A Proposed Methodology to Deal with the Impact of In Vitro Cellular Matrix on the Analytical Performances of a Targeted Metabolomic LC-HRMS Method.

Performances of metabolomic methods have been widely studied on biological matrices such as serum, plasma, and urine; but much less on in vitro cell extracts. While the impact of cell culture and sample preparation on results are well-described, the specific effect of the in vitro cellular matrix on the analytical performance remains uncertain. The aim of the present work was to study the impact of this matrix on the analytical performance of an LC-HRMS metabolomic method. For this purpose, experiments were performed on total extracts from two cell lines (MDA-MB-231 and HepaRG) using different cell numbers. Matrix effects, carryover, linearity, and variability of the method were studied. Results showed that the performances of the method depend on the nature of the endogenous metabolite, the cell number, and the nature of the cell line. These three parameters should, therefore, be considered for the processing of experiments and the interpretation of results depending on whether the study focuses on a limited number of metabolites or aims to establish a metabolic signature.

Heterologous expression of the tetracycline resistance gene tetX to enhance degradability and safety in doxycycline degradation.

Microbial remediation has the potential to inexpensively yet effectively decontaminate and restore contaminated environments, but the virulence of pathogens and risk of resistance gene transmission by microorganisms during antibiotic removal often limit its implementation. Here, a cloned tetX gene with clear evolutionary history was expressed to explore doxycycline (DOX) degradation and resistance variation during the degradation process. Phylogenetic analysis of tetX genes showed high similarity with those of pathogenic bacteria, such as Riemerella sp. and Acinetobacter sp. Successful tetX expression was performed in Escherichia coli and confirmed by SDS-PAGE and Western blot. Our results showed that 95.0 ± 1.0% of the DOX (50 mg/L) was degraded by the recombinant strain (ETD-1 with tetX) within 48 h, which was significantly higher than that for the control (38.9 ± 8.7%) and the empty plasmid bacteria (8.8 ± 5.1%) (P < 0.05). The tetX gene products in ETD-1 cell extracts also exhibited an efficient DOX degradation ability, with a degradation rate of 80.5 ± 1.2% at 168 h. Furthermore, there was no significant proliferation of the tetX resistance gene during DOX degradation (P > 0.05). The efficient and safe DOX-degrading capacity of the recombinant strain ETD-1 makes it valuable and promising for antibiotic removal in the environment.

A nanoplatform based on metal-organic frameworks and coupled exonuclease reaction for the fluorimetric determination of T4 polynucleotide kinase activity and inhibition.

A nanoplatform based on metal-organic frameworks (MOFs) and lambda exonuclease (λ exo) for the fluorimetric determination of T4 polynucleotide kinase (T4 PNK) activity and inhibition is described. Fe-MIL-88 was selected as the nanomaterial because of its significant preferential binding ability to single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA) and its quenching property. The synthesized Fe-MIL-88 was characterized by transmission electron microscope, scanning electron microscope, and X-ray photoelectron spectroscopy. In the presence of T4 PNK, FAM-labeled dsDNA (FAM-dsDNA) is phosphorylated on its 5'-terminal. λ exo then recognizes and cleaves the phosphorylated strand yielding FAM-labeled ssDNA (FAM-ssDNA). The fluorescence of the produced FAM-ssDNA is quenched due to Fe-MIL-88's absorbing on FAM-ssDNA. On the contrary, in the absence of T4 PNK, the phosphorylation and cleavage processes cannot take place. Therefore, the fluorescence of FAM-dsDNA still remains. The fluorescence intensity is detected at the maximum emission wavelength of 524 nm using the maximum excitation wavelength of 488 nm. The assay of T4 PNK based on the fluorescence quenching of FAM-ssDNA achieves a linear relationship in the range 0.01-5.0 U mL-1 with a detection limit of 0.0089 U mL-1 in buffer. The assay exhibits excellent performance for T4 PNK activity determination in a complex biological matrix. The results also reveal the ability of the assay for T4 PNK inhibitor screening. Graphical abstract Schematic presentation of a nanoplatform based on Fe-MIL-88 and coupled exonuclease reaction for the fluorimetric determination of T4 polynucleotide kinase activity. FAM-ssDNA, FAM-labeled single-stranded DNA; cDNA, complementary DNA; λ exo, lambda exonuclease;T4 PNK, T4 polynucleotide kinase.

Sensitive fluorometric determination of glutathione using fluorescent polymer dots and the dopamine-melanin nanosystem.

A bioinspired fluorometric method has been developed for the detection of glutathione (GSH) in biological fluids. It is based on the use of near-infrared fluorescent semiconducting polymer dots (P-dots) and of the dopamine (DA)-melanin nanosystem. The P-dots were prepared from poly(styrene-co-maleic anhydride), the semiconducting polymer poly[(9,9'-dioctyl-2,7-divinylenefluorenylene)-alt-2-methoxy-5-(2-ethyl-hexyloxy)-1,4-phenylene] and the fluorescent dye tetraphenylporphyrin. They have excitation/emission maxima at 458/656 nm, and this enables measurement to be performed with low autofluorescence and scattering background. DA can self-polymerize on the surface of the P-dots to yield a poly-DA coating. This coating, at weak alkaline pH values, causes the quenching of the fluorescence of the P-dots. However, the polymerization of DA is inhibited by GSH. Hence, quenching of fluorescence is prevented. This effect was used to design a fluorometric assay for GSH that has good selectivity and sensitivity. Under optimal conditions, the method has a linear response in the 0.2 to 20 µM GSH concentration range and a 60 nM detection limit. It was successfully applied to the determination of GSH in HepG2 cells and in spiked human serum. Graphical abstract Schematic representation of using a NIR fluorescent P-dots and dopamine (DA)-melanin nanohybrid as a probe for glutathione (GSH) detection. The P-dots were prepared from poly(styrene-co-maleic anhydride) (PSMA), the semiconducting polymer poly[(9,9'-dioctyl-2,7-divinylenefluorenylene)-alt-2-methoxy-5-(2-ethyl-hexyloxy)-1,4-phenylene] (PEPV) and the fluorescent dye tetraphenylporphyrin (TPP).The GSH can inhibit the dopamine self-polymerization and prevented the formation of PDA and fluorescence quenching of P-dots.

A fluorometric method for determination of the activity of T4 polynucleotide kinase by using a DNA-templated silver nanocluster probe.

The authors describe a turn-off fluorometric method for the determination of the activity of the T4 polynucleotide kinase (T4 PNK). It is based on the use of DNA-templated silver nanoclusters (AgNCs). DNA probes with terminal 5' hydroxy groups are used as substrates for DNA phosphatases. If subsequently treated with T4 PNK and Lambda exonuclease (λ exo), the AgNC DNA probes with a modified C-rich sequence and the G-rich sequence is separated. Upon their separation, the strong fluorescence (with excitation/emission maxima at 580/650 nm) that is caused by the proximity of the G-rich region and the C-rich region in the AgNCs decreases sharply. This enabled the fluorometric kinetic determination of the activity of T4 PNK. The assay is characterized by a wide linear range (from 0.01 to 12.5 U·mL-1), a low detection limit (0.01 U·mL-1) and short assay time (typically 60 min). This makes it a promising tool for use in studying processes related to DNA phosphorylation, in drug discovery and in diagnostics. Graphical abstract A turn-off fluorometric method for the determination of the activity of the T4 polynucleotide kinase (T4 PNK) has been developed. It is based on the use of DNA-templated silver nanoclusters (AgNCs). This makes it a promising tool for use in studying processes related to DNA phosphorylation, in drug discovery and in diagnostics.

Towards Understanding Protein Disorder In-Cell.

Investigating the activity and structure of cellular biochemical machinery at atomic resolution has been a point of paramount significance for understanding health and disease over the decades. The underlying molecular mechanisms are primarily studied in vitro. Nuclear magnetic resonance (NMR) is a technique that allows to look into cells and study proteins and other constituents, thanks to careful experimental design and technological advances (spectrometer sensitivity and pulse sequence design). Here we outline current applications of the technique and propose a realistic future for the field.

Measuring Molecular Diffusion in Self-Organizing Xenopus Extracts by Fluorescence Correlation Spectroscopy.

The cytoplasm is densely packed with macromolecules and organelles, displaying viscoelastic properties at various scales. How biochemical reactions function efficiently enough in a seemingly jammed environment remains elusive. Cell-free Xenopus laevis extracts represent a powerful system for investigating the biochemistry and biophysics of living systems. Here we present a protocol for characterizing macromolecular diffusion in self-organizing cytoplasmic extracts using fluorescence correlation spectroscopy (FCS), which measures the motions on a distance scale of ~200 nm. The method can also be used to characterize diffusion in the cytoplasm as it progresses through different phases of the cell cycle.

Comparative toxicology of algal cell extracts and pure cyanotoxins: insights into toxic effects and mechanisms of harmful cyanobacteria Raphidiopsis raciborskii.

Ongoing research on cyanotoxins, driven by the socioeconomic impact of harmful algal blooms, emphasizes the critical necessity of elucidating the toxicological profiles of algal cell extracts and pure toxins. This study comprehensively compares Raphidiopsis raciborskii dissolved extract (RDE) and cylindrospermopsin (CYN) based on Daphnia magna assays. Both RDE and CYN target vital organs and disrupt reproduction, development, and digestion, thereby causing acute and chronic toxicity. Disturbances in locomotion, reduced behavioral activity, and weakened swimming capability in D. magna have also been reported for both RDE and CYN, indicating the insufficiency of conventional toxicity evaluation parameters for distinguishing between the toxic effects of algal extracts and pure cyanotoxins. Additionally, chemical profiling revealed the presence of highly active tryptophan-, humic acid-, and fulvic acid-like fluorescence compounds in the RDE, along with the active constituents of CYN, within a 15-day period, demonstrating the chemical complexity and dynamics of the RDE. Transcriptomics was used to further elucidate the distinct molecular mechanisms of RDE and CYN. They act diversely in terms of cytotoxicity, involving oxidative stress and response, protein content, and energy metabolism, and demonstrate distinct modes of action in neurofunctions. In essence, this study underscores the distinct toxicity mechanisms of RDE and CYN and emphasizes the necessity for context- and objective-specific toxicity assessments, advocating nuanced approaches to evaluate the ecological and health implications of cyanotoxins, thereby contributing to the precision of environmental risk assessments.

The extracts of osteoblast developed from adipose-derived stem cell and its role in osteogenesis.

Cell-based therapy has become an achievable choice in regenerative medicines, particularly for musculoskeletal disorders. Adipose-derived stem cells (ASCs) are an outstanding resource because of their ability and functions. Nevertheless, the use of cells for treatment comes with difficulties in operation and safety. The immunological barrier is also a major limitation of cell therapy, which can lead to unexpected results. Cell-derived products, such as cell extracts, have gained a lot of attention to overcome these limitations. The goal of this study was to optimize the production of ASC-osteoblast extracts as well as their involvement in osteogenesis. The extracts were prepared using a freeze-thaw method with varying temperatures and durations. Overall, osteogenic-associated proteins and osteoinductive potential of the extracts prepared from the osteogenic-induced ASCs were assessed. Our results demonstrated that the freeze-thaw approach is practicable for cell extracts production, with minor differences in temperature and duration having no effect on protein concentration. The ASC-osteoblast extracts contain a significant level of essential specialized proteins that promote osteogenicity. Hence, the freeze-thaw method is applicable for extract preparation and ASC-osteoblast extracts may be beneficial as an optional facilitating biologics in bone anabolic treatment and bone regeneration.

Proteomic profiling of a mouse model of acute intestinal Apc deletion leads to identification of potential novel biomarkers of human colorectal cancer (CRC).

Colorectal cancer (CRC) is the fourth most common cause of cancer-related death worldwide. Accurate non-invasive screening for CRC would greatly enhance a population's health. Adenomatous polyposis coli (Apc) gene mutations commonly occur in human colorectal adenomas and carcinomas, leading to Wnt signalling pathway activation. Acute conditional transgenic deletion of Apc in murine intestinal epithelium (AhCre(+)Apc(fl)(/)(fl)) causes phenotypic changes similar to those found during colorectal tumourigenesis. This study comprised a proteomic analysis of murine small intestinal epithelial cells following acute Apc deletion to identify proteins that show altered expression during human colorectal carcinogenesis, thus identifying proteins that may prove clinically useful as blood/serum biomarkers of colorectal neoplasia. Eighty-one proteins showed significantly increased expression following iTRAQ analysis, and validation of nine of these by Ingenuity Pathaway Analysis showed they could be detected in blood or serum. Expression was assessed in AhCre(+)Apc(fl)(/)(fl) small intestinal epithelium by immunohistochemistry, western blot and quantitative real-time PCR; increased nucelolin concentrations were also detected in the serum of AhCre(+)Apc(fl)(/)(fl) and Apc(Min)(/)(+) mice by ELISA. Six proteins; heat shock 60kDa protein 1, Nucleolin, Prohibitin, Cytokeratin 18, Ribosomal protein L6 and DEAD (Asp-Glu-Ala-Asp) box polypeptide 5,were selected for further investigation. Increased expression of 4 of these was confirmed in human CRC by qPCR. In conclusion, several novel candidate biomarkers have been identified from analysis of transgenic mice in which the Apc gene was deleted in the intestinal epithelium that also showed increased expression in human CRC. Some of these warrant further investigation as potential serum-based biomarkers of human CRC.

Cryo-EM of a heterogeneous biochemical fraction elucidates multiple protein complexes from a multicellular thermophilic eukaryote.

Biomolecular complexes and their interactions govern cellular structure and function. Understanding their architecture is a prerequisite for dissecting the cell's inner workings, but their higher-order assembly is often transient and challenging for structural analysis. Here, we performed cryo-EM on a single, highly heterogeneous biochemical fraction derived from Chaetomium thermophilum cell extracts to visualize the biomolecular content of the multicellular eukaryote. After cryo-EM single-particle image processing, results showed that a simultaneous three-dimensional structural characterization of multiple chemically diverse biomacromolecules is feasible. Namely, the thermophilic, eukaryotic complexes of (a) ATP citrate-lyase, (b) Hsp90, (c) 20S proteasome, (d) Hsp60 and (e) UDP-glucose pyrophosphorylase were characterized. In total, all five complexes have been structurally dissected in a thermophilic eukaryote in a total imaged sample area of 190.64 µm2, and two, in particular, 20S proteasome and Hsp60, exhibit side-chain resolution features. The C. thermophilum Hsp60 near-atomic model was resolved at 3.46 Å (FSC = 0.143) and shows a hinge-like conformational change of its equatorial domain, highly similar to the one previously shown for its bacterial orthologue, GroEL. This work demonstrates that cryo-EM of cell extracts will greatly accelerate the structural analysis of cellular complexes and provide unprecedented opportunities to annotate architectures of biomolecules in a holistic approach.

Recollection of How We Came Across the Protein Modification with Amino Acids by Aminoacyl tRNA-Protein Transferase.

Protein arginylation has been discovered in 1963 as a soluble activity in cell extracts that mediates the addition of amino acids to proteins. This discovery was nearly accidental, but due to the persistence of the research team, it has been followed through and led to the emergence of a new field of research. This chapter describes the original discovery of arginylation and the first methods used to demonstrate the existence of this important biological process.

Cell-Free Therapies: The Use of Cell Extracts to Mitigate Irradiation-Injured Salivary Glands.

Radiotherapy is a standard treatment for head and neck cancer patients worldwide. However, millions of patients who received radiotherapy consequently suffer from xerostomia because of irreversible damage to salivary glands (SGs) caused by irradiation (IR). Current treatments for IR-induced SG hypofunction only provide temporary symptom alleviation but do not repair the damaged SG, thus resulting in limited treatment efficacy. Therefore, there has recently been a growing interest in regenerative treatments, such as cell-free therapies. This review aims to summarize cell-free therapies for IR-induced SG, with a particular emphasis on utilizing diverse cell extract (CE) administrations. Cell extract is a group of heterogeneous mixtures containing multifunctional inter-cellular molecules. This review discusses the current knowledge of CE's components and efficacy. We propose optimal approaches to improve cell extract treatment from multiple perspectives (e.g., delivery routes, preparation methods, and other details regarding CE administration). In addition, the advantages and limitations of CE treatment are systematically discussed by comparing it to other cell-free (such as conditioned media and exosomes) and cell-based therapies. Although a comprehensive identification of the bioactive factors within CEs and their mechanisms of action have yet to be fully understood, we propose cell extract therapy as an effective, practical, user-friendly, and safe option to conventional therapies in IR-induced SG.

Abiotic Stresses Elicitation Potentiates the Productiveness of Cardoon Calli as Bio-Factories for Specialized Metabolites Production.

Cultivated cardoon (Cynara cardunculus L. var altilis) is a Mediterranean traditional food crop. It is adapted to xerothermic conditions and also grows in marginal lands, producing a large biomass rich in phenolic bioactive metabolites and has therefore received attention for pharmaceutical, cosmetic and innovative materials applications. Cardoon cell cultures can be used for the biotechnological production of valuable molecules in accordance with the principles of cellular agriculture. In the current study, we developed an elicitation strategy on leaf-derived cardoon calli for boosting the production of bioactive extracts for cosmetics. We tested elicitation conditions that trigger hyper-accumulation of bioactive phenolic metabolites without compromising calli growth through the application of chilling and salt stresses. We monitored changes in growth, polyphenol accumulation, and antioxidant capability, along with transcriptional variations of key chlorogenic acid and flavonoids biosynthetic genes. At moderate stress intensity and duration (14 days at 50-100 mM NaCl) salt exerted the best eliciting effect by stimulating total phenols and antioxidant power without impairing growth. Hydroalcoholic extracts from elicited cardoon calli with optimal growth and bioactive metabolite accumulation were demonstrated to lack cytotoxicity by MTT assay and were able to stimulate pro-collagen and aquaporin production in dermal cells. In conclusion, we propose a "natural" elicitation system that can be easily and safely employed to boost bioactive metabolite accumulation in cardoon cell cultures and also in pilot-scale cell culture production.

Oral Administration of Mice with Cell Extracts of Recombinant Lactococcus lactis IL1403 Expressing Mouse Receptor Activator of NF-kB Ligand (RANKL).

Receptor activator of NF-kB ligand (RANKL) is known to play a major role in bone metabolism and the immune system, and its recombinant form has been expressed in bacterial systems for research since the last two decades. However, most of these recombinant forms are used after purification or directly using living cells. Here, there were cell extracts of recombinant Lactococcus lactis expressing mouse RANKL (mRANKL) used to evaluate its biological activity in mice. Mice were divided into three groups that were fed phosphate-buffered saline (PBS), wild-type L. lactis IL1403 (WT_CE), and recombinant L. lactis expressing mRANKL (mRANKL_CE). The small intestinal transcriptome and fecal microbiome were then profiled. The biological activity of mRANKL_CE was confirmed by studying RANK-RANKL signaling in vitro and in vivo. For small intestinal transcriptome, differentially expressed genes (DEGs) were identified in the mRANKL_CE group, and no DEGs were found in the WT_CE group. In the PBS vs. mRANKL_CE gene enrichment analysis, upregulated genes were enriched for heat shock protein binding, regulation of bone resorption, and calcium ion binding. In the gut microbiome analysis, there were no critical changes among the three groups. However, Lactobacillus and Sphingomonas were more abundant in the mRANKL_CE group than in the other two groups. Our results indicate that cell extracts of mRANKL_CE can play an effective role without a significant impact on the intestine. This strategy may be useful for the development of protein drugs.

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.

Integrative structure of a 10-megadalton eukaryotic pyruvate dehydrogenase complex from native cell extracts.

The pyruvate dehydrogenase complex (PDHc) is a giant enzymatic assembly involved in pyruvate oxidation. PDHc components have been characterized in isolation, but the complex's quaternary structure has remained elusive due to sheer size, heterogeneity, and plasticity. Here, we identify fully assembled Chaetomium thermophilum α-keto acid dehydrogenase complexes in native cell extracts and characterize their domain arrangements utilizing mass spectrometry, activity assays, crosslinking, electron microscopy (EM), and computational modeling. We report the cryo-EM structure of the PDHc core and observe unique features of the previously unknown native state. The asymmetric reconstruction of the 10-MDa PDHc resolves spatial proximity of its components, agrees with stoichiometric data (60 E2p:12 E3BP:∼20 E1p: ≤ 12 E3), and proposes a minimum reaction path among component enzymes. The PDHc shows the presence of a dynamic pyruvate oxidation compartment, organized by core and peripheral protein species. Our data provide a framework for further understanding PDHc and α-keto acid dehydrogenase complex structure and function.

Elucidating Human Mitosis Using an Anaphase-Like Cell-Free System.

A balanced progression through mitosis and cell division is largely dependent on orderly phosphorylation and ubiquitin-mediated proteolysis of regulatory and structural proteins. These series of events ultimately secure genome stability and time-invariant cellular properties during cell proliferation. Two of the core enzymes regulating mitotic milestones in all eukaryotes are cyclin dependent kinase 1 (CDK1) with its coactivator cyclin B, and the E3 ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Discovering mechanisms and substrates for these enzymes is vital to understanding how cells move through mitosis and segregate chromosomes with high fidelity. However, the study of these enzymes has significant challenges. Purely in vitro studies discount the contributions of yet to be described regulators and misses the physiological context of cellular environment. In vivo studies are complicated by the fact that each of these enzymes, as well as many of their regulators and downstream targets, are essential. Moreover, long-term in vivo manipulations can result in cascading, indirect effects that can distort data analysis and interpretation. Many of these challenges can be circumvented using cell-free systems, which have historically played a critical role in identifying these enzymes and their contributions under quasicellular environments. Here, we describe the preparation of a newly developed human cell-free system that recapitulates an anaphase-like state of human cells. This new toolkit complements traditional cell-free systems from human cells and frog eggs and can be easily implemented in cell biology labs for direct and quantitative studies of mitotic signaling regulated by phosphorylation, APC/C-mediated proteolysis, and beyond.

Chemiluminescent sensors for quantitation of the bacterial second messenger cyclic di-GMP.

Chemiluminescent biosensors have been developed and broadly applied to mammalian cell systems for studying intracellular signaling networks. For bacteria, biosensors have largely relied on fluorescence-based systems for quantitating signaling molecules, but these designs can encounter issues in complex environments due to their reliance on external illumination. In order to circumvent these issues, we designed the first ratiometric chemiluminescent biosensors for studying a key bacterial second messenger, cyclic di-GMP. We have shown recently that these biosensors function both in vitro and in vivo for detecting changes in cyclic di-GMP levels. In this chapter, we present a practical and broadly applicable method for high-throughput quantitation of cyclic di-GMP in bacterial cell extracts using the high affinity biosensor tVYN-TmΔ that could serve as the "Bradford assay" equivalent for this bacterial signaling molecule.

Phosphorylation-Dependent SERS Readout for Activity Assay of Protein Kinase A in Cell Extracts.

Protein kinases are key regulators of cell function, the abnormal activity of which may induce several human diseases, including cancers. Therefore, it is of great significance to develop a sensitive and reliable method for assaying protein kinase activities in real biological samples. Here, we report the phosphorylation-dependent surface-enhanced Raman scattering (SERS) readout of spermine-functionalized silver nanoparticles (AgNPs) for protein kinase A (PKA) activity assay in cell extracts. In this assay, the presence of PKA would phosphorylate and alter the net charge states of Raman dye-labeled substrate peptides, and the resulting anionic products could absorb onto the AgNPs with cationic surface charge through electrostatic attraction. Meanwhile, the Raman signals of dyes labeled on peptides were strongly enhanced by the aggregated AgNPs with interparticle hot spots formed in assay buffer. The SERS readout was directly proportional to the PKA activity in a wide range of 0.0001-0.5 U·µL-1 with a detection limit as low as 0.00003 U·µL-1. Moreover, the proposed SERS-based assay for the PKA activity was successfully applied to monitoring the activity and inhibition of PKA in real biological samples, particularly in cell extracts, which would be beneficial for kinase-related disease diagnostics and inhibitor screening.