopus laevis egg extract spindles.

Regulation of size and growth is a fundamental problem in biology. A prominent example is the formation of the mitotic spindle, where protein concentration gradients around chromosomes are thought to regulate spindle growth by controlling microtubule nucleation. Previous evidence suggests that microtubules nucleate throughout the spindle structure. However, the mechanisms underlying microtubule nucleation and its spatial regulation are still unclear. Here, we developed an assay based on laser ablation to directly probe microtubule nucleation events in Xenopus laevis egg extracts. Combining this method with theory and quantitative microscopy, we show that the size of a spindle is controlled by autocatalytic growth of microtubules, driven by microtubule-stimulated microtubule nucleation. The autocatalytic activity of this nucleation system is spatially regulated by the limiting amounts of active microtubule nucleators, which decrease with distance from the chromosomes. This mechanism provides an upper limit to spindle size even when resources are not limiting.

Normalized Screening of Protein Engineering Libraries by Split-GFP Crude Cell Extract Quantification.

The different expression level and solubility showed by each protein variant represents an important challenge during screening campaigns: Usually, the total activity measurement constitutes the only criterion for identifying improved variants. This hampers the chances of finding interesting mutants, especially if the aim is to improve activity: On the one hand, interesting but poorly soluble variants will remain undetectable. On the other hand, a mutation might not increase activity, but improve expression level or solubility. The split-GFP technology offers an affordable and technically simple manner for overcoming that constraints, making protein library screening more efficient through the normalization of the detected enzymatic activities in relation to the quantified protein contents responsible for them.

A dual-colored ratiometric-fluorescent oligonucleotide probe for the detection of human telomerase RNA in cell extracts.

Human telomerase RNA (hTR), which is one component of telomerase, was deemed to be a biomarker to monitor tumor cells due to its different expression levels in tumor cells and normal somatic cells. Thus far, plentiful fluorescent probes have been designed to investigate nucleic acids. However, most of them are limited since they are time-consuming, require professional operators and even result in false positive signals in the cellular environment. Herein, we report a dual-colored ratiometric-fluorescent oligonucleotide probe to achieve the reliable detection of human telomerase RNA in cell extracts. The probe is constructed using a dual-labeled fluorescent oligonucleotide hybridized with target-complemented Dabcyl-labeled oligonucleotide. In the presence of the target, the dual-labeled fluorescent oligonucleotide translates into a hairpin structure, which leads to the generation of the fluorescence resonance energy transfer (FRET) phenomenon under UV excitation. Compared to conventional methods, this strategy could effectively avoid false positive signals, and it not only possesses the advantages of simplicity and high specificity but also has the merits of signal stability and distinguishable color variation. Moreover, the quantitative assay of hTR would have a far-reaching impact on the telomerase mechanism and even tumor diagnosis research.

Quantifying the cellular NAD+ metabolome using a tandem liquid chromatography mass spectrometry approach.

INTRODUCTION: Nicotinamide adenine dinucleotide (NAD+) is an essential pyridine nucleotide that serves as a key hydride transfer coenzyme for several oxidoreductases. It is also the substrate for intracellular secondary messenger signalling by CD38 glycohydrolases, DNA repair by poly(adenosine diphosphate ribose) polymerase, and epigenetic regulation of gene expression by a class of histone deacetylase enzymes known as sirtuins. The measurement of NAD+ and its related metabolites (hereafter, the NAD+ metabolome) represents an important indicator of cellular function. OBJECTIVES: A study was performed to develop a sensitive, selective, robust, reproducible, and rapid method for the concurrent quantitative determination of intracellular levels of the NAD+ metabolome in glial and oocyte cell extracts using liquid chromatography coupled to mass spectrometry (LC/MS/MS). METHODS: The metabolites were separated on a versatile amino column using a dual HILIC-RP gradient with heated electrospray (HESI) tandem mass spectrometry detection in mixed polarity multiple reaction monitoring mode. RESULTS: Quantification of 17 metabolites in the NAD+ metabolome in U251 human astroglioma cells could be achieved. Changes in NAD+ metabolism in U251 cell line, and murine oocytes under different culture conditions were also investigated. CONCLUSION: This method can be used as a sensitive profiling tool, tailoring chromatography for metabolites that express significant pathophysiological changes in several disease conditions and is indispensable for targeted analysis.