A systematic quantitative approach comprehensively defines domain-specific functional pathways linked to Schizosaccharomyces pombe heterochromatin regulation.

Heterochromatin plays a critical role in regulating gene expression and maintaining genome integrity. While structural and enzymatic components have been linked to heterochromatin establishment, a comprehensive view of the underlying pathways at diverse heterochromatin domains remains elusive. Here, we developed a systematic approach to identify factors involved in heterochromatin silencing at pericentromeres, subtelomeres, and the silent mating type locus in Schizosaccharomyces pombe. Using quantitative measures, iterative genetic screening, and domain-specific heterochromatin reporters, we identified 369 mutants with different degrees of reduced or enhanced silencing. As expected, mutations in the core heterochromatin machinery globally decreased silencing. However, most other mutants exhibited distinct qualitative and quantitative profiles that indicate domain-specific functions. For example, decreased mating type silencing was linked to mutations in heterochromatin maintenance genes, while compromised subtelomere silencing was associated with metabolic pathways. Furthermore, similar phenotypic profiles revealed shared functions for subunits within complexes. We also discovered that the uncharacterized protein Dhm2 plays a crucial role in maintaining constitutive and facultative heterochromatin, while its absence caused phenotypes akin to DNA replication-deficient mutants. Collectively, our systematic approach unveiled a landscape of domain-specific heterochromatin regulators controlling distinct states and identified Dhm2 as a previously unknown factor linked to heterochromatin inheritance and replication fidelity.

Lutein Isomers: Preparation, Separation, Structure Elucidation, and Occurrence in 20 Medicinal Plants.

Lutein and its cis-isomers occur in a lot of plants, including a variety of flowers. In this study, lutein isomers were produced via iodine-catalyzed isomerization, and four cis-isomers (9Z-, 9'Z-, 13Z-, and 13Z') were isolated by means of column chromatography and semipreparative HPLC. The structures of the 9'Z- and 13'Z-isomers were elucidated via NMR measurements. These compounds were used as standards for the HPLC-DAD-MS determination of the carotenoid composition of the flowers of 20 plant species, in which lutein and its geometrical isomers are the main components. The flowers showed great variation in their cis- and trans-lutein content, and also in the presence or absence of other carotenoids, such as violaxanthin, neoxanthin, ß-cryptoxanthin, and ß-carotene. Some of the investigated flowers were found to be rich sources of lutein without zeaxanthin.

Characterization of antibiotic resistomes by reprogrammed bacteriophage-enabled functional metagenomics in clinical strains.

Functional metagenomics is a powerful experimental tool to identify antibiotic resistance genes (ARGs) in the environment, but the range of suitable host bacterial species is limited. This limitation affects both the scope of the identified ARGs and the interpretation of their clinical relevance. Here we present a functional metagenomics pipeline called Reprogrammed Bacteriophage Particle Assisted Multi-species Functional Metagenomics (DEEPMINE). This approach combines and improves the use of T7 bacteriophage with exchanged tail fibres and targeted mutagenesis to expand phage host-specificity and efficiency for functional metagenomics. These modified phage particles were used to introduce large metagenomic plasmid libraries into clinically relevant bacterial pathogens. By screening for ARGs in soil and gut microbiomes and clinical genomes against 13 antibiotics, we demonstrate that this approach substantially expands the list of identified ARGs. Many ARGs have species-specific effects on resistance; they provide a high level of resistance in one bacterial species but yield very limited resistance in a related species. Finally, we identified mobile ARGs against antibiotics that are currently under clinical development or have recently been approved. Overall, DEEPMINE expands the functional metagenomics toolbox for studying microbial communities.

Synthesis and evaluation of a new class of MIF-inhibitors in activated macrophage cells and in experimental septic shock in mice.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with enzymatic activities. Anti-inflammatory effects of MIF enzyme inhibitors indicate a link between its cytokine- and catalytic activities. Herein the synthesis, docking, and bioactivity of substituted benzylidene-1-indanone and -1-tetralone derivatives as MIF-tautomerase inhibitors is reported. Many of these substituted benzylidene-1-tetralones and -indan-1-ones were potent MIF-tautomerase inhibitors (IC50 < 10 µmol/L), and the most potent inhibitors were the 1-indanone derivatives 16 and 20. Some of these compounds acted as selective enolase or ketonase inhibitors. In addition, compounds 16, 20, 26, 37 and 61 efficiently inhibited NO, TNFα and IL-6 production in lipopolysaccharide-induced macrophages. Compound 20, 37 and 61 also inhibited ROS generation, and compound 26 and 37 abolished activation of NF-κB. Compound 37 significantly augmented hypothermia induced by high dose of lipopolysaccharide in mice. The possible mechanisms of action were explored using molecular modelling and docking, as well as molecular dynamics simulations.

Novel cyclic C5-curcuminoids penetrating the blood-brain barrier: Design, synthesis and antiproliferative activity against astrocytoma and neuroblastoma cells.

Novel series of cyclic C5-curcuminoids 17a-j and 19-22 were prepared as cytotoxic agents and evaluated against human neuroblastoma (SH-SY5Y) or human grade IV astrocytoma (CCF-STTG1) cell lines in low (∼0.1 nM - 10 nM) concentrations. Among the tested 21 derivatives, 16 displayed potent antiproliferative activity with IC50 values in the low nanomolar to picomolar range (IC50 = 7.483-0.139 nM). Highly active compounds like N-monocarboxylic derivative 19b with IC50 = 0.139 nM value against neuroblastoma and N-alkyl substituted 11 with IC50 = 0.257 nM against astrocytoma proved some degree of selectivity toward non-cancerous astrocytes and kidney cells. This potent anticancer activity did not show a strong correlation with experimental logPTLC values, but the most potent antiproliferative molecules 11-13 and 19-22 are belonging to discrete subgroups of the cyclic C5-curcuminoids. Compounds 12, 17c and 19b were subjected to blood-brain barrier (BBB) penetration studies, too. The BBB was revealed to be permeable for all of them but, as the apparent permeability coefficient (Papp) values mirrored, in different ratios. Lower toxicity of 12, 17c and 19b was observed toward primary rat brain endothelial cells of the BBB model, which means they remained undamaged under 10 µM concentrations. Penetration depends, at least in part, on albumin binding of 12, 17c and 19b and the presence of monocarboxylic acid transporters in the case of 19b. Permeation through the BBB and albumin binding, we described here, is the first example of cyclic C5-curcuminoids as to our knowledge.

Proteome-wide landscape of solubility limits in a bacterial cell.

Proteins are prone to aggregate when expressed above their solubility limits. Aggregation may occur rapidly, potentially as early as proteins emerge from the ribosome, or slowly, following synthesis. However, in vivo data on aggregation rates are scarce. Here, we classified the Escherichia coli proteome into rapidly and slowly aggregating proteins using an in vivo image-based screen coupled with machine learning. We find that the majority (70%) of cytosolic proteins that become insoluble upon overexpression have relatively low rates of aggregation and are unlikely to aggregate co-translationally. Remarkably, such proteins exhibit higher folding rates compared to rapidly aggregating proteins, potentially implying that they aggregate after reaching their folded states. Furthermore, we find that a substantial fraction (~ 35%) of the proteome remain soluble at concentrations much higher than those found naturally, indicating a large margin of safety to tolerate gene expression changes. We show that high disorder content and low surface stickiness are major determinants of high solubility and are favored in abundant bacterial proteins. Overall, our study provides a global view of aggregation rates and hence solubility limits of proteins in a bacterial cell.

Gene loss and compensatory evolution promotes the emergence of morphological novelties in budding yeast.

Deleterious mutations are generally considered to be irrelevant for morphological evolution. However, they could be compensated by conditionally beneficial mutations, thereby providing access to new adaptive paths. Here we use high-dimensional phenotyping of laboratory-evolved budding yeast lineages to demonstrate that new cellular morphologies emerge exceptionally rapidly as a by-product of gene loss and subsequent compensatory evolution. Unexpectedly, the capacities for invasive growth, multicellular aggregation and biofilm formation also spontaneously evolve in response to gene loss. These multicellular phenotypes can be achieved by diverse mutational routes and without reactivating the canonical regulatory pathways. These ecologically and clinically relevant traits originate as pleiotropic side effects of compensatory evolution and have no obvious utility in the laboratory environment. The extent of morphological diversity in the evolved lineages is comparable to that of natural yeast isolates with diverse genetic backgrounds and lifestyles. Finally, we show that both the initial gene loss and subsequent compensatory mutations contribute to new morphologies, with their synergistic effects underlying specific morphological changes. We conclude that compensatory evolution is a previously unrecognized source of morphological diversity and phenotypic novelties.

Analyzing the Carotenoid Composition of Melilot (Melilotus officinalis (L.) Pall.) Extracts and the Effects of Isolated (All-E)-lutein-5,6-epoxide on Primary Sensory Neurons and Macrophages.

Melilotus officinalis is known to contain several types of secondary metabolites. In contrast, the carotenoid composition of this medicinal plant has not been investigated, although it may also contribute to the biological activities of the drug, such as anti-inflammatory effects. Therefore, this study focuses on the isolation and identification of carotenoids from Meliloti herba and on the effect of isolated (all-E)-lutein 5,6-epoxide on primary sensory neurons and macrophages involved in nociception, as well as neurogenic and non-neurogenic inflammatory processes. The composition of the plant extracts was analyzed by high performance liquid chromatography (HPLC). The main carotenoid was isolated by column liquid chromatography (CLC) and identified by MS and NMR. The effect of water-soluble lutein 5,6-epoxide-RAMEB (randomly methylated-ß-cyclodextrin) was investigated on Ca2+-influx in rat primary sensory neurons induced by the activation of the transient receptor potential ankyrin 1 receptor agonist to mustard-oil and on endotoxin-induced IL-1ß release from isolated mouse peritoneal macrophages. (all-E)-Lutein 5,6-epoxide significantly decreased the percent of responsive primary sensory neurons compared to the vehicle-treated stimulated control. Furthermore, endotoxin-evoked IL-1ß release from macrophages was significantly decreased by 100 µM lutein 5,6-epoxide compared to the vehicle-treated control. The water-soluble form of lutein 5,6-epoxide-RAMEB decreases the activation of primary sensory neurons and macrophages, which opens perspectives for its analgesic and anti-inflammatory applications.

Study on the Synthesis, Antioxidant Properties, and Self-Assembly of Carotenoid-Flavonoid Conjugates.

Flavonoids and carotenoids possess beneficial physiological effects, such as high antioxidant capacity, anticarcinogenic, immunomodulatory, and anti-inflammatory properties, as well as protective effects against UV light. The covalent coupling of hydrophobic carotenoids with hydrophilic flavonoids, such as daidzein and chrysin, was achieved, resulting in new amphipathic structures. 7-Azidohexyl ethers of daidzein and chrysin were prepared in five steps, and their azide-alkyne [4 + 2] cycloaddition with pentynoates of 8'-apo-ß-carotenol, zeaxanthin, and capsanthin afforded carotenoid-flavonoid conjugates. The trolox-equivalent antioxidant capacity against ABTS•+ radical cation and self-assembly of the final products were examined. The 1:1 flavonoid-carotenoid hybrids generally showed higher antioxidant activity than their parent flavonoids but lower than that of the corresponding carotenoids. The diflavonoid hybrids of zeaxanthin and capsanthin, however, were found to exhibit a synergistic enhancement in antioxidant capacities. ECD (electronic circular dichroism) and UV-vis analysis of zeaxanthin-flavonoid conjugates revealed that they form different optically active J-aggregates in acetone/water and tetrahydrofuran/water mixtures depending on the solvent ratio and type of the applied aprotic polar solvent, while the capsanthin derivatives showed no self-assembly. The zeaxanthin bis-triazole conjugates with daidzein and with chrysin, differing only in the position of a phenolic hydroxyl group, showed significantly different aggregation profile upon the addition of water.

Chemical-genetic profiling reveals limited cross-resistance between antimicrobial peptides with different modes of action.

Antimicrobial peptides (AMPs) are key effectors of the innate immune system and promising therapeutic agents. Yet, knowledge on how to design AMPs with minimal cross-resistance to human host-defense peptides remains limited. Here, we systematically assess the resistance determinants of Escherichia coli against 15 different AMPs using chemical-genetics and compare to the cross-resistance spectra of laboratory-evolved AMP-resistant strains. Although generalizations about AMP resistance are common in the literature, we find that AMPs with different physicochemical properties and cellular targets vary considerably in their resistance determinants. As a consequence, cross-resistance is prevalent only between AMPs with similar modes of action. Finally, our screen reveals several genes that shape susceptibility to membrane- and intracellular-targeting AMPs in an antagonistic manner. We anticipate that chemical-genetic approaches could inform future efforts to minimize cross-resistance between therapeutic and human host AMPs.

Novel Cell Wall Antifungals Reveal a Special Synergistic Activity in pbr1 Mutants Resistant to the Glucan Synthesis Antifungals Papulacandins and Echinocandins.

A series of 4-(arylmethylene)-3-isochromanones have been prepared with base-catalyzed Knoevenagel condensation starting from 3-isochromanone and aromatic aldehydes. The outcome of the reaction- the isomeric composition of the products depends on the aromatic aldehyde applied. These reactions afforded mostly the more stable E-diastereoisomer, but some condensations resulted in the Z-diastereoisomer or mixture of the stereoisomers (1-16). The products showed antifungal effect against some pathogenic fungi. We wanted to extend this study and to synthesize a new generation of 4-(arylmethylene)-3-isochromanones. These condensations led mostly to E-diastereoisomers (17-30). The structure verifications were performed by FT IR, 1H and13C NMR methods. Both the 1-16 and the novel 17-30 compounds have been screened against the three yeast models, fission yeast Schizosaccharomyces pombe (wild-type, and pbr1-6 and pbr1-8 mutants resistant to specific cell wall synthesis inhibitors), budding yeast Saccharomyces cerevisiae (wild-type and pbr1-1) and pathogenic yeast Candida albicans (wild-type, ATCC 26555, 90028 and SC5314). Osmotic protection with sorbitol attenuated the in vivo inhibition in living cells suggesting a cell wall-specific antifungal effect. Moreover, the S. pombe wild-type and mutant strains were tested for their resistant or sensitive in vitro ß(1,3)-glucan synthase (GS) activity. We found both in vivo in living cells and in vitro in the enzymatic GS assay a synergistic effect of higher sensitivity of the pbr1 mutants resistant to the specific GS inhibitors papulacandins and echinocandins. These results may provide new insights into new strategies of combined antifungal therapy of GS inhibitors directed against spontaneous mutants resistant to echinocandins.

Limited Evolutionary Conservation of the Phenotypic Effects of Antibiotic Resistance Mutations.

Multidrug-resistant clinical isolates are common in certain pathogens, but rare in others. This pattern may be due to the fact that mutations shaping resistance have species-specific effects. To investigate this issue, we transferred a range of resistance-conferring mutations and a full resistance gene into Escherichia coli and closely related bacteria. We found that resistance mutations in one bacterial species frequently provide no resistance, in fact even yielding drug hypersensitivity in close relatives. In depth analysis of a key gene involved in aminoglycoside resistance (trkH) indicated that preexisting mutations in other genes-intergenic epistasis-underlie such extreme differences in mutational effects between species. Finally, reconstruction of adaptive landscapes under multiple antibiotic stresses revealed that mutations frequently provide multidrug resistance or elevated drug susceptibility (i.e., collateral sensitivity) only with certain combinations of other resistance mutations. We conclude that resistance and collateral sensitivity are contingent upon the genetic makeup of the bacterial population, and such contingency could shape the long-term fate of resistant bacteria. These results underlie the importance of species-specific treatment strategies.

Phylogenetic barriers to horizontal transfer of antimicrobial peptide resistance genes in the human gut microbiota.

The human gut microbiota has adapted to the presence of antimicrobial peptides (AMPs), which are ancient components of immune defence. Despite its medical importance, it has remained unclear whether AMP resistance genes in the gut microbiome are available for genetic exchange between bacterial species. Here, we show that AMP resistance and antibiotic resistance genes differ in their mobilization patterns and functional compatibilities with new bacterial hosts. First, whereas AMP resistance genes are widespread in the gut microbiome, their rate of horizontal transfer is lower than that of antibiotic resistance genes. Second, gut microbiota culturing and functional metagenomics have revealed that AMP resistance genes originating from phylogenetically distant bacteria have only a limited potential to confer resistance in Escherichia coli, an intrinsically susceptible species. Taken together, functional compatibility with the new bacterial host emerges as a key factor limiting the genetic exchange of AMP resistance genes. Finally, our results suggest that AMPs induce highly specific changes in the composition of the human microbiota, with implications for disease risks.

Antibiotic-resistant bacteria show widespread collateral sensitivity to antimicrobial peptides.

Antimicrobial peptides are promising alternative antimicrobial agents. However, little is known about whether resistance to small-molecule antibiotics leads to cross-resistance (decreased sensitivity) or collateral sensitivity (increased sensitivity) to antimicrobial peptides. We systematically addressed this question by studying the susceptibilities of a comprehensive set of 60 antibiotic-resistant Escherichia coli strains towards 24 antimicrobial peptides. Strikingly, antibiotic-resistant bacteria show a high frequency of collateral sensitivity to antimicrobial peptides, whereas cross-resistance is relatively rare. We identify clinically relevant multidrug-resistance mutations that increase bacterial sensitivity to antimicrobial peptides. Collateral sensitivity in multidrug-resistant bacteria arises partly through regulatory changes shaping the lipopolysaccharide composition of the bacterial outer membrane. These advances allow the identification of antimicrobial peptide-antibiotic combinations that enhance antibiotic activity against multidrug-resistant bacteria and slow down de novo evolution of resistance. In particular, when co-administered as an adjuvant, the antimicrobial peptide glycine-leucine-amide caused up to 30-fold decrease in the antibiotic resistance level of resistant bacteria. Our work provides guidelines for the development of efficient peptide-based therapies of antibiotic-resistant infections.

Cotranslational protein assembly imposes evolutionary constraints on homomeric proteins.

Cotranslational protein folding can facilitate rapid formation of functional structures. However, it can also cause premature assembly of protein complexes, if two interacting nascent chains are in close proximity. By analyzing known protein structures, we show that homomeric protein contacts are enriched toward the C termini of polypeptide chains across diverse proteomes. We hypothesize that this is the result of evolutionary constraints for folding to occur before assembly. Using high-throughput imaging of protein homomers in Escherichia coli and engineered protein constructs with N- and C-terminal oligomerization domains, we show that, indeed, proteins with C-terminal homomeric interface residues consistently assemble more efficiently than those with N-terminal interface residues. Using in vivo, in vitro and in silico experiments, we identify features that govern successful assembly of homomers, which have implications for protein design and expression optimization.

Correction: Phenotypic heterogeneity promotes adaptive evolution.

[This corrects the article DOI: 10.1371/journal.pbio.2000644.].

Phenotypic heterogeneity promotes adaptive evolution.

Genetically identical cells frequently display substantial heterogeneity in gene expression, cellular morphology and physiology. It has been suggested that by rapidly generating a subpopulation with novel phenotypic traits, phenotypic heterogeneity (or plasticity) accelerates the rate of adaptive evolution in populations facing extreme environmental challenges. This issue is important as cell-to-cell phenotypic heterogeneity may initiate key steps in microbial evolution of drug resistance and cancer progression. Here, we study how stochastic transitions between cellular states influence evolutionary adaptation to a stressful environment in yeast Saccharomyces cerevisiae. We developed inducible synthetic gene circuits that generate varying degrees of expression stochasticity of an antifungal resistance gene. We initiated laboratory evolutionary experiments with genotypes carrying different versions of the genetic circuit by exposing the corresponding populations to gradually increasing antifungal stress. Phenotypic heterogeneity altered the evolutionary dynamics by transforming the adaptive landscape that relates genotype to fitness. Specifically, it enhanced the adaptive value of beneficial mutations through synergism between cell-to-cell variability and genetic variation. Our work demonstrates that phenotypic heterogeneity is an evolving trait when populations face a chronic selection pressure. It shapes evolutionary trajectories at the genomic level and facilitates evolutionary rescue from a deteriorating environmental stress.

Carotenoid Composition of the Fruit of Red Mamey (Pouteria sapota).

The detailed carotenoid analysis of red mamey (Pouteria sapota) was achieved by HPLC-DAD-MS, chemical tests, and cochromatography with authentic samples. Altogether 47 components were detected and 34 identified from the total extract or after fractionation with column chromatography. The main carotenoids were cryptocapsin, sapotexanthin, and capsanthin 5,6-epoxide. Some further minor components containing the κ-end group with or without a hydroxy group and their 5,6-epoxy precursors were identified. Some comments are made about the biosynthesis of κ-carotenoids in red mamey.

Adaptive evolution of complex innovations through stepwise metabolic niche expansion.

A central challenge in evolutionary biology concerns the mechanisms by which complex metabolic innovations requiring multiple mutations arise. Here, we propose that metabolic innovations accessible through the addition of a single reaction serve as stepping stones towards the later establishment of complex metabolic features in another environment. We demonstrate the feasibility of this hypothesis through three complementary analyses. First, using genome-scale metabolic modelling, we show that complex metabolic innovations in Escherichia coli can arise via changing nutrient conditions. Second, using phylogenetic approaches, we demonstrate that the acquisition patterns of complex metabolic pathways during the evolutionary history of bacterial genomes support the hypothesis. Third, we show how adaptation of laboratory populations of E. coli to one carbon source facilitates the later adaptation to another carbon source. Our work demonstrates how complex innovations can evolve through series of adaptive steps without the need to invoke non-adaptive processes.