The phospho-ferrozine assay: a tool to study bacterial redox-active metabolites produced at the plant root.

Root exudates represent complex mixtures of low-molecular-weight and high-molecular-weight compounds. The former comprise predominantly primary and secondary plant metabolites, the latter mucilage precursors and some proteins. Different collection approaches exist. The traditionally most widely used one (also applied in this study) is soaking roots for several hours in distilled water that have been thoroughly cleaned from soil. Other collection methods comprise hydroponic cultures and rhizoboxes with microsuction devices. The former are more used by molecular biologists in attempts to characterize phenotypes reproducibly, the latter by ecologists in efforts to explore specific regions of the rhizosphere. The classical approach is one that still allows plant to be cultured in soil. This is important because mucilage formation is affected by microbial soil communities and soil physicochemical properties. Primary and secondary plant meta¬bolites provide the majority of low-molecular precursors for mucilage development. Quality and quantity of extractable root-exuded plant metabolites is most probably affected by these parameters which are completely absent in hydroponic cultures. Six model plants were chosen on basis of their status as crop plant and on their tolerance of the uniform culture conditions to which all model plants were subjected to in the only available climate chamber. These included the Brassicaceae Arabidopsis and Rapeseed, the Fabaceae Phaseolus and Pisum, the Solanaceae Tobacco and the grass Maize. In all experi¬ments, the model plants received identical amounts of light, the same water supply and nutrient provision. In attempts to simulate drought stress, one-half of the plants was not deprived of water for two consecutive weeks. One aim was to explore which and to what amounts primary as well as secondary plant metabolites do occur in the root exudates and if they differ from those present in the roots. All six plant species showed similar primary metabolite profiles that, however, varied quanti¬tatively between the plant species. A prominent root exudate metabolite was myo-inositol, a sugar alcohol. Root tissues and root exudates showed different profiles with amino acids showing the most profound differences. The found primary metabolites agree with those reported in the literature. By contrast, secondary metabolites showed characteristic profiles, in which only few com-pounds were common to more than one species. One metabolite that was detected in all species was cinnamic acid. Structure elucidation was focussed especially on those secondary plant metabolites that were pointed out by non-parametric multivariate statistics as substantial contributors to similarity and dissimilarity of root exudates and root tissues. Root exudates were found to contain chalcones, flavanols, isoflavones, cinnamides, a cinnamoyl spermidine, indoles, stilbenes, a hydroxamic acid benzoxazine and a gibberellic acid deri¬vative, amongst others. Notably, no glycosides were detected among the elucidated metabolites and a considerably high proportion of aldehydes was noted. In case of Arabidopsis, an extensive analysis of hydroponically obtained root exudates exists in the literature. Many dimeric structures were reported, most of which could not be detected in the present study. Another explored aspect was the effect of water deficit on root exudation. Primary metabolite patterns changed in a more similar way, sugars such as glucose and sucrose increased and myo-inositol proportions decreased. Amino acid pattern changes, by contrast, were more species-specific. Generally, the amounts of detectable secondary metabolites decreased as shoot: root ratios in the affected plants increased. Only Phaseolus and Maize showed higher shoot: root ratios after water deficit. This suggests a different, more opportunistic strategy to survive stress. Only Pisum exuded a new class of secondary metabolites that was absent in the regularly watered plants. Altogether 24 different root exudate samples were available. Bases on the variability of primary and secondary root exudate metabolites correlations with nutrient supply in leaves was explored by Spearman rank correlation. Interestingly and as once suggested in a previous review, weak correlations between secondary metabolite profiles and leaf nutrients were found. Especially more unsaturated metabolites with vicinal oxygen functions correlated with the uptake of several nutrients, most of them being metal cations. The structural properties of the identified secondary metabolites allows them to act as ligands in coordination complexes in which the nutrient represents the central atom. This chemistry can add to the mobilization and uptake of nutrients by plant roots.

Microbial co-culture: harnessing intermicrobial signaling for the production of novel antimicrobials.

Natural products are substances that are confined from living organisms, they are in the form of primary or secondary metabolites. Secondary metabolites are compounds with varied chemical structures, produced by some plants and strains of microbial species. Unlike primary metabolites (nucleotides, amino acids, carbohydrates, and lipids) that are essential for growth, secondary metabolites are not. Secondary metabolites are produced or synthesized during the stationary stage. In this chapter, we will discuss secondary metabolites from natural products synthesized mainly by plants, fungi, and bacteria. Plants synthesize a large diversity of secondary metabolites; plant secondary metabolites are split into four groups namely alkaloids, phenolic compounds, terpenoids, and glucosinolates. Several classes of fungal and bacterial secondary metabolites, their sources, and pharmacological uses associated with the secondary metabolites are also discussed. Therefore, several classes of secondary metabolites are responsible for the biological and pharmacological activities of plants and herbal medicines.

Secondary metabolites are important facilitators of plant-microbe interactions in the rhizosphere, contributing to communication, competition, and nutrient acquisition. However, at first glance, the rhizosphere seems full of metabolites with overlapping functions, and we have a limited understanding of basic principles governing metabolite use. Increasing access to the essential nutrient iron is oneimportant, but seemingly redundant role performed by both plant and microbial Redox-Active Metabolites (RAMs). We used coumarins, RAMs made by the model plant Arabidopsis thaliana, and phenazines, RAMs made by soil-dwelling pseudomonads, to ask whether plant and microbial RAMs might each have distinct functions under different environmental conditions. We show that variations in oxygen and pH lead to predictable differences in the capacity of coumarins vs phenazines to increase the growth of iron-limited pseudomonads and that these effects depend on whether pseudomonads are grown on glucose, succinate, or pyruvate: carbon sources commonly found in root exudates. Our results are explained by the chemical reactivities of these metabolites and the redox state of phenazines as altered by microbial metabolism. This work shows that variations in the chemical microenvironment can profoundly affect secondary metabolite function and suggests plants may tune the utility of microbial secondary metabolites by altering the carbon released in root exudates. Together, these findings suggest that RAM diversity may be less overwhelming when viewed through a chemical ecological lens: Distinct molecules can be expected to be more or less important to certain ecosystem functions, such as iron acquisition, depending on the local chemical microenvironments in which they reside.

A phenazine-inspired framework for identifying biological functions of microbial redox-active metabolites

The secondary metabolites found in plants represent an extremely rich source of novel chemical diversity for drug discovery and chemical biology programs.1–3 One of the main problems in drug discovery from plant small molecules is the identification of their molecular targets: many compounds have been found to be more promiscuous than originally anticipated, which can potentially lead to side effects, but which may also open up additional medical uses. The drug poly-pharmacological activity can be understood only if its interactions with cellular components are comprehensively characterized. Thus the identification of target proteins and investigation of ligand-receptor interactions represents an essential step in the process of plant drug discovery and development.4,5 However, nature is far more complex, and it is only with multidisciplinary collaborative research encompassing many disciplines that such targets can be successfully studied. Owing to our interest in the field of bioactive plant molecules, we have developed approaches to target identification based on chemical proteomics procedures, supported by spectroscopic and spectrometric data, Surface Plasmon Resonances (SPR) analyses and biochemical tests.5,6 Chemical proteomics is a powerful mass spectrometry-based affinity chromatography approach aimed to identify a set of proteins captured by a small molecules.6,7 The investigated molecule has to be anchored to a solid support through a flexible space arm, and then incubated with a lysate, from cell or tissues, to obtain the interaction between the immobilized compound (bait) and its protein targets. After several washing steps, proteins tightly bound to the beads are eluted and subjected to electrophoresis or gel free separation, followed by enzymatic digestion. The obtained peptide mixtures are then submitted to MS analyses and data base search for protein identification. This study allows the description of all potential macromolecular targets of a small bioactive molecules in a single experiment, leading to a complete and selective target mapping of a drug candidate. We have recently applied this approach to the study of cellular targets of some bioactive plant small molecules showing interesting protein targets; achieved results will be described in this communication.8,9

The comprehensive detection of primary metabolites and secondary metabolites in japonica rice (Nipponbare) and indica rice seeds (93‐11) were clarified by using a combination of targeted and untargeted metabolomics approaches we created. A total of 246 metabolites were identified, encompassing 4 primary metabolite classes (3 sugars, 6 organic acids, 21 amino acids, 5 lipids) and 3 secondary flavonoid metabolite classes (17 flavones, 2 flavonones, 4 flavonols), as well as various other secondary metabolites. We found that japonica rice contains higher levels of sugars (especially fructose), alanine, flavonols, and luteolin complexes, while indica rice contains higher levels of organic acids, amino acids (excluding alanine), flavanones, and apigenin and its complexes. Exploring and comparing the comprehensive nutritional composition of the two types of rice not only enabled individuals to choose their staple food according to their own physical condition but also provided new insights for the development of health‐promoting rice.

In the 21st century, the world is facing persistent global problems that have led to 193 countries to agree on the 17 Sustainable Development Goals (SDGs). The United Nations introduced these goals in 2015 to find solutions that could help end poverty, promote prosperity and protect the planet (United Nations, 2016a). In this brief perspective, we will discuss the potential role of Streptomyces in achieving those SDGs, focusing it in the current strategies applied for discovering novel compounds and in some of the problems that must be faced (Figure 1). Members of the genus Streptomyces are filamentous Gram-positive bacteria belonging to the phylum Actinobacteria. They are ubiquitous microorganisms mainly found in soil but they can also inhabit other niches like seawater or deserts, or living associated with other organisms (Sivalingam et al., 2019). Streptomyces is mainly known for its ability to produce a wide array of bioactive secondary metabolites, which have several interesting applications in different fields (Alam et al., 2022; Demain & Sanchez, 2009; Donald et al., 2022). One of the problems that most concern the United Nations is the existence of a growing demand for food in today's world (Food security information network, 2023). In this context, Streptomyces could play a relevant role in achieving SDG 2 (zero hunger, improved nutrition and sustainable agriculture) and SDG 1 (end poverty). Streptomyces produces several metabolites with significant commercial relevance in enhancing the nutritional value of human food and animal feed, such as vitamins like cobalamin (Rex et al., 2022). Additionally, there is an increasing need for enzymes in the global market (Grand View Research, 2023). Streptomyces due to its wide metabolic potential is used for the sustainable biotechnological production of a broad assortment of enzymes such as proteases, xylanases, amylases, lipases, keratinases, cellulases, dextranases and chitinases among others (Fernandes de Souza et al., 2022; Kumar et al., 2020). These enzymes have applications in several fields, and advantages not only in terms of energy consumption, stability, substrate specificity, purity or reaction efficiency but also in ecological and waste generation, thus contributing to the achievement of sustainable industrialization and innovation (SDG 9) and promoting responsible production and consumption (SDG 12). An example of enzymes with ecological applications is the degradation of lignocellulose and dye decolourization by detergent-stable peroxidases and laccases (Cuebas-Irizarry & Grunden, 2024). These enzymes can be potentially used to treat wastewater resulting from human activities like textile and paper industries, which cause environmental pollution and wastes that affect life below water (SDG 14). Another promising application of Streptomyces is its use to obtain energy from waste resources, what contributes to the pursuit of affordable and clean energy (SDG 7) and climate action (SDG 13). For instance, Muthusamy et al. (2019) were able to produce bioethanol from different agro-residues using an S. olivaceus strain isolated from a mangrove sample. Streptomyces also contributes to the preservation of life on land (SDG 15) because they play a crucial role in sustainable agriculture and plant growth due to its participation in soil fertility (Hozzein et al., 2019). They contribute to phosphate and potassium biosolubilization, nitrogen supply to ecosystems, to stablish beneficial symbiosis with other rhizosphere microorganisms and to produce biocontrol agents such as phytohormones, antimicrobials, antifungals, pesticides, bioherbicides and insecticides (Boubekri et al., 2022; Li et al., 2021). Furthermore, the use of Streptomyces is considered an eco-friendly and promising technology for bioremediation of contaminants like pesticides and heavy metals because they can degrade organic and inorganic compounds more efficiently and safely than chemical agents (Jagannathan et al., 2021). Nevertheless, the greatest contribution throughout history of Streptomyces is as producer of bioactive compounds with applications in clinical, veterinary and agricultural fields, being the most important microbial source of bioactive compounds (Donald et al., 2022). In this context, this microorganism is an incredible force for achieving good health and well-being (SDG 3). During the so-called Golden Age of antibiotic discovery Streptomyces provided humanity with antibiotics, antifungal, anti-parasitic, immunosuppressive agents and antitumor compounds, many of them currently used in clinical (Demain & Sanchez, 2009). Subsequently, limitations in classical search techniques and depletion of traditional habitats have led to the rediscovery of known compounds or the identification of a scarce number of compounds with new scaffolds. This, together with the high costs to develop new compounds for clinical and other uses, resulted in a drastic decline in the discovery of new drugs and the withdrawal of these research departments from some big pharma companies (Genilloud, 2017). Nevertheless, recent screening new approaches, such as the use of pathogenic bacteria conditionally expressing antisense RNA of essential genes, have led to discovering new antibiotics like platensimycin (Figure 2) (Genilloud, 2017). In addition, screening antibiotic active molecules for other activities identified a number of useful natural products (NPs), including some with antitumor activity such as actinomycin D (Figure 2) (Demain & Vaishnav, 2011). Subsequently, advances in -omics and sequencing methods, and the development of synthetic and genomic manipulation techniques in Streptomyces in the last decades, have led to the emergence of new strategies in drug discovery, such as genome mining and combinatorial biosynthesis. These approaches represent promising strategies for discovering novel bioactive NPs, in many cases with high structural diversity. Additionally, these strategies were improved when combined with the isolation of new Streptomyces strains from low explored environments, which produce structurally diverse bioactive NPs with potential clinical applications (Alam et al., 2022; Chen et al., 2021; Donald et al., 2022; Lacey & Rutledge, 2022; Qin et al., 2017; Quinn et al., 2020). Noteworthy, the antibacterial anti-Gram positive chaxalactin (Figure 2) (Castro et al., 2018) produced by Streptomyces leuwenhoeeki from the Atacama desert; or cervimycins produced by Streptomyces tendae strain HKI 0179 from the ancient Italian cave Grotta dei Cervi, with antibacterial activity anti-MRSA, anti-VRE and anti- S. aureus EfS4 (Herold et al., 2005). Notably, in recent years, marine environments have been a prolific source of new NPs with a variety of bioactivities (Alves et al., 2018; Chen et al., 2021; Choudhary et al., 2017; Dharmaraj, 2010; Donald et al., 2022; Yang et al., 2020). Examples include the antibacterial anthracimycin B, produced by Streptomyces cyaneofuscatus M-169 from the Cantabrian sea (Rodríguez et al., 2018); or the cytotoxic neo-actinomycin A produced by Streptomyces sp. IMB094 from a marine sediment (Wang et al., 2017). Another unusual habitat where Streptomyces strains are found is in symbiotic associations with plants, fungi, vertebrates or invertebrate animals, both marine and terrestrial. In these associations, they appear to be a nutritional resource, or to play a protective role for the host against pathogens, parasites or predators, by producing antibiotic compounds (Barka et al., 2016; Batey et al., 2020; Chen et al., 2021; Donald et al., 2022; Qin et al., 2011; Seipke et al., 2012). In this context, it is worth highlighting the role played by some volatile compounds (VOCs) produced by Streptomyces such as geosmine, as an attractant for soil-dwelling arthropods like springtails, to localize them as a food source. In turn, springtails facilitates the dispersal of Streptomyces spores to other niches by these arthropods (Becher et al., 2020). One of the most widespread example is the symbiotic relationship with insects. Thus, new antifungal compounds such as mycangimycins (Scott et al., 2008) or frontalamides A and B (Blodgett et al., 2010) have been isolated. Both are produced by symbiotic Streptomyces strains found in the southern pine beetle (SPB) Dendroctonus frontalis. Another example are the new formicamycins antibiotics (Figure 2) that have shown promising anti-MRSA and VRE activities, which are produced by Streptomyces formicae KY5, isolated from African ants of the Tetraponera genus (Qin et al., 2017). As it has been mentioned before, genome mining has become a useful tool for discovering natural products from the early 2000s (Baltz, 2021; Lee et al., 2020). It can be defined as the set of bioinformatics tools used to detect secondary metabolite biosynthesis gene clusters (smBGCs) and their possible functional and chemical interactions (Albarano et al., 2020). Genome mining has shown that each Streptomyces species possesses about 30 smBGCs, what has supported the hypothesis that most Streptomyces biodiversity is yet to be exploited for NPs discovery (Baltz, 2019; Belknap et al., 2020). In recent years, this strategy has enabled the identification of potentially new secondary metabolites encoded by smBGCs. For example, the antitumor chaxapeptin, identified by mining a S. leuwenhoeeki strain isolated from the Atacama desert (Castro et al., 2018); the antituberculous atratumycin, produced by S. atratus SCSIO ZH16 from the South China Sea (Sun et al., 2019); the new cytotoxic peptide curacozole (Figure 2), isolated from Streptomyces curacoi (Kaweewan et al., 2019); or largimycins, new leinamycin-like compounds identified by mining S. argillaceus (Becerril et al., 2020). Nonetheless, despite some successful examples that can be found in the literature, the enormous diversity of smBGCs identified by genome mining is only partially translated to discovering new bioactive NPs, and identifying and characterizing compounds encoded by these predicted smBGCs still requires substantial laboratory work. Thus, the smBGC can be expressed or low-expressed but the predicted encoded compounds is not detected under standard laboratory conditions (cryptic products), or the smBGC identified is not expressed and the product is unobserved (silent BGC with a cryptic product). All of these scenarios exemplify 'Known Unknowns' secondary metabolites (Hoskisson & Seipke, 2020). Therefore, a key issue for being successful using genome mining as an approach is to find strategies to turn on or to increase the expression of these silent or low expressed smBGCs. For this purpose, there are several genetic strategies that have been used like overexpression of positive regulators; inactivation of negative regulators; heterologous expression of the smBGC; or the insertion of a strong promoter upstream of BGC operons (Olano, García, et al., 2014). Other strategies to alleviate challenge of identifying the cryptic products are OSMAC (one strain of many compounds) (Pan et al., 2019); mimicking the ecological environment of the producer (Cuervo et al., 2022); redirecting precursors to the target biosynthesis pathway (Kallifidas et al., 2018); engineering global regulators (Cuervo et al., 2023); or ribosome engineering (Zhu et al., 2019). Nevertheless, we have to keep in mind that one of the major bottlenecks in drug discovery throughout history was the constant rediscovery of known compounds. From this perspective, some smart bioinformatics genome mining approaches can increase the chances to identifying unknown smBGCs encoding new compounds with potentially clinical applications. For example, several strategies have been used in recent years like mining for resistance genes (Culp et al., 2020), or for Streptomyces Antibiotic Regulatory Protein genes (Ye et al., 2023). Additionally, searching genes involved in the biosynthesis of unusual functional groups has also been used as an approach to select new smBGCs, such as targeting halogenases genes (Prado-Alonso et al., 2022); DNA regions in Polyketide Synthases encoding the didomain DUF–SH specific for sulfur incorporation (Pan et al., 2017); C-terminal thioester reductase (TR) domains and ϖ-transaminases (Awodi et al., 2017); or piperazate synthase encoding genes (García-Gutiérrez et al., 2024; Morgan et al., 2020). Another important application of genome mining is as reservoir of genetic sets and devices for being used in combinatorial biosynthesis strategies. This method squeezes the maximum of synthetic biology techniques, by using different genetic engineering strategies to generate smBGCs with novel gene combinations. These would encode novel biosynthetic pathways that potentially could direct the biosynthesis of new natural products with different or improved properties. Combinatorial biosynthesis encompasses several strategies such as combination of native biosynthetic genes and genes from other smBGCs, expression of genes from other smBGCs into mutants blocked at specific biosynthetic steps, mutasynthesis based on the use of different biosynthetic precursors, or all of the above strategies combined to obtain new structural units (Olano et al., 2009). This method has been successfully used for the biosynthesis of new derivatives of a wide variety of compounds like terpenes (Tang et al., 2022), non ribosomal peptides (Ruijne & Kuipers, 2021), RiPPs (ribosomal synthesized and post-translationally modified peptides) (Sardar & Schmidt, 2016), polyketides (Wang et al., 2022) or nucleosides (Niu et al., 2017). An interesting example was the generation of the new glycosylated analog demycarosyl-3D-β-D-digitoxosylmithramycin SK (Figure 2), derived from mithramycin (Núñez et al., 2012). Production of this compound was achieved by providing the capability to synthesize D-digitoxose to a S. argillaceus strain mutated in a ketoreductase gene of the mithramycin BGC. This analog showed high antitumor activity and less toxicity than the parental compound, and among others, it is able to suppress EWS-FLI1 activity suggesting a potential development in clinical (Osgood et al., 2016). Another example was the production of epirubicin (Figure 2), a less cardiotoxic doxorubicin derivative, which initially was produced by semisynthesis. A new method was designed for its production consisting in expressing avrE or eryBIV from the avermectin and erythromycin gene clusters into a S. peucetius doxorubicin non-producer mutant (Demain & Vaishnav, 2011). To summarize the current state of the art we can highlight a study carried out by Malmierca et al. (2018, 2020), which illustrates the combination of different chromatographic, genome mining, nutritional and combinatorial biosynthesis approaches, as an effective strategy for identifying new NPs. This research was conducted on Streptomyces strains isolated from symbiotic associations with leaf cutters ants of the Attini tribe. These ants maintain close association with Streptomyces that produce bioactive compounds, including antifungals and inhibitors of Escovopsis weberi, a parasitic microfungus of their mutualistic Basidiomycete fungi (Seipke et al., 2011). Malmierca et al. mined those Streptomyces genomes searching for smBGCs encoding glycosylated secondary metabolites, since many therapeutically relevant drugs contain sugar moieties (Salas & Méndez, 2007). By a combination of genome mining, PCR screening, metabolites dereplication, as well as genetic and nutritional approaches, they identified two novel compounds of the cervimycins family (sipanmycin A and B), and two novel members of the warkmycin family (Malmierca et al., 2018). Also, by combinatorial biosynthesis, expressing plasmids for the biosynthesis of deoxysugars into the sipanmycin producer Streptomyces CS149, they generated six different derivatives with altered glycosylation patterns (Malmierca et al., 2020). Recent research, some of them summarized in this Editorial article suggest that Streptomyces remains the leading producer of bioactive compounds. This article emphasizes the contribution of these microorganisms to achieving SDG3. Moreover, recent years have seen the implementation of new methods that have revitalized the discovery of new natural products, accentuating the promising potential of Streptomyces. Even though, despite the discovery of new Streptomyces species and the identification of a large number of hypothetical smBGCs through genome mining research, only a small fraction of them have been characterized so far. This is mainly due to the limitations of these methods. For example, culturing new Streptomyces species from extreme environments under laboratory conditions is usually a challenge, as well as the heterologous expression of smBGCs, which is difficult and time-consuming. Related to genome mining, one of the major issues is the quality of genomic sequences. Most of the sequences in public databases are in draft form. Although incomplete genome sequences may be adequate for assembling many small, non-repetitive secondary metabolites smBGCs, they are unsuitable for large smBGCs like those encoding NRPS or type I PKS. These enzymes are typically involved in the biosynthesis of most compounds identified in drug discovery programs. Consequently, their encoding genes are often predicted to be scattered through several contigs, making challenging to identify the corresponding smBGCs (Baltz, 2021). Another drawback is that although powerful methods for the prediction of product structure from sequences exist, like antiSMASH (Blin et al., 2023), PRISM (Skinnider et al., 2020) or MIBiG (Terlouw et al., 2023) among others, they still have a relative high rate of false positives and generally are limited to identify smBGCs related to known ones. Moreover, once hypothetical smBGCs have been located, it remains a huge challenge to activate them. Additionally, predicting smBGCs is worthless without linking them to their final product and/or expected biological activities (Lee et al., 2020; Olano, Méndez, & Salas, 2014; Ren et al., 2017). On the other hand, it is interesting to note that although the new strategies developed in Streptomyces have shown the potential to discover pharmaceutically important drugs, they have not been successfully integrated into pharmaceutical company pipelines. This could be due to several factors, such as low throughput fermentation, challenges in natural product optimization, and declining return on investment (Baltz, 2021; Ward & Allenby, 2018). Miriam Rodríguez: Writing – original draft; writing – review and editing. Lorena Cuervo: Writing – original draft. Laura Prado-Alonso: Writing – original draft. María Soledad González-Moreno: Writing – original draft. Carlos Olano: Writing – review and editing; funding acquisition. Carmen Méndez: Writing – review and editing; funding acquisition. This work was granted by a grant from the Spanish Ministry of Science and Innovation (PID2020-113062RB-100) to CM, and by a grant from the Spanish Ministry of Science, Innovation, and Universities to CO (RTI2018-093562-B-I00). The authors declare no competing financial interest.

Secondary metabolite biosynthesis genes in fungi are usually physically linked and organized in large gene clusters. The physical linkage of genes involved in the same biosynthetic pathway minimizes the amount of regulatory steps necessary to regulate the biosynthetic machinery and thereby contributes to physiological economization. Regulation by chromatin accessibility is a proficient molecular mechanism to synchronize transcriptional activity of large genomic regions. Chromatin regulation largely depends on DNA and histone modifications and the histone code hypothesis proposes that a certain combination of modifications, such as acetylation, methylation or phosphorylation, is needed to perform a specific task. A number of reports from several laboratories recently demonstrated that fungal secondary metabolite (SM) biosynthesis clusters are controlled by chromatin-based mechanisms and histone acetyltransferases, deacetylases, methyltransferases, and proteins involved in heterochromatin formation were found to be involved. This led to the proposal that establishment of repressive chromatin domains over fungal SM clusters under primary metabolic conditions is a conserved mechanism that prevents SM production during the active growth phase. Consequently, transcriptional activation of SM clusters requires reprogramming of the chromatin landscape and replacement of repressive histone marks by activating marks. This review summarizes recent advances in our understanding of chromatin-based SM cluster regulation and highlights some of the open questions that remain to be answered before we can draw a more comprehensive picture.

Oncology immunotherapy has gained significant advances in recent years and benefits cancer patients with superior efficacy and superior clinical responses. Currently over ten immune checkpoint antibodies targeting CTLA-4 and PD-1/PD-L1 have received regulatory approval worldwide and over thousands are under active clinical trials. However, compared to the rapid advance of Monoclonal Antibody (mAb), studies on immunotherapeutic small molecules have far lagged behind. Small molecule immunotherapy not only can target immunosuppressive mechanisms similar to mAbs, but also can stimulate intracellular pathways downstream of checkpoint proteins in innate or adaptive immune cells that mAbs are unable to access. Therefore, small molecule immunotherapy can provide an alternative treatment modality either alone or complementary to or synergistic with extracellular checkpoint mAbs to address low clinical response and drug resistance. Fortunately, remarkable progress has achieved recently in the pursuit of small molecule immunotherapy. This review intends to provide a timely highlight on those clinically investigated small molecules targeting PD-1/PD-L1, IDO1, and STING. The most advanced IDO1 inhibitor epacadostat have been aggressively progressed into multiple clinical testings. Small molecule PD-1/PD-L1 inhibitors and STING activators are still in a premature state and their decisive application needs to wait for the ongoing clinical outcomes. Since no small molecule immunotherapy has been approved yet, the future research should continue to focus on discovery of novel small molecules with distinct chemo-types and higher potency, identification of biomarkers to precisely stratify patients, as well as validation of many other immune-therapeutic targets, such as LAG3, KIRs, TIM-3, VISTA, B7-H3, and TIGIT.

Interactions of quercetin, curcumin, epigallocatechin gallate and folic acid with gelatin

Fungal secondary metabolism.

Within the last few years the Helmholtz Zentrum München has established several initiatives enabling the translation of basic research results into discovery of novel small molecules that affect pathomechanisms of chronic and complex diseases. Here, one of the main operations is the Assay Development and Screening Platform (ADSP) that has state-of-the-art equipment for compound screening and provides knowledge in a variety of biochemical or cell-based phenotypic assays. In particular, ADSP has a strong focus on complex assays such as high-content screening in stem cells that are likely to provide an innovative approach complementary to biochemical assays for the discovery of novel small molecules modulating key biological processes.

Background:Capsular contracture is a devastating complication that occurs in patients undergoing implant-based breast reconstruction. Ionizing radiation drives and exacerbates capsular contracture in part by activating cytokines, including transforming growth factor-beta (TGF-β). TGF-β promotes myofibroblast differentiation and proliferation, leading to excessive contractile scar formation. Therefore, targeting the TGF-β pathway may attenuate capsular contracture.Methods:A 20,000 small molecule library was screened for anti-TGF-β activity. Structurally diverse anti-TGF-β agents were identified and then tested on primary human capsular fibroblasts. Fibroblasts were irradiated or not, and then treated with both TGF-β and candidate molecules. Resulting cells were then analyzed for myofibroblast activity using myofibroblast markers including alpha-smooth muscle actin, collagen I, Thy1, and periostin, using Western Blot, quantitative real-time polymerase chain reaction, and immunofluorescence.Results:Human capsular fibroblasts treated with TGF-β showed a significant increase in alpha-smooth muscle actin, collagen I, and periostin levels (protein and/or mRNA). Interestingly, fibroblasts treated with latent TGF-β and 10 Gy radiation also showed significantly increased levels of myofibroblast markers. Cells that were treated with the novel small molecules showed a significant reduction in myofibroblast activation, even in the presence of radiation.Conclusions:Several novel small molecules with anti-TGF-β activity can effectively prevent human capsular fibroblast to myofibroblast differentiation in vitro, even in the presence of radiation. These results highlight novel therapeutic options that may be utilized in the future to prevent radiation-induced capsular contracture.

Discovery and characterization of selective small molecule inhibitors of the mammalian mitochondrial division dynamin, DRP1