A Bioinformatic Guide to Identify Protein Effectors from Phytopathogens.

Phytopathogenic fungi are a diverse and widespread group that has a significant detrimental impact on crops with an estimated annual average loss of 15% worldwide. Understanding the interaction between host plants and pathogenic fungi is critical to delineate underlying mechanisms of plant defense to mitigate agricultural losses. Fungal pathogens utilize suites of secreted molecules, called effectors, to modulate plant metabolism and immune response to overcome host defenses and promote colonization. Effectors come in many flavors including proteinaceous products, small RNAs, and metabolites such as mycotoxins. This review will focus on methods for identifying protein effectors from fungi. Excellent reviews have been published to identify secondary metabolites and small RNAs from fungi and therefore will not be part of this review.

Architecture of the dynamic fungal cell wall.

The fungal cell wall is essential for growth and survival, and is a key target for antifungal drugs and the immune system. The cell wall must be robust but flexible, protective and shielding yet porous to nutrients and membrane vesicles and receptive to exogenous signals. Most fungi have a common inner wall skeleton of chitin and ß-glucans that functions as a flexible viscoelastic frame to which a more diverse set of outer cell wall polymers and glycosylated proteins are attached. Whereas the inner wall largely determines shape and strength, the outer wall confers properties of hydrophobicity, adhesiveness, and chemical and immunological heterogeneity. The spatial organization and dynamic regulation of the wall in response to prevailing growth conditions enable fungi to thrive within changing, diverse and often hostile environments. Understanding this architecture provides opportunities to develop diagnostics and drugs to combat life-threatening fungal infections.

Effects of urbanization intensity on glomalin-related soil protein in Nanchang, China: Influencing factors and implications for greenspace soil improvement.

Glomalin-related soil protein (GRSP) is a stable and persistent glycoprotein secreted by arbuscular mycorrhizal fungi that plays an important role in sequestering soil organic carbon (SOC) and improving soil quality. Rapid urbanization disturbs and degrades the soil quality in the greenspace. However, few studies have investigated the effects of urbanization on GRSP and its influencing factors. This study selected impervious surface area as a measure of urbanization intensity. A total of 184 soil samples were collected from the 0-20 cm soil layer in the greenspace of Nanchang, China (505 km2). The GRSP content, soil properties, urban forest characteristics, and land-use configuration were determined. The total GRSP (TG) and easily extractable GRSP (EEG) contents were 2.38 and 0.57 mg g-1, respectively. TG and EEG decreased by 16.22% and 19.35%, respectively, from low to heavy urbanized areas. Moreover, SOC decreased from 39.9 to 1.4 mg g-1, while EEG/SOC and TG/SOC increased by approximately 17% and 34%, respectively, indicating the significant contribution of GRSP to the SOC pool. Pearson and redundancy analysis showed that GRSP was positively correlated with SOC, phosphorus, nitrogen, vegetation richness, and tree height, but negatively correlated with pH, bulk density, and impervious area. The partial least squares path model demonstrated that urbanization affected soil properties, forest characteristics, and land use factors, resulting in GRSP changes. This study clarifies the key factors of urbanization that affect GRSP and provides insight for urban greenspace soil improvement from the new perspective of enhancing the GRSP content.

Proteomics and glycoproteomics of beer and wine.

Beer and wine are fermented beverages that contain abundant proteins released from barley or grapes, and secreted from yeast. These proteins are associated with many quality attributes including turbidity, foamability, effervescence, flavour and colour. Many grape proteins and secreted yeast proteins are glycosylated, and barley proteins can be glycated under the high temperatures in the beer making process. The emergence of high-resolution mass spectrometry has allowed proteomic and glycoproteomic analyses of these complex mixtures of proteins towards understanding their role in determining beer and wine attributes. In this review, we summarise recent studies of proteomic and glycoproteomic analyses of beer and wine including their strategies for mass spectrometry (MS)-based identification, quantification and characterisation of the glyco/proteomes of fermented beverages to control product quality.

Investigation of the Proteomes of the Truffles Tuber albidum pico, T. aestivum, T. indicum, T. magnatum, and T. melanosporum.

Truffles of the Tuber species are known as expensive foods, mainly for their distinct aroma and taste. This high price makes them a profitable target of food fraud, e.g., the misdeclaration of cheaper truffle species as expensive ones. While many studies investigated truffles on the metabolomic level or the volatile organic compounds extruded by them, research at the proteome level as a phenotype determining basis is limited. In this study, a bottom-up proteomic approach based on LC-MS/MS measurements in data-independent acquisition mode was performed to analyze the truffle species Tuber aestivum, Tuber albidum pico, Tuber indicum, Tuber magnatum, and Tuber melanosporum, and a protein atlas of the investigated species was obtained. The yielded proteomic fingerprints are unique for each of the of the five truffle species and can now be used in case of suspected food fraud. First, a comprehensive spectral library containing 9000 proteins and 50,000 peptides was generated by two-dimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC-MS/MS). Then, samples of the truffle species were analyzed in data-independent acquisition (DIA) proteomics mode yielding 2715 quantified proteins present in all truffle samples. Individual species were clearly distinguishable by principal component analysis (PCA). Quantitative proteome fingerprints were generated from 2066 ANOVA significant proteins, and side-by-side comparisons of truffles were done by T-tests. A further aim of this study was the annotation of functions for the identified proteins. For Tuber magnatum and Tuber melanosporum conclusive links to their superior aroma were found by enrichment of proteins responsible for sulfur-metabolic processes in comparison with other truffles. The obtained data in this study may serve as a reference library for food analysis laboratories in the future to tackle food fraud by misdeclaration of truffles. Further identified proteins with their corresponding abundance values in the different truffle species may serve as potential protein markers in the establishment of targeted analysis methods. Lastly, the obtained data may serve in the future as a basis for deciphering the biochemistry of truffles more deeply as well, when protein databases of the different truffle species will be more complete.

The post-translational modification landscape of commercial beers.

Beer is one of the most popular beverages worldwide. As a product of variable agricultural ingredients and processes, beer has high molecular complexity. We used DIA/SWATH-MS to investigate the proteomic complexity and diversity of 23 commercial Australian beers. While the overall complexity of the beer proteome was modest, with contributions from barley and yeast proteins, we uncovered a very high diversity of post-translational modifications (PTMs), especially proteolysis, glycation, and glycosylation. Proteolysis was widespread throughout barley proteins, but showed clear site-specificity. Oligohexose modifications were common on lysines in barley proteins, consistent with glycation by maltooligosaccharides released from starch during malting or mashing. O-glycosylation consistent with oligomannose was abundant on secreted yeast glycoproteins. We developed and used data analysis pipelines to efficiently extract and quantify site-specific PTMs from SWATH-MS data, and showed incorporating these features into proteomic analyses extended analytical precision. We found that the key differentiator of the beer glyco/proteome was the brewery, with beer from independent breweries having a distinct profile to beer from multinational breweries. Within a given brewery, beer styles also had distinct glyco/proteomes. Targeting our analyses to beers from a single brewery, Newstead Brewing Co., allowed us to identify beer style-specific features of the glyco/proteome. Specifically, we found that proteins in darker beers tended to have low glycation and high proteolysis. Finally, we objectively quantified features of foam formation and stability, and showed that these quality properties correlated with the concentration of abundant surface-active proteins from barley and yeast.

Application of PALM Superresolution Microscopy to the Analysis of Microtubule-Organizing Centers (MTOCs) in Aspergillus nidulans.

Photoactivated localization microscopy (PALM), one of the super resolution microscopy methods improving the resolution limit to 20 nm, allows the detection of single molecules in complex protein structures in living cells. Microtubule-organizing centres (MTOCs) are large, multisubunit protein complexes, required for microtubule polymerization. The prominent MTOC in higher eukaryotes is the centrosome, and its functional ortholog in fungi is the spindle-pole body (SPB). There is ample evidence that besides centrosomes other MTOCs are important in eukaryotic cells. The filamentous ascomycetous fungus Aspergillus nidulans is a model organism, with hyphae consisting of multinucleate compartments separated by septa. In A. nidulans, besides the SPBs, a second type of MTOCs was discovered at septa (called septal MTOCs, sMTOC). All the MTOC components appear as big dots at SPBs and sMTOCs when tagged with a fluorescent protein and observed with conventional fluorescence microscopy due to the diffraction barrier. In this chapter, we describe the application of PALM in quantifying the numbers of individual proteins at both MTOC sites in A. nidulans and provide evidence that the composition of MTOCs is highly dynamic and dramatically changes during the cell cycle.

Closed-tube field-deployable loop-mediated isothermal amplification (LAMP) assay based on spore wall protein (SWP) for the visual detection of Enterocytozoon hepatopenaei (EHP).

Hepatopancreatic microsporidiosis (HPM) is an infectious shrimp disease caused by the microsporidian Enterocytozoon hepatopenaei (EHP). In recent years, the widespread occurrence of EHP poses a significant challenge to the shrimp aquaculture industry. Early, rapid and accurate diagnosis of EHP infection is very much essential for the control of HPM crop-related losses. Loop-mediated isothermal amplification (LAMP) is a robust, sensitive, cost-effective disease diagnostic technique. Here, we demonstrate an improved, simple, closed-tube, colorimetric EHP LAMP diagnostic assay. LAMP assay was illustrated with the specific EHP spore wall protein (SWP) gene primers. Naked eye visual detection of LAMP amplicons was achieved using Hydroxy naphthol blue (HNB) or Phenol red dye without opening the tubes. This LAMP assay is efficient in detecting the EHP pathogen in all clinical samples include shrimp hepatopancreas, FTA card samples, feces, pond water, and soil. Also, the elution of EHP DNA from FTA cards was demonstrated within 17 min using a simple dry bath. In clinical evaluation, the visual LAMP assay established 100% diagnostic sensitivity and 100% diagnostic specificity. The visual LAMP assay is rapid, can detect the EHP pathogen within 40 min using a simple dry bath, and does not require any expensive instruments and technical proficiency. In conclusion, this visual LAMP protocol is a user-friendly, specific assay that can be conceivably operated at the farm-site/ resource-limited settings by the farmer himself with simple equipment.

Chemical Composition and Ultraviolet Absorption Activity of an Aqueous Alkali Extract from the Fruiting Bodies of the Tinder Conk Mushroom, Fomes fomentarius (Agaricomycetes).

White rot mushroom Fomes fomentarius is a medicinal fungus with great potential to be explored. This work focused on the chemical composition of a basic aqueous extract from F. fomentarius fruiting bodies. The extract was mostly composed of phenolics, carbohydrates, minerals, and crude fat with a low amount of proteins and chitin. One-third of the total carbohydrates were in the form of beta-glucans with minor amounts of alpha-glucans. The most valuable essential part of the extract was composed of an acid-resistant ultraviolet (UV)-absorbing mixture of phenolic compounds such as melanins, lignins, and humic acids. These compounds, also referred to as melanin-like pigments, provided for the high antioxidant activity of the extract measured in vitro. Moderate sun-protective capacity was observed with regard to UVB rays and also expected in the UVA range. Quantification of melanin-like pigments in the F. fomentarius extract was possible either gravimetrically as acid-insoluble residue or spectrophotometrically in the UV region. Melanin estimation, based on nitrogen measurements, offered misleading results due to the presence of nitrogen-free melanins along with other nitrogen-containing compounds such as proteins and chitin. F. fomentarius water-soluble basic extract, containing beta-glucans and rich in melanin-like substances, could be used, for example, for topical skin application to prevent cell damage caused by excessive UV exposure or cytotoxic free radicals. The bioactive potential, safety, and further applications of the F. fomentarius extract are currently being investigated.

Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry: Protocol standardisation, comparison and database expansion for faster and reliable identification of dermatophytes.

BACKGROUND: Accurate and early identification of dermatophytes enables prompt antifungal therapy. However, phenotypic and molecular identification methods are time-consuming. MALDI-TOF MS-based identification is rapid, but an optimum protocol is not available. OBJECTIVES: To develop and validate an optimum protein extraction protocol for the efficient and accurate identification of dermatophytes by MALDI-TOF MS. MATERIALS/METHODS: Trichophyton mentagrophytes complex (n = 4), T. rubrum (n = 4) and Microsporum gypseum (n = 4) were used for the optimisation of protein extraction protocols. Thirteen different methods were evaluated. A total of 125 DNA sequence confirmed clinical isolates of dermatophytes were used to create and expand the existing database. The accuracy of the created database was checked by visual inspection of MALDI spectra, MSP dendrogram and composite correlation index matrix analysis. The protocol was validated further using 234 isolates. RESULT: Among 13 protein extraction methods, six correctly identified dermatophytes but with a low log score (≤1.0). The modified extraction protocol developed provided an elevated log score of 1.6. Significant log score difference was observed between the modified protocol and other existing protocols (T. mentagrophytes complex: 1.6 vs. 0.2-1.0, p < .001; T. rubrum: 1.6 vs. 0.4-1.0, p < .001; M. gypseum:1.6 vs. 0.2-1.0, p < .001). Expansion of the database enabled the identification of all 234 isolates (73.5% with log score ≥2.0 and 26.4% with log scores range: 1.75-1.99). The results were comparable to DNA sequence-based identification. CONCLUSION: MALDI-TOF MS with an updated database and efficient protein extraction protocol developed in this study can identify dermatophytes accurately and also reduce the time for identifying them.

Intensive pulsed light pretreatment combined with controlled temperature and humidity for convection drying to reduce browning and improve quality of dried shiitake mushrooms.

BACKGROUND: The change of surface color caused by browning during the drying process of shiitake mushrooms seriously affects its market circulation. Intensive pulsed light (IPL) as a non-heat-treatment method can reduce enzyme activity by changing the enzyme structure. Therefore, in this study, the use of IPL pretreatment before drying was aimed to reduce the adverse reactions caused by the browning reaction during the drying processing of shiitake mushrooms. RESULTS: Shiitake mushrooms pretreated with 25 pulses of IPL energy of 400 J reduced the initial polyphenol oxidase enzyme activity, the browning index, and browning degree values by 42.83%, 43.02%, and 47.54% respectively. The IPL pretreatment enhanced the polysaccharides and reducing sugars contents and it reduced 5-hydroxymethylfurfural generation in the dried shiitake mushrooms. The pretreatment also improved the surface color, the antioxidant activity, and retained the umami taste characteristics in the dried shiitake mushroom. CONCLUSION: The IPL pretreatment combined with controlled temperature and humidity for convection drying could be a suitable method to improve the quality of dried shiitake mushrooms. Therefore, this study provides a new pretreatment method for materials that are prone to browning during drying. © 2021 Society of Chemical Industry.

Metaproteomics Analysis of Host-Microbiota Interfaces.

Metaproteomics of host-microbiome interfaces comprises the analysis of complex mixtures of bacteria, archaea, fungi, and viruses in combination with its host cells. Microbial niches can be found all over the host including the skin, oral cavity, and the intestine and are considered to be essential for the homeostasis. The complex interactions between the host and diverse commensal microbiota are poorly characterized while of great interest as dysbiosis is associated with the development of various inflammatory and metabolic diseases. The metaproteomics workflows to study these interfaces are currently being established, and many challenges remain. The major challenge is the large diversity in species composition that make up the microbiota, which results in complex samples that require extended mass spectrometry analysis time. In addition, current database search strategies are not developed to the size of the search space required for unbiased microbial protein identification.Here, we describe a workflow for the proteomics analysis of microbial niches with a focus on intestinal mucus layer. We will cover step-by-step the sample collection, sample preparation, liquid chromatography-mass spectrometry, and data analysis.

Screening Balanites aegyptiaca for inhibitors against putative drug targets in Microsporum gypseum - Subtractive proteome, docking and simulation approach.

Microsporum gypseum is a keratinophilic fungi grouped under dermatophytes infecting skin, hair and nail portions in human and animals causing tinea corporis, tinea facei and tinea capitis. As both human and fungi are eukaryotes, the available drugs for treating dermatophytes produce some side effects due to drug interaction with human also. Apart from this, the gut microbiota has a very big role in the health of human which should not be affected by the drugs. Hence this study focused on finding a target which is unique and essential to M. gypseum and non-homologous to human and gut microbiota, non-homologous to human domain architecture, highly interacting with other proteins, sub-cellular localization of proteins and non-druggability analysis of the targets using subtractive proteomics approach which resulted with 3 novel drug targets from M. gypseum which were modeled using I-TASSER, refined by ModRefiner and validated by PROCHECK. Further these targets were docked with compounds identified through LC-MS of fractioned methanol extract of B. aegyptiaca fruit pulp using Glide module and the stability of the docked complex was analyzed by molecular dynamics simulation using Desmond module of Schrodinger. Cyanidin-3-O-rhamnoside had better interaction with all the targets and Taurocholic acid had better result with ECCP which suggests the multi-targeting potency of these two compounds against M. gypseum which has to be confirmed by in vitro and in vivo studies.

An improved staining method of cell cycle analysis with Sybr Green I for fungi: Cryptococcus neoformans and Saccharomyces cerevisiae.

Cryptococcus neoformans is a pathogenic fungus which causes millions of deaths and infections, especially threatening immunocompromised individuals. During the development of new drugs, the ubiquitination has been found to play an important role in the regulation of the virulence and cell cycle of this fungus. Based on this mechanism, ubiquitination-related mutant strains exhibiting cell cycle arrest have been established for drug development for the fungus. However, flow cytometry detection of the cell cycle in fungi is generally difficult because the thick cell wall and capsule of fungi generally contribute to a nonspecific signal of cytometry. In this study, an improved method, derived from Saccharomyces cerevisiae assays, is developed to specifically stain C. neoformans, in whose cell cycle the G1 and G2 peaks are separated enough to be allowed for cell cycle analysis. As a result, the improved method facilitates the detection of the alterations in the cell cycle of C. neoformans with a mutation that results in cell cycle arrest, which distinctly delays the cell division of C. neoformans. Thus, the improved method reported here provides detailed technical information regarding assays on C. neoformans and, more importantly, offers a solution for assessing the cell cycle in other fungi in the future. Abbreviation: PI: propidium iodide.

A secretion-based dual fluorescence assay for high-throughput screening of alcohol dehydrogenases.

Alcohol dehydrogenases (ADHs) play key roles in the production of various chemical precursors that are essential in pharmaceutical and fine chemical industries. To achieve a practical application of ADHs in industrial processes, tailoring enzyme properties through rational design or directed evolution is often required. Here, we developed a secretion-based dual fluorescence assay (SDFA) for high-throughput screening of ADHs. In SDFA, an ADH of interest is fused to a mutated superfolder green fluorescent protein (MsfGFP), which could result in the secretion of the fusion protein to culture broth. After a simple centrifugation step to remove the cells, the supernatant can be directly used to measure the activity of ADH based on a red fluorescence signal, whose increase is coupled to the formation of NADH (a redox cofactor of ADHs) in the reaction. SDFA allows easy quantification of ADH concentration based on the green fluorescence signal of MsfGFP. This feature is useful in determining specific activity and may improve screening accuracy. Out of five ADHs we have tested with SDFA, four ADHs can be secreted and characterized. We successfully screened a combinatorial library of an ADH from Pichia finlandica and identified a variant with a 197-fold higher kcat /km value toward (S)-2-octanol compared to its wild type.

Endophytic bacteria promote the quality of Lyophyllum decastes by improving non-volatile taste components of mycelia.

Endophytic bacteria are always related to the host different traits, including the secondary metabolites production. However, the effect and mechanism of endophytic bacteria in the mushrooms fruit body on mycelia are still not clear. In this study, we investigated the effect of endophytic bacterial metabolites on the quality of Lyophyllum decastes mycelia. Soluble sugars, starch, protein, free amino acids, 5'-Nucleotides, EUC, and organic acids contents of mycelia were analyzed. We found that endophytic bacterial metabolites significantly increased the contents of soluble sugars, starch, protein, free amino acids, organic acids, and EUC. The present study thus suggests that endophytic bacteria could promote the quality of Lyophyllum decastes by improving non-volatile taste components of mycelia.

Electrochemical analysis of specific catalase activity during development of Aspergillus flavus and its correlation with aflatoxin B1 production.

Aflatoxin B1 (AFB1) contamination causes huge economic losses. To explore the correlation between catalase (CAT) and AFB1 production during fungal development, we fabricated an electrochemical CAT-activity sensor by measuring residual H2O2 after enzymatic degradation. The sensor made by palladium nanoparticles/carbonized bacterial cellulose nanocomposites exhibits a linear range over 0.5-3.5 U/mL and a detection limit of 0.434 U/mL. Both dry weight and CAT activity of mycelia continuously increase. But, the latter shows a greater increase than the former after three days. Specific CAT activity in crude enzyme extract of A. flavus was quantified. It maintains at ~25.00 U/mg for 3 days and enhances to 28.91 and 45.30 U/mg, respectively, on days 4 and 5. AFB1 production follows the same trend. On days 4 and 5, AFB1 concentration reaches 201.35 and 767.9 ng/mL, respectively. The positive correlation between specific CAT activity and AFB1 production suggests that CAT is involved in AFB1 biosynthesis.

Quantitative Data-Independent Acquisition Glycoproteomics of Sparkling Wine.

Sparkling wine is an alcoholic beverage enjoyed around the world. The sensory properties of sparkling wine depend on a complex interplay between the chemical and biochemical components in the final product. Glycoproteins have been linked to positive and negative qualities in sparkling wine, but the glycosylation profiles of sparkling wine have not been previously investigated in detail. We analyzed the glycoproteome of sparkling wines using protein- and glycopeptide-centric approaches. We developed an automated workflow that created ion libraries to analyze sequential window acquisition of all theoretical mass spectra data-independent acquisition mass spectrometry data based on glycopeptides identified by Byonic (Protein Metrics; version 2.13.17). We applied our workflow to three pairs of experimental sparkling wines to assess the effects of aging on lees and of different yeast strains used in the liqueur de tirage for secondary fermentation. We found that aging a cuvée on lees for 24 months compared with 8 months led to a dramatic decrease in overall protein abundance and an enrichment in large glycans at specific sites in some proteins. Secondary fermentation of a Riesling wine with Saccharomyces cerevisiae yeast strain Siha4 produced more yeast proteins and glycoproteins than with S. cerevisiae yeast strain DV10. The abundance and glycosylation profiles of grape glycoproteins were also different between grape varieties. To our knowledge, this work represents the first in-depth study into protein- and peptide-specific glycosylation in sparkling wines and describes a quantitative glycoproteomic sequential window acquisition of all theoretical mass spectra/data-independent acquisition workflow that is broadly applicable to other sample types.

Chemical composition and deterioration mechanism of Pleurotus tuoliensis during postharvest storage.

Pleurotus tuoliensis is a popular edible and medical mushroom, but it is highly perishable during postharvest storage. The quality parameters, chemical composition, malondialdehyde (MDA) concentration, and activity of metabolic enzymes were studied during 12 days of storage at 4 °C and 6 days of storage at 25 °C. Degradation was well described by changes in quality parameters, losses in nutritional value, increased metabolic enzyme activity, the accumulation of MDA concentrations, and the increase of total phenolic (TP) content. The phenylalanine ammonia lyase (PAL) significantly positively correlated with TP, which suggested an underlying mechanism of browning that the increased PAL activity stimulates the biosynthesis of phenols through the phenylalanine pathway. These results suggest that increased activity of laccase, lipoxygenase, PAL, TP and MDA accumulation, together with polysaccharide degradation, are the main factors involved in the deterioration of P. tuoliensis during storage.

Registered report protocol: Quantitative analysis of septin Cdc10-associated proteome in Cryptococcus neoformans.

Cryptococcus neoformans is a pathogenic basidiomycetous yeast that primarily infects immunocompromised individuals. C. neoformans can thrive during infections due to its three main virulence-related characteristics: the ability to grow at host temperature (37°C), formation of carbohydrate capsule, and its ability to produce melanin. C. neoformans strains lacking septin proteins Cdc3 or Cdc12 are viable at 25°C; however, they fail to proliferate at 37°C and are avirulent in the murine model of infection. The basis of septin contribution to growth at host temperature remains unknown. Septins are a family of conserved filament-forming GTPases with roles in cytokinesis and morphogenesis. In the model organism Saccharomyces cerevisiae septins are essential. S. cerevisiae septins form a higher order complex at the mother-bud neck to scaffold over 80 proteins, including those involved in cell wall organization, cell polarity, and cell cycle control. In C. neoformans, septins also form a complex at the mother-bud neck but the septin interacting proteome in this species remains largely unknown. Moreover, it remains possible that septins play other roles important for high temperature stress that are independent of their established role in cytokinesis. Therefore, we propose to perform a global analysis of septin Cdc10 binding partners in C. neoformans, including those that are specific to high temperature stress. This analysis will shed light on the underlying mechanism of survival of this pathogenic yeast during infection and can potentially lead to the discovery of novel drug targets.