生物化学

Molybdenum (Mo) and tungsten (W) are elements that are utilized in biological systems. They are typically incorporated into the catalytic sites of enzymes coordinated to an organic pyranopterin cofactor; Mo may also be present in the form of a FeMo cofactor. While Mo is used by all branches of life, only a few microbes are able to utilize W. In order to study Mo- and W-dependent enzymes, it is important to be able to measure Mo and W in biological samples. Methods for determining Mo and W content in biological samples currently involve expensive and time-consuming processes like inductively coupled plasma mass spectrometry (ICP-MS) and chelation ion chromatography. There are less intensive colorimetric methods for measuring W in abiotic samples, but these have not been adapted to biological samples like cytosolic extracts and purified proteins. Herein, we developed a colorimetric assay based on the complexation of quercetin to molybdate (MoO₄^2-) or tungstate (WO₄^2-), the oxyanion forms of Mo and W that readily form in denatured biological samples. In the assay, the absorbance of quercetin is redshifted proportionally to the concentration of tungsten or molybdenum, which can be measured spectrophotometrically. This protocol provides a rapid method for screening biological samples for both Mo and W, although it does not distinguish between them.

Many small molecules require derivatization to increase their volatility and to be amenable to gas chromatographic (GC) separation. Derivatization is usually time-consuming, and typical batch-wise procedures increase sample variability. Sequential automation of derivatization via robotic liquid handling enables the overlapping of sample preparation and analysis, maximizing time efficiency and minimizing variability. Herein, a protocol for the fully automated, two-stage derivatization of human blood–based samples in line with GC–[Orbitrap] mass spectrometry (MS)-based metabolomics is described. The protocol delivers a sample-to-sample runtime of 31 min, being suitable for better throughput routine metabolomic analysis.

Free radicals, including reactive oxygen species (ROS) and reactive nitrogen species (RNS), induce oxidative stress. This stress plays crucial roles in cellular signaling, stress response, and disease progression, making the quantification of free radicals essential for understanding oxidative stress mechanisms. Here, we present a high-throughput fluorescence-based protocol for measuring the presence of total free radicals, including ROS and RNS, in the whole adult Drosophila melanogaster (fruit fly). The protocol involves homogenizing whole adult flies in PBS and treating only the supernatant of the lysate with dichlorodihydrofluorescein-DiOxyQ (DCFH-DiOxyQ), which then converts into a fluorescent molecule, dichlorofluorescein (DCF), upon reacting with free radicals. The level of fluorescence is directly proportional to the amount of free radicals present in the sample. This protocol offers simplicity, scalability, and adaptability, making it ideal for studying oxidative stress in the model organism Drosophila and its different tissues under different dietary regimes, environmental stresses, genetic mutations, or pharmacological treatments. It is to be noted that the protocol uses a kit from Abcam, which has been used to measure free radicals in mice, rats, human blood, and cell lines. It can also be applied to biofluids, culture supernatants, and cell lysates, making it suitable for a wide range of sample types beyond whole organisms or tissues. However, due to our research focus and expertise, here we describe a detailed protocol to measure free radicals responsible for inducing oxidative stress only in fruit flies.

Cyclic diadenosine monophosphate (c-di-AMP) is a recently discovered second messenger that modulates several signal transduction pathways in bacterial and host cells. Besides the bacterial system, c-di-AMP signaling is also connected with the host cytoplasmic surveillance pathways (CSP) that induce type-I IFN responses through STING-mediated pathways. Additionally, c-di-AMP demonstrates potent adjuvant properties, particularly when administered alongside the Bacillus Calmette–Guérin (BCG) vaccine through mucosal routes. Because of its pivotal role in bacterial signaling and host immune response, this molecule has garnered significant interest from the pharmaceutical industry. This protocol outlines the quantification of c-di-AMP by an HPLC-based assay to enumerate the activity of c-di-AMP synthase from Mycobacterium smegmatis. The following protocol is designed to be generic, enabling the study of c-di-AMP synthase activity from other bacterial species. However, modifications may be required depending on the specific activity of c-di-AMP synthase from different bacterial sources.

This protocol outlines the use of the previously described sodium hypochlorite extraction method for estimating the accumulation of polyhydroxybutyrate (PHB) in bacteria. Sodium hypochlorite (NaClO) is widely used for PHB extraction as it oxidizes most components of the cells except PHB. We assessed the feasibility of using NaClO extraction for the estimation of PHB accumulation in bacterial cells (expressed as a percentage w/w). This allowed us to use a simple spectrophotometric measurement of the turbidity of the PHB extracted by NaClO as a semiquantitative estimation of PHB accumulation in the marine microorganisms Halomonas titanicae KHS3, Alteromonas sp., and Cobetia sp. However, this fast and easy protocol could be used for any bacterial species as long as some details are considered. This estimation exhibited a good correlation with the accumulation measured as dry cell weight or even with the accumulation measured by crotonic acid and HPLC quantifications. The key advantage of this protocol is how fast it allows an estimation of PHB accumulation in Halomonas, Alteromonas, and Cobetia cultures (results are available in 50 min), enabling the identification of the appropriate moment to harvest cells for further extraction, polymer characterization, and accurate quantification using more reliable and time-consuming methods. This protocol is very useful during bacterial cultivation for a quick evaluation of PHA accumulation without requiring (i) large volumes of cultures, (ii) a long time for analysis compared to dry cell weight, (iii) preparation of standard curves with sulfuric acid hydrolysis for crotonic acid quantification, or (iv) specific equipment and/or technical services for HPLC quantification.

Neuroscience incorporates manipulating neuronal circuitry to enhance the understanding of intricate brain functions. An effective strategy to attain this objective entails utilizing viral vectors to induce varied gene expression by delivering transgenes into brain cells. Here, we combine the use of transgenic mice, neonatal transduction with adeno-associated viral constructs harboring inhibitory designer receptor exclusively activated by designer drug (DREADD) gene, and the DREADD agonist clozapine N-oxide (CNO). In this way, a chemogenetic approach is employed to suppress neuronal activity in the region of interest during a critical developmental window, with subsequent investigation into its effects on the neuronal circuitry in adulthood.

Enzyme immobilization offers a number of advantages that improve biocatalysis; however, finding a proper way to immobilize enzymes is often a challenging task. Implanting enzymes in metal–organic frameworks (MOFs) via co-crystallization, also known as biomineralization, provides enhanced reusability and stability with minimal perturbation and substrate selectivity to the enzyme. Currently, there are limited metal–ligand combinations with a proper protocol guiding the experimental procedures. We have recently explored 10 combinations that allow custom immobilization of enzymes according to enzyme stability and activity in different metals/ligands. Here, as a follow-up of that work, we present a protocol for how to carry out custom immobilization of enzymes using the available combinations of metal ions and ligands. Detailed procedures to prepare metal ions, ligands, and enzymes for their co-crystallization, together with characterization and assessment, are discussed. Precautions for each experimental step and result analysis are highlighted as well. This protocol is important for enzyme immobilization in various research and industrial fields.

Key features

• A wide selection of metal ions and ligands allows for the immobilization of enzymes in metal–organic frameworks (MOFs) via co-crystallization.

• Step-by-step enzyme immobilization procedure via co-crystallization of metal ions, organic linkers, and enzymes.

• Practical considerations and experimental conditions to synthesize the enzyme@MOF biocomposites are discussed.

• The demonstrated method can be generalized to immobilize other enzymes and find other metal ion/ligand combinations to form MOFs in water and host enzymes.

Graphical overview

Ion homeostasis is a fundamental regulator of cellular processes and depends upon lipid membranes, which function as ion permeability barriers. Ionophores facilitate ion transport across cell membranes and offer a way to manipulate cellular ion composition. Here, we describe a calcein quenching assay based on large unilamellar vesicles that we used to evaluate divalent cation transport of the ionophore 4-Br-A23187. This assay can be used to study metal transport by ionophores and membrane proteins, under well-defined conditions.

Graphical abstract:

Depending on its local concentration, hydrogen peroxide (H₂O₂) can serve as a cellular signaling molecule but can also cause damage to biomolecules. The levels of H₂O₂ are influenced by the activity of its generator sites, local antioxidative systems, and the metabolic state of the cell. To study and understand the role of H₂O₂ in cellular signaling, it is crucial to assess its dynamics with high spatiotemporal resolution. Measuring these subcellular H₂O₂ dynamics has been challenging. However, with the introduction of the super sensitive pH-independent genetically encoded fluorescent H₂O₂ sensor HyPer7, many limitations of previous measurement approaches could be overcome. Here, we describe a method to measure local H₂O₂ dynamics in intact human cells, utilizing the HyPer7 sensor in combination with a microscopic multi-mode microplate reader.

Graphical abstract:

Overview of HyPer7 sensor function and measurement results.

The ascorbate peroxidase (APX) is a widely distributed antioxidant enzyme. It differs from catalase and other peroxidases in that it scavenges/reduces reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂) to water using reduced ascorbate as the electron donor. It is advantageous over other similar antioxidant enzymes in scavenging ROS since ascorbate may react with superoxide, singlet oxygen, and hydroxyl radical, in addition to reacting with H₂O₂. The estimation of its activity is helpful to analyze the level of oxidative stress in living systems under stressful conditions. The present protocol was performed to analyze the impact of heavy metal chromium (Cr) toxicity on sorghum plants in the form of APX enzyme activity under the application of glycine betaine (GB) and arbuscular mycorrhizal fungi (AMF) as stress ameliorators. Plant defense strategies against heavy metals toxicity involve the utilization of APX and the instigation of AMF symbiotic system, as well as their possible collaboration with one another or with the plant antioxidant system; this has been examined and discussed in literature. In this protocol, an increased APX activity was observed on underlying functions and detoxification capabilities of GB and AMF that are typically used by plants to enhance tolerance to Cr toxicity.

Graphical abstract:

Flow chart of standardized or calibrated enzyme assay with leaf samples of sorghum